accelerated immunological maturation in the chick

Immunology, 1966, 10, 63.

Accelerated Immunological Maturation in the Chick

R. A. MCBRIDE,* L. W. COPPLESON, N. W. NISBET,t M. SIMONSEN,ANNA SKOWRON-CENDRZAK AND H. L. R. WIGZELL§

The McIndoe Memorial Research Unit, East Grinstead, Sussex

(Received 14th June 1965)

Summary. A re-investigation has been made of the earlier claim by Simonsen(1957) that grafted immunologically competent cells (ICC) may proliferate in-definitely as evidenced by unlimited transferability of the graft-host splenomegalyin chick embryos. The observation was repeated, but the original interpretationproved untenable.A method of chimacra analysis was developed, based on neutralization of ICC

with specific serum antibodies. Cells and antisera were mixed in vitro immediatelybefore i.v. injection into the embryo and the neutralization assessed quantitativelyby the reduction of spleen enlargement obtained in comparison with cells plusnormal serum.

It was found that the 17-day embryo recipient of ICC from an adult bird under-goes an accelerated immunological maturation which in 7 days confers upon itsblood and spleen cells a higher degree of immunological competence, per unitnumber of cells, than in relevant controls. The first host is thus enabled to cause aGVH-reaction in a new 17-day embryo which, in turn, matures precociously.Hence a cycle is created which permits a serial transfer of splenomegaly for as longas the new host is antigenic to the previous host. The phenomenon was notrepeatable with hosts of constant genotype.

Accelerated maturation is not a mere antigenic stimulation of the young animal.So far it has only been produced by live cells, and only in conditions where the hostis antigenic to the graft.

INTRODUCTION

However commonplace is now the fact that immunologically competent cells (ICC)which are grafted to a foreign host produce GVH-reactions under appropriate conditions,the fate of the grafted cells remains as intriguing a problem as ever. It is of obviousimportance for transplantation biology, but also one of ramifications in general immuno-logy, pertaining to almost any question of the population dynamics of immunologicallycompetent cells.

Simonsen (1957) claimed that splenomegaly caused by GVH-reactions in 17-day chickembryos could be passaged indefinitely, thus implying an inexhaustible prolification ofICC from the original donor. In contrast, later investigators have failed to propagate

* Present address: Department of Surgery, Mount Sinai Hospital, New York, U.S.A.t Present address: The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire.Present address: Department of Experimental Zoology, Polish Academy of Sciences, Kopernika 7, Krakow,

Poland.§ Present address: Department of Tumour Biology, Karolinska Institutet, Stockholm 60, Sweden.

63C IMMUN

GVH-reactions beyond a very few transfer generations, if at all. (In chicks: Burnet andBoyer, 1960; Papermaster, Bradley, Watson and Good, 1962. In mice: Gorer and Boyse,1959; Dineen, 1961.)Some experiments in mice have shown that the grafted cells may lose reactivity to their

first host though retaining reactivity to a third party representing a new antigen (Simon-sen, 1960; van Bekkum, 1963), which facts are best interpreted as acquisition of specifictolerance on the part of the grafted ICC. There is good evidence that grafted ICC mayhave a very different fate from one strain combination to another (Fox, 1962; Fox andHoward, 1963).The motivation for the present work was mainly that the one system, weekly transfer in

17-day-old outbred chick embryos, where grafted cells seemed to react indefinitely, hadnever been analysed adequately. It began as a limited exercise: to reproduce Simonsen'searly findings, which proved easy. But, attempts at explaining the facts grew considerablyin complexity. They involved the use of chromosomal markers as well as the developmentof a technique for chimaera analysis based on neutralization of the activity of transferredICC with specific serum antibodies.We arrived eventually at the conclusion that GVH-reactions which take place in chicks

around the time ofhatching cause, as a side effect, a maturation of the immune response ofthe host which clearly exceeds what is normal for that age, and which enables its lympho-cytes to launch a GVH-reaction on transfer to new embryonic hosts.

MATERIAL AND METHODSChickens

Most of the embryos used were outbreds, derived from crossing outbred stocks ofRhodeIsland Red and Light Sussex. They were supplied by the Appleby Farm Ltd, Ashford,Kent, as 10-day-old embryos and incubated further in our laboratory in a Secura 1400egg incubator at 37.5°. These RIR x LS hybrids will be referred to as 'O' (outbreds).A sex-linked colour gene makes it easy to sex these birds after hatching.Much more limited supplies of eggs and laying stock came from the highly inbred I and

W strains ofWhite Leghorns maintained by Dr D. G. Gilmour of the School ofAgriculture,University of Cambridge. Laying stock of these strains were kept by us for production of I,W and (I x W) Fi eggs.Towards the end of this work birds and eggs became available by courtesy of Dr M.

McDermid, Thornber Brothers Ltd, which were homozygous for the B locus though nototherwise highly inbred. These 'blood-grouped eggs' belonged to the groups B2, B14, B19and B21. In all four groups the splenomegaly obtained by injection of adult blood from thesame group was very inferior to that obtained by inter-group injections, thus confirmingthe dominant role of the B locus antigens for GVH-reactions (Payne and Jaffe, 1961;Jaffe and McDermid, 1962).

Adult birds used as donors and for production of antisera were of the types already men-tioned, except that a few adult donors were of the (less homogeneous) C line of WhiteLeghorn, or (I x C)F1.

Blood samplingAdult birds were bled from the wing vein, or, for larger volumes (10-30 ml), from the

jugular vein. Clotting was prevented with 3 8 per cent disodium citrate (one part to four

64 R. A. McBride et al.

Accelerated Immunological Maturation in the Chick 65parts of blood). Newly hatched chicks were bled from the jugular vein and yielded from1 *5 to 2 ml. The jugular vein was also used for bleeding at 1 day before hatching, in whichcase the shell was broken over the air-sac and the head pulled out.

Serial transfer of splenomegalyThe technique of i.v. injection of eggs was largely as described by Beveridge and Burnet

(1946). Sterile tuberculin syringes and 30 gauge needles were used.Unless modifications are stated in the text, a serial transfer experiment was conducted

as follows:Whole blood (0-1 ml) from an adult donor was injected i.v. into a batch (usually eight)

of 17-day-old embryos (4 days before normal hatching). Seven days later (3 days afterhatching) the chicks were bled out and killed, and body and spleen weights recorded.Blood for transfer was selected from the chicks with the biggest spleens. It was usuallypooled from two to four chicks, and 0-2 ml injected i.v. into each of a new batch of 17-dayembryos which then, in turn, served as donors 7 days later. For each transfer a batch ofnon-injected chicks was included as negative controls. In short, transfer was made withblood, in groups of 17-day embryos, every seventh day.Preparation of white cell suspensionsfrom blood and spleen

For the analysis with neutralizing antibodies it was necessary to prepare suspensions oflive cells from blood and/or spleen.One volume of citrated blood was mixed ini test tubes with 2 volumes of 1-5 per cent

(w/w) poly-vinylpyrrolidone (PVP) and left at room temperature at an angle of about 600to accelerate the sedimentation of erythrocytes. The supernatant was then centrifugedfor 5 minutes at 2000 rev/min and the sediment resuspended in pH 7-3 phosphate-bufferedsaline. Cell concentration was adjusted as needed on the basis of counts made in a haemo-cytometer after dilution with Trypan Blue (1 mg/ml in isotonic saline). Stained cells wereat most a few per cent. The admixture of erythrocytes varied from about 50 to 90 per centbut did not seem to interfere appreciably with the neutralization of lymphocytes withantibodies, probably because the latter were used in excess.

For the preparation of spleen cell suspensions great care was exercised to avoid asuspension which, perhaps because of dead cells, would kill a majority of the embryosimmediately following injection.The spleens were placed in small mesh sieves, wetted with 25 per cent normal plasma

solution in citrated phosphate-buffered saline, cut with scissors, and pushed through themesh with a round-bottom glass rod under simultaneous addition of extra plasma solution.Cell clumps in the suspension were dispersed to some extent by shaking and were allowedto settle for 5 minutes. The supernatant was pipetted off, and its cellular content washedthree times in plasma containing buffer before the final sediment was resuspended andcounted as described for white blood cells.

In making suspensions from enlarged spleens care was taken to dissect away as many aspossible of the characteristic whitish nodules (which mainly consist of necrotic cells).Enlarged spleens tended on the whole to give more 'toxic' preparations than normalspleens.Preparation ofantisera

Adult birds were immunized by injection of 2-5 ml of whole blood into the pectoralmuscles. No firmly standardized schedule was used, but bleedings were done 3-7 days

after three or more injections which were always spaced at least 1 week apart. In thebeginning sera were collected, but it was soon found that plasma functioned just as well(which was a great advantage in view of the often very slow and incomplete clot retractionof fowl blood).

Technique of antibody neutralizationCell suspensions from blood or spleen of putative chimaeras were analysed by mixing

with equal volume of undiluted antiserum immediately before i.v. injection into testembryos. The latter were 13- or 14-day-old 'O' which were killed 5 days later for weighingofbody and spleen. The origin of active cells was then revealed by the degree of neutraliza-tion given by anti-donor and anti-host antisera as compared to positive controls injectedwith cells plus normal serum. Non-injected negative controls were always included.

In preliminary experiments (not included in the results) we pre-incubated cells andantisera at 370 for 1 hour and added fresh normal chicken serum as a source of extracomplement, but this was found to be unnecessary. In spite of complete inactivation ofpre-incubated cells, Trypan Blue uptake was erratic and usually below 20 per cent. Cellspre-incubated with specific antibodies also showed no leuko-agglutination, whereas thecontaminating red cells formed firm agglutinates. The consideration that mere trappingof donor type ICC in agglutinates of erythrocytes might lead to erroneous neutralizationresults was one of the reasons why pre-incubation was abandoned.

Inactivation of sera by heating at 560 for 30 minutes did not appreciably reduce theiractivity. Ifcomplement is needed for neutralization, as it may well be, it must be provided,at least in part, by the test embryo.

Antisera which were capable of neutralizing the activity of adult 'O' cells did not ontheir own cause changes in the spleen weight of the test embryos.

Technique of chromosomal marker analysisThe same as used by Nisbet and Simonsen (1965).

RESULTS

SERIAL TRANSFER OF SPLENOMEGALY

A total of twenty-eight experiments were made in which transfer of the splenomegalycaused by an initial injection of adult blood (usually 0 1 ml undiluted whole blood) wasperformed. Most of these were designed to investigate specific points which did not requiremore than one or a few passages. In four experiments, however, splenomegaly was passagedthrough ten or more transfer generations.The results are summarized in Table 1, and the longest experiment (ST 28) is also

presented in Fig. 1. In all four experiments the recipients were 'O' embryos, deliberatelychosen on the assumption that each batch of new hosts would present the transferred ICCwith at least one new antigen, regardless of whether the active cells were derived from theoriginal adult donor or from previous hosts. It was in similar conditions that the pheno-menon was originally demonstrated (Simonsen, 1957) and has here been reproduced.A very important control in each experiment is the transfer line labelled NB, started with

normal blood drawn from 3-day-old 'O' chicks, which is the host-type blood with whichthe original donor cells would inevitably be contaminated in the transfer procedure.



.-2 0-

-C:

1 4-c01-! 186

0m 1620. U. K. f

1'I-U- I I I I I I I II _rII I r I I I I I I I I0 5 10 15 20 25 30 35 38

Week of passage

FIG. 1. Serial transfer in 17-day embryos (ST 28). 0, Transfer line initiated with adult IxW blood;*, transfer line initiated with normal 3-day-old 'O' (host type) blood; -- -, negative controls, unin-jected. All transfers made by blood injected i.v. every 7 days. Thus, the spleen weight shown for time 0is the result of the original injection 7 days before, while the result of the first passage is registered attime 1.

TABLE 1

WEEKLY TRANSFER OF SPLENOMEGALY IN 17-DAY OUTBRED ('O') EMBRYOS (SUMMARY OF FOUR SEPARATE EXPERIMENTS)

GroupExperiment Original No. of mean spleen t-test of difference between group means

No. donor transfers weight*(mg) Groups compared t Probability (P)

ST 11 Adult I 10 48 Adult I (NB) 6-73 <0.001Adult C 10 45 Adult C (NB) 7-34 <0-001Adult W 8 29 Adult W (NB) 4-10 <0-001Adult 'O' 10 50 Adult 'O' (NB) 8-79 <0-0013-day 'O' (NB) 10 19 (NB) (NC) 0-00 1-0None (NC) (10) 19

ST 19 Adult I 9 32 Adult I (NB) 4-06 <0-001Adult IxC 13 64 Adult IxC (NB) 14-59 <0-001Adult W 9 31 Adult W (NB) 3-36 <0.013-day 'O' (NB) 13 23 (NB) (NC) 0-56 06 > P> 05None (NC) (13) 24

ST 24 Adult IxW 13 57 Adult IxW (NB) 7-45 <0-0013-day 'O' (NB) 12 23 (NB) (NC) 0-29 0-4 > P > 0 3None (NC) (6) 22

ST 28 Adult IxW 38 60 Adult IxW (NB) 15-64 <0-0013-day 'O' (NB) 38 25 (NB) (NC) 1-87 0-1 > P> 0 05None (NC) (31) 24

* Usually four to eight of the injected embryos survived to the third day post-hatching when the next transfer wasmade. The group means listed are geometric means of all passages of the respective transfer series or of the uninjectedcontrols (NC).

The fact that the spleen weights obtained in the NB lines closely duplicated those ofuninjected negative control groups (NC) or were, at most, insignificantly increased,disproves the possibility that splenomegaly in the main transfer-lines is due to spontaneousimmunological maturation of host cells carried over from previous passages.On the strength of this evidence we first concluded that the original concept most likely

was correct, i.e. that continued proliferation of the initial graft of adult donor cells wasresponsible for the seemingly unlimited propagation of splenomegaly.

It was next decided to study the transfer phenomenon in hosts of constant genotype(i.e. as constant as is available in chickens).

67

Five independent transfer experiments were made with 17-day-old eggs of either the Ior W strains of White Leghorn. Adult donors were either the opposite strain, or outbredbirds. In each experiment splenomegaly was obtained in the original host but disappearedin the first or second passage. If present at all in the first passage hosts, spleen enlargementwas only marginal. These findings were strikingly different from the results in Table 1 andwere first interpreted as evidence of tolerance induction in the graft. However, the trueexplanation is probably different, as we shall see later.

CHROMOSOMAL MARKER ANALYSIS

When we had satisfied ourselves of the reproducibility of the serial transfer of spleno-megaly and, for the reason given above, assumed it to be due to a simple propagation ofICC from the original donor, it was decided to use the chromosomal marker technique asan independent check on that assumption.The obvious plan was to do all transfers, in any given series, with cells from chicks of the

opposite sex to that of the original donor. So, if the latter was a cock, female recipientswere used throughout as donors for the subsequent passage.The usefulness of this method requires that an appreciable part of the proliferating

cells in the enlarging spleen are derived from the donor whether the latter is the originaladult donor or one of the preceding intermediate hosts. As we shall see this requirementwas far from being generally met.

1. Adult donor cells in initial hostsThis was the only situation where high numbers of donor cells were found as a general

rule (though not without exceptions).Twenty-four female 'O' chick spleens were analysed 7 days after they had received (on

the seventeenth day of incubation) 0 1 ml of citrated whole blood from the same adultI xW cockerel. In each spleen 50-100 metaphases were examined. Two spleens failedto show any donor cell mitosis and another four showed 10 per cent or less, but themajority (fourteen) showed 50-90 per cent of dividing cells to be of donor origin. Theaverage percentage of donor cells for all twenty-four spleens was 48 per cent ± 32 (SD).Almost identical results were found in another series, differing from the first one only by

reversal of the donor-host sexes. Female donor cells were diagnosed in all but one out ofthirteen male spleens. The mean contribution of donor cells was 45 per cent ± 36 (SD).

2. Donor cells in subsequent transfersIt soon became clear that dividing cells from the original donor were extremely rare, if

present at all, in host spleens beyond the original injection. This finding was in fact thefirst to cast severe doubt on the hypothesis of continued reaction on passage of the originalgraft cells.

If, alternatively, the marked splenomegaly so persistently found on serial transfer was tobe caused by host cells of previous passages, this fact might also be expected to be revealedby chromosomal markers.

In order to test, in the same transfer line, both for the presence of cells from the originaldonor, and for cells derived from previous hosts, the following basic design was used



(all transfers were done with whole blood or with concentrated leucocyte preparations;all hosts were killed as usual 7 days later):

(a) &adult IxW - $-'O'-- U'O',(b) dT adult IxW + ? O' U'O.

In (a) any male mitosis detected in the last host must be derived from the original donor,whereas in (b) any female mitosis must stem from the intermediate host. In either casethe final hosts are injected identically with a common pool of cells from the intermediatefemale hosts.

Variations of this design consisted in reversing the sex sequence, or in carrying thepassage of splenomegaly through several (up to eight) intermediate hosts of the same sex,or, finally, in starting with blood from 3-day-old 'O' chicks instead of adult blood. Theresults are summarized in Table 2.

TABLE 2IDENTIFICATION BY MEANS OF SEX CHROMOSOMES OF DIVIDING CELLS DERIVED FROM ORIGINAL DONOR AND FROM

INTERMEDIATE HOSTS

Examined spleens Total ofGroup Original donor examined Percentage metaphases of

Origin No. Mean weight metaphases* indicated origin(mg)

I Adult IxW Original 37 90 2500 47 5, original donorhosts

Ia 3-day-old 'O' Original 13 29 625 1 1, original donorhosts

II Adult I xW Subsequent 12 70 773 0.0, original donorhosts

IIa 3-day-old 'O' Subsequent 6 23 366 0 3, original donorhosts

III Adult IxW Subsequent 13 72 975 1-3, hosts ofearlierhosts passage(s)

IlIa 3-day-old 'O' Subsequent 2 26 150 0 0, hosts of earlierhosts passage(s)

* A minimum of fifty metaphases were analysed in almost all of the examined spleens.

The difference between group I and Ia seems easily understandable: the markedsplenomegaly given by adult donor cells but not by 3-day-old donors correlates well withthe contribution of dividing donor cells to the spleen of the primary host.Group II demonstrates the failure to detect a single dividing cell from the original donor

among almost 800 metaphases from twelve enlarged spleens which were all sampled fromthe first to the third transfer generation, after the original inoculation.Group III shows 1-2 per cent of mitoses derived from intermediate hosts, which were of

course all 3-day-old RIR x LS chicks by the time they were killed and served as donors forthe next transfer. This figure is strikingly close to the one found in Group Ia where it isnot, however, associated with splenomegaly. It is obviously no match for the almost 50per cent found in Group I.

It must therefore be concluded that the chromosome analysis has been a poor guide inthe search for the origin of the ICC responsible for the successful passage of splenomegalyin this system. The shortcomings are no doubt rooted in the fact that the host cell multipli-cation which (by an unknown mechanism) is triggered off by the immune reaction of the

69

grafted cells can bring about as much splenomegaly as can a GVH-reaction dominated bydonor cell multiplication. This fact is also apparent from a poor correlation in Group Ibetween splenomegaly and percentage of dividing cells of donor origin: r = 0 189 whichis insignificantly different from 0.Very pertinent in this context is the additional finding that the percentage dividing

donor cells in 17-day 'O' embryos injected with the same adult I x W donor droppedvery markedly (from 94 to 4) over a twenty-fold dose range which had virtually no effecton the degree of splenomegaly. On this background it becomes quite possible that thesmall fraction of cells from intermediate outbred hosts found in Group III (Table 2)reflect the fact that these initiated the spleen enlargement. But the evidence that this is somust surely be provided by other means.

NEUTRALIZING ANTIBODY ANALYSIS

The fact that specific serum antibodies can neutralize the capacity of ICC to produceGVH-reactions was first demonstrated by Siskind and Thomas (1959) in studies on runtdisease in mice. Not unexpectedly, we have found the same to be true in chicks. As thereexists a linear regression of log spleen weight on log dose of grafted cells it is possible toobtain a quantitative estimate of the amount of neutralization obtained. The mean slopeof the regression was 0 43±0 03 in 13-14-day 'O' embryos killed 5 days after injection,which was the test system used for antibody neutralization.

1. The specificity and potency of the antiseraFor analysis of the serial transfer of splenomegaly, ideally two antisera should be avail-

able: one completely specific for cells of the original donor, and another equally specificfor cells derived from either previous hosts or from the very same host from which the cellsto be analysed are harvested.

This ideal could not be realized completely in our continuous long-term transferexperiments (Table 1), as one could predict from a mere look at the common design forST 24 and ST 28 (the two best analysed experiments):

IxW --*'O' - 'O' --'O.*.... '0'Although it is possible to produce an anti-'O' serum by immunizing adult I xW birds

with a pool of 'O' cells there is no guarantee that this will react with any 'O' cell. On theother hand, it should at least fail to cross-react with other I xW cells. Conversely, an 'O'anti-I xW serum (as well as I anti-W, or W anti-I) should react with any I xW cell, butthen there is no guarantee against cross-reactions with 'O' birds other than the antibodyproducer itself.

In spite of these odds, the antisera proved sufficiently specific to yield valid information.Table 3 summarizes the results of all specificity tests done with the antisera which wereemployed in the analysis of serial transfer experiments to be described.

Groups 1 and 4 indicate that three different types of antisera possess the supposedreactivity to I x W cells but do, on the average, also show a marginally significant cross-reaction with 'O' cells. However, detailed examination of the data summarized in Group4 shows that they are obviously inhomogeneous: two 'O' birds cross-react strongly, onemoderately, and the others insignificantly, if at all.


Accelerated Immunological Maturation in the ChickTABLE 3

SUMMARY OF SPECIFICITY TESTS ON EMPLOYED ANTISERA; CELLS+SERUM MIXED BEFORE INJECTION INTO 'O' EMBRYOS

Mean log spleen weightNo after injection of Mean log Neutralization:

Group Antisera Target exper.- No. target cells plus: spleen weight Mean ofcls ments eggs uninjected (NS-AS) -4-SEAntiserum Normal serum controls(AS) (NS)

1 'O' anti-I x W IxW 28 304 1b20 2 00 1-04 0 80+0 03I anti-W (= 16 mg) (= 100 mg) (= 11 mg)W anti-I

2 IxW IxW 16 325 1*91 1-92 1-03 0X01±0-04Anti-'O' (=81 mg) (=83 mg) (= I1mg)

3 IxW '0' 8 99 1-38 2*10 1 02 0 72±0 08Anti-'O' (=24 mg) (= 126 mg) (= 11mg)

4 'O' anti-IxW '0' 12 117 1-61 1-75 1-00 0d14+0-08I anti-W (= 41 mg) (= 56 mg) (= 10 mg)W anti-I

The figures in parentheses are the antilog of the mean log spleen weights.

Groups 2 and 3 show that I xW anti-'O' does not cross-react with other I xW cells butdoes react with eight different 'O' cells tested. One of the latter cell preparations, how-ever, showed a neutralization (NS-AS) of only 0-27. If that value is disregarded Group 3shows a neutralization of 0-82 + 0 05, strikingly close to the very uniform data ofGroup 1.

It thus seems that all the antisera, when tested with the appropriate target cells, reducethe log spleen weight by approximately 0-80. This reduction is remarkably constant overa target cell dose range from 2 x 105 to 4 x 106 white blood cells per egg. The reduction inlog spleen weight from mere dilution of the inoculated cell dose in saline or normal serumwas as mentioned 0*43 ± 0 03 (SE) per ten-fold dilution (determined in separate experi-ments comprising sixty-five eggs in each of three groups covering a twenty-five-fold doserange). Hence the antibody neutralization corresponds to 0.80/0.43, or approximately twoconsecutive ten-fold dilutions. In other words, about 1 per cent of the activity of the ICCremains, 99 per cent having been destroyed by the antisera.

2. Analysis of the transferred blood cellsIt will be recalled that the serial transfer experiments summarized in Table 1 were done

with pooled blood drawn from the preceding group of hosts when they were killed 7 daysafter injection. For the purpose of antibody neutralization tests, a sample of the blood wastreated with PVP as described in 'Material and Methods', and the white cells recoveredwere then tested in 13-14 day 'O' eggs after mixing equal volumes of normal serum orantisera.The results are given in Table 4. They are unambiguous with respect to the neutraliza-

tion caused by anti-host serum (I xW anti-'O'). In every case the latter has eitherneutralized completely (as compared with uninjected controls) or, if a slight residualactivity remains, the degree ofneutralization corresponds to about 99 per cent ofneutraliza-tion (as described above for neutralization of known target cells with the appropriateantisera). As we know from Table 3 this antiserum does not cross-react with the originalI xW donor.The results with anti-donor serum ('O' anti-I x W) are as expected less clean. In most

cases they do not neutralize; in two cases (ST 24 transfers 1 and 8) they do so significantly.

C*

71

R. A. McBride et al.TABLE 4

I X W OU' 0' tO .... '0'. ANALYSIS BY ANTIBODY NEUTRALIZATION OF WHITE BLOOD CELLS (WBC) FROMVARIOUS STAGES OF SERIAL TRANSFER EXPERIMENTS IN 'O' CHICKS FROM THE ORIGINAL HOST TO THAT OF THE TWENTY-

FIFTH TRANSFEROriginal donor adult I x W. All bloods drawn 3 days after hatching (7 days after previous transfer).

Mean log spleen weights* from Neutralization bySerial injection of WBC+sera Mean log

transfer Transfer spleen weightexperiment No. Normal Anti-donor Anti-host uninjected Anti-host Anti-donorserum serum serum controls (NS-AH) ± SE (NS-AD) ± SE

(NS) (AD) (AH)

ST 28 Original 1-97 1-84 1-22 1-08 0 75 0413inJection

ST 28 1 1-81 1*86 1-06 104 0-75 0.05ST 28 3 1-57 1-46 1-11 1-13 0*46 011ST28 15 1*51 145 1*22 1-22 029 006ST 28 25 2-28 2-15 1-22 1-09 106 013ST 24 1 1-59 124 113 113 0 46 0 35ST 24 8 1-90 154 1-19 1 02 0-71 0 36ST 24 12 158 1-56 1.09 1 04 0 49 0*02

Mean 178 1-64 1-16 1-09 0 62±0 08 014±0 05Mean (mg) 60 44 15 13

* Each log spleen weight represents the mean of four to six spleens.

However, the virtually complete neutralization given in the same experiments by anti-host serum strongly suggests that the effect of anti-donor serum is simply due to itsincomplete specificity. The over-all amount of cross-reaction is then the same as knownalready from Table 3, Group 4.

3. Analysis of the original hostThe first experiment of Table 4 suggests that even blood drawn from the original host

at the seventh day after transplantation is active almost completely by virtue of its hostcells. This seemed very surprising to us, not least since the chromosome data showedalmost 50 per cent, on the average, of dividing donor cells in the spleen.

TABLE 5'O' (ANTI-I X W) -- I x W. ANALYSIS BY ANTIBODY NEUTRALIZATION OF WHITE BLOOD CELLS FROM 3-DAY I XW CHICKS

(7 DAYS AFTER INJECTION)Donor: adult 'O' pre-immunized with I xW cells.

Mean log spleen weight* from Neutralization b'O' donors 106 injection of WBC+sera Mean log Y

Experiment (leg-banded 10 spleen weightNo. blue and tWtBd Normal Anti-donor Anti-host uninjected Anti-host Anti-donor

yellow) serum serum serum controls (NS-AH) (NS-AD)(NS) (AD) (AH)

I Blue 4 1-86 1-86 1-41 1-02 0 45 0 002 Blue 1 1-40 1-22 1-06 1-06 0 34 0-183 Yellow 4 1-98 1-88 1415 1-02 0-83 04104 Yellow 1 1-58 1-68 1-25 107 0 33 04105 Yellow 1 1-78 1-70 1-05 1-06 0 73 0-08

Mean ±SE 1-72 1-67 1418 1-05 0-54±0410 0 05±0 05Mean (mg) 52 47 15 11

* Each log spleen weight represents the mean of four to six spleens.

72


It was decided, therefore, to investigate white blood cells from first hosts under con-ditions where both anti-host and anti-donor sera were known not to cross-react. With thematerial available at the time this could only be done by reversing the donor-host com-bination and injecting I x W eggs with blood from the very same 'O' bird which suppliedthe anti-I xW serum.

It will be seen from Table 5 that five experiments done with two pre-immunized 'O'donors supported the impression gained from ST 28: that the active cells are virtually allhost cells. Although the neutralization by anti-host serum in two experiments (1 and 4) isnot quite as marked as to be expected on a 'host-cell alone' hypothesis, there is certainly noindication that the residual activity is due to graft cells.

In four of these experiments spleen cell suspensions were analysed simultaneously inthe same way as white blood cells. In two cases there seemed to be partial neutralizationby both anti-host and anti-donor sera, but the data are incomplete because of highmortality in the test eggs after spleen cell injection.

REQUIREMENTS FOR ACCELERATED MATURATION

A good number of variations in the experimental design were tried out during theinvestigations, sometimes on a wrong working hypothesis. Some were designed, and otherscan serve as a counter-check on the hypothesis of accelerated maturation and, at the sametime, help to define the conditions in which it does, or does not occur.

2-2-

2*0O

-C

0)

1-2

1 23Week of passage

FIG. 2. Serial transfer in 13-day embryos. Technique otherwise as in Fig. 1 (ST 12+ 15). Weighted meansof two identical experiments each involving the same four adult donors (I, C, W and '0') as used in theoriginal injection of ST 11 (Table 1). 0, '0'; A, I; O, C; 0, W; A, transfer line initiated withnormal 3-day-old 'O' (host type) blood; *, negative controls, uninjected.

73

1. Weekly transfer in 13-day embryosEight experiments were performed which were exact replicas of those summarized in

Table 1 (transfer of blood every 7 days), except that all the 'O' embryos were injected onday 13 ofincubation instead ofday 17. Fig. 2 shows (in contrast to Fig. 1) how abrupt andfinal is the loss of splenomegaly on transfer to new hosts. A significant amount of spleenenlargement was obtained only in the first passage from the original host and with onlytwo of the donors. Apparently accelerated maturation has not occurred to a very signifi-cant extent, if at all, before the twentieth day of incubation.

In a similar experiment I x W adult blood was injected into 13-day 'O' embryos andthe recipient spleen was analysed 7 days later with the antibody neutralization technique.Whereas anti-host gave no neutralization whatever, anti-donor gave a value for (NS-AD)= 086, i.e. about 99 per cent neutralization. Hence that is the complete antithesis to theconclusion from Tables 4 and 5, and is moreover supported by similar findings from manyother experiments where host spleen was analysed at 4-5 days after injection of 13- or14-day embryos.These neutralization experiments are significant in two ways: (a) they suggest that

transfer of splenomegaly in younger embryos is due to a true passage of the original donorcells (for as long as transfer is possible) and not to accelerated host maturation, and (b)they remove the otherwise possible criticism of the data in Tables 4 and 5, i.e. that theactive cells are in fact of donor type, but are neutralized by anti-host antibodies becauseof a passive coating with antigens acquired from the host. Were the latter true it wouldsurely also occur when similar material is analysed 7 days after injection at the thirteenthday.

2. Accelerated maturation in newly hatched chicksSince accelerated maturation seemed to occur in the period from day 17 of embryonic

life to day 3 after hatching but was not demonstrable in the first 3 days of that period (asjudged from the attempts ofweekly passage in 13-day embryos), it was to be expected thatthe phenomenon would at least occur in the first 3 days after hatching.Newly hatched 'O' chicks were accordingly injected i.v. with the standard dose of 01

ml of adult I xW blood and bled out 3 days later. None out of six chicks showed spleenenlargement at this early stage. Nevertheless, four of the bloods which were then injectedseparately into batches of 17-day 'O' embryos produced the usual splenomegaly in 7 days,and this was in all cases transferable without decline for another two passages, when theexperiment was discontinued.

3. The role of antigenic stimulation to the hostIn the main body of continuous transfer experiments (Table 1) each injection was done

with material which must be assumed to have been antigenic to the host at the same timeas being able to react itself against host antigens. The realization that accelerated matura-tion seemed a phenomenon of early life post-hatching rather than of embryonic life raisedthe suspicion that no more might be at play than specific immunization of the host withdonor antigens. Chance cross-reactivity with antigens in the next host might then explainwhy a 3-day-old chick which had itself been injected before had more reactive lympho-cytes than normal controls.



Two different sets of data speak strongly against this hypothesis.(a) The fact that spleens in the NB transfer lines of Table 1 stayed so much closer to

non-injected controls than to the adult transfer lines: exactly the same chance ofimmuniza-tion and cross-reactivity should obviously operate in both cases as soon as the originaldonor's cells were eliminated.

(b) The fact that accelerated maturation was not induced in an experiment where bloodfrom the adult I xW donor ofST 28 was injected into 17-day W-eggs. The host blood cellsat 3 days after hatching had exactly the same reactivity as normal 3-day W cells whentested in 13-day 'O' embryos killed after 5 days. Hence the very same I xW cells whichcaused the accelerated maturation seen in Fig. 1 proved inactive in conditions where theywere merely antigenic, but not in the position to react against the host.

Complete confirmation of this result was obtained in three separate experiments eachinvolving 100-200 of the B-grouped Thornber eggs.

Seventeen-day B2 embryos were injected with 0 1 ml of blood (undiluted, or diluted1:25 in saline) from adult heterozygous donors (B2/B14, B2/B19 and B2/B21) and therecipients' blood and/or spleen cells harvested 7 days later and tested in 13-14-day B2,B14, B19 and B21 embryos. In no case was accelerated maturation demonstrated, i.e.the pre-treated B2 chicks caused no more spleen enlargement than untreated B2 agecontrols. Here again, antigenic stimulation itself was incapable of reproducing thephenomenon.

4. The role of GCVH-reaction in accelerated maturationSince host stimulation with histocompatibility antigens caused no immunological

maturation when there was no concomitant GVH-reaction it became necessary to studythe isolated effect of the latter.

This meant initiating the process with parental cells injected into F1 hybrid. As the Wstrain appeared the most homogeneous material available, W -> I xW was chosen for theoriginal injection.

There is the difficulty inherent in this design that the grafted parental cells may beexpected to survive and maintain reactivity in spite of accelerated maturation of the host.Neutralization with specific antibodies becomes again the crucial criterion. Thus, if cellsfrom the I xW host are transferred to 'O' embryos and produce more spleen enlargementsthere than normal I x W controls, this fact will only indicate accelerated maturation if itcan also be shown that their activity can be neutralized with W anti-I as well as with Ianti-W antigen.

Three experiments were performed. In two, the initial dose of adult W cells consistedof 0*05 or 0 10 ml of blood, and the results clearly indicated accelerated maturation. Inthe other, 0-20 ml ofblood from the sameW donor was injected to start with, and then themajority of active cells from the I xW host were neutralized with I anti-W only. Presum-ably, with the higher initial dose, the hybrid host spleen became predominantly repopu-lated with the parental cells.

5. Miscellaneous findingsDuring the course ofST 11 (Table 1) it was repeatedly found that cells from the transfer

line which had been initiated with adult I blood, and thereafter passaged in 'O' eggs, alsoproduced a marked splenomegaly when retransferred to I eggs. This fact was first thoughtcompatible with the extraordinary claim by Burnet and Boyer (1960) that inbred cells can

75

lose their self-recognition during passage through foreign embryos. There are nowindependent reasons not to regard that claim as valid (Warner, 1964; Simonsen, 1965),and the findings above are easily explainable on the accelerated maturation hypothesis:activated 'O' cells should react in I, as well as in other foreign embryos.The old fear, that seemingly odd results may be due to an unknown virus, was usually

alleviated promptly when it came up. For example, the plasma of bloods which possessedspleen enlarging capacity had no activity. Similarly active cell preparations lost theiractivity completely on rapid freezing in liquid nitrogen and re-thawing. Also, the speci-ficity of the neutralizing antisera was such that the 'virus' must have been unbelievablygood at imitating the histocompatibility antigens.Some concern was admittedly caused by the fact that heavy X-ray doses, up to 10,000

R, sometimes failed to inactivate whole blood, or preparations of white blood cells. Thisfinding led to a large-scale investigation of the radiosensitivity of immunologicallycompetent chicken cells which will be reported separately (Coppleson and Simonsen).It suffices to say here that it seems that a small fraction (0.1-1 -0 per cent) of the ICCpopulation is indeed extraordinarily resistant to X-rays.

In a few experiments, sheep red cells, bovine serum albumin, and phytohaemagglutininwere all found to be incapable of producing the accelerated immunological maturation.

DISCUSSIONMany findings in the present work would be compatible with two or more alternative

explanations were it not for the facts revealed by the neutralizing antibodies.These are that the ICC responsible for the unlimited transferability of splenomegaly

in 17-day embryos are cells derived from the previous host(s) and not from the originaladult donor. Furthermore serial transfer in younger embryos (13-14 days) is due, onthe contrary, to a true passage of cells derived from the original donor; but spleen enlarge-ment is then limited to a few passages.Once these facts are known, the rest are either immediately understandable, or become

so when it is further realized that the immunological maturation of newborn chicks isaccelerated when a GVH-reaction takes place in them.The chain of events in the outbred 17-day embryo system, can now be traced as follows.The adult donor cells elicit a classical GVH-reaction in the first host. The enlarging

spleen contains, on the average, equal proportions of dividing donor and host cells althoughthe ratio is subject to great variation, at least in outbred hosts.As a side-effect, which seems to take place in the first 3 days after hatching, the ICC of

the host become stimulated to the extent that both the circulating white blood cells andthe spleen cells possess, per unit cell number, a general heightened reactivity. This issufficient to initiate a GVH-reaction when blood is transferred to new hosts which presentthe previous host cells with foreign antigens. Immunological maturation is then, in turn,accelerated in the second host, and this cycle seems repeatable for ever.

The GVH-reaction given by the precociously matured cells is usually not equal inviolence to that of adult cells. This fact is apparent morphologically: spleen weights are

usually not quite as high, the contribution of dividing donor cells found in the host spleensis very much lower and, finally, few necrotic nodules are usually seen.

If blood from the first host is transferred to another embryo of the same genotype it may or

may not produce splenomegaly. If it does, it may be due to cells of the original donor-


Accelerated Immunological Maturation in the Chick 77

host pair continuing to react against each other; a graft-graft reaction (Warner, 1964;Simonsen, 1965). It may also, depending on the relative antigenic strength of GVH- andHVG-reactions, and on the initiating dose of adult donor cells, be a true propagation ofGVH-reaction caused by the original donor. The main point here is that splenomegaly ineither case will soon disappeai on continued passage. In the first case because of lack ofnew antigenic stimulation to maturing host cells as the original donor cells disappear. Inthe second case, because of the fact that a given population of grafted ICC from blood orspleen can only keep up their reaction for a limited span of time anyway. They are more-over being countered by the maturing immune response of the host. The genuine time-restricted functional capacity is known from passage of GVH-reaction in young (13-14-day) I xW embryos upon initiation with adult W cells (data to be published later).

Thus, in inbred embryos splenomegaly disappears with passage in the younger hosts forone reason and in the older ones for the same, plus the additional reason of acceleratedimmunological maturation. It seems right that it should happen faster in the latter as infact it appears to do. In outbred embryos the same accelerated maturation permits in-definite transfer of splenomegaly in the older hosts and gives the spurious impression ofunlimited proliferation of the original graft cells.

The nature of accelerated maturationHoward and Woodruff (1961) and Blaese, Martinez and Good (1964) have previously

reported that severe GVH-reactions in adult animals can reduce the capacity of the hostto react to antigens which are presumably unrelated to both donor and host cells. It wasquite unexpected to us that GVH-reactions may also produce the opposite effect.Yet there is no doubt that increased immunological reactivity was produced in our

experiments. Given the fact that continuous serial transfer of splenomegaly in outbredchicks (Table 1) beyond the original injection is due to the immunological competence ofhost cells, the accelerated maturation of the latter then becomes apparent from comparisonwith three different controls: NC (negative controls, uninjected), NB (serial transferstarted with normal blood from 3-day-old 'O' chicks) and SM (spontaneous maturation),a total of fifty-eight independent extra groups of 1 7-day 'O' embryos injected with normalblood from a 3-day 'O' donor, (but without further transfer). The overall differencebetween spleen weights of the transfer lines initiated with adult cells and each of the threecontrol groups is most convincing. (The probability is much below 0001 that eitherdifference should be due to sampling error.)

Statistical analysis showed an overall insignificant difference between the spleen weightsof negative controls and those of NB or SM controls, yet the normal 3-day-old birds weresurely not devoid of immunological competence. Their white blood cells regularly pro-duced a significant spleen enlargement when injected into the more sensitive youngerembryos (13-14 days ofincubation), although they produced less than cells from artificially'matured' birds. A few experiments (not included in the data) suggested that the com-petence of the latter may be comparable with that ofnormal 2-week-old birds.

It is clearly an important point whether or not the accelerated maturation is separatefrom mere immunization of the late embryo and/or newborn chick.Solomon (1963) claimed to have found immunization of chick embryos as young as

13-15 days ofincubation although, rather strangely, not of 17-day embryos. The specificityof the apparent immunization was not controlled. His procedure was similar to that used


in newborn mice by Howard and Michie (1962, 1963) who did show a specific immuniza-tion to transplantation antigen with small doses of cells. Brent and Gowland (1963) cameto the same conclusion, applying a different test system in newborn mice, but they did notcontrol the specificity of the immunization. Several other authors (cited by those alreadymentioned) have successfully immunized both foetuses and newborn animals of differentspecies.There is nothing in the present findings which is in conflict with the above-mentioned

results of others and, as already mentioned, we have positive evidence that even ournormal 3-day-old chicks possess some degree of immunological competence. We did,therefore, go to a considerable length in trying to determine if the accelerated maturationinduced by adult blood was reproducible by antigenic stimulation of the host alone,without accompanying GVH-reaction. The results were consistently negative. Heterozy-gous blood injected into homozygous 17-day embryos did not in our test system induce thehost cells to higher reactivity, neither specific nor non-specific.On the other hand, GVH-reactions reproduced the maturation phenomenon even in

the absence of known antigenic stimulus to the host as typified by injection of adult Wblood into I xW embryos. If minor histocompatibility antigens (not determined by theB-locus) have played any role at all in these experiments, they would have had the samechance of doing it whether grafting was done from B-locus homozygotes to heterozygotesor vice versa. The evidence is clearly that B-locus antigens have determined theinduction of accelerated maturation, and that they have done so indirectly, as a non-specific by-product of GVH-reaction.

It is a possibility that this effect is related to the non-specific stimulation which Howardand Michie (1963) found in newborn mice with an endotoxin from Pasteurella pseudotuber-culosis, or with the effect of Freund's adjuvant in newborn rabbits demonstrated by Paraf,Fougereau and Bussard (1963). We have not as yet tried any of these agents. Whether ornot they would happen to work in newborn chicks, it is possible that substances with asimilar effect in chicks are produced as a consequence of GVH-reaction. The commondenominator might be ability to provoke rapid lymphoid cell division.

Accelerated immunological maturation has not really advanced beyond the stage ofbeing found to exist. It would seem to merit further investigation in its own right.

ACKNOWLEDGMENTSOur thanks are due for support from the Medical Research Council, the Leverhulme

Trust and Messrs. Johnson andJohnson (Great Britain) Ltd. The work was also supportedby N.S.P.H.S. Fellowship No. (SP13, 663 (Cl) ) to R. A. McBride.

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