6
Eur. 1.Immunol. 1977. 7: 6-10
C.J. Elson
7 Claman, H., J. Immunol 1976.116: 704. 8 Scott, D.W. and Long, C.A., J. Exp. Med. 1976.144: 1369. 9 Miller, S.D.andclaman, H., Fed. Proc. 1976.35: 789. 10 Howard, J.C., Hunt, S.V. and Gowans, J.L., J. Exp. Med. 1972. 135: 200. 1 1 Rittenberg, MB.and Amkraut, A.A., J. Immunol. 1966. 97: 421. 12 Golub, E.S. and Weigle, W.O.,J. Immunol. 1969. 102: 389. 13 Scott, D.W., J. Immunol. 1973. 1 1 1 : 789. 14 Shearer, G.M., Rehn, T.G. and Carbarino, C.A., J. Exp. Med. 1975.141: 1348. 15 Ornellas, E.P. and Scott, D.W., Cell. Immunol. 1974. 11: 108. 16 Rittenberg, M.G. and Ratt, K.L., Proc. SOC.Exp. Eiol Med. 1969. 132: 575. 17 Handwerger, B.D. and Schwartz, R.H., Tkansplantation 1974. 18: 544. 18 Golub, E.S., Mishell, R.I., Weigle, W.O. and Dutton, R.S., J. Immunol. 1968. 100: 133. 19 Sabbadini, E., Neri, A. and Sehon, A.H., J. Immunol. Methods 1974.5: 9. 20 Scott, D.W., Cell Immunol. 1976. 22: 311. 21 Chiller, J.M., Habicht, G. and Weigle, W.O., Science 1971. 171: 813.
22 Walters, C.S. and Claman, H.N., J. Immunol. 1974.113: 645. 23 Asherson, G.L., Zembala, M., and Barnes, RM.R., Clin. Exp. Immunol 1971. 9: 111. 24 Fidler, J.M. and Golub, E.S.,J. Exp. Med. 1973. 137: 42. 25 Phanuphak, P., Moorhead, J.W. and Claman, H.N., J. Immunol. 1974.112: 115. 26 Moorhead, J.W., in Sighal, S.K. and St. C. Sinclair, H.R. (Eds.) Mode of Action of Suppressor Cells in Contuct Sensitivity to DNFB.Suppressor Cells in Immunity, University of Western Ontario, London, Canada 1975, p. 61. 27 Moorhead, J.W. and D.W. Scott, Cell Immunol. 1977, in press. 28 Naor, D., Mishell, RI. and Wofsy, L., J. Immunol. 1970. 105: 1322. 29 Huber, B.T., Cantor, H. and Gershon, R., Fed. Proc. 1976. 35: 432. 30 Cantor, H. and Boyse, E.A., J. Exp. Med 1975.141: 1376. 31 Forman, J. and Kettman, I., Fed. PKJC.1976.35: 474. 3 2 Koren, H.S., Wunderlich, J.R. and Inman, J.K., J. Immunol. 1976.116: 403. 33 Koskimies, S. and Kkela, O., J. exp. Med. 1976. 144: 467. 34 Naor, D., Saltoun, R. and Falkenberg, F., Eur. J. Immunol. 1975. 5: 220. 35 Borel, Y., Damplant. Rev. 1976. 31: 000.
C.J. Elson
Tolerance in differentiating B lymphocytes
M.R.C. lmmunobiology Group, Department of Pathology, University of Bristol, Bristol
These experiments demonstrate that the response of B lymphocytes t o particulate antigen in vivo can depend on their stage of differentiation: B lymphocytes differentiating from stem cells were rendered tolerant, while mature B lymphocytes were primed under the same conditions. Lethally irradiated mice of one allotype were repopulated with 13- 15 day fetal liver cells from congenic mice bearing another allotype and the effect of antigen on the emergence of responsive B cells determined. B cells, descended from the fetal liver inoculum (identified by their allotype) produced an antibody response t o antigen in the presence of additional T cells, 15 days after transfer. Such a response could be prevented by injecting the recipient with alum-precipitated antigen (but not with deaggregated antigen) shortly after irradiation and reconstitution. This unresponsive state was specific and independent of afferent suppressor mechanisms. Mature B cells, on the other hand, were shown t o be primed by alum precipitated antigen in irradiated reconstituted hosts. It was concluded that a fundamental difference exists between the responsiveness of mature and differentiating B lymphocytes. Tolerance in differentiating B lymphocytes is discussed in relation t o the acquisition of self-tolerance.
1. Introduction
Although it has long been realized that immune reactions t o autologous antigens must be controlled or prevented [I], it is only recently that mechanisms whereby this might occur have come t o light. There are several means whereby exposure [I 15781 Correspondence: C.J. Elson, M.R.C. Immunobiology Group, Department of Pathology, University of Bristol, The Medical School, University Walk, Bristol BS8 ITD, England
Abbreviations: B cells: Bone marrow-derived lymphocytes T cdr: Thymus-dependent lymphocytes HGG: Human I& BSA: Bovine serum albumin DNP: 2,4Dinitrophenyl (A): Alum-precipitated (A€%Alum-precipitated plus E. perfussis PLC Fetal liver cells
to antigen leads to unresponsiveness at the whole animal level. For example, tolerance can be readily induced in T lymphocytes, leaving B lymphocytes unaffected, by exposing adult animals t o small doses of deaggregated protein antigens [2, 31. Unresponsiveness occurs either because the antigen-reactive T lymphocytes are deleted, thus rendering them unavailable t o cooperate with the corresponding B cells or because of the activity of a subclass of T lymphocytes (suppressor T cells) which have the capacity t o suppress the response of other lymphocytes (for review see [4]). It could be argued that deletion of autoreactive clones of T lymphocytes and/or the activity of autoreactive suppressor T cells between them suffice to control the response of autoreactive B cells and that there is no need for these B cells t o become tolerant. This view is supported by the fact that autoantibodies can often be in-
Tolerance in differentiating B lymphocytes
Eur. J. Immunol. 1977. 7 6-10 duced by immunization with antigens which share a common epitope with autologous antigens but also have a foreign epitope which can be recognized by T cells [5,6]. However, exposure to such cross-reactive immunogens does not always lead to autoantibody production, even in a situation where the B cells are unlikely t o be blocked by the corresponding autoantigen [ 71, it is therefore suggested that in these cases the corresponding B cells have in fact been deleted. One mechanism of B cell tolerance originally suggested by Lederberg [8] and elaborated by Nossal[9], is that at some stage in their differentiation from stem cells t o mature antibody-forming precursor cells, lymphocytes go through a phase during which contact with antigen induces only tolerance, not immunity. We therefore embarked on studies designed t o test in vivo whether B lymphocytes differ in their response t o antigen at different stages in their differentiation. This involved the transfer of stem cells of one allotype either alone, or together with mature B lymphocytes from congenic mice bearing another allotype, to lethally irradiated donors. The response of the different populations tested was then identified by the allotype of the antibody they produced.
2.4.
7
T cell depletion
Rabbit anti-mouse brain serum [ 1 I ] was prepared, absorbed and its specificity for T cells tested as described previously [ 121. Cells (5 x 10' ml) were incubated at 37 OC for 1 h with a 1/50 dilution of anti-brain serum and a 1/20 dilution of agarose-absorbed guinea pig complement. After incubation the cells were washed twice. 2.5. General plan of experiments
CBA mice were lethally irradiated (800 r) and restored by intravenous (i.v.1 transfusion of 4 x 106 Igb fetal liver cells. Some mice were given intraperitoneal (i.p.) injections of antigen, with the intention of inducing tolerance in the differentiating B lymphocytes, and others were left untreated as controls. Fifteen days after irradiation and reconstitution the mice were challenged with an i.p. injection of I00 pg alumprecipitated antigen with pertussis as adjuvant and given 5 x 1 O6 CBA spleen cells i.v. as a source of cooperating T cells. They were bled 18 days later and the sera collected. 2.6. Antibody assay
2. Materials and methods 2.1. Animals
CBA/H mice which bear the Iga allotype and the congenic strain CBA/H Igb (referred to as Igb mice) were used. 2.2. Antigens
The human IgG (HGG) used for immunization and tolerance induction was Cohn fraction I1 from Sigma Chemical Co., St. Louis,Mo.,USA. Before coupling to the immunoabsorbent (see Sec. 2.6.) Cohn fraction I1 was further purified by precipitation with 33 % ammonium sulfate and elution from DEAEcellulose in 0.01 M TriaHC1, pH 8.0. Solutions of HGG were deaggregated by centrifugation at 90 000 x g for 2 112 h. The top third was removed and injected into mice immediately. Bovine serum albumin (BSA) was Cohn fraction V from Armour, Chicago, Ill. Batches of dinitrophenylated HGG were prepared by the reaction of sodium 2,4dinitrobenzene sulfonate in sodium carbonate with HGG as described by Little and Eisen [lo]. Antigens were alum-precipitated by adding 10 parts 9 % aluminium potassium sulfate t o 4.6 parts of a solution containing 2-5 mg antigen/ml in 1 M sodium hydrogen carbonate. The resultant precipitate was washed three times in saline. Alumprecipitated antigen is denoted by a suffix e.g. HGG(A). 2.3. Cell suspensions
Igb mice were mated and the females examined for vaginal plugs. Pregnant mice were killed 13- 15 days after impregnation and the fetal livers removed. The livers were disaggregated in a loosely fitting ground glass homogenizer and the cells resuspended in balanced salt solution. They were filtered through wire gauze. Cell suspensions of the spleen and superficial lymph nodes of adult mice were made similarly.
The radioimmunoassay of Klinman and Taylor [ 131 as modified by Elson and Taylor [ 121 was used. Essentially, HGG, DNP or BSA were coupled t o bromacetyl cellulose. Aliquots of this were added t o dilutions of the test sera, incubated for I h at 37 "C and washed. After overnight incubation at 4 OC with either 12SI-labeledpurified anti-allotype (anti-Igb or anti-Iga) antibody or purified sheep anti-mouse Ig antibody, the immunoabsorbent was washed again and the radioactivity bound to it counted. By comparing these counts with those obtained by reacting purified Igb or Iga anti-DNP with trinitrophenylated bromacetyl cellulose it was possible to estimate the antibody concentration in the test sera in pg/ml. 3. Results 3.1. Capacity of soluble and particulate HCC t o induce
tolerance in differentiating and mature B lymphocytes Small amounts of deaggregated hapten-protein conjugates have been found to induce tolerance in differentiating B lymphocytes in vitro [9]. An initial experiment was therefore set up to test the effect of exposing differentiating B lymphocytes t o deaggregated antigen in vivo. From the results recorded in Fig. 1 it is evident that deaggregated HGG had n o effect on the Igb B lymphocytes derived from fetal liver cells (FLC). The above results led the author t o question if the use of soluble antigens t o induce tolerance mimicked the in vivo realities because the antigens t o which one might expect differentiating B lymphocytes t o become tolerant would be those such as major blood group antigens or histocompatibility antigens which are capable of priming mature B cells. These would be multivalent or particulate antigens. Accordingly, the effect of exposing differentiating B lymphocytes t o a particulate form of the same antigen was tested. The results of this experiment (Fig. 2a) revealed that a dose of 1 mg HGG(A) induced almost total unresponsiveness, but smaller amounts had no suppressive effect. Again, deaggre-
8
Eur. J. Immunol. 1977. 7: 6-10
C.J. Elson
Figure 1. Lack of effect of deaggregated HGG on the response of differentiating B lymphocytes. Irradiated fetal liver-reconstituted mice were given four injections at three-day intervals of either 10 or 100 pg of deaggregated HGG or left as controls. They were challenged with HGG(AP) and given CBA spleen cells on day 15. Mean *standard deviation.
It is possible that the tolerogenic effect of HGG(A) o n differentiating B lymphocytes was merely due t o the host environment being conductive t o the induction of tolerance, for example, because of a deficit of helper T cells. I n order to establish, therefore, that there were differences between differentiating and mature B lymphocytes in their response to HGG(A) it was necessary to measure the response of mature B lymphocytes in the same environment as was used for differentiating lymphocytes. Accordingly, the response of lymph node cells, which had been depleted of T cells by treatment with anti-brain serum, were tested in irradiated mice reconstituted with FLC. As can be seen from Fig. 3, lymph node B cells were primed by exposure t o doses of HGG(A) ranging from 10 p g t o 1 mg. Compare this with Fig. 2a in which it is seen that B cells derived from the FLC were unaffected by the lower doses or tolerized by the highest.
gated HGG was without effect even at much higher doses. The injection of HGG(A) into lethally irradiated mice shortly after reconstitution led to severe runting and u p t o 75 % mortality. Experiments were therefore set up t o find ways of reducing this. The results of one such experiment are shown in Fig. 2b. The tolerance-inducing injection of 500 yg HGG(A) was delayed until 5 days after irradiation and reconstitution. This regime reduced the incidence of mortality but still induced tolerance as evidenced by the 20-fold drop in antibody response as compared with controls.
(b)
n
"GG
-
WJ 1Mpo 4
A
"
*ma A
Figure 3. Effect of HGG(A) on mature B lymphocytes in irradiated fetal liverreconstituted recipients. Irradiated mice were given Igb FLC from the same pool as those shown to be tolerized in Fig. 2a. In addition they were injected i.v. with 2 x lo7 antibrain serum-treated lymph node cells from CBA (I@) donors. One day later they were given various doses of HGG(A). On day 15 they were challenged with HGG(AP) and injected with 5 x lo6 Igq Igb spleen cells.
3.2. Specificity of unresponsiveness
f
t HGG
IEEl
n
0
"
"
"
"
lma
lomi
A
A
A
O
O
l o p 1Wpo 1mg
"
Figure 2. Effect of HGG(A) on tolerance induction in differentiating B lymphocytes. (a) Irradiated mice were reconstituted with Igb FLC. A day later they were given various doses of either alum-precipitated (A) or deaggregated (D) HGG or left as controls They were challenged with HGG(A) and injected with 5 x 106 CBA spleen cells on day 15. (b) Mice were exposed to HGG 5 days after irradiation and reconstitution or left as controls.
It was considered that HGG(A) might exert a nonspecific suppressive effect o n the differentiation of B lymphocytes from FLC. This was excluded by the results of the experiment illustrated in Fig. 4.Differentiating lymphocytes were rendered unresponsive to an epitope (DNP) only if they were exposed to that epitope during their differentiation.
3.3. Is tolerance d u e t o suppressor influences Unresponsive states may exist because of active suppressor influences [14-171. In view of this it seemed important to test if the unresponsive state described above was maintained by suppressor mechanisms or if the B cells were truly tolerant. Using the allotype markers, the response of a transferred population of putatively tolerant B cells was measured in the presence of other cells which were responding. As can be seen from Fig. 5 there was n o evidence that the tolerant cells had
Tolerance in differentiating B lymphocytes
Eur. J. ImmunoL 1977. 7: 6-10
I t -
D
I
-
.
”
-
DNP,
BSA
lcxI
HGG (A)
(A)
(A)
Figure 4. Specificity of unresponsiveness. Lethally irradiated mice were reconstituted with Igb FLC and 5 days afterwards injected with either DNP4HGC(A), BSA(A) or HCG(A) or left untreated as controls. On day 15 they were challenged with DNP4HGG(AP) and injected i.v. with CBA spleen cells.
Figure 5. Influence of tolerant cells on responsiveness of normal cells. 2 x 107 spleen cells of Igb origin (see MOW) were transferred to 500 r-irradiated CBA recipients together with 107 normal CBA spleen cells. The recipients were challenged with DNP-HGG(AP) and bled 21 days later. ( 0 ) Igb anti-DNP, (0)total anti-DNP. The Igb cells came from lethally irradiated CBA donors 40 days after reconstitution with Igb FLC and variously left untreated (N), challenged with DNP-HGG(AP) on day 15 (C), or given 5 mg DNP-HGG(A) on day 5 and challenged with DNP-HGG(AP) on day 15 0).
suppressed the response of the normal cells, as judged by the total anti-DNP response, nor that the normal cells had “broken” the tolerance of the Igb population. 3.4. Can soluble multivalent antigen induce tolerance
An experiment was set u p in which irradiated reconstituted mice were given either 1 mg soluble DNP2,HGG o r left untreated as controls. Eighteen days after challenge and injection of CBA spleen cells the Igb anti-DNP response of the control group was 0.63 f 0.49 loglo (pg/ml) and that of the treated group was 0.26 ? 0.68 loglo (pglml). This difference is not significant ( t = 1.23 p 0.2).
>
4. Discussion 4.1. Differentiating lymphocytes
Mature B lymphocytes are here considered as those capable of giving a positive response t o antigen, and differentiating B lymphocytes as their precursors in the process of development from stem cells which lack this ability. After mice have
9
been lethally irradiated and reconstituted with early FLC, mature B lymphocytes are detected by about 8-10 days; thereafter their numbers increase exponentially until they begin t o plateau during t h e third week [ 18, 191. This sequence is consistent with the protocol used t o test the effect of antigen on differentiating B lymphocytes because at the time of exposure only differentiating B cells free from mature cells should be present. I t should be emphasized that adult bone marrow cells may not b e a suitable source of differentiating B lymphocytes f o r use in this protocol because they contain u p t o 50 % cells with detectable surface immunoglobulin [20] some of which are mature B cells [21, 221. 4.2. Tolerance in differentiating lymphocytes
Tolerance independent of active suppressor mechanisms was induced by exposing differentiating B cells t o alum precipitated antigen. Since mature B cells in t h e same environment were primed, a fundamental difference must exist between the responsiveness of mature and differentiating B lymphocytes, a conclusion in accord with t h e Lederberg-Nossal hypothesis [8, 91. Some comment is required o n the finding that at least 10-fold less antigen was necessary t o prime mature B cells than t o induce tolerance in differentiating B cells. This may be merely because some differentiating B cells fail t o come into contact with antigen at low doses and that this likelihood is decreased as the amount of antigen is increased, o r it could indicate that tolerance requires longer persistence of the antigen. It has been reported that exposure of cultured bone marrow cells to monomeric paucivalent antigens induced tolerance probably by preventing the development of the corresponding mature B cells from their progenitors [9]. Here, in contrast, tolerance was not induced b y a similar antigen in vivo. This may merely reflect differences between the behavior of B lymphocyte precursors differentiating in vitro and in vivo. On the other hand, t h e explanation may lie in the fact that thymus-independent antigens and thymus-dependent antigens stimulate different subpopulations of B lymphocytes [23-251. Possibly, t h e precursors of such subpopulations also have different susceptibilities t o tolerance induction. I t may be significant, therefore, that Nossal and Pike [25] used a thymusindependent antigen for challenge, whereas we used a thymusdependent antigen. I t may be asked why alum-precipitated antigen* is so effective in inducing tolerance as compared with deaggregated antigen. Some insight may be gained from recent work of Metcalf and Klinman [26] w h o found that multivalent but not monovalent antigens induced tolerance in developing B cells in vitro. Tolerance was induced more easily in IgM than in IgC precursors. Indeed, the degree of tolerance induced in IgC precursors was comparable t o that induced by soluble multivalent antigen in the present work. Assuming that tolerance cannot be induced in differentiating B lymphocytes until they acquire surface immunoglobulin receptors, we suggest that any factor which increases the avidity of antigen binding t o the receptors promotes tolerance induction, provided that the cells are in an appropriate environment. It is known that increasing the
* The efficiency of alum-precipitated antigen in inducing tolerance in neonatal rats has already been reported [31].
10
Eur. J. Immunol. 1977. 7: 6-10
C.J. Elson
valency increases the avidity of antigen binding t o virgin B lymphocytes [27, 281 and the effect of alum-precipitation of the antigen would be t o increase this. 4.3. Autoimmunity and tolerance in differentiating B cells
If tolerance in differentiating B lymphocytes is a mechanism involved in the control of the immune response t o self-antigens, then it is not immediately apparent why some autoreactive B cells fail t o become tolerant, particularly as these lymphocytes after appropriate stimulation, can cause deleterious autoimmune reactions [29]. Trivial explanations, such as the relevant antigen being sequestered, are reported not t o be a sufficient reason for some autoreactive B cells escaping tolerance-inducing mechanisms [30-321. Instead the difficulty may be resolved by supposing that particular properties of antigens, such as paucivalency, determine whether or not the corresponding clones of lymphocytes are tolerized, As a generalization it seems pertinent t o point o u t that autoimmune reactions are often directed t o what could be considered as relatively weak immunogens, for example, the e antigen in warm-type hemolytic anemia [33]. Antigens of this type may fail t o induce tolerance in differentiating B lymphocytes for t h e same reasons that they are weak immunogens. In this event the B cells are still available t o be activated when other control mechanisms are disturbed.
6 Playfair, J.H. and Clarke, S.M., Nature-New Biol. 1973.243: 213. 7 Reichlin, M., J. Mol. Biol. 1972. 64: 496. 8 Lederberg, J., Science 1959.129: 1649. 9 Nossal, G.J.V. and Pike,B.L., J. Exp. Med. 1975.141: 904. 10 Little, J.R. and Eisen, H.W., in Williams, C.A. and Chase, M.W.
(Eds.) Methods in Immunology and Immunochernistry ,Academic Press, New York and London 1967, Vol. I , p. 128. 11 Golub, E.S., Cell. Immunol. 1971.2: 253. 12 Elson, C.J. and Taylor, R.B., Eur. J. Immunol. 1974.4: 682. 13 Klinman, N.R. and Taylor, R.B., Clin. Exp. Immunol. 1969.1 : 3. 14 Gershon, R.K. and Kondo, K., Immunology 1971,21: 903. 15 Terman, D.S., Minden, P. and Crowle, A.J., Cell. Immunol. 1973. 6: 284. 16 Basten, A., Miller, J.F.A.P., Sprent, J. and Cheers, C., J. Exp. Med. 1974.140: 199. 17 Benjamin, D.C., J. Exp. Med. 1975.141: 635. 18 Nossal, G.J.V. and Pike, B.L., Immunology 1973.25: 30. 19 Sherwin, W.K. and Rowlands, D.T., J. Immunol. 1975.115: 1549. 20 Osmond, D.G. and Nossal, G.J.V., Cell.Immunol. 1973.13: 117. 21 LaFleur, L., Miller, R.G. and Phillips, R.A., J. Exp. Med. 1972. 135: 1363. 22 Stocker, J.W., Osmond, D.G. and Nossal, G.J.V., Immunology 1974. 27: 795. 23 Gorczynski, R.M. and Feldmann, M., Cell. Immunol. 1975. 18: 88. 24 Quintdns, J. and Cosenza, H., Eur. J. Immunol. 1976. 6 : 399.
I thank Dr. R.B. Taylor and Dr. J.D. Naysmith for helpful discussion. Received October 27,1976.
25 Lewis, G.K., Ranken, R., Nitecki, D.E. and Goodman, J.W., J. Exp. Med. 1976. 144: 382. 26 Metcalf, E.S. and Klinman, N.R., J. Exp. Med. 1976. 143: 1327. 27 Wilson, J.D. and Feldmann, M., Nature-New Biol. 1972. 2 3 7 3.
5. References 1 Ehrlich, P., in Himmelweit, F., Marquardt, M. and Dale, H.D. (Eds.)
The Collected Papers of Paul Ehrlich, Pergamon Press, London, 2: 205. 2 Taybr,R.B., Transplant. Rev. 1969. I : 114.
3 Chiller, J.M., Habicht, G.S. and Weigle, W.O.,Proc. Nat. Acad. Sci. US 1970.65: 551. 4 Taylor, R.B. and Basten, A., Brit. Med. Bull. 1976.32: 152. 5 Iverson, G.M. and Lindenmann, J.,Eur. J. Immunol. 1972.2: 195.
28 Klaus, G.G.B., Eur. J. Irnmunol. 1975. 5: 336. 29 Allison, A.C., in Katz, D.H. and Benacerraf, B. (Eds.) Immuno-
logical Tolerance: Mechanisms and Potential Therapeutic Applications, Academic Press, New York 1974, p. 25. 30 Weir, D.M. and Pinckard, R.N.,Imrnunology 1967. 13: 373. 31 Elson, C.J. and Weir, D.M., Clin. Exp. Immunol. 1969, 3: 725. 32 Torrigiani, G., Doniach, D. and Roitt, I.M., J. Clin. Endocrinol. 1969. 29: 305.
33 Dacie, J.V., The Hemolytic Anaemias Part I1 - The Autoimmune Haemolytic Anaemias, Churchill, London 1962, p. 443.
Eur. 1.Immunol. 1977. 7: 6-10
C.J. Elson
7 Claman, H., J. Immunol 1976.116: 704. 8 Scott, D.W. and Long, C.A., J. Exp. Med. 1976.144: 1369. 9 Miller, S.D.andclaman, H., Fed. Proc. 1976.35: 789. 10 Howard, J.C., Hunt, S.V. and Gowans, J.L., J. Exp. Med. 1972. 135: 200. 1 1 Rittenberg, MB.and Amkraut, A.A., J. Immunol. 1966. 97: 421. 12 Golub, E.S. and Weigle, W.O.,J. Immunol. 1969. 102: 389. 13 Scott, D.W., J. Immunol. 1973. 1 1 1 : 789. 14 Shearer, G.M., Rehn, T.G. and Carbarino, C.A., J. Exp. Med. 1975.141: 1348. 15 Ornellas, E.P. and Scott, D.W., Cell. Immunol. 1974. 11: 108. 16 Rittenberg, M.G. and Ratt, K.L., Proc. SOC.Exp. Eiol Med. 1969. 132: 575. 17 Handwerger, B.D. and Schwartz, R.H., Tkansplantation 1974. 18: 544. 18 Golub, E.S., Mishell, R.I., Weigle, W.O. and Dutton, R.S., J. Immunol. 1968. 100: 133. 19 Sabbadini, E., Neri, A. and Sehon, A.H., J. Immunol. Methods 1974.5: 9. 20 Scott, D.W., Cell Immunol. 1976. 22: 311. 21 Chiller, J.M., Habicht, G. and Weigle, W.O., Science 1971. 171: 813.
22 Walters, C.S. and Claman, H.N., J. Immunol. 1974.113: 645. 23 Asherson, G.L., Zembala, M., and Barnes, RM.R., Clin. Exp. Immunol 1971. 9: 111. 24 Fidler, J.M. and Golub, E.S.,J. Exp. Med. 1973. 137: 42. 25 Phanuphak, P., Moorhead, J.W. and Claman, H.N., J. Immunol. 1974.112: 115. 26 Moorhead, J.W., in Sighal, S.K. and St. C. Sinclair, H.R. (Eds.) Mode of Action of Suppressor Cells in Contuct Sensitivity to DNFB.Suppressor Cells in Immunity, University of Western Ontario, London, Canada 1975, p. 61. 27 Moorhead, J.W. and D.W. Scott, Cell Immunol. 1977, in press. 28 Naor, D., Mishell, RI. and Wofsy, L., J. Immunol. 1970. 105: 1322. 29 Huber, B.T., Cantor, H. and Gershon, R., Fed. Proc. 1976. 35: 432. 30 Cantor, H. and Boyse, E.A., J. Exp. Med 1975.141: 1376. 31 Forman, J. and Kettman, I., Fed. PKJC.1976.35: 474. 3 2 Koren, H.S., Wunderlich, J.R. and Inman, J.K., J. Immunol. 1976.116: 403. 33 Koskimies, S. and Kkela, O., J. exp. Med. 1976. 144: 467. 34 Naor, D., Saltoun, R. and Falkenberg, F., Eur. J. Immunol. 1975. 5: 220. 35 Borel, Y., Damplant. Rev. 1976. 31: 000.
C.J. Elson
Tolerance in differentiating B lymphocytes
M.R.C. lmmunobiology Group, Department of Pathology, University of Bristol, Bristol
These experiments demonstrate that the response of B lymphocytes t o particulate antigen in vivo can depend on their stage of differentiation: B lymphocytes differentiating from stem cells were rendered tolerant, while mature B lymphocytes were primed under the same conditions. Lethally irradiated mice of one allotype were repopulated with 13- 15 day fetal liver cells from congenic mice bearing another allotype and the effect of antigen on the emergence of responsive B cells determined. B cells, descended from the fetal liver inoculum (identified by their allotype) produced an antibody response t o antigen in the presence of additional T cells, 15 days after transfer. Such a response could be prevented by injecting the recipient with alum-precipitated antigen (but not with deaggregated antigen) shortly after irradiation and reconstitution. This unresponsive state was specific and independent of afferent suppressor mechanisms. Mature B cells, on the other hand, were shown t o be primed by alum precipitated antigen in irradiated reconstituted hosts. It was concluded that a fundamental difference exists between the responsiveness of mature and differentiating B lymphocytes. Tolerance in differentiating B lymphocytes is discussed in relation t o the acquisition of self-tolerance.
1. Introduction
Although it has long been realized that immune reactions t o autologous antigens must be controlled or prevented [I], it is only recently that mechanisms whereby this might occur have come t o light. There are several means whereby exposure [I 15781 Correspondence: C.J. Elson, M.R.C. Immunobiology Group, Department of Pathology, University of Bristol, The Medical School, University Walk, Bristol BS8 ITD, England
Abbreviations: B cells: Bone marrow-derived lymphocytes T cdr: Thymus-dependent lymphocytes HGG: Human I& BSA: Bovine serum albumin DNP: 2,4Dinitrophenyl (A): Alum-precipitated (A€%Alum-precipitated plus E. perfussis PLC Fetal liver cells
to antigen leads to unresponsiveness at the whole animal level. For example, tolerance can be readily induced in T lymphocytes, leaving B lymphocytes unaffected, by exposing adult animals t o small doses of deaggregated protein antigens [2, 31. Unresponsiveness occurs either because the antigen-reactive T lymphocytes are deleted, thus rendering them unavailable t o cooperate with the corresponding B cells or because of the activity of a subclass of T lymphocytes (suppressor T cells) which have the capacity t o suppress the response of other lymphocytes (for review see [4]). It could be argued that deletion of autoreactive clones of T lymphocytes and/or the activity of autoreactive suppressor T cells between them suffice to control the response of autoreactive B cells and that there is no need for these B cells t o become tolerant. This view is supported by the fact that autoantibodies can often be in-
Tolerance in differentiating B lymphocytes
Eur. J. Immunol. 1977. 7 6-10 duced by immunization with antigens which share a common epitope with autologous antigens but also have a foreign epitope which can be recognized by T cells [5,6]. However, exposure to such cross-reactive immunogens does not always lead to autoantibody production, even in a situation where the B cells are unlikely t o be blocked by the corresponding autoantigen [ 71, it is therefore suggested that in these cases the corresponding B cells have in fact been deleted. One mechanism of B cell tolerance originally suggested by Lederberg [8] and elaborated by Nossal[9], is that at some stage in their differentiation from stem cells t o mature antibody-forming precursor cells, lymphocytes go through a phase during which contact with antigen induces only tolerance, not immunity. We therefore embarked on studies designed t o test in vivo whether B lymphocytes differ in their response t o antigen at different stages in their differentiation. This involved the transfer of stem cells of one allotype either alone, or together with mature B lymphocytes from congenic mice bearing another allotype, to lethally irradiated donors. The response of the different populations tested was then identified by the allotype of the antibody they produced.
2.4.
7
T cell depletion
Rabbit anti-mouse brain serum [ 1 I ] was prepared, absorbed and its specificity for T cells tested as described previously [ 121. Cells (5 x 10' ml) were incubated at 37 OC for 1 h with a 1/50 dilution of anti-brain serum and a 1/20 dilution of agarose-absorbed guinea pig complement. After incubation the cells were washed twice. 2.5. General plan of experiments
CBA mice were lethally irradiated (800 r) and restored by intravenous (i.v.1 transfusion of 4 x 106 Igb fetal liver cells. Some mice were given intraperitoneal (i.p.) injections of antigen, with the intention of inducing tolerance in the differentiating B lymphocytes, and others were left untreated as controls. Fifteen days after irradiation and reconstitution the mice were challenged with an i.p. injection of I00 pg alumprecipitated antigen with pertussis as adjuvant and given 5 x 1 O6 CBA spleen cells i.v. as a source of cooperating T cells. They were bled 18 days later and the sera collected. 2.6. Antibody assay
2. Materials and methods 2.1. Animals
CBA/H mice which bear the Iga allotype and the congenic strain CBA/H Igb (referred to as Igb mice) were used. 2.2. Antigens
The human IgG (HGG) used for immunization and tolerance induction was Cohn fraction I1 from Sigma Chemical Co., St. Louis,Mo.,USA. Before coupling to the immunoabsorbent (see Sec. 2.6.) Cohn fraction I1 was further purified by precipitation with 33 % ammonium sulfate and elution from DEAEcellulose in 0.01 M TriaHC1, pH 8.0. Solutions of HGG were deaggregated by centrifugation at 90 000 x g for 2 112 h. The top third was removed and injected into mice immediately. Bovine serum albumin (BSA) was Cohn fraction V from Armour, Chicago, Ill. Batches of dinitrophenylated HGG were prepared by the reaction of sodium 2,4dinitrobenzene sulfonate in sodium carbonate with HGG as described by Little and Eisen [lo]. Antigens were alum-precipitated by adding 10 parts 9 % aluminium potassium sulfate t o 4.6 parts of a solution containing 2-5 mg antigen/ml in 1 M sodium hydrogen carbonate. The resultant precipitate was washed three times in saline. Alumprecipitated antigen is denoted by a suffix e.g. HGG(A). 2.3. Cell suspensions
Igb mice were mated and the females examined for vaginal plugs. Pregnant mice were killed 13- 15 days after impregnation and the fetal livers removed. The livers were disaggregated in a loosely fitting ground glass homogenizer and the cells resuspended in balanced salt solution. They were filtered through wire gauze. Cell suspensions of the spleen and superficial lymph nodes of adult mice were made similarly.
The radioimmunoassay of Klinman and Taylor [ 131 as modified by Elson and Taylor [ 121 was used. Essentially, HGG, DNP or BSA were coupled t o bromacetyl cellulose. Aliquots of this were added t o dilutions of the test sera, incubated for I h at 37 "C and washed. After overnight incubation at 4 OC with either 12SI-labeledpurified anti-allotype (anti-Igb or anti-Iga) antibody or purified sheep anti-mouse Ig antibody, the immunoabsorbent was washed again and the radioactivity bound to it counted. By comparing these counts with those obtained by reacting purified Igb or Iga anti-DNP with trinitrophenylated bromacetyl cellulose it was possible to estimate the antibody concentration in the test sera in pg/ml. 3. Results 3.1. Capacity of soluble and particulate HCC t o induce
tolerance in differentiating and mature B lymphocytes Small amounts of deaggregated hapten-protein conjugates have been found to induce tolerance in differentiating B lymphocytes in vitro [9]. An initial experiment was therefore set up to test the effect of exposing differentiating B lymphocytes t o deaggregated antigen in vivo. From the results recorded in Fig. 1 it is evident that deaggregated HGG had n o effect on the Igb B lymphocytes derived from fetal liver cells (FLC). The above results led the author t o question if the use of soluble antigens t o induce tolerance mimicked the in vivo realities because the antigens t o which one might expect differentiating B lymphocytes t o become tolerant would be those such as major blood group antigens or histocompatibility antigens which are capable of priming mature B cells. These would be multivalent or particulate antigens. Accordingly, the effect of exposing differentiating B lymphocytes t o a particulate form of the same antigen was tested. The results of this experiment (Fig. 2a) revealed that a dose of 1 mg HGG(A) induced almost total unresponsiveness, but smaller amounts had no suppressive effect. Again, deaggre-
8
Eur. J. Immunol. 1977. 7: 6-10
C.J. Elson
Figure 1. Lack of effect of deaggregated HGG on the response of differentiating B lymphocytes. Irradiated fetal liver-reconstituted mice were given four injections at three-day intervals of either 10 or 100 pg of deaggregated HGG or left as controls. They were challenged with HGG(AP) and given CBA spleen cells on day 15. Mean *standard deviation.
It is possible that the tolerogenic effect of HGG(A) o n differentiating B lymphocytes was merely due t o the host environment being conductive t o the induction of tolerance, for example, because of a deficit of helper T cells. I n order to establish, therefore, that there were differences between differentiating and mature B lymphocytes in their response to HGG(A) it was necessary to measure the response of mature B lymphocytes in the same environment as was used for differentiating lymphocytes. Accordingly, the response of lymph node cells, which had been depleted of T cells by treatment with anti-brain serum, were tested in irradiated mice reconstituted with FLC. As can be seen from Fig. 3, lymph node B cells were primed by exposure t o doses of HGG(A) ranging from 10 p g t o 1 mg. Compare this with Fig. 2a in which it is seen that B cells derived from the FLC were unaffected by the lower doses or tolerized by the highest.
gated HGG was without effect even at much higher doses. The injection of HGG(A) into lethally irradiated mice shortly after reconstitution led to severe runting and u p t o 75 % mortality. Experiments were therefore set up t o find ways of reducing this. The results of one such experiment are shown in Fig. 2b. The tolerance-inducing injection of 500 yg HGG(A) was delayed until 5 days after irradiation and reconstitution. This regime reduced the incidence of mortality but still induced tolerance as evidenced by the 20-fold drop in antibody response as compared with controls.
(b)
n
"GG
-
WJ 1Mpo 4
A
"
*ma A
Figure 3. Effect of HGG(A) on mature B lymphocytes in irradiated fetal liverreconstituted recipients. Irradiated mice were given Igb FLC from the same pool as those shown to be tolerized in Fig. 2a. In addition they were injected i.v. with 2 x lo7 antibrain serum-treated lymph node cells from CBA (I@) donors. One day later they were given various doses of HGG(A). On day 15 they were challenged with HGG(AP) and injected with 5 x lo6 Igq Igb spleen cells.
3.2. Specificity of unresponsiveness
f
t HGG
IEEl
n
0
"
"
"
"
lma
lomi
A
A
A
O
O
l o p 1Wpo 1mg
"
Figure 2. Effect of HGG(A) on tolerance induction in differentiating B lymphocytes. (a) Irradiated mice were reconstituted with Igb FLC. A day later they were given various doses of either alum-precipitated (A) or deaggregated (D) HGG or left as controls They were challenged with HGG(A) and injected with 5 x 106 CBA spleen cells on day 15. (b) Mice were exposed to HGG 5 days after irradiation and reconstitution or left as controls.
It was considered that HGG(A) might exert a nonspecific suppressive effect o n the differentiation of B lymphocytes from FLC. This was excluded by the results of the experiment illustrated in Fig. 4.Differentiating lymphocytes were rendered unresponsive to an epitope (DNP) only if they were exposed to that epitope during their differentiation.
3.3. Is tolerance d u e t o suppressor influences Unresponsive states may exist because of active suppressor influences [14-171. In view of this it seemed important to test if the unresponsive state described above was maintained by suppressor mechanisms or if the B cells were truly tolerant. Using the allotype markers, the response of a transferred population of putatively tolerant B cells was measured in the presence of other cells which were responding. As can be seen from Fig. 5 there was n o evidence that the tolerant cells had
Tolerance in differentiating B lymphocytes
Eur. J. ImmunoL 1977. 7: 6-10
I t -
D
I
-
.
”
-
DNP,
BSA
lcxI
HGG (A)
(A)
(A)
Figure 4. Specificity of unresponsiveness. Lethally irradiated mice were reconstituted with Igb FLC and 5 days afterwards injected with either DNP4HGC(A), BSA(A) or HCG(A) or left untreated as controls. On day 15 they were challenged with DNP4HGG(AP) and injected i.v. with CBA spleen cells.
Figure 5. Influence of tolerant cells on responsiveness of normal cells. 2 x 107 spleen cells of Igb origin (see MOW) were transferred to 500 r-irradiated CBA recipients together with 107 normal CBA spleen cells. The recipients were challenged with DNP-HGG(AP) and bled 21 days later. ( 0 ) Igb anti-DNP, (0)total anti-DNP. The Igb cells came from lethally irradiated CBA donors 40 days after reconstitution with Igb FLC and variously left untreated (N), challenged with DNP-HGG(AP) on day 15 (C), or given 5 mg DNP-HGG(A) on day 5 and challenged with DNP-HGG(AP) on day 15 0).
suppressed the response of the normal cells, as judged by the total anti-DNP response, nor that the normal cells had “broken” the tolerance of the Igb population. 3.4. Can soluble multivalent antigen induce tolerance
An experiment was set u p in which irradiated reconstituted mice were given either 1 mg soluble DNP2,HGG o r left untreated as controls. Eighteen days after challenge and injection of CBA spleen cells the Igb anti-DNP response of the control group was 0.63 f 0.49 loglo (pg/ml) and that of the treated group was 0.26 ? 0.68 loglo (pglml). This difference is not significant ( t = 1.23 p 0.2).
>
4. Discussion 4.1. Differentiating lymphocytes
Mature B lymphocytes are here considered as those capable of giving a positive response t o antigen, and differentiating B lymphocytes as their precursors in the process of development from stem cells which lack this ability. After mice have
9
been lethally irradiated and reconstituted with early FLC, mature B lymphocytes are detected by about 8-10 days; thereafter their numbers increase exponentially until they begin t o plateau during t h e third week [ 18, 191. This sequence is consistent with the protocol used t o test the effect of antigen on differentiating B lymphocytes because at the time of exposure only differentiating B cells free from mature cells should be present. I t should be emphasized that adult bone marrow cells may not b e a suitable source of differentiating B lymphocytes f o r use in this protocol because they contain u p t o 50 % cells with detectable surface immunoglobulin [20] some of which are mature B cells [21, 221. 4.2. Tolerance in differentiating lymphocytes
Tolerance independent of active suppressor mechanisms was induced by exposing differentiating B cells t o alum precipitated antigen. Since mature B cells in t h e same environment were primed, a fundamental difference must exist between the responsiveness of mature and differentiating B lymphocytes, a conclusion in accord with t h e Lederberg-Nossal hypothesis [8, 91. Some comment is required o n the finding that at least 10-fold less antigen was necessary t o prime mature B cells than t o induce tolerance in differentiating B cells. This may be merely because some differentiating B cells fail t o come into contact with antigen at low doses and that this likelihood is decreased as the amount of antigen is increased, o r it could indicate that tolerance requires longer persistence of the antigen. It has been reported that exposure of cultured bone marrow cells to monomeric paucivalent antigens induced tolerance probably by preventing the development of the corresponding mature B cells from their progenitors [9]. Here, in contrast, tolerance was not induced b y a similar antigen in vivo. This may merely reflect differences between the behavior of B lymphocyte precursors differentiating in vitro and in vivo. On the other hand, t h e explanation may lie in the fact that thymus-independent antigens and thymus-dependent antigens stimulate different subpopulations of B lymphocytes [23-251. Possibly, t h e precursors of such subpopulations also have different susceptibilities t o tolerance induction. I t may be significant, therefore, that Nossal and Pike [25] used a thymusindependent antigen for challenge, whereas we used a thymusdependent antigen. I t may be asked why alum-precipitated antigen* is so effective in inducing tolerance as compared with deaggregated antigen. Some insight may be gained from recent work of Metcalf and Klinman [26] w h o found that multivalent but not monovalent antigens induced tolerance in developing B cells in vitro. Tolerance was induced more easily in IgM than in IgC precursors. Indeed, the degree of tolerance induced in IgC precursors was comparable t o that induced by soluble multivalent antigen in the present work. Assuming that tolerance cannot be induced in differentiating B lymphocytes until they acquire surface immunoglobulin receptors, we suggest that any factor which increases the avidity of antigen binding t o the receptors promotes tolerance induction, provided that the cells are in an appropriate environment. It is known that increasing the
* The efficiency of alum-precipitated antigen in inducing tolerance in neonatal rats has already been reported [31].
10
Eur. J. Immunol. 1977. 7: 6-10
C.J. Elson
valency increases the avidity of antigen binding t o virgin B lymphocytes [27, 281 and the effect of alum-precipitation of the antigen would be t o increase this. 4.3. Autoimmunity and tolerance in differentiating B cells
If tolerance in differentiating B lymphocytes is a mechanism involved in the control of the immune response t o self-antigens, then it is not immediately apparent why some autoreactive B cells fail t o become tolerant, particularly as these lymphocytes after appropriate stimulation, can cause deleterious autoimmune reactions [29]. Trivial explanations, such as the relevant antigen being sequestered, are reported not t o be a sufficient reason for some autoreactive B cells escaping tolerance-inducing mechanisms [30-321. Instead the difficulty may be resolved by supposing that particular properties of antigens, such as paucivalency, determine whether or not the corresponding clones of lymphocytes are tolerized, As a generalization it seems pertinent t o point o u t that autoimmune reactions are often directed t o what could be considered as relatively weak immunogens, for example, the e antigen in warm-type hemolytic anemia [33]. Antigens of this type may fail t o induce tolerance in differentiating B lymphocytes for t h e same reasons that they are weak immunogens. In this event the B cells are still available t o be activated when other control mechanisms are disturbed.
6 Playfair, J.H. and Clarke, S.M., Nature-New Biol. 1973.243: 213. 7 Reichlin, M., J. Mol. Biol. 1972. 64: 496. 8 Lederberg, J., Science 1959.129: 1649. 9 Nossal, G.J.V. and Pike,B.L., J. Exp. Med. 1975.141: 904. 10 Little, J.R. and Eisen, H.W., in Williams, C.A. and Chase, M.W.
(Eds.) Methods in Immunology and Immunochernistry ,Academic Press, New York and London 1967, Vol. I , p. 128. 11 Golub, E.S., Cell. Immunol. 1971.2: 253. 12 Elson, C.J. and Taylor, R.B., Eur. J. Immunol. 1974.4: 682. 13 Klinman, N.R. and Taylor, R.B., Clin. Exp. Immunol. 1969.1 : 3. 14 Gershon, R.K. and Kondo, K., Immunology 1971,21: 903. 15 Terman, D.S., Minden, P. and Crowle, A.J., Cell. Immunol. 1973. 6: 284. 16 Basten, A., Miller, J.F.A.P., Sprent, J. and Cheers, C., J. Exp. Med. 1974.140: 199. 17 Benjamin, D.C., J. Exp. Med. 1975.141: 635. 18 Nossal, G.J.V. and Pike, B.L., Immunology 1973.25: 30. 19 Sherwin, W.K. and Rowlands, D.T., J. Immunol. 1975.115: 1549. 20 Osmond, D.G. and Nossal, G.J.V., Cell.Immunol. 1973.13: 117. 21 LaFleur, L., Miller, R.G. and Phillips, R.A., J. Exp. Med. 1972. 135: 1363. 22 Stocker, J.W., Osmond, D.G. and Nossal, G.J.V., Immunology 1974. 27: 795. 23 Gorczynski, R.M. and Feldmann, M., Cell. Immunol. 1975. 18: 88. 24 Quintdns, J. and Cosenza, H., Eur. J. Immunol. 1976. 6 : 399.
I thank Dr. R.B. Taylor and Dr. J.D. Naysmith for helpful discussion. Received October 27,1976.
25 Lewis, G.K., Ranken, R., Nitecki, D.E. and Goodman, J.W., J. Exp. Med. 1976. 144: 382. 26 Metcalf, E.S. and Klinman, N.R., J. Exp. Med. 1976. 143: 1327. 27 Wilson, J.D. and Feldmann, M., Nature-New Biol. 1972. 2 3 7 3.
5. References 1 Ehrlich, P., in Himmelweit, F., Marquardt, M. and Dale, H.D. (Eds.)
The Collected Papers of Paul Ehrlich, Pergamon Press, London, 2: 205. 2 Taybr,R.B., Transplant. Rev. 1969. I : 114.
3 Chiller, J.M., Habicht, G.S. and Weigle, W.O.,Proc. Nat. Acad. Sci. US 1970.65: 551. 4 Taylor, R.B. and Basten, A., Brit. Med. Bull. 1976.32: 152. 5 Iverson, G.M. and Lindenmann, J.,Eur. J. Immunol. 1972.2: 195.
28 Klaus, G.G.B., Eur. J. Irnmunol. 1975. 5: 336. 29 Allison, A.C., in Katz, D.H. and Benacerraf, B. (Eds.) Immuno-
logical Tolerance: Mechanisms and Potential Therapeutic Applications, Academic Press, New York 1974, p. 25. 30 Weir, D.M. and Pinckard, R.N.,Imrnunology 1967. 13: 373. 31 Elson, C.J. and Weir, D.M., Clin. Exp. Immunol. 1969, 3: 725. 32 Torrigiani, G., Doniach, D. and Roitt, I.M., J. Clin. Endocrinol. 1969. 29: 305.
33 Dacie, J.V., The Hemolytic Anaemias Part I1 - The Autoimmune Haemolytic Anaemias, Churchill, London 1962, p. 443.
