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Durham E-Theses
Studies on the thymus and ontogeny of lymphocyteheterogeneity in the clawed toad Xenopus laevis
(Daudin)
Williams, Nigel Howard
How to cite:
Williams, Nigel Howard (1981) Studies on the thymus and ontogeny of lymphocyte heterogeneity in theclawed toad Xenopus laevis (Daudin), Durham theses, Durham University. Available at Durham E-ThesesOnline: http://etheses.dur.ac.uk/7834/
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2
r-
Studies on the thymus and ontogeny of lymphocyte
heterogeneity i n the clawed toad Xenopus laevis (Daudin)
by
Nigel Howcurd Williams B.Sc. (Dunelm)
The copyright of this thesis rests with the author.
No quotation from it should be published without
his prior written consent and information derived
from it should be acl nowledged.
,.. being a thesis presented i n candidatvire
for the degree of Doctor of Philosophy
i n the University of Durham.
November 1981
( i )
Abstract
CONTENTS Page
( i i i )
CHAPTER 1: General Introduction
CHAPTER 2: Lymphocyte r e a c t i v i t y to 'T' and 'B' c e l l mitogens: Studies on thymus and spleen of the adult. Introduction Materials and Methods Results (a) - HTdR incorporation and v i a b i l i t y of
lymphocytes i n mitogen-free cultures (b) P r o l i f e r a t i v e responses of thymocytes
and splenocytes to T and B c e l l mitogens Discussion Tables Figures
CHAPTER 3: Ontogeny of mitogen responsiveness i n thymus and spleen Introduction Materials and Methods Results (a) Preliminary studies comparing miniaturised
and standard culture techniques (b) Ontogeny of mitogen responsiveness (c) Characterisation of thymocyte mitogen
responses ( i ) Autoradiographic evidence of T and
B mitogen stimulation ( i i ) The e f f e c t of nylon wool treatment
on T Eind B mitogen stimulation Discussion Tables Figures
CHAPTER 4: Mitogen and mixed lymphocyte culture responses following thymectomy: studies investigating putative T lymphocyte heterogeneity
Introduction Materials and Methods Results (a) Preliminary studies on mitogen
stimulation of PBL (b) Mitogen studies on lymphocytes from a l l o -
immune toadlets (c) MLC and mitogen studies on lymphocytes from
toadlets thymectomixed a t 3-4 weeks of age
Discussion Tables Figures
12 12 14 17
20 26 31
32 32 33 37
37 38
40
40
41 42 50 61
71
71 73 76
76
77
78 78 83 87
( i i ) Page
CHAPTER 5: Ontogeny of in^ vivo lymphocyte r e a c t i v i t y to the hapten trinitrophenyl conjugated to a thymus-dependent and thymus-independent c a r r i e r Introduction Materials and Methods Results (a) Ontogeny of induced splenocyte PFC
r e a c t i v i t y (b) Ontogeny of induced antigen-binding
c e l l s i n the spleen (c) Antibody forming c e l l s within the
thymus Discussion Tables Figures
CHAPTER 6: Thymus histogenesis over metamorphosis and i n organ culture Introduction Materials and Methods Results (a) Thymus morphology (b) Organ culture experiments Discussion Tables Figures
CHAPTER 7: Concluding remarks
Acknowledgements
Bibliography
89 89 91 95
95
96
98 98
104 110
113 113 114 117 117 119 120 123 124
132
138
139
( i i i )
DECLARATIC»}
Some of the work presented i n Chapters 2,3 and 4 form the
b a s i s of four publications*. Assistance of collaborators i n Chapters
2 and 4 i s g r a t e f u l l y acknowledged.
* ( i ) Horton, J.D., Smith, A.R., Williams, N.H., Smith, A., and
Sherif, N.E.H.S. (1980). "Lymphocyte r e a c t i v i t y to 'T' and
'B' c e l l mitogens i n Xenopus l a e v i s ; studies on thymus and
spleen". Devel. Comp. Immunol. £: 75-86.
Cii ] Horton, J.D., Smith, A.R., Williams, N.H., Edwards, B.F. and
Ruben, L.N. (1980). "B-equivalent lymphocyte developnent i n
the amphibian thymus?". I n : Development and Differentiation
of Vertebrate Lymphocytes, Ed. J.D. Horton. Elsevier/Amsterdam,
pp. 173-182.
( i i i ) Smith, A.R., Williams, N.H. and Horton, J,D. (1980).
" I n v i t r o r e a c t i v i t y of Xenopus lymphocytes to T c e l l mitogens
following a l l o g r a f t r e j e c t i o n emd to sheep erythrocytes
a f t e r i n vivo priming". I n : Phylogeny of Immunological Memory,
Ed. M.J. Manning. Elsevier/Amsterdam, pp. 197-205.
Civ) Williams, N.H. and Horton, J.D. (1981). "Ontogeny of
mitogen responsiveness i n thymus and spleen of Xenopus l a e v i s " .
I n : "Aspects of Developnental and Comparative Immunology I "
Ed. J.B. Solomon. Pergamon: Oxford, pp.493-494.
(iv)
STATEMENT OF COPYRIGHT
"The copyright of t h i s thesis r e s t s with the author. No
quotation from i t should be published without the prior
written consent and information derived from i t shoxild be
acknowledged".
(V)
ABSTRACT
(1) Functionally mature lymphocytes can be demonstrated i n v i t r o
by t h e i r p r o l i f e r a t i o n (measured by s c i n t i l l a t i o n counting) i n response
to mitogens. Studies i n Chapter 2 examine the responsiveness of adult
Xenopus lymphocytes from both the thymus and the spleen to B and T c e l l
mitogens. Optimum mitogen doses and l e v e l s of foetal c a l f servmi
supplementation are established. Good stimulation indices are found when
splenocytes are stimulated with both PHA and Con A. Thymocytes also
respond well to these T c e l l mitogens, but a small response to B c e l l
mitogens (E^. c o l i LPS and PPD) i s also found.
(2) The ontogeny of lymphocyte responsiveness to T and B c e l l mitogens
i s examined i n Chapter 3. Very l i t t l e stimulation i s found i n the l a r v a l
thymus, but shortly a f t e r the end of metamorphosis a thymocyte response
begins to emerge and by 6 months of age good stimulation i s found with
both T and B c e l l mitogens. While good l e v e l s of thymocyte stimulation
with the T c e l l mitogens are maintained i n l a t e r l i f e , the response to the
B c e l l mitogen LPS declines to only a low l e v e l by 12 months of age.
Mitogenic responses emerge i n the spleen af t e r the end of metamorphosis
i n a s i m i l a r manner to thymocytes and good responses were found i n a l l
the subsequent ages tested. The transient response of thymic c e l l s
to LPS, which i s maximal at 6 months of age, was confirmed by auto
radiography and was shown to be dependent on a population of nylon-wool
adherent lymphocytes. ^
(3) The l a r v a l thymus plays a c r i t i c a l role i n the establishment of
T c e l l functions, and Chapter 4 looks at the effect of thymectomy at
d i f f e r e n t stages of l a r v a l development on the subsequent alloimmune
response, mixed lymphocyte r e a c t i v i t y and T c e l l mitogen responsiveness.
The r e s u l t s reveal that (a) skin a l l o g r a f t rejection displayed by
7-day thymectomized animals i s effected i n the absence of spleen and
(vi)
blood lymphocytes reactive to T c e l l mitogens, (b) T-mitogen responsive
c e l l s and MLR T c e l l s ceinnot e a s i l y be separated into d i s t i n c t
populations i n terms of t h e i r degree of thymus dependency.
(4) Chapter 5 looks at the ontogeny of both T helper c e l l and B
c e l l function by examining the i n vivo antigen-binding r e a c t i v i t y
of splenocytes to a T-dependent (TNP-SRBC) and a T-independent (TNP-
LPS) antigen. Good l e v e l s of TtfP binding c e l l s can be induced i n
the spleen when larvae are only 3 weeks old following injection of
TNP-SRBC (after low dose priming with SRBC) or TNP-LPS. The l e v e l
of cuitigen binding i n both cases appears to peak at metamorphosis.
However, when c e l l u l a r antibody production was measured, no plaque
forming c e l l s could be induced i n the spleen u n t i l after the end
of metamorphosis.
(5) Chapter 6 presents (a) a b r i e f h i s t o l o g i c a l study of the thymus
i n l a t e l a r v a l l i f e and at metamorphosis. This work characterises
the dramatic depletion of thymocyte numbers that occurs at the end
of metamorphosis and the renewed lymphopoiesis immediately afterwards,
and (b) preliminary r e s u l t s on the e f f e c t of 7-day organ culture on
thymus histogenesis.
1
CHAPTER 1
General Introduction
Thymus function and lymphocyte heterogeneity i n birds and mammals
I t i s now beyond question that the thymus i s of central immunological
importance (Miller, 1979). The f i r s t major breakthrough that c l e a r l y
linked the thymus with the development of the immvme system came i n
1961, when Mill e r published h i s findings on the effects of thymectomy
i n the newborn mouse. He revealed that neonatal thymectomy (but not
ad<iLt thymectomy) was associated with reduced lymphocyte numbers i n
blood and lymphoid t i s s u e s and abrogated the a b i l i t y of the mouse to
r e j e c t skin a l l o g r a f t s . In the next few years i t became apparent
that neonatal thymectomy i n mammals not only affected c e l l u l a r immunity,
but also impaired antibody responses to some antigens (Miller, 1962,
1963) . I t was r e a l i s e d l a t e r that i t was only the performance,
not the l e v e l of antibody-forming c e l l s which was altered by neonatal
thymectomy (see discussion i n Miller, 1979).
In birds i t became cl e a r early on that two separate 'central'
lymphoid organs were responsible for estcQjlishing humoral and c e l l u l a r
immunity. Thus neonatal removal of the bursa of Fabricius (a
cloacal lymphoid organ found only i n birds) impeiired antibody
production but not a l l o g r a f t rejection i n chickens, whereas early
thymectomy isopaired alloimmune r e a c t i v i t y but had only variable
e f f e c t s on antibody production (Glick, 1956; Szenberg and Warner,
1962; Cooper, Peterson, South and Good, 1966). The search for a
mammalian equivalent of the avian bursa involved protracted
investigations (see Greaves, Owen and Raff, 1973, for discussion).
I t now appears that the foe t a l l i v e r and spleen are centrally
involved i n production of antibody-forming c e l l precursors i n the
mammaliem embryo (Owen, Raff and Cooper, 1975), the bone marrow
assuming t h i s role i n l a t e r l i f e .
These extirpation experiments, backed up with observations
on maumnals with congenital immunodeficient states (such as those on
the nude mouse, with i t s hypoplastic, non-lyii5>hoid thymus - see
Pantelovuris, 1971, 1973; Rygaard, 1973) gave r i s e to the concept
of thymus-dependent (T) and bursa-(or mammalian bursal-equivalent)-
dependent (B) lymphocytes for the 2 ontogenically-distinct c e l l u l a r
popvilations found i n the 'peripheral' lymphoid ti s s u e s (e.g. Iyn5)h
nodes). T and B lymphocytes are c l e a r l y also functionally d i s t i n c t
populations; thus T c e l l s , were shown to be ceiitr a l l y involved i n
c e l l u l a r immunity, while B lymphocytes gave r i s e to antibody-producing
c e l l precursors.
Why the thymus should play a role i n antibody production to
certain antigens ( c a l l e d 'thymus-dependent') remained a inystery u n t i l
the l a t e •60s. Then Claman, Chaperon and T r i p l e t t (1966) revealed
that c e l l s from mouse bone marrow and thymus (each independently capable
of very l i t t l e cintlbody production) could, vrtien injected together into
i r r a d i a t e d r e c i p i e n t s , a ct s y n e r g i s t i c a l l y to give a good antibody
response when challenged with foreign erythrocytes. Mareover, a l l the
antibody i n t h i s type of experiment was shown to be produced by
i n j e c t e d bone marrow c e l l s (Davies, Leuchars, Walls, Marchant and
E l l i o t t , 1967; Mitchell and M i l l e r , 1968). I t appeared that the
T lymphocytes somehow 'helped' bone marrow derived c e l l s to secrete
cintibody. The other l i n e of evidence showing 1/B c e l l cooperation
came from hapten-carrier experiments. A hapten i s a small, chemically
defined substance (e.g. trinitrophenyl (TNP)) which can combine
s p e c i f i c a l l y with antibody, but cannot by i t s e l f induce an immune
response; that i s i t i s antigenic but not immunogenic. To induce
an immune response a hapten must be coupled to a macromolecular
c a r r i e r to form an immunogenic complex. To obtain a good
restoration of secondary anti-hapten IgG antibody response i n
i r r a d i a t e d mice, i t was necessary to have present two separate
lymphocyte populations, one responsive to the c a r r i e r and one
to the hapten (Mitchison, 1971). C a r r i e r - s e n s i t i v e c e l l s were
subsequently shown to be thymus-derived, whereas hapten-sensitive
lymphocytes were bone marrow-derived (Raff, 1970). The precise
mechanism of T c e l l help i s s t i l l under debate, but i t undoubtedly
involves one or more signals from the T lymphocyte that triggers
the B c e l l d i r e c t l y or i n d i r e c t l y v i a macrophages (see Miller, 1979).
In more recent years, extensive research has shown that
mammalian effector T c e l l s (unlike B c e l l s , which seem functionally
f a i r l y homogeneous) are a functionally heterogeneous family of
lymphocytes. Cytotoxic (Miller, Brunner, Sprent, Russell and
Mitchell, 1971), helper and suppressor (Gershon and Kondo, 1972)
T c e l l sub-populations, as well as those involved in delayed-type
hypersensitivity reactions, have been i d e n t i f i e d and, furthermore,
characterised by both t h e i r surface differentiation antigens
(e.g. the Ly antigens described by Cantor and Boyse, 1977) and by
t h e i r expression of antigens encoded by the major histocompatibility
gene complex (MHC). I t i s now beginning to be appreciated that
MHC antigens play an important role i n T lymphocyte r e a c t i v i t y .
Thus i n inbred rodents, and possibly also i n humans and chickens,
r e a c t i v i t y of helper and cytotoxic T c e l l s are believed to be
r e s t r i c t e d by the MHC. I t has been known for some time that MHC
antigens are molecular markers of i n d i v i d u a l i t y : thus when
transplants are performed between MHC-disparate individuals,
graft r e j e c t i o n w i l l occur rapidly, in contrast to the much
better transplant s u r v i v a l i f MHC identity e x i s t s between donor
and host. The more recent finding i s that the MHC i s also of
v i t a l importance i n T c e l l recognition of foreign antigens where
the MHC i s not serving as the immunising difference (Miller, 1 9 7 9 ) .
Thus T helper c e l l collaboration with resting B lymphocytes w i l l ,
i n general, occur only i f these l3^phocytes express the same,
lymphocyte-defined MHC antigens (Katz and Benacerraf, 1975;
Andersson, Schreier cuid Melchers, 1980), and T cell-mediated
c y t o t o x i c i t y s p e c i f i c for virus-altered (Zinkeamagel and Doherty,
1 9 7 4 ) , or hapten-modified c e l l s (Shearer, Rehn and Garbarino, 1975)
requires serologically-defined MHC identity of T lymphocytes with
alt e r e d target c e l l s . Although i t has seemed l i k e l y from mouse
chimera studies that i t i s within the thymus that T c e l l s learn
to become MHC r e s t r i c t e d during development (Zinkemagel, 1978 ) ,
recent experiments suggest that t h i s 'learning' within the thymus
may not alone be responsible for determining the range of MHC
r e s t r i c t i o n (see Howard, 1 9 8 0 ) .
Concomitant with the discoveries of the functional complexity
of the thymus-dependent lymphocyte population i n mammals, and the
possible role of the thymus i n MHC r e s t r i c t i o n , have been investigations
to determine the precise role that the thymus plays i n T lymphocyte
development and d i f f e r e n t i a t i o n (see review by Jordan and Robinson,
1981 and by Owen et a l . 1 9 7 7 ) . i t has become apparent that the
thymus does not play a ' s t a t i c ' role i n t h i s context but, rather.
i s involved i n a precise ontogenetic sequence. Early work by
Moore and Owen (1967) on parabiosed chick embryos showed that
there was an input of haemopoietic stem c e l l s (T and B lymphocytes
are believed to derive from a common stem c e l l source) into the
embryonic thymus. Owen and R i t t e r (1969) showed (using an organ
culture technique) that stem c e l l inflow to the, thymus begins a t
a precise time. Thus explants of 6-day chicken thymuses f a i l e d
to become lymphoid in^ v i t r o , whereas 7-day old thymuses displayed
lynphocyte development when placed i n cultiore. The quail-chick
chimera system has subsequently proved valuable i n analysing the
b r i e f periods of development when the thymus becomes attractive
for stem c e l l s (see Le Douarin, 1977). After entering the thymus,
stem c e l l s p r o l i f e r a t e i n the thymic cortex and then lymphocytes
migrate towards the medulla (Borvim, 1968). Lymphocytes are thought
to leave the thymus through the medullary venules (Saint-Marie, 1973).
I t has been suggested that intrathymic MHC r e s t r i c t i o n of mammcdian
T c e l l s i s dependent on contact of thymus lyn^jhocyte with the thymic
epithelium during ontogeny, and i s determined by the haplotype of
the thymic e p i t h e l i a l c e l l s rather than the thymus-lymphocytes
themselves (Zinkernagel, 1978). This r e s t r i c t i n g property of thymus
e p i t h e l i a l c e l l s i s believed to extend through ontogeny at l e a s t
u n t i l juvenile stages (see Triesman, 1981). Organ culture studies
on the thymus (see reviews by Robinson, 1980; Robinson and Owen,
1976, 1977; Mandel and Kennedy, 1978) and sequential thymectomy
experiments (Shimamoto, 1980) reveal that there i s a sequential
emergence of T lymphocyte populations i n mammals.
Phylogeny of T and B equivalent lymphocytes
The central importeuice of ontogenetic events i n the establishment
of the immune system i n mammals and birds makes the study of the
phylogeny of the immune system p a r t i c u l a r l y important. The
relationship between the development of the individual and the
evolution of species and lineages has been an enduring question
i n evolutionary biology. Ontogeny recapitulates phylogeny was
Haekel's answer i n the nineteenth century (see Gould, 1978).
Although a b e l i e f i n recapitulation i s no longer tenable, further
understanding of the relationship between these two phenomena
i s of continuing i n t e r e s t . Heterochronic differences i n the
ontogeny of diverse inmiune systems highlight the importance of
phylogenetic study, of not only vertebrates but invertebrates
too. Developmental and comparative immunology i s now a well
established area of research, and several symposia (see Hildemann
and Benedict, 1975; Wright and Cooper, 1976; Solomon and Horton,
1977; Horton, 1980; Manning, 1980; Solomon, 1981), review a r t i c l e s
(Du Pasquier, 1973, 1976; Borysenko, 1976; Cohen, 1977) and books
(Meuining and Turner, 1976; Cooper, 1976; Gershwin and Cooper, 1978;
Marchalonis, 1977), have defined the central issues of this research
f i e l d . Primordial cell-mediated immunity, characterised by
cytotoxic r e a c t i v i t y following a l l o g r a f t s e n s i t i s a t i o n and by
s p e c i f i c a l l y inducible memory, has been demonstrated in parazoans,
coelenterates, annelids and echinoderms. Furthermore, mixed
lymphocyte culttare (MLC) r e a c t i v i t y has been demonstrated i n
tunicates. These findings suggest that immunocytes functionally-
equivalent to c e r t a i n mammalian T lymphocytes can e x i s t i n the
absence of a thymus. Thus, although invertebrate immunocytes
often develop within s p e c i a l c e l l u l a r aggregations (such as the
a x i a l organ of echinoderms and the g i l l - a s s o c i a t e d nodules of
tunica tes) , no true thymus e x i s t s i n einy backboneless animal.
Although htamoral factors are known to augment phagocytosis i n many
invertebrates, no immunoglobulin (Ig) molecules have yet been found.
Functionally-equivalent T and B c e l l s , s p e c i f i c immunological
memory and Ig's e x i s t i n a l l vertebrates, including agnathan fishes
which are the only vertebrates where there i s no convincing evidence
for the presence of a thymus. (Good, Finstad, Pollara and Gabrielsen,
(1966) have described cui aggregation of lymphocytes i n the l a r v a l
lamprey which they think may be a protothymus, and Riviera, Cooper,
Reddy and Hildemann (1975) suggest the presence of a protothyimis
within the velar muscle complex of the P a c i f i c hagfish). T c e l l -
equivalent functions, such as acute graft rejection and strong
MLC reactions, are two of the functional assays associated with
dispa r i t y a t the MHC. The presence of these reactions i n 'advanced'
amphibians (anurans), 'advanced' fishes ( t e l e o s t s ) , birds ('advanced'
r e p t i l e s ? ) and mammals, but t h e i r absence i n 'primitive' fishes
(e.g. agnatheuis, chondrichthyans and chondrosteans), 'primitive'
amphibians (urodeles and apodans) and reptileis, has led Cohen and
Co l l i n s (1977) to suggest that the MHC may have evolved independently
on four separate occasions during vertebrate phylogeny, and may
r e f l e c t convergent gene evolution. I t seems l i k e l y , however, that
components of the MHC also e x i s t i n some of the 'primitive' forms,
but a l l e l i c polymorphism may be minimal i n these.
The functional dissociation of the ectothermic immune system
into heterogeneous populations of lymphocytes has been a major
consideration of conparative immunology, and a variety of techniques
have been used for t h i s purpose. D i f f e r e n t i a l mitogenesis has been
of importance i n t h i s respect. I n mammals different mitogens
s p e c i f i c a l l y stimulate either T or B lynphocytes and studies using
8
these mitogens have now been performed i n a l l vertebrate groups
(see Chapter 2 for review).' A second potent method for study of
lymphocyte heterogeneity i n poikilotherms has involved the use of
hapten-carrier experiments (see Chapter 5 for review). Lymphocyte
cooperation (suggesting at l e a s t two different lymphoid populations)
i n himioral immune responses heis been suggested i n both teleost
f i s h and amphibians, but seems to be absent i n agnathans ( F u j i i ,
Nakagawa and Murakawa, 1979).
The extent to which immune maturation i s dependent upon the
thymus throughout the vertberates has been examined i n thymus
ablation experiments. Thymectomy i n the teleost f i s h , the
Mozambique mouth brooder ( S a i l e n d r i , 1973) reveals a slow ontogeny
of T-dependent immune functions i n t h i s species. Thymectomy of
4 month old f i s h s t i l l caused extended surv i v a l of scale a l l o g r a f t s ,
while at 2 months i t suppressed humoral immune r e a c t i v i t y to
subsequent challenge with sheep erythrocytes. The e f f e c t of
thymectomy on T-independent antigens was not examined by Sailendri,
so we cannot conclude that inpaired humoral immunity i s due to
deletion of T c e l l s . I t i s possible that both T and B c e l l s are
spawned i n the thymus of some f i s h (see Chapter 2) . Adult thymectomy
has no e f f e c t on the a l l o g r a f t response of rainbow trout (Botham,
Grace and Manning, 1980), as i n the l i z a r d , Calotes versicolor
(Manickavel, 1972), although a v i t a l role of the r e p t i l i a n thymus
i n lymphocyte maturation i s suggested by the use of a n t i r '-thymocyte
serum i n t h i s species, which r e s u l t s i n a profound lynphopoenia i n
thymus (and spleen) and suppresses a l l o g r a f t rejection.
Amongst the ec to thermic vertebrates, i t i s the amphibian
c l a s s , i n p a r t i c u l a r the anuran order, which has received most
attention with respect to the e f f e c t of thymectomy on immvme
maturation. The thymus i s c l e a r l y the major source of graft
r e j e c t i o n c e l l s i n both urodeles (Charlemagne and Bouillon, 1968;
Cohen, 1969, 1970; Toumefier, 1972, 1973; Charlemagne, 1974;
Fache and Charlemagne, 1975) and anurans (Cooper and Hildemann,
1965; Du Pasquier, 1965; Curtis and Volpe, 1971; Horton and
Manning, 1972; Tochinai and Ka t a g i r i , 1975; Rimmer and Horton,
1977). Although the role of the anuran thymus i n antibocty
production seems broadly oompaurable to that occurring i n mammals
(see below), the situation i n urodeles seems more uncertain in
t h i s respect. Thus responses to mammalian T-dependent antigens
are e i t h e r minimally impaired (Toumefier and Charlemagne, 1975)
or increased (Charlemagne and Ttoumefier, 1977) following thymectomy.
Possibly humoral immune regtilation i n urodeles i s not effected
by helper T c e l l s . Indeed i t may be that urodeles recognise
a l l cintigens as T-independent (see Ruben and Edwards, 1980, for
discussion).
The clawed toad (actually a pipid frog - see Deuchar, 1972),
Xenopus l a e v i s , has proved p a r t i c u l a r l y useful i n the study of
functional dichotomy of the anuran immune system into T-dependent
and T-independent con4>onents (see Cohen and Turpen, 1980, for review).
Thymectomy early i n l i f e can e a s i l y be achieved i n this species, which
does not runt a f t e r the operation (Horton and Manning, 1974).
I t has been shown that early thymectomy impairs a l l o g r a f t rejection
(Horton and Manning, 1972), abrogates humoral immune r e a c t i v i t y to
10
c l a s s i c a l T-dependent antigens (K>chinai and Katagiri, 1975;
Turner and Manning, 1974; Horton, Rinnner emd Horton, 1976) and
abolishes i n v i t r o mixed leucocyte culture (MLC) r e a c t i v i t y
CDu Pasquier and Horton, 1976) and responsiveness to the T c e l l
mitogens phytohaemagglutinin (PHA) and Concanavalin A (Con A)
(Du Pasquier and Horton, 1976; Manning, Donnelly and Cohen, 1976;
Donnelly, Manning and Cohen, 1976), whereas i t has no apparent
e f f e c t on antibody production to c l a s s i c a l T-independent antigens
( C o l l i e , Turner and Manning, 1975; Tochinai, 1976) or on i n vitro
lyii5)hocyte stimulation by the putative B c e l l mitogens
E . c o l i lipopolysaccharide (LPS) and p u r i f i e d protein derivative
of tuberculin (PPD) (Manning e t a l . , 1976). Horton and Sherif's
(1977) sequential thymectomy studies have suggested the p o s s i b i l i t y
of sequential establishment of different T c e l l subsets i n Xenopus
(see Chapter 4 ) .
The work i n t h i s Thesis atten5>ts to gain further insight into
the development of thymus function and lymphocyte"' heterogeneity
i n Xenopus. Chapter 2 examines the culture conditions necessary
for obtaining good mitogen responses i n thymus and spleen to putative
T and B c e l l mitogens, and forms the basis for work i n the next
Chapter. Chapter 3 investigates the ontogeny of mitogen reactive
c e l l s i n thymus and spleen, to gain d i r e c t insight into the time
when lymphocytes responsive to putative T and B c e l l mitogens f i r s t
appear, and to follow t h i s responsiveness over the immunologically-
i n t e r e s t i n g period of metamorphosis. The p o s s i b i l i t y of T lymphocyte
heterogeneity i n Xenopus i s assessed i n thymectony experiments
performed i n Chapter 4. This work centres on the i n v i t r o
11
p r o l i f e r a t i v e c a p a b i l i t i e s (mitogen and MLC r e a c t i v i t y ) of
lyaaphocytes taken from toadlets thymectomised i n early or mid-
l a r v a l l i f e . Chapter 5 looks a t the i n vivo ontogeny of antigen-
reactive T-and B-eq\iivalent c e l l s i n the spleen with the aid of
T-dependent and T-independent hapten-carrier coii5>lexes. As i n
Chapter 3, i t i s the l a r y a l and perimetamorphic periods which
are the main focus of study. The ontogeny of splenic antibody-
secreting c e l l s , and the p o s s i b i l i t y of antigen production within
the thymus are also b r i e f l y considered i n Chapter 5. The
penultimate Chapter i s concerned with the h i s t o l o g i c a l changes
occurring i n the thymus over metamorphosis. I t also examines
the h i s t o l o g i c a l outcome of organ culturing the l a r v a l thymus.
The major findings from the work i n t h i s Thesis are outlined
i n Chapter 7.
12
CHAPTER 2
Lymphocyte r e a c t i v i t y to 'T' and 'B' c e l l mitogens ; Studies on Thymus
and Spleen of the adult
Introduction
Hie d i f f e r e n t i a l responsiveness of lyn^jhocytes to mitogens has
been a major means of distingviishing T and B c e l l s i n mammals and birds
Greaves and JanOssy, 1972). The phytomitogens PHA, an extract of
the red kidney bean Phaseolus vulgaris (Nowell, 1960), and Con A,
an e x t r a c t of the Jack bean Canavadia ensiforois (Powell and Leon,
1970), exclusively stimulate T c e l l s . In contrast LPS, an extract
of the bacterium Escherichia c o l i , euid PPD induce proliferation
exclusively i n B c e l l s . Pokeweed mitogen (PWM), an extract of the
stem of Phytolacca americana (Borejeson et a l . , 1966), seems to
stimulate both T and B lyn5)hocytes.
The use of these mitogens i n a variety of endothermic vertebrates
has demonstrated the presence of both T- and B-mitogen responsive
c e l l s i n the peripheral lymphoid system. In contrast, the thymus
was shown to contain lymphocytes responsive only to T c e l l mitogens
(Robinson and Owen, 1976) suggesting that t h i s central lymphoid organ
i s e x c l u s i v e l y a T c e l l domain. Furthermorer the iu^xjrtance of
the thymus early i n ontogeny for the subsequent establishment of
functional T c e l l s was confirmed by the absence of T mitogen
responses i n the periphery following early thymectomy.
The phylogeny of lymphocyte heterogeneity and, in p a r t i c u l a r ,
the role of the thymus i n the establishment of functionally-distinct
lynphocyte populations, i s an important question in immunology.
13
D i f f e r e n t i a l mitogenesis i s a useful means of detecting lymphocytes
functionally-equivalent to mamnalian T and B c e l l s throughout the
vertebrates. Of the agnathan f i s h e s , both the hagfish (Riviere
e t a l . , 1975) and Icuiprey (Cooper, 1971) possess peripheral
lyn^hocytes that are stimulated by PEA. Amongst the elasmobranch
f i s h e s , nurse shark peripheral blood leucocytes (PEL) respond
rather poorly to T c e l l mitogens. They vuidergo blastogenesis to
Con A, but only when large amounts of mitogen (lmg/10^ c e l l s ) are
used, and the response to PHA i s found only i n a population of
gradient-separated PEL (Lopez, Sigel and Lee, 1974; Sigel, Lee,
McKinney and Lopez, 1978). I t has recently been shown, however,
that nurse shark PEL are stimulated by PHA, Con A and LPS to become
cytotoxic to xenogeneic target c e l l s (Pettey and McKinney, 1981).
In the teleosts (Osteichthyes) responsiveness to both T and B
mitogens can readily be demonstrated i n the periphery. Thus
lymphocytes from spleen and pronephros of the bream respond to
PHA, Con A and LPS (Cuchens, McLean and Clem, 1976). These l a t t e r
workers also foiond that the bream thymus contains lymphocytes reactive
to both T and B mitogens (see also Cuchens and Clem, 1977). In
contrast, the rainbow trout behaves more l i k e the mammalian thymus
i n that i t responds to Con A but not to LPS (Etlinger, Hodgins
and C h i l l e r , 1976; Chilmonczyk, 1978).
Recent studies on mitogen responsiveness i n anuran amphibians
have revealed similourities between poikilotherm and endotherm lymphoid
systems, to the extent that normal mitogen r e a c t i v i t y of Xenopus
lymphocytes to PHA and Con A i n the periphery i s dependent on the
presence of the thymus early i n development, i n contrast to the
thymic independence of LPS and PPD responsiveness (Du Pasquier and
Horton, 1976; Manning, Ebnnelly and Cohen, 1976). The work i n this
14
Chapter was designed to conplement these studies by investigating
the i n v i t r o p r o l i f e r a t i v e behaviour of advilt Xianopus lymphocytes
from the thymus to mammaliem T and B c e l l mitogens. Additionally
i t compares thymocyte responses with those of lynphocytes from
the spleen, the l a t t e r being the major peripheral lynphoid organ
i n t h i s species. The experiments investigate optimal culture
conditions, i n p a r t i c u l a r mitogen concentration and l e v e l of serum
supplementation - for mitogen r e a c t i v i t y of thymocytes and splenocytes.
Materials and Methods
(a) Rearing and care of animals
A l l animals used i n t h i s Thesis were bred and reared i n the
laboratory. Spawning was induced by in j e c t i o n of chorionic
gonadotrophin (Xenopus Ltd.) into the dorsal lymph sac of male
and female Xenopus l a e v i s (Daudin). Animals mated overnight and
the eggs l a i d were transferred to aerated, standing water and
maintained at 23°C - 2°C. After hatching, the tadpoles were
distributed a t a density of about 10 per tank (one per l i t r e ) and
were fed on nett l e powder twice a week up u n t i l metamorphosis,
afte r which toadlets were kept 4 per tank and were fed on Tubifex
worms and l a t e r on minced meat ( l i v e r or heart) twice a week.
A l l animals used i n t h i s Chapter were aged 10-15 months.
(b) Lymphocyte cultxire
Thymus and spleen were removed from the animal a s e p t i c a l l y
i n a Icuninar flow hood, following anaesthesia with MS 222 (Sandoz) .
Organs were transferred to watch glasses containing modified Leibovitz
L-15 culture medium (Flow Laboratories). For each 58 cm^ of L-15
medium the following additions were made:- 24 cm^ double-distilled 3 2- 3 water, 1.6 cm 2mM mercaptoethanol, 0.8 cm IM Hepes buffer.
15
3 3 0.8 cm penicillin/streptomycin (Flow), 0.5 cm fungizone (Flow)
and 0.5 cm^ L-glutamine (Flow). This amphibian medium was
s t e r i l i s e d by f i l t r a t i o n through a 0.22 ym 'Millex' f i l t e r (MLllipore)
Tissues were teased apart under a steremicroscope using watchmaker's
forceps. The teased tissues were then transferred to glass
centrifuge tubes and the contents allowed to s e t t l e for a minute.
The supernatant c e l l suspension, devoid of a l l large clunks, was
washed twice (using a refrigerated centrifuge at 350 x g for 10 mins)
cuid resuspended i n medium. Lymphocytes were counted i n a Neubauer
American Optical counting cheimber and their concentration adjusted
to 5 X 10^ cm .
T r i p l i c a t e lymphocyte cultures prepared from individual
adults were set up i n microtest plates (M25 ARIL, S t e r i l i n ) , which
have V-shaped wel l s . Forty y l c e l l suspension (2 x 10^ lyii5)hocytes)
were distributed to each well. Ten y l mit:ogen (or medium in control
cultures) and f i n a l l y 10 y l heat-inactJLvated feetal c a l f serum
(FCS, batch 427006, Flow) were then added (both mitogen and serum
being freshly diluted with an^jhibian medium). The extent to which
the l e v e l of serum supplementation affected lymphocyte v i a b i l i t y
and r e a c t i v i t y to various mitogens was examined:- FCS was added
to give a f i n a l concentration of either 10% or 1%; serum-free
cultures were also set up, the ten y l being replaced by 10 y l medium.
Plated cultures were placed i n a humidified incubator a t 28°C
for 2 days. At t h i s time the cultures were pulsed with 10 y l (lyCi)
t r i t i a t e d thymidine (" HTdR, s p e c i f i c actJ.vity 5 Ci mmol ,
Radiochemical Centre, Amersham). The c e l l s were then incubated
fcr a further 18-24 hours at 28°C before they were harvested with
a Skatron c e l l harvester (water wash model. Flow). Ihe glass fibre
16
d i s c s containing the recovered c e l l s from individual wells
were placed into separate glass s c i n t i l l a t i o n v i a l s , a i r dried
for 30 minutes, 0.5 cm^ tissue s o l u b i l i z e r (Protosol, New England
Nuclear) was then added to each v i a l and incubated at 55-60°C for
1 hour. F i n a l l y 5 cm" s c i n t i l l a t i o n f l u i d ( 42 cm^Liquifluor
(NEN) per l i t r e of toluene) was added. ^HTdR incorporation was
measured with a Nuclear Enterprise l i q u i d s c i n t i l l a t i o n counter.
Stimulation indices ( S . I . ' s ) for each individual experiment
were measured by dividing the mean counts per minute (cpm) for the
t r i p l i c a t e cultures with mitogen added by the mean cpm obtained
with control (no mitogen) cultures. Stimulation indices from,
on average, 8 separate escperiments provided each mean S.I. given
i n Figures 2.1-2.4. S.I.'s were considered positive only when
they reached 1.5, since the mean (- SE) coeff i c i e n t of variation
(SD/mean) i n cpm for t r i p l i c a t e cultures was 23% - 3.5%, the
highest variation being 47%.
(c) Lymphocyte v i a b i l i t y
The e f f e c t of FCS supplementation on lymphocyte v i a b i l i t y
was examined i n 72 hour mitogen-free cultures. C e l l s from both
thymus and spleen from 5 separate experiments were stadned i n
0.2% nigrosine for 10 minutes and the percentage of dye-excluding
lymphocytes calculated from the mean of t r i p l i c a t e cultures.
(d) Mitogens
The following mitogens were used:- PHA M, Con A, E . c o l i LPS
055 :B5 ( a l l from Difco), PPD 298 (Ministry of Agriculture, Fi s h e r i e s
and Food, Weybridge) and PWM (Flow Laboratories).
17
Results
(a) ^HTdR incorporation and v i a b i l i t y of lyiig>hocytes i n mitogen-
free ciiltures.
The l e v e l of ^HMR uptake after 3 days i n culture by thymocytes
and splenocytes i n the absence of mitogen i s given i n Table 2.1.
Addition of 1% FCS to spleen and thymus cultures increased mean
background cpm 2-3 fold when compared with serum-free cultures.
A further 3-4 fold increase i n cpm occurred when splenocytes
were cultured with 10% PCS. In contrast, thymocytes in 10%
FCS displayed only marginally elevated cpm when cooipared with
1% FCS cultures. Hiymocyte background cpm were noticeably
lower than splenocyte cpm for each FCS concentration. With
respect to 0% and 1% serum supplementation, this covild well be
related to the poorer v i a b i l i t y of thymus lymphocytes (35% and 55%
viable respectively) when compared with spleen lymphocytes
(65% and 79%) at 72 hours of culture. Ten per cent FCS supple
mentation resulted i n improved v i a b i l i t y of thymus lymphocytes
(84%), d i r e c t l y con5)arable to spleen lymphocyte v i a b i l i t y (86%).
This equally good v i a b i l i l y of lymphocytes from both thymus and
spleen i n 10% FCS does not explain the very hic^i levels of ^HTdR
incorporation seen i n tihe splenocyte cultures at this l e v e l of
servim supplementation. This feature m i ^ t be related to antJ.genic
stimulation of spleen lymphocytes by FCS, a stimulation not readily
apparent i n thymus cultures. Or i t could be that 10% PCS culture
conditions are s t J . l l not optimum for thymocyt:e prol i f e r a t i o n .
(b) P r o l i f e r a t i v e responses of thymocytes and splenocytes to
T and B c e l l mitogens
The dose-response curves (based on mean S.I.'s) of thymocytes
and splenocytes to Con A, PHA and LPS are shown i n Figs. 2.1-2.3
18
respectively. Table 2.1 shows d e t a i l s of cpm recorded i n
these experiments, but only for optimally-stimulated and mitogen-
free cultures.
Con A; With 1% FCS stj^jplementation the response of
thymocytes and splenocytes to t h i s mitogen was almost i d e n t i c a l .
Maximal S.I. 's of j u s t over 12 were recorded with c e l l s from
both orgems when 0.5iig cm Con A was used. With 10% FCS, more
mitogen (2.5yg cm~^) vaa required to optimally stimulate splenocytes
and thymocytes, and maximal S.I.'s were lower. The p a r t i c u l a r l y
low mean S.I. (4.3) found with splenocytes cultured i n 10% FCS
appeared to be primarily related to the high background counts
under these serum conditions. Con A responses i n serum-free
media were not examined.
PHA: In contrast to Con A, where substantial stimulation
occurred over only a narrow dose range of mitogen, a wider range
of PHA concentrations effected good l e v e l s of proliferation.
With 10% FCS supplementation, thymocytes and splenocytes displayed
i d e n t i c a l dose-response curves. For both c e l l types 40yg cm ^ PHA
proved optimal, with meem S.I.'s of around 7. The maximal
stimulation of thymic lymphocytes was unchanged when cultured i n
1% FCS or i n the absence of serum, although under these conditions
8yg cm"" PHA also proved to be 'optimal'. Maximal stimulation of
splenocytes cultured i n 1% FCS or with no serum was almost doubled
when compared with the 10% FCS spleen cultures. Forty }ig cm ^ and
8yg cm~^ proved optimal for the 1% FCS cultures, whereas 8yg cm ^
achieved 'maximal' stimulation of serum-free cultures.
19
LPS: Splenocytes responded vigorously, especicdly i n
serum-free conditions to t h i s B c e l l mitogen, displaying a mean
maximal S.I. of 22 with the highest concentration of LPS used
(2mg cm ^ ) . Addition of FCS resulted i n a s i g n i f i c a n t l y lower
maximal S.I. (7.4). In both serum-free and 1% PCS spleen c e l l
cultures, the more LPS added, the greater the upteike of HTdR.
Thymus lymphocytes of these 10-15 month old animals responded
r e l a t i v e l y poorly to LPS when conpared with spleen cultures. In
serum-free or 1% FCS conditions, mean maximal S.I.'s were only
j u s t greater than 2, with only 4/8 and 8/10 thymus cultures
(respectively) displaying s i g n i f i c a n t S . I . ' s . However, with
10% FCS eill 13 thymocyte cultures set up consistently displayed
significeint S.I.'s with optimal LPS concentration (1 mg cm ^ ) .
The mean S.I. of these maximally-responding cxiltures was 4
(range 2.2 - 8.8) .
The potential of thymus lymphocytes to respond to 2 other
mitogens - PPD and PWM - was also b r i e f l y examined (see Fig.
2,4).
PPD: With 10% ser\am supplementation, the maximal mean
S.I. (3.3) was attained with 1.0 mg cm"" PPD, with only one culture
f a i l i n g to respond. The background mean cpm i n these experiments
was 811 - 145, whereas cpm following PPD stimulation rose to
2,365 - 270.
PWM: With 1% FCS supplementation the maximal mean S.I. of
thymocytes was 4.6 when 25 yg cm ^ PWM was used. Background mean
cpm was 790 - 206 i n contrast to the mean of 2631 - 292
20
cpm recorded i n the PWM stimulated cultures.
Combined PHA and LPS: In order to gather some circumstantial
evidence that the T and B mitogens used here were, i n fact ,
stimulating d i s t i n c t populations of Xenopus lymphocytes, an
additional experiment was carried out with splenocytes.
Uiymocytes were not used because of their r e l a t i v e l y poor
response to LPS. In t h i s expexlmsnt, cultures from the same
spleen were set up with (a) PHA (20 yg cm"" ) alone, (b) LPS
(2 mg cm~^) alone and (c) PHA and LPS combined i n the same well.
One % FCS supplementation was losed. The r e s u l t s are shown
i n Table 2.2. The stimulations achieved when the two mitogens
were combined i n culture proved to be approximately that es^jected
from adding the S.I.'s achieved by use of PHA or LPS i n separate
w e l l s . This suggests that these two mitogens may well be
stimulating separate splenocyte populations, i . e . T and B-
equivalent c e l l s respectively.
Discussion
The experiments have shown that both thymocytes and splenocytes
from 10-15 month old Xenopus respond well to the T c e l l mitogens
PHA and Con A, PHA having a broader dose-response curve than
that of Con A. The p r o l i f e r a t i v e response of c e l l s from these
2 organs to Con A was v i r t u a l l y i d e n t i c a l i n terms of optimal
mitogen dose, l e v e l of stimulation and e f f e c t of serum supplementation.
PHA r e a c t i v i t y was also comparable i n the two organs, although
maximal stimulation of thymocytes was lower than that recorded
i n splenocyte cultures v*ien serum-free or 1% FCS sv^jplementation
21
conditions were used, i . e . under conditions that allow only
poor thymocyte v i a b i l i t y . In terms of mitogen r e a c t i v i t y ,
therefore, i t appears that functionally-mature T-equivalent
c e l l s e x i s t i n good numbers i n both organs i n Xenopus.
Reactivity of anuran lyophocytes to these T c e l l mitogens
has been examined i n some d e t a i l (Bufo marinus, Goldshein and
Cohen, 1972; Rana pipiens, Goldstine, C o l l i n s and Cohen, 1976;
Xenopus l a e v i s , Du Pasquier and Horton, 1976; Manning, Donnelly
and Cohen, 1976; Donnelly, Manning and Cohen, 1976; Horton and
Sherif, 1977; Green and Cohen, 1979). In terms of optimal
mitogen dose, these studies are comparable with mammalian
findings (see for example, Coutinho, Moiler, Andersson and
Bullock, 1973; Janossy and Greaves, 1971). The present
experiments on Xenopus highlight the requirement for higher
doses of mitogen to optimally stimulate lymphocytes when these
are cultured with increasing l e v e l s of PCS. This finding i s
well characterised i n mammals and i s thought to be related to
binding of mitogen to PCS proteins (see Coutinho et a l . , 1973).
The finding of good responsiveness to T c e l l iritogens i n the.
anuran thymus contrasts with the r e s u l t s from the urodele,
Ambystoma mexicanum, where no response to T c e l l mitogens was found
i n the thymus (Collins and Cohen, 1976). This i s a good example
of the d i s t i n c t differences that e x i s t between anuran and urodele
immune systems (Jurd, 1978).
The demonstiration of a subst:antial response of Xenopus
thymocytes to Con A and PHA contrasts to some extent with the
work of Donnelly, Manning and Cohen (1976), Green and Cohen (1979),
22
who reported only minimal stimulation of adult Xenopus
thymocytes with these mitogens. The use of a higher
concentration of lymphocytes per culture i n the experiments
reported here may, to some extent, explain t h i s dispeirity.
Thus experiments reported i n the thi r d Chapter of t h i s Thesis,
attempting to reduce c e l l nvmibers i n microtest plate mitogen
cultures, were not successful i n procuring good S.I.'s.
Donnelly et a l (1976) also noted that detection of residual
Con A r e a c t i v i t y i n spleens of early-thymectomized Xenopus
was dependent upon maintaining a s u f f i c i e n t l y high concentration
of c e l l s i n v i t r o . In contrast to unfractionated thymocytes,
t h e i r experiments did demonstrate an excellen'^ Con A and PHA
response i n the low density fraction of bovine serum albiimin
separated thymus lyii5)hocytes, revealing heterogeneity of T-
equivalent c e l l s i n the anuran thymus, which has been previously
demonstrated i n mammals and birds (Stobo and Paul, 1973).
Work in t h i s Chapter reveals that Xenopus thymocytes
are stimulated by the mitogen PWM, although t h i s response i s
r e l a t i v e l y poor con^jared with that to PHA and Con A. Work on
mammals suggests that PWM stimulates a subset of T lyn$>hocytes
that have helper activity,and that t h i s response can be influenced
by suppressor T lymphocytes (Greaves, 1972; Hirano, 1977;
Keigthley et a l . , 1976). In humans PWM stimulation of helper
T c e l l s can lead to polyclonal B c e l l activation (Hammerstr6m
et a l . , 1979; Lanzavecchia et a l . , 1979).
23
Under optimal conditions, the splenic response of Xenopus
to LPS was better than t±at seen with PHA or Con A. Manning
e t a l . (1976) revealed quite the opposite i n their studies.
However they added 1% FCS i n their experiments, which we have
shown are suboptimal for LPS r e a c t i v i t y . The fact that very
high concent:rations (1 mg cm ^) of LPS are required to 'optimally'
stimulate Xenopus splenocytes was, however, also demonstrated
i n t h e i r experiments. Less LPS (100-400 yg cm ^ 055 : B5
preparation) i s required to optimally stimulate splenocyt:es
i n other anphibians (Goldstine, Collins and Cohen 1976;
C o l l i n s and Cohen, 1976), and fishes (Etlinger, Hodgins and
C h i l l e r , 1976), higher doses of LPS proving to be suboptimal.
The reasons for these differences are unclear. A p a r a l l e l
between the findings i n Xenopus and experiments performed i n
mammals e x i s t s , however. Thus the more LPS (055 : B5) added
to mouse spleen c e l l s (maximum concentration used = 100 yg cm ' ^ ) ,
the greater the degree of induced p r o l i f e r a t i o n (Coutinho
e t a l . 1973).
In contrast to spleen c e l l s , Xenopus thymocytes respond
r e l a t i v e l y poorly to LPS. Nevertheless, the positive stimulation
of mciny thymocyte preparations with t h i s putative B c e l l mitogen
and also with PPD suggests the p o s s i b i l i t y of a population of
B-equivalent c e l l s witJiin the anuran thymus. However, one must
be cautious in drawing t h i s conclusion since the highest leve l s
of stimulation with LPS were seen in those thymocyte cultures
supplemented with 10% PCS. I t could therefore be that the
induced r e a c t i v i t y was achieved by synergistic interaction of
foreign serum components with mitogen, possibly effecting alteration
24
of the s p e c i f i c i t y of t h i s mitogen for B-equivalent c e l l s (see
E t l i n g e r et al^, 1976, for discussion). However, the s i g n i f i c a n t
response to t h i s mitogen i n h a l f of the serum-free thymocyte
cultures implies that the better response of thymocytes i n 10% FCS
might simply be related to increased v i a b i l i t y of LPS-reactive c e l l s
under these culture conditions. I t should also be pointed out
that LPS may be i n d i r e c t l y stimulating Xenopus T lymphocytes. Thus
LPS can, v i a interaction with macrophages, cause triggering of
some T c e l l s i n mice (Roelants, 1978). LPS-reactive Xenopus
thymocytes do, however, appear to be a d i f f e r e n t population to
PHA and Con A responsive thymocytes (see nylon wool separation
experiments performed i n Chapter 3). Although the double mitogen
stimulation experiments with Xenopus also suggest that PHA and LPS
stimulate separate spleen lymphocyte populations, similar studies
with thymocytes would ascertain that t h i s holds for the thymus.
Experiments are currently i n progress (Cribbin, Horton and
Zettergren) i n the laboratory to determine whether B-equivalent c e l l s
can be i d e n t i f i e d d i r e c t l y i n LPS stimulated thymus c e l l cultures.
These experiments involve the use of anti-Xenopus Ig reagents to
detect the appeareince of cytoplasmic-Ig-labelled lymphocytes.
LPS i s used i n mammals to stimulate B c e l l s to develop into antibody
secreting plasma c e l l s (see Melchers, 1979). In the teleost, the
trout, LPS stimulation i n v i t r o does induce B-equivalent c e l l production
i n the spleen (assayed by induction of PFC's and by the appearance of
c e l l s with the u l t r a s true ture of plasma c e l l s ) but not in the thymus
(EUinger, Hodgins and C h i l l e r , 1978) . "The finding of a B c e l l a c t i v i t y
within the anuran thymus would not be novel since functioning a n t i
body forming c e l l s have been found i n the thymus of diverse vertebrate
25
species, e.g. mice (Herzenberg and Herzenberg, 1974; Bosma
and Bosma, 1974; Micklem elt a l . , 1976); rabbits (Chou, 1966; Jentz,
1979), chickens (Seto, 1978), sneikes (Kawaguchi, Kina and Muramatsu,
1978), f rogs (Moticka, Brovm and Cooper, 1973; Minagawa, Ohnishi and
Murakawa, 1976) and f ishes (Sai lendri and Muthukkaruppan, 1975;
Ortiz-Muniz and S ige l , 1971).
In endotherms intrathymic antibody forming cel ls may we l l
be spawned i n the periphery and then have migrated to the thymus,
since a clear separation o f T and B c e l l systems i s believed to
ex i s t early i n t h e i r development, the thymus e f f e c t i n g T c e l l
maturation, the bursa o f Fabricius or bursal-equivalent being
the primary s i t e of B lymphocyte d i f f e r e n t i a t i o n . There i s no
d i r e c t bursal equivalent i n mammals since B c e l l d i f f e r e n t i a t i o n
i s m u l t i f o c a l , beginning i n the placenta and then l a t e r emerging
i n other s i t e s , e.g. the f o e t a l l i v e r and spleen and eventually
i n the bone marrow (Melchers, 1979; Owen, Raff and Cooper, 1975).
In lower vertebrates there i s substantial evidence that the
thymus i s the primary s i t e o f T-equivalent lynphocyte d i f f e r e n t i a t i o n
(see the Introduct ion to t h i s Thesis) but uncertainties remain
concerning the o r ig ins o f B-equivalent lymphocytes. This i s
discussed i n the next Chapter i n the l i g h t o f experiments invest igat ing
the ontogeny o f mitogen r e a c t i v i t y to putat ive T and B mitogens i n
thymus and spleen o f l a r v a l and young adult Xenopus, since these
studies provide addi t ional characterisation o f the thymocyte response
to LPS.
26
TABLE 2 .1 Comparison o f cpm obtained i n mitogen-free and
opt imally-st imulated thymus and spleen c e l l
cultvires under d i f f e r e n t conditions o f FCS
supplementation.
Mitogen Organ FCS cone. %
Optimal mitogen cone, yg cm"^
Background mean cpm - S.E.
Stim\^ated mean cpm - S.E.
Spleen 1 0.5 2,555 + 737 . 21,500 + 5,296
10 2.5 10,016 + 1,251 41,000 + 5,489
Con A 1 0.5 823 + 127 8,336 + 3,735
Thymus 0.5
+ 9,831 10 2.5 1,063 390 12,365 9,831
0 8 1,651 + 452 17,302 + 667
Spleen 1 40 3,522 +. 887 32,426 + 6,031
10 40 9,131 + 1,294 56,115 + 6,268
PHA 0 8 496 + 72 4,815 + 3,513
Thymus 1 8 1,677 + 494 7,578 + 1,793
10 40 1,936 + 633 12,726 + 4,030
0 2000 822 + 166 12,568 + 4,085 Spleen + 3,473 Spleen
1 2000 2,341 489 19,206 3,473
LPS 0 2000 302 + 33 540 + 130
Thymus 1 1000 957 + 238 2,217 + 611
10 lOOO 1,073 304 4,065 + 1,084
Although maximal S . I . ' s can be calculated from the Table, such indices
w i l l not agree precisely wi th those shown i n Figs. 2.1-2.3, since the
l a t t e r are based on differences between background and stimulated cpm
recorded i n i nd iv idua l experiments, rather than on pooled cpm data
(see Methods).
27
TABLE 2.2 E f f e c t on spleen lymphocyte p r o l i f e r a t i o n o f
combining PHA and LPS i n the same culture
Spleen 1
Mitogen cpm Mean S . I . ( ind iv idua l cpm
wells)
Spleen 2 cpm Mean S . I .
( in d i v i dual cpm wells)
1,825
2,298
3,497 2,540
1,831 1,129 1,480
29,077
LPS _3 28,389 (2mg cm ) 31,222 29,563 11.6
19,263
23,188
16,221 19,557 13.2
17,358
PHA _2 36,643 (20 yg cm ) 12,547 22,182 8.7
14,100
9,831 10,669 11,533 7.8
2mg cm LPS^ 53,979
-3 72,045 20yg cm
68,316 64,780 25.5
41,328 28,500 30,579 33,469 22.6
Figs. 2 .1 - 2.4
O no FCS supplementation
• 1% "
A 10% "
Each poin t represents the mean S . I . ( - S.E.)
calculated from (an average) 8 separate
experiments.
F i g . 2.1
28
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32
CHAPTER 3
Ontogeny of mitogen responsiveness i n thymus and spleen
In t roduct ion
P r o l i f e r a t i v e r e a c t i v i t y to both T and B c e l l mitogens i s not
an inherent property of embryonic lynphocytes. Indeed the
emergence o f mitogen r e a c t i v i t y i n metmmals i s considered to be a
marker f o r a cer ta in l eve l of funct ional maturity (Janossy & Greaves,
1972; Stobo and Paul, 1973; MDsier, 1974; Kruisbuek, 1979). The
mitogen studies i n ' t h i s Chapter were designed to investigate the
ontogeny o f T- and B-equivalent lymphocytes w i t h i n the anuran
thymus during l a r v a l and post-metcunorphic l i f e . The competence
o f in t ra- thymic l a r v a l and young toadlet lymphocytes to respond i n
v i t r o has been examined by others wi th respect to MLC r eac t i v i t y
(Du Pasquier & Weiss, 1973), but there have been no reports concerning
the development of mitogen-reactive ce l l s w i th in th i s organ. The
emergence of thymocyte r e a c t i v i t y during the f i r s t two years of
l i f e to Con A, PHA and LPS are examined here and p r o l i f e r a t i v e
responses are compared, wherever possible, w i th those occurring
i n the spleen. The experiments on ontogeny of mitogen r eac t i v i t y
pay p a r t i c u l a r a t ten t ion to the thymocyte LPS response, which
proves to be maximal i n the young toadlet . The ce l l s involved i n
the p r o l i f e r a t i v e response to th i s mammalian B c e l l mitogen are
compared w i t h thymocytes stimulated wi th PHA fo l lowing autoradiography
and nylon wool separation. The e f f e c t of metamorphosis on emergence
o f thymocyte mitogen r e a c t i v i t y i s also discussed i n some d e t a i l .
33
Materials and Methods
(a) Miniaturised c e l l cul ture technique
Larval lymphoid tissues possess r e l a t i v e l y few lynphocytes
compared wi th the adul t , so the standard technique of lymphocyte
cul ture described i n Chapter 2 had to be miniaturised. I n i t i a l
experiments wi th toadlet lymphocytes showed that reducing c e l l
nvanbers i n the standard microtest plate reduced the subsequent
s c i n t i l l a t i o n counts to an unacceptably low and variable l e v e l .
However, i t was possible t o reduce c e l l numbers and volume by
4
25% (5 x 10 lymphocytes i n lOyl) i n flat-bottomed Terasaki
microcultvure plates ( S t e r i l i n ) : t h i s yielded S . I . ' s comparable to
the standard technique (2 x 10^ lymphocytes i n 40yl) . These
ejcperiments are described below. Success wi th miniatur isat ion
made i t possible to work wi th ind iv idua l l a r v a l thymuses, but
l a r v a l spleens s t i l l proved to contain too few lymphocytes f o r
i n d i v i d u a l analysis. The standard c e l l culture technique described
i n Chapter 2 was used wi th toadlet t issues.
The miniaturised technique i s i l l u s t r a t e d i n F ig . 3 . 1 .
The l a r v a l thymus was dissected out and placed i n a microtest plate
containing medium (see Chapter 2) where i t was broken up mechanically
under a stereomiciroscope using tungsten needles. Thymocytes were
then washed by cen t r i fuga t ion f o r 10 minutes at 350 x g. The
resuspended ce l l s were counted and adjusted to 5 x 10^ lymphocytes
cm~^. Ten y l of the ce l l s and 2.5 y l o f mitogen (medium i n control
cultures) were added to each we l l o f a Terasaki p la te . F ina l ly
2.5 y l of FCS was added to give a f i n a l concentration of 1%.
34
One per cent FCS supplementation was used throughout a l l the
ontogeny escperiments, since t h i s helped keep background counts
down (althouc^ l a r v a l thymocyte backgrovmd cpm were s t i l l very
high i n the TerasaJci plates - see Results). The mitogen
concentrations used are given i n the Tables. T r ip l i ca t e lymphocyte
cultures were set up.
A f t e r 48 hours at 28°C the ce l l s were pulsed wi th 0.25 yCi
3 -3 3 HTdR (2.5 y l o f a 1 : 10 d i l u t i o n ImCi cm HTdR, spec i f ic
a c t i v i t y 5 Ci mmol ^ ) . 18 - 24 hours l a t e r the contents from
i n d i v i d u a l wel ls were t ransferred to separate posit ions on
harvesting mats of the Skatron c e l l harvester by manual p ipe t t i ng .
The mats were then inserted i n to the head o f the harvester and
the c e l l s washed thoroughly and prepared f o r s c i n t i l l a t i o n counting
as i n the previous Chapter. The c o e f f i c i e n t o f var ia t ion using
the miniaturised technique was comparable w i t h that i n the standard
technique. Therefore S . I . ' s o f 1.5 and above were considered
p o s i t i v e .
(b) Autoradiography
At the end o f the 3 day cu l tu re , instead o f harvesting the
radioact ive wells the l a t t e r were washed wi th eunphibian phosphate-
buf fe red saline (PB) and then lyiii)hocytes counted. Approximately
1 X 10^ thymus lymphocytes were resuspended i n 50 y l FCS and a
smear o f ce l l s prepared using a cytocentr ifuge (Shandon) as fo l lows .
The ce l l s were inserted i n t o cuvettes and centrifuged fo r 4 minutes
a t 600 rpm, which forces the ce l l s onto a c i r cu la r area of a
microscope s l i d e . The sl ides were then f i x e d i n methanol and a i r -
d r i ed . I n the darkrxjom they were dipped i n I I fo rd K5 nuclear
35
emulsion, fo l l owing the technique described by Rogers (1973),
and incubated f o r 1 week at 4°C to expose the s i l v e r grains.
Following photographic development they were stained wi th
Leishman's s t a i n , and a permanent mount prepared.
The method f o r counting labe l led and b l a s t lymphoid ce l l s
was as f o l l o w s : - F i f t y f i e l d s , using the microscope at xlOOO
magnif ica t ion , from the centre o f the smear were examined and a l l
lymphoid ce l l s counted and i d e n t i f i e d . Coded slides were used
and gave consistently-repeatable resu l t s . Cells were in t en t iona l ly
heavily-labelled to f a c i l i t a t e counting.
(c) Nylon wool separation
Thymocytes were separated on a nylon wool column using the
method o f Blomberg, Bernard & Du Pasqxiier (1980) . B r i e f l y 1 gm
nylon wool ("Leuko-Pak", Fenwal Laboratories - obtained from
Travenol) was washed by b o i l i n g 6 times i n d i s t i l l e d water.
I t was then dr ied and teased to remove knots, loosely packed in to
a 10 cm^ syringe and autoclaved. Lyii4>hocyte separation then
proceeded as fo l lows . The column was saturated wi th s t e r i l e
amphibian strength PBS containing 10% FCS f o r at least 1 hour
p r i o r to use. The thymocyte suspension was then added i n 1-2 cm^
medium. Two to three cm^ o f the PBS/FCS solut ion was added and
the column incubated at 30°C f o r 1 hour. A f t e r t h i s time the
non-adherent ce l l s were flushed through wi th about 20 cm^ o f
PBS/FCS, centr i fuged and resuspended i n medium and the lyn$)hocyte
concentration readjusted to 5 x 10^ cm~" f o r cul ture . The nylon
wool-passaged ce l l s were compazed f o r mitogen r e a c t i v i t y wi th
unseparated ce l l s from the same thymocyte suspension. In these
mitogen ej^eriments the miniaturised technique was used throughout.
36
(d) Experimental design
( i ) Preliminary stutiies on minia tur isa t ion
Mitogen responses and background cpm o f ind iv idua l thymus
and spleen c e l l suspensions (taken from 4-8 month o ld toadlets)
were examined using the microtest and Terasaki plates, wi th
standard and miniaturised techniques respectively.
( i i ) Ontogeny o f mitogen responsiveness
This major series o f e3q>eriments was designed to investigate
the ontogeny o f responsiveness o f thymocytes, from thymuses taken
from ind iv idua l larvae o f stage 52 (3 weeks old) and continuing
v m t i l about 2 years o f age, to the T c e l l mitogens PHA and Con A
and the B c e l l mitogen LPS. Figure 3.2 shows the relat ionship
between age and developmental stage, that has been used throughout
the thesis , and fol lows the normal table o f Xenopus laevis
development o f Nieuwkoop & Faber, 1956. As already mentioned,
the very low lymphocyte numbers found i n l a r v a l spleens precluded
mitogen studies on t h i s organ u n t i l a f t e r metamorphosis.
( i i i ) Characterisation o f the thymocyte mitogen response
This involved autoradiography and nylon wool separation
ej^eriments. The autoraciiography provided morphologic evidence
o f s t imula t ion w i t h T and B c e l l mitogens. Autoradiographs were
prepared from thymocyte cultures (taken from 6 month o ld animals)
t reated wi th the mitogens PHA and LPS. The nylon wool experiments
looked at the e f f e c t o f removing nylon wool-adherent ce l l s on
mitogen responses. Thymocyte suspensions from 4 animals (6 or
12 months o l d - see Results) were separated on nylon wool columns.
A f t e r separation, the passaged ce l l s were stimulated wi th e i ther
PHA, Con A or LPS.
37
Results
(a) Preliminary studies comparing miniaturised and standard culture
techniques.
Figiare 3.3 examines mitogen s t imulat ion o f thymus and spleen
ce l l s from ind iv idua l toadlets w i t h standard and miniaturised
techniques. Although levels o f mitogen st imulat ion of thymocytes
proved to be rather poor at 4 months o f age (see subsequent
f ind ings below), the microtest (standard technique) and Terasaki
(microtechnique) systems nevertheless proved comparable. With the
spleens (taken from 8 month o l d toadlets) good and conparable
s t imulat ions were seen w i t h both techniques. Findings wi th
thymocytes suggested that the miniaturised technique could be
en^Jloyed f o r the ontogenetic studies on the larvae.
Analysis o f background counts i s given i n Table 3 . 1 . I t
had been expected that the l eve l o f t r i t i u m counts i n the Terasaki
plates would be 25% o f those wi th the microtest system. This
proved to be the case f o r splenocytes, whereas thymocyte counts
i n the Terasaki plates were, on average, more than s i x f o l d
higher than expected from the counts recorded i n the microtest
p la tes . This d i f ference also occurred when 10% FCS supplementation
was used ( t h i s concentration o f FCS promotes greater v i a b i l i t y
o f thymocytes i n microtest plates compared wi th 1% supplementation -
see Chapter 2 ) . I t i s not known why the miniaturised technique
promotes elevated " HTdR uptake only i n the thymocyte cul tures .
Perhaps thyme cultures are more sensi t ive than spleen cultures
to i n v i t r o conditions (e.g. plate shape and c e l l number per wel l)
because the thymus contains (at the outset o f culture) a much higher
38
propertjanof rapid ly p r o l i f e r a t i n g lymphocytes than does the
spleen.
(b) Ontogeny o f mitogen responsiveness
( i ) Studies w i t h Con A
Thymus. The resxilts are given i n Tables 3.2 and 3.3 and F ig .
3.4. At the f i r s t stage examined (stage 52/3, = 3 weeks old) no
response was found; i n f ac t the wells containing mitogen a l l gave
coiants lower than that found i n control we l l s . The next stage
examined was stage 54/5 (= 4 weeks old) and again the cultures
containing Con A generally gave a depressed cpm conpared wi th
unstimulated c e l l s . Thymocytes from 2 animals i n th i s group
d id display a S . I . o f j u s t >1.5, possibly ind ica t ing the emergence
o f Con A reactive thymocytes a t t h i s time. By stage 56/7
(6 weeks old) a small response to Con A was found i n a l l ciiLtures
tested, w i t h a mean S . I . o f 1.71. This response disappears at
the onset o f metamorphosis (stage 58/9, - 7 weeks old) and recovers
only slowly a f t e r metamorphosis. At 4 months o f age s t imulat ion
was pos i t ive i n only one animal o f 6 tested, but a f t e r th i s age a l l
toadlet thymuses responsed to Con A. The ma-ximum mean leve l o f
s t imula t ion seen (mean S . I . = 10) occurred i n 1 year o ld toadlets .
Spleen. The resul ts are given i n Table 3.4 and F ig . 3.4.
At 4 months o f age, which was the ea r l i e s t tested, 3 out of 4
toadlets responded wi th mean S . I . = 2.3. Splenocyte responsiveness
to Con A remained constant (mean S . I . = 6) i n 6-9 month o ld animals,
higher levels being recorded only i n older animals. The highest
S . I . (17.6) was seen i n a 2 year o l d animal.
39
( i i ) Studies wi th PHA
Thymus. The resul ts are given i n Tables 3.5 and 3.6 and
F ig . 3.5. With t h i s mitogen, no consistent p r o l i f e r a t i v e response
was found i n the 4 l a r v a l stages tested (3-8 weeks of age). Just
one stage 56/7 larva gave a s i g n i f i c a n t ( S . I . = 1.6) response.
At 3^ months o f age ( i . e . 6 weeks a f t e r the end of metamorphosis)
2 out o f 4 animals showed a pos i t ive PHA thymocyte response. As
wi th Con A, the PHA response then increased slowly with age,
g iv ing a maximum mean l eve l ( S . I . = 18) by about 9 months of age.
This l e v e l of response appears to decline i n l a t e r l i f e , since
the mean S . I . at 2 years o f age was jus t under 6.
Spleen. The resul ts are given i n Table 3.7 and Fig . 3.5.
The youngest toadlets tested (3.5 months) gave negative responses,
but a constant pos i t ive PHA response appeared-soon afterwards, by
4 months o f age. A high l eve l of PHA stimulaUon (S.I.'=^ 18)
was seen i n spleens taken from 6-7 month o l d toadlets. In 9 and
12 month o l d animals the mean S . I . o f splenocytes to PHA was reduced,
but some ind iv idua l animals s t i l l displayed S . I . ' s o f > 10.
( i i i ) Studies w i t h LPS
Thymus. The resul ts are given i n Tables 3.8 and 3.9 and F ig .
3.6. There was no mitogenic response to LPS at the ea r l i e s t stages
tested (stages 52-55). The mitogen-treated thymocytes from these
young larvae a l l showed a depressed cpm leve l compared wi th unstimulated
c e l l s . At stage 56/7 a small response was detected i n a l l 3 larvae
tested (mean S . I . = 2.1) . LPS r e a c t i v i t y o f thymocytes could no
longer be detected a t the onset o f and jus t a f t e r the completion of
metamorphosis, but re-emerged i n 2 out of 3 toadlets tested a t 4 months
40
o f age. A f t e r t h i s time c o n s i s t e n t LPS responsiveness o f
thymocytes was seen i n young t o a d l e t s , r i s i n g to a peak mean S.li
o f = 6 iat about s i x months o f age. The thymocyte LPS response
g r a d u a l l y diminished a f t e r 6 months and by 12 ronths o f age i t had
dwindled to a low l e v e l (mean S . I . = 1.6) and t h i s was a l s o fovind
i n l a t e r l i f e . These ontogenetic s t u d i e s notonly confirm t h a t LPS
re s p o n s i v e n e s s o c c u r s i n post-metamorphic Xenopus thymus, but
r e v e a l e d t h a t the response i s found c o n s i s t e n t l y i n young t o a d l e t s ,
r e a c h i n g l e v e l s t h a t compare favouredily w i t h r e a c t i v i t y to T c e l l
mitogens.
Spleen. The r e s u l t s a r e given i n Table 3.10 and F i g . 3.6.
The youngest animals t e s t e d f o r splenocyte responsiveness to LPS
were t o a d l e t s o f 3 3 and 4 months o l d . Only 1 o f the 4 animals
t e s t e d responded. A f t e r 4 months a response r a p i d l y emerged, with
a mean S . I . a t 6 months being 7.8. IMl i k e the thymus, t h i s l e v e l
o f LPS r e s p o n s i v e n e s s i s maintained i n o l d e r animals.
(c) C h a r a c t e r i s a t i o n o f thymocyte mitogen responses
( i ) Autoradiographic evidence o f T and B mitogen s t i m u l a t i o n
These r e s u l t s confirm those obtained by s c i n t i l l a t i o n counting
i n as much as s t i m u l a t i o n with both PHA and LPS r e s u l t s i n i n c r e a s e d
l e v e l s o f ^HTdR uptake and b l a s t c e l l transformation i n thymocytes.
S i m i l a r s t u d i e s on a few spleen c e l l p r e p a r a t i o n s a l s o provided
morphologic evidence f o r enhanced " HTdR uptake i n mitogen-treated
c u l t u r e s . The percentage o f tritium-lc±)elled c e l l s i n the unstimulated
c u l t u r e s was about 1% (see Table 3.11 and F i g . 3.7) . I n the LPS-
stimvilated c u l t u r e s from the 6 month o l d animals, the percentage o f
41
l a b e l l e d c e l l s i n c r e a s e d to 5.5%. The m a j o r i t y o f these l a b e l l e d
c e l l s were l a r g e r than lOym i n diameter. I n the PHA s t i m u l a t e d
c u l t u r e s the percentage o f l a b e l l e d thymocytes i n c r e a s e d to 11.2%.
Again the m a j o r i t y o f l a b e l l e d c e l l s were o f lymphoblast morphology
(>10 m). T h i s v a l u e f o r PHA f i t s q u i t e w e l l w i t h mammalian work
where 15% o f murine thymocytes respond to Oon A (Gerhart e t a l . ,
1976). F i g u r e 3.8 (a-c) shows c y t o c e n t r i f u g e p r e p a r a t i o n s o f
thymocytes ( c u l t u r e d i n medium, LPS or PHA) t h a t have been prepared
f o r autoradiography. I t can be seen t h a t thymocytes are a g g l u t i n a t e d
i n the mitogen t r e a t e d c u l t u r e s , p a r t i c u l a r l y w i t h PHA. P r o l i f e r a t i n g
lymphoblasts were g e n e r a l l y h e a v i l y l a b e l l e d (see F i g . 3.8,d)
although some b l a s t s , w i t h fewer exposed s i l v e r g r a i n s , were a l s o
found.
( i i ) The e f f e c t o f nylon wool treatment on T and B mitogen
is t i m u l a t i o n
These experiments were designed to look a t the e f f e c t o f removing
nylon wool adherent c e l l s on the p r o l i f e r a t i v e response of thymocytes
to p u t a t i v e T and B mitogens. Of the 4 animals used i n these
experiments, 2 were s i x months o l d ( i . e . a t an age when the maximum
response o f thymocytes to LPS o c c u r s ) and 2 were twelve months o l d
(when thymocytes respond w e l l only to T c e l l mitogens) . The r e s u l t s
a r e shown i n F i g . 3.9. I n the younger animals a good response to
LPS was found i n c o n t r o l , non-nylon wool t r e a t e d c e l l s ( S . I . ' s = 6.5
and 3 . 5 ) . Mitogen responses to Con A and PHA were a l s o as expected.
Passage through nylon wool e l i m i n a t e d the response to LPS ( S . I . ' s
now 0.9 and 0 . 3 ) . I n t e r e s t i n g l y responses to both T c e l l mitogens
were a l s o a p p r e c i a b l y reduced, but r e a c t i v i t y to these g e n e r a l l y
remained a t around 50% the l e v e l found i n unseparated thymocytes.
42
I n the thymocyte c u l t u r e s prepared from the 2 o l d e r animals
the l e v e l o f LPS response i n unseparated c e l l s was f a i r l y low
( S . I . ' s = 2.5 and 2.1) but the response to the T c e l l mitogens
was s t i l l c o n s i s t e n t l y good. Nylon wool passaged thymocytes from
these o l d e r animals showed no response to LPS. Thymocyte r e a c t i v i t y
to PHA was now e i t h e r u n a f f e c t e d or i n c r e a s e d by nylon wool passage.
Con A r e a c t i v i t y i n t h e s e 1 y e a r o l d animals was only s l i g h t l y
lowered by nylon wool passage.
D i s c u s s i o n
By about m i d - l a r v a l development, anuran l a r v a e are competent
to r e j e c t s k i n a l l o g r a f t s (Horton, 1969) and respond to c i r c u l a t i n g
thymus-dependent a n t i g e n s (Kidder, Ruben & Stevens, 1973). S i n c e
both o f t h e s e immune mechanisms n e c e s s i t a t e thepresence o f T
lymphocytes (see Horton & Manning, 1972; Horton, Rimmer & Horton,
1977) i t might be expected t h a t f u n c t i o n a l T c e l l s would be found
w i t h i n the thymus i t s e l f during l a r v a l l i f e . I n the mouse the
a b i l i t y o f thymic lymphocytes to respond to T c e l l mitogens occurs
by p a r t i o r i t i o n and i s used as the f i r s t hallmark f o r the d i f f e r e n t i a t i o n
o f f u n c t i o n a l T c e l l s (Robinson & Owen, 1976), However, the r e s u l t s
o f thymocyte mitogen r e a c t i v i t y i n developing Xenopus suggest, a t
b e s t , only a minimal response of l a r v a l thymocytes to Con A,
and no response a t a l l to PHA.
The r e s u l t s p r e s e n t e d here show t h a t an appreciable and
c o n s t a n t T mitogen response i n the Xenopus thymus emerges only a f t e r
metamorphosis and maximum l e v e l s a r e not seen i n thymus o r spleen
u n t i l about 6 months or l a t e r . T h i s seems to be r a t h e r slow when
43
compared to mammals, s i n c e s u s t a i n e d a d u l t l e v e l s of mitogen
repo n s i v e n e s s i n the thymus to Oon A and PWM a r 3 found by 3.5 days
a f t e r b i r t h i n the mouse (Robinson, 1976; Mosier, 1977). Adult
l e v e l s o f r esponsiveness i n the thymus to T c e l l mitogens are
found i n the guinea p i g by mid-gestation ( L e i p e r and Solomon,
1977) as i s a l s o the case w i t h sheep (Leino, 1978) and humans
( S t i t e s e t a l . , 1 9 7 4 ) . I n Xenopus PHA responsiveness (but rot
Con A r e a c t i v i t y ) d e c l i n e s i n o l d e r animals. This d e c l i n e i n
PHA r e s p o n s i v e n e s s w i t h age has been reported f o r mammals (0n(5dy
e t a l . , 1980).
The low and t r a n s i e n t response of l a r v a l Xenopus thymocytes
to Con A and the apparent l a c k o f t h e i r r e a c t i v i t y to PHA could be
due to s e v e r a l f a c t o r s . F i r s t , sub-optimal m i c r o c u l t u r e c o n d i t i o n s
used w i t h the l a r v a l thymus may be r e s p o n s i b l e . As mentioned
i n the R e s u l t s , t h e . T e r a s a k i p l a t e s d i d encourage high thymocyte
backgroimd cpm, which may have tended to mask any p r o l i f e r a t i o n
induced by the T c e l l mitogens. However these p l a t e s were used
s u c c e s s f v i l l y i n s e v e r a l experiments on thymocytes. The use o f an
i n a p p r o p r i a t e dose o f mitogen to s t i m u l a t e l a r v a l thymocytes seems
u n l i k e l y , s i n c e a t the o u t s e t of these ontogenetic' experiments other
doses o f Con A and PHA were t e s t e d (without any g r e a t e r s u c c e s s ) .
Secondly, the f i n a l s t a g e s o f f u n c t i o n a l maturation of T c e l l s may,
i n the l a r v a , occur predominantly i n the p e r i p h e r y , perhaps achieved
by thymus humored f a c t o r s t h a t are known to e x i s t i n amphibians
(Dardenne e t a l . , 1973). Although t h i s may be true for mitogen-
r e a c t i v e T c e l l s , Du P a s q u i e r and Weiss (1973) s t u d i e s on Xenopus
showing t h a t MLC-reactive thymocytes are p r e s e n t a t l e a s t by stage 54,
i n d i c a t e t h a t t h i s i s not the case f o r a l l lymphocyte populations.
44
I n mammals, PHA r e s p o n s i v e n e s s o f spleen c e l l s occurs p r i o r
to PHA r e a c t i v i t y o f thymocytes, suggesting post-thymic maturation
o f t h i s p o pulation (Kruisbeek, 1979). Horton and S h e r i f (1977)
found t h a t an i n t a c t thymus was necessary i n Xenopus u n t i l stage
54 to e s t a b l i s h PHA responsiveness i n the a d u l t spleen. S i m i l a r
f i n d i n g s have a l s o been shown f o r Con A (Manning and C o l l i e , 1977).
P e r i p h e r a l T c e l l s capable o f r e a c t i n g to T c e l l mitogens could
t h e r e f o r e be e j ^ e c t e d to be found i n the spleen during l a t e l a r v a l
l i f e . (Experiments were i n f a c t c a r r i e d out on pooled l a r v a l s p l e e n s ,
but the r e s u l t s were too i n c o n s i s t e n t f o r conclusions to be drawn,
probably due to mixed lymphocyte r e a c t i v i t y between a l l o g e n e i c
c e l l s ) . However, the r e s u l t s w i t h the spleen a f t e r metamorphosis
suggest t h a t i f t h i s i s so, then t h e i r numbers probably remain
s m a l l u n t i l w e l l i n t o a d u l t l i f e . S t u d i e s on s e v e r a l . l a r v a l and
post-metamorphic lymphoid organs are obviously required to i n v e s t i g a t e
t h i s i s s u e , although these e:5)eriments w i l l req'oire the pooling o f
lymphocytes from h i s t o c o n ^ a t i b l e animals to y i r l d s u f f i c i e n t c e l l s
f o r a n a l y s i s . T h i r d l y , Con A and PHA might, i n anurans, s e l e c t i v e l y
a c t i v a t e only h e l p e r T c e l l s , r a t h e r than c y t o t o x i c and MLC r e a c t i v e
s u b s e t s . T h i s i s d e a l t w i t h i n more d e t a i l i n the next Chapter,
but alloimmune lymphocyte populations are known to be e s t a b l i s h e d
i n the p e r i p h e r y e a r l y i n l i f e , whereas those h s l p e r subsets
i n v o l v e d i n humoral immianity are dependent upon an i n t a c t thymus
f o r a much longer p e r i o d o f development - even u n t i l a f t e r metamorphosis
when low doses of antigen are used (Gruenewald and Ruben, 1979) .
Indeed s i g n i f i c a n t l e v e l s o f I g "G" antibody production (which
depends h e a v i l y on T c e l l help) occur only i n the a d u l t anuran
(Du P a s q u i e r & Hainovlch, 1976).
45
D i f f e r e n c e s i n the emergence o f Con A and PHA r e a c t i v e
thymocytes i n Xenop\xs a r e p o s s i b l y i n d i c a t e d from the data
p r e s e n t e d here. I t seems t h a t the PHA responsive population
i s s l o w e r to mature, f i r s t appearing only a f t e r metamorphosis.
T h i s c o u l d be because the two T c e l l mitogens a r e s t i m u l a t i n g
d i f f e r e n t T c e l l svibsets. T h i s has been shown to occur i n
inammals ( gayry e t a l . , 1976). Stobo and Paul
(1973) have shown i n mice t h a t only mature thymic lynphocytes
respond to T c e l l mitogens, w i t h one subset responding e q u a l l y
w e l l to Con A and PHA, whereas another s u b s e t responds almost
e x c l u s i v e l y to Con A, A d d i t i o n a l work (Paetkau e t a l . , 1976;
L a r s s o n e t a l . , 1980) has shown t h a t i n some s t r a i n s o f mice
thymocytes respond w e l l to Con A, but only poorly to PHA.
However i t was found t h a t the a d d i t i o n of Con A to thymocyte
c u l t u r e s causes an i n c r e a s e i n t h e r a t e o f s y n t h e s i s o f a
c o s t i m u l t o r f a c t o r ( I n t e r l e u k i n ) , which i s r e q u i r e d f o r e f f e c t i v e
s t i m v i l a t i o n o f Con A r e s p o n s i v e lymphocytes. Moreover, a d d i t i o n
o f t h i s c o f a c t o r (taken from the supernatant of Con A s t i m u l a t e d
c e l l s ) to thymocyte c u l t u r e s enabled these to become good responders
to PHA. Whether o r not c o f a c t o r s are i n v o l v e d i n anuran lymphocyte
mitogen responses i s not y e t known.
The emergence o f LPS r e a c t i v e c e l l s i n the l a r v a l and
young a d u l t thymus (and t h e i r disappeeurance over metamorphosis)
p a r a l l e l s the ontogenetic p a t t e r n o f thymocyte responsiveness to
Con A. T h i s suggests, perhaps, t h a t the LPS response i s manifested
by c e l l s a c t u a l l y spawned w i t h i n the thymus r a t h e r than through
c e l l s c a s u a l l y m i g r a t i n g through t h i s organ from the periphery.
46
LPS r e s p o n s i v e n e s s i n the thymus reaches a maximum l e v e l a t
about s i x months and then d e c l i n e s , i n c o n t r a s t to the gradual
e l e v a t i o n o f LPS r e a c t i v i t y d i s p l a y e d by splenocytes taken from
o l d e r t o a d l e t s .
Morphologic confirmation t h a t LPS r e s u l t s i n thymocyte
s t i m u l a t i o n i n s i x month o l d t o a d l e t s was provided i n the auto
r a d i o g r a p h i c experiments presented. Moreover, the l i k e l i h o o d
t h a t liPS r e a c t i v e thymocytes are a subset d i s t i n c t from PHA and
Con A r e a c t i v e thymocytes was suggested by the nylon wool s e p a r a t i o n
s t u d i e s . Thus, under c o n d i t i o n s which are known to s u b s t a n t i a l l y
reduce s\irface I g + ve lymphocytes ( i . e . B - equivalent c e l l s )
from the s p l e e n (Blomberg e t a l . , 1980), thymocyte r e a c t i v i t y
remains o n l y to T c e l l mitogens. I n t e r e s t i n g l y a t s i x months o f
age, b u t not a t 12 months, T mitogen r e a c t i v i t y o f thymocytes i s
cilso a f f e c t e d by nylon wool passage, in5>lying t h a t some T-equivalent
c e l l s a r e a l s o removed by t h i s procedure. T h i s could be explained
by the f i n d i n g t h a t thymocytes o f young a d u l t Xenopus are s t i l l
r i c h i n s u r f a c e I g (Du P a s q u i e r & Weiss, 1973).
Organ c u l t u r e s t u d i e s i n mamnals have c l e a r l y shown th a t B
c e l l d i f f e r e n t i a t i o n i s m u l t i f o c a l (Owen, R a f f and Cooper, 1975).
Thus B c e l l s develop f i r s t i n the p l a c e n t a (Melchers, 1979),
then i n the f o e t a l l i v e r and s p l e e n , and l a t e r i n the bone marrow
i n mice. I n Xenopus, 19Sand 7S immunoglobulins are f i r s t found
a t s t a g e 35 (2 days old) when the l a r v a i s j u s t hatching (Leverone
e t a l . , 1979), before a thymic rudiment has even appeared (Manning
& Hbrton, 1969). Furthermore, cytoplasmic Ig"*^ c e l l s a r e found
i n l a r v a l Rana kidney very e a r l y i n ontogeny (Zettergren e t a l . ,
1980). These and o t h e r f i n d i n g s (showing normal LPS r e a c t i v i t y
47
i n early-thymectomized Xenopus - see Turner and Manning, 1974)
i n d i c a t e an important extra-thymic o r i g i n o f c e r t a i n B -equivalent
p o p u l a t i o n s . B - e q u i v a l e n t lymphocyte development i n anurans
may w e l l be m u l t i f o c a l as i n mammals; i t appears t h a t the bone
marrow i s a s i t e o f B c e l l development i n a d u l t frogs ( Z e t t e r g r e n
e t a l . , 1980).
I t i s p o s s i b l e however, t h a t some B-equivalent c e l l s ubsets
d i f f e r e n t i a t e w i t h i n the anuran thymus. A number o f experimental
o b s e r v a t i o n s support t h i s h y p o t h e s i s . Following e a r l y thymectomy
i n Xenopus, both IgG and IgM antibody production to thymus-dependent
a n t i g e n s i s abrogated and a l s o r e a c t i v i t y to TN P - F i c o l l i s d r a m a t i c a l l y
i m p a i r e d (Horton, Edwards, Ruben & Mette, 1979). This hapten-
c a r r i e r complex was, u n t i l r e c e n t l y , thought to a c t i v a t e a subset
o f mammalian B lymphocytes cuid was considered to be a thymic
independent antigen(Mosier, Mond & Goldings, 1977). However,
r e c e n t s t u d i e s suggest a degree o f thymic-dependence of T N P - F i c o l l
r e a c t i v i t y i n mammals (Mond e t a l . , 1980) . C e r c a i n embryological
experiments, i n which g i l l bud regions ( c o n t a i n i n g thymic rudiments)
were t r a n s f e r r e d to p l o i c ^ - d i s t i n c t h o s t s , suggested the p o s s i b i l i t y
t h a t v i r t u a l l y a l l p e r i p h e r a l lymphocytes i n leopard frogs
n o r m a l l y o r i g i n a t e i n the thymus (Turpen, Volpe & Cohen, 1975;
Turpen & Cohen, 1976). However, more r e c e n t f i n d i n g s on the
p o t e n t i a l i t y o f the g i l l bud region suggest t h a t i n a d d i t i o n to
housing the thymus anlage, i t a l s o c o n t a i n s the pronephric rudiment,
which may be an important source o f lymphoid stem c e l l s , p a r t i c u l a r l y
of B lymphocytes (Volpe e t a l . , 1981; Zettergren e t a l . , 1980).
48
The p o s s i b i l i t y t h a t the mammalicui thymus spawns some B
c e l l s has a l s o been considered. Micklem e t a l . (1976) d i s c u s s
the p o s s i b i l i t y t h a t m u l t i p o t e n t i a l stem c e l l s e x i s t i n the murine
thymus which are capable of d i f f e r e n t i a t i n g i n s i t u i n t o B c e l l s .
J e n t z (1979) has found t h a t , by c e l l t r a n s f e r experiments, B
c e l l s i n the thymus have d i f f e r e n t r a t e s of f u n c t i o n to B c e l l s
i s o l a t e d from ot h e r organs (34 times more productive of antibody
than s p l e e n B c e l l s ) , i n r a b b i t s .
Other experimental o b s e r v a t i o n s suggest t h a t the amphibian
thymus i s not a source o f cintibody-producing c e l l s . Thus
thymocyte r e c o n s t i t u t i o n estperiments on thymectomized Xenopus,
both in^ v i v o ( K a t a g i r i e t a l _ . , 1980) and in_ v i t r o (Ruben e t a l . ,
1977 i n d i c a t e t h a t the thymus provides only helper a c t i v i t y
r a t h e r than antibody producing c e l l s .
One major f e a t u r e o f these experiments has been to demonstrate
t h a t metamorphosis e f f e c t s a temporary d i s r u p t i o n of the emergence
o f thymocyte mitogen r e a c t i v i t y . T h i s i s not s u r p r i s i n g s i n c e t h i s
p e r i o d of development i s a s s o c i a t e d w i t h severe d e p l e t i o n of
thymocyte numbers (Du P a s q u i e r and Weiss, 1973; see a l s o Chapter 6) .
Dvuring metamorphosis a d u l t - s p e c i f i c a ntigens are appearing and
t h e r e c o u l d be a danger t h a t these antigens w i l l be destroyed by
an immunologically mature immune system. However i t has been
shown t h a t t h e r e i s an i n c r e a s e d s u s c e p t i b i l i t y of the metamorphosing
anuran to t o l e r a n c e i n d u c t i o n o f MHC antigens, probably achieved
through the a c t i v i t y o f (T) suppressor c e l l s , which have been
demonstrated a t t h i s time i n Xenopus (Du Pasquier and Bernard, 1980).
A c t i v e suppression of lymphocyte r e a c t i v i t y appears to develop
i n the l a r v a e a t the very time t h a t mitogen responsiveness i s
49
beginning to emerge. The gradual diminution of suppressor
c e l l a c t i v i t y w i t h i n the thymus a f t e r a d u l t antigens are
e s t a b l i s h e d may e f f e c t , i n p a r t , the gradual i n c r e a s e i n thymus
(and spleen) mitogen re s p o n s i v e n e s s seen i n e a r l y a d u l t l i f e .
The c o - e x i s t e n c e o f mdLtogen-responsive and suppressor subsets
i n the same lymphoid t i s s u e has been demonstrated i n r a t s by
d e n s i t y g r a d i e n t s e p a r a t i o n (Rocha e t a l . , 1979). There i s
c i r c u m s t a n t i a l evidence f o r suppressor a c t i v i t y w i t h i n the
thymus of a d u l t Xenopus, s i n c e Donnelly, Manning & Cohen (1976)
were a b l e to d e t e c t T mitogen r e a c t i v e c e l l s only i n a c e r t a i n
f r a c t i o n o f s e p a r a t e d thymocytes, whereas unseparated c e l l s
f a i l e d to respond to Con A and PHA. A c t i v e suppression w i t h i n
the thymus during l a r v a l l i f e i s a l s o suspected (Du Pasquier,
p e r s o n a l communication) and t h i s could account f o r the poor
mitogen responses d i s p l a y e d by thymocytes p r i o r to metamorphosis.
50
TABLE 3.1 comparison o f HTdR cpm obtained i n unstimulated
c u l t u r e s i n T e r a s a k i and m i c r o t e s t p l a t e s
Splenocyte c u l t u r e s from 8 month o l d t o a d l e t s
40Ml i n m i c r o t e s t p l a t e
l O p l i n Terasedci p l a t e
480 3355 2963 3367 1292 1994
260 847 347 647 634
1665
T e r a s a k i m i c r o t e s t
%
Observed Expected
54.2 25.2 11.7 19.2 49.1 83.5 40.4 - 24.6% 25.0%
Thymocyte c u l t u r e s f o r 4 month o l d t o a d l e t s
M i c r o t e s t p l a t e
T e r a s a k i p l a t e
T e r a s a k i m i c r o t e s t %
177 202 501 705 485 249 724
207 116.9 209 103.5
1029 205.4 1789 253.8 754 152.5 344 142.1
1103 152.3 Observed 161.9 - 51.0% Expected 25.0 %
Each h o r i z o n t a l row o f data r e f l e c t s cpm obtained f o r the same c e l l suspension.
51
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VO
VO ^ 00 o 'n 00 rH
O + 1 CN 00
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CN VO 00 in VO 'd' CO 00 O H
CT> O ro CM VO "a" O in VD rH
rH .H rH rH
rH CM ro
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cn CO CO in I
55
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•T <n in o 00 CN rH in vo CTl rH rH
00 rH cn cn O <n CN 00 00 <n o ro CM H rH
rH CN ro "d*
in
r- CM ID cn 00 ii3
cn
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rH CM
CN cn CN CN 00 rH CN rH T
cn CT^ in 00 vo 00
rH
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+ 1 O O O O O O 0|r^ in CO lO 1^ vo CO inlcn
00 n o m n CT» rH CN
rH in 00 "d" rH in vo vo rH ro CN vo
rH ro 00 VD vo vo 0^ rH CN vo '3' ro O
T rH rH rM
00 vo'd' CO 'a' CO rH m CO CTl r~ CM T O en O rH CM 00
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rH CN ro -3- in
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cn CO vD ro ro CN r-- rH
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56
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CN 00 H rO O O CN CO CN •^f ID CN C en 19 iH in
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57
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59
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rH CM ro
o o o o CM CM ro fO
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o o o o cvi m in a» • • • • rH ro •"3' in CM
+ 1
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00
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00 CM 00 ro CTi in CM
in CM vp 00 in CM Q ro O CM O OJ H rH CM CM
rH CM ro
VO
CM vo o cn vo vo O
O O vo en H CM vo
rH CM ro ^
o o o o ro CM in CO • « • •
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CO • -
o
CTl CTl CD CM in r» ^ ro rH ro rH CM ro CM rH H ro rH
CO rH r- o rH CM H VD CM a» CM CM rH ro H
rH CM ro ^
60
TABLE 3.11 Autoradlograph Data
% of l a b e l l e d and unlabelled thymocytes in relation
to c e l l s i z e following mitogen stimulation
Proportion (%) of unleibelled c e l l s
Proportion (%) of labelled c e l l s
Control (unstimulated)
LPS
PHA
D-iOym >10iJm O-lOpm >10ym Total
98.9 1.1 0 0 0 99.1 0.9 0 0 0 99.9 0.1 O 0 0 98.4 0.4 0.4 0.8 1.2 78.0 17.8 0.5 3.7 4.2 94.3 4.8 0.9 0 0.9
1.05
96.9 0.6 0 . 2.5 2.5 95.8 0.8 1.1 2.3 3.4 95.5 0.9 0.4 3.2 3.6 92.8 0.8 2.7 3.7 6.4 77.7 11.4 0 10.9 10.9 93.8 0.3 0.5 5.4 5.9
5.45
84.3 0.6 5.3 9.7 15.0 82.4 2.7 5.4 9.5 14.9 86.4 0 6.1 7.5 13.6 95.5 1.2 2.2 1.2 3.4 74.6 14.7 1.1 9.6 10.7 87.7 2.8 2.6 6.8 9.4
11.17
0.55
45 - 1.25
,81
F i g . 3.1
Organs were transferred to a Microtest plate
where a lymphocyte suspension was prepared and
washed by centrifugation. Following counting
and adjusting to a concentration of 5 x 10^
lymphocytes cm ^,the c e l l s were distributed into
a Terasaki plate for culture. After 2 days,
the c e l l s were pulsed for 24 hours and then
hand harvested onto a glass f i b r e mat and
prepared for s c i n t i l l a t i o n coiinting.
61 F i g . 3.1
Technique of cell culture in Terasaki plates
DayJ)
10nl cells 2-5^1 mediun)/
mito>?en
^ y 2 _ 2-5ul " H th>'midine
DayJ . cells harvested
62 Fig. 3.2
I
O c O
HI
i n
i n
00
o
i n rsi
00
I / )
CO Q
63
Fig. 3.3
c
1/)
I CO Cl .
.2 "« % 2
E ^ : cfl U
I s .3 -s
3 E
CM CM
C/5
F i g s . 3.4 - 3.6
Each point represents the mean S.I. (- S.E.)
For number of animals used, see Tables 3.2-
3.10.
64
F i g . 3.4
< c I
I I
CM CN
65
Fig. 3.5
' T
I <
CM
CM
ee
c
I
CM
CM CM
cn
66
Fig. 3,6
in
BO <
C/5
Fig. 3.7
67
12
11
- Total percentage of ^ T d R labelled cells
\ :entage of labelled cells
over 10pm tlittmettr.
Percentage of
labelled cells
4 f
• ( • • • l f i j . r ' i | .- -r-:.. illilillli
Ji''it.j,a|:t4ir.r|{
l«<4')"i-' WiU'irMDii'i"'':!
-.4-. t i J i i C H
i iU i i i ' ;•>' ' | " ( " - I , • •
II <N
illfilll fiiiiiiiiilpi iiiiiii lllllli l i i l i l n i l i Pl i
Control LPS PHA
F i g . 3.8
Autoradiographs of control and mitogen stimulated
thymus c e l l s from 6 month old animals.
(a) Control c e l l s showing background l e v e l s
of l a b e l l i n g i n the absence of mitogsn.
(L-labelled c e l l ) .
(b) Thymus c e l l s stimulated with LPS.
Some clumping of the c e l l s has occurred.
Several thymocytes with exposed s i l v e r
grains can be c l e a r l y seen.
(c) Thymus c e l l s stimulated with PHA.
Considerable agglutination has occurred.
^HTdR incorporation i s extensive, with
many thymocytes s i l v e r - l a b e l l e d .
(d) Higher magnification micrograph of PHA-
stimulated thymocytes.
Note that the majority of labelled lymphocytes
are b l a s t c e l l s . Most b l a s t s are heavily-
la b e l l e d , while others possess fewer exposed
s i l v e r grains.
•* - • • •
1cm=50^d
68
69
F i g . 3.9
A l l mitogens were used a t the optimum doses .
used f o r the ontogenetic s t u d i e s . Cultures
were supplemented w i t h 1% FCS.
Animals 1 and 2 were 6 months o l d .
Animals 3 and 4 were 12 months o l d .
70 C/5
CD
• o
I
o
QTG
3-< g c o n
IS9
• • (It
71
CHAPTER 4
Mitogen and mixed lymphocyte c u l t u r e responses f o l l o w i n g
thymectomy ; Studies i n v e s t i g a t i n g p u t a t i v e T lymphocyte heterogeneity
I n t r o d u c t i o n
I n t h i s Chapter a d i f f e r e n t approach to the study o f the
ontogeny o f th^Tnus f u n c t i o n i n the Xenopus larvae i s used and two
issues, emanating from previous studies on thymectomised Xenopus,
are i n v e s t i g a t e d . A few years ago i t was demonstrated t h a t f o l l o w i n g
thymic a b l a t i o n by microcautery a t 7 o r 8 days (Horton & Manning,
1972; Rimmer & Horton, 1977), and even as e a r l y as 5 days o f age
(Horton and Horton, 1975)(when the thymus contains, i n t o t a l ,
<500 c e l l s ) , a chronic f i r s t - s e t a l l o g r a f t r e j e c t i o n eventually
occurs, u s u a l l y several months p o s t - g r a f t i n g a t 23°C. Moreover,
second-set g r a f t s , a p p l i e d a t various i n t e r v a l s (3-112 days)
a f t e r f i r s t - s e t r e j e c t i o n , were r e j e c t e d r a p i d l y w i t h i n 2-3
weeks by these early-thymectomized ( t x ) Xenopus. I t was
suggested t h a t t h i s r e j e c t i o n capacity i s probably achieved by a
thymus-independent component, p o s s i b l y one t h a t i s not normally
a c t i v e i n the non-tx animal, or o n l y o f secondary importance.
However, thymectomy experiments performed a t even e a r l i e r stages
by others (Tochinai and K a t a g i r i , 1975; Ton?>kins and Kaye, 1981)
p o i n t t o the d i s t i n c t p o s s i b i l i t y t h a t very e a r l y seeding from
the thymus o f a few lymphocytes might have occurred p r i o r t o
thymectomy a t 5-8 days o f age. I f t h i s were t r u e , the memory
response displayed by e a r l y - t x toads could w e l l be achieved by
expansion o f t h i s small T c e l l p o p u l a t i o n , p o s s i b l y induced by
h i s t o c o m p a t i b i l i t y antigens o f the g r a f t e d s k i n . I n an attempt
72
t o i n v e s t i g a t e whether such r e s t o r a t i o n occurs, experiments
were s e t up to look f o r the reappearance o f T c e l l markers,
such as r e a c t i v i t y t o the T c e l l mitogens (PHA and Con A), i n
a l l o g r a f t e d , e a r l y - t x toads, since these markers are v i r t u a l l y
undetectcible i n non-grafted, e a r l y - t x animals (Ho'rton and
S h e r i f , 1977; Manning and C o l l i e , 1977; Du Pasquier and Horton,
1976; Manning, Donnelly and Cohen, 1976; Green and Cohen, 1979).
The i n v e s t i g a t i o n concentrates on s p l e n i c lymphocytes, but
p e r i p h e r a l blood lymphocytes (PBL) are also used, since i t
has p r e v i o u s l y been shown (Horton, Horton and Rimmer, 1977)
t h a t the spleen i s p o s s i b l y not c e n t r a l l y involved i n s k i n
g r a f t d e s t r u c t i o n i n t x animals ( i . e . may not be a s i t e t h a t
houses any r e s t o r e d T c e l l p o p u l a t i o n ) , i n c o n t r a s t t o i t s
c e n t r a l involvement i n alloimmunity i n c o n t r o l toads.
The second p a r t o f t h i s i n v e s t i g a t i o n concerns a r e
examination o f the e f f e c t s o f l a t e r l a r v a l thymectomy on in^ v i t r o
p r o l i f e r a t i v e responses. I n a study o f the e f f e c t o f sequential
thymectomy on the a b i l i t y o f Xenopus splenocytes t o d i s p l a y mixed
lymphocyte c u l t u r e (MLC) r e a c t i v i t y and t o respond to T c e l l
mitogens, Horton and S h e r i f (1977) i n d i c a t e d t h a t the thymus may
be r e q u i r e d f o r a longer p e r i o d o f l a r v a l l i f e to e s t a b l i s h
mitogen responsiveness than i s necessary f o r the establishment
o f MLC r e a c t i v i t y . For example they showed t h a t thymectomy
a t 3-4 weeks o f age (stages 52-54) no longer abrogates s p l e n i c
MLC r e a c t i v i t y (compared to the e f f e c t o f 7 day thymectomy) ,
whereas PHA r e a c t i v i t y o f spleen c e l l s taken from other 3-4 week-
t x t o a d l e t s was abolished. They suggested the p o s s i b i l i t y
73
that MLC and PHA reactive lymphocytes i n Xenopus may therefore
be d i s t i n c t T c e l l popiHations. In order to determine more
rigorously whether MLC positive lymphocytes, that are PHA
negative can be detected within the spleen, splenocytes from
individual toadlets thymectomlzed at 3-4 weeks of age were here
tested i n both MLC and mitogen assays. Mitogen studies on
PBL i n these animals were also c a r r i e d out to examine whether
other peripheral lymphocyte populations also require a prolonged
thymus presence i n vivo to permanently establish their PHA
r e a c t i v i t y .
Materials and Methods
(a) Thymectomy
Thymectomy was performed by microcautery, following the
technique described by Horton and Manning (1972), either at
stage 47 (1 week) or at stages 52-54 (3-4 weeks). At one v;eek
of age the thymus i s a small translucent organ, approximately
lOOym i n diameter, thymus lymphocyte differentiation has begun,
as revealed by l i g h t and electron microscope studies (Horton
and Manning, 1972; Nagata, 1977; Rimmer, 1977), but few small
lymphocytes are found. By 3-4 weeks of age the thymus has
enlarged to a diameter of about 1mm, displays cortico-medullary
d i f f e r e n t i a t i o n , and contains large numbers.of small lymphocytes
(p a r t i c u l a r l y in the cortex). Thymic ablation was confirmed
when the animals were k i l l e d . Non-operated animals served as
controls.
(b) Grafting
The grafting technique of Horton and Manning (1972) was
followed. Dorsal skin (2mm^) from a non-sibling donor was transplanted.
74
After grafting the toadlets were kept at 26°C to encourage
more rapid a l l o g r a f t rejection i n the tx animals than occurs
at 2 3 ° C .
(c) Separation of PBL
A mixture of 34% T r i o s i l ("Isopaque," Vestric) and 9%
F i c o l l 400 (Pharmacia) was prepared which gave a f i n a l density
of 1 . 0 9 4 . This mixtvure was then distributed .'.nto aliquots,
autoclaved, stored i n the dark at 4°C u n t i l use. Blood was
colle c t e d under s t e r i l e conditions from anaesthetised animals
by cardiac puncture, and was transferred by pipette into
amphibian L 15 medium containing 20 linits heparin (Roche) cm ^.
The blood/medium mixture was made up to 2 cm^ by adding more medium
and was then c a r e f u l l y layered onto 2 cm^ of tl.e F i c o l l / T r i o s i l
mixture i n a 10 cm^ centrifuge tube. This was centrifuged
for 2 minutes at I60x g. The top 1 cra' of medium was then
discarded and the bottom 1 cm" containing lymphocytes at the
interface was c a r e f u l l y pipetted off. The c e l l s were f i n a l l y
washed 3 times i n medium, prior to th e i r use i n the mitogen .
assays.
(d) Mixed lyn^hocyte culture
Preparation of c e l l s for MLC i s similar to that described
for the standard mitogen culture i n Chapter 2 . Control cultures
contained 40vil spleen c e l l s from one animal at a concentration of
5 X 10^ lymphocytes cm . The mixed lymphocyte cultures used
2 0 p l of c e l l s from each animal and consisted of splenocytes from
either 2 control or 2 tx animals. Control and thymectomized
75
t o a d l e t s belonged t o the same f a m i l y . I n a d d i t i o n to 1%
FCS supplementation, l O y l Xenopus serum ( f i n a l concentration
0.5%} was added. Xenopus serum has been shown to lower
background counts, so r e s u l t i n g i n b e t t e r S.I.'s (Du Pasquier
and Horton, 1976). P i l o t experiments t e s t e d the e f f e c t o f
3 d i f f e r e n t pools o f Xenopus serum on MLC r e a c t i v i t y and the best
pool was used throughout a l l experiments described here. The
c u l t u r e s were pulsed a t 3 days w i t h 1 pCi " HTdR and c e l l s
harvested 18-24 hours l a t e r , p r i o r t o processing f o r s c i n t i l l a t i o n
counting. S t i m u l a t i o n i n d i c e s were c a l c u l a t e d by d i v i d i n g
mean cpm o f mixed c u l t u r e s by the average o f the mean cpm o f
the corresponding c o n t r o l c u l t u r e values. .S.I.'s were considered
p o s i t i v e when they reached 1.5.
(e) Experimental design
( i ) P r e l i m i n a r y studies on mitogen s t i m u l a t i o n o f PBL
Ihe a b i l i t y o f PBL t o respond t o PHA and LPS w i t h our c u l t u r e
technique had t o be assessed p r i o r t o the use o f these c e l l s i n the
2 major i n v e s t i g a t i o n s . The standard c u l t u r e technique was used
and i n v o l v e d separated PBL a t 5 x 10^ cm w i t h 1% FCS supplementation.
Both 7-day-tx and c o n t r o l t o a d l e t s (8-10 months old) were used.
PHA was added a t a f i n a l concentration o f 20 yg cm ^ and LPS
a t 2 mg cm"" . LPS was used p r i m a r i l y t o check f o r f u n c t i o n a l
v i a b i l i t y o f lymphocytes i n t x animals.
( i i ) Mitogen s t u d i e s on lymphocytes from c o n t r o l and
thymectomized alloimmune t o a d l e t s .
C o n t r o l and 7-day-tx t o a d l e t s were g r a f t e d when 8-10 months
o l d . (Grafts f o r t x hosts came from c o n t r o l or t x donors but t h i s
d i d not a l t e r t h e i r f a t e l ) Grafts were inspected every 2-3 days.
76
Eighteen weeks p o s t - g r a f t i n g splenocytes and PBL were examined
f o r mitogen r e a c t i v i t y by standard c i i l t u r e technique. The
f o l l o w i n g concentrations o f mitogens were used:- PHA - 20yg cm -3 -3 Con A - 1 yg cm ; LPS - 2 mg cm . The 18 week i n t e r v a l
between g r a f t i n g and the mitogen e^qseriments Afas selected
because second-set g r a f t s a p p l i e d t o e a r l y - t x animals a t t h i s
p o s t - t r c i n s p l a n t a t i o n i n t e r v a l should be r e j e c t e d r a p i d l y - i . e .
any T c e l l r e s t o r a t i o n o c c u r r i n g should be detectable by t h i s
time.
( i i i ) Mitogen and MLC studies on lymphocytes from t o a d l e t s
thymectomized a t 3-4 weeks o f age.
Animals used i n these studies were 8 months o l d . PBL wCfi used
o n l y f o r mitogen experiments and were c u l t u r e d w i t h the standard
technique. Splenocytes from i n d i v i d u a l animals were u s u a l l y
t e s t e d f o r mitogen r e a c t i v i t y ( m i n i a t u r i s e d technique) and i n
MLC (standard technique). LPS was again used to check f u n c t i o n a l
lymphocyte v i a b i l i t y . Doses o f mitogen used were as i n ( i ) above.
Results
(a) P reliminary studies on mitogen s t i m u l a t i o n o f PBL
Results are i l l u s t r a t e d i n Table 4.1 and Fig. 4.1. They
show t h a t although the response o f PBL from c o n t r o l boadlets t o
PHA was ge n e r a l l y low (mean S.I. - S.E. = 5.5 - 2.8), a l l the
cultxares gave p o s i t i v e S.I.'s. I n c o n t r a s t PHA stimulated
PBL c u l t u r e s on thymectomized animals gave negative S.I.'s
throughout (mean S.I. = 0.9 - 0.2. With LPS both c o n t r o l and
t x PBL gave p o s i t i v e S.I.'s (means o f 6.0 - 3.4 and 12.0 - 3.0
r e s p e c t i v e l y ) . These r e s u l t s confirm t h a t responsiveness t o T
c e l l mitogens i s abrogated by e a r l y t x , vAiereas r e a c t i v i t y to B
77
mitogens; i s l e f t i n t a c t . The data i s i n agreement with recent
findings (Green and Cohen, 1979) on mitogen r e a c t i v i t y of tx
Xenopus PBL.
(b) Mitogen studies on lymphocytes from alloimmune toadlets
Counts per minute are given in Table 4.2. S.I.'s and graft
r e j e c t i o n times are i l l u s t r a t e d i n Fig. 4.2. At 26°C the 7
control toadlets on average rejected allografts in 16 - 0.8 days.
The 7 tx animals took, on average, nearly three times as long
(43 - 8.3 days).
Following graft r e j e c t i o n , splenocytes from controls
displayed good l e v e l s of r e a c t i v i t y to both PHA and.Con A
( S . I . ' s were 15.4 (-4.4) and 7.4 (-3.2) respectively. PBL
from these animals responded well to both PHA (mean S.I. =6)
and LPS (mean S.I . = 12.2). A l l the spleens from early-tx
allografted toadlets f a i l e d to respond to Con A (mean S.I.
= 0.5 (-0.2)). Reactivity to PHA was also abolished in spleen
c e l l cultures from 4 out of 5 animals (mean S.I. = 0.9 - 0.3) ,
the other showing a dramatically impaired response ( S . I . = 1.8).
The p o s s i b i l i t y that s i t e s other than the spleen could contain
restored populations of T-mitogen-reactive c e l l s seems unlikely
i n view of the r e s u l t s with PBL from 2 tx allografted animals.
Thus PBL from these f a i l e d to respond to PHA, although they showed
good mitogen r e a c t i v i t y to LPS. I t appears, then, that a l l o g r a f t
r e j e c t i o n occurs i n tx animals in the absence of lymphocytes able
to respond to T c e l l mitogens.
78
(c) MLC and mitogen studies on lymphocytes from toadlets
thymectomized a t 3-4 weeks o f age
MLC datcK. f o r splenocytes o f c o n t r o l and t x animals i s given
i n Table 4.3. A l l 9 alloge n e i c combinations between c o n t r o l s
r e s u l t e d i n enhanced ^HTdR uptake compared w i t h background
(autologous) c u l t u r e s (mean S.I. - S.E. = 3.4 - 0.5; range
1.5-5.9). I n c o n t r a s t , only h a l f (6/12) MLC combinations
between the 11 t x animals used displayed p o s i t i v e S.I.'s. ( o v e r a l l
the mean S.I. was 1.6 - 0.3, ranging from 0.6-3.4). I t appears
t h a t i n these ea^periments thymectony during m i d - l a r v a l l i f e
has impaired the normal a a t i i r a t i o n o f MLC-reactive lymphocytes
i n some, but not a l l , t o a d l e t s .
Mitogen responses o f splenocytes and PHA o f 8 o f these
same t x animals are given i n Table 4.4. Only 3/8 splenocyte
c u l t u r e s responded t o PHA (mean S.I. = 2.2 - 0.6), the best S.I.
being 6.0. Likewise only 2/7 PBL c u l t u r e s displayed PHA
r e a c t i v i t y (mean S.I. = 1.1 - 0.3). R e a c t i v i t y o f splenocytes
and PBL t o LPS was, as expected, unaffected by m i d - l a r v a l
thymectomy (mean S.I.'s were 7.6 - 1.9 and 9.4 - 1.9 r e s p e c t i v e l y ) .
Since only one i n d i v i d u a l case o f p o s i t i v e MLC r e a c t i v i t y
(animals 10 x 11) but negative PHA r e a c t i v i t y o f both partners
occurred i n the t x group, i t seems t h a t t x a t 3-4 weeks o f age
has here impaired both PHA and MLC r e a c t i v i t i e s t o such an ex t e n t
t h a t the experiments provide no c l e a r evidence t h a t these functions
are a f f o r d e d by d i s t i n c t T lymphocyte populations.
Discussion
One major o b j e c t i v e o f t h i s Chapter was to determine
whether lymphocyte r e a c t i v i t y t o T c e l l mitogens occurs i n e a r l y -
79
t x animals t h a t have r e j e c t e d s k i n a l l o g r a f t s . Responsiveness
t o T c e l l mitogens i s o f t e n considered t o be a hallmark o f a l l
T c e l l popvilations, one which transcends t h e i r s p e c i f i c
inmunologic f u n c t i o n . Indeed T c e l l mitogens are widely used
as an i n d i c a t o r t h a t competent T lynphocytes are present i n an
i n d i v i d u a l (see Chapter 3 ) . I n the present experiments on t x
Xenopus, these mitogens have been used t o i n v e s t i g a t e the
p o s s i b i l i t y o f a general r e s t o r a t i o n o f T-equivalent c e l l s .
Considering f i r s t the speed o f g r a f t r e j e c t i o n i n the
7-day-tx t o a d l e t s , i t i s i n t e r e s t i n g t o note t h a t keeping the
animals a t 26°C r a t h e r than 23°C a f t e r t r a n s p l a n t a t i o n considerably
reduces the time taken t o e f f e c t g r a f t r e j e c t i o n ( c f . Rimmer and
Horton, 1 9 7 7 ) . E l e v a t i n g the temperature by 3°C r e s u l t s
i n a more r a p i d and uniform alloimmune r e j e c t i o n i n e a r l y - t x
toads. Perhaps acute g r a f t r e j e c t i o n a t 23°C i s c r i t i c a l l y
dependent on a n p l i f i e r T c e l l s (MLC-positive lymphocytes are
absent from t x , non-grafted animals - see Du Pasquier and Horton,
1 9 7 6 ) , whereas a t higher temperatures these c e l l s assume less
inportance.
E a r l y - t x animals t h a t have r e j e c t e d g r a f t s were shown
t o be unresponsive t o 2 T c e l l mitogens, a t l e a s t w i t h respect
to t h e i r splenocytes and PBL. As these p e r i p h e r a l popialations
o f lymphocytes responded w e l l t o LPS, the n o n - r e a c t i v i t y seems
s p e c i f i c f o r T, r a t h e r than B c e l l mitogens. The mitogen
assays were performed 18 weeks p o s t - f i r s t - s e t g r a f t i n g - i . e .
a t a time when second-set g r a f t s should be r e j e c t e d r a p i d l y -
80
t h e r e f o r e i t seems l i k e l y t h a t even acute a l l o g r a f t d e s t r u c t i o n
i n t x animals i s e f f e c t e d by lymphocytes t h a t are not able t o
respond t o PHA o r Con A. This i n t e r p r e t a t i o n f i t s w e l l w i t h
previous sequential t x data, where thymic a b l a t i o n as l a t e as
30 days s t i l l abrogates s p l e n i c PHA r e a c t i v i t y , but leaves
a l l o g r a f t r e j e c t i o n capacity t o develop q u i t e normally (Horton
and Manning, 1972; Horton and S h e r i f , 1977; Manning and C o l l i e ,
1977).
I t w i l l now be important t o determine whether MLC r e a c t i v i t y
( p a r t i c u l a r l y towards donor MHC antigens) i s restored f o l l o w i n g
a l l o g r a f t d e s t r u c t i o n i n t x animals. I f i t i s not, then i n
order t o argue t h a t a l l o g r a f t immunity i n e a r l y - t x t o a d l e t s i s
mediated by a thymus-dependent lymphocyte subset ( r a t h e r than
a thymus-independent component), one would have to suggest
t h a t t h i s i s achieved by an early-seeded T-equivalent c e l l
p o p u l a t i o n t h a t i s PHA and MLC negative. This l i n e o f research
should be a u s e f u l approach i n determining whether c y t o t o x i c T
lymphocytes ( r e c e n t l y denonstrated i n Xenopus by Bernard,
Bordmann, Blomberg and Du Pasquier, 1979) represent a separate
T-equivalent subset from MLC- and PHA-responsive lymphocytes
i n amphibians. I n t e r e s t i n g l y i t has been shown i n mammals t h a t
MLC r e a c t i v e populations can respond t o PHA and Con A, but t h a t
c e r t a i n a l l o r e a c t i v e T c e l l s are non-responsive t o these T c e l l
mitogens (Watenabe e t a l . , 1977). Indeed Hayry e t a l . (1976)
have found, by long term s e l e c t i v e c u l t u r e , t h a t there i s only
a small overlap between these f u n c t i o n s i n mice.
The second major i n v e s t i g a t i o n performed i n t h i s Chapter
i n v o l v e d a f r e s h look a t the p o s s i b i l i t y t h a t thymectomy i n mid-
l a r v a l l i f e could reveal t h a t T-mitogen-reactive c e l l s are, a t l e a s t
81
to some extent, a population of lymphocytes d i s t i n c t from
MLC-reactive c e l l s . However the studies on in vitr o p r o l i f e r a t i v e
behaviour of lymphocytes from toadlets tx at 3-4 weeks of age
have provided no c l e a r d i s t i n c t i o n between these two populations
i n terms of their degrees of thymus-dependency. The establishment
of both PHA- and MLC-reactive lymphocyte populations in the
periphery would appear to require a prolonged thymus presence
i n vivo compared with the more rapid, but equally gradual,
establishment by the thymus of i i i vivo alloimmune r e a c t i v i t y
(with respect to the l a t t e r , see Tompkins and Kaye, 1981).
Although a d i s t i n c t i o n between the duration of thymus-dependence
of MLC and PHA reactive populations was suggested by Horton and
Sherif (1977) (as noted i n the Introduction), these workers
did indicate some impairment of MLC responses regularly occurs
following thymectomy at the stages of development studied here.
Further investigations on sequential thymectomy and MLC
r e a c t i v i t y should make use of defined strai n s of Xenopus, using
MLC combinations where the mixed lymphocytes come from toadlets
with strong MHC differences. Under such cond-.tions MLC r e a c t i v i t y
of control c e l l s would be far more uniform than seen here in
experiments which, unfortunately, had to make use of si b l i n g
combinations. In Xenopus, antigens stimulating in the MLC
reaction segregate as i f controlled by a single genetic locus,
and acute graft r e j e c t i o n seems to be governed by t h i s same region.
This polymorphic genetic locus has been c a l l e d the XLA MHC system
(Du Pasquier and Kobel, 1977) and bears certain homology with
mammalian MHC's. In Xenopus, some erythrocyte antigens segregate
with MLC determinants, while others belong to different linkage
groups (Du Pasquier and Kobel, 1977). As i n endotherras, there
82
must also be minor HC euitigens i n Xenopus, since MLC identical
toadlets are s t i l l capable of rejec t i n g grafts exchanged between
each other. Du Pasquier and Kobel (1977) have shown that
graft r e j e c t i o n times can be predicted by MHC haplotype differences,
and that the l e v e l of MLC r e a c t i v i t y i s also related to t h i s
MHC d i s p a r i t y (2 haplotype differences giving greater MLC
S.I.'s than 1 haplotype difference). This linkage betv/een
MLC r e a c t i v i t y and speed of a l l o g r a f t rejection i s discussed
by Cohen and C o l l i n s (1977) who conclude that there I s some
correlation between these two functions throughout a l l vertebrates .
so far investigated, although Cohen and Horan (1977) found no such
correlation i n the newt.
83
TABLE 4.1
Controls
Mitogen stimulation of peripheral blood lymphocytes i n non-grafted toadlets ; the effect of 7-dav thymectomy
-3 PHA 20ygcm Background counts per min.
124 377 78
630 593 626 146
Stimulated counts per min. SI
LPS 2 mg cm -3
680 146 593
284 1604 1370 1037 1147 2067 464
Mean - S.E,
2220 1885 1213
2.3 4.3 22.0 1.7 1.9 3.3 3.2
- 5 7 5 ^
3.2 12.8 2.1
2.8
Mean - S.E. 6.0 ± 3.4
7-day thymectomized -3 PHA 20ygcm
599 1241 1315 1180 457 657
675 195 1196 1159 726 558
Mean S.E.
1.1 0.2 0.9 1.0 1.5 0.9 0.9 3 0.2
LPS 2 mg cm 599
1241 183
1315 1180 457 1967
16852 8570 1928 17108 v3350 2399 2945
Mean - S.E.
28.1 6.9
10.3 13.0 5.4 5.2
12.0 - 3.0
Controls
84
TABLE 4.2 Mitogen stimulation of splenocytes from control and 7-day thymectomized toadlets following graft rejection
Unstimulated cpm PHA Stimulated cpm Con A stimulated cpm
1 4034 42765 30176
2 3193 16818 13308
3 438 11960 7120
4 841 20665 nd
5 1428 13261 2122
7 day thymectomized
Unstimulated cpm
188 1
2
3
4
5
2807
2790
1426
327
PHA stimulated cpm Con A stimulated cpm
185
1427
802
1639
597
181
593
623
287
253
85
TABLE 4.3 The e f f e c t of thymectomy at stage 52-54
on MLC r e a c t i v i t y of splenocytes
CONTROLS THYMECTOMIZED
3275 20272 18612 1 271 709 794 (4.7) (3.7) (3.1) (0.9)
5328 9260 2 182 1466 (1.5) (1.9)
6919 3 1356
5 6 4 5 6
4 897 3027 2793 (2.5) (2.8)
5 1560 4903 (3.7)
6 1101
7 8 9
7 2938 56921 8361 (5.9) (4.5)
8 16295 13428 (1.6)
9 802
4 1053
7 133
10
11
5035 6512 (3.4) . (1.1)
1930 . 8283 (1.3)
10677
8 9
21.9 nd (0.7)
446 nd
5257
10 11
nd 1780 (0.9)
nd 1159 (0.6)
8074 7321 (2.0) (1.5)
2428 5702 (1.8)
3864
S.I.'s given i n parentheses
86
TABLE 4.4 The e f f e c t of thymectomy at stage 52-54 on
r e a c t i v i t y of splenocytes and PEL to PHA and LPS
Animal No. Spleen Blood PHA LPS PHA LPS
4 1.9 11.9 0.9 3.3
5 1.2 8.4 2.6 17.7
6 1.1 5.9 0.8 10.6
7 3.9 0.5
.8 1.1 1.3 1.6 5.2
9 6.0 17.6 0.2 10.3
10 1.3 7.8 0.9 13.0
11 1.3 7.6 1.0 5.4
Animals 4-11 are the same tx toadlets as i n the MLC experiments
i n Table 4.3.
F i g . 4.1
Mitogen responsiveness of peripheral
blood leucocytes (PBL) from control and
thymectomized animals. PHA was at 20iigcm
and LPS 2.0mg cm^. One per cent FCS
supplementation was used throughout.
87 CO
CO
CO
-o V 1/1
1
£
ee CM
r
eo
c o
CM CMI
a.
< X Ou
Q-
<
F i g . 4.2
Mitogen responsiveness of control and
thymectomized animals that have rejecte'^.
a l l o g r a f t s .
m PHA at 20Mgcm~'
^ Con A at l.Oygcm ^
I I LPS at 2 mg cm"'
88
E c
E
89
CHAPTER 5
Ontogeny of i n vivo lymphocyte r e a c t i v i t y to the hapten t r i n i t r o -
phenyl conjugated to a thymus-dependent and thymus independent c a r r i e r .
Introduction
Central to the fvinctioning of a l l immune systems i s the a b i l i t y
to recognise antigenic determinants, and c r u c i a l to this i n i t i a l
recognition phase i s the binding of antigen to s p e c i f i c receptors
on the surfaces of c e l l s of the immune system. An immune response
to c i r c u l a t i n g antigen can be characterised by an increase i n both
number of lymphocytes capable of binding antigen and the a f f i n i t y
of t h i s binding capacity. Although some antigens stimulate B c e l l s
d i r e c t l y to produce antibody, most ('thymus-dependent') antigens
require the involvement of helper T c e l l s before a response can be
e l i c i t e d . The importance of t h i s collaboration between T and B
c e l l was f i r s t investigated i n thymectomized and restored mice,
using xenogeneic erythrocytes as a thymus-dependent antigen (Claman
et. a l , 1966; Davies, e t a l , 1967; Mitchell and Miller, 1968).
Hapten-carrier experiments have also been centrally involved in
t h i s respect (see Mitchison, 1971). Lymphocyte collaboration has
since been demonstrated (through hapten-carrier studies) on diverse
vertebrates that include teleost f i s h (Yocum et^ a i . , 1975; Stolen
& Makela, 1975, 1976; Ruben, Warr, Decker & Marchalonis, 1977),
urodeles (Ruben, van der Hoven & Dutton, 1973) and anuran amphibians
(Ruben, 1975; Ruben & Edwards, 1980).
This chapter exploits the hapten-carrier system to investigate
the i n vivo ontogeny of T-helper c e l l a c t i v i t y and B-equivalent
90
lymphocytes i n the Xenopus spleen. In the presenc studies sheep red
blood c e l l s (SRBC) have been used as a thymus-dependent c a r r i e r , and
LPS as a thymus-independent c a r r i e r for the hapten trinitrophenyl
(TNP). Recent studies on Xenopus (Horton, Edwards, Ruben and Mette,
1979) have confirmed the thymus-dependent/independent status
respectively of these two c a r r i e r s . Carrier pre-immunisation i s
required to achieve good l e v e l s of anti-TNP responses following
TNP-RBC i n j e c t i o n s . In contrast TNP-LPS requires no such c a r r i e r
pre-immunisation and so one can examine functional B c e l l ontogeny
d i r e c t l y .
While at metamorphosis many anuran immune responses seem
to decline or disappear (e.g. thymocyte mitogen r e a c t i v i t y ) (Chapter
3) i t has recently been reported that i n vivo induced antigen-binding
splenocyte r e a c t i v i t y to a thymus-dependent hapten-carrier complex
act u a l l y increases a t t h i s time (Ruben eit al^., 1980). These authors
suggest that t h i s elevation i n 'humoral' immune r e a c t i v i t y may be
caused by an enhancement of helper T c e l l a c t i v i t y at metamorphosis,
necessary perhaps as a counterbalance to the suppressed c e l l u l a r
immunity found at t h i s time (Du Pasquier and Bernard, 1980). The
present experiments further investigate the influence of metamorphosis
on the ontogeny of humoral immune r e a c t i v i t y , and examine the p o s s i b i l i t y
that both T- cuid B-equivalent lymphocyte r e a c t i v i t y i s elevated at
t h i s time.
The work begins with a comparison of plaque-forming c e l l (PFC)
responses i n spleens taken from dif f e r e n t aged animals to three antigens •
SRBC, TNP-SRBC and INP-LPS. The ontogeny of splenocyte r e a c t i v i t y
to the 2hapten-carrier conjugates (using the more sensitive immuno-
91
cytoadherence (ICA) assay) i s then considered in d e t a i l . The
chapter ends with a consideration of the thymus as a s i t e containing
antibody-forming c e l l s , since mitogen studies e a r l i e r i n the
Thesis suggest t h i s organ contains some B-equivalent lymphocytes.
Materials and Methods
(a) Antigens and i n j e c t i o n s
Sheep red blood c e l l s (SRBC) and horse red blood c e l l s (HRBC)
i n Alsever's solution (Flow Laboratories) were washed three times
i n s a l i n e p r i o r to use i n i n j e c t i o n and/or assays).
Toadlets received a single i n j e c t i o n of 10% SRBC, 50yl per gram
body weight, v i a the intraperitoneal (i.p.) routT. Larvae v;ere
immxanized with a single i n j e c t i o n of Syl 50% SRBC i.p. Larval
i n j e c t i o n s were performed with the aid of a micropipette, drawn
from a 50yl microcap (Shandon). The t i p of the micropipette was
inserted through the ventral t a i l musculature into the peritoneal
cavity. When the pipette was withdrawn, the muscle prevented
leakage of injected material from the peritoneal cavity.
Haptenation of erythrocytes with INP followed the procedure
of Rittenberg and Pratt (1969). TNP-SPBC for i n j e c t i o n were prepared
by adding 2.5 cm^ of packed SRBC to 7 cm^ cacodylate buffer (pH 6.9),
containing 0.065g TNBS (BDH). The mixture was s t i r r e d constantly
i n a foil-covered f l a s k for 30 minutes at room temperature, and then
15 cm^ s a l i n e were added to stop the reaction. These 'heavily'-
conjugated c e l l s were then washed i n glycyl-glycine (4.5 x 10 'M) to
remove any unbound TNP, and following t h i s were washed three times
i n s a l i n e . These haptenated c e l l s were injected at the same
92
concentration and dose as the unhaptenated SRBC. Carrier
pre-immunisation was necessary to obtain a good RFC response to
TNP-SRBC (see R e s u l t s ) . Here 0.0025% SRBC were injected 2 days
p r i o r to the TNP-SRBC i n j e c t i o n , 50yl (g.b.w.) of this low c a r r i e r
dose for toadlets, 5yl for larvae.
TNP-LPS was prepared as described elsewhere (Jacobs and
Morrison, 1975) and was injected at a concentration of SOyg cm ^
for toadlets (50yl g.b.w.) and O.Smg cm for larvae (5pl).
For the PFC and ICA assays, HRBC were used as TNP-carrier,
but with a l i g h t e r haptenation protocol than used for TNP-SRBC
in j e c t i o n ; only O.0025g 1NBS was used and the reaction was stopped
a f t e r 10 minutes.
(b) PFC assay
The s l i d e haemolytic plaque assay, o r i g i n a l l y described
by Cunningham and Szenberg (1968) and modified by Du Pasquier (1970)
for amphibian studies, was used. C e l l suspensions were prepared
i n culture medium as described i n Chapter 2, but here c h i l l e d on
i c e , and adjusted to a maximum of 5 x 10^ lymphocytes cm ^. Each
PFC assaly mixture consisted of 200JJ1 spleen c e l l s mixed with 20IJ1
of 25% assay RBC and 50yl 1:10 guinea pig serum (Wellcome) as a source
of complement. Tests of several different complement batches were
made and the best was selected for use throughout a l l the experiments.
Control assays were also set up for each experiment, using 50ul
medium instead of complement; these assays were uniformly negative.
The assay mixtures were prepared on i c e , but then allowed
to reach room temperature to prevent formation of a i r bubbles when
inserted into the PFC chambers. After thorough resuspension, duplicate
lOOyl assay samples were gently pipetted into PFC s l i d e chambers
93
(Wild and Dipper, 1974). Chambers were sealed with molten
par a f f i n wax and vaseline and were then incubated for 2-3 hours
at 28°C. After t h i s time plaques could be seen macroscopically
as d i s t i n c t c l e a r areas, but they were checked under the microscope.
Only when a central Xenopus lymphoid c e l l could be seen surrounded
by mammalian red c e l l ghosts was a PFC scored. PFC were expressed
as the number 10 ^ lymphocytes. Anti-TNP PFC were estimated
from the difference i n numbers of PFC v. TNP-HKBC mini& HRQC (stC
Resultis).
Cc) ICA assay
C e l l suspensions were prepared exactly as described for
the PFC assay. Two tubes were set up for each assay, each txibe.
containing lOul t e s t RBC ( 1 % ) , and 50]il of the lymphocyte suspension.
Tubes were coded (to avoid bias when counting RFC's) and incubated
overnight at 4°C. Following gentle resuspension, the assay
suspensions were scanned microscopically using the whole .9 mm
of an American Optical Neubauer coxinting chamber. RFC were scored
when more than 3 t e s t RBC adhered to the lymphocyte membrane
(see F i g . 5.3). The number of RFC, eacpressed as RFC/10^ lymphocytes,
was calculated from at l e a s t 4 haemacytometer counts (2 from
each duplicate tube s e t up).
However, anti-TNP RFC were usually estimated from the difference
i n numbers v. TNP-HRBC-HRBC. In a few ICA experiments (see Results),
the s p e c i f i c i t y of anti-TNP r e a c t i v i t y was checked by incubating
the assay suspensions i n either TNP-glycine (10 M) or glycine (10 M)
alone.
94
(d) Experimental design
( i ) Ontogeny of induced splenocyte PFC r e a c t i v i t y
The k i n e t i c s of the PFC response of toadlets to a single
i n j e c t i o n of SRBC were examined f i r s t to ascertain optimum times
to assay. Injected animals were kept at the elevated temperature
of 26°C to t r y and increase the uniformity of the antibody
response (which had been r e a l i s e d with the a l l o g r a f t rejection
studies performed i n Chapter 5 ) . Sixteen animals, aged 9-15
months, were given a single i n j e c t i o n of SRBC and spleen c e l l
suspensions from a t l e a s t 2 separate animals were assayed on
days 4,6,7,8,9,12 and 14.
These i n i t i a l k i n e t i c experiments were preliminary to a
second s e r i e s of experiments (again carried out at 26°C), which
compared ontogeny of PFC responsiveness to the T-dependent antigens
SRBC and TNP-SRBC with that occurring to the T-independent antigen
TNP-LPS. For each l a r v a l assay i t was necessary to pool 10-20
individual spleens (depending on age) due to the very low lymphocyte
nvmibers. In these experiments some of the assays on toadlet
spleens were performed on pooled spleen suspensions, to ensure
that the l a r v a l r e s u l t s were not an a r t i f a c t of the pooling of
allogeneic c e l l s i n the PFC assay. These pooling experiments
are marked i n the Tables.
( i i ) Ontogeny of induced antigen-binding c e l l s in the spleen
La r v a l assays consisted of a pool of 10-20 spleens. I n i t i a l
experiments on larvae excunined TNP-SRBC immunisation schedules
required for good an t i TNP-HRBC binding a c t i v i t y in the spleen.
These studies included an experiment to check whether or not
95
low-dose priming with the erythrocyte c a r r i e r was necessary
to e f f e c t elevated l e v e l s of RFC's over background (assays from
sa l i n e injected animals). Ontogenetic ICA experiments ( a l l carried
out a t 23°C) with TNP-SRBC and also with TNP-LPS were then examined,
with p a r t i c u l a r emphasis on events at metamorphosis. Again some
of the toadlet assays were performed on pooled splenocyte suspensions,
to ensure that l a r v a l antigen binding r e s u l t s were not an a r t i f a c t
of pooling allogeneic lymphocytes.
( i i i ) Antibody forming c e l l s within the thymus
I n the t h i r d study, the thymus i t s e l f i s b r i e f l y examined as
a s i t e of PFC a c t i v i t y following immunisation with TNP-SRBC.
Procedures were exactly as for the spleen, but each assay was
prepared from an individual thymus. The thymuses came from
6 month old toadlets, which had been kept at 26°C after i n j e c t i o n .
Results
(aI Ontogeny of induced splenocyte PFC r e a c t i v i t y
The r e s u l t s showing the k i n e t i c s of the toadlet response to a
single i n j e c t i o n of SRBC show (see Table 5.1) that moderate le v e l s
of PFC were induced i n the spleen. In contrast to the absence of
splenic PFC recorded i n non-injected toadlets kept at 26°C, by 4 days
post-SRBC i n j e c t i o n PFC had appeared. Maximum PFC l e v e l s were
found by 6-8 days post-injection, after which time the l e v e l of
response declined. The k i n e t i c s and l e v e l of PFC responsiveness
correspond quite well with PFC r e s u l t s from other poikilotherms
(e.g. cyprinid f i s h , R i j k e r s and Muiswinkel, 1977).
Table 5.2 shows the r e s u l t s of experiments comparing the
PFC responses of different aged animals to the three antigens SRBC,
96
TNP-SRBC and TNP-LPS. No splenic PFC to any of the antigens
were seen here before 3 months of age - i . e . u n t i l post-metamorphic
l i f e . By 4 months of age (two months after the end of metamorphosis)
small numbers of PFC were fovmd to SRBC, and by 8 months, adult
anti-SRBC PFC l e v e l s were seen. With regard to TNP-SRBC, a n t i -
TNP PFC were found i n 7 month old animals, although the numbers were
rather low. The assay on the 12 month animals was performed on a
pool of spleen c e l l s and these again showed a (low level) anti-TNP
PFC response, suggesting that the lack of PFC responses to TNP-
SRBC before and over metamorphosis i s not an artefact of pooling
c e l l s . The r e s u l t s with TNP-LPS are similar to those with the T-
dependent antigens, i n that up to 3 months of age no splenic PFC
response was found. At s i x months, however, reasonable l e v e l s of
anti-TNP PFC were found after TNP-LPS inje c t i o n , as they were at
12 months.
I t appears from these r e s u l t s that a s i g n i f i c a n t change in
some immune componentts) takes place after the end of metamorphosis
which allows the subsequent detection of splenic PFC. However,
a second s e r i e s of ontogenetic experiments was undertaken using the
more s e n s i t i v e ICA assay.
(b) Ontogeny of induced antigen-binding c e l l s i n the spleen
Antigen-binding r e a c t i v i t y to TNP-SRBC and TMP-LPS during l a r v a l ,
metamorphic and adult l i f e i s shown i n Tables 5.4 and 5.5, also i n
Figures 5.1 and 5.2. Table 5.3 shows the r e s u l t s of i n i t i a l
experiments investigating the TNP-SRBC response with pooled l a r v a l
spleen c e l l suspensions. These experiments confirmed the need for
low dose c a r r i e r priming (as demonstrated by Rviben and Edwards, 1977)
for adult Xenopus, given 2 days before the main injection of TNP-
erythrocyte complex to produce good l e v e l s of TNP-HRBC splenic RFC.
97
Lack of such c a r r i e r pre-immunisation - i . e . giving TNP-SRBC
alone did, however, appear to give a low l e v e l aati-TNP-HRBC response
Cc.f. s a l i n e injected c o n t r o l s ) . Furthermore, in j e c t i o n of 50%
SRBC alone f a i l e d to induce TNP-HRBC binding above background
l e v e l s . Background l e v e l s of an t i TNP-HRBC RFC's following NaCl
i n j e c t i o n were found to be low throughout ontogeny with a mean l e v e l
of 782 - 204.
Table 5.4 and F i g . 5.1 show the r e s u l t s of the complete
ontogenetic study on anti-TNP-SRBC responses, using the standard
immunisation ( i . e . low dose priming) schedule. Anti-TNP binding
lymphocytes are found i n the e a r l i e s t - i n j e c t e d animals (injected
a t stage 52/3). I n these larvae modest RFC numbers are also seen
with HRBC: high background a c t i v i t y to erythrocyte antigens early
i n ontogeny i s expected i n view of Du Pasquier's findings on
Alytes obstetricans (1970). The mean l e v e l of anti-TNP RFC's
following TNP-SRBC i n j e c t i o n increased through ontogeny, reaching
maximum l e v e l s of 6418 - 3641 and 7684 - 3776 RFC/10^ lymphocytes
a t stages 56/7 and 58/9 respectively. Animals injected a t stage
65/6 (which i s a t the end of metamorphosis, when lymphocyte numbers
are at th e i r lowest) showed a lower mean l e v e l of anti-TNP response
than did those larvae injected a t stages 56-59 (although S.E.'s
are very high at these stages of maximal response, which precludes
any s t a t i s t i c a l significance of this, apparent difference). The
anti-TNP RFC l e v e l C3402 - 260) at stage 65/6 TNP-SRBC injected
animals i s similar to the mean anti-TNP l e v e l s found in the juvenile
and adult animals assayed.
98
Table 5.5 and F i g . 5.2 show the ontogeny of splenocyte
RFC a c t i v i t y to TNP when the hapten i s coupled to the T-independent
c a r r i e r LPS. Again a reasonable anti-TNP response i s seen i n the
e a r l i e s t stage larvae injected (52/3) 4622 - 755 RFC's). A rather
high backgrovind RFC count against HRBC was again seen in one spleen
pool at t h i s stage. By the onset of metamorphosis (stage 56/7)
ttxe l e v e l of anti-TNP RFC following TNP-LPS inj e c t i o n was s i g n i f i c a n t l y
elevated (11,937 - 3579) and t h i s high l e v e l was sustained right
through metamorphosis. At stage 65/6 mean anti-TNP RFC numbers
were 13,203 - 357. Anti-TNP RFC l e v e l s i n juvenile and adult stages
tested were 7276 - 554 and 8396 - 462 respectively, which were
s i g n i f i c a n t l y lower than the l e v e l s found over metamorphosis. Some
assays were incubated with TNP-glycine (see Tcible 5.4) to demonstrate
the s p e c i f i c i t y of the anti-TNP RFC response.
(c) Antibody forming c e l l s within the thymus
Table 5.6 shows the r e s u l t s of a b r i e f investigation of the
thymus as a centre of PFC a c t i v i t y following TNP-SRBC injection.
A low l e v e l of response was consistently found i n thymocyte suspensions
assayed 10 days af t e r low dose c a r r i e r priming, although the PFC
response was afforded almost exclusively to the c a r r i e r (SRBC).
Thus there were no PFC's i n 5/6 thymic suspensions to TNP-HRBC
and no thymic PFC a t a l l to HRBC. The thymic, a n t i - c a r r i e r PFC
response appeared to have disappeared by 14 days. Other preliminary
findings (not reported) with the ICA assay support these PFC r e s u l t s .
Thus low l e v e l s of a n t i - c a r r i e r RFC's are seen on day 10, but l i t t l e ,
i f any, response to the hapten occurs.
Discussion
The i n i t i a l experiments reported i n t h i s Chapter demonstrated
that splenic PFC can consistently be induced i n 9-15 month old toadlets
99
following i n j e c t i o n with the thymus-dependent antigen SRBC.
However, when PFC responses to SRBC, TNP-SRBC and TNP-LPS were
examined i n younger toadlets and larvae, no antibody forming c e l l s
were found i n the spleen vintil 1-2 months after metamorphosis.
This seems rather surprising, since servmi immunoglobulins in Xenopus
have been reported to be present from a very early stage of
development (stage 35 - see Leverone et al^., 197S) and because PFC
have been found i n other anuran larvae (Alytes oustetricans, Du
Pasquier, 1970); Rana catesbeiana, Moticka et ca,, 1973).
There are a number of possible reasons why PFC could not be
detected i n spleens of yoving Xenopus. F i r s t , i t i s possible that
the spleen i s not established as a s i t e of antibody production u n t i l
a f t e r metamorphosis, but t h i s seems unlikely as RFC's are found i n
t h i s organ from an early age, and antigen binding i s a prerequisite
for PFC a c t i v i t y . Perhaps the k i n e t i c s of PFC production d i f f e r
before metamorphosis and hence the l a r v a l assays were performed
a t the 'wrong' times a f t e r i n j e c t i o n . Secondly, i t seems that
metamorphosis i n Xenopus signals major changes with respect to
immunoglobulin production; these changes include alterations i n both
surface Ig expression on thymocytes and the capacity for Ig 'G'
antibody production. With respect to the l a t t e r , i t has been found
that although l a r v a l serum contains Ig'G'-like antibodies, such
antibody i s not detected as r e a d i l y as i n the adult (Du Pasquier
and Haimovich, 1976). Although deficient Ig'G' antibody production
would not appear to be a causative factor for the lack of PFC' s
encountered here (the assays measured d i r e c t plalques, presiomably
IgM-secreting c e l l s ) i t i s possible that l a r v a l Xenopus IgM antibody
i s , for some reason, unable to f i x mammalian complement e f f i c i e n t l y .
100
Possibly amphibian complement could be used successfully here,
although i t may be necessiary to use adult serum. I t has been
shown that some genes of the mammalian MHC code for, and control
expression of, complement components (see Schreffler, 1978 for
review). I f the Xenopus MHC also controls some aspects of
complement activation, i t could be that the complement genes
involved are activated only i n the adult. In t h i s respect
Du Pasquier £t al_ (1979) suggest that serologically-detectable
MHC antigens i n Xenopus are detected at or only af t e r metamorphosis.
Although no PFC were found u n t i l after metamorphosis, antigen
binding c e l l s can readily be induced i n the Xenopus spleen from
ea r l y i n ontogeny. Thus Kidder et a l . (1973) demonstrated
anti-SRBC binding c e l l s i n lairval spleens from animals injected
at stage 50 onwards. The experiments reported here employed
the hapten-carrier system to examine the ontogeny of the RFC
response of spleen c e l l s to TNP-SRBC and to TNP-LPS. Good levels
of induced antigen-binding c e l l s i n the larva to TNP after TNP-
SRBC i n j e c t i o n required low dose c a r r i e r priming. In the adult
amphibian such c a r r i e r - r e a c t i v e lymphocytes are believed to
represent T-helper c e l l s i n view of t h e i r antigen-binding c h a r a c t e r i s t i c s
(they are minimal-binding c e l l s of the non-secretory type - see
Rxiben, 1975) and t h e i r thymus-dependency (Horton £t al_., 1979).
Therefore i t would appear from the present experiments with TNP-
SRBC that some T-helper c e l l populations are fvinctioning at the
e a r l i e s t stages injected (stage 52/3). This finding i s i n
agreement with the work of Ruben, Welch and Jones, 1980. On the
other hand i n order to explain the deficient Ig'G' antibody
responses displayed by anuran larvae (see above), f u l l establishment
of T helper c e l l a c t i v i t y may develop only af t e r metamorphosis.
101
The experiments here reveal that stage 52/3 Xenopus larvae also
contain antigen-reactive, B-equivalent, splenic lymphocytes,
since antigen binding r e a c t i v i t y to TNP occurrec following TNP-LPS
administration to such smimals.
Maximum anti-TNP RFC l e v e l s following low dose SRBC priming
and TNP-SRBC i n j e c t i o n 2 days l a t e r were seen i n the spleen at the
onset of metamorphosis. This finding i s i n agreement with similar
experiments on Xenopus reported by Ruben, Welch and Jones (1980).
These workers suggest that the elevated TNP response found i n the
spleens of metamorphosing animals may be due to the effect of
metamorphosis on a population of c e l l s that normally suppresses
helper function i n l a r v a l and adult Xenopus: i f t h i s suppressor
population was s e l e c t i v e l y l o s t or blocked early i n metamorphosis,
then higher l e v e l s of anti-TNP binding c e l l s i n the spleen following
TNP-RBC in j e c t i o n might be expected, due to unrestrained T c e l l
help. However, the elevated response to TNP-LPS at metamorphosis
demonstrated here does not altogether f i t with t h e i r suggestion.
Thus the TNP-LPS r e s u l t s suggest that elevated B c e l l r e a c t i v i t y may
be occurring at metamorphosis. Alternatively, their could simply
be a temporary increase i n proportion of (antigen-binding) B c e l l s
over T c e l l s i n the small spleen found at metamorphosis (Du Pasquier
and Weiss, 1973), possibly related to reduced T c e l l migration from
the thymus that may be occurring at t h i s time (see Chapter 6) .
Whether the RFC's recorded i n the spleen against TNP-HRBC represent
B- rather than T-equivalent antigen-binding lymphocytes i s uncertain,
although i t i s pertinent to point out that the majority of such
RFC's were of the secretory type which, i t has been suggested
elsewhere (Ruben and Edwards, 19 80), are antibody-secreting
lymphocytes. f<f S C I E N C E
SECTION LibrarV
102
Very recently Jones and Ruben (1981) have re-examined the
e f f e c t of metamorphosis on the splenic antigen-binding response
to TNP when conjugated to an erythrocyte c a r r i e r . They reveal
that stage 57/8 i s a unique period i n which a (low level) a n t i -
TNP RFC response (3.5 x 10^ RFC*s/10^ lymphocytes) can occur
i n the absence of c a r r i e r (erythrocyte) pre-immunisation.
(Interestingly a low l e v e l anti-TNP response was also seen here
i n stage 57/8 euiimals injected with TNP-SRBC without low dose
SRBC priming (see Table 5.3). Jones and Ruben suggest that
an i n t e r n a l histoincompatibility between l a r v a l and adult lymphoid
c e l l s , which i s l i k e l y to be occurring at t h i s time (see Du Pasquier,
Blomberg and Bernard, 1979), provides a substitute for c a r r i e r
priming ) i . e . overrides the absolute necessity for T c e l l help).
I n t e r e s t i n g l y t h e i r experiments provide no evidence for elevated
B c e l l r e a c t i v i t y at metamorphosis, since although anti-TNP
splenic RFC numbers following TNP-LPS in j e c t i o n at stage 57/8
were higher =10,500 RFC/10^ lymphocytes) than i n the adult =4,000
RFC/10^ lymphocytes), they were no different from RFC numbers i n
the group of animals injected with TNP-LPS at stages 51-54 (10,000
RFC) . This contrasts with the findings presented here, where larvae
injected with TNP-LPS at stage 52/3 yielded s i g n i f i c a n t l y fewer
TNP-binding splenocytes than those animals injected a t stage 57/8.
The stage 52/3 larvae used i n t h i s Chapter were a l l 3 weeks old,
whereas the ages of the stage 51-54 larvae used by Jones and Rviben
were not given. I f the i r stage 51-54 animals were i n fact older
than the stage 52/3 larvae used here- i . e . had a more developed
lymphoid system (closer to metamorphosis?) than t h e i r external
stage suggested (see Ruben, Stevens and Kidder, 1972 for discussion
of t h i s age/stage effect) - t h i s could conceivably provide an
explanation for the discrepancies encountered.
103
The f i n a l experiment i n t h i s Chapter revealed that
( i n vivo) the young adult thymus contains anti-SRBC PFC (and RFC)
a f t e r TNP-SRBC immunisation, whereas anti-TNP plaques i n t h i s
organ were not detected. Although further experiments are
obviously required to investigate the significance of positive
a n t i - c a r r i e r , but negative anti-hapten antibody production, the
findings do suggest that antigen administration can achieve
PFC appearance i n the thymus. This has previously been demonstrated
i n thymuses of several vertebrate species (see Discussion i n Chapter
2 ) . The experiments here do not define whether the thymus
i s i t s e l f a s i t e for reaction to the hapten-cairier complex,
but the r e s u l t s do suggest that c e l l u l a r antibody production
i n t h i s organ i s not achieved simply by a random recirculation of
PFC, as only PFC against the c a r r i e r were found in the thymus.
Recent findings (Hsu and Du Pasquier, 1981) demonstrating the
high l e v e l s of PFC production in^ v i t r o by Xenopus thymocytes
Cfollowing i n vivo priming with DNP-KI£ and reimmunisation
i n vitrc> point again to the thymus as a major s i t e of B-equivalent
c e l l s i n t h i s species.
104
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111
I 1
CM "5"
C c E
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F i g . 5.3
Cytocentrifuge preparations of c e l l s from
an ICA assay using splenocytes from toadlets
immunised against sheep erythrocytes (SRBC).
Rosette forming c e l l s (RFC) can be seen xn
each micrograph and comprise a central
lymphoid c e l l (small or large lymphocyte)
binding one or additional layers of SRBC.
XRBC = Xenopus erythrocyte.
XL = Xenopus lymphocyte Scale 1cm = 25vim
112
113
CHAPTER 6
Thymus histogenesis over metamorphosis ana i n organ culture
Introduction
U n t i l quite recently, studies on the ontogeny of the immune
system i n anurans have concentrated on events during embryonic and
l a r v a l l i f e . Both c e l l u l a r and humoral immunity develop prior to
metamorphosis, the acquisition of immunocompetence being concommitant
with the d i f f e r e n t i a t i o n of lymphoid ti s s u e s , p a r t i c u l a r l y the
thymus (see Horton, 1969; Kidder et a l . , 1973). In Xenopus early
thymus histogenesis has been studied i n considerable d e t a i l .
Thus u l t r a s t r u c t u r a l investigations have shown the appearance of
large basophilic c e l l s within (between the thymic e p i t h e l i a l c e l l s )
and immediately adjacent to the thymus at 3 days (stage 40) (Nagata,
1977) . Recent experimental embryological evidence on ploidy-
l a b e l l e d Xenopus reveal that the extra-thymic origin of these stem
c e l l s may well be the nephric region of the embryo (Volpe et^ a l . 1981) .
The basophilic stem c e l l s gradually increase i n number within the
thymus and are thought to be the precursors of the f i r s t small lymphocytes
seen a t around 7 days of age (Manning and Horton, 1969; Rimmer,
1978) . An e x t r i n s i c o r i g i n of thymic lymphocytes in other vertebrates
has also received considerable experimental support (see Le Douarin,
1977).
In Xenopus the immune system as a whole i s thought to undergo
major chajiges at metamorphosis (which occurs about 7-8 weeks after the
f i r s t appearance of small lymphocytes i n the thymus and the i n i t i a t i o n
of immunocompetence). Thus the number of recoverable thymocytes
i s known to drop dramatically at t h i s time (Du Pasquier and Weiss,
114
1973), alloimmune r e a c t i v i t y i s 'suppressed' (Chardonnens, 1976)
eind regulatory T c e l l a c t i v i t i e s (e.g. help and suppression) are
altered (see Discussion i n Chapters 3 and 5 and also Du Pasquier
and Bernard, 1980). Details of histologic changes in the thymus
over metamorphosis are, however, not available (see Manning and
Horton, 1969) . The f i r s t part of t h i s Chapter therefore looks
at the e f f e c t of metamorphosis on thymus histogenesis and attempts
to correlate the s t r u c t u r a l changes observed during l a r v a l ,
metamorphic and early post-metamorphic l i f e with the numbers of
thymic lymphocytes that can be recovered when the organ i s teased
apart iri v i t r o .
The second part of t h i s Chapter i s a preliminary investigation
on the e f f e c t of organ culture on l a r v a l thymus lymphopoiesis.
Organ culturing of the amphibian thymus should, i f technically
f e a s i b l e , prove to be a powerful tool to examine development and
d i f f e r e n t i a t i o n of thymic lymphocytes (e.g. i n early l a r v a l l i f e and
at metamorphosis) i n the absence of in^ vivo c e l l u l a r migrations or
humoral e f f e c t s . I t has been shown i n mice (Robinson, 1980;
Mandel and Kennedy, 1978) and chickens (Sallstrom and Aim, 1973) that
transfer of the thymus early i n ontogeny into organ culture does not
prevent p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of lymphocytes. Precursor
c e l l s , present i n the embryonic thymus before removal, continue to
d i f f e r e n t i a t e normally with respect to the i r surface antigenic
markers and T c e l l functions, t h i s d i f f e r e n t i a t i o n p a r a l l e l i n g the
developmental sequence observed i n vivo.
Materials and Methods
Ca) Thymus morphology
The f i r s t experiment considered the number of thymic lymphocytes
recoverable from animals of various stages of development, including
115
the period over metamorphosis. The c e l l s were prepared as
described i n Chapter 3. For each stage examined, an average of 10
animals was used.
For the h i s t o l o g i c a l studies, 3 animals were selected at
the following stages; stage 54/5 (4 weeks), stage 58 (7 weeks),
stage 60 (8 weeks) , stage 66 (9 weeks, the end of metamorphosis),
one week post-metamorphosis, 2 weeks post-metamorphosis and 3 weeks
post-metamorphosis. The selected animals were heavily anaesthetised
i n MS 222 then fixed i n Bouins f i x a t i v e . After dehydration, the
specimens were wax embedded cind Bum sections cut and stained with
haematoxylin and eosin. Thymuses from two additional stage 66 animals
were prepared for l\im sectioning. These thymuses were fixed in cold
gluteraldehyde (5% gluteraldehyde i n O.IM sodium cacodylate) overnight,
then washed i n O.lM cacodylate buffer for 24 hours and post-fixed
i n osmium tetroxide for half an hour. After washing for ha l f an
hour i n O.IM cacodylate buffer, the specimens were dehydrated i n
methanol, cleared i n propylene oxide and then transferred to a
1:1 mixture of epon/epoxypropane overnight. They were f i n a l l y
embedded i n epon, the p l a s t i c being allowed to polymerise at 60°C
for 48 hours. lym sections were cut using a Reichert ultramicrotome
and were stained i n toluidine blue. To assess the location of
p r o l i f e r a t i n g lymphocytes within the young adult thymus, two 3-week
post-metamorphic toadlets were injected with t r i t i a t e d thymidine
(S.A. 22.4 Ci/mmol) 4 hours before k i l l i n g . Thymuses were then
prepared for autoradiography as described in Chapter 3.
Obi Thymus organ culture
The technique used followed that developed by Robinson and
Owen (1976) for mammalian thymus organ culture. Thymuses were
116
dissected out of anaesthetised larvae a s e p t i c a l l y , care being
taken to remove as much connective tissue as possible from around
the thymic capsule. Thymuses were placed in a watchglass containing
amphibian culture medium (see Chapter 2) but with the addition
of sodium bicarbonate (0.085 g cm • ) . Ten cm^ culture medium .
was added to a Falcon s t e r i l e p l a s t i c P e t r i dish, which was
supplemented with 1.5 cm^ decomplemented FCS. Several batches
of FCS were t r i e d i n preliminary organ culture experiments,
but the batch (purchased from Gibco) showing maximum lymphocyte
recovery was selected and used i n a l l experiments presented here.
On the surface of the medium were floated 13mm diameter Nucleopore
polycarbonate f i l t e r s ( S t e r i l i n ) with d.4iim pore s i z e . These had.
been boiled i n d i s t i l l e d water, with 3 changes, and then autoclaved
before use. Each pair of thymuses was placed on the prepared
f i l t e r , as i n the diagram below.
thymuses on f i l t e r .
Such thymus organ cultures were incubated i n a humidified incubator
at 28°C with 5% CO2 i n a i r . Two stages of development were selected
for experimentation; stage 52, as a young thymus where lymphocyte
numbers are rapidly increasing, and stage 58, when metamorphosis
has j u s t begun. At both stages 9 animals were used. Thymocyte
suspensions were prepared d i r e c t l y from 3, as described i n Chapter
3, except that a f t e r the mechanical disruption the thymus was broken
up by incubation i n trypsin/EDTA for half an hour. (Trypsinisation
117
proved to be necessary for releasing lymphocytes from organ-cultured
thymuses). Three animals were kept as controls and allowed to
develop normally to determine the extent of i n vivo changes i n
thymus morphology over the experimental period. The f i n a l 3
animals provided thymuses for organ culture. On day 7 the organ
cultured thymuses were taken off the f i l t e r s and c e l l suspensions
prepared by teasing and try p s i n i s a t i o n . Thymocytes were then
counted i n a haemacytometer and a cytospih preparation made (see
Chapter 3 ) . The cytospins were used to count the numbers of
lymphocytes and e p i t h e l i a l c e l l s present. Thymocyte cytospin
preparations from the 3 control animals were also made at 7 days.
One pair of 7-day organ-cultured thymuses from a stage 52 larvae
were plastic-embedded, sectioned at 1pm and stained with toluidine
blue (as described i n Ca) above).
Results
(a) Thymus morphology
Figure 6.1 shows the number of thymic lymphocytes that
can be recovered a t various stages of development from stage 5o/51
(2 weeks old) up to 3 weeks a f t e r .the end of metamorphosis ( i . e .
12 weeks t o t a l age) . The data show that i n the larva a maximum
l e v e l of about 10^ thymic lymphocytes occurs at the very beginning of
metamorphosis (stage56/7(6 weeks)) . . This l e v e l i s similar to that
found by Du Pasquier and Weiss (1973) at t h i s stage. Du Pasquier
and Weiss recorded that thymocyte numbers decline to 3 x 10^ over
the peri-metamorphic period, whereas the r e s u l t s here show a sharper
f a l l to only 3.5 x lo'^ a t 9 weeks (the end of metamorphosis).
Thymocyte numbers increase dramatically within 3 weeks post-metamorphosis,
when 1.2 x 10^ lymphocytes are recoverable.
118
The histology of the thymus i n l a r v a l , metamorphosing
and post-metamorphic Xenopus i s shown in Figs. 6.2-6.5. At
stage 54 (Fig. 6.2a) and stage 58. (Fig. 6.2b) the thymus i s
highly lymphoid. At stage 60 the thymus (Fig. 6.2c) i s s t i l l
large and continues to be an extremely lymphoid structure.
Cortex and medulla are c l e a r l y defined at a l l the above 3 stages,
with a greater density of lymphocytes i n the cortex. Lymphoblasts
can be seen i n the subcapsular region of the cortex (Fig. 6.3a)
but these become l e s s evident by stage 60 (Fig. 6.3b). By the.
end of metamorphosis (stage 66) the thymus i s not reduced i n
s i z e but h i s t o l o g i c a l l y now looks quite different (Fig. 6.4a).
There i s a large, e s s e n t i a l l y e p i t h e l i a l , medullary region,
i n which many basophilic granulocytes are found. The cortex
appears narrower than at early stages. Overall the number of
small lymphocytes appears greatly reduced. Furthermore, there
i s now no indication of a subcapsular layer of lymphoblasts (Fig.
6.4a). By one week post-metamorphosis, the thymus appears to be
s l i g h t l y reduced i n s i z e and i s again sparsely populated with,
lymphocytes (Fig. 6.4b). However, there now appears a broad and
d i s c r e e t band of lymphoblasts beneath the thymic capsule (Fig. 6.5a).
Towards the medulla there i s a small c o r t i c a l band of lymphocytes,
and lymphocytes are now seen again i n the medulla. Two weeks after
the end of metamorphosis, the discreet peripheral band of lymphoblasts
i s p a r t i c u l a r l y noticeable (Fig. 6.4c and 6.5b)., By t h i s
time the area occupied by c o r t i c a l small lymphocytes has enlarged
and the thymus as a whole has grown. Increasing numbers of
lymphocytes are v i s i b l e i n the medulla. At 3 weeks post-metamorphosis
the thymus has enlarged considerably (Fig. 6.4d) and takes on i t s
'adult' form; i t i s highly lymphoid i n both corcex and medulla.
119
with a narrow band of lymphoblasts around the periphery of
the cortex. Autoradiographs of such thymuses (Fig.
6.6) show that i t i s the lymphoblasts of the peripheral cortex
which are a c t i v e l y synthesising DNA. This zone therefore
represents the region where thymic lymphocyte pro l i f e r a t i o n i s
occurring.
(b) Organ culture experiments
Development of the stage 52 thymus i n organ culture does not
mirror develoEment occurring i n vivo (see Table 6.1). After
culture, the proportion of lymphocytes to e p i t h e l i a l c e l l s decreases
when compared with the thymuses prepared d i r e c t l y from the control
larvae. Many large e p i t h e l i a l c e l l types are noticeable i n the
7r-day cultured thymus (see Fig.6.7-6.8) . The t o t a l number of thymocytes
also declined after 7 days in culture, i n rela t i o n both to the
nvunber at the s t a r t of the culture period ( i . e . at stage 52) and the
number i n the control thymuses, which remained in vivo for the
duration of the experiment. However, the c e l l s remaining aft e r
7 days i n culture showed good v i a b i l i t y (70-80%, as assessed
by dye exclusion).
The r e s u l t s were e s s e n t i a l l y the same when stage 58 thymus was
cultured. Although thymocyte nxambers i n the in vivo developing
controls declined (as expected) at t h i s time from 0.94 x 10^ to
0.40 X 10^, i n organ culture the decline was greater (0.17 x 10^
c e l l recovered at 7 days of c u l t u r e ) . The proportion of lymphocytes
to e p i t h e l i a l c e l l s was only marginally decreased in organ-cultured
thymuses compared with i n vivo development.
120
Figure 6.7 shows a lum section of a 7-day organ cultured
thymus taken from a stage 52 animal. I t can be seen that the
cortico-medullary structure becomes rather i n d i s t i n c t . Further
more, compared with the i n vivo picture expected at t h i s time
(see F i g . 6.Xa), there are large v e s i c l e s i n the medulla, an overall
reduction i n number of lymphocytes and a flattening of the thymus
onto the f i l t e r . Melanin deposits are noticeable following organ
culture.
Discussion
The work i n the f i r s t part of t h i s Chapter investigated
morphologic events occurring i n the thymus at metamorphosis.
The data on c e l l recovery confirms previous findings (Du Pasquier
and Weiss, 1973) that t h i s period of development i s associated
with a considerable diminution of thymocyte niambers but highlights
the end of metamorphosis as being the most dramatic period i n t h i s
respect. The h i s t o l o g i c a l study of thymus ontogeny also reveals
that the end of metamorphosis i s associated with signi f i c a n t
s t r u c t u r a l a l t e r a t i o n s i n t h i s organ. Thus thymic medullary
lymphocytes are v i r t u a l l y absent at t h i s time and the subcapsular
lymphoblast zone, normally found i n the cortex, has disappeared.
Int e r e s t i n g l y these morphologic events occur at about the stage
when serologically-detectable MHC antigens can f i r s t be detected
on leucocytes i n Xenopus (Du Pasquier, Blomberg and Bernard, 1979).
I f the thymus i s c e n t r a l l y involved i n MHC r e s t r i c t i o n of certain
T lymphocyte subsets i n Xenopus (see Bernard, Bordmann, Blomberg
and Du Pasquier, 1981) then perhaps substantial reduction in
thymocyte nvmibers, and discontinuity of normal lymphopoiesis,is
an e s s e n t i a l prerequisite to reprogramming a new population of T
121
c e l l s to i n t e r a c t c o r r e c t l y with, and become tolerant to, the
new adult MHC antigens emerging at, or soon afte r , metamorphosis.
Certainly -the data presented here suggest a new wave of thymus
lymphocyte p r o l i f e r a t i o n occurs rapidly j u s t a f t e r the completion
of metamorphosis, since the peripheral c o r t i c a l lymphoblast zone
reappears and lymphocyte numbers throughout the thymus increase
at t h i s time. Thymus lymphopoiesis i n Xenopus may well be similar
to that seen in mice, to the extent that lymphocytes at the periphery
of the cortex divide rapidly and produce a maturing population
which moves toward the centre of the organ (Weissman, 1967).
Whether or not the surge i n thymus lymphopoiesis seen immediately
afte r metamorphosis i s dependent.upon a new wave of stem c e l l
input into the organ at t h i s time (from newly emerging bone marrow?)
i s unknown (see below).
The organ culture experiments on Xenopus thymus, revealing
r e l a t i v e l y low lymphocyte/epithelial c e l l r a t i o s and low overall
thymocyte nxambers compared to thymuses developing i i i vivo, appear
to c o n f l i c t with findings i n birds and mammals, which show that
removal and organ culture of the embryonic thymus does not prevent
p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of functional T lymphocytes
(see Introduction). Before considering the p o s s i b i l i t y of r e a l
physiologic differences, technical factors must be considered.
Although many amphibian organs have been cultured (Monnickendam and
B a l l s , 1973), t h i s i s the f i r s t time that intact Xenopus thymus has
been kept i n v i t r o and examined h i s t o l o g i c a l l y after several days
in organ culture. Although the culture technicjue used was adapted
from a successful mammalian system (Robinson and Owen, 1976), and it
one used successfully for culturing the chicken thymus (Sallsffom
and Aim, 1973), i t may be unsuitable for the amphibian thymus.
122
I t must be remembered that i n i t i a l attempts at thymus organ
culture i n the mouse led to largely e p i t h e l i a l cultures. Improved
cultvire techniques led to the f i r s t demonstration of thymic
lymphopoiesis i n v i t r o (see Robinson, 1980, for review). In
the present experiments on the Xenopus thymus, lymphocytes were
found at the end of the culture period and t h e i r v i a b i l i t y was
quite good. However, the decrease i n thymus lymphocyte numbers
revealed a f t e r culture might suggest that a constant input of
precursor c e l l s into the amphibian thymus i s required. Proliferation
of a small number of stem c e l l s that enter the thymus early in
ontogeny may be s u f f i c i e n t for lymphopoiesis i n mammals but not
in amphibians. Although t h i s hypothesis could help to explain
the rather gradual establishment of T-dependent functions in Xenopus,
further work on the organ culture system i s obviously required.
01 a •H ft (0 3 >i o m o w •M CO >i H §
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F i g . 6.1
Number of recoverable thymocytes at various
stages of development from 2-12 weeks of
age. The end of metamorphosis i s at 9 weeks.
X l A
3 z
i n
124
a; a; 3C so
CO CM
CO ^
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m
CO CM in
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in
Fig. 6.2
Thymus histology during l a r v a l and e a r l y
metamorphic l i f e showing the increase i n
s i z e and the highly lymphoid structure
of cortex (c) and medulla (m)
(a) Stage 54
(b) Stage 58
(c) Stage 60
8pm sections
Stain H and E
125
m
5^
F i g . 6.3
(a) This micrograph shows the band of
lymphoblasts (L) found at the periphery
of the cortex at stage 54.
(b) By stage 60, although the thymus i s s t i l l
lymphoid, t h i s layer of large, rapidly
dividing lymphocytes, i s no longer so
read i l y apparent.
8vim
Stain H and E
126
a
/
F i g . 6.4
Thymus histology during l a t e metamorphosis
and early post-metamorphic l i f e .
Ca) Stage 66 - the end of metamorphosis, lym
(b) 1 week a f t e r the end of metamorphosis
(c) 2 weeks "
(d) 3 weeks "
I I I I
I I I I
8 ym
H and E
127
•
5^.
128
F i g . 6.5
Micrographs of the thymus
(a) 1 week and
(b) 2 weeks a f t e r the end of metamorphosis.
A discrete band of lymphoblasts (L) can
be c l e a r l y seen i n the peripheral c o r t i c a l
region. Lymphocyte numbers are increasing
i n both cortex and medulla.
8ym
129
F i g . 6.6
Autoradiograph of thymus from 3-week
toadlet following in^ vivo i n j e c t i o n
of t r i t i a t e d thymidine.
LL= Labelled lymphocytes i n peripheral
cortex.
130
F i g . 6.7
lym p l a s t i c section of a thymus ( s t a i n
toluidine blue) from a stage 52 animal
following orgcui cultvire for 7 days.
The organ has reduced lymphocyte numbers
with many large non-lymphoid elements i n
the medulla (M) membrane to which thymus
i s attached. n»^. 3C (OO
Fi g . 6.8
Cytospin preparations of thymocytes taken
from larvae that were a t stage 52 at the
s t a r t of the experiment.
(a) control c e l l s prepared d i r e c t l y from
the l a r v a a f t e r 7 days i i i vivo.
(b) c e l l s following organ culture
i n v i t r o for 7 days. Although lymphocytes
(L) can be seen, there are many large
e p i t h e l i a l c e l l s (E) present.
7%
•
132
CHAPTER 7
Concluding remarks
The major findings of t h i s Thesis are outlined in
Figure 7.1 and can be considered i n four sections, representing
the main technical approaches used.
CD Mitogen r e a c t i v i t y of lymphocytes i n v i t r o
I n i t i a l culture experiments investigated the capacity of
adult Xenopus thymus and spleen c e l l s to p r o l i f e r a t e following
treatment with an array of mitogens which, i n mammals, s e l e c t i v e l y
activate either T or B lymphocytes. Good p r o l i f e r a t i v e responses
to putative T c e l l mitogens were recorded (using s c i n t i l l a t i o n
counting) for both thymocytes and splenocytes.. These c e l l s required
comparable optimal PHA eind Con A doses and displayed similar l e v e l s
of stimulation. Splenocytes responded well to high doses of the
B c e l l mitogen LPS. Interestingly thymocytes also displayed
a consistent, a l b e i t low l e v e l , p r o l i f e r a t i v e response to LPS
and PPD, suggesting the p o s s i b i l i t y that a population of B-
equivalent lymphocytes resides within the adult anuran thymus.
4
The use of a miniaturised culture technique (5 x 10 lymphocytes
per well) allowed, for the f i r s t time, mitogen experiments to be
performed on thymocytes taken from individual larvae and
metamorphosing Xenopus. During these stages of development
thymocytes appear to display l i t t l e sign of enhanced ^HTdR uptake
following Con A, PHA or LPS stimulation. A low l e v e l response
to Con A and LPS was, however, recorded j u s t prior to metamorphosis,
when the thymus reaches i t s maximal l a r v a l s i z e . Metamorphosis
133
effected a temporary disruption of the emergence of thymocyte
mitogen r e a c t i v i t y . Appreciable and constant T and B mitogen
responses i n the Xenopus thymus and spleen gradually
emerge a f t e r metamorphosis. Maximiim l e v e l s of LPS responsiveness
of thymocytes were seen at 6 months of age (when the mean S.I.
reached 6) . LPS r e a c t i v i t y of thymocytes at t h i s time was confirmed
by autoradiography; moreover, nylon wool separation experiments
showed that t h i s responsiveness l i e s within, or i s dependent upon,
a nylon wool adherent population of thymocytes. The d i s t i n c t
ontogenetic pattern of thymocyte responsiveness to LPS suggests
that these B-equivalent c e l l s may actually be spawned within the
thymus rather than be casually migrating through t h i s organ from
the periphery.
The capacity of l a r v a l lymphoid c e l l s other than those from the
thymus to react to mitogens was not examined i n rny d e t a i l here.
Such experiments are needed to determine the s i t e s where functionally-
equivalent T and B lymphocytes f i r s t emerge i n the anuran. These
experiments w i l l require the use of histoccanpatible animals where cell
pooling w i l l allow study of very early stages . (and during metamorphosis),
when lymphocyte numbers are small. In Xenopus some hybrid l a e v i s / g i l l i
females lay a proportion of diploid eggs, which can be activated
by i r r a d i a t e d sperm into gynogenetic development (Kobel and Du Pasquier,
1975) . LG hybrids from a p a r t i c u l a r clone produced in t h i s way have
been shown to be i d e n t i c a l to one another i n a variety of immunological
assays (e.g. grafting, MLC r e a c t i v i t y and antibody patterns (see
Kobel and Du Pasquier, 1977). Gynogenetic development of anuran eggs
through pressure (Tompkins, 1978) or cold shock treatment (Kawahara,
1978) following activation with irradiated sperm are other procedtares
that are being successfully used to procure (by suppressing elimination
134
of the second polar body) MHC compatible animals for immunological
study (see discussion i n Kobel and Du Pasquier, 1977) .
Histocompatible colonies of Xenopus occurring naturally have
also been described i n some laboratories (Katagiri, 1978). The
increasing a v a i l a b i l i t y of histoconpatible s t r a i n s of Xenopus
increases the value of t h i s model system for future immunologic studies,
C2) Thymectomy
Thymectomy was used i n conjunction with lymphocyte culture
and grafting studies to investigate the extent to which different
T c e l l functions are dependent upon an i n t a c t thymus. Xenopus
thymectomized at 7 days of age are often s t i l l capable of rejecting
skin a l l o g r a f t s , a l b e i t i n very chronic fashion. Spleen and blood
lymphocytes taken from such thymectomized and alloimmune animals were,
however, shown to be unresponsive to the T c e l l niitogens Con A and
PHA, although they responded normally by p r o l i f e r a t i o n following
LPS treatment. Lymphocytes involved i n graft rejection and T-mitogen-
reactive c e l l s might therefore represent d i s t i n c t T c e l l populations
in Xenopus, a suggestion which f i t s with previous sequential thymectomy
studies (Horton and Sherif, 1977). Experiments i n t h i s Thesis
involving thymectomy during mid-larval l i f e f a i l e d to provide support
for Horton and Sherif"s suggestion that T mitogen reactive c e l l s and
MLC reactive lymphocytes represent separate T c e l l lineages.
E a r l y thymectomy i n Xenopus w i l l continue to be a valuable
technique with which to probe the developing immurs system. With
isogeneic animals, reconstitution experiments are readily feasible
to further d i s s e c t possible T lymphocyte heterogeneity. The role
135
of the thymus i n MHC r e s t r i c t i o n Ceui also be profitably examined
i n t h i s anuran: thus thymuses of known MHC haplotype can be implanted
to early-thymectomized animals, whose subsequent cytotoxic T c e l l
r e a c t i v i t y and T-B c e l l interactions can be assessed. The lack
of runting or wasting disease i n early-thymectomized Xenopus
suggests, perhaps, that natural k i l l e r (NK)-like c e l l s assvmie
considerable importance i n t h i s anurah. This i s certainly an
inte r e s t i n g area for future work.
C3) Hapten-carrier immunisation
Three main findings came from these experiments, which
employed the hapten TNP conjugated eo either a T-dependent (SRBC)
or T-independent c a r r i e r (LPS). F i r s t l y , i t was shown that
responsiveness to these injected antigens can be detected (by
antigen binding) i n the spleen from an early l a r v a l age, revealing
that T-helper c e l l s and B-equivalent lymphocytes are functioning
from t h i s time. The l e v e l of induced RFC's against TNP reached
adult l e v e l s by the onset of metamorphosis, irrerpective of whether
the iiapten was presented on the T-dependent or T-independent c a r r i e r .
I n f a c t the RFC response to TNP-LPS during metamorphosis was elevated
when compared with the adult. Secondly, antibody-secreting PFC to
injected SRBC, TNP-SRBC and TNP-LPS were detected in the spleen only
aft e r the end of metamorphosis. Thirdly, small numbers of anti
SRBC PFC were found i n the adult thymus following TNP-SRBC administration.
This work suggests that metamorphosis i s associated with
altered humoral immune regulation. Further experiments are required
to characterise the TNP respqnse at metamorphosis to determine the
basis for the enhanced splenic r e a c t i v i t y recorded at t h i s time.
The apparent lack of PFC response u n t i l after metamorphosis i s
136 intriguing and also warremts further investigation.
(4) Histologic studies on thymus development at metamorphosis
The h i s t o l o g i c a l studies highlighted the dramatic drop i n
lymphocyte numbers that occurs at the end of metamorphosis. A
dearth of lymphocytes throughout the thymus occurs at th i s time,
j u s t p r i o r to a rapid reappearance of lymphocytes i n the peripheral
cortex. Three weeks a f t e r the end of metamorphosis the thymus i s
considerably enlarged and i s again highly lymphoid. These
findings correlate with altered immune r e a c t i v i t y demonstrated
both i n t h i s Thesis and elsewhere during metamorphosis.
The concept of s p e c i f i c suppressor c e l l s as a discrete T
c e l l s\ibpopulation, which faced some resistance from immunologists
when f i r s t proposed, has now come of age (Golub, 1981), and advances
i n vmderstanding immunoregulation i n the next few years w i l l , i n
part, focus on the central involvement of these c e l l s i n modulating
immune responses. Amphibian metamorphosis (a time when an internal
histoincompatibility i n the form of the emergence of new adult-
s p e c i f i c antigens i s presented to a competent immune system)
presents a unique opportvinity for examining immunoregulatory T c e l l
populations, which may have significance outside of purely
phylogenetic consideration.
137 Figure 7.1 Summary of major findings i n Thesis concerning the development
of the iiamune system i n Xenopus -laevis.
Chronic a l l o g r a f t r e j e c t i o n c a p a c i t y but not r e a c t i v i t y to T c e l l mitogens — call develop i f the thymus i s removed a t t h i s stage. Antigen binding responsiveness to T-dependent and T-independent antigens i s — found i n the spleen. ' Thymectomy a t t h i s stage causes impairment of both T mitogen r e a c t i v i t y and HLC responsiveneiss Small response to both T and B c e l l mitogens f i r s t found: the thymus. I j a r v a l thymic lymphocyte numbers reach maximum l e v e l .
Age (weeks
0
1
Thymic lymphocyte n-jmbers are declining-Mitogen responsiveness no longer •observed i n the thymus. Elevated level.s of RFC r e a c t i v i t y to TNP-SHBC and TNP-LPS are found i n the spleen.
Thymic lymphocyte numbers are a t a minimum. No mitogen responsiveness observed i n the thymus. El e v a t e d l e v e l s of antigen binding to TNP-LPS found i n the spleen.
Thymic lymphocyte numbers reach maximum adult l e v e l s . Good mitogen responsiveness to both T and B c e l l mitogens nov? found i n both thymus and Spleen. iMaximum l e v e l s of thymus response to LPS , found. PFC responsiv'sne.Ga to T-dependent and T-independent antigens i s e s t a b l i s h e d . PFC'a detectable i n thymus.
24
138
ACKNOWLEDGEMENTS
I should l i k e to express my thanks to toy supervisor
Dr.J.D. Horton for h i s unstinting guidance and help during
the course of t h i s study and i n part i c u l a r my appreciation
of h i s enthusiasm and commitment to t h i s work and develop
mental and comparative immunology i n general.
Thanks are due to Professor D. Barker and the
Department of Zoology for providing the f a i c i l i t i e s to make
t h i s study possible. i • •
I would also l i k e to thank those who have provided
technical assistance at various times throughout t h i s study,
i n p a r t i c u l a r Miss Judith Chambers, Miss Margaret I'Anson
and Mrs. Christine Richardson. f
|I am es p e c i a l l y indebted to Mrs. Pauline Bransden
and Mr, David Hutchinson for the es^pert help received in
the preparation of t h i s t h e s i s .
F i n a l l y I should l i k e to thank Durham University
for providing my Research Studentship for the duration I
of these studies. I
139
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