digitool.library.mcgill.cadigitool.library.mcgill.ca/thesisfile57434.pdf · human...
TRANSCRIPT
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Immunosuppression by Human Alpha-fetoprotein
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Studies on the Immunosuppressive Effect " ~ 1 ~
. of Human ~lpha-Fetoprotein on Cell-Mediated
Immune Re'act,ions .!!lyitro
bI/ J
Gary O'Neill
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A "thesis submitted to the F'aculty of Graduate Studies and " Res e arc h ; 'n par t j al f u 1 fi 1,r men t 0 f the r e qui rem e n t s
for the degree of Haster of Sciénce,
Department of Microbiology and Immunology McGi1l University Montréal, Québec Canada
Decembc,r 1981
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Abstraèt
Studies on the Immunosuppressive E'fect of Human Alpha
Fetoprotein on Ce11-Mediated Immune Rea~tions In Vitro ''1
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G. O.' Ne il1
M.Sc.
.Department of Microbio1ogy and lmmuno', ogy o
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Human a1pha-feto,pr.otein (AFP), isolated fram fetal and
hepatoma source material by p,hysic'ochemiçal and immunoadsor'bent ,
purifiçati~n te.chniques, was tested in the autologous and . al log en e i c m i xe d 1 Y mp hoc j te. r e a c t i an ( ML R ). 0 u r fin d i n 9 5
, indicate that ~ different AFP's con$istently show strong dose-
" ~ d e pen den tin h i bit 1 0 n ( ..> 80% s u p pre 5 5 ion) 0 f the a u ta log 0 U 5 ~1 L R'
at a final ,concentration of 200 ug/ml. In contrast, the effect
of AFP on allogeneic MLR's was variable with a mu ch weaker
degree of inhibition in camp,arisan ta that obtained on
autologous MLR. Autalogous MLR's could ,be efficie~ntly blocked i
by addition of anti-Ia anti60dies, whil~ allogeneic MlR's were
shown ta be significantly less sensitive to ~nhi~ition by thes~
r e age n t 5 • T h i 5 S U 9 g'e s t s t ha t d i f fer e n c sin the sen s i t i v.. i t Y t a
AFP-mediated suppression by al~o- versu auto-reactive T cells ~ !
may be part~ally attributable to the relat~ve contribution of
l'a molecules as the stimul,ating antigens. These findings have
i m po r tan t i m pli ca t 1 ans w i th r e s pee t t 0, u r und ers tan d i n 9 0 f ha w
se1f-tol~ra~ce is maintained durin9 ... early ontogeny .
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?ommajre
' .. , Etude de l'effet 'immuQDsuppress~ur de) 'al pha-foetopl;,'ptéine humaine sur
la réponsé ilnmunitaire de la médiation· lymphocytaire in vitro!
, G. O'Neill
M.Sc.
Département de microbi?logie et d'imfllunologie
L'alpha-foetoprotéine (AFP) humaine, isolée au, moyen de techniques
phys i co-chimi ques ou immunoadsorbantes du fl ui de prél evé sur l e foetu~
ou sur l'hépatome, a fait l'objet de tests dans le prO'Cessus de réaction
lymphocytaire mixtè (RLM), autologue et allogénique.· Les résultats
obtenus démontrent que, pour 8 échantillons d'alpha-foetoprotéine' diffé
rents, dont la concejtration finale é~.ait de 200 mg/ml, l~S inhibition.
allogéniques (>80%) de la réaction lymphocytaire mixte auto1ogue sont ,
directement 'proportionnelles à la concentration. Par contre, dans la
réaction lymphocytaire mixte allogénique, le degré d'inhibitions varie ) - , ,
avec beaucoup moins d'intensité que dans la réaction auto1oglje.~ Les
réactions lymphocytaires mixtes autologues pou~raient donc êtr~ effi
. cacement arrêtées par l'addition d'anti-ânticorps la, alors que les 1
réactions lymph.ocytaires allogéniques montreraient beaucoup moins de
senslbilité à ces réactifs. Il appe~t que la dHférence de sensibllité
des, lymphocytes T observée ~ntre 1 es deux types de r~actions lymphocyta ires
mixtes (autologue et allogél1ique) pourrait en partie être attribuable . ~ -. , aux molécules la agissant comme antigènes. Ces découvertes ont des . -répercussions importantes sur la compréhension du phénomème de tolérance
, . de l'organisme humain à ses propres cellules durant l'ontogenèse.
Traduit' par D. P. Bir,on
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Ackn1wledgements ~~ \. ,
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l am especially grateful to Drs. "-R.A. f'1urgita and P. Gold " \ ~... ~
i ."for thei r gui d~nc1nd cont inuous' encourjge~ent ;hroughout m~ : s~udies at McGilll. 1 wish to thank former colleagues r~r. D.c'.
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Hooper; Mr. D. Ho~kins, Mr. K. Kaplan, Dr. B. Pomerantz, and
M r s. S. 0 1 Ne i 1 l far va III ab l e dis eus s ; ans and ta Mary Wa 1 1 s .f a r
typing this manuscript. .
~ This wark ~as supparted by a fello~ship fram the Canadian -- ~~.-
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C a n c erS 0 cie t y, l ne., and b Y the M e d'i cal Res e a r en Cou ne; lof
Canada grants MA-6470 ,nd MT-4598 ta Ors. R.A. M~rg;ta and P.
Gold.
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Table of Contents
ABSTRACT
SOMMAIRE . . . .. ACKNOWLEDGEMENTS
TABLE OF eoNTENT~ •.
LI"S T 0 F 'TA BLE S . .
LI ST 0 FFI G UR ES. •
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.. . LIST OF ABBREVIATIONS ••
INTRODUCTION
LITERATURE REVIEW. . .
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1.' Normal Expression of AFP. .1..
A. Species Distribution .. . B. Sites of Synthesis ..
C. Levels of AFP in Body Fluids. 1
D. AFP-producing Cell Type
II. AFP in disease .. . ~. . . . III. Purificatton of AFP ....
A. Immunochemica1 Techniques o
B. Physicochemica1 Techniques.
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1. Concanavalin A-Af~inity Chromatography. . 11
2. Sepharose Blue Dextran Affinity Chromatography. 12
3. Ion-Exchange Affin'ity Chromatography .•
C. Assessment of Purity of AFP isolates.
IV. Quantitation of AFP .... ' ..•
V. Physicochemica1 P'roperties of AFP
A. Mo1ecu1ar Size Heterogeneity.
B. Charge Heteroge~ity .....
C. Lectin-binding Heterogeneity.
D. Amino aci.d composition and sequence
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. Table of Cont'ents (continued)
VI. Functional Biological Prop""erties of AFP. "
A.-Sinding of Various Substances ~
B. Immunoregulatio'n .. r •••••• : ••
1. Immunosuppression by AFP in vitro ..• • -,4' •
, 2. Selective Nature oJ AFP-metliated effects '.
a. Ce 1,1 u 1 a r . '. . . . . . . . . '. . .
b. G en e tic . . . , . , . . , . " . .
3, ,Factors tha t Infl ~ence AFP 1 s Act; vi ty
a, Methods of Purification , ,"
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c. Cultur conditions .. " ' .. '/' , b. sou~ce of. AFP '. , " ,
.!.!!. v i v 0 e mon s t rat ion 0 f' l mm un 0 s u pp r ek s ion b y
AFP , .:- , , . ,
VII. Allogeneic MLR.
vnI. Autq.logous MLR ..
A. ~ntroduction. B" Res p 0 n der C e 1,1 ,
CI Stimu1atory Cel1.
p. Functional Properties of Autologous MLR Responding , \,
Ce 11. . . . . . . . . '.
1. Cytotoxic Activity.
2. Suppressor .Activity "
3. Hel p e y.4' Act i vi ty \ '. u • 4 •
, E. Stlmu1us in Autologous MLR.
F. Auto1ogous MLR in Dis~ase
G. Murine Autologous MLR .
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Table of Contents (continued)
MATERIALS AND METHODS , , ,,41
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!. AFP Source Material , II.
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IV,
Preparâtion of Antisera.
Assays of AFP, ... • 'l Purification of Af~.
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A. Physicoc~emica1 Purification of AFP.
1. Ion-exchange Chrom~atography "fi '1t
2. Chromatography on Sephadex G-2iO , .. ,'i •. 3. Sepha rose- 8 1 ue Dex t ran Cc:> 1 umn Chroma togra phy
4. Concanavalin A-Sepharosè 48 affinity
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oC h rom a t 0 9 ra p h y, . . .' . 4 6
5. C h rom a t 0 9 r a p h Y 0' n Sep h a d ex G - 2 0 0 4 7
B. ,urification by Immunoadsorbent ~hromatography 47
1. Prepa~ation of Substituted Affinity Matrices 47
2. l mm u n 0 a d sor ben t Pur i f i. c a t ion 0 f AFP.
V. Preparation of Newborn Serum Supplements.
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• e' • 49
50 VI. Analytical Procedures. . '
A. Ouchterloney Immunodiffusion .' 50
B. Polyacrylamide Gel Electrophoresis
1. ,A PAG E .
2. SDS- PAGE
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3. Analytical Isoelectric Focusing in PAG 52
/Vi'!. ,Assays for Lymphocyte-inhibitory Activ-;,ty, 53
A. Cell Preparation . 54
1. Isolation of Peripheral Blood Lymphocytes~ 54 , .
2. Separation of T and non-T cells from PBL..... 54'
a. E-rosette Fractionation. 54
b. ~~ll Affinity FractiGnation'on Ig anti-Ig
Columns for the Selectibn of T and non-T
Ce 11 s . 56
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fable of Contents (continued) 1
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B. Identifièation of Lymphocyte Subpopu)ations
C. Irradiation of Cells .•.. _ ...
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D. Ce" C~ul ture Sys tem \. O,... ' "'. E: Data .Analysis
RE S UL TS. . ... 1. Purification of AFP
II.
III.
A. Physicochemical Purification. \
B. l mm U ri 0 a d sor ben t Pur i fic a t ion •
C. AFP Yields. , . . . . .. . D. Assessmeot of Purity of AFP Iso1ates.
C y t 0 t 0 x i c j _t Y 0 f AFP -P ~ par a t ion s' . •
The Differential Effect of AFP on Autologous and
A1logeneic MLR ...
A. Autologous.MLR.
1. Preparation of Responding and Stimulating
Cell Populations. . . •• . ..... .
2. Kinetics and Dose-Response Investigation of
AFP-mediated Suppression on Autologous MLR.
3. Con sis t e'n t Su pp r es s ive Act ion 0 f Var i 0 u 5
AFP's on Auto1ogous MLR 'p' •
B. A110geneic MLR.
1. Compari son of the Effects of AFP on
Autologous and Allogeneic MLR~ ...
2. Serum-free Culture Condïtion Requirement for .1
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the Su p p r'e 5 s ion 0 f· A 11 0 9 e ne i c ML R . 70
IV. Effect of anti-Ia and anti-T Aritiseru~ on. the T-ceoll
Prol i fer a t ive Res po n set 0 Au t 01 0 go usa n d Allo 9 en e"i c , non-T Stimu1ating Cel1~ . . . . . 71
V. Demonstration- of the Inhibitory Potency of AFP
Contained in Newborn Serum on the Autologous ~LR. 73
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DI SCUSS ION
REFERENCES
TABLES
.. FIGURES.
Table of Contents (continued)
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List of Tables
Page
Yields of AFP obtained by the phYSicochemical versus immunoadsorbent purification methods . 104,
Effect of AFP on cell viability. 105
A comparison of the responding and stimulating potential of unfractionated T and non-1 lymphocytes~ 106
Surface characteristics and mitogen reactivity of E.:.rose'tte fractionated cell populations, '107
Summary of the suppressive effects of etght different AFP isolates on the autologous and a110geneic MLR. 108
Proliferative response of allo-antigen activat€d lymphocytes cultured in various concentrations of serum or human serum albumin 109
Effect of auto10gous. allogeneic, and heterologous sera on t~e proliferative response of autoactivated T lymphocytes. . . . 110
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List of Figures " 1
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Figures Page
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Physicochemical and immunoadsorbent purification schemes ... : ................. 111
2. Ion-exchange chromatogra'phy on D~ Sephadex A-50 of crude}ascitic fluid .D • • • • • •• •••••• • '112
3. Molecu1ar':si,eve column chromatography on Sephadex G-200 of void volume frDm IEC . . . . . . . . .. 113
4. Sepharose Blue Dextran affinity of chromatography of AFP-containing fractions from G-200 chromatography .. 114
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Con A-5epharose affinity chnomatography of the poo1ed AFP-containing fracti~ns from SBD affinity chromatography ................. , 115
Gel filtration on Sephadex G-200 of purified AFP.
Elution pattern/of co rd serum on an anti-AFP Sepharose 4B immunoadsorbent co1umn
116
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Elution pattern of AFP preparations on an anti-who1e normal human 'sa~um Sepharose 48 immunoadsorbent co1umn ........................ 118
Demonstration of the pUfity ,of AFP by ,;mmuno-electrophoresis .............. .
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Demonstration of the purity of AFP by a1ka1ine PAGE
Gel ouchterloney a~alysis of p9rified AFP.
SOS-PAGE analysis of purifl'ed feta1 d~rived AFP
Isoelectric focusing gel -of purified AFP: . "
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T cell prolifera(ion in response to irradiated auto us non-T cel1s cu1tured in various , con c e n t r a_ t i 0 rfs 0 f PU,r1' u man AFP. . .. .... 1 2 4 ~
Demonstration of the consistent suppressive j of eight different hepatoma and feta-l derived AFP's------1 on autoactivated T lymphocytes ............. 1?5
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20.
21.
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, List of Figures (continued)
Page 0>
Comparative effects of human AFP on T cel1 prolifer~tion induced by au~ologous versus allogenelc non-T cells ......... . . 126
Suppressive effects of seven different purifie~ AFP's derived from different sources on the in vitro lymphocyte response to al10geneic ce1Js in albumin suppl emènted cul tures ......... '. . . .. 127
AFP-mediated suppress7'OJ'l of allogeneic MtR's in' album;n and whole serum supplemented cultures. . 128
Bl.ocking effects of anti-Ia and'anti-T cell reagents o n a u t 0 log 0 u s ver sus a 1 log e n e i c ~1 L R-:--~ . . . . . 1 29
Suppression of autologous MLR by newborn ~èrum ... 130
Selective dep1etion of AFP from n:~born serum~ removes most of its'~uppressive activity dn autologous MLR .. . ............... 131
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AFP . anti~gamma anti-HSA anti-NHS anti-TF
,APAGE AS A, TH anti AH
ATS
Bis BUdR
CFA C r~ l CNBr Con-A cpm
DEAE
EBV EL! SA
Fes HEPE 1 5_
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HSA HS-PBS 3H-TdR
IEF 19 H1
r~A F MHC MLF MLR
'ï.)
Abbreviations
alpha-fetoprote1n rabbit antisera a9~inst human gamma globulfn rabbit antisera against human serum albumin rab bita n t i se ra" a g 'a i n 5 t no rm al huma n s ~r u m rabbit anti5era against human transferrin alkaline polyacrylamide gel electro~ho~esis adul t human' serum
xiii
a murine alloanti5erurn which ;5 cro5sreactive with ' human "la" antigens antithymocyte serum
N, N'-methylene bisacrylamide bromodeoxyuridine
complete Freunds adjuvant cell-mediated immunity cyanogen bromide concanavalin A counts per minute
dle~hylaminoethyl
Epstein-Barr virus Enzyme~iinked immunosorbent assay
Fetal calf serum }
N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid . human serum albumin <
high salt phosphate buffered ~aline methyl-3H-thymidine
isoelectric focusing' immunoglobulin infectious mononucleosis
mouse amnoitic fluid major hi5tocompatfbility complex macrophage inhibitory factor mixed lymphocyte reaction
.,
f
NBS NBS-AFP NHS NHS
PBl PBS PHA
. pl , PlC
PWM
RIEP ,
SBO SOS-PAGE
se S4B
_ sIg SlE SR'BC
TD TEMED TI TN P:ï Tris
~ new'born serum newborn serum depleted of AFP norma 1 huma n serum normal mouse serum
peri pheral blood lymphocytes phosphate .,buffered saline phytohemagglutinin i spo l e c tri c po i nt primary liver cance~
~ pokeweed mitagen ..
rocket immunoelectrophoresis
Sepharose Blue Dextran sodium dodecyl sulfate polyacrylqmi~e gel electrophoresis standard error Sepharose 4B surface immunoglogul in
1 tsystemic lupus erythematosus ~heep red blood cells
thymus dependent ,N, N, N', N'-tetramethylenediamine
thymus independent trinitrobenzene sulfonic acid tri s (hydroxymethyl) -ami no-methane , .
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INTRODUCTION
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The deve~opment of the mammali·an fetus 'presents immuno-
l'ogisÜ with' s~veral puzz1i~g problems. O'ne question that aris~es II> •
is why t~e human fetus does not no~mally experience immunologie
attack ,by the maternal immune system (-1 ,2.). The fetus can be ,
regarded as a potential allograft due to the possession of ..
paternal transplantation antigens. While experimental evidence
indicates_ that at least several mechanisms may be involved in the 1
successful maintenance of the fetal allograft, t~ey have not been
pre C i sel y d e fin e d (1, 2 • 3 /4 ) .
The possibilities that have been proposed include 1) the
physi~al protection of the fetùs achieved by the anatomical
separatlon of the fétijs and mother; 2) a decrease in the anti-
9 e ni c pro p e r t i es 0 f the f et al - pla c e n t al uni t; 3) i nt e Ybf e r e n c e, b y
blocking factors of maternal immune responses directed against
the f e tus; and 4) m 0 d u l a ft ion 0 f m~t e r n a 1 i mm une r e s p 0 n ses (1, 2 • 3 ,
4). t1echanisms. 1) and 02) may only partially contribute to t~e
mai~ance of the fetal allograft. Thus, there is good evidence
for the transfer of fetal antigen to the mothe'r an'd for her
response to those antigens (5). The maternal response to fetal
anttgens may have a profound effect in interfering with oth~~_ \ \.;:
maternal effector mechanisms directed against the fetus (6). \-,
Following maternal sens;ti zation to fetal antigens, antibodi~s···;
produced by the,mother 'a~d/or their complexes \~ith 'antigen may
prevent the manifestation of_harmful maternal reactions., There
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, is a1so evidenee to suggest- that protection of' the fetu,s may .. occu'r by depression of ce'rtain materna1 immune funçtions '(1,2,3,
4). Th;", modu1a'tion of materna1 responses may be brought ab'out
Dy specifie and ~ons~ecific immunoregulatory mechanisms, ,
including cellular.processes and humoral,facjors (1,2,3,4).
One humoral' factor that has been postulated by Murgita and
Tomasi (7,8,9) to play a ro1e in the protection of the fetus from
harmful maternal lmmune reactions is alpha-fe.toprotein (AFP),'
AFP is ~ normal fetal serum protein that is present in high . concenOtrations ear1y in fetal life (10). 'It is a1so present in
am n i 0 tic f lui dan d f et al - der ive d AFP ca n b e fou n d ,i n the se ru m or c
pregnant v/amen (11). During normal 'life its synthesis is almost
completely suppressed, except in certa'in maligriant-;diseas~s, yo1k . • sac tumo~s, as we11 as sorne conditions involving ext~nsive
r.egenera~ion of the 1iver (11). Murgita'and Tomasi (7,8)' based ,
their original hypothesls for AFP-mediated immunoregulation dn
in vit>rô experiments performed in the murine system. Their
studies revea1ed that AFP has hig~ly selective and potent f
1 ", immun,oruppressiv~effects on ,..,certai.n in .vitro ass,ays of humoral
and ce'll-mediated .immunlty (CMI),
Subseqilently, several investigators have expanded, these ~ .
findings ~nd have provided further evidenee to support the
concept that 'AFP has 'immunoregu1atory properties which may be
important in the control of maternal immune reactions ~gainst
the fetus (9).
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1 nad d i t.to n t 0 t h i s con CI'! pt, i s the h y pot h e 5 i s t h a t AFP 1 '. ~.
" Dl ay se a 'r 0 1 e_' i ry the r e 9 u 1 a t ion 0 f a u t 0 sen s i t i z a t ion _ in' the f e tus
and newbOrn (7). A c1assical dogma of immuno1o~y states that .;1
lymphocyte reactions against se1f-components do not occur in
normal ind'ividua1s (12). Alt(hou9h seveY'êll th..eo~i.gs have been
postulated to explain the achievement of to1eranc~ to auto
antigens in the fet~s (13), it is unclear how this state is
a tt ai ne d . 1 n ~ i sel'o n a 1 sel e ct ion the 0 r y, Bu r net (1 2) po st u 1 a te d
that during the differentiation of the immune system, self-. to1erance results from the el imination of clones of lymphocytes
capable of recognizlng s~lf or autoantlgens.,
During the past decade, however, certain new data which
suggest that se1f-reactive ce1ls are normal constituents of the r
immu,~system have challenged Burnet's hypothesis. For example,
human B celJs have been' shown to bind auto1ogous antigens
i'nc1uding thyroglobulin and DNA (14,15). Cohen and Uerkele (16)
- have observed the in vitro i~duction cif s~lf-reactive cytotoxic . _. lymphocytes against syngeneic fibrob1asts Dr autologous
epithe1ium ce,lls. Thus, self-tolerance appears ta' be a far mor~e
comp1ex process than previo~sly assumed. 1
Sin cee 1 i m l n a t ion c a n no t b eth e sol ~ exp 1 a na t ion for s e l'f -
tô,\erance, other mechanisms that regulate expression of self-
react'ivity must also be present. Se~vera1 authors maintain that
the normal state of tolerence to se1f-antigens may in-sorne cases
bp due to a~state of active suppression (17,18) Th,e 1
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manifestation of autoreactive phenomenon may then repr sent
effector cell activation. resulting from a j:ircumventi n of" active
suppression (17). Inhibition of competent lymphocy es may be
achieved by cellular mechanisms (i.è. suppressor T/cel1s) and -- 1
liu/moral fact,or! (17,18)' .. Thus, con.tro1 of immU1-l functions would
hé critical, particularly during the perlnatal eriod when h
control and-establishment of the T lymphocyte repertoire is
.accomplished (3,13). Ev'rdence that humoral and ce1l-mediated ;w
immune responses are ~récisaly regulated in the fetus and newborn l'
h a s b e e n 0 b t a i n e d b Y s t u ct. i es, 5 h 0 ~(.. i n 9 t h a t f e t a 1'/ n e w b 0 r n i mm une
responses to m~st antigens are depressed in comparison ta the .
adult in spite of significant numbers of immunocom~etent ce11s \
(3). Hooper and Murgita (19) have documented evidence showing o
that AFP is a potent regulator of anti-self-reactions medipted
by newborn mouse lymphocytes. This raises the possibility that
human AFP may regulate autologous reactivity.
We have conducted an ana1ysis of the effects of human AFP '1
on two in vitro correl~tes of a110- a~d auto-reaqtlvity: 1) the
• allogeneic mixed lYiJ1phocyte reaction {MLR), (20); and 2) the " t' \
~ u t 0 log 0 u s ML R (21 .• f. 12 ) . The lit e ra tu r e r e vie win c 1 u de 5 .. discus~i~n5 on the al1ogenei~ and autolagous MLR, the occurrence
of AFP in biological system~ and its proposed physiological . -
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LITERATURE REVIEW
I. Normal Express,ion of AFP
A. Species distribution
Plasma alpha-globulins, which share anti~enic and bio
cne~al simi1~rities with human AFP, are synthesized in sharks,
birds and all mammalian species thus far st~died (23). An . archaic homologue of AFP probab1y existed at leas~t-400~45e
• mi1)ion years ago, in anima1s that ~ere eomman ancestors to the
species mentioned above (23). rmmunodiffusion analysis has
demonstrated that human AFP wi'~ cross-reaet with that of at .r
least eleven other mamm~lian species (24). During evo1ution,
therefore, AFP apparently was maintained in several divergent
evolutionary 1ineages (23). Despite this cro~s-reactivity,
hetero1ogous AFP elicits a strong antibody response in certain
spec;es (25).
B. Sltes of s!flJ.hWs
AFP ~ in high coneentrafions in feta'l serum, yet
------on1y nanogram 1ev!ls are detectable in the normal adult (10), , ~
In the human fetus, AFP is synthesized at three ~natomi~l sites:
the f et a 1 1 ive r, the f 8't a l 9 a s t roi nt est i na l t ra ct, and the y 01 k
sac (26,29t. AFP production by the fetal yolk sac and liver can
occur as early as the fourth weekrof gestation. The major site ~
1
1
,1
iP 1
r , 1 i i
4 =
(
(
...... -..---.-------- -~-
6
of ~FP synthesi~ 1s the fetal )iver, which can sy~thesize AFP at ~~. ,
a rate approaGhing 30mg/day in the second trimester of .pregnaQcy,-~
(28). By this time"production of AFP by th-e yolk'sac is i ,
declining. The total quantity of AFP synthesized in.the- fetu's
increases until 20 weeks of gesta~ion; it then remains constant .
u n t i 1 3~ 3 2 w e e k s,' a t w hic h t i met 0 t a 1 pr-o duc t ion éI e c 1 i n e s
( 10) .
/
C. Levels of AFP in Body'Flu1d "
During gestation, AFP synth~sized by tbe fetus is 1
distributed into four main fluid ~ompartments: fetal serum,
amniotic fluid, maternal serum,and 'cerebrospinal fluid (11) .• The , '
levels in amniotic fluid are maintained by the AFP excreted in,
fetal urine and that which is formed by yolk sac elements (11). >c
On the basis of studies in the pregnant rat u~ing radiolabelled
A.FP, Sell (29) demonstrated that AFP ;n maternal serum. is
1 ",
..
l J l , ,
p r i m a ri 1 y 0 f f et a 1 0 r i gin. The t r a' n s fer 0 f AFP, f rom f et u 5 t 0 ; .
mother by transamniotic :and transplacental' diffusion. probably ~
accounts for its ilevated maternal serum fevels during pregnancj
( 1 1 ) .
.' The s e r u m AFP con c e n' t rat ion l n the hum an' e ni b r y 0 ris e 5
rapidly from a leveT of approximately 67ugJml at, 6.5 weeks to ~
m~ximum that averages 2-3mg/ml between l~ and 13 weeks post~
conception. AFP levels then declin~ exponentially ta an average
of 50ug/ml at birth (30). Although serum~ lev-els decline during r
t ,
'.'
, ,
) "
1
7
th~ second and third trimesters, the total quantity of AFP
s y n t fi e 's i z e d b Y the f e tus i seo n s tan t f ltO m 2 0 t 0 3 2 w e ~ k S (2 3 ) .
The decre?se in serum AFP concentrations is due to the increase
in the rate ,of fetal d~velopment e-;xceed ,ing the total amount of ""'--;-..::J
AFP sy.nthesized (23).
, In the nl~born, serum AFP levels can range from 4 ~o 188ug/
ml (30,31). During the, first l'to 2 weeks. fol'lowing birth, se'fu1]l
AFP levels'decrease with a half-life of 3.5 days (32). Adult
, lev el S 0 f 4 t 0 2 5 n 9 / m 1 are no t r'e a che d un t i 1 2 ye ars ' 0 f 1 i f e (29,
.331). The nanogra.m leve,ls pre,sent throughout adu,lt Jife i'ndica'te
that suppression of AFP synthesis 1s not ~amplete fo116wing birth
(31') . ' , " .. ,
Levels Of AFP in amnioUc f(ui,d parallel the rlse and fal1
of 'fetaT ,serum' c'oncentration~ (11,2'3)., At fQ.urt,een ~/eeks 0'f51
ge~tation)\'FP preseQt in,amniotic flu-i-d reac":hes a ma~i~m .
con'ce'ntration of 10 to 20ug/ml, whi~h i-s' a.-pp'ro'xfm,atei'y 100 to 3"QO )1' ~
times lower than feta,l serum le,y'els (11,23), ~ \ '- ~
P e a k 1 ev e " s' are '"
fol1owed by an exponenti(~~l decliné ta 3-ccmcent'rat'ion of 10 to
,200 n 9 / m 1 a t b i. r t h (n, 23)'.
Elevations of fetal-deriv~d AFP in ma!ernal serum are
detectable by, the: seventh week po'stconception, and rise steadily 1 , .--
t"o a manmum of 150 to 250~g/m1 atta:ined 9Y the 28th·to ~&th week
( 2 3 ). ,AF P lev e 1 s f a l 1 b e f o' rel a b 0 r t 0 il con c e n t r ,a t ion 0 f 2 0 t 0 , '
l 0 0 n 9 / ml. a p pro x i mat e 1 y 2 0 0 t 0 6 0 0 t i mes l a w e r t h a f·l', the /, , (;>
corresponding fetal' level ('ll,ti33). Following parturition, ArlP in
\ 1
P 1
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'! i
1 1
1 ~ 1 l
.,
1
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8
.. '
the materna1 circulation decrines with a half-life o~ 4 to 5' days
until normal levels are reached (11). • i
, (
,/ D. AFP-Producing Cell Type
In th,e fetal liver, up to 80% of tbe hepatocyte's, but not • , 1
the hematopCiietic cells, hà've been demon'strated to synthesize AFP ,
C 3 4 ) . l t i sun c l e a r if ,t h e f e t a l hep a toc y tes i m u l tan e ° u s l y
synthesizes albul1)in, __ or if its precursor can differentiate into
both AFP and alb.umin praducing cells (ln. The adult liver does
not contain defectqble AFP-producing cells (34).
" '
\I. AFP in Di seas,
The dis co ver y b Y Abe l e IJ, (35) a f l hep ~ e sen c e of AFP i n' mi ce
\'1 i t h l ive r Q\a n c e r h a s 1 e d toi t s c l a ~ s i fic a t ion a san 0 n c 0 -
. development~ gene product (11 J. Tatarinav subsequently
d e man 5 t rat e d a 5 i mil a r pro t e i n i n s e r a f ra m hum ans \'1 i t h
hepatocellular carcinoma '(36). Extensive screening programs have
revealed elevated serum AFP levels assoclated 'wlth a variety of 1
non-ma1ignant conditions, int1uding acute and chronic hepatitis . (37)" ataxia telengiectasia,,(38), tyrosinosis (39), and infant
, -;. - '
neural ttrf)ê defects (40). Aside fram pregnant women, elevated
AFP level§ are most often found ln patients with beriign and
malignant d~seases of t~e liver (41). Eighty percent of those
suffering fram primary liver cancer (PlC) can expres,s increases'
G
, : '
!
1 -
r
-----
9 '
in' ~ e r u m AFP lev e l 5-- w,h i ch vary f rom na no gr a m p e r ml t 0 se ver a l
milligramsoper ml (41). A high serum AFP level. is considered to "
Dea rel a t ive lys p e c i fic dia 9 nos i s for PL C 'w h en se ru m con c en t r a -
tions exceed 2ug/ml (41). It is interesting to note the
geographie.ineidence of PLC and e1evated serum AFP levels. For ,
eX,ample, 'a greater frequency of AFP-producing PLC's oecur in
~ertain African populations than i~ populations in the United
States and Europe (11 ,41 ,42,43). Temporary elevations of serum
AFP 1evels éan be shown in up ta 24% of patiénts suffering from
benign 1iver diseases inc1uding liv~r cirrhosis, acute viral
h' e pat i t i s, c h ~c a e t ive ; ive rd; s e a s e '( Ch r 0 n i c a 9 9 r e s, s ive and /'
persistent hepatiti~), al~oholie hepatitis, and drug induced
1iver diseùe (41). The serum AFP content in these diseas~s can
range fr,om 20 [ta 200ng/m1 (41). In the rat, investig-atfons
emp10ying immunohisto1ogic staining techniques,have studied the J
AFP-oroducing ce11 in ehemically induced hepatomas (11). The
ob s e r vat ion S 0 f s ev e ra 1 i n v est i g a t 0 r 5 h a ver e. v e ~ 1 e d 0 val e e 11 s
and transitional basophilie hepatocyte-1ike ee11s to be
responsible for AFP-production (11).
III. Purification of AFP
" A. Immunoehemical Techniques
AFP has been suecessfully purified from se~era1 animal l
species uSlng various combinations of biochemica1 'and
o
1
1 1 1
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,f j .1 J
j
l
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10
immunochemical techniques (11). Since the purification condi
tions ca~ apparently affect the physical and biological
properties of the protein (44,45), the selection of methodology
ts important. In addition is the problem of~lbumln in serum
samples trom which AFP is to be isolated. Albumin can comprise
up to 50% of serum protein and shares many physiochemical
similaritles wlth AFP. Therefore, most purification schemeso take
advantage. of the antigenic differences between AF~ 'and albumin.
Immunoprecipitation was the earliest t~chnique used to
obtain human AFP (40). In this method. AFP is precipitated ~
adsorption to a specifie anti-AFP antiserum. The antigen-. , antibody precipitate is dissociated at low pH and fractionated by
gel fl1tratlon, WhlCh separates the gamma globulin and AFR
fractions. More recent protocols involve antibody-agarose
immunoadsorbent affinity çhromatography (470,48). - In this type of
chromatograph~. immunoadsorbent columns consisting of antl-AFP
antibodies or antl-normal human serum (NHS) antibodies ;
immobilized by covalent linkage to an insoluble agarose' matrix
are used. When AFP-containing materiaT is passed over an anti~c
AfP column,' it is specificàll y and reversibly adsorbed by the , ~'j
4!lt) an t i - AFP an t i b 0 die s . C om po n e nt 5 ~ i cha r.e no f b 0 und m a y b e
,easily washed a~ay. Solutions coritilning chaotropic agents such
a 5 g'lY c i ne - Hel 0 r s 0 d i u m th i 0 c y a-n a te are the n 'r e qui r e d 't 0
11 l "
dis5bciate the AFP from the matrix~bound immun6~10bul~n (47,48).
The types of eluting buffers as well as fheir pH and ionie
, rr
o " ,
,
\ l -~ ~-: . ,
1
1 j
(
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l l
strength has been reported to be critiëal in preserving the
biologica1 activity of,AFP (44,45), as will be discussed. Smal1 .. amounts of contaminating substances which may remain in these AFP
preparations can be removed by passage over co1umns of anti-NHS J
antibody-agarosé affinity co1umns. The use of low pH eluting
buffers and chaotropiC agents can be avoided by using t~e
fo11owing methods: 1) antibodies of low affinity obtained early
ln the immunization period (49);' 2) anti-AFP antiserum raised
against AFP of a species different from that which is intended to
be purified (~ the use of anti-A\; antibodies raised agai~st
rat AFP for the purification of mouse AFP) (48,49); and
'3) presaturatlon of the anti-AFP immunoadsorbent column with AFP
in order to occupy high affinity antibodies leaving only the
lowest affinity antibodies available for the subsequent
fractionation of the samp1e (51). ~
B. Physicochemical Technigues
1. Concanavalin-A Affinity Chromatography
Affinity chromatography on columns of the plant lectin . con c a n a v 9 lin - ~ (C 0 n - A) des cri b e d b Y Pa 9 e (52)'~ ca n pra v 1 de
S e ver a l 'h und r e d fol d pur i fic a t ion 0 f AFP - con t a i n i n 9 mat e ria l 1 na, ! single step. Lectins are a group of proteins which possess the
abjl~ty to react reverslbly with specifie sugar residues.
Oligosacchande ~n~its which c'omprise 4% of the AFP molecule are &
not found in albumin (53). Thus, the presence or absence of
,-
"
(
, 1 2
" ~
/~ carbohydrate moieties allows the separa.tion of serum albu'min and
\, 0î--...._ other mole,cules from AFP,by passage of AFP-eo:ntAtnirry~material
. , "-, "- .
over a·column containing Con-A immobi.lized on;a 'sQli-d mat~ 1
J'
J . . ,~. ~ ...... .... ." ~ 'i
(52). The Con-A will reversibly bind the sugar r~siduês-~.on .A:FP .... ~-L
( 52 ) . Following extensive washing of the column, elution of
bound AFP is accompli shed by application of alpha-methyl-D-
glucoside, the leetin's c.ompetltive binding sugar (52). Although
Con-A Sepharose chromatJgraphy is highly specifie for the,sugar
residues of AFP, the use of this method is limite9f1>y the 01
presence of Con-A reactive and non-reaetive AFP species (54). /
~ith and Kelleher have reported con-~ reactive and non-reactive
species in a hepatoma patient's serum, .whieh have subsequently
been found h most AFP-containing'body fluids (54): Thus, '
selective depletion or ènrichme~t of Con-A réactive and non-~
reactive AFP species may oecur during purification,
2 , Sep h a r 0 s e B lue' 0 e x t r a n A f tin i t Y C h rom a t 0 9 r a p h Y
A T b u min b i~ b 1 u e d y e s a n.d t h i sas we l 1 h a s b e e nus e d for
the separation- of AFP and albumin (55), To remove albumin fram
AFP-containing fluids they are passed over columns containing a
blue dye linked to Sepharose. AFP 1S recovered in the unbound
fraction and the album1n bound to,the column can be eluted wlth
urea (55):
c
J ......
1
1 l
(1
1 3
3 . Ion - ex cha n 9 e Aff i n it y C h rom a· t 0 gr a ph Y
Ion-~xchange èhromato{raphy lPas been used, to separate
. substa~t~quantities of albumin, transferrin, and beta and .
ga"mma globulins from AFP-containing source material (55). "This,
procedure separates the molecules on the basis of molecular
charge. Chromatography columns packed with ~nion e~change ~esins
"'---!'Jill bind AFP while cationic and uncharged molecules are"not ~ ,
---. r ~ t a rnè<t-{ ill--=- A f ter ex t en s i v.e wa s fi ; n 9 0 f the col u m n, b 0 und . --. ~
molecule's, includi-;gp;r?, can be eluted by application of a
linear gradient of increasïng ionic strength (55). In addition,
ampholyte displacement chromatography (56), isoelectric focusing
(53), and pr~parative polyacrylamide gel electrophoreisis (57) . '
have been previously described for the purification of AFP.
C. Assessment of Purity of AFP Isolates
AFP preparations isolated by the abave methods have been
shown to meet strict criteria for purity as revealed by immuno-
chemical and biochemical techniques inclûding polya'crylamide gel
electrophore.isis in alkal ine conditions (AP~GE) (49,58), and in .f'
reducing conditio~s containing sodium dodecyl sul fate (SDS-PAGE) ~
(49,59). Since the ~olecular weight of AFB [losely resembles
that of albumin (53), these two proteins cannot·be d1stinguished o
by SOS-PAGE. However, AFP ;s more negat;vely charged than
a l b u min (5 2 ) . The r e for e, A - 'p AGE, wh; c h sep a rat e son the bas; s 0 f
molecular ICharge is used ta verify the absence of albumin in AFP
"
,
i -------1
..
(
l'
(
14 , .
, , , .
'samples. ImmunoelectrO'phoreisis (60) and.:;Ouchterloney gel , 1
diffusion,(6l) techniques, against relevant antisera (anti-AFP, , ,
anti-albumin, anti-NHS) have been emoloyed ta confirm' the purity
of the preparations ta a sensitivity of approximate1y 2.5ug/ml
( 1 l ) .
IV. Quantîtation of AFP
A variety of methods for th~ quaniitation of AFP have been
u t i 1 i z e d (11). V i rt u a l 1 Y a 1 1 t e c h n i que sem plo Y mon 0 s p e' c i fic a n t i -!
A Fi' an t i se ra., Wh e n AFP con c e n t rat ion s ex cee d 2. 5 u 9 / ml,
qualltative measurements can be~made by Ouchter1oney double
diffuslon techniques (11). Standard rocket immunoelectro-'
phoreisis, normal1y with a sensitivity approaching 1ug/ml, can be
used to measure lower concentrations with a sensitivity -of 20ng/
ml by incorporating radioacetilTe label and autoradiogra~~' (60).
Enzyme-linked immunosorbent assay (ELISA) (62) and radioimmuno
assay (RIA) (63) tèchniques have sensitivfties of approximately
3ng/ml (11). ELISA has the advantage of using stable reilgents <\
and does not require radioactive materials. ~
V. Ph~sicochemical Properties of A,FP
~he chara~terization Qf AFP has included irivestigati~ns on
its mo1ecular weight, charge, amino acid composition and r , 0
..
1
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1 r
f 1 l 1 " j
1 ]
. ( ,
1
..
,"
.... r
(
1 5
\ sequence, tertiary structure, and the composition of the attached
carbo,hydrate rTlreiety (11, 49). These studies have sho\'1n that
human A~P ex~ts as a group of c10sely related molecular
variants (11,54) . Differences in the structure of ~he prot'ei,rl
and i t s ca rboRyd rà,t~ 110rt i'; n h a v e been i.mpl i cated as the basis \;"~ . '. \
for the three types of heterQgeneity demons~rated in human AFP: l,
, .. size" charge/.and 1ectin bi.nding (54). Genetic polymorphism and
r 1
postsynthetic modifications of AFP have been suggested as the
basis for thé,r heterogeneity (54). The proportion of
in~ividual ~ariants has been reported to correlate with various 'J>
P h Y sic al, and pat h 0 log i c a )1 s t a tes, and d i f fer e n ces i n b i 0 log i cal
activity (54).
A. Molecular Size Heterogeneity
Human AFP has been reported to consist of several molec~lar
size populations (54,61). Molecular weight determinations by
SDS-P«GE under den9turing conditions yields a single band
corresponding to a moleeular weight of JO,OOO, equivalent to that
of albumin (49). Molec.ular weight variants, obse,rved ,on APAGE in
non-denaturing conditions, are probably due to the formation of
AFP oligomers, including dimers and trimers (64): Upon exposure "
of AFP oligomers to 2-mercapto~thanol they appear to dissociate
tom 0 nom ers (6 4 ) " T h i s s u 9 9 est s ,t h a' t the 0 1 i 9 0 mer ~ r e sul t f rom
intermolecular disulfide bond formation( (64). Manipulations of
the AFP pfeparation, such as lyophilization and freeze thaw f
. , " '
1
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1
1
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1 !
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1
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( cycles, may increase oligomer formation.
1
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Charge heterogenei ty of AFP can be demonstrated b~oion-
exchange chromatography (65), zone electrophoresis in agarose
gels (66,67), and fsoelectric focusing' in ampholytes (68,69).
Charge variants may reflect differences in the polypep'tide ,
portion and the carbohydrate mo1ety, which accounts for 4% of the
AFP molec,ule (67). Lester ~~ (68) have demonstral:ted 3
molecular charge variants on extended ag~rose gel electrophoresi~,
and isoelectric focusing (IEF). The isoelectric pOin~s (pl)
• • ra n 9 e d b e t w e e n 4. 6 and 5. 2. Th e.s e W 0 r k ers (68) and A 1 p e r t ~.LÈl.!..
(66) maintain ~hat the variation in sia1ic acid content of the
AFP molecule cou1d only partial1y account for ~he heterogeneity,
s~nce oesialy1ation did not result in a comp1e,te 1055 of
heterogenei ty. ~
If IEF is performe~d in SM urea, at least 6 major
charge isomers can be identified (68). On the basis of this result,
Les ter ~~ _~l: (68) s u g 9 est t h a t the cha r 9 e d; f fer e n ces d e pen d u po n
vadations in pr;imary molecular structure as opposed to differences
-in conformati~nal heterogeneity (68,70) .• Recently, however, Lester
1
~.L~.! (71) conc1uded· that heterogeneity observed in IEF may be
d~e to the formation of'proteln-ampholyte complexes. These workers
separated charge varlants by IEF in the absence of urea. When o
analyzed by cro-ssed immunoef'ectrophoresis, the iso1ated
variants refocused over a range of isoelectric point? and still 1
displayed three charge variants.
r,
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1
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1 \ - \
,
•
1 7
The quantition,of AFP molecu1ar variants ;n various normal
and p Cft ~ 0 log i cal con ci i t ion s ha s r ev e ale d cha ra ete ris t iof pat ter n s
_ (11.,54). For example,'AFP isolates obtained f"rom fetal liver and
hepatoma tissue contain almost exclusive1y variant 3, which ;s
t'he most negati.vély charged species (68,70,73). AFP derived from,
the serum or ascitic fluid is electropho~tical1y more
heterDgeneous than tissue-derived AFP. L"ester et al. suggested l"
that' the charge differences were due to postsynthetic modifica
tions of the AFP molecule (73). That is, AFP synthesized by the
tissues'\\as the more neQative1y-charged variant is modified upon
secretion ta a less negative1y charged species. This scheme was
based dn evidencw obtained from the electrophoretic patterhs of
AFP isolated from the serum,! ascitic f1uid, and tumor of~ a single \ P
hepatoma patient (73). The tumor extract was rich in variant 3, ..,
while no variant l was observed. The proportion of variant 3 ~~s
diminis~ed in the serum isolate and was almost absent in AJP
deriv'ed from )\Scl*tic fluid. The decrease in the species 3, was 1
paralle.led l3y a parallel inc;-ease in variant 1, the most .
electropositive species. Furthermore. Smith and Kel1eher (54)
suggest. that differences in the proportions of the molecular
variants may be due to dîfferent catabolic rates of the several Q •
forms of AFP.
/
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, !
, j j
1 . i 1
(
(
.....,....
,18
c. Lectin-bjnding heterogeneity
The heterageneity of AFP with respect ta lectin binding is
,due ta differences in t he terminal sugar residues of the
carbohydrate moeity, which include the sugars gal~ctose, fucose,
mannQse, N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid
(17,54). The lectins cancanavalin-A and Lens culinaris"resolve
AFP into 2 and 4 variants dep~nding pn the affinity o'f the lectin ,
for the sugar residu~s of each molecular species .(74,75,76).
Lens culinaris lectin expreises the same specificity as ton-A,
but. b i n d s ale s s e r po r t i· 0 n a f AFP t han Con - A (7 6 ) . The Con - A
variants, reactive and non-r~active, have been demonstrated in
different proportions in AFP from hepatoma patient's serum,
abortion fluid, amniotic fluid, and yolk sac tumors (54).
D. Amino acid composition and seguence
Analysis of AFP's from variaus mammalian species has shown
significant similarities and hom~logies in amino acid'composition • \ .
and sequence (11). In addition, ~her~ are several pieces of data
t 0 s u 9 9 est' t h a t AFP .a n d a l b u min s h are a c 0 m mon a n ces t r a 1 9 e ne.
Ru'oslahti (77) has r~rted that AFP and albumin share at least
50% homology in their amine acid sequences of the peptide
fragments' they studied. Furthermore, following denaturation of'
AFP and albumin, the peptides revealed significant immunological
cro,ss-rea~tivity (78). <"
\
\ \
i
1
1 ; ;
f l j , i
,1 1
l
( (
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, ,
19
VI. Functional Bi.o1ogical Properties of AFP .. The appearance of ~FP at an ear1y evo1utionary stage, its
<
retention through evolution over a wide species range, and its
complex biochemical nature suggest it plays an ïmportant
bio1ogica1 ro1e. The precise function(s) of AFP remain to be
estab1ished with certainty. Several current proposed functions
inc1ude immvnoregu1ation, binding o'f hormones and other
substances, and maintenance of fetal osmotic pres~ure (9,11,17).
E v ide n ces u p po r fi n 9 the sep 0 s tu 1 a tes c a n b e fou n d i n s e ver a 1
mamma1ian species, su ch as humans, mice, rats and pvgs (9,11).
A. Binding of Various Substances
The ability of human AFP to bind hormones and sma11
1 1
\ i f 1 î
1
~----------------~--
substances suggests a carrieYfunction (11): Human AFP can bind
fatty acids~ copper, and bilirubin, but not estrogens or:
prostaglandins (11,80,81,82). However, estrogen binding has been
reported to be quite strong and specifie in the rat and mouse
(83}. Plapinger interprets this finding as a possible mechanism
. by which the rodent fetal brain is protected against the effects
'of ~cessive amounts of circu1ating estrogens (84). This concept
ieS °based on the observations that the conversion of andr'ogeris ta
e ~ t r 0 g:e n s cau ses mas cul i ni ,2 a t ion 0 f the r 0 den t br a ; n ( 85 ) .
Presumably AFP binds the excess~estrogen as it is produced, thus
preventing its effects on the brain (84).
1 1 i
(
t
20.
B. Immunoregulation,
1. Immuhosuppression by AFP In Vitro
, The possibility that AFP regulates immune funct;ons was , , ,
initially investigated in the murine system (7,8,86). Based on
a postulated rela~fo'n"*hip between AFP and immunoglobulin ontogeny
during development, ~everal studies investigated the e"ffect of
AFP containing fluids on\ertain lymphocyte functions (7,9,86).
Preliminary evidence was obtained by Ogra et al. (86) by
injecting murine amniotic fluid (t-1AF) into tnîce from
birth ta adulthood. Upon challenge Dt these mice with the ~hymus)
dependent (TD} antigen, sheep red blood cells (SRBC), a
1 1 \ l
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1 1 loi
1 i l !
--------deficiency in antibody forl]1ation, predominantly in the Ig.trand (
IgG classes. \'Ias observed. Murgita and Tomasi (7,8) further
developed these findings using in vitro culture systems to test
the effects Dt r~AF and its several components on humoral' and
cell-mediated immune f>unctions. These' authors observed that r1AF
suppressed bath the primary and secondary ~ vitro antibody
response to SRBC (7 J. The most pro;four)d eTfect was on the IgA
and IgG antibody classes (7). MAF also exe~ted inhibitory
effects on the prol iferative response of Con-A and' phitohemag~
glutin (PHA)-activated lymphocytes, but had no effect on ':he
mixed lymphocyte reaction (8). The, theory that AFP was the
suppressive substanee in amniotic fluid ~as confirmed by
experiments using pure AFP preparations isolated from MAF by
imm.ulmadsorbent affinity chromatography (7,8). The purified
, 21
, (
_~~~~~ mouse AFP preparations were tested for their influence ôn the
primary and secondary antibody response .of spleen cells to SRBC '.
(7). Both primary and secondary antibody ~esponses were markedly
inhibited. IgG and IgA responses, well kn0wn to have thymus
influence (87), were apparently more sensitive to AFP-mediated
suppression than IgM res~onses (7). In addition. mouse AFP
suppressed tne mitagenic effects of PHA, ~~A:_!)td--lrpOP01Y-_ ---sac cha ri d e (L P S) 0 n mou ses p 1 fi! e n cel 1 s a's-- We,ll _as the pro 1 . ~-
- tion---i-n-d-u-e-e-&-i-n ~m u r me- aJ~l--o 9 e n è i c ML R (8). " vitro experiments Murgita and W' extended th'ese '
~ _______________ ~servafi ons. These workers cQnfi rmed the i nhi bi tory' e'ffect -of
1
\
murine ,AFP on the TO antibody response ta SRBC, but reported that
thymus independent (TI) antibody respanses ,ta dinitrophenyl-,1 ,
substituted Fieoll and the LPS-stimulated polyelonal B cell
antibody synthesis w~re unaffected by AFP. On the basis of these
r e 5 u l t s " i t \'1 as. 5 u 9 9 est e d t h a t AFP - m e dia t e d e f f e c t s i ri'~"'v it ra, we r e
selective"with suppressive effects restricted to TD reactfons
( 88 ) . y a c h n in, L ,e ste r., and Mill e r (89, 90 , 91) r: e po rte d sim i l a r
~mmunosuppressive activitles associated with human AFP. Purified
human"AFP was shown by these workers to suppress the mitogenic
" response of human lymphocytes to PHA, Con-A, anti-human thymoeyte
serum (ArS), and the human r~LR, but did ,not inhibit human
lymphocyte rosette formation with SRBC (89,90,91). Fetal AFP was
1 to 3 orders more potent than hepatoma derived AFP in )
inhibiting human lymphocyte responses (90.91), possibly ~s a
1
22
result of variations in the relative proportions of the di.f .. ferent ..
mol ecu 1 ars p e cie s 0 f AF P ( 8 9),. . --
Sin c e Mur 9 i t a é t al. (7, 8) and Y a c h n ; n and Les ter (8~, 9 0 ,
91) reported these data, a number of ot,her investigators (9,11,
92,93,~4, ha~e documented evidence supporting [44,45,65,00,72,73, i
________ 82,95,96,97-109) or showing a number of va~ts (39,75;-~---- 1
110-115). The effects documented~r human AFP on irlyitro
~crc-.vt<e functions inrclude 1) suppression of mitogen stimulatld ~
(PHA, Con-A, pokeweed mitogen (PWM); lymphocyte proliferation
1
1 • C6 5 , 7 0 , 7 2 , 8 2 , 8 9 , 9 0 , 9 1 , 9 5 , 9 9 , 1 0 4 ,', d8 , , , 3 ); 2) i n h ; b it ion 0 f
al10antigen induced proliferation in the one-way allogeneic MLR ,
(44,45,82,90,95,104); 3) depression of the lymphocyte prolifera-
tive response to sodium periodat~, and the antigen SK-SD (65,89-
91 ) .
Those assays of lymp-hocyte funltion \·/hichare apparently\
not inh...ibited by human AFP include 1)1ymphocyte proliferation
i n duc e d b Y pro te i n A 0 f the St a ph Y 1 0 C 0 ecu 5 au r eus C 0.\'/ an l
organism, general1y considered to be aB cell response (104),
2) Hie formation of active T rosettes (95,99',3) macrophage
inhibltory factor UtIF) p~oducti'on (102)r 1 ,1 À -
These results'.indicate, as in the murine syst.em, that AFP f suppresses ce'rJain immune re~ctions-, and is not a general ,1
,1
A numb~r- o~~ ___________ ---+~ ; n Il i bit 0 r y a~ e nt'" f 0 -r a lli mm un 01 0 9 i c" f uri ct ion s . -- ~
investi ators maintain that AFP does no-t ex.b-i-b-H-oTo1ogically l
~ 1 However, "as 're1evant unoregulatory effects (39.75,110-115).
" .
1 1
(
---------------
(
• <
discussed below, recent studies have clearly shown that these
reported differences may relate to several"factors which can
influence effects mediated by AFP (9): ,
2\ Selective Nature of AFP-mediated Immu,.nosuppression
a. Cellular
Of primary importance 'lS the concept that AFP exerts
selective and restricted effects on certain immuhe functions (9).
As described abave in the human and murine systems, differential
effects of AFP on certain T and B cell immune functions are ...
obvious. Thus, in bath mice and humans, TI B cell functians f
appear ta be refractary ta AFP-mediated effects (7,9,88,104).
The concept that AFP acts selectively has been expanded by
studies in th.e murine system (106,107). These investigations
have revealed that AFP apparently has genetic and cellular
r est r i c t ion s (1 0 6 1 l 0 7.) . N uri n e AFP h a s b e e n a b 5 e r v e d t 0 h a v e
selective ~ffects on individual T-cell subsets (88,106,lO7). '(
Murine T lymphocytes can be subdivided ;nto subse'ts that have
bath functionally distlnct posi~ve and negativ"e regulatory
.,.,i'nfluences (116). T lymphocytes Vlhich. provide ~ 'J ______ ------------
nctions
Uî r 0 u 9 h bot h s p e ~J:-à-flâ--m:rnspe c if; c sig n a 1 s ----------- ............. ________ ------sujJpressar cell L activation are defined by the
and can i nduce /1
~"'+' -Ly 1 2 ce 11
surface phenotype (116). Experimental evidence indicates that
+ -certain funct;ans ofthe-Lyl 2 subset are particularly sensitive ~
to AFP-mediated effects (106,107): In contrast to the negative
(
1 ,
~.
j
influences of AFP, severafreports in the human (9,96,10J) and
mouse'(105,117) show that 'AFP' can induce suppressor T le-lls. ,
A c cor d i n 9 toM u r g i t a e t aJ: (l a 5 ) " mur i n e s u p pre s sor T c e 11 s-
24
activated by mouse AFP in culture, can inhibit the TD primary
antibody response to SRBC and dinitrophenyl- keyhole limpet
hemocyanin (DNP-KLH) but not the TI antibody response to DNP
Ficoll and DNP-substituted polymerized flagellin frdm Salmonella
adelaide (POL). The conclusion implied from these exp~riments
is that positive and I)egative influences mediated by AF.P are
selective for certain immune functions"and ce11 types (9).
Thus, the effects of AFP wi 11 vary with different in vitro assay
systems according to the ce 11 function invo1ved (9J.
", b . Génetic
-
An additional level of camp1 ex i ty has been observed at the ~?'
genetic l eve l by Peck et al. (106,107), in that AFP has genetic
restriction. These authors induced MLR a c,t i vat ion across 4
sifferent murine major histocampatibility complex (MHC) ,
disparities (~differences at the I-region, K,D-region, M1s
locus. and non-MHC), AFP exerted its most profound suppressive
effect on Ia:,a,s,",ociated T-ce11 proliferative responses (106,107), :.,
Reactions involving non-MHC and K,D-region differences were
refractory to the inhibitory effects of AFP (106,107). Although
AFP did not prevent K,D-region induced T cell proliferation, the ..
\ 1
D i
-;:
. J
.1 • " 4 i ; , -.,
J 1 • , ~
1
1
l' J ,
,1
.' . ,
(
i
generation of cytotoxic T cells which normally occurs in such 1
cult ure °s w a 5 a b sen t, i n the pre sen c e 0 f AFP (1 06 , l 0 7) . Hum a n
MLR's involve complex MHC differences (20). The genetic
restrictions invQlving AFP in human al10antigen inducecl cell
25
pro1 if e rat ion h a ven 0 t b e e n in v est i 9 a t e d . The ,p os s i b il i ty th a t
human AFP may have genetic specificity could account for the lack
of significant suppression of allogeneic MLR observed by
Charpentier et al. (110).
3. Factors that Influence AFP-mediated Effects
a. Methods of Purification
Goeken and Thompson (45) have reported that the immuno-
suppressive properties of AFP are re1ated to the manner in which
it is iso1ated. They found.that AFP eluted from immunoadsorbent
affinity co1umns with buffers of low ionic strength ,resulted in
a partial or complete 105S of suppressive activity. Two reports
(44,95) have documented that elution of AFP from an immuno
adsorbent column at pH 10 fol1owed by treatment of the columnr
with sodium thiocyanate resulted in two AFP isolates, AFP-I and
AFP-II, respectively. AFP-I exerLed strong suppressive e~fects
on MLR~ in contrast ta AFP-II which did not show a comparable
effect. The authors comment that the 10ss of biologica1
activity from AFP-II may be due to the use of the sodium
thiocyanate elution techni9~e (44,95).
\
'j
j ,
r 1 },
jj
(
,;26
b. Source of AFP
Variations in t~e inhibitory potency of indiv'dual AFP
isolates from human sources but not murine sources have been
observed by several investigators (9,89,104,108). Yachnin et al,
(89) studied the relationship between AFP lmmunosuppressive
potency'and its orlgin from fetal liver and serum. and the "
ascitic fluid, serum, and 1 iver extracts of hepatoma patients.
Their observations demonstrated the suppressive,potency of fetal 1
AFP ta be 3 orders of magnitude greater when cornpared with AFP .
derived from patients with hepatomas (89,90,91). The variation (
in biologie activity was not limited to a distinction between
fetal and hepatoma AFP, but was apparent in heoatoma AFP's as a
class (72,73). In studles using AFP isolated from the serum,
as c, i tic fl u id, and tu m 0 r 0 f a sin 9 lep a t i en t w i th· a h ,e pat 0 ma, a
100-f..old variation \.,as found i~ the potency of each AFP
preparation (~3). Tumor AFP was the most potent preparat1on,
serum AFP had iniermediate poiency, and ascitic.fluid AFP was
the least immunosuppressive (73) .. In similar· studies, Murgita et
al. (104) observed effects ranging from complete suppression ta
augmentation of PHA-stimulated lymphocyte responses with 9
different isolates ~f fetal-derived ~FP. As discussed abQve,
h u m'a n AFP dis pla y s mie r 0 h ete r 0 9 e n e i t Y w i t h r e s p e c t toc /1 a r 9 e
(71). Yachnin and Lester (65,70,72) studied the charge
differences of AFP isolates from various sources and their 0
biological potency, These authors show a relationship between 1
-.
1
27 j
, /'
the immunosuppressive effect of ,certain AFP preparations and the 1
proportion of specifie AFP isomers. \
Fetal and tumor derived AFP, which contai~ almost
exclusively AFP-3 are highly suppressive. Intermediate ta low
levels of immunosuppressive poténcy are seen with serum and
ascitic f1ùid derived AFP's which ,cQ,ntain lesser amounts of AFP-3
and a higher proportion of the electropositive species, AFP-l and
AFP-2 (70). The 1mmunosuppressive potency of each isolate
correlaied with its relative proportion of AFP-3 (70). They
concluded that the capacity of a given AFP preparation to inhibit
lymphocyte transformation correlates with the proportion of the
most electronega~ive isomer (70).
Zimmerman et al. (109) reported that the sialic acid
residues in murine AFP were essential for inhibition of in vitro
antibody production by murine lymphocytes, since desialyation
removed lmmunosuppressive properties. In contrast to these
findings, Lester et al. found no difference in immunosuppressive
potency between sialylated and aSlalylated AFP. Further~ore,
following enzymatic desial1'Jatio'n of AFP, complex charge
heterogeneity persisted in AFP preparations (89).
c. Culture conditions .
Studles ln the human system indicated that culture medium
components can modify the effects of AFP on lymphocyte responses
(104). In serum-free conditions, human AFP had a potent
o
.
, , ,
~ , ~ j
.' , , '1 J ~ '1
~ ~
1
1
\
--y
28
suppressive effect of PHA-induced lymphocyte proliferation (lb4).
Equivalent AFP concentrations were less inhibitory in cultures'
supplemented with 10% pooled human AB serum, and not suppressive
in cultures containing 5% fetal calf serum (FCS) (104).
4. In Vivo Demonstration of AFP-mediated ~mmunosuppression
While not as extensive as in vitro investigations, there is
evidenc~ i1ra,t AFP is immun?logic'a1ly active l!!. vivo (97,98).
Gersh\'Jin et al. (97,98) have document.ed evidence describing the
ability 'of mouse AFP ta alter the qualitative and quantitative
characteristics of Mo1oney sarcoma virus-(MSV), induced tumors
and pristane induced plasmacytomas. MSV-treat~mice which had
r e c e ive ct d ail yin j e c t ion s 0 f v AFP d e v e l 0 P e d l a r 91€ r~, t u m 0 r s ,
required a longer period for regression, and nad a signifi~antly j.
higher mortality (97). In addition, the transfer of splénic T -'. c e 11 5 0 f A F p'- t r e a t e dm; ce; n t 0 U n man; p 1r1 a te d m i c e cau s e d a
depression in I~G and IgA sec~ndary antibody respon es to SRBC
(97) . The con01usion reaihed by these authgrs was 0'
hat AFP 1
induced the generation of a nonspecific spl~ic sup ressor T
or A FP l'la s cell (97), Additional evidence of an 'in vivo ro1e ---,
obtained in pristane-primed mice (98). The appeara ce of
p1asmacytomas in these mice was acce1erated if the ice had been
treated wlth AFP (98),
)
\ .\
r
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l ! ,
10
1
)
(
1
29
VII. Allogene,ic MLR
Initial studies by Bain et al. (118) i'n 1964 revealed tfjat
the .i.!l vitro co-culture of peripheral blood lymphocytes (PBl)
frbm two unrelated individuals results in the blastic
transf~rmation of some cells. This ~ vitro proliferative \,
response of lymphocytes following challeng~ with allogeneic cells . has been termed the allogeneic mix~d lymphocyte reaction (MlR)
(20). The jntensity of the allogeneic MlR correlates r~asonably
~ll with differences among the strong transplantation antigens
of the two individuals (20,119). There is general agreement that
the ree,ognition of sudaee antigens on the stimulating cells
whic~ are foreign to the,responding cel1s is the stimulus (20).
The relevant target antigens appear to be the produets of the "-
HLA-D region of the MHC in man and the MHC-I 'region ln mice (20, f' / \
120,121). The genetie disparity at the HLA-D locus of the
responding and stimulating l~~phocytes is reflected by the
magnitude of the~allogenelc response (20). In this reaction the
primary responding and stimulating cells are consï~erfd to be T
and B lymphocytes'rE!speetively (20,122,123). The proliferat(ve (
\
... ph a seo f the a l log e n e i é ML R 9 ive sri set 0 s e ve yi a,} 0 the r ~ ,
components of the reaction. Investigations on the geneties of
alloreactions by Bach et al. (t24,125) revealed that cytolytic ~
eells a~e generated in the murine reaction and are primarily
directed against the K
complex. furthermore,
and/or(D regions of the murine H-2
both he~per and suppressor activities
"
have
, .
1
l 1 l'
1
i ~ \
l 1 !
J
1 •
\
(
been reported to be generated in the al10geneic MLR,(116).
The allogeneic MLR has,been us~d clinica1ly for ihe
evaluation of"histocompatibi1ity between potential dORors and
recipients of transpla~tation organs (20).
VII!. Auto1ogous MLR
A. Introduction . '"
1 •
30
Normally, l,ymphocytes rlf identical HLA type do not respond
hn allogeneic MlR~ (20). Ho-wever:, severa1 groups (21,22,126-132)
have reported~the existenc# of a population of T cells which can
be induced to prolîferate when they are cocultured with
autologous non-T cells. Injti.al studies (126-132) revealed that ~
PBllS can be stimu1ated in culture by auto1ogpus cultured ,
1ymphoblasts. More recent reports (21,22) document the a,bility
of normal freshly seif)arated adult non-T cells ta stimul.ate . '
auto1ogous T cells. This latter reaction, termep the autologous
~r syngeneic MLR, has tur-ned out to be an important immunolo,gic
reactio~. While the b;ologi~ role of autologous MLR is uncleàr,
severa1 possibilities have been proposed. Weksler and ~irnbaum·
(132) postu1ated that the autologous MLR represented a natu~al <\
'" '" defense mechanism against neoplastic transformation. ~1or e
recently Kuntz et al. (,22) have interpreted this form of
autoreactivity as part of the overall process throu~h which T 1
, f
..
..
1 , !
1 ) ,
':
" ) .
/
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" ..... _--------,--~--~~--~-----~-~- -~--- - --
31
• cells modulate B cell functions. This concept'of regulation 1$ , ,
o consistent with mountiog experimental evidence describing that
>.
autolcgous MLR 1) genera~es cel~s which regulate antibody
pro duc t ion (1 33- 1 36) and the c lt 0 t 0 xie T cel 1 r e s po n s e (1 3 7 ), and
2) it is deficient in patie.ntsJWith autoim~~'ne diseases and 1
conditions involving immu(nological disorders (138-148).
Apparentl,Yt ,sel f-recognition const'itutes a central
requirement for the expression of immunity (149) Therefore
recognition of self-antigens need not be detrimental to the host
(150). The reéognition of one class of molecules, the r~HC
antigens, has been shown to be essential for several types ot .?
immunological responses, including normal cell to cell
communication and immunoregulation (149). The requirement ~~r
recognition of self-MHC antigens has been documented for T cell
responses against chèmically modified cells (151), virus infected .. . cells (152) l'Ieak histocompatibility antigens (153), and male
\ 1 ,J " ,
specifie H-Y antigens -(154). -Effective cel}-=cell communication \ ..
is a1so dependent on beneficial autorecognition of self-MHC
molecules (149). This is shown' by studies on the role of ~1HC .r
gene products in cell-cell interactioÎls between 1) T and B 1 ~
lymphocytes, Z) T cells and macrophages, and 3) J lymphocyte
subsets (149). Therefore it appears that self-recognltlon, and ~
. possibly the self-reactivity observed in autologous MLR, "',
constitutes an esse~tial requirement for immune functions.
The human autologous MLR involves the co-culture of T cells f
\
(
o ,
32
wi th inYctivated autologous non-T lymphocytes, obtained 'from the
per;Pher:01ood of 'normal adult d,onors (il ,22). The T and non
T fr~ons are obtained by methods inclading E-rosette - .
formation ~ith SRBC and cell affinity chromatography (155).
Normally reaétions are carried out in cultures supplemented with
autologous serum, in the complet~ absence of foreign antigens , ( 22) .
(J
Weksler and Kozak (156) demonstrated that hu~n autologous >
MLR induces both memory and specificity. Following a primary
autologous MLR, responding cells proliferate in a typically
accelerated secondary MLR when challenged with autologous non-T
cells but not allogeneic non-T cells (156). Since memory and
s~ecificity are generally considered attributes of immunological ., responses, these findings support the autologous MLR as such
(15~
B. Responder:Cel1 •
Experimental e~idence in humans suggests that the respond-
ing cell population in ~utol.ogous NLR is of T cell origin (21, _
22,134,157,158). Characteristics of the autoreactive T cell th·at
:, a v e b è e n r e po rte d ; ne 1 u de: / 1) the 1 a c k 0 f r e cep t 0 r 5 for the F'c ,1
portion of IgG (157,158), 2).(a ,sensltivity to hydrocortisone
(112,160,161),3) a responsiveness to Con-A (135,15~,162) and
4) the ability to form autologous rosettes (158,159). Recent'
data (134,162) indicates that the T cel1 population reactjve in
o /
\ } J ~ ,
-1
1 •
33
autologous MLR is apparently separate from those involved in
allogeneic reactions. This conclusion is based on ~ ~itro
analyses of T cell populatipns which have been selectively
depleted of autoreactive or alloreactive responding cells. The
removal of the responqing cell in the autologous MLR, by suicide
with bromodeoxyuridine (BDdR)' and light treatment, from total T cells
renders these unresponsive in avtologous MLR. Conversely,
alloreactive c.ell depleHon results in a T lym,phocyte population
found to have a normal response in autologous MLR (134,162). ,
Further. Hausman and Stobo (134) hav~ shown that cells depleted of - ... autologous MLR reactive types were deficient in T helper cell
activity, while ~atane and Green-(162) demonstrated a de~letion of
T suppressor function.
C. Stimulatory Cell
Several studies have explored the nature of the"stimulatory
cells in autologous MLR (163-166). Although their nature is n~t
fully understood, there is agreement that they do not appear to be
T cells (163-166). Since the non-T population i~ V~fY heterogeneous,
the cell type has been evaluated by fraétionating the non-T
lymphocytes -into subpopulations of ~ cells, monocytes, and a
subpopulatlon of lymphocytes lacking the cel1-~urface markers and
biologie aetivittes of T and B cells ternied null or K cells (163-
164). Three recent studies (;63-165) maintain that monocytes were
the best stimulating cell tjpe, whereas B cells produced no
s t i mu 1 a t ion. 1 n con t ras t. Sa kan e _ ~_LÊ.l~ (1 45 )
\ 1
1
j l 1 1
(,
. 34
and ~mith (166) have reported that B cells stimulated autologous
T cells and that monocytes fàiled or triggered only moderate
proliferation of T cells. The studies of Kuntz et al. (22) have
d~awn further attention to the complexity of the prob1em by
demonstrating that K lymphocytes but not B lymphocytes were the
major stimulators in autologous MLR. They also found that the
stimulatory capacity of the hon-T cel1s was augmented after the
depletion of monocytes (22). It appears that the pr~se nature
of the stimulatory cel1 in ~he human autologous MLR remains
controversial.
D. Functional Properties of the Autologous MLR Responding Cel1 \
1. Cytotoxic Activity
Several groups have observed that activation of autoreact
ive T cells in autologous MLR can result in the generation of
cel 1 5 \1 it h c Y t 0 t 0 x i c (1 6 7 - l 7 0 ), s u p pre s sor (1 3 5 , 1 3 7 , 1 6 2 , 1 7 0 ), and
helper functions (134,136,170). functional
. activities of these proliferating not well understood .
In general', T cells stimulated in autologous ,~1L'R have been
reported to manifest cytolytic activity against a variety of ~ "" ?
tumor cells, allogeneic cells, and autologous cells (167-170).
There is disagreement among several authors as, to the specificity
of the cytotoxic T cel1. On the one hand, Tomonari et al: (168)
and t1iller and Kaplan (167} observed autologous ,MLR-activated
t
1
\ ,
1
/
35
'---- ,é cy totox i c ce 11 s were capable of lysing mitQ,gen a'ctivated
• autologpus lymphocytes~ On the other hand Van de S touwe et al.
(169).have reported that T cel1s stimu1ated ; n ~utologous MLR
fai1ed ta lyse auto1ogous ce11s. ;
2. Suppressor Activity
There is increasing evid-ence that the respondin,9 c'e11 in
a u ta log 0 u ~ ML Ris pa r tic u 1 a r 1 yen r i ch e d i nie e 11 s ,w i t1L-p.-r-e-p-eTt:leS
of a suppressar T ~e11 (135,137,162,170), Smith and Know1ton
(137) have s~own that T cells activated in autologous MLR ~ay
exhibit suppressive effects on both the prol.iferative and
1 ·1
1 •
t .t 1
cytotoxic response~ of fresh unstimu'lated T ce11s to a110geneic 1--
cells in t1LR.
3. Helper Activity
Ho w ev e r i n a-d dit ion ta the 9 e n e rat ion 0 f T cel 1 s w i th
cytotoxic and suppressor ,functions, T cells \'1ith helper
i nf1 uences are a1s,o reported to be acti va.fea in auto l o'gous t~LR
(134,136,170). The auto1ogous response appar~ntly results in the
productlon of helper factors. These factors were shown by
-Chiorrazzi et al. (133) to indu~e mononucle~r ce11s to,synthesize
immunoglobuli~. In a subsequent paper this grDup reporte~ that
helper faitors derived fro~ supernatants of auto1ogous reactions
could provide a signal to induc~ the development of cytotoxic
ce11s against trinitrobenzene sulfonic acid (TNPS) ,modified
1 \ Î 36~
n
~ 1
autalogous lymrthJ1Jlyt"es (171). Shin et al. (136) have observed . . that autoreactive T lcells are triggered ta pro1iferate by HLA-D "'
• antigen on PWM-pUlsed non-T cel1s and that a subpgpulation of
.these Prolifi~a-~ing cens in turn helPs~WM-primed B ce1'ls to
"diffe-rentiate inta 'Ig-secretingS-eJ-+-s-;-~e helper T cell in
------------------the"se 'ex~~sêlTffe,red fram ether praliferating T cells i'n ____ ------' l
~ ____ -------- that they reàched 'thé peak of prol iferation on day 2 of cul,ture,
, • j
, '----
(
three days prior to t~ peak of the tota,l c~lt'ure respo~~~ __ ,~----~
-----'----------------------------
------- . -----~Iogous MLR E. Stimulus , ======= ------_______________ -î'h e na tu r e' . a f the tri 9 9 e r i n 9 st i.m u 1 us ..-------- ,
responsible for
autolagous proliferation has been investigated in severa1 species
(134,136,172,173,174). Because T cells and B ce'lls are in l ,
autalagous combination, and since rec~gnition of MHC-l region
'(HLA-O in man) is necessary for several types 9f immune ~ell ; q
interQctions (149), several lnvestigators reasoned that la "
anti'gens might play a ro1e.;-.in sltimu1ating auto1ogous reactive T
cel1s (134,136,172,173). The ,question of wheth.:er t~HC-l regio'n
gene produots provide actlvation signa1s in the auto1ogous MLR is
amSI'lered by data from sevêral sources, such 'as: ~) ,ceH 5 bearing
HLA-D are reported ta be necessary for auto1ogous Mt~ (134),
2) ,th,e addl'tion of anti-Ia antibodies into cultures severe1;}'
depletes the autologous ~LR (136,172.1~73). 3) cytotoxic remova1
+ of la ce11s from stimu1a~or popu1atlons abrogated the
autologous res.ponse (172,1?4). 'The·se data imply·that the
, ,
.",
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i ......
• .1;'
37
, 1
a u t 0 log 0 u s r e a c t ion . s-- d-ep'e n den t u po n r e c 0 9 nit ion 0 f the
,~l - locus products (134.136,172-174).
------------- • \<.
H0\tever, the
- mechanism by which autologous MLR-p,rimed cens recognize self and
how MHC antigens are 'related to self:recognition in this reaction
have not been e1ucidated.
F. Autplogous MLR in Oisease
It has been suggested that the humari autologous MLR ~ay be
an in vitro manifêstation of a normal' reg"ùlatory mecha~ism by
\1lhic\h T and B lymphocyte functions are controlled (22). Thus,
abnormal levels of autologous MLR may signal immune dysfun'~tion. "
Autologous MLR has been investigated ;n var;ous pathological
condi~ions. Chronic 1ymphocytic 1eukemia (147,148) is
characterizep by an accumulation of B ce11s in the péripheral
blood. Patients with this ma1ignancy fail tp demonstrate any
activity in auto1ogous MLR (147,148). The 1ack of autologous MLR
in these patients was reported to be que,to defective stimulation,
by ~h~ leukemlC ~ cells (147). The auto1ogous reaction was a1so
s h 0 \'1 n 0 t 0 b e d e f e c t ive Jin the J y m p hoc y tes 0 f pat i e n t s \11 i t h ,
~yst~mic 'lupus erythematosus (SLE) (142,145,146). ho promil1.efl.t .... features of thlS auto-immune dlsorder are autoantibodies .. i,n the
, serum and T cell defects lnc1uding an inability of lymphocytes
t 0 9 e n e rat e 5 U P p,r e s sor T cel l fun c t f 0 n s (1 4 2 , l 4 5 , l 4 6 ) . A s ,
pointed out by Sakane et al. (145,146), the autologous MLR
r e s po n der Tee l 1, wh' i chi s de f e c t ive i n S LEp d i e·n t s 1 i sen r i che d
\ 1
p
(
38
in lymphocytes capable of becoming suppressor T cells innormal
individuals. lmmunological disordêrs have been associated with I/It
infectious mononucleoils (lM), which fol1ows infection wirh
Epstein-Barr VlrUS (EBV) (144). Ouring acute lM, impaired ., autologous MLR an~ development of transient autoimmune phenomena
such as production of autoantibodies to T and B lymphocytes and
réd blood cells have been reported (144). Palacios'(170) has
found that EBV infected cells are sensitive ta cytotoxic cells
activated in autologous MLR. In patients with Hodgkin~s disease,
i m pa ire d au t 0 log 0 IJ' s ./1 L R ha s b e end e mon s t rat e d byE n 9 1 e man e t al.
(139). The defect responsible for decreased a~tologous MLR in
Hodgkln's disease ,ppf.rs to be in the responder T cell population
rat h e r t han l n the s t 1 m u 1 a tin g" cel 1 5 (1 3 9 ). T h i 5 i s c 0 m pat i b 1 e
lIith eVl~ence showing that in Hodgkin's disease, B cell functions
are normal, whereas T t:ell-mediated immune functians are
impalred (139). Similar deflciencles have been observed in
Down's syndrome (140), Sjogren'" syndrome (143), and prima,ry
b, i 1 1 a r y c 1 r r il 0 S 1 S' (1 4 l ) .
G. MUrlne Autologous MLR
Slmllar1y, several types of autologous 11LR have been
reported ln the murlne system (175-179). The capaclty of murine
neonatal thymocytes to pro11ferate when cocultured with
autologous splenlc bone marrow-derived B cells has been termed
the T y p e [ 5 Y n 9 e n e i c ~4 L R (1 7 6 ) . Ho VI e e t al. r e p 0 rte d t h a t 1 :1 1 S
J i ,
'>
i
\ 1
1 1 1
1
(
, .
39
. 1
autoreactlve potential is not present ln the adu1t thymus (176).
According to these ~uthors, the in vitro proliferative response
of the thymus derived cells rlses sharply follo\ving birth, and
starts to decllne in the flrst week of llfe. Vo~ Boehmer and
Byr'd '(178) observed that much of the Typè l reaction w,as due to
ne6?atal thymocytes interacting wlth an antigen present on
syngeneic adult cells and not on syngenelc spleen cells of neo-
natal animals. Recent~y however, Hooper and Murgita (19) showed
evidence that autologous reactions occur in newborn thymocyte
.anti-adult syngeneic spleen cell co-cultures, newborn thymocytes
ant17'newborn non-T autochthonous spleen cell culture and in adult
lymph no de antl;-adult autochthonous spleen cell mixtures. The
latter reactlon, prol1feratlon of untreated adult lymph node
c € 1 T 5 t 0 a d u 1 t 5 P 1 e n l c t}s t l m u lat 0 r ce l 1 s h a s b e e n t e rm e d the T y p e
II reaction (176,179). Hooper and ~1urgita (19) reasoned that
control of self-reactivl ty ln the fetal and newborn mouse, may
depend on lmmunoregu1atory factors capable of blocking self-
reco9nitlon~ Thus, they investlgated the effects of murine AFP ,
on several types of autologpus MLR. Addition of murine AFP, we11
below levels considered physiolog1C with respect to the fetus and
n e w b 0 r n, Iv a 5 s 11 01'/ n t 0 e f f e c t l V e l y b l 0 C k pro l i fer a t ion 0 f t} e 0 n a t a l
th y m 0 c y tes 1'/ h en co cul tu r e d 'I~ l the l the rad u l t s pl e e n 0 r '< fi. .
autochthonous newborn spleen, and th-e response of adult lymph node
ag,pinst adult autochthonous spleen cells (19). These results
1
1 1 1 1
, \1
1 •
indicate that during' early ontogeny, AFP may be an important
regulatory agent in the control of autoreactivity (9).
oC
40
1 f
41
MATERIALS AND METHODS
I. AFP Source Material
Human AFP was purified from cord serum collected at birth 1
and the ascltlc fluid of patients with primàry hepatomas. Cord
serum was lsolated from blood samples by centrifugation at lOOOG
for ZOrnln and then frozen at -20°C. AS~ltic fluid was clarlfied
bye e n t rl f u 9 a t ion a t l 0 0 0 G for 2'0 min and 5 t 0 r e d f r O"ZI'e n a t - 2 0 0 C .
II. Preparation of Antlserum
Rabbit ant~sera against human alpha-fetoprotein (a~tl-AFP),
no r mal hum ans e r u,m (a n t 1 - Nil S ). and the hum ans e r u m pro te i n s
transferrin (anti-Tf), album;n (anti-HSA), a~ gamma globuHn
(anti-gammaJ were prepared for use in these studles by a
pro ce dur e sim l l art 0 th a tus e d b"y Ha r b 0 e et al. (1 80 ). An t i se r a
\1 e r e pre par e d b Y l n J e c' tin 9 ad u lt mal e rab bits atm u l t i.p 1 e l n t r a -
,J1l u S cul ars i tes 'of i t h 1. 0 m 9 0 f e i the r AFP, Tf, H SA, 0 r 9 a mm a }
globulin, emulslfled in an equa1 volume of complete Freund's
adjuvant (CFA). Antisera to normal human serum (Çlnti-NHS)~\'ias
produced ln rabblts which had been lmmunized ,dth O.4mls of NHS,
(total mg protein::::approx. Z6mg) in 0.6 mls of' PBS and 1 Dml of
CFA. Booster lnjectlons of 300-500ug protein)were given at 2
weeks postlmmunization. The rabbits were bled from an ear artery
at 7-10 day intervals, t\10 weeks after the boost. The serum was ~.,
1 --,
,
\
\y' ,-
I~. , ._ ..... ", ....
42 1
separated from the blood by'"àllowing a clot to form and decanting ,
off the serum. The IgG fractions of serum were isolated by
precipitation using 18% sodium sulfate (180).· The precipitate
was dialyzed against phosphate buffered saline, pH 7.2, (PBS) and
stored at -200 C. The ~ntisera were shawn ta be monospecif~c for
their relevant antigen(s) by immunodiffusion (61) and immuno
electrophoreSls (Jal) agalnst their relevant antigen and NHS.
Three other antisera were purcha~ed: A.TH anti-A.TL, anti/
1 a, and a nt i - Ly t . A. TH an t i - A . TL an t i se r<\J m (C e d a r', an e
Laboratories, Hornby, Ontario) is a murine alloantiserum prepared
by immunlzation of A.TH mice with lymphocytes from congenic A.TL
mice. This antiserum is cross-repctive with a determinant
present on both mouse la and human HLA-Dr antigens (182,183,184).
The antiserum, received as lyophilized material, was reconsti
tuted with lml distilled w~ter, aliquoted and frozen at -70 0 C.
Prior to use, an aliquot was thawed, diluted ta the working
concentration and sterilized by passage through a sterile O.45u
Millipore fl.lter U~il1ipore Corp., Bedford, ~~A). Anti-Ia Il.
(#NEI-Oll, New England Nuclear, Boston, MA) and anti-human T-
lymphocyte Lytl; #NEI-012, New England Nucle~~) '.
are murine
immunoglobulins of the IgG class. 'Thesè antlsera viere prepared
by hybridoma cell-fusion technlques as ~escribed by Hansen et aL. o
(185). The anti-Ia antibody reacts against human G cells but not
against human T cells (185). The a~ti-Lyt' antibody reacts with
50 to 80% of peripheral blood T lymphocytes but not with B
1 1
,
(
.. . "
cell~ (lB5). The antisera upon receipt, were dil~ted 1/10 in
RPMI-1640 m€dium (Flow Laboratories, Mjssissauga, Ontario)~
aliquoted and stored frozen at -70 0 e.
III. Assays of AFP \
43
o "
AFP concentrations were estimated bj rocket immunoelectro-
phoresis (RIEP) according to the method of Axelsen (60).: This t
• method has a sensitivity of 1-5ug/ml (60). The immunoelectro-
D h 0 r e 5 i s wa 5 p e r f 0 r,Jd . i n a 1 % ( w / v ), a 9 a r 0 se gel con t a i n i n g 2 %
(v/v) rabblt aati-AFP a n t i s e rad i s sol v e d in' a T r ; s - bar b ft] t •
strength = 0.05. Ten mls .of this gel "
glass plates (82 X lOOmm). Ten m1ç...ro1iters,
.buffer, pH8.6, ionic
s~lution was cast on
of the test sample and the referen~e standards were a~pli~d ta
w e l 1 s p une h e d i n t 0 the gel. P a p e r w i c k S \'1 e r e e m plo y e d toc 0 n nec t
the gel to the buffer compartments of t~e el,~ctroPhores;s
'" r apparatus. Immunoelectrophoresis wa~ then performed for 2hrs at
a constant vol'dge of 250V. Affer termination of the electro-f IJ i
phoresis. the p~ates were pressed under fllter paper for 15min,
washed for 15min ln O.H1 NaCl and for 2 X 15min in l'Jater, pressed
as before, and'dried under a heat lamp. The plates wer~ stalned
for (om,n in a sol;tion containing o/.% Coo',ssie Brilliant Blue
(Biorad' Laboratories, Mississauga, Ontario) dissolved in ~thanol: >
acetic acid: water (45:10:45. v/v/v). A semilogarjthmic s.tandard
curve, rocket peak helght versus log (concentration (ug/ml)) was
", ...
44
( plotted bas~d on the 4 kno\oJn dilutions of the reference standard;
IV. pJrification of AFP
Human AFP was Durified from cord serum and ascitic fluid
b Y t w 0 d i f fer e n t rn eth 0 d s,ou t 1 i n e d i n Fig." 1: 0 n e met h 0 d
utilized a physicochem;cal" approach which has been previously
described in detail ,(55). In addition, sorne AFP isolations were
carried out usirlg an ant1body-agarose, immunoadsorbent affinity
chromatography procedure (186).
, A. Physicoch:emical Purification of AFP
1. Ion-exchange Chromatography on DEAE Sephadex-A50
In a standard batchwise procedure, 200ml fractions of the
starting material were used for AFP isolation. The material was
dialyzed at 4°C for l8hrs against D.1M phosphate buffer, pH7.0
(initial buffer). Approximate1y 90g of diethylarninoethyl (DEAE)
Sephadex-A50 (Pharmacia, Canada Ltd., Dorval, Quebec)'was
equilibrated with the initial buffer and packed in a coarse
sinte.red glass funnel (12cm diamet.er). The dialyzed source ~
material was loaded on top, and chromatography was performed by
gravit y flow. The column was \vashed with 3L of D.1M phosphate
buffer, pH 7~0, and 5L of O.lM phosphate buffer, pH 5.5, and then
eluted with 2.5L of D.1M sodiam acetate- D.5M NaCl buffer, pH
4.0. Rocket·immunoelectrophoresis assays revealed that the AFP
/
1
\
\ j j~
, ..i j
(
j ,
\
, 45
was contained in the fractions eluted with O.lM sodium' acetate-
-a,5M NaCl buffer, These frac'tions were pooled, concentrated in
an Amicon Model 2000 concentrator (Amicon Corp., Mississauga, ,
Ontario) fitted with a PM-30 filter membrant (exclusion limit = 30,000 daltons) and dialyzed against~lM phosphate buffer,
pH 7.~. at 4°f: in preparation for column ion-exchange
c.hromatography on DEAE Sephad'ex-A50. His preparation \~as ","
des i g n a te d 1 r;,C - 1 .
Fraction IEC-l was applied to a 5 X ~9cm chromatography
column packed with DfAE-Sephadex A'-50, prepared as described
above. The column was elute.d with a linear pH - ionic strength
gradient generated by gradient mixihg of'2.0L Qf O.lM phosphate
buffer, pH 5.5, in the mlxing chamber and 2.0L of O,lM sodium
a ce t a te - 0 . 5 M Na Cl, pH 4. a, i n the r es e r V 0 i r . The col u m nef f1 u en t
was monitored by measuring its absorbance at 280nm.' On the basis
of rocket immunoelectrophor.esis assays; the major-ity of AFP was
eluted bet'ween conductivity of 7 and 20 mmhos. Se1ected AFP-
containing fractions were then concentrated as described above,
and deslgnated fraction IEC-2.
?, 2. Chromatography on Sephadex G-200
Ali quo t S 0 f f r a c t ion lE C - 2 VI e r e. ch rom a ta gr: a ph e don Sep h a d !\ G - to 0 col u m n 5 (P Il a r mac; a) e qui 1 i br a te d w i th O. 0 S M T r' i 5
(tris(hydroxymethyl)-amino-methane; Sigma Chemical Co .• St.
Louis, Missouri)- O.SM NaCl bufter. The same buffer was alsD f
r
')
\
46
," used for elution and dialysis o.f the sample'. ,The column was
calibrated l'llth the serum proteins IgG and albumin 'at 'a flovi rate
of 40ml/hr. Fraction IEC-2 \'las loaded onto the "prepared column,
and protein was monitored continuou~ly at 280nm. Fracti,ons were ~"_ ......
• sayed for.;.AFP activity, and on the basis of these assays, the
AFP-containing fractions were pooled, and subjected to further
pur; fication.
3 . .?epharose-Blue Dextran Column Chromatography'
Fractions containing AFP from Sephadex G-200 chromatography
were concentrated by ultrafiltration on Amicon PM-3D membranes, ,
dialzyed against D.05t~ Tris- D.SM NaCl buffer, pH 8.0, and
fractionated on a Sepharose-Blue Dextran column (SBD;
Pharmacia) preequilibrated with the same buffer. The column l'las
eluted at a flow rate of 20Dmls/hr with approXimatélY~ bed
volumes of buffer .. On the basis of .,ssars perform.ed, the AFP-l'la~
con t a i n e d i n the v a i d vol ume . Bou n d a l b u min w as, e l ut e d w i t h the
sam e 'b u f fer con t a i n i n 9 6/4 ure a , The col u m n VI a s the n r e e qui 1 l -
br a t e d \>J it h 't h est art i n 9 bu f fer.
4. Concanavalin-A-Sepharose 4B Affinity Chromatographye.
Sodium acetate buffer at pH 6.0, O.lM, containing 1.0r~
• f NaCl, 1.OM CaC1 2 , 1.Omr~ ~lgC12' and 1.Om~ MnC1 2 vias used for
dlalysis of the concentrated AFP-contairiing fractions from the
1 1
1
(
47
SI3D cplumn,' and equilibration of a Concanava1in-A-Sepharose 48 )
(Con-A S4B; Pharmacia). The AFP-containing fractions from SBO
chromatography were loaded onto the co1umn and eluted with the
same buffer at 25m1jhr elution rate. All fractions were
-#Ii" monitored at 280nm and for AFP concentration. The elution l'las
discontinued \'1hen there was no measurab1e absorbance. The bound
material was· then e1uted with 2% (wjv) alpha-methyl-D-glucoside
dissolved in the equilibrating buffer.
J 5. Chromatography on Sephadex G-200
The AFP-containing material from Con-A ~4B chromatography
w a s c Jn c e n t rat e d a ri d dia l y z e d a gai n s t O. 0 5 M Tri 5 - 0 . 5 M N a C 1 a·n d ,. chromatographed on Seohadex G-200 as described above. AFP-
containing material \'las collected, dia1yzed against PBS.
o concentrated, and stored at -20 C.
B. Purification of AFP by Immunoadsorbent Chromatography
1. Preparation of Substltuted Afflnlty Matrices
Purified IgG antibody preparations \'/ere conjugated to
cyanogen bromide (CNBr; Sigma) activated Sepharose 4B (S48;
Pharmacia) according to the meth9d of Cuetracas et al. (95,187),
but ~Jith sorne modifications. Sepharose 4B~was washed extenslvely
\·lith water on a Buchner funne', under suction. lihe decanted gel '!>
was weighed, and then mixed with an equa1 volume of wat~r and
CNBr (15~ CNBr per 30g of 548). The CNBr was dissolved by gentle
1 1
i
J
(
(
48,
\
stirring. The pH was then adjusted to, and maintained at 11, by ...
titration of 50% (w/v) Na OH into the gel. slurry. The temperature . \
of the slurry was held constant at 20°C by addition of crushed
ice. As the reaction neared completjon, the pH stabilized at
11. v/hen the réâction had ended, the activate~ S4B (S4B a ) was
washed with 3l of cold .water and 2L of D.1M NaHCD 3 .
IgG preparations of ânti-AFP and anti-NHS were diluted to
a final concentration of 5mgjml in O.lM NaHC0 3-0.5M NaCJ. The ~
antiserum preparations were added ta the S4B a in a ratio of 19
protein per 30g of S4B a , and stirred gently at 4°C for 24hrs. ~
The gel was then wfs"hed ~ith 2l of high salt P8S (HS-PBS; D.5M).
Til e ex c,e s sac t ive gr 0 u p s we r e b l 0 c k e d b Y a l ter n a te wa shi n g 5 w i th, '" ~.
o . 2~ 9 l Y ci ne - H Cl, pH 2. 8, and lM eth an 0'1 am 1 ne. The an t i b 0 d y-
agarose gel was then washed extensively with HS-PBS, packed into
chromatography columns (80-H10g of gel per co1umn), and stared
a t 4 C.
2. Immunoadsorbent Puriflcation of AFP
Immunoadsorbent chromatography purification consisted of
sequential passage of source material ovér anti-AFP antibody-S4B
(anti-AFP-S48) and ~~ti-normal human serum a~tibody-
Sepharose 48 (anti-NHS-S4S) affinity columns. Cord serum or
ascitic fluid was loaded onto tHe antl-AFP-S4, solumn. Elution ,
of nonbound pro,teins was performed by gravit y flow I·Ji·th 1-2L of
Il S - P B'S . Bou n d mat e ria l Iv a sel u te d b Y a P pli c a t ion 0 f O. 2 ~1
'f
,
, ,
j
....
(
,\
'-
.1
glycine-Hel, pH 2.8, into tubes containing crystalline Tri5·fo~ .
immediate nèutralization. The column was regenerated by washin~
with 0.5L of 4.0M guanidine, followed by 1.5-2.0L of HS~PBS. On
the basis of AfP assays, the fraction eluted with O.2M glycine
HC1, pH 2.8, was dJalyzed against PBS, concentrated by ultra-
• filtration on PM-3D membranes and sUbjected to further
purification.
The s "m a 11 am 0 u n t 0 f rem a i n i n 9 con t ami n a n t sin the AFP
~ preparation were removed by p~ssage over the rabbit anti-NHS
antibody-agarose column. Serum proteins retained by the column
were then eluted with O.5L of 4.0M guanidine, followed
immediate]y ~ith 1 .5-2.0L of HS-PBS. Rocket immunoelectro-,
phoresis assays revealed that the AFP was contained in the void
,volumn. These fractions were poole~, concentrated, dialyzed
against' PBS, and stored at -20°C.
V. Prep~ration of Newborn Serum Supplements
Cord serum l'las selective~y depleted of AFP (NBS-AFP) by
passage ~ver a rabbit anti-AFP immunoadsorbent column. To
control for nonspecific protein losses an aliquot of the newborn
se r u m (N B S) \'1 ii 5 pas se d .0 ver a n i ne r t Se p"h a r 0 S e 4 B col u m n (N B S -
S48) .. The column passed fractions were concentrated to their
original volumes by Amicon filtration. The AFP concentration of
NBS"ana NBS-S4B was approximately 80ug/ml. AFP was not
, . l
c
(
'. __ .-.- _. "._. r --- "-~-'-- .--.--~.'-,
, , 50 1
d ete c t a b lei n N B S - AFP 0 rad u 1 t s e rab y roc k e t i mm u n 0 e l e c t r 0 -
phoresis. The'serum preparatîons wer.e sterilized by Mil1ip.ore
filtration and stored at -20°C.
VI. Analytical Procedures
,.~ A. Ouchterloney Immunodiffusion
Immun~d;ffusion in 1% (w/v) agarose in 60mm petri plates
was.pertformed according to the method of Oucht.erloney ,(61).
Antiserum used in the analyses included anti-AFP, anti-HSA, anti-
Tf, and anti-NH-S.
B. Pol y~l amide Gel El ectrophores i s
" Regular' analytical polyacrylamide ~el electrophoresis "
(APAGE) was performed in the discontinuous buffer system .of ô
Davis (58) and the sodium dodecyl sul fate, containing buffe.r . ~
system of Lugtenbèrg (SOS-PAGE) (188).
1. APAGE
For the pre par a't ion 0 f ru n n i n 9 and s tac k j n 9 gel s the
fol l 0 \oJ i n 9 sol u t ion s we r eus e d : r) sep a rat i n 9 9 e l mon 0 mer
sol u t i o.n con t a i n 1 n 9 289 a c .-:y l ami d e (B i 0 rad (C a nad a) L t d . ,.
t1 i s sis sa u 9 a, 0 n boa ri 0) plu s O. 735 9 N, N 1 - met h Y 1 en e bis a cr l y ami de
(Bis; B;orad) dissolved in lOOml of double distilled water; • ' "Q Jo
\ -, 2)'-c the' s tac k i n 9 gel II) 0 nom è r sol u t ion con t a i n e cl 2 0 9 a cry l ami d e
"
1
1 j
, 1
1
/
(
. '
(
, "_ ... ~ ........ 4-~ ... _....,.. ........ ,, ___ , ... _ .....,.,~~,,""I ___ l'I"".....,... '"'J J
51
plu s 5 9 Bis dis sol v e d i n 1 0 Om lof do U b l e dis t il l e d \'1 a te fi' • The se , 0
solut'ions were stored in the dark at 4°C,. The separating gel was
prepared by mixing: 7ml o(separating gel monomer solVtion. 7ml
of O.15M Tris-Hel, pH 8.9, and l4ml of ammonium persulfate
solution (1.4mg/ml). The gel solution was deaerated, ,~nd th en
polymerization VJC\S initiated by th,e addition o{'N,N,N' ,N'-tetra
methylenediamine (TEt1ED) in a final concentration of 0.2% (v/v), ,
The staçking gel was prepared by mixing: O.4ml of stackfng' gel
monomer_ solutiort', O.4ml of 0:05M Tris-Hel, pH 6.1. 1.6ml of . ,
ammonium persulfate solution (1.4mg/ml), and D.8ml of water,'
After deaerafion, TEt4ED in a final concentration of 0,,2% (v/v) ~ --
was add ed· to 5 ta rt po 1 ymeri za t ; on. f-l~phores i s l'la s perfo rmed C>
in either a Buchler tube gel (Buchler Instruments, Fisher) or ~'
Pharmacia slab gel/tube gel electrophoresis apparatus (P~~rmacia).
Samp1es were applied in lO-50ul aliquots containing approximately
, l 0 ~ 5 0 u 9 , pro t e i n a n' d 3 u lof O. 0 5 % b rom 0 p h e n 0 l b 1 u e . E l e c t r 0 -, ' \
phoresis was conducted at 3mA constant current per tube gel for
apnroximately 2hrs. The gels viere stained for' l2hrs at 37°C in
0.42% (w/v) Coomassie Bri1liant Blue R-250 (Biorad) in a mixture
of 50% met han a 1 and 9 1 a c i a lac e tic a C'1 d (8 5 : 1 S , v / v) and the n
destained in a mixture of water,' acetic acid, and methanol
(85:7.5:7.5, v/v/ov).
Following APAGE, certain replicate gels viere l&ft unstained . -
for immunological characterization by Gel Ouchterloney. The gels
were embedded in 1% (w/v) agarose, o
Troughs paralle1 to the gel ....
, !
r
" ". 1
1 1 1
, \ . '1
j
. .
' . . ~'f
(
..
52
were eut and filled with antisera of different speeifieities.
The protein in the APAGE gel and the antibodies in the trougtv'
were allowed to diffuse toward one another f6r 18hrs at 20 C.
2. SOS-PAGE
The separating gel' sol ution for SOS:'PAGE \vas prepared by
mix'ing 6.25mls of separating gel monomer solutfon (44g acryl.\
amide plus 0·.8g Bis in 100mls water), 12.5mls of 0.75M Tlris-HC1, 1
pH 8.8, 5.12ml<s water, 0.50mls of 10% (\'1/v) SOS, and O.613mls of
ammonium persulfate soluti·on (lOmg/ml). The staeking gel /
sol ut ion" \'1 as pre par e d b Y ml x f n 9 1. 0 ml 0 f st a C k i n 9 gel mon 0 mer
solution (30g aery1amide r p1us 0.89 Bis in lOOmls water), 5.0m1s
of O,25M Tris-Hel, pH 6.8, 3\6mls water., 8.1Gmls of 10% (w/v)
SOS, and 0.24mls of ammonium persulfate solution (lOmg/m1). The i
protein sampl,e (1-2mg/ml) was solubi,lized in the following . solution: 0.0625M Tris-Hel, pH 6.8', contai"ning ,2% (w/v) SDS,
10% (v/v) glycerol, 0.001% (w/v) b.romo,phenol b"lue, and 5% (.v/v) ç
2-merçapto~thanol (2-~1E). Protei~ samples (20-40ug protein/gel)
were heated in a water bath at 100De fo'r 5min and ele,ctropho,resect,
stained, and destained as aesèribed above.
1 3 . A n a 1 y t l cal 1 5 0 e l e c tri c Foc u S l n g. i n PA G E
Isoe1ectric focuSlng (IEF) of the purified AfP preparations
,\oJas performed accordlng to the. method of Righetti and Drysdale 1 ~
(189). Four percent po1yacrylamide gels containing 2% , ~
, r 1
\
....
(
I:J 1
ampholytes, ~H range of 4-6 (LKB, Fisher Scientific Co., f
Montréal, Canada)-were cast. Gels were preelectrophoresed at \
250V for 45min at SoC to establish the pH gradient. Thirty to
53
(
<)
one hundred micrograms of the sample in '20% glycerol and 0.005%
br 0 m 0 p h en 0 l b 1 u e 'we rel a y e r e don t 0 the gel b ,e n e a ct hab u f fer Z 0 n e
of 10% glycerol with 2% ampholytes', pH range of 4-6. Anodic and
cathodic buffers contained 20mM NaOH and 10mM H3P0 4 , respectively.
Electrophoresis was resumed at a constant voltage of 400-450V for ,
3hrs' r Upon completion of focusing the gels were dialyzed in 5%
trichloroacetic acid- 5% sulfosalicylic acid for 2hrs, washed in
3L of water, and stained for 4hrs in 0.05% Coomassqe Brilliant
Blue R-250, 0.1% cupric sulfate, in a,mixture of acetic acid,
ethanol, and water (16:25:65, v/v/v)~and destained ln acetic
acid, ethanol, and water (10:10:80, v/v/v). The pH gradient of
unstained gels was "determined using a surface pH _electrode
(Fisher) .
VI!. Assays for Lymphocyte-inhibitory Activity
The affects of purified AFP prepfrations, NBS supplements,
antl-I,a, anti-Lytl, A.TH anti-A.TL a~tiserum, and HSA on the
proliferative response of human T cells to autologous or allo
ginelc challenge in m1xe~ lymphocyte culture (MLC) were
determined as previously described (22).
1 f
1
1 1 l
!
(
'.
54 1
A. Cell Preparation
1. Isolation of Peripheral Blood lymphocytes
Human peripheral blood lymphocytes (PBl) were isolated from
the whole blood of normal adult volunteers by density centri
fugation as described by Boyum (190). B100d was collected on ,
preservative free he'parin (Abbott laboratories, ~1ontrêal, QUébec),
diluted 1:1 with PBS, 1ayered on Ficoll-Paque (Pharmacia), and
centrifuged at 400 G for 35min at room temperature. Cells .
removed from the interf~ce were washed three times in PBS and
resuspended at 10 X 106/ m1 in RPMI-1640 medium (Flow).' Viability
as assessed by Trypan Blue dye exclusion was greater than 95%.
2. Separation of T and non-T cells from PBl
Ta prepare r~sponding T enriched and stimulating non-T cel1
populations for MlC reactions, two techniques were used:
rosette formation with SRBC (191), and 2) cell affinity
fractionation on Ig anti-Ig columns (192,193).
a. E-rosette fractionation
1) E-
Unseparate~ populations of PBl were fractionated into Tf
enriched, referred to as T cells, and T-depleted, referred to as
non-T cel1s, subpopu1dtions according to their capaclty to form
rosettes with SRBC. Sheep red blood ce11s (Institute Armand-'
Frap~ier. lava1, QUébec) were w~s~ed three times with PBS and
1 ,
1
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1 . 1 --
j
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- •• •
resuspended at500 X 106/ ml in RPMI-1640 medium containing 20%
fetal calf f~um (FCS, Flow). PBL's. from Ficoll-Paque gradient
eentrifUgat~on, at a e'oneentrat;on CJf 10 X l06/ ml in RPMI-1640
. r. ~ 1 R were mlxed ~~~h a: equa volume of the S Be susoension',and
incubated at 37°C for 15 min. The cell suspensions wer~ ,
centrifuged at 50 X G for 5min and ineubated for lBhrs at 4°C.
The cell mixtu'res were then 9e..ltlY resusp'erided. with ei~her a
wide-bore pipette or by inverting the tube. Th"e e'~ll suspension r
was then l~yered over Ficoll-Paque and centrifuged at 400 X G for \
The i n ter f ace con t a i n i n 9 n.o n - E - r 0 set tin 9 cel l s
was removed, resuspended in RPMI-1640 medium and referred ta as
-T-depleted (non-Tros) eells. The pe11et of E-rosetting eells was "-
resuspended in RPMI-1640 medium and referred t6 âs Tros cells. '/' ' SRBC were lysed by two me'thods: 1) hypotonie shock' with sterile
distilled water, immediately followed by the addition o~ a
sufficient volume of a lOXPBS solution to rèturn the eell
suspension to an isotonie condition; 2) by washing the ,eells in ..l,
O. 1 4 M Tri s - a mm on i u m ch l 0 r ide, pH 7 . 2 . Alle e 1 1 pop u lat ion s \o.f e r e
then washed 3 times ln PBS and resuspended in RPMI-~640 medium
containing 20mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
aCld (HEPE's; Flow), 5X lO-SM 2-ME, 4mM L-glutamine, 20U/ml
(p~ cil 1 in .. and 20 u 9 / m l 5 t r e p tom y c ; n .
iJ
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51
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III
b. Cell Affinity Fractionation on 19, anti-Ig Columns for the Selection of T and non-T Ce" s
Human Ig -rabbit anti-human I~ (HuIg/rab anti-HuIg) coated
o 9 1 a s s b e a d col u m n s \'1 e r eus e d t 0 sep a rat e P B \ 1 sin t 0 Tan d r1 0 n - T
subpopulatlons by the method of Wigze11 (1920. Glass beads were
aCld cleaned for l~hrs in a solution containing equivalent
vol u rn'e s 0 f H Cl and H N 03 , wa s he d i n PB S, and ste ri l t z e ~ b Y
autoclavlng. These beads were t-ben coated with human 19 by
incubation of the beads for 18hrs in PBS containing purified
human IgG (5mg/rn1). Unattached proteins were removed by packing ~
the beads ln a chromatographie column and washing with PBS. ,The . \ bead columns were then incubated at 37°C with rabblt Ig anti-
human 19 antiserum for 60min and then washed with PBS. These
colurnns specific plly bind ce11s bearing surface Ig (sIg) (192,
193). PBl (10-20 X 10 6/m1) were applied to the top of the '"
co1urnn (3-5 X 10 6 cells/ml· of bea'ds) and collected by elution
with PBS 'unti1 the affluenfwas cel1 free. The unretained cells
were referred to as Tco1 ~ells. Bound ce11s were obtained by
vlgorous pipetting of the glass beads in a sterile petri plate.
The b e a d s we r e d'e c a n t e d F the s u p ~ r n a t' a n t wa s ce n tri f u g e d
(200 X G for lOrnin) ta dbtain a cell pel-let. These ce11.s \'iere
washed three tirnes in PBS and resuspended in the supp1emente~
RPf11-1640 medlurn descrlbed above. Viabilities of all' è'ell'
populations prepared by the rnethods descrlbed above exceeded 90%
as judged by trypan blue dye exclusion.
..
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57
....,.-J
B: Identific~tion of lymp'hocyte Subpopulations
Cell populations obtained by Eicol1-Paque density
centrifugation, E-r~?ette formation, and cell affinity fractiona-'"
t ion. t e c h n i que s. w e r est u die d w i t h r e s pee t t 0 sIg and the a b i lit Y
The percentaqe of cells forming
rosettes with SRBC was determined by the method of Mendes et al.
(191), described in detail above, with the modification of a
60min incubation at 4°C. One hundred lymphocytes were counted
and only rosettes'having 3 or more adherent SRBC were counted.
Surface immunoglobu1in was detected with fluoresceinated
rabbit anti-human 19 (Cedar1and laboratories, Hornby, Ontario)
in the ~irect f1uoresceinated antibody technique (194). Cel1s
were brought to a concentration of 5 xC0 4/ml in RP~1I-1640 ..
m e d i u m con t a i n i n 9 5 % F CS. T 0 O. 2 m l s. 0 f cel l s l'la sad d e dO. 2 m lof
f1uoresceinated rabbit anti-human Ig, and the~ incubated on ice ;
for 30 min. Cells were washed three times wit\, RP~l1-1640 media
containing 5% FCS and resuspended' in PBS containing 30% glycero1.
Ce11s wertviewed with. a fluorescence microscope. One hundred
ce11s were counted and the percentage staining with f1uorescein
conjugated rabblt anti-human-1g w~s efiumerated. \..
C. 1rradlation of Cel1s •
Certain populations of PBl'S and non-1 cel1s were
irradiated with 200-2500 rads in a gamma ce11 irradiator. After
, irradiation mortthan 90% of cells l'iere still viable as judged
1
(
(
":
• • ..
58 1
, by trypan dye exclusion.
D. Cel1 Culture System (
One-\l/ay auto1ogous and a1logeneic ~1lR'S \"lere p~rformed in
triplicate in 96-well round bottom micr?titer plates (Flow).
Responding T lymphocytes (lOS/well) and allogeneic o~ autologous
irradiated stimulator non-T cells (1-2 X lOS/I"ell) were mixed in
a total volume of O.2mls. Tissue culture media consisted of
RPMI-1640 medium supplemented as described above, with the
addition, where indicated, of serum autologous to the T cell
donor, allogeneic serum, 2mg/ml purified human serum albumin, or
newborn serum preparations. Various concentrations of purified
AFP, HSA, murine monoclonal IgG preparatlons dlrected against the ,~
human'Ia antigen (anti-Ia) or against human T cells (anti-Lytl),
A.TH anti-A.Tl, murjne alloantiserum, or normal mouse serum (NMS)
were added at the inltiation of cultures. Cell cultures were
incubated for 3-9 days at 37°C ln a humldified atmosphere of
5% CO 2/95-% air. One microcurie of methyl-3H-thymidine (3 H- TdR ; "-
speclfic activlty=55Ci/mM. ICN Pharmaceutlcals, Montreal. Quebec)
was added to each well six Kours before harvesting. At the end
J of the culture period, the cells were harvested onto glass flber
filters wiÙ the aid of a multlple sample harvester (Skatron,
F 1.0 W ) • The f i ber f l 1 ter s we r e ad de d ro l l qui d sei n t i l 1 a t 1 0 n
countlng cocktall obtained from New England Nuclear. The amount
~ of 3H- TdR inc~rporated 1nto ONA was measured as counts per minute
"
•
59
• 1
(cpm) on a Beckman 8000 liquid scintillation counter (Beckman
Instruments, Fullerton, California).
E. Data Analysis • Results are expressed as mean values t the standard error
of the mean. The ~tatistical significance of differences between
mean values was determined by Student's t test.
1 .,
) 1 i
1 f
t
i
60
RESULTS
J. Puriflcation of AFP
A . ~i co che m i cal Pur i fic a t ion
Gold et al. (55) have described a physicochemical procedure 1
for the purification of milligram quantities of human AFP. This
method, outlined in Fig. l "las used\\for the purification of AFP
from poole~ human cord sera and varidus primary liver carcinoma
ascitic fluids. The results obtained during the physicochemical " (
purification of AFP from 640 mls ofoascitic fluid ~re presented
in FlgS. 2-6. The other 50"uree materials behavèd similarly at
each stage of ~e purification procedure. Constant amounts of
AFP ln vanous volumes of ascitic fluïd l.oJere {iibjected to bulk ~ ----1 -.
r" ion-exchange chromatography at pH 7.0. On the basis of AFP
assays, the retained proteins, eluted \"/ith an increasing linear ~\
pH-lonic strength gradient. contained most of the AFP (results
no t s h o\v n ). Th i 5 f ra c t ion wa s des i 9 na t e dIE C - 1 .
However, fraction 1EC-l was found to be heavily
contaminated with other serum proteins including albumln.
Further purification by columfl chromatography was peÇformed on a 1
DEAE-Sephadex A-50 column ps shown in Fig. 2. The void volume,
which consisted of all non-blnding substances, contained albumin,
transferrln, and several other serum components, but no AFP as
detected by O~chterloney double immunodiffusion. The bound
protein, later eluted after the application of the linear
('
,-
(
61
gradient of O.IM sodium acetate- O.SM NaCI buffer, pH 4.0,
con t a i n e d the AFP a c t ive mat e ria 1, a 1 b u mi", t r{~n s fer r in, a 1'). d
other serum components identified by immunodiffusion and immuno
electrophoresis. ;.
The bound protein peak elyted between conductance val~es of
7-20 mmhos, was coJcentrated and subjected to gel filtration on
Sephadex G-200, as shown in Fig. 3. Rocket immunoelectrophoresis
(REIP) examinat.ion o,f the G-200 fractions revealed that most' (}f
the AFP appeared in 825-l12Sml of eluent volume. This elution
volume corresponded to that of purified human serum albumin, with
which the column had been previously calib~ated. The AFP
containing fractions from the Sephadex G-200 column were applied
to a column of SBD to separate AFP from albumin. The "fall
through ll (non-retained) p~.ak contained all the AFP activity. The
binding of serum albumin, however, to the SBD column was not
complete as assessed by double immunodiffusion. Chromatography
of the f~Jl-through peak through a regenerated SBO column (Fig.
4) resulted in the removal of albumin, as based on immunbdiffu-
sion analysis.
Con A-Sepharose ~olumn chromatography of nonbound
material fram the SBD column resulted in the elution proflle
shown in Fig. 5. RIEP assays revealed t~t the fall-through oeak
'yielded no AFP activity~ Elution of AFP from the Con A-Sepharose
column was accomplished with 2% alpha-methyl-D-glucoside.
--&Q..ntaminanp were not de,tecte{! in the materi~l eluted \'dth alpha-
1
;
1
1 l
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62
methyl-D-glucoside by double diffusio~ analysis against anti-NHS.
However, APAGE revealed in addition to AFP sorne minor
contaminants.
On molecular-sie~e chromatography of the AFP-containing
fraction from the Con A-Sepharose column of G-200, a single peak
was obtained, as shown i.n Fig. 6. in the molecular \~eigh't zone of
HSA. The homogeneity of the fraction was indicated by a single
band on APAGE.
B. Immunoadsorbent Purification
An immunoadsorbent purification scheme, outl~ned in Fig. l,
was also employed for the isolation of AFP from crude sou~ce
material. Starting material was first passed over a column
A packed \'1ith anti-AFP immunoadsorbent, prepared by coupling 3g of
,r:)
rabbit anti-AFP IgG to 90g of CNBr-activated 54B. A typical run
is depicted in Fig) 7. The retained proteins were eluted
5 e que n t i'à l l Y , first by buffer, pH 2. B, and then by a 9 l Y c i n e - Hfl
4M guanidine. The glycine-Hel elution ~uffer gave a sharp peak,
teqned peak I. In the initial runs, peak 1 cGntained most of the 1.
AFP, with a minor AFP peak, termed peak II, appearing aftt'r '/
application of 4M guanidine. Runs in which the column was
intentionally overloaded \'Iith prote;n indicated that the maximum
binding capacity was 20-30 mg of AFP. Extraction of AFP from
'source material was virtuallx complete if the total amount of AFP
applied did not exceed ,the maximum binding capacity of the ,
..
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1
i i
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63
immunoadsorbent. Peaks 1 and II were pooled separately and • analyzed by double ;mmunodiffus;on. Pe'aks 1 and II usually "
reacted with anti-AFP, but gave no reaction when tested with •
anti-Tf, anti-gamma, anti-KSA, an~ anti-NHS. APAGE revealed a
band electrophoretically more negative than albumin and several
minor contaminants.
the removal of remaining contaminants was accomplished by
negative-affinity €hromatography on an anti-NHS immunoadsorbent
column (Fig. 5). This column was prepared by conjugating 2g of
rabbit anti-NHS IgG to 60g of CNBr-activated S4B. AFP was not
retained by this column, and appeared in the void volume. AFP
isolates were consider~d pure wh en a single band was revealed on
APAGE. "Normal human serum components in the AFP samples were
usually removed by a single passage ov~r'the anti-NHS i~muno-
adsorbent column.
. .. C. AFP Yields
Shown in Table lare typical yields of AFP obtained by the
ohysicochemical and immunoadsorbent purification methods. The ,l .
overall yields by immunoadsorbent chromatography ranged from 34-
59%. Physicochemical purification resulted 1n yields of 9-19%~
/ aDproximately h/o fo]d lower thèln immunoadsorbe'nt purification.
o
-r
(
64
apparent differences in e1ectrophoretic mobi1ity as judged by
immunoelectrophoresis between fetal and hepatoma derived AFP and
beh/een AFP iso1aze,s purified by the phys;cochemical and , immunoadsorbent purification schemes.
Shown in Fig. 10 is a typical pattern on APAGE for a o
purified hepatoma derived AFP isolate. AFP samp1es appear as a ,
single band in the post-albumin area after electrophoresis in
7.5% polyacryl"amide gels. The band was identified as human AFP
using a gel Ouchterlen~y' technique. For this test an unstained
gel' \Vas immobilized. in 1% agar beh/een t\"I0 parallel troughs
containing a·nti-AFP and anti-HSA. A sï~gle precipitin tine \'las
formed agalnst anti-AFP at the Dosition which corresponded to
the stained protein band as sho\oJn in Fig. 11. No immuno-
precipitat10n arc was observed against anti-albumin.
E1ectrophoresis O~ SOS-PAGE revealed faint Coomassie Blue
stained contaminants in the AFP ~reparations (Fig. 12), visible f
1 1 "
(
only when the gels were Joaded with 30 or more micrograms of
protein.
Since AFP has been shown to be heterogeneous by several
techniques", we analyzed our isolates for molecular heterogeneity
by iso"electric fol:u'sing. 'In basic agreement with Lester et al.
(68), IEF resolutiori was increased in the presence of eM urea. ,
• A t Y pic a lIE F pat ter n foc use d i n 0 the pre 5 e n c e 0 f 8 Mur e ais s..h 01" n
in Fig. 13. F1ve bands with isoelectric points (pl) ranging from
5.8-6.2 are"present in varying proportio~~. The number of bands
detectable in the AFP isolates by IEF and 8M urea varied from
3-6.
II. Cytotoxicity of AFP Preparations
The pure AFP prepar"ations \'1·-er-e--ttst~---.a..l~i-tttlWl1ïlln Jable 2
for a possible cytotoxic effect on PBl's. Ficoll-Paque purified
PBl's were cultured'in kPMI-16AO medium cont~ini~g eith~r one of
six different AFP preparations, HSA, ~r gamma glo~ulin, Cell
viability (trypan blue dye exclusion) in the various cultures,
including those to which AFP had been adde~, was sl~ilar to the
{v,iabllity in cultures with HSA, gamma globulin and<without'any
~ protein addition. It therefore appears that the AFP isolates do
not exert cytotoxic effects 'on PBl 1 s, o
t
•
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III. The Differential Effect of AFP on Auto~ogous and Allogeneic t4l R
Previous studi~s have .-sholtn that AFP does exert variable
suppressive influences on certain immune functions in vitro.,
The results are °apparently fnfluenced by.the type of assay
employed, th~ source of AFP, the method of purification, and the
concentration of AFP tested (9,94). Initial experiments loJere Q
performed ta determine if our AFP prepàrations possessed immuno-
~egulatory properties on two types of T cell-mediated tmmune , '
reactions: 1) autologous ~1lR, and 2) allogeneic MlR.
A. Autologous MLR
1. Preparation of Responding and Stimulating Cell Populations
We were particularly interested in determining the effects
of AFP on the autologous MlR, since this system is considered to
reflect T cell prol.jferation a~ainst autologous antigens. 'This
prol i ferati ve p~ase of the 'human autologous Ml~ resùl ts from the
coculture of T and noo-T cells obtained from the peripheral bJood . . of a single adult. ~xperiments were first conducted to determine
the effectiveness of PBl'S, T cells, and non-T cells obtained by ~
. " ( E-rosette formation al1~ cell afflnity chromatography as ... .
responders and stimulators in the autologous MLR. \
The r e s'u 1t sin Ta b l e 3 s h.ow
PBl,
that vigorous proliferation of
by 3H- TdR incorporation on day . x
6 of
Tros' and Tcol as determined
culture occurred with either irradiated non-Tros or . ' "'-
i
. \ \ 1
1 î 1
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ôI 67
T x . r • x non- col' but not with lrradiated PBL. On the basis df
5 t i mu 1 a t ion, i n d i ces T r 0 S 1'1 e rem a r gin a l 1 Y b e t. ter r e s p 0 n der s t han ,.. 1
Tcol and PSL. PBL's respbnded vigorously to autologDus non-T
stimulation, however, the 3H- TdR incorporation of unstimulated,
PBL's \Vas 4-5 fold higher than Tcol and Tros' All further
experiments employed E-rosette fractionation techniques to
prepare populations of T and non-T cells.
The Tros and non-Tros populations prepared by E-rosette )/
fractionation were characterized by rerosetting, presence of sIg,
and reactivity to phytohemagglutinin (PliA) as shown in Table 4. ,
Analysis of the E-rosetting properties of these cell populatiùns . demonstrated that greater than 80% of the Tros reformed rosettes
with SRBC, while only 6% of the non-Tros cells formed rosettes .
Greater than 40% of the non-Tros cells expressed sIg, in ~ntrast
ta the non-Tros ~hich lacke? sIg as detectable by the direct / fluorescent antibody technlque. The lymphocyte proliferation
induced by mitogenic stimulation with PHA of unseparated PBl was 't:1t "-
compared ta Tros and non-Tros' Table 4 shows the mean results of ~
seven experiments. The non-Tros PHA resp6nse was approximately
five-fold less than the response of PBL or T cells.
2. Kinetics and Dose-Response Investigation of A~P-Mediated Suppression on Autologous MLR -h
Initial experiments were designed ta examine the effects of . . .
human AFP on T cell activation in the primary human autologous
\ 1 1 1 ~ ! 1
68
MlR. The kinetics of th~ T cell response to irradiated
autologous non-T cells, cultured in the presence and absence of ",
cord serum derived AFP added at the initiation of culture was
me as ure don d a y s 4·, 5 , 6 ,7 , 8, and 9. As show n i n Fig. 14 , th i s
c 0 cul t u r, e pro duc e d a s t r 0 n 9 p r Ji m a r y a u t 0 log 0 u s ri L R \'i i t h P e a k
3H- TdR incorporation on days 6 and 7. The results show that the
a u t 0 log bus pro l if e rat ive r e s po n sei s r e duc e d "b Y a'b 0 u t 80 % i n the
presence of 200ug/ml AFP, with.,lsignificant s.uppression observed
with as little as 5ug/ml AFP. Dose-dependent inhibitory effects
\'i e rem 0 s t a p par en t a t p e a k pro l i fer a t ion . S u'p pre s s ion 'b Y AFP q
wa-s' strong throughout th,e timecourse; a sh{ft in the kinetics of . .. ....... ~"
cultures \'Iith AFP was not obser;ved. Additio'n of 5-200ug/ml HSA
" had no significant effects. These data' indicate that human AFP
-exerts str-ong inhibitory effects on the primary autologous MLR.
3. Consistent Suppressive Action of Various AFP's on Autologous MLR
In the human syStem, the ability of different AFP
preparations to suppress al~ogeneic MLRocan vary'widEly (7,89,
i-.. :-1 02 , l 06 ) . Th us, \'1 e sou 9 h t t 0 e x ami net h e e f f e c t S 0 f d if fer e nt
preparations of AFP on aiJt-o-:logous MLR. The results of one
experiment are shown in Fi.!t ,,1'5, in which,3 feta'l and 5 hepatoma $.
derived AFP's were tested at a conc.entratiQn of lOOug/ml, on day . ,
6 of an autologous MLR. The results demonstrate that all eight
AFP 's e x e r t -$ U P pre 5 s ive e f f e c t son a u t 0 log 0 u S /4 L R . A t a-.... ....." . ,v
c<o n c en t rat i 011 0 f 200 u 9 / m 1 a gr e a ter de 9 r' e e 0 f con 5 ; 5 te n ,c y , n . "
~ 1
, " \'
J
1 l
1 1 \ 1 ,
(
suppressioin ,
iS observed, as shown in Table 5.
B. Allogeneic MlR
1. Comparison of the Effects of AFP on Autologous and Allogeneic r~lR
Next, experiments were perforrned to determine \·/hether an ,
69
allogeneic MLR displayed a similar sensitivity to AFP. Parallel
autologous and ~al1ogeneic MLR's were incubated for 6 days in
RPMI-1640 imedium supp1emet:lted \.,ith 10% human serum in the,.
presence of 5-200ug/ml AFP or albumin. The mean results of three
separate exper,iments are shown in Fig. 16. The addition of AFP
at a fin,aJ concentration of 200ug/ml suppressed over 80%
(p<O.005) of the proliferation in the autologous MLR, whereas
allogeneif MLR's remained "unaffected or ~/ere o~nl'y Sli9~tlY inhibited!bY addition of AFP. The apdition of 5-200ug/ml HSA to.
cultures rad no significant effects on the autologous or
allogeneic MLR. The results indicate thjt in serum-supplemented
cultures, a c1ear difference in sensitivity to AFP-mediated
suppression bet~"een autologous ,and allogeneic NLR's is apparent.
The data displaved in Table 5 are based on similar J -,' , experimeflt,~ and summarize Orur experience in comparinq the potency
of diffe1ent AFP isolates. Fet~l and hepato~a derived AFP's
consistïtly/supp(.;sed autologous MlR's, whereas similar effects
'were notlobse,~ved o~ .allogeneic t1LR's. The supp'ress;ve effect
of AFP or aute,logous ~1LR appears, to be unrelat;d ta the,magnjtude
f (
~
1 j
,
\ i f l j l i
\
of proliferation, since 10w', medium, and high levels of
a u t 0 log o,u s pro 1 ; fer a t ; 0 n we rem a r k e d 1 Y s u p pre s s e d '.
70
2. Serum-Free Culture Condition Requirement for the Suppression of Allogeneic MlR
Several studies (44,45,82,90,95) have demonstrated that AFP
strongly suppresses allogeneic MlR in serum-supplemented media.
In contrast, other studies (104) have reported that AFP is most
suppressive in serum free conditions and only moderately
inhibitory in serum-supplemented cultures. We have examined the
possibility that the weak or minor inhibitory influence of AFP on
serum-supplem~nted allogeneic MlR noted in these latter studfes
may be due ta culture conditions. ~ An initial study was condu,cted
to.compare the effect of various concentrations of human serum
and HSA on their growth~supportive effects for allo-activated
lymphocytes. The results· in Table 6 indicate that the growth
promoting effect of serum can be partiall~ replaced by HSA.
The optimum concentration 9f HSA for lymphocyte prolifer~tion was
1 •
~
1 l ! , ,
1
"
2mg/ml. However, allogeneic MLR responses supplemen~ed with low A
con c e n t rat ion S 0 f 5 e r u m ' (~ O. 5 %) \'1 e rem 0 r eth a n fou r fol d
higher than those obtai~ed with RPMI-1640 mediu~ supplemented
\'iith 2mg/ml HSA .. "" No significant proliferat,ive response vias
observed in protein-free medium. < l
On the basis of these results, we next sought to determine
the effect of AFP on alloactivated lymphocytes cultur~d in HSA-
(
supp1emented media. The mean resu1ts of three experiments
emp10ying 7 different AFP preparations are presented in Fig.17
The AFP iso1ates were indistinguishab1e as assessed by immuno-\
e~ectrophoresis and APAGE: However, the AFP preparations
displayed a wide range of effectiveness in inhibiting
alloactivated lymphocyte pro1tferation in albumin-supplemented
conditlons. Addition of albumin to the cultures as a proteln
control did not result in any suppressive effects .
71
., 1
Shown in Fig.1B is a 'comparison of the effects of ·AFP on
day 6 alloge'neic MLR's incubated in the presence of 2mg/ml..sA
" or 10% autologous sera. The AFP preparation used in these
studies, AFP-l, was pr~viously shown to'be a potent inhibitor of
il10geneic MLR's in albumin supplemented cultures. Marked
suppressio'n of l cultures supplemented with 2mg/m1 albumin occurred
with 200ug/ml AFP. In contrast, this AFP concentration did not
substantially reduce the a1logeneic MLR in serum-supplemented
conditions.
• Effect of Anti-Ia and Anti-T Antiserum on the T Cell Prol{ferative Response p Autologous and Al10geneic Non-T Stimulating Ce1ls •
IV.
The 0 b s e r vat ion b y 5 h ,i n e t al. (1 3 6) t h a tan t 1 - 1 a a n t i s e r u m
blacks the humqn auto1ogous MLR, suggests that auto1ogpus MLR
stimulation is linked to I-region antigens. In addition it has
been shown that AFP exerts a highly selective suppressive effect
"
\
l )
-
1,
"
72
on I-region associated murine MLR's (106.107). In v;ew of these ~
observations, 'we sought to determine if the sensitivity of the
.f tit 0 log 0 U 5 M L R ver sus the a l log en e i c t4 L R t 0 AFP - m e dia t e d \,
suppression was related to a greater dependence on Ia-antigen as
the lymphooyte stimulating determinant in these reactions.
Experiments were designed to directly compare the effects of
anti-Ia reagents on autologous and alrogeneic MLR's. Parallel 1
auto1ogous and a110geneic cultures were incubated for 6-7 days in
serum supp1emented medium with or ~ithout various concentrations
of 1) A.TH anti-A,TL, a murine anti-Ia a110antis_erum known to
recognize human la antigens (182,183,184); 2) mur-ine monoclonal
anti-human IgG (185); 3) murine motoclonal ant;-human T cell IgG
(185); and 4) normal mouse serum (NMS). Fig'ure19 shows that
'80% blocking of the autologous MLR occurred in the presence of a
final concentration of 2% A.TH anti-A.TL aJloantiser·um. In
comparison, the addition of an equivalent concentration of A.TH
anti-A.TL to the al10geneic N~R resu1ted in 30% blockage. Since
A.TH anti-A.TL antiserum is the serum'derived from A.TH mice,
NMS was a~ded to MLR's as a control for possib)e serum-induced
effects: The presence of NMS in MlR's resu1ted in stronq
augmentati~n of the proliferatlve response. Murine monoclonal
anti-human la revealed blocking effects simi1ar to A.TH antl-A,TL
an t i 5 e r u m . The s e - r e sul t sin die a t e a gr e a ter con tri but; 0 n of ~I a
antigens in the initiation of autologous MlR versus the allo-
geneic MlR. Thi~ supports but does not prove that the
~
1
1 \
1 1 ! , 1 1
1
(
"
73
differential sensitivity of auto1ogous versus allogeneic HLR to
AFP-mediated suppression is associated with the greater
contribution of lymphocyte stimulating la molecules in autologous
than in allogenelc reactions.
V. Demonstration of the Inhibitory Potency of AFP Contained in Newborn Serum dn the Autologous MLR
Because purified AFP was shown to markedly suppr~ss
autologous MLR as demonstrated in the present studies, it was -
conceivable that newborn serum, which contains from 20-100ug/m1
of AFP at birth (30), would b.e able to similar1y inhibit
autologous proliferation. This question was investigated by
culturing adult auto1ogous t4LR's in concentrations of newbcrrn and
adu1t serum ranging from 1-50%, as shown in Fig.29 Peak
autologous proliferation in adult serum-supp1emented cultures
o c c u r r e d a t l 0 -; 0 % fi n a l s e r u m c-o n c en t rat; 0 n . l n dis tin c t
contrast, NBS in final concentrations of 10-50% did not support
significant autologous proliferation. These results may be due \
to several pqssibilities which ift'"c'~ude: 1) that autologous and
allogenelc serum supplements have different growth supportlve
effects in au.tologous:~MLR; 2) newborn serum contains a
suppressive substance; and 3) that newborn serum'lacks the
components necessary to support autologous proliferation.
Ne examln~d whether the depressed response of ~utologous
(
,
(
L (
•
74
MLR in NBS cou1d be due to t~e use of NBS as an allogeneic serum
supDlement. As seen in Table 7, no consistent differences were
apparent in the growth sijpportive effects of auto10gous sera
suppl ements as compared \~ith aHogeneic serum supplements.
H 0 W e v ë r a u t 0 log 0 u s pro l i fer a t ion w a s 'ct s u p po rte d i n pro t e i n -! '11
free medium or in cultures containing NMS, or 2mg/m] HSA. These
resul ts suggest that the absênce of auto10gous MLR in -the
presence of concentrations of NBS 10% and higher was Rot the
re~ult of the use of allogeneic sera as the media supplement.
It has been previous1y reported that NBS contains an
immunosuppressive substance(s) (212). We investigated ,the
possibility that AFP is the suppressive factor in NBS by
comparing the effects of addition to cultures of NBS and NBS
which had been selectively depleted of AFP by immunoadsorbent
c~romatOgraPhY. The results' in fig. 21 demoy;strated tflat
lYmphocyte proliferation in cultu~es supple~ented with NB~ which
had been depleted of AFP, was not significant1y different fram
cultures supplemented with adult sera. NBS that had been passed
over an inert S4B column was shown to retain most of its
suppressive activity, in9icating that manipulations of NBS ~uring
chromatography did not alter its suppresslve qualities. These
data indlcate that t~e la~k of growth support by NBS is 1arge1y
due to the presence of a suppresslve substance which can be
removed by anti-AFP immuno·adsorb.en,t'-chromatography.
l'
1· 1
j
l ,1 1 J.
\.
..
, '
75
Discussion
The purpose of the present experiments was to examine the
e ff e c t s 0 f hum a n, a l p h a - f e top rot e i non the a u t 0 log 0 usa n d
allogeneic mixed lymphocyte reactions'. The data demonstrate ,. that fetal and hepatoma derived AFP's exert consist'en,t1y strong
immunosuppressive, action on ~utolOgous reactions fn ,distinct
contrast to its range of effects on allogeneic MlRs. Dose
response experiments (Fig. 14 and 16) reveal that apP!",oximately
200 ug/ml AFP' yields maximal suppression of the autologous MLR.
Furthermore. as depicted in Fig. 14', 200 and 100 ug/ml AFP do
not differ sign~ficantly in the degree of suppression exerted
on autologous MLR. This suggests that maximal inhibition
Dccurs within this concentration range. In addition, 13 of 17
experiments show that 200 ug/ml af several different ~FPs
produced 80-90% inhibition of aut01ogous MLR (Table 5).
l' lf e l d et al. (11 2) r'e p 0 r t th a t lev e 1 s 0 f 1 - 20 n 9 / m l AFP
in normal adult serum do not exert any physiologically relevant
effects on autologous MLR. The;r conclusion was derived from ;-
the observation that 10-10,000 ng/m1 of pure AFP did not
S~gnificant1y decrease autologous,proliferation.:!J:!. vitro. We
are in general agr'eement with these authors that the 1-20 ng/ml
of AFP, in normal adult serum would not have immunosuppressive
activity since the minimà1 concentration of AFP \\Ihich can ~
exert a significant negative influence acc~rding to our reS'ults
"
" .' \ j1
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1
i ' r 1
t (
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76
is 5 ug/ml (Fig. 14 and 16). Thus, on the basis of our studies
i t i 5 e v ide n t t h a t -1 lf e 1 d e t al. (11 2) w e r e i n ve s t i 9 a tin g
suppression of autolôgous MLR by AFP at the lower limit of its
in vitro effects. It is unlikely, that AFP plays any significant
regulatory role in the control of autologous MLR in the adult.
Our investigation has been concerned wtth serum AFP levels in
the fetus and newborn whi.ch are 5-100 X 10 3 greater than normal o •
adult serum concentrations (30). The 1evel s 0/ AFP shown here
to suppress autoreactions are 10-20 fold lower than the
physiological levels in the fetus and newborn. ....
With respect to a1logeneic reactions, the action of AFP
ranged from 99% suppression of lymphocyte proliferation to no
effect, depending on the particular AFP preparation used and~
the culture conditions employed. For example, allogeneic
reactions conducted in media supp1emented with 10% human serum
were unaffected or weak1y inh{bited by 200 ug/ml AFP (Fig. 16
and Table 5). However, in the, absence of human serum \"', .
supplements. eight differen~ fefal a~d ~epatoma derived alpha-
fetoprotein preparations produced effects on the proliferation
of alloactivated lymphocytes ranging from 99% suppression to no
effect (Fig. 17). A wide range of AFP-induced effects on
allogeneic reactions has previously been described. Charpentie,r,
et al. (110f observed that 5 ng/ml to 2 mg/ml of a single , "1'
hepatoma-derived AFP had no signlficant immunosuppressive . .. ,activity on human allogeneic MLR. On the other hand, several
" '
. ' 1 •
1
.J
1 f
1 1
1
1
. '
..
77
r
studies show that strong suppression of a110reactivity can be
obtained with 200-600 ug/ml of fetal and hepatoma AFP's (44,45,
104'; Yachnin and Lester, in a detailed series of
investigations (70,73,8~,90,<1,l08) report that AFP's db exertQ
a range of suppressive effects on allogeneic MLR. The authors
found that when the action of several AFP's on a110reactivity
wa s co m par e d, u P t 0 a l 000 f 0.1 d d i f fer en c e i n i n h i b it 0 r y -
potency was observed. They concluded that human AFP prepara-
tions vary in their ability to suppress alloantigen-induced
lymphocyte transformation depending on their source. For
example, to obtain 50% inhibition of al10geneic MLR. greater
than 2.5 mg/ml of a hepatoma isolate was required. An
equivalent degree of suppression was obtained usihg 3 ug/ml of
a fetal AFP. The inhibitory potency can vary even within
hepatoma AFPs. This was shown by examining alpha-fetoproteins
isolated from the tumor tissue,. serum, and ascitic fluid from
a single patient with hepatocellular carcinoma (73).
The question as to why fétal and hepatoma AFPs differ in
their'inhibitory effect on allogeneic MLR was examined by
Yachnin and Lester (70,89). Employing the technique of
isoelectric focusing (IEF) they found that AFP's could consist
of up to 6 closely related charge variants. Fetal AFP's
contain a higher proportion of the most electronegative
subspecies. In contrast, hepatoma AFP's have a lower ratio of
this s~bspecies. The inhibitory potential of AFP on allogeneic
\ If.
~ oo>l
1 i 1 1 j
1 ! , • i , f
1
, i l
• 1
1
-, , ,
reactions ~orrelated ~ the proportion of a'particular .~;/
electronegative subspecies. On the basis of these findings w~
studied our fetal and hepatoma isolates using isoelectric
focusing. Our 1nvestigations revealed differences in the lEF
patterns of the AFP isolates. We found that the number of
subspecies could range from 3-6. Thus', the possibil ity exists /-
<.
78
that the variation of AFP-mediated inhibition of allogeneic MLR
which we have observed is partly due to'~he differences in
subspecies composition of our AFP isolates. However in
au t 0 log 0 u s r e a c t ion s ill AFP 1 s. reg a rd les s,. 0 f the i r s u b s p e cie s
composition, possessed the same degree of suppressiye action.
In addition tlh the source and microheterogenei ty of AFP,
there are other variables which may contribute to the varied
effects of alpha-fetoprotein on allogeneic reactions.
Purification conditions is one factQr that has been reported ta
alter the activity. of AFP on allogeneic MLR. Goeken and
Thompson (45) have observed that AFP eluted from immunoadsorbent
columns with O.5M NaCl abrogated proliferation of alloantigen -
activated lymphocytes, whereas preparations eluted with O. 15M
NaCl lacked significant inhibitory properties. Auer and Kress 1
(44) report that alpha-fetoprotein purified by using mild
. elution buffers, su~h as glycine-NaOH, suppress alloantigen
indlIced lym.phocy,te proliferation,. However, when harsh buffers l,
suc h a 5 s 0 d i u m t h i 0 c yan a t e are use d AFP i' sol a tes 1 a c k i n 9 a n y •
inhibitory activity are obtained. The authors conclude that
(
... •
chemical conditions could alter the AFP molecu,le rendering it 4}
immunologically in.active. In our studies we isolate~AFPls
using two purificaiion methods (fig. 1). ~hese inc1uded a·
physicochemica1 approach described by Gold' et al. (55) and an
79
" immunoadsorbent method based on the work of Ruos1ahti (f47)".
physicochemical techniques employed buffers of pH 4,.8 and
solutions with molarities ranging from'O.05-0.5M. The
The
'(,
immunoadsorbent procedure required only a brief exposure of
source material to O.lM glycine-Hel, pH 2.8. Since certain
,chemical conditions have been shown to affect AFP1s inhibitory .
potency on allog-eneic reactions, it lS also possible that our
purification procedures contributed to the varied effects of AFP
on alloreactivity which we observed. The consistently strong 1 "
suppressive influence of all AFP's tested on autologous MLR1s
suggests that the'act'ivity of alpha-fetoprotein on auto-
reactivity is independent of the influence of purificatien
conditions.
There, is a possibility that'AFP i"~ an immunologically
1 inert molecule, but purification methops render it
immunosuppressive.' The data in Fig. 2 ~ould tend to exclude
this hypothesis. The results demonstrate that newborn ~erum has )
an inhibitory influence on proliferation induced in a~àlogous
MLR. • v ~ ~ ,:-,
The selectlve removal of AFP from newborn serum abrogates
its inhibitory effect on a'utologous reactions. This suggests " .
that the AFP contained in fres-h, unmaniptJlate<l newborn 'serum has
- __ ~A ....... _ ~<
-
1 > f
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80
intrinsic suppressive pro~~rties.
Several o'ther observations concerning the purificat~ "'i
" techniques we used bear mentioning. Both methods yielded Afp , )
, " preparations \t,Q~;jch..1.ere' Jree of contaminating subHances a's
,/. . judged by APAGÉ (Fig. 10) 'and immu'noe'lectr'ophoresis (Fig, 9).
However, approximately two fold higher yields were obtained with .
t~e i~m~oa,.dsorben~. t~chnique ,~e,:~s;r~ t~~ ~.~Y,~~:co~hemi6:a1 isolatio method (Table 1). Secondly, the two-step immuno-
• 1 , ~
adsorbent purification approach required 2-3 days to complete, 1
as compared. to 10-15 days for the six-step physicochemical
method. Since immunoadsorbent purifi0ation requires bath a , "
shorter time peri,od and' fewer manipulations' of source material,
the chances of 1) bacterial contamination and 2) degradation of , . AFP by prote~lytic enzymes i~ reduced. The prevention of
contamination and degradation is of obvious paramount importance . ./ , - li
For instance, there is evidence that the immunosuppressive
effects of murine AfP are eliminated by treatment with
neuraminidase (109). \."" ThlS enzyme is found in body fluids and
can be produced by cer'tain bacteria wfr.ich may contaminate AFP
preparations. Moreover, bacteria1 products suc,h as lipopolYï
saccharlde and prote;n A of Stà'hylocaccus ?ureus campete with
the immunosuppressive a'ctivity·o} AFP by theïr capacity to l,
induca iigorous lymphocyte proliferation. ~
In ~ddition to the AFP source and purifica~ion methods,
we have a1so observed that culture conditions influence the
\
1 - ~ !
..
; " \ 1 , ,
·i
J
(
81
act-ion of AFP or al1o-activated, but not-auto-activated
lymphocytes. The results in Fig. 18 indicate that allogeneic
o MLR 1 sare suppressed, by AFP only 'in the a'bsence<..of 'h%uman serum'
!! u p P l ê m e n;t s . Mur 9 i ta e t al." ( l 04) r e p d'r t sim i l a r fin d i n 9 s . l) 'v
They observed that maximal AFP-mediated suppression of mitogen-
fnduced lymph~cyte transform~~ion occurred in serum-free medium. , ,
~oderate inhibition was C).btained in cultures supplemented with
human ser;um. No effect was noted in cultures containing fetal
calf serum. \.
In contrast, auto-logous MLRts conducted in the presence of " 'v-J
lO~ human serum 'are abrogated_by the addition of AFP (Fig. 16).
Moreover the concentration of serum in autologous ,cultures do es
n 0 t a p p e art 0 a f f e ct' AFP - me dia t e d i n h, i bit ion (F î g. 2 0 ) . .t;~ A s o ( ... or) ~
discussed above the negative influenc~ of newborn s~rum on
autologous MLR i~ eVerted primarily by AFP. Thus. the
suppressio~ ~f ~Ol090US reactions by newborn serum can be \.
considered to reflect AFP-mediated inhibition. Autoactivated
,lymphocytes cultured in media supplemented with 1-50% newborn
serum were .QUtrkedly suppressed as compared to cultures
containing adult serum.
An importaht question 1's \'Jhy serum present in allogeneic
MLRls interf-eres l'Ji th AFP suppression .. It is possible that a
factor(s) present in serum binds ta AFP and obstruct's its
immunosuppresslve activity. Human AFP's have been shown to bind ~
~ ,to several serum ~ubstances, including' fatty acids (80) and
;'
1
1 0
( ,
82
c,opper (81). Alternatively, AFP may bind and thus neutralize ./
AFP receptors on'target lymphocytes.
~tudies to date offer several possible explanations to , ,
reconcile the differential sensitivity of 'autol,o~ous and . .
a 11 0 9 e n e i c ML R t 0 AFP - m e dia t \d e f f e c t s . P e c k et a 1. Cl 0 7) r e p 0 r t
that murine AFP shows genetic restri,ction. They found that
murine MLR induced against l-region determ~nants are markedly
1
\
\ '\
i 1 1
,1
'il" suppressed by AFP, whe"reas T cell prol iferation activated 'by MHC
K and D alloantigens a~e refractory.to AFP-me~iat~d ~ffects.
-It is possible that human IIIa"-dependent T cell responses are
preferentially suppressed by AFP. S e ver a l r'e po r t s (1 34 , 1. 3 6 , 1 9 6 ) .t. ,
demonst~ate that humaIT IIIa ll antigens are primarily responsible
for stimulqtion of T cells in the autologous MLR. Our
experiments (Fig. 19) reveal that the murine alloanotiserum, A.TH
anti':A.Tl, whicK is cr'oss-reactive with human IIIall.determinants
(185), blocked 80% iff autolo9?,us MLR and only 30% 0f the J allogeneic response. Thus, the differential effect of Inti-la
a:tibodiêS may reflect a greater co'ntribution of the' 'la molecule f as the stimulating antigen in autologous .versus allogeneic MLR.
Alpha-fetoprotein has also been shown ta exhibit cellular 1
restriction .. In the murlne and human systems, AFP exerts
selective effects on certain T cell subsets (7,8,9,88,'104,106,
1 07 ) . Mur i ,n e AFP s u P pre s ses ce r t a i n th Y mus - d e pen den t i mm une
respanses (9). The primary target for "AFP-mediated immuna-
~uppression has been shown ta be a helper T cell of the Ly 1+2-
r"""""------'l..--------;;,.....----.--~-----------:'-~-,~-------
, \
. '
83
~
p h e n 0 t y'p ê (1., 7). " 1 n ,t h ~ )u man s y ~' t e m, M ttr 9 i F a e t al. (1 0 4) h a v e
shown tha t AFP uPP,~s-ses both ~i togen and a 11 oant i gen
st i mu lat e d T l ym p ho GY t e p·r 0 l if e rat i oOn , ' bu"t f a il s toi n hi bit the
mitogenic r\ponse 0: 8 cell-s to protein A'of ~taphy·lococcus
aureu'S bacteria. These, authors a1so noted a dissociation in the
sensit~vity of PHA- and allo-antigen induced lymphocyte
transformation to inhibitio~ by alpha-feto'protein. There is some c- .~
evidence that auto- and allo-reactive lymphocytes differ in the v
sense ~hat these cells may be generated from separate cell
-surface receptors for MHC-coded antigen~ on the cell surface of
'. target cells, and are not distinct cell types.
An1intriguing observation in our studies is that AFP can be
mitogenic for unstimu1ated T 1ymphocytes. When pu~ified resting
.T cells are cultured alone in t~e presence of 200 ug/ml AFP ,the
DNA syn(hesis of these cells is up to two times higher than that
"" of unstimulated ,T ~lS in the absence of AFP as shown in Fig.
14 a'nd 6 of 1'5 autologou"s reacti'on-s in Table 5. The f.act that
AFP can be mitogenic for resting T lymphocytes is interesting , ,
with respect td the Teports.tha~ alpha-fetop~oteln can induce
s u p p r'e s sor T c e Tl s .:!..D- vit r 0 ( 9 • 9 6 • 1 0 l ) . 1 n the, hum ans y ste m the
proliferative response of mitogen and allo-antigen activated . lymphocytes are'markedly suppressed by the addition to these
cultures of human lymphocytes which have been prei~cubated for
3-4 days in medium containing'AFP.
There)s general agreement that immune,recognition of
"
, : 1
. ,
1
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84
i:t
autologous cells is ~fundamèn~a~prereqUisite for cooperation
and communication between lymphocytes. (149,150). However, ,
abnorma1 autorecognition ean resu1t in destruction of host tissue ,
during autoimmune diseases (1504. Thus, mechanisms must normally
be"present to control self-reactive cells. lrainin et al. (197)
have oreported that autosensitization may be reguhted by a
'humoral mechanism. Their studies revealed that autbcy~otoxic
responses were suppressed by the additio~ of thymie humoral
factor. It is tempti~g to speculate that the suppression of • autoreactiv'Ï'):y by a humoral substance such as"alpha-fetoprotein
represents a biolo.gic de-fense mechanism through which the fetus
is Pl"bvided with .effective protection against harmful self-
reactivity.
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1,
References
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• 7. il u r 9 i ta, R. A ., a n ct T. B. Tom as i . 1 975 . Su p pre s s ion 0 f the J i mm une r e s po n s e b y a 1 ph a - f e top rot e in. 1 . T h,e ,e ff e c t 0 f mou s e
alph~-fetoprotein on the primary and secondary antibody response. J. Exp. r~ed. 141: 269.
8; "1 u r 9 i ta", R " A ., and T. B. Ta mas i . 1 9 7 5 . Su p pre s s ion 0 f the immune response by al pha-fetoprotein. II. The effect of' mouse alpha-fetoprotein on mixed lymphocyte reactiviry and
,mitagen induced lymphocyte transformation. J. Exp. r1ed. ,.!.il: 440. ,)
9. Mur 9 i ta, R. A ., and \~ i 9 z e 1 ,1. 1 979 , Se) e ct ive i mrlU n J reg u 1 a t 0 r y • properties of alpha,-fetopro.tein. Rie. cl 11'1. Lab. 9: 327. '
" 1 O. G it 1 in, D., and ~~. Boe 5 m a ,n . 1 9 6 6 . 5 \ r u mal p h a - .f e top rot e in, a 1 ~ u m 1 n~, and 9 am ma - G - 9 lob u l, i n in. the hum a n con cep tus . J . C lin. f n v est. 4 5: 1 82 6',
- . 11. Ruos1ahti, E., and +1, Seppala. 1979. Alpha-fetoprotein in
oance( and fetal de~elopment. 1
Adv. Cane. Res. 29: 275.
1 2'. B'u r net, M. 1 mm u.n i t y" .
1959, "The clonaI selection theory of acquired Vanderbilt University Press, Nashvil1e, Tennessee.
\
•
·1
1 1
l .
r .... ',
, '
86
'. ..
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Parmely, M.H., and J.S. Thompson. 1976. Eff~ct of alphafetoprotein and other serum factors derived from'hepatoma-, bearing rats on the mixed lj'mphocyte response. J., Immunol. ll1.: 18.;32.
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(
"
. .
• , ; 1
\ î j
t i
96 1
120. Thor by, E., Bondevik, H., and S.G. Solheim. 1973. The in bitory effect of HL-A antibodies on lymphocyte tr nsformation in vitro. Transplant. Proc . .§.: 343.
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)
1 1
) 1 t 1.
i
components of the major histocompatibility comp1ex in the ~ mouse. J. Exp. r~ed. 136: 1430.
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l, .
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-- . . 97
, _../
r
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1
'.
i j
" l
1 i t .~
t
143. "Hfyasaka, N~ Talal. mixed lymphocyte react,on in 28: 77A. ('
98
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- ,}
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dl \
154.
'~\
Gordon, R.D., ,Simpson, -E., and L.E. 'Samelson. 1975. In vitro ce1l-mediated immune respO'n.e to the male specifTë" (H-V) antigen in mice. 'J.' Exp. Med. 142: 1108.
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• 1
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Q
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\
, ,
1
J .. < •
,"
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l '
1 , i
1 , 1
J i .!;
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l ~ ,1 i
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(
175. ,
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i ~ ~ 1 { i
~' 1 )
,'\
,1 l
1
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'02
!
186. Ruos1ahti, E. 1976. Antigen-antibody precipitates and immunoadsorbents. Application ta purification of a1pha-fetopro..tein. Scand. J. Immunol. suppl. 3: 39.
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194.
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..
f f
,\ 1
1 ~ ,
\ ,\ ! J 1
103
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Table ,\
, Yields of AFP obtained by the physicochemical versus
immunoadsorbent purification m~thocfs'.
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Table l
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. Starting Material Sample Method of
PurificatlOn AFP(mg/ml) Total AFP(mg)' 0
.r/7
'"' physicochemical 0.01 30
2 physicochemical O. l 64 3 physicochemical 0.78 '15.8 4 immunoadsorbent 0.05 20
\. 5 immunoadsorbent 0.05 9.4 6 immunoadsorbent 0.043 ' .... 12.9
~
1Iit_rtw~~"!îiJIÔ~"""'"':o.~~.o..1.,. _r """"h<-I - ~--- ~ .~ ... ~- 4'
----
Purified AFP Preparation
AFP(mg/ml) Total AFP (mg) "
0.4 3.2
2.0 6.0
1.0 3.1 2.~ 8.0
1.0 3.2 2.0 7.6
"
-:
-.;c
Yleld(%),
11
9
19
40
34
59
.
o ~
_._-~..-....
(
./
Table 2.
E f f e ct 0 f AFP 0 n' ce 11 . v i ab i 1 i ty . Tri pli ca te we 11 S con sis tin 9
of 2 X 10 5 lymphocytes in O.2ml RPMI-1640 medium containing
200ug/ml ~FP, human serum albumin, human gamma globulin, or 1
media \'Iere incubated for 6 days. Ce11 viability Ivas, the.n
determined by trypan blue dye exclusion.
,
0 "-
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Cens incubated
in medium
containing: a
AFP-l AFP-2 , AFP-3
AFP-4
AFP-5 , IX'FP- 6 .
albumin human gamma
globulin
J
1
Table 2
Cell concentration
b'
(cells/ml)
0.39 X 10-6
0.34 'X 106
0.30 X 106
0.28 X 106
0.24 X 106
0.24 X,l06
0.56 X 106
0.44 X 106
0.50 X 106
• i Y
." o
1
105 ,
%'.
viability c
43 0
43
48 /
45
50
46
49
41 ~
50
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Table 3.
A c 0 m par i SeO n 0 f the r e s p 0 n d i n 9 a, n d ,s t i m u lat i n 9 pot e n t i a lof
unfractionated cells, and T and non-T cel1 populations orepared
by E-rosette formation with SRBC (denoted,by subscript ros.)'
an~ceTl affinity fractionation on human immunoglobulin (19)-· . rabbit anti-human Ig coated glass bead columns (denotèd by
subscript coL). Responder cells (1 X 10~) were cultured with
2 X 10 5, irradiated (2,500rads),autologous stimulator cells, in
RPMI-1640 medium· containing 10% autologous se,rum.
puls~d with 3H- TdR 6 hours before termination on
Cultures were
day 7. Results , i
repre~ent the me an counts per minute ± s.e.m. of triplicate ,a
cultures. Stimulatlon indices (S.I.) represent the r.atio of the
mean cpm in stimulated cultur~s over the mean cpm in unstimulated
cultures.
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Res-pand i ng lymphocyte
population
PBL T col. T ras.
?
8,,308 ± 907 562 ±. 275
:- + 232 - 87
Re Me Mc Nt: %.u._:r téS; *.. 1*' t ebkCiM --~~~ ........ ....i::.IGt.~-!"<'4~ ~
o
PSL • x
(>
650 ± 64 '
11 ,685 ± ~2, 1 02
l ,859 ± 509
1,739 ± 534
-;:
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Table 3
Stimulating Cell Population 3H-thymidine incor~oration (cpm ±
S-. l . non-T ras. x S. 1 .
1.3
1.3 Q
1.5
2.0
284 ± 87
67,317 ± ~,633
52,632 ± 4,893
35,768 ± 4,078
,;
7.8
62
69
A
,
s. e.m.)
non.Jco1 .x
139 ± 45 1,9,642 ± 4..116
15,592 ± 5,300
11 ,689 ± 6,815
-----.
'"'
~
--"'- ~II:! ... --, .,~,~ .. ""-.,,.-" ~ ,. ... ~""'-
~ ,
~
S. I.
2.3
22 32
,
..... C> 0\
~~. --
. ~
"(
(
,"
Table 4. '
Surface characteristics and mitogen reactivity of E-rosette
fractionated cell populations. Techniques used to determine
surface characteristics are described "in the Materials and
Methods section. For mitogen induced 3H- TdR incorporation
studies,5 X 104 cells in o.lml were mixed with O.lml RPMI-
1640 media containing 20% autologous sera with and without PHA
a t 4 u 9 / ml. 3 H - T d R . in cor po ra t i on i n tri pl ,i ~ a t e we 11 5 \'la s
assessed' after 3 days in culture. Results are expressed as the
mean cpm ± s.e.m. for seven expe·riments.
aAverage of the mean for 7 (seven) experiments
L.,
{
• j
!
1
" . ~
107
r .. 1 •
~ , r
Table 4 1 .. ,1
j
1 J
l
j 1
Cell % % PHAa ,
1 population s19+ cells Urosette cell s 2ug/ml
1 , . ,
PBL 12 68 4.679 ± 710 176.803 ± 4.137 :. T 0 84 5,449 ± 787 151,071 ± 3,569
non-T 43 6 1,250 ± 99 24 ~ 152 ± l ,570 '
"'
(
,"
(
, '
,/
fable 5.
Summary of the suppressive effects of eight different AFP
lsolates on the aut~logous and allogeneic MLR: Enriched T and
non-T cell populations were isolated as descrt"bed in the' text., ,
Responder T ce1ls (1 X 10 5) and irradiated (2,500,rads)
autologous or al~oge~eic stimulator non-T ce1ls (1-2 X 10 5 )
were incubated in 0.2m1s RPMI-1640 medium supplemented with 10%
'serum autologous' to the responding cell population. Purified
hepatoma (hep) and fetal (fet) derived AFP was added at the
initiation of cultures to obtain a final culture concentration "
of 200ug/ml. Cultures were pulsed with 1.OuCi 3H"-TdR six hours
before termination. The results représen..t~-the mean counts per
minute ± the standard error of the mean of triplicat~
determinations. SignifiC~ce of dlfferences ~etween mean
values of control responses and cultures containlng AFP uSlng
the Student's t test are indlcatep.
\
1· !
"
\ , ,
~ 1
1
\.
~
'\
Table 5
T cells A1102eneic MLR AFP :t Source -AFP +AFP -AFP +AFP SUP.
hep. 1 ,21~ ± 219 1,743 ± 7,639 75,478 ± 7,639 61,72a ± 7,024 18 " 5,487 ± 1,647 2,412 ± 409 38,177 :! 2',480 23,970 ± 1,190 35
6,832 ± 2,423 3,693 ± 1 ,168 703 ± 76 919 ± 383 728 ± 98
hep. 6,970 ± 2,395 2,D39 ± 336 40,869 ± 3,305 47,934 ± 9,719 0 4,112 ± 2,382 1,052 ± 390 53,235 ± 3,699 49,876 ± 9,216 2 6,832 ± 2,423 7,982 ± 4,656
\ 703 ± 76 1,718 ± 339 728 ± 98
hep. 1,031 ± .61 '2,842 ± 647 40,039 ± 4,314 25,181 :t 745 40" 728 ± 98 -/ fet. 10,480 :!: 1,410 15,954 ± 2,678 51,988 ± 4,366 48,447 ± 1,512 22
4,643 ± 613 12,601 ± 2,797 81.306 ± 4,833 89,966 ± 5,096 0 350 :! 87 8,249 ± 2,534
fet. 1,191 ± 443 1,699 ± 506
= fet. 325 ± 81 153 ± 56
fet. 207 ± 74 405 ± 262
fet. 728 ± 98
.&
-" ---)
W?&*;u~ .,.Ui~j "&" ........... \ ~ .... 1+._~ ...... ~
~ 6
p -AFP
n.s. 46,6B ± 7,474 p<O.OSO 26,306 ± 2,969
61,504 ± 8,851 18,716 ± 2,825 28,815 ± 3,942
n.s. 28,123 ± 4,895 n.s. 50,437 :t 10,283
67,253:t 13,465 20,171 ± 2,904 37,395 ± 5,313
23,719 ± 1,030 33,604 ± 5,141
n.s. n. s.
136,1~6 ± 10,098
46,603 ± 1,910
23,532 :! 1,841
20,917 ± 1.248
33,604 ± ?,141
~
Autologous MlR
+AFJ>
1,158 ± 430 4,260 ± 2,969 9,295 ± 2,656 3,913 ± 200 , 6,235 ± 1,317
12,331 -± 2,020 4,lS8 ± 1,076
19,955 :t 3,161 3,318 ± 1,347 5,327 ± 2,895
11,486 ± 5,350 12,894 :!: 1,695
41,953 :t 6,041
%
. -,-
SUP. P Q
99 pcO.OOS -85 1'<0,005 90 p<O.005 83 p<D.Ol 81 p<O.005
54 p<O.05 92 pcO.005 80 pco.os 92 pc().OOS 86 peO.OOS
66 p<.0.050 61 p<.0.050
75 p<0.005
3,187 ± 1,314 - 96 1'<0.005
1 ,684 ± 821 93 p<O.OOS •
2.886 ± 1.041 88 p<0.OO5 \
2,109 ± 1,296 93 p<O.005
J
o C>
" go
----
t)
i
. ,..
(
('
f
Table 6.
Proliferative response of 'allo-antigen activated lymohocytes
cultured in various concentration of serum autologous to the
responding cell population or purified human serum albumin.
Cultures 'c~ntaining 10 5 responding lymphocytes and 2 X 10 5 .,
irradiated (2,500rads) allogeneic lymphocytes \vere incubated for
6 days in RPMI-1640 medium supplkmented with autologous serum of
purified human serum album;.)l ln the final concentrations
indicated. 3H- TdR incorporation was measured during the f~nal
6 hours of incubation. Results represent the mean of trt~icate ...
cultures '± t'rie standard error of the mean. The stimulation
index (S.!.) is the ratio of the mean cpm of stimulated'cultures
(allog,eneic mix) over unstimulated responder cells a.LOne.
'/
, , (
, ' i
(
l'
Cgncent~ation of Supplement
Autologous Sera(%) \
J \ 20
10
5
l
0.5
Human Serum Al bumi J1,.. (mg/ml)
8
4
2
l
0.5
0.25
e·
rabl e 6
3H-thymidine incorporatlOn ± S.E.M. a
Responder cells a10ne
323 ± 86
2,445 ± 785
2,643 ± 78
4,457 ± 2.264
4,228 ± 815'
1 ;381 ± 377
778 ± 176
1 ,283 ± 672
. 605 ± 57
832 ± 170
634 ± 19
287 ± 42
\
All ogenei c mix
1,457 ± 370
113,179 ± 27,215
106,439 ± 13,094
100,285 ± i5,542
50,679 ± 9,525
69,227 ± 10.331
10,164 ± 3,238
10,787 ± 3,407
12,652 ± 1,603
5,923 ± 2,189
5,674 ± 1,700
3,037 ± 1,642
> •
..
S. I.
4.5
46
40
22
12
50
13
8.4
~'1
7.2
9
11
- 109
r ,
. ) .
1
1 1
.;
j
(
(,
·,
."..
Table 7.
Effect of autologous. allogeneic, and heterologous sera on the
proliferative response of auto'activated T lymphocytes. QCultures
containing 10 5 responding T cells and 10 5 irradiat'ed (2,500
rads) autologous non-T cells were indubated for 7,days in RPMI-
164~ medium supplemented with'lO% sera derived from various
sources as indicated. Source .of allogeneic sera is denoted by
the initials of the donor in parentheses. 3H- TdR incorporatidn
was measured during the final 6 hours of incubation. Results
represent the mean of trlpl icate cultures ± the standard error , '
of the mean. The stimulation index (5.1.) is the ratio of the . , . mean cpm of stimulated cultures (AMLR) over unstimulated
responder T cells alon~.
f
110 . 1
Table 7
/
3H- TdR incor~oration ± S.E.M. , i <.
i Source of serum Responder T ce11s AMLR
a10ne
auto 1 ogous serum 3,830 ± 777 22,832 ± 9584
a 11 ogenei c serum (RK) 6,573 ± 3653 14",115 ± 4200 .
a110geneic serum (JZ) \ 4,942 ± 1479 50,825 ± 18,501
(RV) ,
a110geneic serum 1,365 ± 214 1 24,171 l 5850
a110geneic serum (MS) 1 ,141 ± 37 28,515 ± 4356
allogeneic se~m (PM) 610 ± 34 14,410 ± 96]
a110geneic serum (GO) 3,606 ± 1455 27,200 ± 6542 , a110geneic serum (AB) 6,763 ± 1128 21,098 ± 2754
feta1 calf serum 70,064 ± 7374 126,283 ± 6954
norma 1 mouse serum ( .j
1,309±155 918 ± 116
human serum a1bumln 1 ,555 ± 34 1,695 -± 283
RPMI-1640 medium 679 ± 78 697 ± 96
-
/
c , ..........
)0),
Figure
Starting material: a) human cord serum b) ascitic fluid of
pati~nts suffering fram primary hepatocarcinomas
~ 1 ~ ~-
A PHYSICOCHEMICAL PURIFICATION
,) Ion exchange chromatography (DEAE-SephQdex A-50)
a) Bulk IEC
b) Column IEC
t ii) Moleçular-sieve chromatography
(Sephadex G-200)
t iii) Sepharose Blue Dextran Affini~y
Chromatogniphy
f iv) Concanavalin-A ~ffinity
Chromatography
l v) Molecular-Sleve chromatography
(Sephadex G-200)
;"rl"'i"f;Fni 'Î"! ~*>Mrmortm"i:: cl.... __ .. u ...... - ' .... ~
~ IMMUNOCHEMICALJPURIFICATION
i) rabbit anti-human AFP Sepharose 4B immuno
adsorbent chromatography
t· ii) rabbit anti-normal human
serum Seph-arose 4B immunaadsorbent chromatogr~
,
.... 11'
AFP content assayed by rocket immunoelectrophoreisis
Purity assessment of AFP preparations:
- i) immunoelectrophoreis;s ii) analytical PAGE
iii) SOS-PAGE iv) Gel Ouchterloney
v) Isoelectric focusing
,/
-,-, ............ ~-< ..... .,.,- .. ,--
/
" .. ..
\
(
Figure 2.
1on-exchange chromatography pf crude ascitic fluid on DEAE-
Sephadex A-50. Ten ml fractions were collected with optical
density monitored at 280nm. The void peak (elute:d between
fractions 1-50) consisted of a,lbumin, transferrin, and normal
serum components, with no detectable AFP. The AFP-containing \.
eff2uent peak was eluted after the appllcation,of the linear . gradtent, at co'nducta,nce values betv/een 7 and 20mmhos. Detail.s
1 •
of el ut ion are 9 ive n. in the tex t .
, ,
" 1
(
(04WW ) À11l\LiJnONOJ ( .... )
2.;:-______ Q:r:------7ï1g
, .
ct l{)
.c... 0
(0-0) wuà8G lO 3JN\18~OS8\1
z o ru
0 4 100::
lL.
r 112 1
1 ., i
, , 'r.,)
( 1
l
.... Il , , ~-
. • l'
Figure 3.
./
Molecular-sieve column chromatography on Sephadex G-200 of the
-eluted void volumn from IEC chromatography. The AFP containing'
fractio~s detected by rocket immunoelectrophoreists were
conl:ained ;n fractions 55-'75. Details of elution are given in ,
the text. c
,.,
\. , ,
.. , .
o
(
f
r ,
i !
\ , 1 1
1 1
, , , '"
{ 1 \
l
--'.
~ ; (lWj 5n ) d.:J'V ( .... ) lO 0 lO
l ,..... 10 '"
"
1 1 1
(!) -m. ~ . ,
~ 0 0
(0-0) WU082 ~D 3JNtr8~OS8'V
(
. . ..." ~,", .. " ~ .. ~ ~
113 '
0 m
er. w CD ~
~~ 0 Z (D
0 r-()
« er. I.L
01
, '
1 , : i
j 1 ,
\
1
, !
1 '---}--
{ ,
1-- , , 1
, '1 1
~
i 1
(
- -,..----.
Figure 4 . ...
, Affinity chromatography of the AFP-containing material from
( . G-200 chromatography on Sepharose Blue Dextran. AFP is not
, . . bound by SBD and is cODtained in the void volume (fractions
5-35). Alpumin is retained by the SBO column and is .eluted
with 6M urea. Details of elution are given in the ~ext.
"
1 f
r
. "
1
114
,
li
0, C\J
- --~ -", --,1 !
'""'-~
0:: 0 c::( Q) w w lI) 0::: ~ ::>
:::J ~ Z CD
0 Z <.D 0 ~ U <!
0 0:: r<> LL
t !
LO '0 . . - -< 1
wU082 ~o 38N\t8tjOS8\t
\ ( \
(
•
Figure - -,,-
\ "', 5 .
Affinity chromatography on Con A-Sepharo~e of the po61ed AFP
containing fractions from SBO affinity chromatography. Unbound
albumi~ was eluted with starting buffer and AFP retained on t~e
column was eluted with alpha methyl O-glucoside. See text for
deta,i l s.
\
~
r ,
r 1
1
1
j j
l
1 1
\ ..
l-i
115
\ 1
0 <1",
,.~
n. ..... 0:: <t .. lJJ al
.....
Q) ~ ." ::>'
- fi >-0 "z ~(J ) 0 Cl) :J E"6J C\Jz lrô ,0 -f-
t) cd: a:: u...
\
an . o
(
wUOB2 ~ 3~N~8~OS8~
\
\ . 1
}
.-
0'
"
Figure 6. /
Gel filtration oi' purified AFP on S~phadexJJ-2007 'AFP'
'"
..
concentrations were determined by a rocket immunoelectropho~eisis
technique. Details of elution are given in tHe text .
) r
.".. l
\
..
'/ .
1
(1~/6w) d.:rv ( .... ) Ln •
lQ C\J o d 0 ~--------~~------------ . lf.)
0 V-
1
6 ro
0 (\J
J-"' .... . . Ln • o
o •
(0-0) wU082 . ~o . 3JNV8èiOS8V
116
0::: W CD -~ ( =:>. z
z ""v 0 -t-U <!: 0::: LL
.;
" ,v
"-- ""'-' ~"
\
-
f r l
, J 1 1
I 1 ,
l ~ .{
.1
(
... ' 1
. •.
Figure 7.
E1ution pattern of cord serum on an a~ti-AFP Sepharose 48
immunoadsorbent co1umn. The effluent peaks, e1ut-ed 'wit'h D.1M
~C1-g1ycine (pH 2.8) (fractions 185-210) and 4.0M guanidine .. . (fractions 241-257) consisted of AFP and a sma11 amount of ,
notimal serum components. T-he large void peak (fractions ,10-97)
did not contain AFP detectable Iby rocket immunoe1ectro/phoreisis /
which has a sensitivity o·f lug/m1 (Norgaard-Pedersen and Gaede,
l 9 75) .
o
, 1 i ,
\ , i , ,
).
î
1
/
117
l ' Q)
0 c: -0 ,10 Oc
N 0 ::J i Cl
1 :?!~
v 0 c:: . V
W 0
al '-
:E 0 C\J ::::>
Z Q) c: 0 - ~~ C\ 0 Z 1
0 0 0 l - -1-
U <l: 0::: l.L
0 1.0
---O---___ -.J '. --o~--~---o--------o--~-----o------~·o
• • If •
(~ ~ N -
,wu 08Z ~D 38N'18~OS8V
Figure 8.
Removal of remaining normal serum components from AFP
preparations by affinity chromatography on an anti-whole j ,.''' ......
normal human serum IgG Sepharose-4B immunoadsof6ent column.
AFP was contained in fractions e1uted witb the starting buffer.
Normal human serum compo'nents are retained by the immuno-
adsorbent and may be subsequently eluted with 4M guanidine.
1
.. '
1
\
\ . j
! ! 1 j
(
0 co
....
0 U')
~ "
c
); :s "2 0 0 =' 01 t\I
:e-4 -' . 0'
<i
, ' ~ __________ ~ __________ ~o
v. ~ o 0
WU08Z '0 3~N\18~OS8V
(
118
Cl: 0
IJJ CIl ~ ::J z Z 0 -~ « a: l1..
:'
1
i..
4 j
1 1 i !
\ 1 !
, 1 ~
1
~ j j
-------
1
~
-----------~---------~- -,
,-
1 Î
1
..
Figure 9.
Demonstration of the purity of AFP by 'immunoelectrophore1sis
performed as described in the Materials and Methods section.
1
1 1
1 1 1
, ;
i 1
• ,j
1 "
'c
119 1
\ <
\
l J
1
,f
)\ i j ,)
1 •
i I-
l J
) )
, ' 1 1
!
\
/ \ '
n '
(,
'J
(
1
1
Figure 10.
Demonstr:ation of the purity,of AFP by alka1ine PAGE using 7.5%
polyacrylamide separat~ng gels performed as describ~d in the
Materia1s and Me.tho~section. (A) Pu'rified human serum
a1bumin. (B) Feta1 derived AFP purified by antibody-ag~rose
immunoadsorb~~t chromatography.
-"
J ,
. '"
C:,o.
L ___ . __ ._,~_
,0
120
" ' • v
1 1 ,
-, ,
j 1 ,) ')
~
v ! u
A J ~ ~
( "
1 :a
J
[' ,
---
-)
o
Figure 11. u Gel Ouchterloney analysis of purified AFP. AFP was electro-
. pho,r es ed on duyl icate polyacrylamide gel s .. One gel \'Jas stained
vJith Coomassie Blue (see Fig. 10), and the second gel o ~ ~,
immobilized in 1% agar. Monospecific anti-AFP or anti-albumin
antiserums were added ta traughs eut parallel to the po1yaeryl-
amide gel'. Control wells contained· cord serum or purified human
serum albumin as indicated.
(
'\.,
,
1
:--P
l
1 1 î' ;
121
1 '1
, 1 '.
(
:'
Figure 12. ..
S D S - PA G E a n à lys i s 0 f pur i fie d f et a l der ive d AF P (A), and
purlfied human serum albumin (B). Details provided in the
tex t.
"
1
---
..
\-
,
j. (
(
122
o
! .
-1 i 1
1
f
(
("
Fig u re l 3 . )
Microheterogeneitj of AFP ~s djsplayed by isoelectric focusing
, i n pol Y a cry l am {de gel' con ta; -n ; n 9 2 % a m p h 0 l Y tes, p H r a n 9 e 4 - 6 . 5
and 8M urea". Iso-electric focusing was performed at 40 C for
5.0 hours with a final pbwer of 500V and 1.5mA'. The Coomassie
blue-stained gel displayed in the ~hotographic inset is
positioned to indicate the position of the AFP subspecies
rel atiye to the pH gradient (e) across the gel. and the
spectrophotometric scan of the gel at 480nm (solid 1 ine). The
anodal end of the gel is at the left. The isoelectric points
(pr's),of the subspecies are indicated.
i ' ,
\ t t i A l
J 1 L
~~----------------------~----~----~~----~---- -~- ~~-- ----, -- ---..,.
(
(-)WU0817 tD -a:lUOqJOSq'if
v o
N o
o o
o tD
0 III
0 <t
0 If)
0 I\J
O~--------O±---------~Q--------~O ~ ~ ~ 0
<t
123 l 1
! \
E E -Q)
"0 0 c:::: 0
E 0 .... -Q)
u c:::: 0 --ln
.0
\
"
,Figure 14. -' Kinetics of T cel' proliferation in response ta irradiated
autologous non-T cells cultured in ~arious concentrations of
purified human AFP. lbS T cells were cocultured with 105
irradiated (2,500 rads) autologous non-T ce11s in ·a.2'rn1 RPMI-
1 6 4 a m e d i u Il] con ta; n i n 9 5 % a u t 0 log 0 U s. s e ra. Hum a n AFP i sol a t e d
from cord serum was added at the initiation of autologous
cultures in the fo1lowing concentrati.(Ûns: ., 200ug/ml; ('
0, lOOug/ml; e, 50ug/m1; 0, 25ug/ml ... , \Oug/ml; 6, 5ug/m1;,
). 1ug/m', , no AFP added. Dashed 1ines repr.esent T ce11s
alone incubated in the presence and ab-sence of AFP. Six hours
prior ta harvest,ing, cul tures were pulsed wlth 3H- TdR and,
assayed dal1y. Each point represents the mean ± s.e.m. of
tri pli c a te cul tu r e·s. T h Y m 1 d i n e i n cor po rat ion b y cul tu r e s
containing lOOug/ml of purified human se'rum albumin was 120,580
± 14,158 at 144 haurs.
1 i
il i 1
~ \' , 1
l
\ i , 1
t j
1
1 1
---124
..J'
"",. ~ .
...
b '" ~IOO J -~
\ i 7 ': .~
0:: !50 '0 t-
1
:I: If)
2~
. .::. ::::::t: ::.:::::::~~ .-_~~ ~ :: .. _._~ ~ ~:~:::.:::: .. ; 96 120 144 168 192 216
HOURS OF CUL TURE
(
)
r " , '
"
Figl!lre 15.
Demon~tration of t~consistént ~uppressive effects of eight
different hepatoma and feta1 derived AFP's on autoactivated T
jy m ph 0 C y tes . Exp e r i men t ale d'n ct i t ion sas. des cri b e ct i n Fig ure
16. Human AFP isolated from feta1 and hepatoma sources was
added at the initiation of autologous cultures in a final
con c e n t rat ion 0 f 1 0 0 u 9 / ml. . Re sul t s r e pre sen t the m e a n ± s. e . m .
" of triplicate cultures of one experiment.
"
) r
f 1 \ J
1 /
.... \
1
Cl
Protein addition
albumin
AFP-I AFP-3
_AFP-4 AFP-6 AFP-7 AFP-8 AFP-9 AFP-IO
'1>
Source
human -serum
hepatoma
hepatoma hepotoma
fefal hepotoma
fetal fetal hepatomo
i1ii: PFsTAIZ ret •• rte, 'tne,rdfflt;a's" ~,...:ld .............. ~ ___ .... ,_~_ .. ~~ ............. > ~
:-....--- ..
"Il
~~
, .. "
AUTOLOGOUS MIXED LYMPHOCYTE REACTION i
1 • '"
o 10 20 30 40 . 3 - ~ H-TdR . incorporation (cpm x, 10 )
~
,..
i 1 ,-
1 1
1 1
.~ ,
,
1-'
"'" (J1
,,;
">-
- ___ • ~ __ ~h __ ' ____ ' ___ --- -- - ---- .. ----. __ __
'" ~
( \
\.
, \
Figure 16.
Corn par a t ive e ff e c t s 0 f hum a n AFP 0 n T cel 1 pro 1 if e'r a t ion i~ ~e d
by autologous'versus allogeneic non-T cells. ~-enriched and
non-T ce11 populations,were iso1ated from normal adult peripheral
"" blwo d by E-rosette formation as described in the Materials and
ri eth a d s sec t ion . Var i a u s con c e n t rat ion s a f pur if i e d hep a t a m a -
der ive d AFP 0 r pur if i e d hum ans e r u mal b u m ; n \~ e r e ad d e d ton j
cultures containing 10 5 T lymphocytes with 2 X 10 5 autologous or
allogenelc 'irr~ted (2,500 rads) non-T cel'ls in RPMI-1640 1
medium sUPP" f\!llented with 10% fresh sera autologous ta the T cell
donor. Allageneic and autologous MLR's were harvested after 144 ,
and 168 hours respectively, following a 6 hour pulse with luei
of 3H- TdR . Results are expressed as the percentage of the
control response, calculated as described in the Materials and
Methads se'ction. The data are p,resented as the average of the J
1
mean proliferati've responses of T cells obtalned from three
indiviu,dals .stlmulated in paralloel autologous or allogeneic . ,
r~ LR ' s . Ho riz 0 nt a l l i. n e s ln die a t eth e ra n 9 ~ 0 f st and a rd e r r 0 r s ,
and rel e v a rrt p val u es of st a t i s t l cal 1 y sig nif l c a n t d i f fer e n ces
{ ~ 0 < 0 . 0 5 0) b e t 1-' e e n exp e r i men t a l and con t r 0 1 r e s po n ses are
shown.
,
125
(
1
1
, i. t
(
(
1
• 1
) '"
i '\
! 1
'-
Figure 17.
Suppressive effects of seven fiffere~t purified AFP's derived ,
from.different sources on~the ~ vitro lym~hocyte'response to
allogeneic cells in al~umin supplemented cultures. Allogene{c
~.1lR'S consisted of 1 X l05\tdult PBl'S resp,on~ing against 2 X , . 10 5 irradiated (2,500 rads) allogeneic PBl'S incubatèd in RPMI;
1640 m~dium containing 2mg/ml human ser~m albumin. AFP iso1ates
'purified by immunoadsorbent chromatography (AFP':1,5,6) and
p hy sic 0 c h e,m i cal met h 0 d s (A F P - 2 , 3", 4 , 7 y,..~ s des cri b e d a b 0 v e w e r e
added'at the initiation of cultures in a final concentration of
200ug/ml. 3H- TdR incorpo~ation was measured during the final 6 , hours of incubation ,on day 6. The data are pres~nted as the
mean proliferative response of three different responder-
stimulator allogeneic MLR combinations. Horizont~l lines ,
indicate the range of standard errors.
, ,
or - --- .~
} 1 , 1
1 -1
i , , , ,
. '
--
Proteln Final concentratfon addition Source ln culture
.: AFP-I hepotoma 200 ug/ml
AFP-2 hepatoma 200 ug/ml
AFP-3 hepotoma 200 ug/mi
AFP-4 hepatomo 200 ug/ml AFP-5 hepatoma 200 uglml
AFP-6 fetal 200ug/ml
AFP-7 hepatoma 200 uglml
albumin human sera 200 ug/ml
J
~'1o...'IlI:&' 1# II b.f:IéJI~w*, ikimt,",thrkr~mC4 .,~_~~"'._""'~
~
.. .
(j
~}i- -~--
3H-tbymidine incorporation in allogeneic MLR (epmlt 163)
9 Ip 2P 39 4;.0
...
~,_~-_.."u~ ......... ~,,-_-.;;' .... _ • ..q... • ..--.""' ... -;;. ... -"' ........ - ...... _.<: ........... _ ....... --~ .............. ,~-"" ~-- ...... ----
.......
1-' 1\)
-..J
-~- ----'-----
~
(
!
\
\ \
\ \
Figure 18.
AFP mediated ~uppre$$ion df allogeneic MLR's in albumin and whole
serum supplemented cultures. 10 5 responding lymphocytes wHh 2 X
10 5 irradiated allogeieic stimulating lymphocytes were cultured
for 144 hours in media containing 10% human sera autologous to the
responding cells or 2mgjml purified human serum albumin. Human
AFP isolated from hepatoma ascitic fluid was added at the
fnitiation of cultures f.or a final concentration of 200ug/ml.
Thymid~ne incorporation was assessed during the final 6 hours of
culture. Results' repre,sent the mean ± s.e.m. of three individual
experiments. ,1
J l
.~ 1
MLR type
~
allogeneic
ollogeneic
~'''"'
Culture media ~ supplement
J
~ ,
;:;
2 mg/ml albumin
10°/0 human sero
)-...... "'nIi>#Q t 1'.... 'D"1 .... -~~~ ... -.I ... < ............... ~>- .............
;.
, ~"
\ \
Protein addition
no addition
AFP (200ug/ml)
albumin (200ug/mJ)
no addition
AFP {200ug/ml} - ,
olbumin (200ug/mt)
~
\
1#
À,
ALLOGENEIC MLXED LYMPHOCYTE REACTION r i
o
'---,
20 ,3H- TdR
"
----,
40
incorporation (cpm X-103
)
",
,/
-~~ ~-- ......... - ........ __ ~ __ -1"" _ '''''~''''' ~ .. _M___ ~ __ ......... ~ ~ ........... ""- ............... _ .... _ .... , '-
60
4
1-' N ID
, .
Figure 19.
Blocking effects of anti-Ia and anti-T cell reag~nts'on , autologous versus allogeneic MLR. Cultures c6ntaining 10
5 T
cells and 2 X 10 5 irradiated autologous or allogenei~ non-T cells
separated from the peripheral blood of, healthy adult donors as . described in the Materials and Methods section were incubated in
O.2ml of RPMI medium su~~lemented with 10% fresh autoldgous sera. ,.
Normal mouse serum, A.THaA.TL mouse a110antiserum (Cedarland
Laboratories, Hornby, Ontario)~ a murine monoclonal 19G antihuman , ..
la preparation (New England Nuclear, Boston, Mass.), and a murine
monoclonal IgG antihuman T lymph~cyte antiserum (NEN) were
added at the in.itiation 0ra r,a 11 el allogeneic (v) or
in the various concentrations indicated. - ,
autologous
3H-thymi-
dine uptake \>Jas measured at 168 hourso The percent bTocking
represents the ratio of th~fference between th~ mean cpm of
trlplicate st~mulated and Jnstimu1ated cultures in the presence
of blocking reagents over 'the difference between the,mean cpm of
triplicate stlmulated and unstimulated cultures in the bb~en~e of
blocking reagents. The ~tanda:d error of the means were in all
cases less than 12% of the mean.
.. .
1 1
1
1
1 f 1
---..--~~------------.J .,-.
r--.
'+500 100 100 1100
+3251- \ /' 751- \ 75t- '\. 75
r- Dl \ III c/\\ / \l' .. c
t3 +250, 50t 1 \~ 5O~ li 50 o m . l '1 / \\ î \\ . J
~ + l25l \ \ 2sl d \ 2J ~ 25f \, L
, 1 -
--1-- -;- -, - ,1 l", 1 1 1 1 1 ! Il 2 1 0.5 0 2 1 0.5 0 5 2.51.2 0 5 25 1.2 0
% serum ~ ~'jI. serum ~ ug/ml IgG i uglml IgG \ ~
Normal mouse ATH èi A.TL a la a Lyf! \0
serum ~
---------,--.. =---.... "'.~.~*--.. -.. -.~.-,._· ....... ~4_.~ ..... ~ ...,. ......... ...- ~~~-"'----- ... ~ .... ,-...... ~ .. --- ~~ .... ~~ ......... .- ~ ~ -.;> ............. _ .. ""'~l~~~ ..... "'"'~ __ ~_~....,.~~ __ .-. __ .. _._._<L.~._ .. __ ----- -----.,.------
" , , (
l"
l l -
1
Figure 20.
Suppression of At~-LR by newborn serum, T enriched an.~non-~ cell
populations were prepared by the fractionation of peripheral l
blood from a single adult donor usinq a SRBc' rosetting technique. j',
Cultures containing J0 5 T cells (.) or 10 5 T lymphocytes with 10 5
.
. irradiated autologous non-T cell s (iJ) were ill1cubated for 6 days
ln RPMI-1640 medium supplemented with fresh newborn serum or
fresh autologous adult serum in final concentrations ranging from
l t 0 5 0 % • 3 H' - T d R i n ~ 0 r po rat ion w a s me a sur e d dur i n 9 fil e -f i n a l 6
hours of incubation. Results repre-sent th~ me an of triplicate
cul tures ± the standard error of the mean.
"
-rra )C
E Du -c: o +-Cl ~
o Cl. ~ o~-
30
20
g la
0:: -0 l-
I
l r<>
~ o
NBSAS
1
"'
NBS AS
5
. -_.Plia,· g Vi tt., ... ·~&""" ... .. ,..'-' .... ~"'n_ ............. "_ -'"- _ ~ ,.'.
NBS AS
la NBS AS
20
'<
NBS AS 30
% serum supplement '\.
~ ~.-,.~'"----._---_~~ ,.. ... ~.l' -
~
NBS= fresh newborn serum
AS = QutorogouS adult serum
NBS AS
40 NBSAS
5'0
-- ___ ~ __ k ~ ............. -...--'-- __ _
~ u CJ
•
-_.--
r
Figure 21.
\.
Selective dep1etion of ~FP from newborn serum removes most of its
. suppressive activity on AMLR. T lymphocytes from hea1thy human
adu1t perip,heral blood were cultured with irradiated (2,500 rads)
autologous non-T cell preparations in RPMI-1640 medium
supp1emented \.",th ,(a) 20% newborn sera (NBS), (b) 20% newborn
sera passed over an inert Sepharose-4B column (NBS-S4B), (c) 20% . ~
newborn sera se1ective1y depleted of AFp by passage over a rabbit-
anti-human AFP-Sepharose 4~ column (NBS-AFP), or (d) 20% auto
logou's adult sera (AS). Serum fractions "Jer-e concentrated to
thelr original volumes by ultrailtratlon (Minlcon-B15 concen
trator, exclusion limit = 1.5,000 ~1W, Amlcon CO'rp., Oakvllle,
Ontario). '/ The AFP concentration in newborn serum a~ a~sayed by
focket immunoelectrophoreisls was determined to be BOug/ml in
N3S and NBS-S4B, \."ith no detectable AFP (.>1- to 5ugjml) in either
NBS-AFP or AS. The proliferative response at day 7 culture was
measured as 3H-thymidine incorpora~ion and the data expressed as \ \
the arithm~tic mean cpm of triplicate cultures plus or minus one 7
s tan d a rd e r r 0 r .
f 1
\
1 1 Î
-~o )(
E C-o -Z 0 -~ «
/ ~ a. ~ u ~
W Z ë5 ~ >-J: ~
1 J: ro
"
. •
30
20
10
'r
, p " , . ~ - .. _t"',"f"".l'r'ltJl"'t~f ""tl~ ,') < \.>t.-l ~" ""'tI.-... ~ ... ~~, ,-.... 1 .... ..,..,..,.,...,..... .... ~' ..... • ~"r..\~. -...."""~ .. ...,~"'"" ....... _ ... " .....
/
Q
.':'lc9Tcells O-I05Tcells +, 2xI05
irradioted autologous non-T cells
(
131
\ !
1 { 1