Transact. Colleg. int. Allerg., JOth Symp., Copenhagen 1974 Int. Archs Allergy appl. Immun. 49: 230-240 (1975)
T-Cell Control of B-Cell Responsiveness1 J. F. A. P. M iller Melbourne, Victoria
B cells2 are antibody-forming cell precursors and antigen-activated B cells will differentiate to antibody-secreting cells [M iller and M it c h e l l , 1969]. The antibody response to the so-called ‘T-dependent’ antigens is, however, defective in the absence of appropriately stimulated T cells [M iller and O so b a , 1967], It has been postulated that specific B-cell triggering requires at least 2 signals: the binding of antigenic determinants to membrane immunoglobulin receptors and some other signal from cooperating T cells, macrophages or from some intrinsic property of the antigen itself. ‘T-independent’ antigens which, by definition, can trigger B cells in the absence of T cells, have been assigned one or more of the fol lowing properties: a polymeric structure of high molecular weight, high epitope density but restricted epitope heterogeneity [B asten and H o w a r d , 1973]; poor degradability and hence persistence [M it c h ell et al., 1972a, b]; mitogenicity for B cells [C o u t in h o and M ö ller , 1974]; ability to activate C3 [D u k o r and H artm a n n , 1973], and immunogenicity for IgM but not for IgG precursors [B asten and H o w a r d , 1973]. Epitope density alone is not a sufficient condition for T-independent B-cell trig-
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1 Publication No. 2030 from the Walter and Eliza Hall Institute. 2 Abbreviations used in this paper: B-cell, bone marrow-derived antibody-form ing cell precursor; B/z-cell, immediate precursor of IgM-secreting B cells; By-cell, immediate precursors of IgG-secreting B cells; DNP, 2,4-dinitrophenyl; DNP-D-GL, DNP conjugate of a synthetic random copolymer of D-glutamic acid and D-lysine; DRC, donkey erythrocytes; Fla, flagellin of Salmonella adelaide; FyG, fowl immu noglobulin G; Ig, immunoglobulin; KLH, keyhole limpet hemocyanin; MRC, mouse erythrocytes; MSH, Maia sqtiinada hemocyanin; NIP, 4-hydroxy,3-iodo,5nitrophenylacetyl; PFC, plaque-forming cells; SIN, type III polysaccharide from Streptococcus pneumoniae; T-cell, thymus-derived cell.
M iller
231
gering. Thus, anti-hapten antibody responses to both lightly and heavily conjugated albumins arc T-cell dependent, even though the highly substi tuted conjugates evoke a ‘T-independent’ type of response (only IgM) [K la u s and H u m ph r e y , 1974], Not all T-independent antigens are C3 bypass activators nor B-cell mitogens: for example, some DNP-conjugates (such as DNP-ficoll) are not able to activate complement and are not mitogenic for B cells but can evoke T-independent antibody responses [P a ul et al., 1973], Although IgG responses are in general highly T-cell dependent, there arc some exceptions. DNP-ficoll has been claimed to produce both IgM and IgG responses in the absence of T cells [P a ul et al., 1974], Furthermore, antigens, such as DNP.KLH, can stimulate primed B cells, but only in low doses, to give rise to small amounts of IgG antibody in the absence of carrier-primed T cells [K linm an and D o u g h t y , 1973], It would appear, therefore, that T cells are not obligatory for triggering IgM and even IgG-precursor B cells. Their function in cell cooperation is more likely to be an immunorcgulatory one, as has been recently emphasized [M il l e r , 1974],
A number of experimental situations suggest that T cells facilitate B-cell responses to antigen if this exceeds a certain critical concentration effective presumably within the microenvironment of the B cell. (1) The antibody response to a hapten on an immunogenic carrier in mice previously exposed to the same hapten on a carrier which does not activate T cells is negligible and there is no evidence of priming. Thus, no IgG anti-DNP antibody could be detected in mice challenged with DNP.MSH one week after pretreatment with DNP.SIII, the carrier Sill being unable to activate T cells [M it c h e l l et ai., 1972b], (2) The antibody response to sheep erythrocytes of athymic nude mice given T cells was markedly reduced if the mice had been pretreated one week before with the antigen given alone. Pretreatment with horse eryth rocytes did not affect the anti-sheep response, indicating the specificity of the effect. It was also shown that the lymphoid cell population of sheep crythrocyte-pretreated nude mice was defective in its capacity to adop tively transfer IgG antibody responsiveness to sheep erythrocytes when injected together with T cells in irradiated BAB/14 mice 1-3 weeks after pretreatment. In the one host system, any IgM antibodies produced after
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Facilitation of B-Cell Responses by Appropriately Activated T Cells
232
M iller Table I. Paralysis of By cells by NIP-conjugatcd mouse erythrocytes
Syngeneic cells tranferred to irradiated mice
4 x 107 normal spleen cells'
NIP PFC per spleen at day 7 in irradiated recipients challenged with NIP. FyG direct PFC
indirect PFC
11,000
27,000
4 x 107 spleen cells from NIP. MRC-pretreated mice'
4,100
4 x 107 spleen cells from NIP. MRC-pretreated mice + I07 normal B lymphocytes-
6,500
0
16,000
1 Data from H amilton et al. [1974], 2 Unpublished data of H amilton and M iller; B cells were separated from T cells by cell electrophoresis of normal thoracic duct lymphocytes.
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the first injection with sheep erythrocytes might well have inhibited B« cells from responding subsequently when sheep erythrocytes were given together with T cells, but the adoptive transfer system established that By cells must have been inhibited by the first contact with antigen in the ab sence of T cells [M it c h e l l et al., 1974], (3) The IgG antibody response of normal mice to a second challenge of a hapten on an immunogenic carrier when the first challenge was to the hapten on a nonimmunogenic carrier, such as autologous erythrocytes, is markedly reduced and often not detectable. Thus, for instance, a com plete suppression of the IgG, but not the IgM, antibody response to NIP conjugated to an immunogenic protein, FyG, was observed in mice pre treated with NIP-coated syngeneic erythrocytes (NIP.MRC). This oc curred within 5-7 days, persisted for at least 2 months and was evident on adoptive transfer of spleen cells to irradiated mice (table I). It is inter esting to recall that syngeneic mouse erythrocytes persist for at least 2-3 months, and it is probable that there are no T cells with reactivities di rected towards the mouse’s own erythrocytes. Thus, in the absence (or functional absence) of carrier-specific T lymphocytes, the interaction of excessive amounts of persisting antigen with By lymphocytes must specifi cally suppress tiie potential of these cells to produce IgG antibody, thus
T-Cell Control of B-Cell Responsiveness
233
Tabic //. T cell dependent humoral antibody responses Highly T cell dependent
Marginally or not T cell dependent
High-affinity antibody1 IgE2 IgG3' 4
low affinity antibody1 IgM34
1 2 3 4
G ershon and Paul [1971]. H amaoka et at. [1973], M iller et al. [1967], M itchel et al. [1972a],
leading to a state of stable and specific B-cell tolerance [H a m ilto n and Further experiments have shown that even in NIP-primed mice, the IgG anti-NIP response could be inhibited by treatment with NIP-coated syngeneic erythrocytes 1 week before secondary challenge with NIP.FyG [H a m il to n et al., 1974], Likewise with DNP.SIII, DNP-primed cells were much more susceptible to inhibition than unprimed DNP-sensitive cells [M it c h e l l et al., 1972a]. In terms of antibody affinity, it has been shown that the production of high-affinity antibodies is more easily sup pressed during tolerance induction than is the production of low-affinity antibodies, and likewise IgG antibodies are more easily suppressed than IgM antibodies [S isk in d and B e n a c e r r a f , 1969]. In keeping with these observations it is likely that B cells with high intrinsic antigen-binding ca pacity will be more susceptible to receptor blockade if antigen tends to persist than B cells with lower antigen-binding potential. This may be the reason why persistent, poorly degradable antigens generally elicit only IgM responses, not IgG. On the other hand, degradable, nonpersistent an tigens elicit both IgM and IgG responses and it is the IgG response and the high-affinity antibody responses which are markedly diminished or abolished in the absence of T cells, the IgM or low-affinity antibody re sponses being either unaffected or reduced significantly but not abolished (table II). These observations would appear to link: (a) antigen persisten ce; (b) susceptibility of potential IgG producers to paralysis, and (c) ab sence of a T-cell influence [M it c h e l l , 1974; M il l e r , 1974]. Recent experiments by M it c h ell [1974] have further substantiated these correlations. A conjugate of DNP on rabbit anti-SIII antibody could
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M il l e r , 1973a, 1974].
234
Miller
Table III. Effect o f ‘digestible' spacer between S ill and DNP on adoptive DNP antibody response in irradiated mice1 Spleen cells transferred rabbit Ig-primed DNP. Flaprimed
Antigens given DNP-rabbit Ig anti-SIlI2
+
+ +
+ -
+ + + + (anti-
T-Cell Control of B-Cell Responsiveness1 J. F. A. P. M iller Melbourne, Victoria
B cells2 are antibody-forming cell precursors and antigen-activated B cells will differentiate to antibody-secreting cells [M iller and M it c h e l l , 1969]. The antibody response to the so-called ‘T-dependent’ antigens is, however, defective in the absence of appropriately stimulated T cells [M iller and O so b a , 1967], It has been postulated that specific B-cell triggering requires at least 2 signals: the binding of antigenic determinants to membrane immunoglobulin receptors and some other signal from cooperating T cells, macrophages or from some intrinsic property of the antigen itself. ‘T-independent’ antigens which, by definition, can trigger B cells in the absence of T cells, have been assigned one or more of the fol lowing properties: a polymeric structure of high molecular weight, high epitope density but restricted epitope heterogeneity [B asten and H o w a r d , 1973]; poor degradability and hence persistence [M it c h ell et al., 1972a, b]; mitogenicity for B cells [C o u t in h o and M ö ller , 1974]; ability to activate C3 [D u k o r and H artm a n n , 1973], and immunogenicity for IgM but not for IgG precursors [B asten and H o w a r d , 1973]. Epitope density alone is not a sufficient condition for T-independent B-cell trig-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:06:18 PM
1 Publication No. 2030 from the Walter and Eliza Hall Institute. 2 Abbreviations used in this paper: B-cell, bone marrow-derived antibody-form ing cell precursor; B/z-cell, immediate precursor of IgM-secreting B cells; By-cell, immediate precursors of IgG-secreting B cells; DNP, 2,4-dinitrophenyl; DNP-D-GL, DNP conjugate of a synthetic random copolymer of D-glutamic acid and D-lysine; DRC, donkey erythrocytes; Fla, flagellin of Salmonella adelaide; FyG, fowl immu noglobulin G; Ig, immunoglobulin; KLH, keyhole limpet hemocyanin; MRC, mouse erythrocytes; MSH, Maia sqtiinada hemocyanin; NIP, 4-hydroxy,3-iodo,5nitrophenylacetyl; PFC, plaque-forming cells; SIN, type III polysaccharide from Streptococcus pneumoniae; T-cell, thymus-derived cell.
M iller
231
gering. Thus, anti-hapten antibody responses to both lightly and heavily conjugated albumins arc T-cell dependent, even though the highly substi tuted conjugates evoke a ‘T-independent’ type of response (only IgM) [K la u s and H u m ph r e y , 1974], Not all T-independent antigens are C3 bypass activators nor B-cell mitogens: for example, some DNP-conjugates (such as DNP-ficoll) are not able to activate complement and are not mitogenic for B cells but can evoke T-independent antibody responses [P a ul et al., 1973], Although IgG responses are in general highly T-cell dependent, there arc some exceptions. DNP-ficoll has been claimed to produce both IgM and IgG responses in the absence of T cells [P a ul et al., 1974], Furthermore, antigens, such as DNP.KLH, can stimulate primed B cells, but only in low doses, to give rise to small amounts of IgG antibody in the absence of carrier-primed T cells [K linm an and D o u g h t y , 1973], It would appear, therefore, that T cells are not obligatory for triggering IgM and even IgG-precursor B cells. Their function in cell cooperation is more likely to be an immunorcgulatory one, as has been recently emphasized [M il l e r , 1974],
A number of experimental situations suggest that T cells facilitate B-cell responses to antigen if this exceeds a certain critical concentration effective presumably within the microenvironment of the B cell. (1) The antibody response to a hapten on an immunogenic carrier in mice previously exposed to the same hapten on a carrier which does not activate T cells is negligible and there is no evidence of priming. Thus, no IgG anti-DNP antibody could be detected in mice challenged with DNP.MSH one week after pretreatment with DNP.SIII, the carrier Sill being unable to activate T cells [M it c h e l l et ai., 1972b], (2) The antibody response to sheep erythrocytes of athymic nude mice given T cells was markedly reduced if the mice had been pretreated one week before with the antigen given alone. Pretreatment with horse eryth rocytes did not affect the anti-sheep response, indicating the specificity of the effect. It was also shown that the lymphoid cell population of sheep crythrocyte-pretreated nude mice was defective in its capacity to adop tively transfer IgG antibody responsiveness to sheep erythrocytes when injected together with T cells in irradiated BAB/14 mice 1-3 weeks after pretreatment. In the one host system, any IgM antibodies produced after
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:06:18 PM
Facilitation of B-Cell Responses by Appropriately Activated T Cells
232
M iller Table I. Paralysis of By cells by NIP-conjugatcd mouse erythrocytes
Syngeneic cells tranferred to irradiated mice
4 x 107 normal spleen cells'
NIP PFC per spleen at day 7 in irradiated recipients challenged with NIP. FyG direct PFC
indirect PFC
11,000
27,000
4 x 107 spleen cells from NIP. MRC-pretreated mice'
4,100
4 x 107 spleen cells from NIP. MRC-pretreated mice + I07 normal B lymphocytes-
6,500
0
16,000
1 Data from H amilton et al. [1974], 2 Unpublished data of H amilton and M iller; B cells were separated from T cells by cell electrophoresis of normal thoracic duct lymphocytes.
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the first injection with sheep erythrocytes might well have inhibited B« cells from responding subsequently when sheep erythrocytes were given together with T cells, but the adoptive transfer system established that By cells must have been inhibited by the first contact with antigen in the ab sence of T cells [M it c h e l l et al., 1974], (3) The IgG antibody response of normal mice to a second challenge of a hapten on an immunogenic carrier when the first challenge was to the hapten on a nonimmunogenic carrier, such as autologous erythrocytes, is markedly reduced and often not detectable. Thus, for instance, a com plete suppression of the IgG, but not the IgM, antibody response to NIP conjugated to an immunogenic protein, FyG, was observed in mice pre treated with NIP-coated syngeneic erythrocytes (NIP.MRC). This oc curred within 5-7 days, persisted for at least 2 months and was evident on adoptive transfer of spleen cells to irradiated mice (table I). It is inter esting to recall that syngeneic mouse erythrocytes persist for at least 2-3 months, and it is probable that there are no T cells with reactivities di rected towards the mouse’s own erythrocytes. Thus, in the absence (or functional absence) of carrier-specific T lymphocytes, the interaction of excessive amounts of persisting antigen with By lymphocytes must specifi cally suppress tiie potential of these cells to produce IgG antibody, thus
T-Cell Control of B-Cell Responsiveness
233
Tabic //. T cell dependent humoral antibody responses Highly T cell dependent
Marginally or not T cell dependent
High-affinity antibody1 IgE2 IgG3' 4
low affinity antibody1 IgM34
1 2 3 4
G ershon and Paul [1971]. H amaoka et at. [1973], M iller et al. [1967], M itchel et al. [1972a],
leading to a state of stable and specific B-cell tolerance [H a m ilto n and Further experiments have shown that even in NIP-primed mice, the IgG anti-NIP response could be inhibited by treatment with NIP-coated syngeneic erythrocytes 1 week before secondary challenge with NIP.FyG [H a m il to n et al., 1974], Likewise with DNP.SIII, DNP-primed cells were much more susceptible to inhibition than unprimed DNP-sensitive cells [M it c h e l l et al., 1972a]. In terms of antibody affinity, it has been shown that the production of high-affinity antibodies is more easily sup pressed during tolerance induction than is the production of low-affinity antibodies, and likewise IgG antibodies are more easily suppressed than IgM antibodies [S isk in d and B e n a c e r r a f , 1969]. In keeping with these observations it is likely that B cells with high intrinsic antigen-binding ca pacity will be more susceptible to receptor blockade if antigen tends to persist than B cells with lower antigen-binding potential. This may be the reason why persistent, poorly degradable antigens generally elicit only IgM responses, not IgG. On the other hand, degradable, nonpersistent an tigens elicit both IgM and IgG responses and it is the IgG response and the high-affinity antibody responses which are markedly diminished or abolished in the absence of T cells, the IgM or low-affinity antibody re sponses being either unaffected or reduced significantly but not abolished (table II). These observations would appear to link: (a) antigen persisten ce; (b) susceptibility of potential IgG producers to paralysis, and (c) ab sence of a T-cell influence [M it c h e l l , 1974; M il l e r , 1974]. Recent experiments by M it c h ell [1974] have further substantiated these correlations. A conjugate of DNP on rabbit anti-SIII antibody could
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:06:18 PM
M il l e r , 1973a, 1974].
234
Miller
Table III. Effect o f ‘digestible' spacer between S ill and DNP on adoptive DNP antibody response in irradiated mice1 Spleen cells transferred rabbit Ig-primed DNP. Flaprimed
Antigens given DNP-rabbit Ig anti-SIlI2
+
+ +
+ -
+ + + + (anti-
