immunological attack by adult cells in the developing ... · splenomegaly produced by implantation...

/ . Embryol. exp. Morph., Vol. 11, Part 1, pp. 119-134, March 1963Printed in Great Britain

Immunological Attack by Adult Cells

in the Developing Chick Embryo: Influence of the

Vascularity of the Host Spleen and of Homograft

Rejection by the Embryo on Splenomegaly

by j . B . SOLOMON and D. F . TUCKER1

From the Chester Beatty Research Institute, London

INTRODUCTION

T H E immunological attack by adult cells introduced into the embryo is firstmanifest by splenomegaly. The extent of this splenomegaly depends upon manyfactors. There must be antigenic differences between the donor and host inthat 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 ofthe donor cells (Ebert, 1951; Simonsen, 1957; Solomon, 1960, 1961a), thenumber 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 ageof the host embryo and the method of administration of the donor cells.Danchakoff (1916) first showed that the histology of the spleen during splenome-galy varied with the age of the host without being aware of the nature of thetransplantation reactions involved. Recently Isacson (1959) has shown thatthe age that the embryo was injected with donor cells affected the splenomegalyas it was more pronounced in 13- and 15-day-old embryos than those injectedat 11 or 20 days of incubation. Isacson (1959) postulated that some immuneresponse on the part of the embryo would account for the reduced splenomegalyin older embryos. We have examined the effect of embryo age over a muchwider range of development with donor cells administered both as implantsand as intravenous (i.v.) injections. It appears that the vascular differentiationof the embryonic spleen markedly affects the colonization of donor cells in thisorgan so that only slight splenomegaly is obtained when donor cells areintroduced before the host spleen is well vascularized. Evidence that the

1 Authors' address: Chester Beatty Research Institute, Pollards Wood Research Station,Nightingales Lane, Chalfont St. Giles, Buckinghamshire, U.K.

120 J. B. SOLOMON AND D. F. TUCKER

embryo is not as immunologically defenceless as has previously been supposedis presented and discussed. A preliminary report of some of this work has beenpublished (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 wereincubated and some hatched in a Westernette Incubator at 37 • 5°C and removedat 6-21 days of incubation for implantation (chorio-allantoic grafting) orintravenous (i.v.) injection.

Spleens from chicks or adult chickens were removed under aseptic conditionsand placed in a Petri dish containing normal saline. The spleen capsule wasremoved and the spleen pulp macerated with curved forceps. Implants (10-15 mg. wet weight) were teased out from the macerated spleen pulp and keptice-cold until implanted. Donor chicken spleen was implanted on the chorio-allantoic membrane (CAM) of chick embryos (8-15 days of incubation) usingthe procedure recently described in full by Solomon (1961a). In each implanta-tion experiment the spleen from one adult donor was introduced into abouteighty embryos. Each experiment also included sham-operated eggs whichunderwent the same procedure as the experimental eggs except that they hadno tissue implanted. Eggs from control and experimental groups were placedon their sides with the windows facing uppermost and returned to the in-cubator. About 80 per cent of these embryos survived the immediate post-operative period. Similar experiments with donor spleen and embryos of partially inbred chickens gave little or no splenomegaly (Mun, Kosin & Sato, 1959).

Cell suspensions were prepared from spleens removed from adult femalechickens using the technique described by Solomon (1962). Embryos wereinjected 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 x 106 spleen cells in 0-05 ml. of spleen-cellsuspension. Control embryos in each experiment underwent the same operationas the experimental eggs except that they received 0-05 ml. of Ringer-phosphatesolution (0-01M, pH 7-4). Embryos which had died from post-operativetrauma were removed at one day after injection and the survivals were about90 per cent of those injected. Newly hatched chicks were injected into a legvein within 12 hours of hatching. Chicks were then kept under infra-red lampswith water and chick crumbs ad lib for a further 6 days.

A few embryos from each experiment were killed daily after implantation orinjection. The embryos were sexed by examination of their gonads, theirspleens were removed, placed on moist lint and then weighed to the nearest0 • 1 mg. Larger numbers of embryos from the experimental group were killedat the time of onset of splenomegaly and for 3-4 days afterwards.

SPLENOMEGALY IN THE DEVELOPING CHICK EMBRYO

RESULTS

121

Similarity of the degree of immunological competence of cells from different donorsOnly 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 donorsproduced an almost identical degree of splenomegaly when injected intotwenty-nine 15-day-old embryos. Moreover, while typing Rhode Island Reddonor bloods for other experiments we have rarely found exceptional degreesof 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 spleencells have required the use of twenty-five donors.

Individual variation ofantigenic stimulus by the host

The wide range of embryonic spleen weights obtained after the introductionof immunologically competent cells necessitates the use of about twentyembryos in each experimental group to obtain an adequate measure of thesplenomegaly. Intravenous injection of adult cells into 173 15-day-old embryos

20 40 60 80 100

Host spleen weights (mg.) grouped at each 5 mg.

TEXT-FIG. 1. Distribution curves of spleen weights from embryosimplanted with juvenile spleen.

produced spleen weights which conformed to a normal bell-shaped distributioncurve. However, in previous experiments in which spleen cells from juvenilechicken were implanted on to the CAM of 8-day-old embryos whose spleenswere removed 10 days later, the spleen weights of each sex showed a skeweddistribution (Text-fig. 1). Burnet & Boyer (1961) obtained similar skew dis-tribution curves of counts of foci of proliferating cells in individual embryosfollowing inoculation of adult blood on the CAM. The skewness of such

122 J. B. SOLOMON AND D. F . TUCKER

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 failureof some of the implants to 'take' on the CAM (even though the implantswere placed over the most vascular portion) as well as failure of sufficientimmunologically competent cells to escape from the implant. Another complica-tion in splenomegaly assay is that female hosts often bear larger spleens thanthe males (Solomon 1961&, 1962); when such a sex difference was found in thepresent work the spleen weights were adjusted to equal numbers of each sex tocorrect for this effect.

Determination of onset of splenomegaly

The failure of many implants to 'take' on the CAM, as well as the widerange of antigenic stimulus to donor cells, makes the statistical determinationof the onset of splenomegaly rather difficult. The onset is presumably firstdetectable in the embryos possessing the most antigens which are absent fromthe donor and these embryos will later bear the largest spleens. The onset ofsplenomegaly in each experiment was the first sample of spleens to show anincrease (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 thecommencement 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 shellwindows removed or were injected i.v. with Ringer) for the period of develop-ment covered in these experiments are shown in Text-fig. 2. The wet weightof the embryonic spleen increased four-fold during 13-17 days of incubationand then remained nearly constant until after hatching.

Splenomegaly produced by implantation of adult spleen into embryos from8-15 days

The course of the splenomegaly in embryos implanted with adult chickenspleen at 8, 9, 11, 13 and 15 days of incubation is shown in Text-fig. 3. Twoseparate experiments were performed at each of the host ages and altogetherspleens from ten hens were implanted, in all, to 495 embryos. The course ofthe splenomegaly is the same whether embryos were implanted at 8, 9, 11 or13 days (Text-fig. 3); in each experiment the onset of splenomegaly was after14 days of incubation and there was no case of increased spleen weight in anindividual embryo before this time. The splenomegaly produced by implanta-tion at 8 and 9 days had reached its maximum at 3 days after its onset. Im-plantation at 15 days produced only a very slight increase in host spleen weight.

SPLENOMEGALY IN THE DEVELOPING CHICK EMBRYO

5o

40

££ 30CD

53co

•S 20

123

10

II 13 15 17 19 21 1 2 3 4 5 6

thatching

Days of incubation ' Days after hatchingTEXT-FIG. 2. Control chick embryo spleen weights. The verticallines represent the standard error of the mean spleen weights afterhatching. The range of the standard error of the mean spleen weights

from 11-20 days of incubation was ± 0 • 5-+ 1 -0 mg.

100

11

>

75

50

oX 25

Time of implantation of donor spleen

A 7 O O A

1 1 i I I

10 12 14 16 18 20

Age of host (days of incubation)

TEXT-FIG. 3. Course of the splenomegaly syndrome afterimplantation of adult spleen at 8(A), 9(V), 11(D), 13(O) and

15(A) days of incubation.


Comparison of the extent of splenomegaly at 3 days post-onset shows thatit was greatest after implantation at 13 days (Table 1).

Biggs & Payne (1961a) believe that the reticulum cell foci seen in the chimaericspleens are donor cells and Biggs (private communication) found that the fociappeared in spleens from our experiments, just before, or at the same time as,

TABLE 1

Comparison of the extent of splenomegaly produced by splenic implantation

Age at implantation(days of incubation)

89

111315

Number ofhosts

1210252621

Host mean spleen wt.at 3 days post onset

mg ± S.E.

41±965+12

*78+14t99+1326+3

* Greater than 26 mg. (p <0 01). t Greater than 26 or 41 mg. O <0 01).Variance ratio (analysis of variance) = 6-2 exceeds the value (3 • 5) which occurs by chance

in 1 per cent of trials.

Age of host (days of incubation : days after hatching)

TEXT-FIG. 4. Course of the splenomegaly syndrome after intravenous in-jection of adult spleen cells at 11 (GX 13(o), 15(A) and 18(#) days of

incubation.

SPLENOMEGALY IN THE DEVELOPING CHICK EMBRYO 125

the onset of splenomegaly. Biggs detected foci at 8 days after implanting spleenat 8 days of incubation; 4 days after implanting at 9 and 11 days, 3 days after13-day implanting and 2 days after 15-day implanting.

Splenomegaly produced by intravenous injection of adult spleen cells into embryosfrom 6-21 days

The course of the splenomegaly after i.v. injection of spleen cells fromfifteen adult hens into embryos (424) at 11, 13, 15 and 18 days is shown in Text-fig. 4. After the onset of splenomegaly (usually 3 days after injection) the slopesof the splenomegaly on a log plot are essentially similar, indicating a uniformrate of spleen weight increase. The splenomegaly produced by injections at 11and 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 followeddue 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

I 140•oc

_ £

120

S" 80o

C

o

60

40

20

10 12 14 16 18 20 t ' ""hatching

Time of administration of donor spleen cells (days of incubation

TEXT-FIG. 5. Effect of the age of the host embryo on the extent of thesplenomegaly syndrome with i.v. injection (#) or implantation (•) of adultspleen 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)


just commenced. There was no case of splenomegaly before 15 days of incuba-tion even when 8-day-old embryos were i.v. injected.

The extent of splenomegaly 3 days after onset is shown in Text-fig. 5; onlyinjections between 8 and 18 days have been followed in detail so that the onsetcould be determined. For comparison, the host spleen mean weights obtained6 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 daysis also included (assuming an onset at 15 days). An analysis of variance on the8 to 18 days group gave a variance ratio of 18-1, which greatly exceeds thevalue of 3 • 5 (which occurs by chance in 1 per cent of the trials) and similaranalysis on the 6-day-old embryo to 1 day chick group gave a variance ratioof 14 • 5 (control 4 • 3 at 1 per cent level).

When donor cells were injected i.v. reticulum cell foci could generally bedetected 2 days later; foci were not profuse in the chimaeric spleens until 5 daysafter 11-day injection and 6 days after 18-day injection but were profuse at only3 days after injection at 13 or 15 days of incubation (Biggs, private communica-tion).

Massive cell doses at different ages

Between 13 and 17 days the normal embryonic spleen weight increases four-fold and between 13 and 20 days the embryo body weight increases five-fold;

TABLE 2

Effect of increasing the dosage of donor cells at different host ages.

Age of hostat injection

(days ofincubation)

89

11151 71 /

4- to 16-h-oldchick

2-day-oldchick

i<51 <J

201-day-old

chick

Number ofexperimental

hosts

8215819f816J718

11

("2017516

16

Cell dose

0-02 or 0-1 ml0-02 or 0-1 ml0 0 1 , 0 0 2 , 0 0 5 or 0-1 ml0-1 ml0 02 ml0-1 ml0.1 ml0-5 ml

0 1 or 0-5 ml

1 x 10'2-5 x 10'1 x 10' or 4 x 10'

1 x 10' or 4 x 10'

Increase in hostmean spleen weightat 3 days post-onset

of splenomegaly(Control = 1 - 0 )

blood 3-94-87-3

17-32-43-11-62-7

1 0

spleen cells 8-4„ „ H-1

2-0

1-3

Blood from one RIR, spleens from WL donors. Variance ratio for bloods = 12-7,exceeds the value (3 • 3) which occurs by chance in 1 per cent of trials. All values greaterthan 1 -0 are significantly greater (p<0-01) than control spleen weights.


the blood volume per unit body weight remains constant during this period(Barnes & Jensen, 1959). The number of donor cells per host unit bloodvolume, spleen weight or body weight would thus diminish during this periodwith a constant dose of donor cells. Accordingly, much larger doses of donorcells were injected into embryos at various ages (Table 2). Splenomegaly wasnot greatly increased by giving much larger doses of donor cells and therelationship between variation of splenomegaly and the age of the embryopreviously obtained with low doses of spleen cells was not disturbed. Nosplenomegaly occurred when 2-day-old chicks were injected, as had previouslybeen found by Simonsen (1957).

DISCUSSION

Possible reasons why embryos of other ages than 13 days of incubation areless suitable hosts for the proliferation of allogeneic cells will be discussedfrom two points of view—that before 13 days the host spleen is not fullyvascularized and differentiated and that after 13-15 days the embryo is nolonger 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 vascu-larized with venous sinuses and granulocytopoiesis commenced. At the twelfthto thirteenth day intense arterial vascularization began and at the fifteenth toseventeenth day small lymphocytes appeared. Sandreuter (1951) noted lym-phocytic activity in the 12-day-old spleen, and both he and Biggs & Payne(1961a) found that the normal spleen changed from a predominantly granulo-cytopoietic to a lymphopoietic role from about 15 days. Whereas granulocyto-poiesis was the main function of the 15-day-old spleen, by the eighteenth daygranulocytopoietic tissue had begun to disappear and had nearly completelydisappeared by the first day post-hatching when the lymphocytic activity waspronounced.

Danchakoff's finding (1916) that intense arterial vascularization of the spleendoes not begin until the twelfth to thirteenth day offers an explanation for theabsence 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 periodbefore the donor cells began to proliferate, coincides with the onset of spleno-megaly in our work at 15 days.

DeLanney & Ebert (1959) found that the earliest splenomegaly occurred at12 days of incubation. Ebert (1957) implanted adult spleen into the coelomof 3- to 4-day-old embryos which at 13-14 days had a mean spleen weightfive times that of the controls. These experiments do not agree with our resultsin which a longer (6 day) latent period was observed when 8-day-old embryoswere implanted or injected and no splenomegaly ever occurred in embryosyounger than 15 days. There are indications that splenomegaly in embryos

128 J. B. SOLOMON AND D. F . TUCKER

injected before 15 days of incubation reached its maximum at 19-20 days ofincubation. Biggs & Payne (19616) showed that some splenomegaly stillpersists up to 11 days after injecting 15-day-old embryos, a similar time-courseto splenomegaly in mice (Simonsen & Jensen, 1959). Brandly, Thorp &Prickett (1949) first showed that i.v. injection of donor cells was superior toother methods in eliciting splenomegaly. In our experiments this is only trueafter 11 days, and the optimum time for the introduction of cells by implantationor i.v. injection is at 13 days to obtain maximum splenomegaly. To avoiddeath of the embryos due to other manifestations of the graft-against-the-hostreaction we have found that it is best to remove the host spleens 6 days afteri.v. injection at 13-15 days.

There is evidence that the iso-antigens responsible for transplantationimmunity 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 producedby injection or implantation of young embryos is due to lack of antigenicstimulus 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 dayslater. This indicates that implantation of tissue at 15 days may be less efficientthan at early stages of development. This may be due to a weak homograftrejection by the host embryo or to the regressive vascular changes occurring inthe 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 cellswere i.v. injected into embryos older than 15 days is in agreement with theresults of Simonsen (1957) who injected 18-day-old embryos, Cock & Simonsen(1958) who used newly hatched chicks and Isacson (1959) who injected 17- to20-day-old embryos. This suggests that the environment of older embryos isunsuitable for the proliferation of adult cells and recalls the work of Dixon &Weigle (1957) who were unable to detect antibody synthesis by sensitized adultrabbit cells transferred to 5- to 6-day-old rabbits. However, Sterzl (1957) andHolub (1958) were able to obtain satisfactory antibody synthesis under similarconditions. Sibal & Olsen (1958), Trnka (1958), Trnka & Riha (1959), Trnka &Sterzl (1960) and Isacson (1959) have all obtained evidence that adult chickenspleen cells from previously sensitized chicken inoculated into embryos, willproduce antibody while dividing in the embryo. High agglutinin titres undersuch conditions were always associated with splenomegaly (Papermaster,Bradley, Watson & Good, 1959). All this work strongly indicates that theembryonic environment will support normal functioning of adult cells. How-ever, Isacson (1959) has shown that 11- to 15-day-old chick embryos werebetter 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 at13 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 ofproliferation of donor cells because Isacson found weaker production of anti-body in the older embryos. Isacson (1959) attributed the lesser splenomegalyin the older embryos to a partial immune rejection of donor cells by the hostand described certain specific histological changes in the chimaeric spleens tosupport this idea. Other workers have suggested that the chick embryo becomescapable of immunological rejection of cells before hatching (Murphy, 1913,1914; Humphreys, 1960; Trnka & Riha, 1959), although the chick embryo haspreviously been considered to be incapable of the immune reactions of homo-graft rejection (Billingham, Brent & Medawar, 1953).

The production of immunological tolerance depends upon the establishmentof cells at certain sites where the host cells responsible for transplantationimmunity will eventually arise. As the maximum splenomegaly is obtained byinjection at 13 days, then this indicates that donor cells will become mostreadily established in the host spleen at this age. This idea is partially confirmedby the results of Cannon, Terasaki & Longmire (1957) which showed thattolerance to skin grafts, produced by injection of embryonic blood, was slightlyless at 10-11 days than when 12- to 18-day-old embryos were injected, but bothgroups showed better homograft survival than hosts injected at 1-3 days afterhatching. Further confirmation is given by the work of Billingham, Brent &Medawar (1956) who obtained a poorer survival of skin homografts whenlarge doses of adult blood were injected into newly-hatched chicks than whenembryonic blood was injected into 10- to 11-day-old embryos.

The histological appearance of lymphocytes in the chick embryo spleen atthe fifteenth to seventeenth day of incubation (Danchakoff, 1916; Sandreuter,1951; Biggs & Payne, 1961a) coincides well with the beginnings of decrease ofsplenomegaly in our work. Kalmutz (1962) states that 'physiologically matureand immunipotent lymphopoietic tissue' exists practically as soon as lymphoidelements can be identified in the opossum embryo. If reduction in the extentof splenomegaly with increasing age of the embryo is connected with the im-munological status of the host then there are indications that a steady increasein the population of cells capable of homograft rejection occurs in the embryofrom 15 days of incubation. The presence of immunologically competentlymphocytes in the 19-day-old chick embryo has recently been confirmed, as itis possible to induce transplantation immunity to skin homografts at this andlater ages but not in the 15-day-old embryo (Solomon, unpublished results).The possible ability of the chick embryo to reject foreign cells recalls earlierwork when foetal sheep were shown to be able to reject homografts long beforebirth (Schinkel & Ferguson, 1953), calf neonates were capable of rejectinghomografts (Billingham & Lampkin, 1957) and guinea-pigs and rabbits could

9


reject homografts at 1-3 days before birth (Egdahl, 1957). Recently Howard &Michie (1962) have produced evidence for the presence of immunologicallycompetent cells in newborn mice, as they could produce either tolerance orimmunity in such mice depending upon the dose of antigen. The number oflymphocytes in the chick have probably increased sufficiently by 2 days afterhatching to completely prevent the establishment and proliferation of inoculateddonor cells in the spleen of normal chicks and thus both the production ofsplenomegaly and the maintenance of tolerance to homografts. After this thelymphocytes of the chick probably increase rapidly so that at 10 days afterhatching (Simonsen, 1957; Solomon 1960, 1961a) there will be sufficient toelicit either splenomegaly when transferred to a foreign host or to reject ahomograft.

SUMMARY

1. Adult chicken spleen was implanted on to the chorio-allantoic membraneof chick embryos at 8,9,11,13 and 15 days of incubation. The initial manifesta-tion of graft against the host attack by these adult cells was an increase of hostspleen weight (splenomegaly). No splenomegaly appeared in the embryonichosts before 15 days of incubation. The appearance of splenomegaly roughlycoincided with the appearance of reticulum cell foci in the chimaeric spleens.Varying latent periods between the time of introduction of donor cells and thefirst appearance of increased spleen weights necessitated comparison of thehost spleen weights 3 days after the onset of splenomegaly. This showed thatimplantation at 13 days produced the greatest splenomegaly.

2. Only very slight splenomegaly was obtained after implantation of spleenat 15 days. A possible homograft rejection by the host and poor 'take' of thesplenic implant on the chorio-allantoic membrane are both thought to beresponsible.

3. Intravenous injection of adult chicken spleen cells into embryos at 6, 8,11 and 13 days of incubation again produced no splenomegaly before 15 daysof incubation. Comparison of the host spleen weights 3 days after onset ofsplenomegaly showed that injection at 13-15 days gave the greatest splenome-galy. Injection before this time gave very slight splenomegaly and injectionsafter 13 days produced a decreasing degree of splenomegaly with increasing ageof embryo injected.

4. Increasing the number of adult spleen cells at 15 days or giving largedoses 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 theabove pattern.

5. When 1-day-old chicks were injected with allogenic blood only slightsplenomegaly occurred and when 2-day-old chicks were injected there was nosplenomegaly 6 days later.

6. The suitability of the chick embryo as an environment for the prolifera-tion of immunologically competent cells is discussed.


7. The reduction in splenomegaly when embryos older than 13-15 days areinjected is discussed in relation to the appearance from this time of a weakhomograft response by the embryo and to the greater difficulty of inductionof both splenomegaly and tolerance to skin grafts soon after hatching.

8. It is proposed that very small numbers of immunologically competentcells appear in the embryo soon after 15 days and progressively increase fromthis 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 arediscussed.

RESUME

Attaque immunologique de Vembryon de poulet par des cellules adultes; influencede la vascularisation de la rate de Vhote et du rejet de Vhomogreffe par Vembryon

sur la splenomegalie

1. De la rate de Poulet adulte fut greffee sur la membrane chorio-allantoi-dienne d'embryons de Poulet de 8, 9, 11, 13 et 15 jours d'incubation. Lapremiere manifestation de l'attaque de l'hote par la greffe, c'est a dire par lescellules adultes de celle-ci, fut l'augmentation du poids de la rate de l'hote, ousplenomegalie. La splenomegalie n'apparut jamais chez l'embryon hote avantle 15ieme jour d'incubation. Le debut de la splenomegalie coincide a peu presavec l'apparition de foyers de cellules reticulaires dans les rates chimeres. Desperiodes de latence variables entre l'implantation des cellules donneuses et ledebut de l'accroissement du poids de la rate amenerent a comparer les poidsdes rates chez les embryons notes trois jours apres le debut de la splenomegalie.II en est apparu que l'implantation a 13 jours d'incubation causait la splenome-galie la plus importante.

2. Une splenomegalie seulement tres discrete fut observee apres implantationa 15 jours d'incubation. On peut penser qu'un eventuel rejet de l'homogreffepar l'hote ainsi qu'une mauvaise reprise de la greffe sur la membrane chorio-allantoidienne en soient les causes.

3. Des injections intraveineuses de cellules de rate de Poulet adulte a desembryons de 6, 8, 11 et 13 jours d'incubation n'ont pas produit non plus desplenomegalie avant le 15ieme jour d'incubation. L'etude du poids de la ratedes notes trois jours apres le debut de la splenomegalie montra que l'injectioneffectuee a 13-15 jours d'incubation causait la plus forte splenomegalie. Lesinjections effectuees plus tot n'ont produit qu'une tres legere splenomegalie, etles injections effectuees apres le 13ieme jour ont donne des degres de splenome-galie qui decroissent a mesure que l'embryon injecte est plus age.

4. Les resultats precedents ne furent pas modifies et la splenomegalie ne futpas notablement augmentee par l'accroissement du nombre des cellules derate adulte, ni en injectant des doses massives de sang a 8, 9, 11, 15 et 17 joursd'incubation, ainsi qu'un ou deux jours apres l'eclosion.


5. L'injection de sang etranger a des Poulets d'un jour ne causa qu'unelegere splenomegalie. Chez des Poulets de deux jours aucune splenomegaliene fut constatee six jours apres une telle injection.

6. Les avantages de l'embryon de Poulet en tant que milieu de proliferationde cellules immunologiquement competentes sont discutes.

7. La splenomegalie plus faible causee par l'injection chez des embryonsplus ages que 13-15 jours d'incubation est discutee, en relation avec Fapparitiona partir de ce moment d'une faible reponse de l'embryon a rhomogreffe, etavec la plus grande difficulte pour obtenir la splenomegalie ainsi que la toleranceinduite aux greffes de peau sitot l'eclosion.

8. II est avance qu'un tres petit nombre de cellules immunologiquementcompetentes apparaitraient chez l'embryon peu apres le 15ieme jour d'incuba-tion et se multiplieraient progressivement jusqu'a la competence de l'hote,realisee 10 jours apres l'eclosion. Les eventuelles consequences de ce develop-pement sur les degres de tolerance induite sont discutes.

ACKNOWLEDGEMENTS

We would like to thank Professor A. Haddow, F.R.S., for his interest in this work andMiss J. P. Collis for technical assistance. We are deeply indebted to Dr P. M. Biggs andDr L. N. Payne of Houghton Poultry Research Station for the histological examination ofmany 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 CancerInstitute of the National Institutes of Health, United States Public Health Service.

REFERENCES

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(Manuscript received 19th June 1962)

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