ABSTRACT
Adult chicken spleen was implanted on to the chorio-allantoic membrane of chick embryos at 8,9,11,13 and 15 days of incubation. The initial manifestation of graft against the host attack by these adult cells was an increase of host spleen weight (splenomegaly). No splenomegaly appeared in the embryonic hosts before 15 days of incubation. The appearance of splenomegaly roughly coincided with the appearance of reticulum cell foci in the chimaeric spleens. Varying latent periods between the time of introduction of donor cells and the first appearance of increased spleen weights necessitated comparison of the host spleen weights 3 days after the onset of splenomegaly. This showed that implantation at 13 days produced the greatest splenomegaly.
Only very slight splenomegaly was obtained after implantation of spleen at 15 days. A possible homograft rejection by the host and poor ‘take’ of the splenic implant on the chorio-allantoic membrane are both thought to be responsible.
Intravenous injection of adult chicken spleen cells into embryos at 6, 8, 11 and 13 days of incubation again produced no splenomegaly before 15 days of incubation. Comparison of the host spleen weights 3 days after onset of splenomegaly showed that injection at 13–15 days gave the greatest splenomegaly. Injection before this time gave very slight splenomegaly and injections after 13 days produced a decreasing degree of splenomegaly with increasing age of embryo injected.
Increasing the number of adult spleen cells at 15 days or giving large doses of blood at 8, 9, 11, 15 and 17 days of incubation and in the 1-and 2-day-old chick failed to increase the splenomegaly greatly or markedly to alter the above pattern.
When 1-day-old chicks were injected with allogenic blood only slight splenomegaly occurred and when 2-day-old chicks were injected there was no splenomegaly 6 days later.
The suitability of the chick embryo as an environment for the proliferation of immunologically competent cells is discussed.
The reduction in splenomegaly when embryos older than 13-15 days are injected is discussed in relation to the appearance from this time of a weak homograft response by the embryo and to the greater difficulty of induction of both splenomegaly and tolerance to skin grafts soon after hatching.
It is proposed that very small numbers of immunologically competent cells appear in the embryo soon after 15 days and progressively increase from this time until onset of competence of the host at 10 days post-hatching. Possible effects of such cell growth on the degree of induction of tolerance are discussed.
INTRODUCTION
The immunological attack by adult cells introduced into the embryo is first manifest by splenomegaly. The extent of this splenomegaly depends upon many factors. There must be antigenic differences between the donor and host in that the host must possess antigens absent in the donor (Cock & Simonsen, 1958; Mun, Kosin & Sato, 1959; Burnet & Boyer, 1961; Jaffe & Payne, 1961). The degree of splenomegaly also depends upon the immunological maturity of the donor cells (Ebert, 1951; Simonsen, 1957; Solomon, 1960, 1961a), the number of donor cells injected into the embryo (Isacson, 1959; Terasaki, 1959a; Solomon, 1962) and, in some cases, upon the sex of the host (Solomon, 1962).
In this paper two further factors are shown to affect splenomegaly—the age of the host embryo and the method of administration of the donor cells. Danchakoff (1916) first showed that the histology of the spleen during splenomegaly varied with the age of the host without being aware of the nature of the transplantation reactions involved. Recently Isacson (1959) has shown that the age that the embryo was injected with donor cells affected the splenomegaly as it was more pronounced in 13-and 15-day-old embryos than those injected at 11 or 20 days of incubation. Isacson (1959) postulated that some immune response on the part of the embryo would account for the reduced splenomegaly in older embryos. We have examined the effect of embryo age over a much wider range of development with donor cells administered both as implants and as intravenous (i.v.) injections. It appears that the vascular differentiation of the embryonic spleen markedly affects the colonization of donor cells in this organ so that only slight splenomegaly is obtained when donor cells are introduced before the host spleen is well vascularized. Evidence that the embryo is not as immunologically defenceless as has previously been supposed is presented and discussed. A preliminary report of some of this work has been published (Solomon & Tucker, 1961).
MATERIALS AND METHODS
Adult (non-inbred) utility White Leghorn and Rhode Island Red chickens, chicks and White Leghorn eggs were used in these experiments. Eggs were incubated and some hatched in a Westernette Incubator at 37·5°C and removed at 6–21 days of incubation for implantation (chorio-allantoic grafting) or intravenous (i.v.) injection.
Spleens from chicks or adult chickens were removed under aseptic conditions and placed in a Petri dish containing normal saline. The spleen capsule was removed and the spleen pulp macerated with curved forceps. Implants (10–15 mg. wet weight) were teased out from the macerated spleen pulp and kept ice-cold until implanted. Donor chicken spleen was implanted on the chorioallantoic membrane (CAM) of chick embryos (8–15 days of incubation) using the procedure recently described in full by Solomon (1961 a). In each implantation experiment the spleen from one adult donor was introduced into about eighty embryos. Each experiment also included sham-operated eggs which underwent the same procedure as the experimental eggs except that they had no tissue implanted. Eggs from control and experimental groups were placed on their sides with the windows facing uppermost and returned to the incubator. About 80 per cent of these embryos survived the immediate postoperative period. Similar experiments with donor spleen and embryos of par tially inbred chickens gave little or no splenomegaly (Mun, Kosin & Sato, 1959).
Cell suspensions were prepared from spleens removed from adult female chickens using the technique described by Solomon (1962). Embryos were injected in a vein of the CAM according to the method described by Solomon (1962), which is a slight modification of that used by Beveridge & Burnet (1946). All such embryos received 6–9 × 106 spleen cells in 0·05 ml. of spleen-cell suspension. Control embryos in each experiment underwent the same operation as the experimental eggs except that they received 0·05 ml. of Ringer-phosphate solution (0·01M, pH 7·4). Embryos which had died from post-operative trauma were removed at one day after injection and the survivals were about 90 per cent of those injected. Newly hatched chicks were injected into a leg vein within 12 hours of hatching. Chicks were then kept under infra-red lamps with water and chick crumbs ad lib for a further 6 days.
A few embryos from each experiment were killed daily after implantation or injection. The embryos were sexed by examination of their gonads, their spleens were removed, placed on moist lint and then weighed to the nearest 0·1 mg. Larger numbers of embryos from the experimental group were killed at the time of onset of splenomegaly and for 3–4 days afterwards.
RESULTS
Similarity of the degree of immunological competence of cells from different donors
Only slight variation in the competence of spleen cell preparations to elicit splenomegaly (either implanted or injected i.v.) at a given age has been found (Solomon, 1961a, 1962). Samples of blood from three of the twenty-five donors produced an almost identical degree of splenomegaly when injected into twenty-nine 15-day-old embryos. Moreover, while typing Rhode Island Red donor bloods for other experiments we have rarely found exceptional degrees of competence to produce splenomegaly. In a similar study Isacson (1959) used cells from only one donor thus avoiding any variation between donors, but the use of much larger numbers of hosts in this work and the use of spleen cells have required the use of twenty-five donors.
Individual variation of antigenic stimulus by the host
The wide range of embryonic spleen weights obtained after the introduction of immunologically competent cells necessitates the use of about twenty embryos in each experimental group to obtain an adequate measure of the splenomegaly. Intravenous injection of adult cells into 173 15-day-old embryos produced spleen weights which conformed to a normal bell-shaped distribution curve. However, in previous experiments in which spleen cells from juvenile chicken were implanted on to the CAM of 8-day-old embryos whose spleens were removed 10 days later, the spleen weights of each sex showed a skewed distribution (Text-fig. 1). Burnet & Boyer (1961) obtained similar skew distribution curves of counts of foci of proliferating cells in individual embryos following inoculation of adult blood on the CAM. The skewness of such distribution curves, due to a high proportion of unenlarged host spleens (sometimes as much as 40 per cent of the total), is probably due to failure of some of the implants to ‘take’ on the CAM (even though the implants were placed over the most vascular portion) as well as failure of sufficient immunologically competent cells to escape from the implant. Another complication in splenomegaly assay is that female hosts often bear larger spleens than the males (Solomon 1961b, 1962); when such a sex difference was found in the present work the spleen weights were adjusted to equal numbers of each sex to correct for this effect..
Determination of onset of splenomegaly
The failure of many implants to ‘take’ on the CAM, as well as the wide range of antigenic stimulus to donor cells, makes the statistical determination of the onset of splenomegaly rather difficult. The onset is presumably first detectable in the embryos possessing the most antigens which are absent from the donor and these embryos will later bear the largest spleens. The onset of splenomegaly in each experiment was the first sample of spleens to show an increase (usually P < 0·01) in mean spleen weight above that of the controls. The younger the embryo at the time of introduction of donor cells and the commencement of their proliferation (as measured by increase in spleen weight), the shorter was the latent period.
Growth of the normal embryonic spleen
The mean spleen weights of control chick embryos (which had their shell windows removed or were injected i.v. with Ringer) for the period of development covered in these experiments are shown in Text-fig. 2. The wet weight of the embryonic spleen increased four-fold during 13–17 days of incubation and then remained nearly constant until after hatching.
Control chick embryo spleen weignts. The vertical lines represent the standard error of the mean spleen weights after hatching. The range of the standard error of the mean spleen weights from 11–20 days of incubation was + 0·5-± 1·0 mg.
Splenomegaly produced by implantation of adult spleen into embryos from 8–15 days
The course of the splenomegaly in embryos implanted with adult chicken spleen at 8, 9, 11, 13 and 15 days of incubation is shown in Text-fig. 3. Two separate experiments were performed at each of the host ages and altogether spleens from ten hens were implanted, in all, to 495 embryos. The course of the splenomegaly is the same whether embryos were implanted at 8, 9, 11 or 13 days (Text-fig. 3); in each experiment the onset of splenomegaly was after 14 days of incubation and there was no case of increased spleen weight in an individual embryo before this time. The splenomegaly produced by implantation at 8 and 9 days had reached its maximum at 3 days after its onset. Implantation at 15 days produced only a very slight increase in host spleen weight.
Course of the splenomegaly syndrome after implantation of adult spleen at 8(▵), 9(▿), 11(□), 13(○) and 15(▴) days of incubation.
Comparison of the extent of splenomegaly at 3 days post-onset shows that it was greatest after implantation at 13 days (Table 1).
Biggs & Payne (1961a) believe that the reticulum cell foci seen in the chimaeric spleens are donor cells and Biggs (private communication) found that the foci appeared in spleens from our experiments, just before, or at the same time as, the onset of splenomegaly. Biggs detected foci at 8 days after implanting spleen at 8 days of incubation; 4 days after implanting at 9 and 11 days, 3 days after 13-day implanting and 2 days after 15-day implanting.
Splenomegaly produced by intravenous injection of adult spleen cells into embryos from 6–21 days
The course of the splenomegaly after i.v. injection of spleen cells from fifteen adult hens into embryos (424) at 11, 13, 15 and 18 days is shown in Textfig. 4. After the onset of splenomegaly (usually 3 days after injection) the slopes of the splenomegaly on a log plot are essentially similar, indicating a uniform rate of spleen weight increase. The splenomegaly produced by injections at 11 and 13 days appeared to have reached a maximum at 6 days after injection. The course of splenomegaly after injection at these early ages was not followed due to the failure of many embryos to hatch and consequent dangers of selection. Hatching was successful after injection at 18 days as the splenomegaly had only just commenced. There was no case of splenomegaly before 15 days of incubation even when 8-day-old embryos were i.v. injected.
Course of the splenomegaly syndrome after intravenous injection of adult spleen cells at 11(□), 13(○), 15(▴) and 18(•) days of incubation.
The extent of splenomegaly 3 days after onset is shown in Text-fig. 5; only injections between 8 and 18 days have been followed in detail so that the onset could be determined. For comparison, the host spleen mean weights obtained 6 days after injection at 19 and 20 days of incubation, and in the day-old chick, are included and the mean spleen weight 10 days after i.v. injection at 6 days is also included (assuming an onset at 15 days). An analysis of variance on the 8 to 18 days group gave a variance ratio of 18-1, which greatly exceeds the value of 3·5 (which occurs by chance in 1 per cent of the trials) and similar analysis on the 6-day-old embryo to 1 day chick group gave a variance ratio of 14·5 (control 4·3 at 1 per cent level).
Effect of the age of the host embryo on the extent of the splenomegaly syndrome with i.v. injection (•) or implantation (▄) of adult spleen cells. Statistical differences (p <0·01) : 13 day is greater than rest (except for 15 day) 15 day is greater than rest (except for 13 & 18 day) 18 day is greater than rest (except for 6, 8, 11 & 21 day) 19 & 20 day is greater than rest (except for 6, 8 & 11 day)
Effect of the age of the host embryo on the extent of the splenomegaly syndrome with i.v. injection (•) or implantation (▄) of adult spleen cells. Statistical differences (p <0·01) : 13 day is greater than rest (except for 15 day) 15 day is greater than rest (except for 13 & 18 day) 18 day is greater than rest (except for 6, 8, 11 & 21 day) 19 & 20 day is greater than rest (except for 6, 8 & 11 day)
When donor cells were injected i.v. reticulum cell foci could generally be detected 2 days later; foci were not profuse in the chimaeric spleens until 5 days after 11-day injection and 6 days after 18-day injection but were profuse at only 3 days after injection at 13 or 15 days of incubation (Biggs, private communication).
Massive cell doses at different ages
Between 13 and 17 days the normal embryonic spleen weight increases fourfold and between 13 and 20 days the embryo body weight increases five-fold; the blood volume per unit body weight remains constant during this period (Barnes & Jensen, 1959). The number of donor cells per host unit blood volume, spleen weight or body weight would thus diminish during this period with a constant dose of donor cells. Accordingly, much larger doses of donor cells were injected into embryos at various ages (Table 2). Splenomegaly was not greatly increased by giving much larger doses of donor cells and the relationship between variation of splenomegaly and the age of the embryo previously obtained with low doses of spleen cells was not disturbed. No splenomegaly occurred when 2-day-old chicks were injected, as had previously been found by Simonsen (1957).
DISCUSSION
Possible reasons why embryos of other ages than 13 days of incubation are less suitable hosts for the proliferation of allogeneic cells will be discussed from two points of view—that before 13 days the host spleen is not fully vascularized and differentiated and that after 13–15 days the embryo is no longer completely immunologically defenceless.
The histology of normal spleen differentiation has been described by Dan-chakoff (1916) who found that at the eleventh day the spleen became vascularized with venous sinuses and granulocytopoiesis commenced. At the twelfth to thirteenth day intense arterial vascularization began and at the fifteenth to seventeenth day small lymphocytes appeared. Sandreuter (1951) noted lymphocytic activity in the 12-day-old spleen, and both he and Biggs & Payne (1961a) found that the normal spleen changed from a predominantly granulocytopoietic to a lymphopoietic role from about 15 days. Whereas granulocytopoiesis was the main function of the 15-day-old spleen, by the eighteenth day granulocytopoietic tissue had begun to disappear and had nearly completely disappeared by the first day post-hatching when the lymphocytic activity was pronounced.
Danchakoff’s finding (1916) that intense arterial vascularization of the spleen does not begin until the twelfth to thirteenth day offers an explanation for the absence of any splenomegaly in this work until the host was at least 15 days old. The arterial vascularization at 12 days together with the 3 day latent period before the donor cells began to proliferate, coincides with the onset of splenomegaly in our work at 15 days.
DeLanney & Ebert (1959) found that the earliest splenomegaly occurred at 12 days of incubation. Ebert (1957) implanted adult spleen into the coelom of 3-to 4-day-old embryos which at 13–14 days had a mean spleen weight five times that of the controls. These experiments do not agree with our results in which a longer (6 day) latent period was observed when 8-day-old embryos were implanted or injected and no splenomegaly ever occurred in embryos younger than 15 days. There are indications that splenomegaly in embryos injected before 15 days of incubation reached its maximum at 19–20 days of incubation. Biggs & Payne (19616) showed that some splenomegaly still persists up to 11 days after injecting 15-day-old embryos, a similar time-course to splenomegaly in mice (Simonsen & Jensen, 1959). Brandly, Thorp & Prickett (1949) first showed that i.v. injection of donor cells was superior to other methods in eliciting splenomegaly. In our experiments this is only true after 11 days, and the optimum time for the introduction of cells by implantation or i.v. injection is at 13 days to obtain maximum splenomegaly. To avoid death of the embryos due to other manifestations of the graft-against-the-host reaction we have found that it is best to remove the host spleens 6 days after i. v. injection at 13–15 days.
There is evidence that the iso-antigens responsible for transplantation immunity and the erythrocyte antigens are present at about 4 days of incubation (Burke, Sullivan, Petersen & Weed, 1944; Levi & Schechtman, 1954; Terasaki, 19596). It is therefore highly improbable that the poor splenomegaly produced by injection or implantation of young embryos is due to lack of antigenic stimulus by the host.
The developmental end-point for implantation is at 17 days, as when Murphy (1914) implanted rat tumour at this time it had not become vascularized 2 days later. This indicates that implantation of tissue at 15 days may be less efficient than at early stages of development. This may be due to a weak homograft rejection by the host embryo or to the regressive vascular changes occurring in the CAM at this time causing restriction of nutrition to the implant (Vulpian, 1857; Sandstrom, 1932; Oakley, 1938).
The lower host spleen weights obtained in our experiments when donor cells were i.v. injected into embryos older than 15 days is in agreement with the results of Simonsen (1957) who injected 18-day-old embryos, Cock & Simonsen (1958) who used newly hatched chicks and Isacson (1959) who injected 17-to 20-day-old embryos. This suggests that the environment of older embryos is unsuitable for the proliferation of adult cells and recalls the work of Dixon & Weigle (1957) who were unable to detect antibody synthesis by sensitized adult rabbit cells transferred to 5-to 6-day-old rabbits. However, Sterzl (1957) and Holub (1958) were able to obtain satisfactory antibody synthesis under similar conditions. Sibal & Olsen (1958), Trnka (1958), Trnka & Riha (1959), Trnka & Sterzl (1960) and Isacson (1959) have all obtained evidence that adult chicken spleen cells from previously sensitized chicken inoculated into embryos, will produce antibody while dividing in the embryo. High agglutinin titres under such conditions were always associated with splenomegaly (Papermaster, Bradley, Watson & Good, 1959). All this work strongly indicates that the embryonic environment will support normal functioning of adult cells. However, Isacson (1959) has shown that 11-to 15-day-old chick embryos were better hosts for antibody synthesis by transferred adult cells than 17-to 20-day-old embryos. Isacson also studied the splenomegaly produced in these embryos and found that when host spleens were removed 6 days after i.v. injection at 13 or 15 days they were larger than those from embryos injected at 11 days, which were in turn larger than those from embryos injected at 17-20 days. These differences in splenomegaly can be related to the different degrees of proliferation of donor cells because Isacson found weaker production of antibody in the older embryos. Isacson (1959) attributed the lesser splenomegaly in the older embryos to a partial immune rejection of donor cells by the host and described certain specific histological changes in the chimaeric spleens to support this idea. Other workers have suggested that the chick embryo becomes capable of immunological rejection of cells before hatching (Murphy, 1913, 1914; Humphreys, 1960; Trnka & Riha, 1959), although the chick embryo has previously been considered to be incapable of the immune reactions of homograft rejection (Billingham, Brent & Medawar, 1953).
The production of immunological tolerance depends upon the establishment of cells at certain sites where the host cells responsible for transplantation immunity will eventually arise. As the maximum splenomegaly is obtained by injection at 13 days, then this indicates that donor cells will become most readily established in the host spleen at this age. This idea is partially confirmed by the results of Cannon, Terasaki & Longmire (1957) which showed that tolerance to skin grafts, produced by injection of embryonic blood, was slightly less at 10–11 days than when 12-to 18-day-old embryos were injected, but both groups showed better homograft survival than hosts injected at 1-3 days after hatching. Further confirmation is given by the work of Billingham, Brent & Medawar (1956) who obtained a poorer survival of skin homografts when large doses of adult blood were injected into newly-hatched chicks than when embryonic blood was injected into 10-to 11-day-old embryos.
The histological appearance of lymphocytes in the chick embryo spleen at the fifteenth to seventeenth day of incubation (Danchakoff, 1916; Sandreuter, 1951; Biggs & Payne, 1961a) coincides well with the beginnings of decrease of splenomegaly in our work. Kalmutz (1962) states that ‘physiologically mature and immunipotent lymphopoietic tissue’ exists practically as soon as lymphoid elements can be identified in the opossum embryo. If reduction in the extent of splenomegaly with increasing age of the embryo is connected with the immunological status of the host then there are indications that a steady increase in the population of cells capable of homograft rejection occurs in the embryo from 15 days of incubation. The presence of immunologically competent lymphocytes in the 19-day-old chick embryo has recently been confirmed, as it is possible to induce transplantation immunity to skin homografts at this and later ages but not in the 15-day-old embryo (Solomon, unpublished results). The possible ability of the chick embryo to reject foreign cells recalls earlier work when foetal sheep were shown to be able to reject homografts long before birth (Schinkel & Ferguson, 1953), calf neonates were capable of rejecting homografts (Billingham & Lampkin, 1957) and guinea-pigs and rabbits could reject homografts at 1–3 days before birth (Egdahl, 1957). Recently Howard & Michie (1962) have produced evidence for the presence of immunologically competent cells in newborn mice, as they could produce either tolerance or immunity in such mice depending upon the dose of antigen. The number of lymphocytes in the chick have probably increased sufficiently by 2 days after hatching to completely prevent the establishment and proliferation of inoculated donor cells in the spleen of normal chicks and thus both the production of splenomegaly and the maintenance of tolerance to homografts. After this the lymphocytes of the chick probably increase rapidly so that at 10 days after hatching (Simonsen, 1957; Solomon 1960, 1961a) there will be sufficient to elicit either splenomegaly when transferred to a foreign host or to reject a homograft.
RÉSUMÉ
Attaque immunologique de /’embryon de poulet par des cellules adultes; influence de la vascularisation de la rate de Vhôte et du rejet de l’homogreffe par Vembryon sur la splenomegalie
De la rate de Poulet adulte fut greffée sur la membrane chorio-allantoi-dienne d’embryons de Poulet de 8, 9, 11, 13 et 15 jours d’incubation. La première manifestation de l’attaque de l’hôte par la greffe, c’est a dire par les cellules adultes de celle-ci, fut l’augmentation du poids de la rate de l’hôte, ou splénomégalie. La splénomégalie n’apparut jamais chez l’embryon hôte avant le 15ième jour d’incubation. Le début de la splénomégalie coïncide à peu près avec l’apparition de foyers de cellules réticulaires dans les rates chimères. Des périodes de latence variables entre l’implantation des cellules donneuses et le début de l’accroissement du poids de la rate amenèrent à comparer les poids des rates chez les embryons hôtes trois jours après le début de la splénomégalie. Il en est apparu que l’implantation à 13 jours d’incubation causait la splénomégalie la plus importante.
Une splénomégalie seulement très discrète fut observée après implantation à 15 jours d’incubation. On peut penser qu’un éventuel rejet de l’homogreffe par l’hôte ainsi qu’une mauvaise reprise de la greffe sur la membrane chorio-allantoidienne en soient les causes.
Des injections intraveineuses de cellules de rate de Poulet adulte à des embryons de 6, 8, 11 et 13 jours d’incubation n’ont pas produit non plus de splénomégalie avant le 15ième jour d’incubation. L’étude du poids de la rate des hôtes trois jours après le début de la splénomégalie montra que l’injection effectuée a 13–15 jours d’incubation causait la plus forte splénomégalie. Les injections effectuées plus tôt n’ont produit qu’une très légère splénomégalie, et les injections effectuées après le 13ième jour ont donné des degrés de splénomégalie qui décroissent à mesure que l’embryon injecté est plus âge.
Les résultats précédents ne furent pas modifiés et la splénomégalie ne fut pas notablement augmentée par l’accroissement du nombre des cellules de rate adulte, ni en injectant des doses massives de sang à 8, 9, 11, 15 et 17 jours d’incubation, ainsi qu’un ou deux jours après l’éclosion.
L’injection de sang étranger à des Poulets d’un jour ne causa qu’une légère splénomégalie. Chez des Poulets de deux jours aucune splénomégalie ne fut constatée six jours après une telle injection.
Les avantages de l’embryon de Poulet en tant que milieu de prolifération de cellules immunologiquement compétentes sont discutés.
La splénomégalie plus faible causée par l’injection chez des embryons plus âgés que 13–15 jours d’incubation est discutée, en relation avec l’apparition à partir de ce moment d’une faible réponse de l’embryon à l’homogreffe, et avec la plus grande difficulté pour obtenir la splénomégalie ainsi que la tolérance induite aux greffes de peau sitôt l’éclosion.
Il est avancé qu’un très petit nombre de cellules immunologiquement compétentes apparaitraient chez l’embryon peu après le 15ième jour d’incubation et se multiplieraient progressivement jusqu’à la competence de l’hôte, réalisée 10 jours après l’éclosion. Les éventuelles conséquences de ce développement sur les degrés de tolérance induite sont discutés.
ACKNOWLEDGEMENTS
We would like to thank Professor A. Haddow, F.R.S., for his interest in this work and Miss J. P. Collis for technical assistance. We are deeply indebted to Dr P. M. Biggs and Dr L. N. Payne of Houghton Poultry Research Station for the histological examination of many of the chimaeric spleens.
This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research, Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Anna Fuller Fund, and the National Cancer Institute of the National Institutes of Health, United States Public Health Service.