Proc. Natl. Acad. Sci. USA
Vol. 73, No. 7, pp. 2462-2466, July 1976
Immunology
Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes (differentiation/tolerance)
C. BRUYNS, G. URBAIN-VANSANTEN, C. PLANARD, C. DE VOS-CLOETENS, AND JI URBAIN Laboratory of Animal Physiology, Universit6 Libre de Bruxelles, 67, rue des Chevaux, 1640, Rhode-St-Genese, Belgium
Communicated by J. Brachet, April 6,1976
ABSTRACT Taking advantage of recent findings about membrane fluidity, we have studied and compared the biosynthetic capacities of fetal or neonatal mouse B (bone-marrow derived) lymphocytes (until 10 days after birth) and adult B lymphocytes. Although both early and adult lymphocytes can synthesize surface immunoglobulins, they have a different physiological behavior after interaction with a ligand (antiimmunoglobulin sera or antigen), either in vivo or in vitro. Fetal and neonatal lymphocytes bearing surface immunoglobulins do not reexpress their membrane receptors after capping and endocytosis promoted by anti-immunoglobulin sera. On the other hand, adult lymphocytes resynthesize completely their receptors after the same treatment. Furthermore, intrafetal injections of hemocyanin in pregnant mice lead to a striking decrease in the number of hemocyanin-binding cells. It seems plausible that this non-reexpression of surface immunoglobulins could be the first step in tolerance establishment. The mechanisms by which the immunological repertoire is developed are still largely unknown. Useful information may be obtained from studies of lymphocyte differentiation. In some species, for example, the mouse, immunological maturity is attained only after birth (1). Several studies, however, indicate that immunoglobulin receptors appear on the membrane of some lymphoid cells before the animal is able to synthesize antibody in response to antigenic stimulation (2-4). The following experiments were designed to investigate the rate of appearance of B (bone-marrow-derived) lymphocytes and the physiological effects of treating early B lymphocytes with anti-immunoglobulin sera or early fetal antigen-binding cells with antigen. The main conclusions drawn from this study are that B lymphocytes and keyhole limpet hemocyanin- (KLH) binding cells can be detected early in fetal liver and spleen and that, until approximately 10 days after birth, the interaction of lymphocytes with anti-immunoglobulin sera or with antigen leads to the inactivation (no receptor reexpression) of the lymphocytes that bind the ligand. No such inactivation occurs after that period. Furthermore, intrafetal injections of KLH at 15 days' gestation lead to a marked decrease in the number of KLH-binding cells.
MATERIALS AND METHODS Antisera and Membrane Immunofluorescence. Spleen and liver were removed from fetuses of timed pregnant BALB/c mice at different days of gestation, and from newborn mice. Cell suspensions were made and processed for membrane immunofluorescence using fluorescein- (Fl-) or rhodamine- (Rho-) labeled, polyspecific anti-Ig, whose properties have been described in detail previously (5). The results were unaffected by Abbreviations: B lymphocytes, bone-marrow-derived lymphocytes; KLH, keyhole limpet hemocyanin; Fl-, fluorescein; Rho-, rhodamine; TMV, tobacco mosaic virus; FCS, fetal calf serum; MIg, membrane
immunoglobulin.
adsorption of these antisera with adult liver and kidney cells. Furthermore, when adult spleen cells are first treated, in capping conditions, with tobacco mosaic virus (TMV) and Rholabeled anti-TMV and then, in non-capping conditions, with Fl-labeled anti-Ig, TMV-binding cells with coincident fluorescence can be detected (5). Thus, contamination of the anti-Ig iera by anti-membrane antibodies seems highly unlikely. Cells bearing immunoglobulin receptors of types IgM or IgA were detected by indirect immunofluorescence. The first step was the incubation of cells with antisera raised against mouse myeloma protein MOPC 104 (IgM) or MOPC 315 (IgA) in rabbits or with batches of commercially available anti-IgM sera (goat-anti-IgM and IgA obtained from Meloy). In a second step, cell suspensions were incubated with Fl-anti-rabbit Ig or Flanti-goat Ig. Control slides were always made, omitting the first reagent. The purity of anti-IgM sera was checked by immunodiffusion, immunoelectrophoresis, and Laurell electrophoresis. The anti-IgM sera used in these studies react strongly with the mouse myeloma protein MOPC 104 (IgM), but not at all with MOPC 173 (IgG) or with purified mouse IgG. The detection of immunoglobulin-bearing cells and of antigen-binding cells (for TMV or KLH) on adult spleen cells was performed as described (5). For adult cells, incubation at 370 or 40 gave the same number of antigen-binding cells. Immunoglobulin-bearing cells are detected by incubation of fetal and newborn cells in phosphate-buffered saline-1% fetal calf serum (FCS)-0. 1 M NaN3 with rabbit Rho-anti-mouse Ig for 15 min at 370 as described in ref. 2. IgM and IgA are detected by incubation first with goat anti-mouse IgM or antimouse IgA (Meloy GAM/IgM-GAM/IgA; 40 Ml of serum for 107 cells) for 6 min at 370 and then, after two washes, by incubation with a rabbit Fl-anti-goat Ig (RAG/FITC, Nordic Tilburg) for a further 15 minat 370. After two washes in phosphate-buffered saline-1% FCS-0.01 M NaN3, cells are mounted on microscope slides. TMV- or KLH-binding cells are detected after incubation of fetal and neonatal cells with TMV or KLH (100 ,g per 107 cells) for 6 min at 370 and then, after one wash, incubation for a further 15 min at 370 with rabbit Rho-anti-TMV or rabbit Fl-anti-KLH. After two washes in phosphate-buffered saline-1% FCS-0.01 M NaN3, cells are mounted on microscope slides. Without sodium azide or at very low concentration, capping and endocytosis of fetal and neonatal receptors were extremely rapid. Incubation of fetal and neonatal cells with Rho-anti-TMV or with Fl-anti-KLH antisera alone did not reveal non-specific u'ptake of labeled antisera. Resynthesis Experiments. Spleen cells suspensions are made in phosphate-buffered saline-1% FCS; after two washes, the cells are resuspended in Hanks' balanced salt solution and incubated for 1 hr at 370 either with rabbit anti-mouse Ig serum, heat-inactivated at 560 for 20 min and extensively adsorbed on 2462
Procl Natl. Acad. Sci. USA 73 (1976)
Immunology: Bruyns et al.
2463
202 20
20 10 30 105 Days FIG. 1. Detection of MIg cells in fetal liver and spleen and in spleen of newborn BALB/c mice. (A) Fetal cells; (B) newborn cells. 0, MIg cells in liver; *, MIg cells in fetal and newborn spleen; *, IgA-bearing cells; A, IgM-bearing cells. 13 14 15 16 17 18 19
kidney and liver cells of normal BALB/c mice (40 1A of serum per 2 X 107 cells), or with Pronase (1 mg of Pronase per 2 X 107 cells + 0.1 mg of DNase per 2 X 107 cells). Control cells are incubated in Hanks' balanced salt solution for the same length of time without any treatment. After incubation, the cells are washed once in Hanks' balanced salt solution and twice in M 199 medium, containing Hanks' salts, Bactocilline, and 10% heat-inactivated FCS. Cell suspensions are counted and cultured (107 cells per ml) in Kimax test tubes at 370 in an atmosphere of 5% CO2. After 1-S hr and after 24 hr, aliquots are taken and washed twice in phosphate-buffered saline-1% FCS; live cells are counted and prepared for immunofluorescent staining. Culture of Liver Explants. Fetal livers of BALB/c embryos of different ages are removed, cut into small fragments, and placed in Falcon organ culture dishes on HAWP 025 Millipore filters floating on 1 ml of RPMI 1640 medium containing 10% decomplemented FCS. Cell suspensions of part of the fetal livers are prepared in phosphate-buffered saline containing 10% FCS and 0.1 M NaN& Cells are stained for membrane 1g, IgM, 0, and KLH receptors, using direct or indirect immunofluorescent techniques. Ig and IgM are detected after incubation at 370 for 15 min with rabbit Rho-anti-mouse Ig or with goat Fl-antimouse IgM (Meloy). For the indirect technique, cells are first incubated with goat-anti-IgM (Meloy, 40 Al per 107 cells), rabbit anti-mouse 0 [prepared following Golub (25), 15 Ml per 107 cells], or KLH (100 Mg per 107 cells) for 6 min at 370; the cells are then centrifuged at 100 X g, washed once, and reincubated for 15 min at 370 with Fl-anti-goat Ig (RAG/FITC), Fl-antirabbit Ig (GAR/FITC), or Fl-anti-KLH serum. The stained cells are washed twice in phosphate-buffered saline-10% FCS-0.01 M NaN3 and mounted on microscope slides. The percentagesof fluorescent cells are determined with a Leitz Orthoplan microscope equipped with an Opak Fluor vertical illuminator. After 4 or 6 days, explants are pooled and cell suspensions are washed and tested for viability using the typan blue exclusion method. Cell suspensions are then prepared for membrane immunofluorescent staining. Intrafetal Injections. Timed pregnant BALB/c mice are operated at 15 days' gestation. Each fetus is injected either with 1 Al of rabbit anti-mouse Ig or goat anti-mouse IgM, heat-in-
activated for 20 min at 560 and extensively adsorbed on kidney and liver cells from normal BALB/c mice, or with 1 gg of KLH (1 gl containing 1 mg of KLH/ml of 0.9% NaCl). Sham-operated females receive 1 Mul of 0.9% NaCl in each fetus. After 4 days, i.e., 19 days' gestation, the mice are killed and the liver and spleen of every surviving fetus are removed. Cell suspensions are washed in phosphate-buffered saline-1% FCS and processed for immunofluorescence staining in phosphatebuffered saline-1% FCS containing 0.1 M NaN3. Membrane .immunoglobulins (MIgs) are detected by adding 1 drop of Rho-anti-Ig for 15 min at 370; KLH-binding cells are detected by a two-step method: a first incubation with KLH (0.1 mg of KLH per 107 cells) for 6 min at 370 and then, after one wash, a second incubation with a rabbit Fl-anti-KLH for a further 15 min at 370. After two washes, the cells are mounted on microscope slides.
RESULTS AND DISCUSSION As shown in Fig. 1, cells bearing MIgs can be detected by day 16 or 17 in fetal spleen and still earlier in fetal liver. The percentage of Ig-bearing cells increases during the first few days after birth. After that period, there is always a decline in the percentage of cells bearing 1g. About 10 days after birth, there is a new increase in the percentage of Ig-bearing cells, which reaches adult values by the 30th day after birth. As soon as immunoglobulin-bearing cells are detectable, some cells bearing receptors that bind KLH or TMV can be detected (0.03% of KLH-binding cells in fetal liver on day 13; 0.17% in fetal spleen on day 17; 0.57% at birth in the spleen). These results are in, agreement with those of Decker et al. (4) and those of Spear et al. (2). The percentage of hemocyanin-binding cells seems to be quite large. However, similar frequencies were found by Dwyer and MacKay (6) using a method of labeling cell receptors with iodinated hemocyanin. The binding of hemocyanin seems to be specific, since the percentage of hemocyaninbinding cells does not decrease if cells are first incubated with other antigens. Furthermore, removal of MIgs eliminates the antigen-binding capacity (see below). The diversity of the fetal receptor repertoire seems considerable since cells binding various antigens have been found
2464
Immunology: Bruyns et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
A
B 5-6 days
*2 I1
days
010
adu It
12
24
12
24
Hours
FIG. 2. Resynthesis of MIg and TMV-binding receptors in newborn (5-day-old), young (16-day-old), and adult BALB/c mice after treatment of spleen cells with anti-Ig serum or after stripping of receptors with Pronase. (A) stripping of receptors after endocytosis; (B) after Pronase treatment. MIg cells in control cultures (0 0) and cultures after incubation (0-0); TMV-binding cells in control (-) and incubated (-A) cultures. ---
- --
(KLH, TMV, f3-galactosidase, dinitrophenol, trinitrophenol.. .. In addition, it has been shown that fetal trinitrophenol-binding cells exhibit the same range of avidities for antigen as adult lymphoid cells (7). It therefore seems unlikely that antibody diversity could originate from.somatic mutations occurring in a small number of germ-line genes coding for antibodies of maximum protective value for the species (e.g., antibodies against the carbohydrates of common pathogens) (8). To show that these immunoglobulin receptors are indeed synthesized by the cells bearing them and are not maternal immunoglobulins passively adsorbed on the membrane, the biosynthetic capacities of these lymphocytes were evaluated after the removal of MIgs by capping and endocytosis promoted by anti-immunoglobulin sera or after stripping of superficial proteins with Pronase as described by Loor et al. (9). The cells were then cultured for 24 hr and tested for the reappearance of MIgs. The results of these experiments are shown in Fig. 2. The two types of treatment give very similar results. While a resynthesis is observed in adult spleen cells, there was practically no immunoglobulin reexpression in cells taken from mice 5 days after birth. Intermediate values were obtained for cells taken from 16-day-old mice. Essentially similar results were obtained for TMV-binding cells. There is a critical period (around 10
days after birth) before which lymphocytes are unable to reexpress receptors after treatment with anti-immunoglobulin sera or stripping with Pronase. This critical period seems to coincide with the decline in the percentage of immunoglobulin-bearing cells in the spleen. It must be stressed that removal of MIgs eliminates the antigen-binding capacity. Sidman and Unanue (10) have likewise shown that early lymphocytes from C57B1/6 mice treated with anti-immunoglobulin sera do not reexpress their receptors. In contrast with our results, however, they showed that after stripping of superficial proteins with Pronase, early lymphocytes were able to resynthesize their receptors. The difference between our results and those of Sidman and Unanue could be due to the quality of FCS. Some batches of FCS can act as polyclonal B cell activators (11) which can interact with young B lymphocytes (12). Our results are also in nice agreement with those of Raff and coworkers (13). Raff et al. showed that anti-Ig antibodies inhibit totally the development of B cells bearing IgM receptors in explants of fetal liver. IgM suppression in adult lymphocytes required much higher antibody concentration and was reversible. At this stage, two hypotheses could account for our results: either the immunoglobulins detected on early lymphocytes are passively adsorbed, or early and mature lymphocytes show different physiological behavior following interactions with a ligand or stripping with Pronase. To decide between these two possibilities (adsorption of maternal Ig or no immunoglobulin reexpression), fragments from fetal liver were prepared and cultured following the method described by Owen et al. (3). The results given in Table 1 show that the number of immunoglobulin-bearing cells increases during culture. These results, in complete agreement with those of Owen et al. (3), strongly suggest that the immunoglobulin receptors detected on early lymphocytes are indeed synthesized by the cells bearing them. In vivo inactivation of receptor reexpression on antigenbinding cells was attempted by intrafetal injection of KLH in pregnant mice at 15 days' gestation. The peritoneal cavity of mice 15 days pregnant was opened and 1 Mg of KLH was injected directly into each fetus through the uterine membrane. After the peritoneal cavity had been closed, the mice were allowed to rest in darkness at 280. Four days later, the mice were killed and suspensions of fetal spleen and liver cells were prepared and processed for membrane immunofluorescence in order to detect receptors for KLH. As controls, sham-operated animals were injected with 1 ,l of saline. Other pregnant mice were similarly treated by intrafetal injections of anti-immunoglobulin sera. The results given in Table 2 show that, in fetuses injected by day 15 of fetal life with 1 Mg of KLH, there is a strong decrease (82% inhibition) of the number of lymphocytes detected with KLH and Fl-rabbit anti-KLH. No such decrease is observed in sham-operated animals injected with saline. Injection of anti-immunoglobulin sera or anti-IgM sera led to a decrease in the number of immunoglobulin-bearing cells and the same decrease in the number of KLH-binding cells. These results confirm that KLH is bound to IgM receptors of fetal cells. Adult animals, even when injected with doses as high as 100 ,g, show no decrease in the number of antigenbinding cells 4 days after injection. It seems, therefore, that early lymphocytes are inactivated following interactions with antigen or anti-immunoglobulin sera. The fate of these inactivated cells is unknown and its elucidation would require further experiments.
The difference in the physiological behavior of early and late
lymphocytes seems also to be present in adult animals. Mature
B -cells originate from bone marrow stem cells. Nossal and Pike
Immunology: Bruyw et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
2465
Table 1. Appearance of membrane receptors on fetal liver cells in culture fragment experiments
Age of fetus, days
Culture time, days
13
0 4 6 0 4 6 0 4 6 0 4 6
14
15
16
IWgM- 1direct)
IgM (direct)
Total cells 3,045 2,000 6,600 4,500 2,500 3,100 Not done
11,652 7,275 14,985
Hemocyanin
0
%
Total cells
%
Total cells
%
Total cells
%
3,045
0.36
19,228
4,400 2,000
4,505
0.8 3.1
0.06 0.12 1.9
3,700 4,620 4,500 4,092 2,910 Not done
1.7 4.7 0.24 1.5 0.6 0.3 0.6
0.03 0.22 0.92 0.04 0.12 0.32 0.9 1.3
4,298
1.3
0.06 0.12 0.05
7,380 7,000 1,612
2,600 12,464 5,753 4,970 2,438 2,970
Not done
3,259 5,000
7,321 5,080 5,812
2.2 2.4
Not done
4,252 6,295
Not done 0.36 0.47 0.69
(14) have shown that, during their differentiation, B lymphocytes pass through a stage in which contact with an antigen induces clonal abortion. The different physiological behavior of early and late lymphocytes is probably related to differences in membrane components. It has been shown that receptors for the third complement component (C3) appear only at 15 days after birth (15). No IgD is detectable on fetal and neonatal lymphocytes (16). Moreover, 15-day-old fetal liver cells respond to dextran sulfate but require a further 6 days of differentiation to respond to bacterial lipopolysaccharide and a further 11 days to become responsive to tuberculin purified protein derivative (PPD) (12). Our results are in agreement with the time models for the self-non-self discrimination phenomenon (17). Following this model, immature B lymphocytes (in ontogeny or in adult bone marrow) are paralyzed, and not induced, by any contact of an antigen with the immunoglobulin receptors. These findings are difficult to reconcile with the "one non-specific signal hypothesis" which states that no signal is generated after interactions of MIgs with a fitting ligand (11). Although we think
3,900
1.4 2.5 6.1 3.5 1.9 8.3
3,200 0.13 0.42 0.19
1,722 6,095 1,400
that our findings are related to the ease of tolerance induction in neonates [tolerance to grafts (18, 19), allotypic suppression (20), idiotypic suppression (21), IgM suppression by in vivo injection of anti-IgM (22)], it is clear that the disappearance of receptors due to the interaction of MIgs with a ligand cannot fully account for tolerance phenomena. Repeated injections of antigen or anti-immunoglobulin sera are probably necessary to inhibit the differentiation of new waves of stem cells and to obtain a durable state of inactivation. It has been shown that normal healthy animals have lymphocytes with receptors recognizing self antigens (23). Ada and Cooper (24) have shown that the establishment of tolerance to KLH by injection in neonates is accompanied by the appearance of suppressor T (thymus-derived) cells. It seems plausible
to suggest that, once early lymphocytes have been inactivated by contact with an antigen, new emerging B lymphocytes bearing the same receptors will be recognized as foreign by T
cells. It is tempting to speculate that these suppressor T lymphocytes possess receptors that recognize the idiotypic specificities of the surface immunoglobulins of inactivated B lymphocytes. Such a mechanism could operate for self antigens,
Table 2. Inhibitions of MIg and of KLH-binding cells in fetal liver and spleen 4 days after intrafetal injection of anti-Ig, anti-IgM, or KLH
Spleen Injection Untreated fetus
Receptors
MIg Anti-KLH
MIgM 1
pm of 0.9% NaCl or phosphate-
MIg Anti-KLH
Liver
Total cells
%
5,268 9,778 2,402 1,633 4,815
3.78 ± 0.82 ± 2.94 ± 3.89 ± 0.88 ±
0.65 0.30 0.01 0.20 0.22
0 0
3,892 7,649 2,524 4,113
2.76 ± 0.16 ± 1.70 ± 0.33 ± 0.62 ± 0.87 ±
0.13 0.03 0.66 0.27 0.06 0.08
27 82 55 60 85 72
% inhibition
Total cells
5,288 12,529 3,662
1,449 8,666
buffered saline 1 pg of KLH
MIg
Anti-KLH 1
A of anti-Ig
MIg
Anti-KLH 1 1 of anti-IgM
MIg
MIgM
2,710 2,755
3,758
10,346 6,728 6,084
4,485 4,455
% inhibition
%
1.78 ± 0.72 ± 2.10 ± 1.80 ± 0.55 ±
0.44 0.20 0.20 0.08 0.15
0 24
1.73 ± 0.61 0.23 ± 0.09 0.31 ± 0.11 0.12 ± 0.28 0.37 ± 0.24 0.64 ± 0.34
3 68 83 84 79 70
The percentages of MIg and of KLH-binding cells are compared with the percentages of cells obtained in normal untreated fetuses. Mean values were obtained from at least four different experiments for the untreated fetuses. Results indicated for injected fetuses are the mean values obtained from two to four different experiments.
2466
Immunology: Bruyns et al.
since Cunningham has shown that tolerance to some self components is maintained by an active suppressor mechanism (26). Such a hypothesis would fit nicely with the recent network idea of the immune system (27). In summary, we have shown that fetal and neonatal lymphocytes bearing surface immunoglobulins and antigen receptors do not reexpress their receptors after interaction with a multivalent ligand (antigen or anti-immunoglobulin antibody), either in vitro or in vivo. This is in striking contrast with adult lymphocytes, which are able to resynthesize completely their surface immunoglobulins after stripping with Pronase or capping and endocytosis promoted by anti-immunoglobulin sera. Since tolerance is readily achieved by antigen injection in neonates, we suggest that this non-reexpression of surface immunoglobulins could be the first step leading to establishment of natural tolerance. New emerging B lymphocytes bearing the same receptors would be recognized as foreign by specific T cells. We thank Prof. J. Brachet and Professor R. Jeener for critical reading of the manuscript. We are indebted to Mrs. Storms for her expert technical assistance. The help from Mrs. Verleyen has been greatly appreciated. This work was made possible by a financial contract between University of Brussels and Euratom (Contract 099-72-1BIAB-1972). C.D.V.-C.is Charge de Recherches of the Fonds National de la Recherche Scientifique (FNRS), and J. U. is Chercheur qualifie FNRS. 1. Spear, G. P. & Edelman, G. M. (1974) J. Exp. Med. 139,249-263. 2. Spear, G. P., Wang, A., Rutishauser, V. & Edelman, G. M. (1973) J. Exp. Med. 138,557-573. 3. Owen, J. T., Cooper, M. D. & Raff, M. C. (1974) Nature 249, 361-63. 4. Decker, J. M., Clarke, J., MacPherson, A. & Sercarz, E. E. (1974) J. Immunol. 113, 1823-1833.
Proc. Nati. Acad. Sci. USA 73 (1976) 5. Urbain-Vansanten, G., Richard, C., Bruyns, C., Hooghe, V., Van Acker, A. & Urbain, J. (1974) Ann. Immunol. (Paris) 125C, 885-900. 6. Dwyer, J. M. & MacKay, I. R. (1972) Immunology 23,871-879. 7. d'Eustachio, P. & Edelman, G. M. (1975) J. Exp. Med. 142, 1078-1091. 8. Cohn, M. (1974) Prog. Immunol. 2,261-284. 9. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J. Immunol. 2, 203-212. 10. Sidman, C. L. & Unanue, E. E. (1975) Nature 257,149-151. 11. Couthino, A. & Mller, G. (1975) Adv. Immunol. 21, 113-236 12. Gronowicz, E., Couthino, A. & M6ller, G. (1974) Scand. J. Immunol. 3,413421. 13. Raff, M. C., Owen, J. T., Cooper, M. D., Lawton, A. R., Megson, M. & Gathings, W. E. (1975) J. Exp. Med. 142,1052-1064. 14. Nossal, G. J. V. & Pike, B. (1975) J. Exp. Med. 141, 904-917. 15. Gelfand, M. C., Sachs, D. H., Lieberman, R. & Paul, W. E. (1974) J. Exp. Med. 139,1142-1153. 16. Vitetta, E. S. & Uhr, J. W. (1975) Science 189,964-969. 17. Lederberg, J. (1959) Science 129, 1649-1653. 18. Medawar, P. B. (1961) Nature 189, 14-17. 19. Billingham, R. E. & Brent, L. (1957) Proc. R. Soc. London 146, 78-90. 20. Harrison, M. R. & Mage, G. (1973) J. Exp. Med. 138, 764774. 21. Strayer, D., Cosenza, H., Lee, W. M., Rowley, D. A. & K6hler, H. (1974) Science 186, 640-643. 22. Lawton, A. R. & Cooper, M. D. (1974) Contemp. Top. Immunobiol. 3, 193-225. 23. Cohen, I. R. & Feldman, M. (1975) Ann. N.Y. Acad. Sci. 249, 106-115. 24. Ada, G. L. & Cooper, M. G. (1975) in Immunological Tolerance, eds. Katz, D. H. & Benacerraf, B. (Academic Press, New York), pp. 87-106. 25. Golub, E. S. (1971) Cell. Immunol. 2,353-361. 26. Cunningham, S. J. (1975) Nature 254, 143-144. 27. Jerne, N. K. (1974) Ann. Immunol. (Paris) 125C, 373-389.
Vol. 73, No. 7, pp. 2462-2466, July 1976
Immunology
Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes (differentiation/tolerance)
C. BRUYNS, G. URBAIN-VANSANTEN, C. PLANARD, C. DE VOS-CLOETENS, AND JI URBAIN Laboratory of Animal Physiology, Universit6 Libre de Bruxelles, 67, rue des Chevaux, 1640, Rhode-St-Genese, Belgium
Communicated by J. Brachet, April 6,1976
ABSTRACT Taking advantage of recent findings about membrane fluidity, we have studied and compared the biosynthetic capacities of fetal or neonatal mouse B (bone-marrow derived) lymphocytes (until 10 days after birth) and adult B lymphocytes. Although both early and adult lymphocytes can synthesize surface immunoglobulins, they have a different physiological behavior after interaction with a ligand (antiimmunoglobulin sera or antigen), either in vivo or in vitro. Fetal and neonatal lymphocytes bearing surface immunoglobulins do not reexpress their membrane receptors after capping and endocytosis promoted by anti-immunoglobulin sera. On the other hand, adult lymphocytes resynthesize completely their receptors after the same treatment. Furthermore, intrafetal injections of hemocyanin in pregnant mice lead to a striking decrease in the number of hemocyanin-binding cells. It seems plausible that this non-reexpression of surface immunoglobulins could be the first step in tolerance establishment. The mechanisms by which the immunological repertoire is developed are still largely unknown. Useful information may be obtained from studies of lymphocyte differentiation. In some species, for example, the mouse, immunological maturity is attained only after birth (1). Several studies, however, indicate that immunoglobulin receptors appear on the membrane of some lymphoid cells before the animal is able to synthesize antibody in response to antigenic stimulation (2-4). The following experiments were designed to investigate the rate of appearance of B (bone-marrow-derived) lymphocytes and the physiological effects of treating early B lymphocytes with anti-immunoglobulin sera or early fetal antigen-binding cells with antigen. The main conclusions drawn from this study are that B lymphocytes and keyhole limpet hemocyanin- (KLH) binding cells can be detected early in fetal liver and spleen and that, until approximately 10 days after birth, the interaction of lymphocytes with anti-immunoglobulin sera or with antigen leads to the inactivation (no receptor reexpression) of the lymphocytes that bind the ligand. No such inactivation occurs after that period. Furthermore, intrafetal injections of KLH at 15 days' gestation lead to a marked decrease in the number of KLH-binding cells.
MATERIALS AND METHODS Antisera and Membrane Immunofluorescence. Spleen and liver were removed from fetuses of timed pregnant BALB/c mice at different days of gestation, and from newborn mice. Cell suspensions were made and processed for membrane immunofluorescence using fluorescein- (Fl-) or rhodamine- (Rho-) labeled, polyspecific anti-Ig, whose properties have been described in detail previously (5). The results were unaffected by Abbreviations: B lymphocytes, bone-marrow-derived lymphocytes; KLH, keyhole limpet hemocyanin; Fl-, fluorescein; Rho-, rhodamine; TMV, tobacco mosaic virus; FCS, fetal calf serum; MIg, membrane
immunoglobulin.
adsorption of these antisera with adult liver and kidney cells. Furthermore, when adult spleen cells are first treated, in capping conditions, with tobacco mosaic virus (TMV) and Rholabeled anti-TMV and then, in non-capping conditions, with Fl-labeled anti-Ig, TMV-binding cells with coincident fluorescence can be detected (5). Thus, contamination of the anti-Ig iera by anti-membrane antibodies seems highly unlikely. Cells bearing immunoglobulin receptors of types IgM or IgA were detected by indirect immunofluorescence. The first step was the incubation of cells with antisera raised against mouse myeloma protein MOPC 104 (IgM) or MOPC 315 (IgA) in rabbits or with batches of commercially available anti-IgM sera (goat-anti-IgM and IgA obtained from Meloy). In a second step, cell suspensions were incubated with Fl-anti-rabbit Ig or Flanti-goat Ig. Control slides were always made, omitting the first reagent. The purity of anti-IgM sera was checked by immunodiffusion, immunoelectrophoresis, and Laurell electrophoresis. The anti-IgM sera used in these studies react strongly with the mouse myeloma protein MOPC 104 (IgM), but not at all with MOPC 173 (IgG) or with purified mouse IgG. The detection of immunoglobulin-bearing cells and of antigen-binding cells (for TMV or KLH) on adult spleen cells was performed as described (5). For adult cells, incubation at 370 or 40 gave the same number of antigen-binding cells. Immunoglobulin-bearing cells are detected by incubation of fetal and newborn cells in phosphate-buffered saline-1% fetal calf serum (FCS)-0. 1 M NaN3 with rabbit Rho-anti-mouse Ig for 15 min at 370 as described in ref. 2. IgM and IgA are detected by incubation first with goat anti-mouse IgM or antimouse IgA (Meloy GAM/IgM-GAM/IgA; 40 Ml of serum for 107 cells) for 6 min at 370 and then, after two washes, by incubation with a rabbit Fl-anti-goat Ig (RAG/FITC, Nordic Tilburg) for a further 15 minat 370. After two washes in phosphate-buffered saline-1% FCS-0.01 M NaN3, cells are mounted on microscope slides. TMV- or KLH-binding cells are detected after incubation of fetal and neonatal cells with TMV or KLH (100 ,g per 107 cells) for 6 min at 370 and then, after one wash, incubation for a further 15 min at 370 with rabbit Rho-anti-TMV or rabbit Fl-anti-KLH. After two washes in phosphate-buffered saline-1% FCS-0.01 M NaN3, cells are mounted on microscope slides. Without sodium azide or at very low concentration, capping and endocytosis of fetal and neonatal receptors were extremely rapid. Incubation of fetal and neonatal cells with Rho-anti-TMV or with Fl-anti-KLH antisera alone did not reveal non-specific u'ptake of labeled antisera. Resynthesis Experiments. Spleen cells suspensions are made in phosphate-buffered saline-1% FCS; after two washes, the cells are resuspended in Hanks' balanced salt solution and incubated for 1 hr at 370 either with rabbit anti-mouse Ig serum, heat-inactivated at 560 for 20 min and extensively adsorbed on 2462
Procl Natl. Acad. Sci. USA 73 (1976)
Immunology: Bruyns et al.
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202 20
20 10 30 105 Days FIG. 1. Detection of MIg cells in fetal liver and spleen and in spleen of newborn BALB/c mice. (A) Fetal cells; (B) newborn cells. 0, MIg cells in liver; *, MIg cells in fetal and newborn spleen; *, IgA-bearing cells; A, IgM-bearing cells. 13 14 15 16 17 18 19
kidney and liver cells of normal BALB/c mice (40 1A of serum per 2 X 107 cells), or with Pronase (1 mg of Pronase per 2 X 107 cells + 0.1 mg of DNase per 2 X 107 cells). Control cells are incubated in Hanks' balanced salt solution for the same length of time without any treatment. After incubation, the cells are washed once in Hanks' balanced salt solution and twice in M 199 medium, containing Hanks' salts, Bactocilline, and 10% heat-inactivated FCS. Cell suspensions are counted and cultured (107 cells per ml) in Kimax test tubes at 370 in an atmosphere of 5% CO2. After 1-S hr and after 24 hr, aliquots are taken and washed twice in phosphate-buffered saline-1% FCS; live cells are counted and prepared for immunofluorescent staining. Culture of Liver Explants. Fetal livers of BALB/c embryos of different ages are removed, cut into small fragments, and placed in Falcon organ culture dishes on HAWP 025 Millipore filters floating on 1 ml of RPMI 1640 medium containing 10% decomplemented FCS. Cell suspensions of part of the fetal livers are prepared in phosphate-buffered saline containing 10% FCS and 0.1 M NaN& Cells are stained for membrane 1g, IgM, 0, and KLH receptors, using direct or indirect immunofluorescent techniques. Ig and IgM are detected after incubation at 370 for 15 min with rabbit Rho-anti-mouse Ig or with goat Fl-antimouse IgM (Meloy). For the indirect technique, cells are first incubated with goat-anti-IgM (Meloy, 40 Al per 107 cells), rabbit anti-mouse 0 [prepared following Golub (25), 15 Ml per 107 cells], or KLH (100 Mg per 107 cells) for 6 min at 370; the cells are then centrifuged at 100 X g, washed once, and reincubated for 15 min at 370 with Fl-anti-goat Ig (RAG/FITC), Fl-antirabbit Ig (GAR/FITC), or Fl-anti-KLH serum. The stained cells are washed twice in phosphate-buffered saline-10% FCS-0.01 M NaN3 and mounted on microscope slides. The percentagesof fluorescent cells are determined with a Leitz Orthoplan microscope equipped with an Opak Fluor vertical illuminator. After 4 or 6 days, explants are pooled and cell suspensions are washed and tested for viability using the typan blue exclusion method. Cell suspensions are then prepared for membrane immunofluorescent staining. Intrafetal Injections. Timed pregnant BALB/c mice are operated at 15 days' gestation. Each fetus is injected either with 1 Al of rabbit anti-mouse Ig or goat anti-mouse IgM, heat-in-
activated for 20 min at 560 and extensively adsorbed on kidney and liver cells from normal BALB/c mice, or with 1 gg of KLH (1 gl containing 1 mg of KLH/ml of 0.9% NaCl). Sham-operated females receive 1 Mul of 0.9% NaCl in each fetus. After 4 days, i.e., 19 days' gestation, the mice are killed and the liver and spleen of every surviving fetus are removed. Cell suspensions are washed in phosphate-buffered saline-1% FCS and processed for immunofluorescence staining in phosphatebuffered saline-1% FCS containing 0.1 M NaN3. Membrane .immunoglobulins (MIgs) are detected by adding 1 drop of Rho-anti-Ig for 15 min at 370; KLH-binding cells are detected by a two-step method: a first incubation with KLH (0.1 mg of KLH per 107 cells) for 6 min at 370 and then, after one wash, a second incubation with a rabbit Fl-anti-KLH for a further 15 min at 370. After two washes, the cells are mounted on microscope slides.
RESULTS AND DISCUSSION As shown in Fig. 1, cells bearing MIgs can be detected by day 16 or 17 in fetal spleen and still earlier in fetal liver. The percentage of Ig-bearing cells increases during the first few days after birth. After that period, there is always a decline in the percentage of cells bearing 1g. About 10 days after birth, there is a new increase in the percentage of Ig-bearing cells, which reaches adult values by the 30th day after birth. As soon as immunoglobulin-bearing cells are detectable, some cells bearing receptors that bind KLH or TMV can be detected (0.03% of KLH-binding cells in fetal liver on day 13; 0.17% in fetal spleen on day 17; 0.57% at birth in the spleen). These results are in, agreement with those of Decker et al. (4) and those of Spear et al. (2). The percentage of hemocyanin-binding cells seems to be quite large. However, similar frequencies were found by Dwyer and MacKay (6) using a method of labeling cell receptors with iodinated hemocyanin. The binding of hemocyanin seems to be specific, since the percentage of hemocyaninbinding cells does not decrease if cells are first incubated with other antigens. Furthermore, removal of MIgs eliminates the antigen-binding capacity (see below). The diversity of the fetal receptor repertoire seems considerable since cells binding various antigens have been found
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Immunology: Bruyns et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
A
B 5-6 days
*2 I1
days
010
adu It
12
24
12
24
Hours
FIG. 2. Resynthesis of MIg and TMV-binding receptors in newborn (5-day-old), young (16-day-old), and adult BALB/c mice after treatment of spleen cells with anti-Ig serum or after stripping of receptors with Pronase. (A) stripping of receptors after endocytosis; (B) after Pronase treatment. MIg cells in control cultures (0 0) and cultures after incubation (0-0); TMV-binding cells in control (-) and incubated (-A) cultures. ---
- --
(KLH, TMV, f3-galactosidase, dinitrophenol, trinitrophenol.. .. In addition, it has been shown that fetal trinitrophenol-binding cells exhibit the same range of avidities for antigen as adult lymphoid cells (7). It therefore seems unlikely that antibody diversity could originate from.somatic mutations occurring in a small number of germ-line genes coding for antibodies of maximum protective value for the species (e.g., antibodies against the carbohydrates of common pathogens) (8). To show that these immunoglobulin receptors are indeed synthesized by the cells bearing them and are not maternal immunoglobulins passively adsorbed on the membrane, the biosynthetic capacities of these lymphocytes were evaluated after the removal of MIgs by capping and endocytosis promoted by anti-immunoglobulin sera or after stripping of superficial proteins with Pronase as described by Loor et al. (9). The cells were then cultured for 24 hr and tested for the reappearance of MIgs. The results of these experiments are shown in Fig. 2. The two types of treatment give very similar results. While a resynthesis is observed in adult spleen cells, there was practically no immunoglobulin reexpression in cells taken from mice 5 days after birth. Intermediate values were obtained for cells taken from 16-day-old mice. Essentially similar results were obtained for TMV-binding cells. There is a critical period (around 10
days after birth) before which lymphocytes are unable to reexpress receptors after treatment with anti-immunoglobulin sera or stripping with Pronase. This critical period seems to coincide with the decline in the percentage of immunoglobulin-bearing cells in the spleen. It must be stressed that removal of MIgs eliminates the antigen-binding capacity. Sidman and Unanue (10) have likewise shown that early lymphocytes from C57B1/6 mice treated with anti-immunoglobulin sera do not reexpress their receptors. In contrast with our results, however, they showed that after stripping of superficial proteins with Pronase, early lymphocytes were able to resynthesize their receptors. The difference between our results and those of Sidman and Unanue could be due to the quality of FCS. Some batches of FCS can act as polyclonal B cell activators (11) which can interact with young B lymphocytes (12). Our results are also in nice agreement with those of Raff and coworkers (13). Raff et al. showed that anti-Ig antibodies inhibit totally the development of B cells bearing IgM receptors in explants of fetal liver. IgM suppression in adult lymphocytes required much higher antibody concentration and was reversible. At this stage, two hypotheses could account for our results: either the immunoglobulins detected on early lymphocytes are passively adsorbed, or early and mature lymphocytes show different physiological behavior following interactions with a ligand or stripping with Pronase. To decide between these two possibilities (adsorption of maternal Ig or no immunoglobulin reexpression), fragments from fetal liver were prepared and cultured following the method described by Owen et al. (3). The results given in Table 1 show that the number of immunoglobulin-bearing cells increases during culture. These results, in complete agreement with those of Owen et al. (3), strongly suggest that the immunoglobulin receptors detected on early lymphocytes are indeed synthesized by the cells bearing them. In vivo inactivation of receptor reexpression on antigenbinding cells was attempted by intrafetal injection of KLH in pregnant mice at 15 days' gestation. The peritoneal cavity of mice 15 days pregnant was opened and 1 Mg of KLH was injected directly into each fetus through the uterine membrane. After the peritoneal cavity had been closed, the mice were allowed to rest in darkness at 280. Four days later, the mice were killed and suspensions of fetal spleen and liver cells were prepared and processed for membrane immunofluorescence in order to detect receptors for KLH. As controls, sham-operated animals were injected with 1 ,l of saline. Other pregnant mice were similarly treated by intrafetal injections of anti-immunoglobulin sera. The results given in Table 2 show that, in fetuses injected by day 15 of fetal life with 1 Mg of KLH, there is a strong decrease (82% inhibition) of the number of lymphocytes detected with KLH and Fl-rabbit anti-KLH. No such decrease is observed in sham-operated animals injected with saline. Injection of anti-immunoglobulin sera or anti-IgM sera led to a decrease in the number of immunoglobulin-bearing cells and the same decrease in the number of KLH-binding cells. These results confirm that KLH is bound to IgM receptors of fetal cells. Adult animals, even when injected with doses as high as 100 ,g, show no decrease in the number of antigenbinding cells 4 days after injection. It seems, therefore, that early lymphocytes are inactivated following interactions with antigen or anti-immunoglobulin sera. The fate of these inactivated cells is unknown and its elucidation would require further experiments.
The difference in the physiological behavior of early and late
lymphocytes seems also to be present in adult animals. Mature
B -cells originate from bone marrow stem cells. Nossal and Pike
Immunology: Bruyw et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
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Table 1. Appearance of membrane receptors on fetal liver cells in culture fragment experiments
Age of fetus, days
Culture time, days
13
0 4 6 0 4 6 0 4 6 0 4 6
14
15
16
IWgM- 1direct)
IgM (direct)
Total cells 3,045 2,000 6,600 4,500 2,500 3,100 Not done
11,652 7,275 14,985
Hemocyanin
0
%
Total cells
%
Total cells
%
Total cells
%
3,045
0.36
19,228
4,400 2,000
4,505
0.8 3.1
0.06 0.12 1.9
3,700 4,620 4,500 4,092 2,910 Not done
1.7 4.7 0.24 1.5 0.6 0.3 0.6
0.03 0.22 0.92 0.04 0.12 0.32 0.9 1.3
4,298
1.3
0.06 0.12 0.05
7,380 7,000 1,612
2,600 12,464 5,753 4,970 2,438 2,970
Not done
3,259 5,000
7,321 5,080 5,812
2.2 2.4
Not done
4,252 6,295
Not done 0.36 0.47 0.69
(14) have shown that, during their differentiation, B lymphocytes pass through a stage in which contact with an antigen induces clonal abortion. The different physiological behavior of early and late lymphocytes is probably related to differences in membrane components. It has been shown that receptors for the third complement component (C3) appear only at 15 days after birth (15). No IgD is detectable on fetal and neonatal lymphocytes (16). Moreover, 15-day-old fetal liver cells respond to dextran sulfate but require a further 6 days of differentiation to respond to bacterial lipopolysaccharide and a further 11 days to become responsive to tuberculin purified protein derivative (PPD) (12). Our results are in agreement with the time models for the self-non-self discrimination phenomenon (17). Following this model, immature B lymphocytes (in ontogeny or in adult bone marrow) are paralyzed, and not induced, by any contact of an antigen with the immunoglobulin receptors. These findings are difficult to reconcile with the "one non-specific signal hypothesis" which states that no signal is generated after interactions of MIgs with a fitting ligand (11). Although we think
3,900
1.4 2.5 6.1 3.5 1.9 8.3
3,200 0.13 0.42 0.19
1,722 6,095 1,400
that our findings are related to the ease of tolerance induction in neonates [tolerance to grafts (18, 19), allotypic suppression (20), idiotypic suppression (21), IgM suppression by in vivo injection of anti-IgM (22)], it is clear that the disappearance of receptors due to the interaction of MIgs with a ligand cannot fully account for tolerance phenomena. Repeated injections of antigen or anti-immunoglobulin sera are probably necessary to inhibit the differentiation of new waves of stem cells and to obtain a durable state of inactivation. It has been shown that normal healthy animals have lymphocytes with receptors recognizing self antigens (23). Ada and Cooper (24) have shown that the establishment of tolerance to KLH by injection in neonates is accompanied by the appearance of suppressor T (thymus-derived) cells. It seems plausible
to suggest that, once early lymphocytes have been inactivated by contact with an antigen, new emerging B lymphocytes bearing the same receptors will be recognized as foreign by T
cells. It is tempting to speculate that these suppressor T lymphocytes possess receptors that recognize the idiotypic specificities of the surface immunoglobulins of inactivated B lymphocytes. Such a mechanism could operate for self antigens,
Table 2. Inhibitions of MIg and of KLH-binding cells in fetal liver and spleen 4 days after intrafetal injection of anti-Ig, anti-IgM, or KLH
Spleen Injection Untreated fetus
Receptors
MIg Anti-KLH
MIgM 1
pm of 0.9% NaCl or phosphate-
MIg Anti-KLH
Liver
Total cells
%
5,268 9,778 2,402 1,633 4,815
3.78 ± 0.82 ± 2.94 ± 3.89 ± 0.88 ±
0.65 0.30 0.01 0.20 0.22
0 0
3,892 7,649 2,524 4,113
2.76 ± 0.16 ± 1.70 ± 0.33 ± 0.62 ± 0.87 ±
0.13 0.03 0.66 0.27 0.06 0.08
27 82 55 60 85 72
% inhibition
Total cells
5,288 12,529 3,662
1,449 8,666
buffered saline 1 pg of KLH
MIg
Anti-KLH 1
A of anti-Ig
MIg
Anti-KLH 1 1 of anti-IgM
MIg
MIgM
2,710 2,755
3,758
10,346 6,728 6,084
4,485 4,455
% inhibition
%
1.78 ± 0.72 ± 2.10 ± 1.80 ± 0.55 ±
0.44 0.20 0.20 0.08 0.15
0 24
1.73 ± 0.61 0.23 ± 0.09 0.31 ± 0.11 0.12 ± 0.28 0.37 ± 0.24 0.64 ± 0.34
3 68 83 84 79 70
The percentages of MIg and of KLH-binding cells are compared with the percentages of cells obtained in normal untreated fetuses. Mean values were obtained from at least four different experiments for the untreated fetuses. Results indicated for injected fetuses are the mean values obtained from two to four different experiments.
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Immunology: Bruyns et al.
since Cunningham has shown that tolerance to some self components is maintained by an active suppressor mechanism (26). Such a hypothesis would fit nicely with the recent network idea of the immune system (27). In summary, we have shown that fetal and neonatal lymphocytes bearing surface immunoglobulins and antigen receptors do not reexpress their receptors after interaction with a multivalent ligand (antigen or anti-immunoglobulin antibody), either in vitro or in vivo. This is in striking contrast with adult lymphocytes, which are able to resynthesize completely their surface immunoglobulins after stripping with Pronase or capping and endocytosis promoted by anti-immunoglobulin sera. Since tolerance is readily achieved by antigen injection in neonates, we suggest that this non-reexpression of surface immunoglobulins could be the first step leading to establishment of natural tolerance. New emerging B lymphocytes bearing the same receptors would be recognized as foreign by specific T cells. We thank Prof. J. Brachet and Professor R. Jeener for critical reading of the manuscript. We are indebted to Mrs. Storms for her expert technical assistance. The help from Mrs. Verleyen has been greatly appreciated. This work was made possible by a financial contract between University of Brussels and Euratom (Contract 099-72-1BIAB-1972). C.D.V.-C.is Charge de Recherches of the Fonds National de la Recherche Scientifique (FNRS), and J. U. is Chercheur qualifie FNRS. 1. Spear, G. P. & Edelman, G. M. (1974) J. Exp. Med. 139,249-263. 2. Spear, G. P., Wang, A., Rutishauser, V. & Edelman, G. M. (1973) J. Exp. Med. 138,557-573. 3. Owen, J. T., Cooper, M. D. & Raff, M. C. (1974) Nature 249, 361-63. 4. Decker, J. M., Clarke, J., MacPherson, A. & Sercarz, E. E. (1974) J. Immunol. 113, 1823-1833.
Proc. Nati. Acad. Sci. USA 73 (1976) 5. Urbain-Vansanten, G., Richard, C., Bruyns, C., Hooghe, V., Van Acker, A. & Urbain, J. (1974) Ann. Immunol. (Paris) 125C, 885-900. 6. Dwyer, J. M. & MacKay, I. R. (1972) Immunology 23,871-879. 7. d'Eustachio, P. & Edelman, G. M. (1975) J. Exp. Med. 142, 1078-1091. 8. Cohn, M. (1974) Prog. Immunol. 2,261-284. 9. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J. Immunol. 2, 203-212. 10. Sidman, C. L. & Unanue, E. E. (1975) Nature 257,149-151. 11. Couthino, A. & Mller, G. (1975) Adv. Immunol. 21, 113-236 12. Gronowicz, E., Couthino, A. & M6ller, G. (1974) Scand. J. Immunol. 3,413421. 13. Raff, M. C., Owen, J. T., Cooper, M. D., Lawton, A. R., Megson, M. & Gathings, W. E. (1975) J. Exp. Med. 142,1052-1064. 14. Nossal, G. J. V. & Pike, B. (1975) J. Exp. Med. 141, 904-917. 15. Gelfand, M. C., Sachs, D. H., Lieberman, R. & Paul, W. E. (1974) J. Exp. Med. 139,1142-1153. 16. Vitetta, E. S. & Uhr, J. W. (1975) Science 189,964-969. 17. Lederberg, J. (1959) Science 129, 1649-1653. 18. Medawar, P. B. (1961) Nature 189, 14-17. 19. Billingham, R. E. & Brent, L. (1957) Proc. R. Soc. London 146, 78-90. 20. Harrison, M. R. & Mage, G. (1973) J. Exp. Med. 138, 764774. 21. Strayer, D., Cosenza, H., Lee, W. M., Rowley, D. A. & K6hler, H. (1974) Science 186, 640-643. 22. Lawton, A. R. & Cooper, M. D. (1974) Contemp. Top. Immunobiol. 3, 193-225. 23. Cohen, I. R. & Feldman, M. (1975) Ann. N.Y. Acad. Sci. 249, 106-115. 24. Ada, G. L. & Cooper, M. G. (1975) in Immunological Tolerance, eds. Katz, D. H. & Benacerraf, B. (Academic Press, New York), pp. 87-106. 25. Golub, E. S. (1971) Cell. Immunol. 2,353-361. 26. Cunningham, S. J. (1975) Nature 254, 143-144. 27. Jerne, N. K. (1974) Ann. Immunol. (Paris) 125C, 373-389.
