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Rosette forming cells in neonatal animals
Mechanisms of Ageing and Development, 3 (1974) 191-201
191
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
R O S E T T E F O R M I N G CELLS IN N E O N A T A L ANIMALS
R. E. CALLARD* School of Tropical Medicine, University of Sydney, Sydney, New South Wales 2006 (Australia)
(Received May 11, 1974)
SUMMARY Spleen cells, syngeneically grafted from neonatal and adult mice into lethally irradiated hosts, were challenged with Sheep Red Blood Cells (SRBC) and the resuiting immune responses compared. The maximum Plaque Forming Cell (PFC) response obtained from 108 syngeneically grafted adult spleen cells was approximately five times that obtained from 10s syngeneically grafted spleen cells from 7 day old animals. The immune potential of spleen cells obtained from mice at different times after birth correlated well with the number of spontaneous RFCs formed at either 4°C or 37°C by nucleated cells prepared from these spleens. Neonatal spleen cells were far superior to adult spleen cells in their ability to repopulate irradiated host spleens, and to prevent radiation sickness artd death. This suggests a higher proportion of pluripotent stem cells in the neonatal spleen. These cells did not, however, mature into immunocompetent cells after 7 days in the host spleen.
INTRODUCTION The onset o f immune function is first detectable in most animals late in foetal life (Solomon1). Although there is some variation between strains, a detectable immune response can usually be elicited in mice within a few days of birth, with full immunocompetence being reached two or three weeks later (Playfair2,3; Takeya and Nomoto4). Chiscon and Golub 5 have shown that the cells o f the immune system, and not the neonatal cellular environment, are probably responsible for the lack of immunocompetence of the neonatal mouse. In their experiments, combinations of thymus and bone marrow cells obtained from neonatal mice, and grafted into adult X-irradiated hosts, gave a much lower PFC response to SRBC than did cells obtained from adult animals. Further, although spleen cells from adult mice gave a strong PFC response * Present address: School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
192 to SRBC in vitro, no PFC response was elicited from spleen cells obtained from mice younger than ten days (Fidler, Chiscon and Golub6). Some authors have suggested that the lack of neonatal immunocompetenee is due to an absence of functional macrophages (ArgyrisV,S; Bendinelli, Senesi and Falconeg). However, Fidler et al. 6 by separating spleen cell preparations into glass-adhering macrophage-rich fractions and non-adhering lymphocyte-rich fractions, were able to attribute the lack o f response of neonatal spleen cells in vitro to the lymphocytes rather than the macrophages. Miller 1° was able to impair the immune development of several strains of mice by neonatal thymectomy. Although T cell function reaches adult levels in the thymus within a few days of birth (Chiscon and Golub 5) full T cell function may not be realised in the spleen for up to twenty days (Chiscon and Golub5; Howe and Manziel1011; Joel, Hess and Cottier12). This suggests that the maturation and release of T lymphocytes by the thymus, followed by their migration to the spleen and other peripheral lymphoid organs, is an important aspect of post-natal immune development. It is also known that neonatal lymphocytes do not respond as strongly to the mitogen PHA as do adult lymphocytes (Ayoub and Kasakurala; Howe and Manziel1011). Since it is thought that T cells respond better to soluble PHA than do bone marrow derived (B) cells (Greaves and Bauminger14; Rodey and Goodl5), this probably also reflects a T cell deficiency in the neonatal mouse. This accumulated evidence clearly implicates at least the thymus derived T lymphocytes as being largely responsible for the lack of neonatal immunocompetence. Little is known however, of the growth of B cell function over the period of neonatal immune development. Rosette-Forming Cells (RFC) are formed by the binding of indicator Red Blood Cells (RBC) to lymphocytes via specific cell bound antibodies (Wilson TM) and, therefore, may represent immunologically committed lymphocytes. The exact status of RFC in the immune response is however, uncertain. It has been shown that RFC are necessary for the initiation of an immune response to the particular red cell antigen which was used for their formation (Bach, Muller and Dardenne17). Brody is showed that RFC alone were insufficient for the initiation of an immune response and that T cell synergism was required for a PFC response. This suggests that RFC are in fact antibody forming cell precursors (AFCP) and are exclusively part of the B cell population. However, various proportions of RFC from the spleens of non-immune animals have been shown to be T cells; ranging from 0 ~o (Schlesinget 19) to 5 0 ~ (Argyris, Haritou and Cooney2°). Recently, Wilson and Miller zl in an attempt to settle this dispute, established the ratio o f T cells to B cells in the RFC population of non-immune animals to be 1:5. They argue that the T rosette-forming cells largely represent cells mediating a delayed hypersensitivity response to RBC. In the present work evidence is submitted to show that the number of spontaneous RFC found in l0 s neonatal spleen cells correlates well with the immune potential of this number of cells after transfer into an irradiated host. This suggests that the lack of immunocompetence in the neonatal mouse is due to an absence of immunologically committed lymphocytes in the peripheral lymphoid organs.
193 MATERIALS AND METHODS
Animals An inbred population of Albino mice has been reared at the School of Public Health and Tropical Medicine, Sydney, by twenty generations of brother-sister matings. The colony is now maintained as a closed inbred strain with random mating. This strain has not yet been named in the literature. In appreciation of Mr. Keith Stuart who originally bred the strain and now maintains it, these mice will be referred to as the KS strain. The mice were fed exclusively on Rat and Mouse Kubes obtained from Allied Stock Feeds, Rhodes, N.S.W. Australia. Food and water were available
ad libitum. Antigen Sheep RBC were obtained from the Department of Surgery at the University of Sydney. Heparinised sheep blood was mixed with an equal volume of modified Alsevers solution (Kabat and Meyer22,) and stored for one week before use. Cells were washed 4 × in phosphate buffered 0.14 M saline at p H 7.2 and resuspended to obtain the required concentration.
Irradiation Mice were subjected to 800 rads whole body irradiation from a therapeutic 6°Co source. Up to six mice were irradiated simultaneously in a circular, perforated perspex chamber. One half of the desired dose was administered from each side of the irradiation chamber.
Spleen cell transfers Spleens were excised from donor mice and the cell mass gently expressed through the split end of the spleen capsule and washed through an 80 mesh stainless steel sieve with I 0 ml of Medium 199 (with added 10 ~ Foetal Calf Serum and adjusted to p H 7.2 with 1 M NaOH). The cell suspension was passed three times through a 21 gauge needle fitted to a 10 ml syringe and any remaining clumped material was allowed to settle out. The supernatant, containing mainly single cells in suspension, was centrifuged at 400 × g for 7 minutes. The cell pellet was resuspended in 2 ml of Medium 199/10 ~ FCS and an aliquot removed for counting on a Neubauer Improved Haemocytometer. Cell viability was checked by Trypan Blue dye uptake and was found consistently to be between 90 and 95 ~ . Host mice, irradiated 24 hours previously, were grafted with l0 s nucleated cells in 0.5 to 1 ml of Medium 199 injected into the tail vein by means of Tuberculin syringe fitted with a 26 gauge needle.
Rosette formation A suspension of single spleen cells was obtained as described above. The cells were washed twice in Medium 199/5 ~o FCS and the final cell pellet resuspended in 2 ml of medium. This cell suspension was layered onto 7 ml of 100 K FCS in a 12 ml conical centrifuge tube and allowed to stand for 15 minutes to remove any remaining
194 cell clumps. The cell suspension was then removed and centrifuged through another 7 ml of 100~ FCS at 400 × g for 7 minutes. The resuspended pellet consisted of single, debris free cells (Nossal et al.23). Rosettes were formed as follows: 0.2 ml of spleen cell suspension, 1.6 ml of Medium 199/5 ~ FCS and 0.2 ml of 25 ~ SRBC were pipetted into a 76 mm × 10 mm (i.d.) serology tube. The inside diameter was checked to ensure consistency since the diameter of the tube may influence the number of rosettes formed (WilsonZ4). The cell suspension was mixed by inversion and then centrifuged at 500 × g for 10 minutes. After centrifugation the cell pellet was left for 30 minutes and then gently dispersed with a Pasteur pipette followed by 3 seconds of agitation on a Vortex-Genie (Model K-550GE). The rosettes were counted on a haemocytometer and the number of rosettes per 106 nucleated spleen cells calculated. Only those cells completely surrounded by SRBC were counted as rosettes. The entire procedure was carried out at room temperature or at 4°C as required.
PFC assay Direct plaque-forming cells were assayed by the method of Cunningham and Szenberg 25. Weights All mouse spleen and body weights were routinely recorded. RESULTS
1. Time response of syngeneically grafted spleen cells immunised with SRBC In order to demonstrate that the depressed immune response of the neonatal mouse is dependent on the cells of the immune system and not the neonatal cellular environment, the following procedure was adopted. 108 nucleated spleen cells obtained from either 7 day old or eight week old mice were grafted intravenously into adult irradiated hosts which were then immunised with 0.5 ml of 4 ~o SRBC administered intraperitoneally. Spleens were removed from the hosts at 1 day intervals and the number of PFC's per spleen determined (Fig. 1); each point represents 3-12 mice. The general shape of the time response curve obtained from the neonatal cells is the same as that obtained from adult cells. This suggests that the kinetics of both responses are very similar. In both cases the peak response was at 5 days. 108 adult spleen cells from 12 mice elicited a mean peak response of 104 PFC per spleen which was five times the mean peak response of an equal number of neonatal (7 day old) cells also taken from 12 mice (P < 0.02). All PFC assays in the experiments to be described were performed five days after immunisation. 2. Development of immune potential over the neonatal period The immune potential of 108 nucleated spleen cells obtained at different times over the neonatal period was assessed. Spleen cells were prepared from neonatal mice of ages 3, 7, 11 and 16 days, and from adult (2 month old) mice, and grafted intravenously into 12 week old host mice which had received 800 rads whole body irradia-
195
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Fig. 1. PFC time response o f neonatal and adult spleen cells transferred into an irradiated host.
Irradiated, twelve week old, host mice were grafted with 10s nucleated spleen cells from either 7 day old or 8 week old donor mice. Immunisation with 0.5 ml of 4% SRBC was carried out within 1 hour of the spleen cell transfer. Spleens were excised at daily intervals from the host mice and the PFC's per spleen determined. Between 3 and 12 mice were used at each sample point. Seven day old spleen cells elicited a response significantly lower (P < 0.02) than 8 week old cells at each sample time. PFC response of 108 transplanted 7 day old spleen cells, (Q O) and 8 week old spleen cells
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m i n i s t e r e d i n t r a p e r i t o n e a l l y w i t h i n 1 h o u r o f the s p l e e n cell t r a n s f e r , a n d t h e P F C r e s p o n s e was a s s a y e d a f t e r five days. T h e n u m b e r o f R F C in 10 s n u c l e a t e d spleen cells f r o m d o n o r m i c e was also d e t e r m i n e d a n d p l o t t e d w i t h t h e P F C (Fig. 2).
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Fig. 2. The growth of immune potential in neonatal mice. Irradiated, 12 week old, host mice were grafted with 108 nucleated spleen cells obtained from mice of ages 3, 7, 11, 16 and 56 days. Host mice were immunised with 0.5 ml of 4 ~ SRBC within 1 hour of the spleen cell transfer. Spleens were excised from the host mice 5 days after immunisation and the PFCs per spleen determined (O 0). The number of RFC per 104 nucleated spleen cells was also determined for each spleen cell preparation obtained from the neonatal mice ( O - - - © ) . It can be seen that the RFCs present in the neonatal spleen cell preparations reflects the immune potential of these cells when transferred into an irradiated host.
196 The ability of grafted neonatal spleen cells to respond to SRBC and elicit a PFC response increased rapidly from the third day of life to reach adult levels by day 16. The spontaneous RFC in 108 spleen cells also increased rapidly from day three to a maximum, adult level, at day 16. This correlation between spontaneous RFC and immune potential is consistent with RFC being at least representative of specifically committed lymphocytes and possibly identical to AFCP specific to SRBC. The suggestion that the lack of neonatal immunocompetence is due to a deficiency of specifically committed lymphocytes in the peripheral lymphoid organs follows naturally from this. It is not known however, whether neonatal spleen cells are as capable as adult spleen cells of repopulating the irradiated spleen. Nor is it known whether the transplanted spleen cells are capable of maturation in the host animal into immunocompetent cells. An attempt has been made to answer these questions in the next two sections.
3. Repopulation of irradiated spleens following syngeneic grafting with neonatal and adult spleen cells The repopulation of irradiated spleens was followed by the increase in spleen weight following grafting with syngeneic spleen ceils (Fig. 3). Spleen tissue consistently yielded 106 nucleated cells per mg wet spleen weight. Twenty four hours after 800 rads whole body irradiation, the average spleen weight of 3 month old mice had dropped from 151 mg to 72 rag. Thereafter, it decreased steadily to reach 50 mg within five days. Few mice survived longer than 10 days. Intravenous grafting of 108 eight week old spleen cells, 24 hours after irradiation, reversed this weight loss and caused the spleen to slowly increase in weight to
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Fig. 3. Spleen weights of twelve week old mice at different times after 800 rads whole body irradiation. The stippled area represents the normal ( ± one standard deviation) adult spleen weight. ( 0 O), spleen weights of irradiated mice which did not receive syngeneic spleen cells. All had died within 8 days. ( O - - O ) , spleen weights of irradiated mice grafted with 108 adult spleen cells 24 hours after irradiation. ( ÷ - - + ) , spleen weights of irradiated mice grafted with 108 neonatal spleen cells 24 hours after irradiation.
197 reach an average maximum of 120 mg six days after grafting (seven days after irradiation). This was significantly lower than the average normal spleen weight of 151 mg (0.05 > P > 0.02). Again few mice survived longer than 10 days. Three mice which were still alive after 14 days showed no sign of acute radiation poisoning (loss of weight, rhinitis, diarrhoea). They had however, a spleenomegaly with spleen weights ranging from 200-250 mg showing an induced, hyperactive spleen regeneration. It was observed in the course of this work that even without syngeneic grafting very few mice had survived six months following 800 rads whole body irradiation. These mice appeared healthy, with body and spleen weights within the normal range. It was supposed that sufficient bone marrow pluripotent stem cells may occasionally survive 800 rads irradiation allowing spleen regeneration. In the case where adult spleen cells were grafted into irradiated hosts, it is also possible that some pluripotent stem cells may have been transferred with the differentiated spleen cells thus allowing regeneration of the irradiation-destroyed cell populations. Reconstitution with 108 neonatal (3-12 day old) spleen cells resulted in a rapid increase in the weight of the irradiated host spleen. Five days after grafting the average host spleen weight was 189 mg which was significantly higher (P < 0.001) than the average normal spleen weight of 151 mg. This rapid increase continued for 8-10 days to reach a plateau of an average of 250 mg, which is very much greater than the range of normal spleen weights (125-175 mg, P < < 0.001). Furthermore, grafting of neonatal spleen ceils significantly prolonged the lifespan of the irradiated mice. Only two mice out of forty eight had died after 14 days. This suggests that pluripotent stem cells exist in the preparations of spleen cells from neonatal mice. These are capable of rapid division and differentiation, thus regenerating the depleted cell populations and apparently overpopulating the formerly depleted irradiated spleen. If this were in fact the case, it might be expected that reconstitution with neonatal spleen cells would result in an equally rapid growth in immunocompetence of these cells in the host animal. This possibility is investigated below. 4. Maturation of neonatal spleen cells in a lethally irradiated host In this experiment 108 spleen cells obtained from either adult (8 week old) or 7 day old mice were grafted into 12 week old hosts irradiated with 800 rads 24 hours previously. The host mice were challenged with 0.5 ml of 4 ~ SRBC at times of 0, 3, 7 and 14 days after grafting, and the PFC response was assayed after a further 5 days. It has been shown (Fig. 2) that with this KS strain of mice, an adult response to SRBC is acquired at 2-3 weeks of age. It was thought that if the neonatal spleen contains immature immunocytes, these may develop into fully immunocompetent cells in the irradiated host spleen. Thus, spleen cells obtained from 7 day old mice, grafted into an adult irradiated host and challenged with SRBC 7 days later, might be expected to elicit a PFC response approaching that of adult cells. If, however, full immunocompetence is attained in the neonatal spleen by the migration of immunocompetent cells from other organs, then no such maturation in the host spleen would be expected. All irradiated animals grafted with adult spleen cells died within 9 days. However, those animals grafted with neonatal (7 day old) spleen cells were still alive
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Fig. 4. The immune potential of syngeneically grafted adult and neonatal spleen cells challenged with SRBC various times after grafting. The hatched areas represent the immune response of l0 s adult spleen cells challenged with 0.5 ml of 4% SRBC at 0, 7 and 14 days after transfer into an irradiated host. The stippled areas are the same for 10s neonatal (7 day) spleen cells. (A), spleen cells challenged on the same day as the spleen cell transfer. (B), spleen cells challenged 7 days after transfer and (C), 14 days after transfer.
after 2 weeks. It can be seen that within 7 days at least, no maturation of the neonatal cells had occurred in the host spleen (Fig. 4). In fact, the immune potential of both the adult and the neonatal spleen cells had diminished while in the host spleen. After 14 days in the host spleen however, the neonatal cells were capable of eliciting a response approaching that of adult cells. As mentioned above, it appears that neonatal spleen cells contain pluripotent stem cells capable of repopulating irradiation depleted lymphoid tissues. It is suggested therefore, that this late maturation of the immune potential of neonatal cells in the irradiated host is due to the repopulation of the host bone marrow followed by lymphopoiesis and reconstitution of the hosts immune system, rather than simple maturation of committed immunocytes already present in the neonatal spleen cell preparation. It is considered unlikely that the observed delay was due to experimental trauma. No such delay was observed in the kinetics of the immune response of transplanted spleen cells immunised at the time of transfer, as compared to the intact animal. In both cases the peak responses were observed by this author to be at five days. DISCUSSION
The lack of immunocompetence in the neonatal mouse has been investigated in many different strains (Hechtel et al.~6; Playfair2,a; Solomon1; Takeya, and Nomoto4), however the cause of this deficiency has not yet been clearly shown. Several workers have significantly increased the response of neonatal mice to SRBC with syngeneically grafted adult macrophages, thereby suggesting a lack of competence in macrophage cells in the neonatal immune mechanism. (Argyris7,8; Bendinelli et al.9). These authors however, used peritoneal exudate as a source of macrophages which also contains many other monocytes including T and B lymphocytes. Fidler et al. 6 have demonstrated the functional maturity of glass adhering cells, but
199 the lack of immunocompetence of non adhering cells, obtained from the neonatal mouse spleen. They suggest that the lack of immunocompetence of young spleen cells is associated with the presence of pluripotent stem cells and an absence of committed lymphocytes. Immunocompetent lymphocytes may be divided into two components; the bone marrow derived or bursa equivalent (B) cells which are the direct precursors of antibody producing cells, and thymus influenced helper (T) cells. MacGillivray e t al. 27 have compared the ability of thymocytes from mice of different ages to restore immunocompetence to neonatally thymectomised mice. Foetal thymocytes had no effect. At birth, the ability of thymocytes to restore the immune capacity of the thymectomised mice was low, but increased rapidly to reach a maximum at 4 weeks. Similarly, Chiscon and Golub 5 demonstrated that at birth T cell function in CBA mice was 1 ~ that of the adult. T cell function in the thymus approached that of the adult after 48 hours, but did not appear in the spleen for 4 days. This suggests that T cell function is acquired in the thymus and later migrates to the spleen. The position with B cell function is not so clear. The bursa of Fabricius is the organ in chickens in which B cell function is attained. In mammals however no equivalent organ has been identified. B cell function has been observed in the mouse spleen one day after birth (Chiscon and GolubS), but it was not clear whether it was acquired in the spleen, or elsewhere, the mature cells later migrating to the spleen. Many authors have attributed the lack of neonatal immunocompetence to T cells. However, few have investigated the possibility of an absence of AFCP (B) cells. It has been suggested that spontaneous RFC formed at 4°C represent AntibodyForming Cell Precursors (AFCP) (Wilson16,24; Miller and Phillips28). Bach, Muller and Dardenne 17 have demonstrated that the removal from spleen cell preparations of RFC formed with SRBC, specifically eliminated the ability of these cells to respond to SRBC in an irradiated host. The ability of these cells to respond to chicken RBC remained however, intact. The majority (80~) of non immune RFC are B cells (Wilson and Miller zl) and these probably represent AFCP. Thus, from the results shown in Fig. 2 demonstrating a correlation of the immune potential of neonatal spleen cells with the presence of spontaneous RFC in the neonatal spleen, it is concluded that the low response of neonatal spleen cells to challenge with SRBC is due to a deficiency of committed B lymphocytes (AFCP). It might be suggested that the lack of a functional microenvironment in the neonatal spleen prevents the maturation of stem cells into committed immunocytes. Indeed the importance of the micro-environment of lymphoid tissues in the differentiation and maturation of pluripotent stem cells has been amply demonstrated (ThomasZg). However, the results illustrated in Fig. 4 indicate that, although the neonatal spleen contains stem, or precursor cells, no immunocompetent cells appear in the host spleen for more than 7 days after the transfer of neonatal cells into an adult irradiated host. This suggests that neonatal cells in an adult environment do not acquire immunocompetence any more rapidly than they do in a neonatal environment. In fact, although it might be expected that spleen cells obtained from 7 day old mice, transferred into an adult host and challenged with SRBC after a further 7 days, would
200 elicit a response equivalent to spleen cells from 14 day old mice, this was not observed. This is consistent with stem cells acquiring i m m u n o c o m p e t e n c e elsewhere later migrating to the spleen. It would appear from the superior ability of n e o n a t a l spleen cells to repopulate the depleted irradiated spleen and, in contrast to adult spleen cells (Fig. 3), to considerably p r o l o n g the life span of the lethally irradiated mouse, that the neonatal spleen contains a much higher p r o p o r t i o n o f p l u r i p o t e n t stem cells t h a n the adult spleen. P r e s u m a b l y the p l u r i p o t e n t stem cells present in the neonatal spleen are capable of r e p o p u l a t i n g l y m p h o i d a n d p r o b a b l y erythroid tissues in the irradiated mouse. This would lead eventually to the r e p o p u l a t i o n o f the spleen a n d t h y m u s thus restoring full i m m u n o c o m p e t e n c e to the irradiated animal. The initial decrease in the i m m u n e potential o f the t r a n s p l a n t e d neonatal spleen cells (Fig. 4) followed by an eventual restoration of i m m u n e function supports this concept, rather t h a n the acquisition of functional m a t u r i t y in the t r a n s p l a n t e d spleen cells within the host spleen.
REFERENCES 1 J. B. Solomon, Unification of foetal and neonatal immunology, Nature, 227 (1970) 895. 2 J. H. L. Playfair, Strain differences in the immune response of mice. 1. The neonatal response to sheep red cells, Immunology, 15 (1968) 35. 3 J. H. L. Playfair, Strain differences in the immune response of mice. I1. Responses by neonatal cells in irradiated adult hosts, Immunology, 15 (1968) 815. 4 K. Takeya and K. Nomoto, Characteristics of antibody response in young or thymectomized mice, J. Immunol., 99 (1967) 831. 5 M. O. Chiscon and E. S. Golub, Functional development of the interacting cells in the immune response. I. Development of T cell and B cell function, J. lmn,unol., 108 (1972) 1379. 6 J. M. Fidler, M. O. Chiscon and E. S. Golub, Functional development of the interacting cells in the immune response. II. Development of immunocompetence to heterolcgous erythrccytes in vitro, J. lmmunol., 109 (1972) 136. 7 B. F. Argyris, Role of macrophages in immunological maturation, J. Exp. Med., 128 (1968) 459. 8 B. F. Argyris, Transplantation of adult peritoneal cells into newborn mice, Transplantation, 8 (1969) 241. 9 M. Bendinelli, S. Senesi and G, Falcone, Effect of adult peritoneal cells on the antibody response of newborn mice to sheep red blood cells, J. lmmunol., 106 (1971) 1681. 10 J. F. A. P. Miller, Effect of neonatal thymectomy on the immunological responsiveness of the mouse, Proc. Roy. Soc. London Ser. B, 156 (1962) 415. 11 M. L. Howe and B. Manziello, Ontogenesis of the in vitro response of murine lymphoid cells to cellular antigens and phytomitogens, J. Immunol., 109 (1972) 534. 12 D. D. Joel, M. W. Hess and H. Cottier, Magnitude and pattern of thymic lymphocyte migration in neonatal mice, J. Exp. Med., 135 (1972) 907. 13 J. Ayoub and S. Kasakura, In vitro response of foetal lymphocytes to PHA, and a factor plasma which suppresses the PHA response of adult lymphocytes, C/in. Exp. hmnunol., 8 (1971) 427. 14 M. F. Greaves and S. Bauminger, Activation ofT and B lymphocytes by insoluble phytomitogens, Nature New Biol., 235 (1972) 67. 15 G. E. Rodey and R. A. Good, The in vitro response to phytohaemagglutinin of lymphoid cells from normal and neonatally thymectomized adult mice, Int. Arch. Allergy Appl. lmmunol., 36 (1969) 399. 16 J. D. Wilson, immunoglobulin determinants on rosette-forming cells: their changing nature during an immune response, Aust. J. Exp. Biol. Med. Sei., 49 (1971) 415. 17 J. F. Bach, J. Y. Muller and M. Dardenne, In vivo specific antigen recognition by rosette forming cells, Nature, 227 (1970) 1251. 18 T. Brody, Identification of two cell populations required for mouse immunocompetence, J. hnmunol., 105 (1970) 126.
201 19 M. Schlesinger, Anti O antibodies for detecting thymus-dependent lymphocytes in the immune response of mice to SRBC, Nature, 226 (1970) 1254. 20 B. F. Argyris, H. Haritou and A. Cooney, Density gradient fractionation of mouse lymphoid tissues. 1. Plaque forming and rosette forming cells in normal, sensitized and tolerant spleen, Cell. Immunol., 3 (1972) 101. 21 J. D. Wilson and J. F. A. P. Miller, T and B rosette-forming cells, Eur. J. lmmunol., 1 (1971) 501. 22 E. A. Kabat and M. M. Mayer, Experimental lmmunoehemistry, 2nd Edn., Thomas, Springfield, 1961, p. 149. 23 G. J. V. Nossal, N. L. Warner, H. Lewis and J. Sprent, Quantitative features of a sandwich radioimmunolabelling technique for lymphocyte surface receptors, J. Exp. Med., 135 (1972) 405. 24 J. D. Wilson, The relationship of antibody-forming cells to rosette-forming cells, Immunology, 21 (1971) 233. 25 A. J. Cunningham and A. Szenberg, Further improvements in the plaque technique for detecting single antibody forming cells, Immunology, 14 (1968) 599. 26 M. Hechtel, T. Dishon and W. Braun, Hemolysin formation in newborn mice of different strains, Proc. Soc. Exp. Biol. Med., 120 (1965) 728. 27 M. H. MacGillivray, B. Mayhew and N. R. Rose, A comparison of the immunologic function of thymus cells at varying stages of maturation, Proe. Soc. Exp. Biol. Med., 133 (1970) 688. 28 R. G. Miller and R. A. Phillips, Sedimentation analysis of the cells in mice required to initiate an in vivo immune response to sheep erythrocytes, Proe. Soe. Exp. Biol. Med., 135 (1970) 63. 29 D. B. Thomas, The differentiation of transplanted haemapoietic cells derived from bone marrow, spleen and foetal liver. J. Anat., 110 (1971) 297.
191
© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
R O S E T T E F O R M I N G CELLS IN N E O N A T A L ANIMALS
R. E. CALLARD* School of Tropical Medicine, University of Sydney, Sydney, New South Wales 2006 (Australia)
(Received May 11, 1974)
SUMMARY Spleen cells, syngeneically grafted from neonatal and adult mice into lethally irradiated hosts, were challenged with Sheep Red Blood Cells (SRBC) and the resuiting immune responses compared. The maximum Plaque Forming Cell (PFC) response obtained from 108 syngeneically grafted adult spleen cells was approximately five times that obtained from 10s syngeneically grafted spleen cells from 7 day old animals. The immune potential of spleen cells obtained from mice at different times after birth correlated well with the number of spontaneous RFCs formed at either 4°C or 37°C by nucleated cells prepared from these spleens. Neonatal spleen cells were far superior to adult spleen cells in their ability to repopulate irradiated host spleens, and to prevent radiation sickness artd death. This suggests a higher proportion of pluripotent stem cells in the neonatal spleen. These cells did not, however, mature into immunocompetent cells after 7 days in the host spleen.
INTRODUCTION The onset o f immune function is first detectable in most animals late in foetal life (Solomon1). Although there is some variation between strains, a detectable immune response can usually be elicited in mice within a few days of birth, with full immunocompetence being reached two or three weeks later (Playfair2,3; Takeya and Nomoto4). Chiscon and Golub 5 have shown that the cells o f the immune system, and not the neonatal cellular environment, are probably responsible for the lack of immunocompetence of the neonatal mouse. In their experiments, combinations of thymus and bone marrow cells obtained from neonatal mice, and grafted into adult X-irradiated hosts, gave a much lower PFC response to SRBC than did cells obtained from adult animals. Further, although spleen cells from adult mice gave a strong PFC response * Present address: School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
192 to SRBC in vitro, no PFC response was elicited from spleen cells obtained from mice younger than ten days (Fidler, Chiscon and Golub6). Some authors have suggested that the lack of neonatal immunocompetenee is due to an absence of functional macrophages (ArgyrisV,S; Bendinelli, Senesi and Falconeg). However, Fidler et al. 6 by separating spleen cell preparations into glass-adhering macrophage-rich fractions and non-adhering lymphocyte-rich fractions, were able to attribute the lack o f response of neonatal spleen cells in vitro to the lymphocytes rather than the macrophages. Miller 1° was able to impair the immune development of several strains of mice by neonatal thymectomy. Although T cell function reaches adult levels in the thymus within a few days of birth (Chiscon and Golub 5) full T cell function may not be realised in the spleen for up to twenty days (Chiscon and Golub5; Howe and Manziel1011; Joel, Hess and Cottier12). This suggests that the maturation and release of T lymphocytes by the thymus, followed by their migration to the spleen and other peripheral lymphoid organs, is an important aspect of post-natal immune development. It is also known that neonatal lymphocytes do not respond as strongly to the mitogen PHA as do adult lymphocytes (Ayoub and Kasakurala; Howe and Manziel1011). Since it is thought that T cells respond better to soluble PHA than do bone marrow derived (B) cells (Greaves and Bauminger14; Rodey and Goodl5), this probably also reflects a T cell deficiency in the neonatal mouse. This accumulated evidence clearly implicates at least the thymus derived T lymphocytes as being largely responsible for the lack of neonatal immunocompetence. Little is known however, of the growth of B cell function over the period of neonatal immune development. Rosette-Forming Cells (RFC) are formed by the binding of indicator Red Blood Cells (RBC) to lymphocytes via specific cell bound antibodies (Wilson TM) and, therefore, may represent immunologically committed lymphocytes. The exact status of RFC in the immune response is however, uncertain. It has been shown that RFC are necessary for the initiation of an immune response to the particular red cell antigen which was used for their formation (Bach, Muller and Dardenne17). Brody is showed that RFC alone were insufficient for the initiation of an immune response and that T cell synergism was required for a PFC response. This suggests that RFC are in fact antibody forming cell precursors (AFCP) and are exclusively part of the B cell population. However, various proportions of RFC from the spleens of non-immune animals have been shown to be T cells; ranging from 0 ~o (Schlesinget 19) to 5 0 ~ (Argyris, Haritou and Cooney2°). Recently, Wilson and Miller zl in an attempt to settle this dispute, established the ratio o f T cells to B cells in the RFC population of non-immune animals to be 1:5. They argue that the T rosette-forming cells largely represent cells mediating a delayed hypersensitivity response to RBC. In the present work evidence is submitted to show that the number of spontaneous RFC found in l0 s neonatal spleen cells correlates well with the immune potential of this number of cells after transfer into an irradiated host. This suggests that the lack of immunocompetence in the neonatal mouse is due to an absence of immunologically committed lymphocytes in the peripheral lymphoid organs.
193 MATERIALS AND METHODS
Animals An inbred population of Albino mice has been reared at the School of Public Health and Tropical Medicine, Sydney, by twenty generations of brother-sister matings. The colony is now maintained as a closed inbred strain with random mating. This strain has not yet been named in the literature. In appreciation of Mr. Keith Stuart who originally bred the strain and now maintains it, these mice will be referred to as the KS strain. The mice were fed exclusively on Rat and Mouse Kubes obtained from Allied Stock Feeds, Rhodes, N.S.W. Australia. Food and water were available
ad libitum. Antigen Sheep RBC were obtained from the Department of Surgery at the University of Sydney. Heparinised sheep blood was mixed with an equal volume of modified Alsevers solution (Kabat and Meyer22,) and stored for one week before use. Cells were washed 4 × in phosphate buffered 0.14 M saline at p H 7.2 and resuspended to obtain the required concentration.
Irradiation Mice were subjected to 800 rads whole body irradiation from a therapeutic 6°Co source. Up to six mice were irradiated simultaneously in a circular, perforated perspex chamber. One half of the desired dose was administered from each side of the irradiation chamber.
Spleen cell transfers Spleens were excised from donor mice and the cell mass gently expressed through the split end of the spleen capsule and washed through an 80 mesh stainless steel sieve with I 0 ml of Medium 199 (with added 10 ~ Foetal Calf Serum and adjusted to p H 7.2 with 1 M NaOH). The cell suspension was passed three times through a 21 gauge needle fitted to a 10 ml syringe and any remaining clumped material was allowed to settle out. The supernatant, containing mainly single cells in suspension, was centrifuged at 400 × g for 7 minutes. The cell pellet was resuspended in 2 ml of Medium 199/10 ~ FCS and an aliquot removed for counting on a Neubauer Improved Haemocytometer. Cell viability was checked by Trypan Blue dye uptake and was found consistently to be between 90 and 95 ~ . Host mice, irradiated 24 hours previously, were grafted with l0 s nucleated cells in 0.5 to 1 ml of Medium 199 injected into the tail vein by means of Tuberculin syringe fitted with a 26 gauge needle.
Rosette formation A suspension of single spleen cells was obtained as described above. The cells were washed twice in Medium 199/5 ~o FCS and the final cell pellet resuspended in 2 ml of medium. This cell suspension was layered onto 7 ml of 100 K FCS in a 12 ml conical centrifuge tube and allowed to stand for 15 minutes to remove any remaining
194 cell clumps. The cell suspension was then removed and centrifuged through another 7 ml of 100~ FCS at 400 × g for 7 minutes. The resuspended pellet consisted of single, debris free cells (Nossal et al.23). Rosettes were formed as follows: 0.2 ml of spleen cell suspension, 1.6 ml of Medium 199/5 ~ FCS and 0.2 ml of 25 ~ SRBC were pipetted into a 76 mm × 10 mm (i.d.) serology tube. The inside diameter was checked to ensure consistency since the diameter of the tube may influence the number of rosettes formed (WilsonZ4). The cell suspension was mixed by inversion and then centrifuged at 500 × g for 10 minutes. After centrifugation the cell pellet was left for 30 minutes and then gently dispersed with a Pasteur pipette followed by 3 seconds of agitation on a Vortex-Genie (Model K-550GE). The rosettes were counted on a haemocytometer and the number of rosettes per 106 nucleated spleen cells calculated. Only those cells completely surrounded by SRBC were counted as rosettes. The entire procedure was carried out at room temperature or at 4°C as required.
PFC assay Direct plaque-forming cells were assayed by the method of Cunningham and Szenberg 25. Weights All mouse spleen and body weights were routinely recorded. RESULTS
1. Time response of syngeneically grafted spleen cells immunised with SRBC In order to demonstrate that the depressed immune response of the neonatal mouse is dependent on the cells of the immune system and not the neonatal cellular environment, the following procedure was adopted. 108 nucleated spleen cells obtained from either 7 day old or eight week old mice were grafted intravenously into adult irradiated hosts which were then immunised with 0.5 ml of 4 ~o SRBC administered intraperitoneally. Spleens were removed from the hosts at 1 day intervals and the number of PFC's per spleen determined (Fig. 1); each point represents 3-12 mice. The general shape of the time response curve obtained from the neonatal cells is the same as that obtained from adult cells. This suggests that the kinetics of both responses are very similar. In both cases the peak response was at 5 days. 108 adult spleen cells from 12 mice elicited a mean peak response of 104 PFC per spleen which was five times the mean peak response of an equal number of neonatal (7 day old) cells also taken from 12 mice (P < 0.02). All PFC assays in the experiments to be described were performed five days after immunisation. 2. Development of immune potential over the neonatal period The immune potential of 108 nucleated spleen cells obtained at different times over the neonatal period was assessed. Spleen cells were prepared from neonatal mice of ages 3, 7, 11 and 16 days, and from adult (2 month old) mice, and grafted intravenously into 12 week old host mice which had received 800 rads whole body irradia-
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Irradiated, twelve week old, host mice were grafted with 10s nucleated spleen cells from either 7 day old or 8 week old donor mice. Immunisation with 0.5 ml of 4% SRBC was carried out within 1 hour of the spleen cell transfer. Spleens were excised at daily intervals from the host mice and the PFC's per spleen determined. Between 3 and 12 mice were used at each sample point. Seven day old spleen cells elicited a response significantly lower (P < 0.02) than 8 week old cells at each sample time. PFC response of 108 transplanted 7 day old spleen cells, (Q O) and 8 week old spleen cells
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Fig. 2. The growth of immune potential in neonatal mice. Irradiated, 12 week old, host mice were grafted with 108 nucleated spleen cells obtained from mice of ages 3, 7, 11, 16 and 56 days. Host mice were immunised with 0.5 ml of 4 ~ SRBC within 1 hour of the spleen cell transfer. Spleens were excised from the host mice 5 days after immunisation and the PFCs per spleen determined (O 0). The number of RFC per 104 nucleated spleen cells was also determined for each spleen cell preparation obtained from the neonatal mice ( O - - - © ) . It can be seen that the RFCs present in the neonatal spleen cell preparations reflects the immune potential of these cells when transferred into an irradiated host.
196 The ability of grafted neonatal spleen cells to respond to SRBC and elicit a PFC response increased rapidly from the third day of life to reach adult levels by day 16. The spontaneous RFC in 108 spleen cells also increased rapidly from day three to a maximum, adult level, at day 16. This correlation between spontaneous RFC and immune potential is consistent with RFC being at least representative of specifically committed lymphocytes and possibly identical to AFCP specific to SRBC. The suggestion that the lack of neonatal immunocompetence is due to a deficiency of specifically committed lymphocytes in the peripheral lymphoid organs follows naturally from this. It is not known however, whether neonatal spleen cells are as capable as adult spleen cells of repopulating the irradiated spleen. Nor is it known whether the transplanted spleen cells are capable of maturation in the host animal into immunocompetent cells. An attempt has been made to answer these questions in the next two sections.
3. Repopulation of irradiated spleens following syngeneic grafting with neonatal and adult spleen cells The repopulation of irradiated spleens was followed by the increase in spleen weight following grafting with syngeneic spleen ceils (Fig. 3). Spleen tissue consistently yielded 106 nucleated cells per mg wet spleen weight. Twenty four hours after 800 rads whole body irradiation, the average spleen weight of 3 month old mice had dropped from 151 mg to 72 rag. Thereafter, it decreased steadily to reach 50 mg within five days. Few mice survived longer than 10 days. Intravenous grafting of 108 eight week old spleen cells, 24 hours after irradiation, reversed this weight loss and caused the spleen to slowly increase in weight to
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197 reach an average maximum of 120 mg six days after grafting (seven days after irradiation). This was significantly lower than the average normal spleen weight of 151 mg (0.05 > P > 0.02). Again few mice survived longer than 10 days. Three mice which were still alive after 14 days showed no sign of acute radiation poisoning (loss of weight, rhinitis, diarrhoea). They had however, a spleenomegaly with spleen weights ranging from 200-250 mg showing an induced, hyperactive spleen regeneration. It was observed in the course of this work that even without syngeneic grafting very few mice had survived six months following 800 rads whole body irradiation. These mice appeared healthy, with body and spleen weights within the normal range. It was supposed that sufficient bone marrow pluripotent stem cells may occasionally survive 800 rads irradiation allowing spleen regeneration. In the case where adult spleen cells were grafted into irradiated hosts, it is also possible that some pluripotent stem cells may have been transferred with the differentiated spleen cells thus allowing regeneration of the irradiation-destroyed cell populations. Reconstitution with 108 neonatal (3-12 day old) spleen cells resulted in a rapid increase in the weight of the irradiated host spleen. Five days after grafting the average host spleen weight was 189 mg which was significantly higher (P < 0.001) than the average normal spleen weight of 151 mg. This rapid increase continued for 8-10 days to reach a plateau of an average of 250 mg, which is very much greater than the range of normal spleen weights (125-175 mg, P < < 0.001). Furthermore, grafting of neonatal spleen ceils significantly prolonged the lifespan of the irradiated mice. Only two mice out of forty eight had died after 14 days. This suggests that pluripotent stem cells exist in the preparations of spleen cells from neonatal mice. These are capable of rapid division and differentiation, thus regenerating the depleted cell populations and apparently overpopulating the formerly depleted irradiated spleen. If this were in fact the case, it might be expected that reconstitution with neonatal spleen cells would result in an equally rapid growth in immunocompetence of these cells in the host animal. This possibility is investigated below. 4. Maturation of neonatal spleen cells in a lethally irradiated host In this experiment 108 spleen cells obtained from either adult (8 week old) or 7 day old mice were grafted into 12 week old hosts irradiated with 800 rads 24 hours previously. The host mice were challenged with 0.5 ml of 4 ~ SRBC at times of 0, 3, 7 and 14 days after grafting, and the PFC response was assayed after a further 5 days. It has been shown (Fig. 2) that with this KS strain of mice, an adult response to SRBC is acquired at 2-3 weeks of age. It was thought that if the neonatal spleen contains immature immunocytes, these may develop into fully immunocompetent cells in the irradiated host spleen. Thus, spleen cells obtained from 7 day old mice, grafted into an adult irradiated host and challenged with SRBC 7 days later, might be expected to elicit a PFC response approaching that of adult cells. If, however, full immunocompetence is attained in the neonatal spleen by the migration of immunocompetent cells from other organs, then no such maturation in the host spleen would be expected. All irradiated animals grafted with adult spleen cells died within 9 days. However, those animals grafted with neonatal (7 day old) spleen cells were still alive
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Fig. 4. The immune potential of syngeneically grafted adult and neonatal spleen cells challenged with SRBC various times after grafting. The hatched areas represent the immune response of l0 s adult spleen cells challenged with 0.5 ml of 4% SRBC at 0, 7 and 14 days after transfer into an irradiated host. The stippled areas are the same for 10s neonatal (7 day) spleen cells. (A), spleen cells challenged on the same day as the spleen cell transfer. (B), spleen cells challenged 7 days after transfer and (C), 14 days after transfer.
after 2 weeks. It can be seen that within 7 days at least, no maturation of the neonatal cells had occurred in the host spleen (Fig. 4). In fact, the immune potential of both the adult and the neonatal spleen cells had diminished while in the host spleen. After 14 days in the host spleen however, the neonatal cells were capable of eliciting a response approaching that of adult cells. As mentioned above, it appears that neonatal spleen cells contain pluripotent stem cells capable of repopulating irradiation depleted lymphoid tissues. It is suggested therefore, that this late maturation of the immune potential of neonatal cells in the irradiated host is due to the repopulation of the host bone marrow followed by lymphopoiesis and reconstitution of the hosts immune system, rather than simple maturation of committed immunocytes already present in the neonatal spleen cell preparation. It is considered unlikely that the observed delay was due to experimental trauma. No such delay was observed in the kinetics of the immune response of transplanted spleen cells immunised at the time of transfer, as compared to the intact animal. In both cases the peak responses were observed by this author to be at five days. DISCUSSION
The lack of immunocompetence in the neonatal mouse has been investigated in many different strains (Hechtel et al.~6; Playfair2,a; Solomon1; Takeya, and Nomoto4), however the cause of this deficiency has not yet been clearly shown. Several workers have significantly increased the response of neonatal mice to SRBC with syngeneically grafted adult macrophages, thereby suggesting a lack of competence in macrophage cells in the neonatal immune mechanism. (Argyris7,8; Bendinelli et al.9). These authors however, used peritoneal exudate as a source of macrophages which also contains many other monocytes including T and B lymphocytes. Fidler et al. 6 have demonstrated the functional maturity of glass adhering cells, but
199 the lack of immunocompetence of non adhering cells, obtained from the neonatal mouse spleen. They suggest that the lack of immunocompetence of young spleen cells is associated with the presence of pluripotent stem cells and an absence of committed lymphocytes. Immunocompetent lymphocytes may be divided into two components; the bone marrow derived or bursa equivalent (B) cells which are the direct precursors of antibody producing cells, and thymus influenced helper (T) cells. MacGillivray e t al. 27 have compared the ability of thymocytes from mice of different ages to restore immunocompetence to neonatally thymectomised mice. Foetal thymocytes had no effect. At birth, the ability of thymocytes to restore the immune capacity of the thymectomised mice was low, but increased rapidly to reach a maximum at 4 weeks. Similarly, Chiscon and Golub 5 demonstrated that at birth T cell function in CBA mice was 1 ~ that of the adult. T cell function in the thymus approached that of the adult after 48 hours, but did not appear in the spleen for 4 days. This suggests that T cell function is acquired in the thymus and later migrates to the spleen. The position with B cell function is not so clear. The bursa of Fabricius is the organ in chickens in which B cell function is attained. In mammals however no equivalent organ has been identified. B cell function has been observed in the mouse spleen one day after birth (Chiscon and GolubS), but it was not clear whether it was acquired in the spleen, or elsewhere, the mature cells later migrating to the spleen. Many authors have attributed the lack of neonatal immunocompetence to T cells. However, few have investigated the possibility of an absence of AFCP (B) cells. It has been suggested that spontaneous RFC formed at 4°C represent AntibodyForming Cell Precursors (AFCP) (Wilson16,24; Miller and Phillips28). Bach, Muller and Dardenne 17 have demonstrated that the removal from spleen cell preparations of RFC formed with SRBC, specifically eliminated the ability of these cells to respond to SRBC in an irradiated host. The ability of these cells to respond to chicken RBC remained however, intact. The majority (80~) of non immune RFC are B cells (Wilson and Miller zl) and these probably represent AFCP. Thus, from the results shown in Fig. 2 demonstrating a correlation of the immune potential of neonatal spleen cells with the presence of spontaneous RFC in the neonatal spleen, it is concluded that the low response of neonatal spleen cells to challenge with SRBC is due to a deficiency of committed B lymphocytes (AFCP). It might be suggested that the lack of a functional microenvironment in the neonatal spleen prevents the maturation of stem cells into committed immunocytes. Indeed the importance of the micro-environment of lymphoid tissues in the differentiation and maturation of pluripotent stem cells has been amply demonstrated (ThomasZg). However, the results illustrated in Fig. 4 indicate that, although the neonatal spleen contains stem, or precursor cells, no immunocompetent cells appear in the host spleen for more than 7 days after the transfer of neonatal cells into an adult irradiated host. This suggests that neonatal cells in an adult environment do not acquire immunocompetence any more rapidly than they do in a neonatal environment. In fact, although it might be expected that spleen cells obtained from 7 day old mice, transferred into an adult host and challenged with SRBC after a further 7 days, would
200 elicit a response equivalent to spleen cells from 14 day old mice, this was not observed. This is consistent with stem cells acquiring i m m u n o c o m p e t e n c e elsewhere later migrating to the spleen. It would appear from the superior ability of n e o n a t a l spleen cells to repopulate the depleted irradiated spleen and, in contrast to adult spleen cells (Fig. 3), to considerably p r o l o n g the life span of the lethally irradiated mouse, that the neonatal spleen contains a much higher p r o p o r t i o n o f p l u r i p o t e n t stem cells t h a n the adult spleen. P r e s u m a b l y the p l u r i p o t e n t stem cells present in the neonatal spleen are capable of r e p o p u l a t i n g l y m p h o i d a n d p r o b a b l y erythroid tissues in the irradiated mouse. This would lead eventually to the r e p o p u l a t i o n o f the spleen a n d t h y m u s thus restoring full i m m u n o c o m p e t e n c e to the irradiated animal. The initial decrease in the i m m u n e potential o f the t r a n s p l a n t e d neonatal spleen cells (Fig. 4) followed by an eventual restoration of i m m u n e function supports this concept, rather t h a n the acquisition of functional m a t u r i t y in the t r a n s p l a n t e d spleen cells within the host spleen.
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201 19 M. Schlesinger, Anti O antibodies for detecting thymus-dependent lymphocytes in the immune response of mice to SRBC, Nature, 226 (1970) 1254. 20 B. F. Argyris, H. Haritou and A. Cooney, Density gradient fractionation of mouse lymphoid tissues. 1. Plaque forming and rosette forming cells in normal, sensitized and tolerant spleen, Cell. Immunol., 3 (1972) 101. 21 J. D. Wilson and J. F. A. P. Miller, T and B rosette-forming cells, Eur. J. lmmunol., 1 (1971) 501. 22 E. A. Kabat and M. M. Mayer, Experimental lmmunoehemistry, 2nd Edn., Thomas, Springfield, 1961, p. 149. 23 G. J. V. Nossal, N. L. Warner, H. Lewis and J. Sprent, Quantitative features of a sandwich radioimmunolabelling technique for lymphocyte surface receptors, J. Exp. Med., 135 (1972) 405. 24 J. D. Wilson, The relationship of antibody-forming cells to rosette-forming cells, Immunology, 21 (1971) 233. 25 A. J. Cunningham and A. Szenberg, Further improvements in the plaque technique for detecting single antibody forming cells, Immunology, 14 (1968) 599. 26 M. Hechtel, T. Dishon and W. Braun, Hemolysin formation in newborn mice of different strains, Proc. Soc. Exp. Biol. Med., 120 (1965) 728. 27 M. H. MacGillivray, B. Mayhew and N. R. Rose, A comparison of the immunologic function of thymus cells at varying stages of maturation, Proe. Soc. Exp. Biol. Med., 133 (1970) 688. 28 R. G. Miller and R. A. Phillips, Sedimentation analysis of the cells in mice required to initiate an in vivo immune response to sheep erythrocytes, Proe. Soe. Exp. Biol. Med., 135 (1970) 63. 29 D. B. Thomas, The differentiation of transplanted haemapoietic cells derived from bone marrow, spleen and foetal liver. J. Anat., 110 (1971) 297.