Clin. exp. Immunol. (1978) 34, 59-62.
Prevention of tolerance in differentiating B lymphocytes by T cells C. J. EL SON Immunobiology Group, Department of Pathology, University of Bristol (Received 13 February 1978)
SUMMARY
Normal, but not tolerant, adult thymocytes prevented particulate antigen inducing tolerance in differentiating B lymphocytes. This was demonstrated by measuring the emergence of responsive B cells (identified by their allotype) developing from a foetal liver innoculum in irradiated hosts with or without the addition of antigen, thymocytes or tolerant thymocytes. The relevance of these findings to the recruitment of B lymphocytes in adult animals and to the development of those autoreactive B cells found in normal animals is discussed. INTRODUCTION Nossal & Pike (1975) elaborated the hypothesis that B lymphocytes go through a phase in their development during which they are particularly sensitive to tolerance induction. It has now been established that soluble multivalent hapten protein conjugates (some of which have no tolerizing effect on mature B cells) can induce tolerance in differentiating thymus-dependent B cells (Cambier et al., 1977; Metcalf & Klinman, 1976, 1977; Szewczuk & Siskind, 1977). This has suggested to a number of workers that soluble autologous protein antigens which bear repeating epitopes may prevent the emergence of mature B cells reactive with the epitopes (for review see Elson, Naysmith & Taylor, 1978). Perhaps, even particulate autoantigens, which are immunogenic for mature B cells, may induce tolerance in differentiating cells. This was tested for extrinsic antigens by repopulating lethally irradiated mice of one allotype with 13-15 day foetal liver cells from congenic mice bearing another allotype, and determining the effect of antigen on the emergence of responsive B cells. The B cells which had descended from the foetal liver inoculum (identified by their allotype) produced an antibody response to antigen in the presence of additional T cells, 15 days after transfer. Such a response was prevented by injecting the recipient with alum precipitated antigen shortly after irradiation and reconstitution. The unresponsive state was specific and independent of afferent suppressor mechanisms. Mature B cells, on the other hand, were primed by alum precipitated antigen in the same environment (Elson, 1977). Other workers (Bruynes et aL, 1976) have shown that keyhole limpet haemocyanin, a particularly immunogenic antigen for mature B cells, also induces tolerance in differentiating B cells. It follows that the injection of immunogens into adult animals, whilst stimulating the corresponding mature B cells, could render their immature counterparts tolerant. Alternatively, the immature cells may be protected from tolerance inducation by the environment of adult animals. One respect in which the environment of normal adult and lethally irradiated foetal liver reconstituted mice differ, is in the presence of T cells. The purpose of the experiments described here was to test whether the addition of T cells affected the induction of tolerance in T-cell dependent B cells developing from their progenitors in irradiated hosts. Correspondence: Dr C. J. Elson, Immunobiology Group, Department of Pathology, University of Bristol, The Medical
School, University Walk, Bristol BS8 1TD.
0099-9104/78/0100-0059$02.00 ©0 1978 Blackwell Scientific Publications 59
60
C. J. Elson 100
( a)
( b)
a-
0%
10~~
+
*~~~~~~~~~
I
C
C +Thy
Tol
TOI+ Thy
Tol
TolI ToI+ Thy To + Thy D
D
FIG. 1. Effect of thymocytes on tolerance induction by DNP5HyG (A) in differentiating B lymphocytes (a). Irradiated Igb mice reconstituted with CBA (Iga) foetal liver cells were given alum precipitated DNP-HyG (Tol) on day 5 or left as controls (C). In addition, half the mice in each group were given 2 x 107 Igb adult thymocytes intravenously on day 0 (Thy) (b). Irradiated foetal liver reconstituted mice were exposed to DNP5 HyG (A) on day 5 (Tol). Some of these mice were injected with 0-8 mg deaggregated HyG (D) per mouse on day 0, others with thymocytes (Thy), others with both deaggregated HyG and thymocytes, and the remainder left as controls. All mice were challenged with DNPiHyG on day 16 and the Iga response measured. The mean and standard deviation are shown.
MATERIALS AND METHODS Animals and antigens. CBA/H mice which bear the Iga allotype and the congenic strain CBA/H Igb (referred to as Igb mice) were used. The human gamma globulin (HyG) used was Cohn fraction II from Koch light. Dinitrophenylated (DNP) HyG was prepared by the reaction of sodium 2,4-dinitrobenzene sulphonate in sodium carbonate with HyG as described by Little & Eisen (1967). HyG was absorbed with mouse spleen cells and deaggregated by centrifugation as described previously (Elson & Taylor, 1977). Antigens were alum-precipitated by adding 10 parts 9%O aluminium potassium sulphate to 46 parts of a solution containing 2-5 mg antigen per ml in 1-0 M sodium hydrogen carbonate. The resultant precipitate was washed three times in saline. Cell suspensions. CBA (Iga) mice were mated and the females examined for vaginal plugs. Pregnant mice were killed 13-15 days after impregnation and the foetal livers removed. The livers were disaggregated in a loosely fitting ground glass homogeniser and the cells resuspended in balanced salt solution. They were filtered through wire gauze. Cell suspensions of the thymus and spleen of adult mice were made similarly. Cell transfers and general plan of experiments. Igb mice were lethally irradiated (800R) and restored by intravenous transfusion of 4 x 106 CBA foetal liver cells. Five days later some mice were given intraperitoneal injections of 500 Pg alum precipitated antigen, with the intention of inducing tolerance in the differentiating B lymphocytes, whilst others were left untreated as controls. Sixteen days after irradiation and reconstitution, the mice were challenged with an intraperitoneal injection of 100 jug alum precipitated antigen with B. pertussis as adjuvant and given 5 x 106 Igb spleen cells intravenously as a source of cooperating T cells. They were bled 18 days later and the sera collected. Radioimmunoassay. This was performed as described previously (Elson & Taylor, 1974). Briefly, the immunoabsorbant
Prevention oftolerance in differentiating B cells
61
was first incubated with the mouse serum to be tested, washed and then incubated with 125I-labelled purified anti-Iga allotype antibody. The immunoabsorbant was washed again and counts made of the radioactivity bound to it. By comparison with results obtained with standard purified anti-DNP it was possible to estimate antibody concentrations in test sera in terms of pg/ml.
RESULTS The results of two experiments are shown in Fig. 1. It is evident that the addition of thymocytes prevented the induction of hapten specific tolerance in differentiating B cells. This effect required the presence of carrier specific T cells because B cell tolerance was not prevented if the thymocytes were first exposed to small doses of deaggregated HyG, a procedure known to induce T cell tolerance (Chiller, Habicht & Weigle, 1972).
DISCUSSION The finding that the tolerance which is normally induced in developing B cells by exposure to particulate hapten-protein conjugates does not take place if T cells are added, is reassuring for two reasons. The first is that without the preventive effect of T cells the emergence of newly matured B cells could be suppressed by an extrinsic immunogen, thus preventing their recruitment (Ford, 1968; Elson & Sullivan, 1977) into antibody responses. There is some evidence that this may occur when the corresponding T cells are tolerant. If adult rats were first rendered unresponsive at the T cell level by multiple injections of soluble human serum albumin (HSA) (Bell & Shand, 1973; Shand & Bell, 1976) then B cell tolerance to HSA (and not priming) was induced by injecting alum-precipitated HSA plus adjuvant (Bell, Shand & Gradwell, 1977). It is reassuring also because any autoreactive B cells which might develop later in life would still become tolerant to autoantigens as long as the corresponding T cells were also tolerant. On the other hand, in the presence of self reactive T cells the corresponding autoreactive B cell may emerge unscathed. This may be one way by which those autoreactive B cells found in normal animals escape elimination (Elson, et al., 1978). It should be emphasized that the question of whether autoreactive helper T cells do exist is currently being debated (Esquivel, Rose & Kong, 1977). Even if they do, there may be insufficient of them either quantitatively or qualitatively to enable the corresponding B cells to respond to the autoantigen. It may be asked how T cells prevent the induction of tolerance in differentiating B cells. Some insight may be gained from the recent work of the Sherr, Szewczuk & Siskind (1978) who showed that the functional maturation of foetal liver B cells in irradiated hosts was accelerated by the addition of adult thymocytes. As B cells are only hypersusceptible to tolerance induction for a limited period during their development, and as tolerance induction is a time-dependent process (Metcalf & Klinman 1976, 1977; Cambier et al., 1977), then it could be that in the presence of T cells, B cells pass through the hypersusceptible phase too rapidly for tolerance to be induced. REFERENCES BELL, E.B. & SHAND, F.L. (1973) Cellular events in protein tolerant inbred rats. I. The fate of thoracic duct lymphocytes and memory cell during tolerance induction to human serum albumin. Eur. 7. Immunol. 3, 259. BELL, E.B., SHAND, F.L. & GRADwELL, S. (1977) Cellular events in protein tolerant inbred rats. IV. The mechanism of immunogen-maintained tolerance. Eur. .. Immunol. 7, 406. BRUYNES, C., URBAIN-VANSANTEN, G., PLANARD, C., DE VOS-CLEOTENS, C. & URBAIN J. (1976) Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes. Proc. Nat. Acad. Sci. (Wash.), 73, 2462.
CAMBIER, J.C., VITETTA, E.S., UHR, J.W. & KETTMAN, J.R. (1977) B-cell tolerance II TNP17 HgG induced tolerance in adult and neonatal B cells responsive to thymus dependent and independent forms of the same hapten. 3. exp. Med. 145, 778. CHILLER, J.M., HABICHT, G.S. & WEIGLE, W.O. (1971) Kinetic differences in unresponsiveness of thymus and bone marrow. Science, 171, 813. ELSON, C.J. (1977) Tolerance in differentiating B lymphocytes. Eur. 3. Immunol. 7, 6. ELSON, C.J., NAYSMITH, J.D. & TAYLOR, R.B. (1978) B-cell tolerance and autoimmunity. Int. Rev. Exp. Path. 19.
62
C. J. Elson
ELSON, C.J. & SULLIVAN, C.P. (1977) A suppressor mechanism controlling lymphocyte recruitment. Immunology, 33, 369. ELSON, C.J. & TAYLOR, R.B. (1974) The suppressive effect of carrier priming on the response to a hapten carrier conjugate. Eur. 5. Immunol. 4, 682. ELSON, C.J. & TAYLOR, R.B. (1977) Cellular basis of persistent tolerance induced by an aggregate free heterologous immunoglobulin. Immunology, 33, 635. ESQUIVEL, P.S., ROSE, N.R. & KONG, Y.C.M. (1977) Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. 3. exp. Med. 145, 1250. FORD, W.L. (1968) Duration of the inductive effect of sheep erythrocytes on the recruitment of lymphocytes in the rat. Immunology, 15, 609. LITTLE, J.R. & EISEN, M.W. (1967) Methods in Immunology and Immunochemistry (ed. by C.A. Williams & M.W. Chase), Vol. 1, p. 128. Academic Press, New York & London.
METCALF E.S. & KLINMAN, N.R. (1976) In vitro tolerance induction of neonatal murine spleen cells. J. exp. Med. 143, 1327. METCALF, E.S. & KLINMAN, N.R. (1977) In vitro tolerance induction of bone marrow cells: A marker for B cell maturation. J. Immunol. 118, 2111. NoSsAL, G.J.V. & PiKE, B.L. (1975) Evidence for the clonal abortion theory of B-lymphocyte tolerance. J. exp. Med. 141, 904. SHERR, D.H., SZEWCZUK, M.R. & SIsKIND, G.W. (1978) Ontogeny of B-lymphocyte function V. Thymus cell involvement in the functional maturation of B-lymphocytes from fetal mice transferred into adult irradiated hosts. 3. exp. Med. 149, 196. SHAND, F.L. & BELL, E.B. (1976) Cellular events in proteintolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin. Cell. Immunol. 21, 20. SZEWEZUK, M.R. & SIsKIND, G.W. (1977) Ontogeny of Blymphocyte-function. III. In vivo and in vitro studies on the ease of tolerance induction in B lymphocytes from fetal, neonatal, and adult mice. 7. exp. Med. 145, 1590.
Prevention of tolerance in differentiating B lymphocytes by T cells C. J. EL SON Immunobiology Group, Department of Pathology, University of Bristol (Received 13 February 1978)
SUMMARY
Normal, but not tolerant, adult thymocytes prevented particulate antigen inducing tolerance in differentiating B lymphocytes. This was demonstrated by measuring the emergence of responsive B cells (identified by their allotype) developing from a foetal liver innoculum in irradiated hosts with or without the addition of antigen, thymocytes or tolerant thymocytes. The relevance of these findings to the recruitment of B lymphocytes in adult animals and to the development of those autoreactive B cells found in normal animals is discussed. INTRODUCTION Nossal & Pike (1975) elaborated the hypothesis that B lymphocytes go through a phase in their development during which they are particularly sensitive to tolerance induction. It has now been established that soluble multivalent hapten protein conjugates (some of which have no tolerizing effect on mature B cells) can induce tolerance in differentiating thymus-dependent B cells (Cambier et al., 1977; Metcalf & Klinman, 1976, 1977; Szewczuk & Siskind, 1977). This has suggested to a number of workers that soluble autologous protein antigens which bear repeating epitopes may prevent the emergence of mature B cells reactive with the epitopes (for review see Elson, Naysmith & Taylor, 1978). Perhaps, even particulate autoantigens, which are immunogenic for mature B cells, may induce tolerance in differentiating cells. This was tested for extrinsic antigens by repopulating lethally irradiated mice of one allotype with 13-15 day foetal liver cells from congenic mice bearing another allotype, and determining the effect of antigen on the emergence of responsive B cells. The B cells which had descended from the foetal liver inoculum (identified by their allotype) produced an antibody response to antigen in the presence of additional T cells, 15 days after transfer. Such a response was prevented by injecting the recipient with alum precipitated antigen shortly after irradiation and reconstitution. The unresponsive state was specific and independent of afferent suppressor mechanisms. Mature B cells, on the other hand, were primed by alum precipitated antigen in the same environment (Elson, 1977). Other workers (Bruynes et aL, 1976) have shown that keyhole limpet haemocyanin, a particularly immunogenic antigen for mature B cells, also induces tolerance in differentiating B cells. It follows that the injection of immunogens into adult animals, whilst stimulating the corresponding mature B cells, could render their immature counterparts tolerant. Alternatively, the immature cells may be protected from tolerance inducation by the environment of adult animals. One respect in which the environment of normal adult and lethally irradiated foetal liver reconstituted mice differ, is in the presence of T cells. The purpose of the experiments described here was to test whether the addition of T cells affected the induction of tolerance in T-cell dependent B cells developing from their progenitors in irradiated hosts. Correspondence: Dr C. J. Elson, Immunobiology Group, Department of Pathology, University of Bristol, The Medical
School, University Walk, Bristol BS8 1TD.
0099-9104/78/0100-0059$02.00 ©0 1978 Blackwell Scientific Publications 59
60
C. J. Elson 100
( a)
( b)
a-
0%
10~~
+
*~~~~~~~~~
I
C
C +Thy
Tol
TOI+ Thy
Tol
TolI ToI+ Thy To + Thy D
D
FIG. 1. Effect of thymocytes on tolerance induction by DNP5HyG (A) in differentiating B lymphocytes (a). Irradiated Igb mice reconstituted with CBA (Iga) foetal liver cells were given alum precipitated DNP-HyG (Tol) on day 5 or left as controls (C). In addition, half the mice in each group were given 2 x 107 Igb adult thymocytes intravenously on day 0 (Thy) (b). Irradiated foetal liver reconstituted mice were exposed to DNP5 HyG (A) on day 5 (Tol). Some of these mice were injected with 0-8 mg deaggregated HyG (D) per mouse on day 0, others with thymocytes (Thy), others with both deaggregated HyG and thymocytes, and the remainder left as controls. All mice were challenged with DNPiHyG on day 16 and the Iga response measured. The mean and standard deviation are shown.
MATERIALS AND METHODS Animals and antigens. CBA/H mice which bear the Iga allotype and the congenic strain CBA/H Igb (referred to as Igb mice) were used. The human gamma globulin (HyG) used was Cohn fraction II from Koch light. Dinitrophenylated (DNP) HyG was prepared by the reaction of sodium 2,4-dinitrobenzene sulphonate in sodium carbonate with HyG as described by Little & Eisen (1967). HyG was absorbed with mouse spleen cells and deaggregated by centrifugation as described previously (Elson & Taylor, 1977). Antigens were alum-precipitated by adding 10 parts 9%O aluminium potassium sulphate to 46 parts of a solution containing 2-5 mg antigen per ml in 1-0 M sodium hydrogen carbonate. The resultant precipitate was washed three times in saline. Cell suspensions. CBA (Iga) mice were mated and the females examined for vaginal plugs. Pregnant mice were killed 13-15 days after impregnation and the foetal livers removed. The livers were disaggregated in a loosely fitting ground glass homogeniser and the cells resuspended in balanced salt solution. They were filtered through wire gauze. Cell suspensions of the thymus and spleen of adult mice were made similarly. Cell transfers and general plan of experiments. Igb mice were lethally irradiated (800R) and restored by intravenous transfusion of 4 x 106 CBA foetal liver cells. Five days later some mice were given intraperitoneal injections of 500 Pg alum precipitated antigen, with the intention of inducing tolerance in the differentiating B lymphocytes, whilst others were left untreated as controls. Sixteen days after irradiation and reconstitution, the mice were challenged with an intraperitoneal injection of 100 jug alum precipitated antigen with B. pertussis as adjuvant and given 5 x 106 Igb spleen cells intravenously as a source of cooperating T cells. They were bled 18 days later and the sera collected. Radioimmunoassay. This was performed as described previously (Elson & Taylor, 1974). Briefly, the immunoabsorbant
Prevention oftolerance in differentiating B cells
61
was first incubated with the mouse serum to be tested, washed and then incubated with 125I-labelled purified anti-Iga allotype antibody. The immunoabsorbant was washed again and counts made of the radioactivity bound to it. By comparison with results obtained with standard purified anti-DNP it was possible to estimate antibody concentrations in test sera in terms of pg/ml.
RESULTS The results of two experiments are shown in Fig. 1. It is evident that the addition of thymocytes prevented the induction of hapten specific tolerance in differentiating B cells. This effect required the presence of carrier specific T cells because B cell tolerance was not prevented if the thymocytes were first exposed to small doses of deaggregated HyG, a procedure known to induce T cell tolerance (Chiller, Habicht & Weigle, 1972).
DISCUSSION The finding that the tolerance which is normally induced in developing B cells by exposure to particulate hapten-protein conjugates does not take place if T cells are added, is reassuring for two reasons. The first is that without the preventive effect of T cells the emergence of newly matured B cells could be suppressed by an extrinsic immunogen, thus preventing their recruitment (Ford, 1968; Elson & Sullivan, 1977) into antibody responses. There is some evidence that this may occur when the corresponding T cells are tolerant. If adult rats were first rendered unresponsive at the T cell level by multiple injections of soluble human serum albumin (HSA) (Bell & Shand, 1973; Shand & Bell, 1976) then B cell tolerance to HSA (and not priming) was induced by injecting alum-precipitated HSA plus adjuvant (Bell, Shand & Gradwell, 1977). It is reassuring also because any autoreactive B cells which might develop later in life would still become tolerant to autoantigens as long as the corresponding T cells were also tolerant. On the other hand, in the presence of self reactive T cells the corresponding autoreactive B cell may emerge unscathed. This may be one way by which those autoreactive B cells found in normal animals escape elimination (Elson, et al., 1978). It should be emphasized that the question of whether autoreactive helper T cells do exist is currently being debated (Esquivel, Rose & Kong, 1977). Even if they do, there may be insufficient of them either quantitatively or qualitatively to enable the corresponding B cells to respond to the autoantigen. It may be asked how T cells prevent the induction of tolerance in differentiating B cells. Some insight may be gained from the recent work of the Sherr, Szewczuk & Siskind (1978) who showed that the functional maturation of foetal liver B cells in irradiated hosts was accelerated by the addition of adult thymocytes. As B cells are only hypersusceptible to tolerance induction for a limited period during their development, and as tolerance induction is a time-dependent process (Metcalf & Klinman 1976, 1977; Cambier et al., 1977), then it could be that in the presence of T cells, B cells pass through the hypersusceptible phase too rapidly for tolerance to be induced. REFERENCES BELL, E.B. & SHAND, F.L. (1973) Cellular events in protein tolerant inbred rats. I. The fate of thoracic duct lymphocytes and memory cell during tolerance induction to human serum albumin. Eur. 7. Immunol. 3, 259. BELL, E.B., SHAND, F.L. & GRADwELL, S. (1977) Cellular events in protein tolerant inbred rats. IV. The mechanism of immunogen-maintained tolerance. Eur. .. Immunol. 7, 406. BRUYNES, C., URBAIN-VANSANTEN, G., PLANARD, C., DE VOS-CLEOTENS, C. & URBAIN J. (1976) Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes. Proc. Nat. Acad. Sci. (Wash.), 73, 2462.
CAMBIER, J.C., VITETTA, E.S., UHR, J.W. & KETTMAN, J.R. (1977) B-cell tolerance II TNP17 HgG induced tolerance in adult and neonatal B cells responsive to thymus dependent and independent forms of the same hapten. 3. exp. Med. 145, 778. CHILLER, J.M., HABICHT, G.S. & WEIGLE, W.O. (1971) Kinetic differences in unresponsiveness of thymus and bone marrow. Science, 171, 813. ELSON, C.J. (1977) Tolerance in differentiating B lymphocytes. Eur. 3. Immunol. 7, 6. ELSON, C.J., NAYSMITH, J.D. & TAYLOR, R.B. (1978) B-cell tolerance and autoimmunity. Int. Rev. Exp. Path. 19.
62
C. J. Elson
ELSON, C.J. & SULLIVAN, C.P. (1977) A suppressor mechanism controlling lymphocyte recruitment. Immunology, 33, 369. ELSON, C.J. & TAYLOR, R.B. (1974) The suppressive effect of carrier priming on the response to a hapten carrier conjugate. Eur. 5. Immunol. 4, 682. ELSON, C.J. & TAYLOR, R.B. (1977) Cellular basis of persistent tolerance induced by an aggregate free heterologous immunoglobulin. Immunology, 33, 635. ESQUIVEL, P.S., ROSE, N.R. & KONG, Y.C.M. (1977) Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. 3. exp. Med. 145, 1250. FORD, W.L. (1968) Duration of the inductive effect of sheep erythrocytes on the recruitment of lymphocytes in the rat. Immunology, 15, 609. LITTLE, J.R. & EISEN, M.W. (1967) Methods in Immunology and Immunochemistry (ed. by C.A. Williams & M.W. Chase), Vol. 1, p. 128. Academic Press, New York & London.
METCALF E.S. & KLINMAN, N.R. (1976) In vitro tolerance induction of neonatal murine spleen cells. J. exp. Med. 143, 1327. METCALF, E.S. & KLINMAN, N.R. (1977) In vitro tolerance induction of bone marrow cells: A marker for B cell maturation. J. Immunol. 118, 2111. NoSsAL, G.J.V. & PiKE, B.L. (1975) Evidence for the clonal abortion theory of B-lymphocyte tolerance. J. exp. Med. 141, 904. SHERR, D.H., SZEWCZUK, M.R. & SIsKIND, G.W. (1978) Ontogeny of B-lymphocyte function V. Thymus cell involvement in the functional maturation of B-lymphocytes from fetal mice transferred into adult irradiated hosts. 3. exp. Med. 149, 196. SHAND, F.L. & BELL, E.B. (1976) Cellular events in proteintolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin. Cell. Immunol. 21, 20. SZEWEZUK, M.R. & SIsKIND, G.W. (1977) Ontogeny of Blymphocyte-function. III. In vivo and in vitro studies on the ease of tolerance induction in B lymphocytes from fetal, neonatal, and adult mice. 7. exp. Med. 145, 1590.
