- Email: [email protected]
B cell interaction in man
HOW
T CELLS HELP B CELLS
427
References.
AMOROSO,K. & LIPs~:v, P.E. (1990), Frequency of human B cells differentiating in response to anti-CD3-activated T cells. (Submitted for publication). AMOROSO,K., KELLYP. & LIPSKY,P.E. (1990), Induction of immunoglobulin isotype switching in human B cells stimulated with anti-CD3oactivated T cel~s. Fed. J. (in press). GEPPERT,T.E. & LIPSKY,P.E. (1987), Accessory-cell-independence of the promotion of human B-cell differentiation by IL-6 (BSF-2). J. ImmunoL, 142, 1569-1575. GEPPER'I,T.E. & LiPs~:v, P.E. (1989), Antigen presentation at the inflammatory site. CRC Critical Rev. Immunol., 9, 313-362. HIROHATA,S. • L1PSKY,P.E. (1989), T-cell regulation of human B-cell proliferation and differentiation. Regulatory influences of CD45R ÷ and CD45R- T4-cell subsets. J. Immunol., 142, 2597-2607. HIROHATA,S., JELINEK,D.F. & LIPSKY,P.E. (1988), T-cell-dependent activation of B-cell proliferation and differentiation by immobilized monoclonal antibodies to CD3. J. Immunol., 140, 3726-3744. HIROHATA,S., PATEL,S.S. & LIPSKY,P.E. (1990), Regulation of human B-cell responsiveness by CD8 + T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen. Ceil. Immunol., 127, 35-46. PISETSKY,D.S., JELINEK,D.F., MCANALLY,L.M., REICH,C.F. & LIPSKY,P.E. (1990), In vitro autoantibody production by normal adult and cord blood B ceils. J. Clin. Invest., 85, 899-903. SPL~,WSK~,J.B., JELINE~:,D.F. & Linsey, P.E. (1989), Immunomodulatory role of interleukin 4 on the secretion of immunoglobulin by human B cells. J. ImmunoL, 142, 1569-1575. SPLAWSra, J.B., MCANALLV,L.M. & L~s~Y, P.E. (1990a), IL-2-dependence of the p~omotion of human B-cell differentiation by IL-6 (BSF-2). J. Immunol., 144, 562-569. $OLAWSr~, J.B., JEL~NEK,D.F. & Lmsr:v, P.E. (1990b), Decreased T cell-B cell collaboration by human neonatal lymphocytes reflects functional deficiencies of both T cells and B cells (submitted for publication). TOHMA, S. & Lms~:v, P.E. (1990), Polyclonal activation of human B cells induced by antiCD3-stimulated fixed T cells. Fed. J. (in press). TOHMA,S., HIROHATA,S. & Lli~SKY,P.E. (1990), The role of CDI Ia/CDI8-CD54 interactions in human T-cell-dependent B-cell activation. (Submitted for publication).. VERN~NO, L., MCANALLY, L.M., AMOROSO, K. & LIPsrV, P.E. (1990), Generation of nondividing high-rate immunoglobulin-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells. Fed. J. (in press).
COGNATE T H E L P E R C E L L / B C E L L I N T E R A C T I O N IN MAN M.K. Crow and S.M. Friedman
From the Departments o f Medicine, The Hospital f o r Special Surgery and The New York Hospital-Cotnell University Medical College, New York, N Y 10021 (USA)
Since Mitchison first established the need for " l i n k e d " recognition of hapten-carrier conjugates by T and B cells in order to generate an anti-hapten antibody response (Mitchison, 1971), the nature of the activation signals transmitted from T helper (Th) to B cell have been studied with great interest.
When B-cell populations were depleted of lymphokine-responsive activated cells, the small, resting B cells which remain clearly required close physical contact with Th cells prior to progression to proliferation and differentiation (Julius et aL, 1982; Anderson et aL, 1980; Singer and Hodes, 1983). As the
428
32 nd F O R U M I N I M M U N O I ,
T-B cell contact was MHC-restricted, requiring recognition of antigen/MHC class I! conjugates on the B-cell surface by the TCR of specific T h cells, a logical prediction was that the MHC class II molecule itself might transduce the crucial activation signal when crosslinked by the "cognate" Th/B ceil interaction. Documentation of such a role for B-cell surface MHC class II molecules has been sought in a number of systems, but remains elusive. Indeed, convincing evidence has emerged which shows cell-cell interaction between resting responder B cell and T h cells, preactivated via the TCR, obviates the requirement for MHC restriction (Riedel et al., 1988; Julius and Rammensee, 1988). While ouz ow~ experiments and those from other laboratories strongly support a requirement for intimate physical contact between antigenactivated T h cell and B cell for the generation of proliferating and Igsecreting B cells, the precise events occuring at the Th/B cell interface have yet to be defined. Our own experiments with the human system have utilized T-cell clones specific for allogeneic MHC class II molecules, or for the hapten trinitrophenyl (TNP) in association with an autologous MHC class II molecule. Alternatively, freshly isolated peripheral blood T cells, activated via the TCR-T3 complex with anti-CD3 mAb, have been used. Each of these experimental systems generates a polyclonal B-cell response, readily assayed in at least 4 ways: 1)by the generation of second. messenger molecules, specifically the products of inositol lipid hydrolysis (Chartash et al., 1988); 2) by the induction of B-cell surface CD23, an activation molecule expressed by most sIgM÷ IgD÷ cells after productive T h / B cell interaction (Crow et al., 1986); 3) B-cell proliferation (Goldberg et al., 1985); and 4) B-cell differentiation into Ig-secreting cells (Friedman et aL, 1985; Principato et aL, 1983). As the latter 2 assays require 5-10 days of culture and reflect multiple lymphokinemediated signals, as well as direct T hmediated signals, we have focused our efforts on the assays of second mes-
OG Y
senger molecule generation and CD23 expression. The alloreactive human Th-cell clones were used to show that allospecific Th/resting B cell interaction does not result in inositol lipid hydrolysis, or an increase in intracellular Ca ÷2 within the responder B cells, while antihuman IgM antibody does activate this second messenger cascade (Chartash et al., 1988). As a positive control for these studies, we determined that within 12 h of culture, the target B cells were induced to express cell surface CD23 by the allospecific T h / B cell interaction, but not by sIg cross-linking. These results suggest that antigen and T h cells induce different functional B-cell responses by activating distinct second messenger systems within the B cell. This issue has been addressed in studies by Noelle and Snow, which demonstrate a brief rise in B-cell cAMP after physical interaction with fixed antiCD3-activated T h cells (Bartlett and Noelle, 1990). Moreover, Cambier has shown that anti-MHC class II antibodies stimulate increased B-cell cAMP levels. It appears then that enzymes such as protein kinase A, which generate elevated intracellular cAMP, should be targets of investigation of the biochemical mediators of cognate T-cell help /'C"~ml~,-~ ~,...~,~t~,~
- ,,~. , , ~ ! ~
~g.
,
1~'7~
~ ,~'~.~ 1 ~ .
As IL-4 is known to induce B-cell CD23, we have performed studies in order to distinguish B-cell activation that results from soluble mediators versus physical cell-ceil contact. We have utilised 2-chamber-culture wells and either allospecific T-cell clones or peripheral blood T cells activated with anti-CD3 mAb (Crow et al., 1989). Culture of either group of T cells with unfractionated tonsil or blood non-T cells generates sufficient IL-4 secretion to stimulate CD23 expression on B cells separated by a membrane permeable to lymphokines. However, if the T cells are activated in the presence of resting B cells, strictly depleted of monocytes and low-density B cells, CD23 expression is induced on those resting B cells in physical contact with the activated T h cells, but not on those B cells physically separated. Moreover, IL-4 could not be de-
HOW
tected in supernatants cultures.
T CELLS HELP B CELLS
from those
Gamma interferon has several functions that oppose those of IL-4. Among these activities is gamma interferon's capacity to inhibit IL-4-induced B cell CD23. We found that, while gamma in. terferon inhibited IL-4-induced CD23 expression on resting B cells by about 50 %, it usually resulted in no or slight inhibition of activated Th-cell-induced CD23 expression by resting B cells in close physical proximity. Taken together, the two-chamber and gamma interferon experiments support the requirement for physical Th cell/resting B cell interaction in the induction of an early stage of B-cell activation. While we do not see a need to postulate a second, non-IL-4-mediated mechanism of CD23 induction, it is clear that B-cell activation can be stimulated by unmeasurably low levels of IL-4 delivered at the T-B cell interface, or perhaps by a Tcell surface-bound form of IL-4, present on an activated but not resting T cell. Our studies of CD23 expression induced by human Th cells, when taken together, support a sequence of events in which (1) the T cells are first activated through the TCR, and then (2) efficient cell-mediated B activation is ~.mtatgu, out omy wn~n the ~ and B cells are in close physical proximity. An interesting hapten altered-self reactive human Th-cell clone, Ell, has been useful in approaching these issues (Friedman et al., 1986). Clone Ell proliferates in response to TNP bound to macrophages or B cells of the DR5 haplotype. We found that Eli's requirements for generating help for B-cell activation (CD23 expression) are less stringent than its own requirements for proliferation. Ell will stimulate CD23 expression on TNP-conjugated B cells of any individual, regardless of DR haplotype. Moreover, TNP coupled to BGG or to Sepharose beads can induce B cell CD23 expression when present in cultures of Ell and untreated B cells. Experiments in which Ell cells were pretreated with TNP Sepharose beads for 2 h, fixed C,-,,-,:!:~.,~-~,-I
L..*-
~_1
.....
L
----
~
429
with paraformaldehyde and washed extensively also were successful in stimulating CD23 expression on cocultured B cells. These data suggest that once activated through their TCR, Ell cells undergo a change which facilitates their capacity to provide an activation signal to any B cell in an MHCunrestricted manner. The effect of TNP in this system could include the induction of a confermational change in the TCR, of increased expression or a conformational change .in accessory T-cell ~urface molecules or insertion of lymphokines such as IL-4 into the T-cell membrane. As most investigators have not been successful in stimulating B-cell differentiation using !ymphokines alone (including IL-4), we continue to search for an additional T-cell-mediated signal which permits implementation of the full Bcell program. In recent preliminary studies, we have cultured resting tonsil B cells for 16 h with medium or various combinations of mediators which might mimic the initial B-cell exposure to antigen and activated Th cells. We have used (1) phorbol dibutyrate, the watersolub!~ protein kinase C activator, .~a orovitie a signal analogous to that which might result from antigen interaction with slg; 2) IL-4, the early-acting T-cell Jymphokine; and 3) forskoiin, a cAMP analog, which might mimic a T-cell ~urface-mediated B-cell activation signal. A~ter these analogs were washed from the B cells, the cells were cultured with medium or Staphylococcus aureus Cowan strain I (SAC) for 10 days and IgM secretion measured by ELISA. SAC did not induce any detectable secretion of IgM in B-cell cultures pretreated with any one or two of the stimuli in combination. However, B cells pretreated with the combination of PDB, IL-.4 and forskolin were rendered competent to respond to SAC with active IgM secretion. This data provides further suggestion that B-cell triggering through a molecule that generates cAMP might be responsible for the enigmatic phenomenon of cognate Tcell help.
430
32 'ld F O R U M I N I M M U N O L O G Y
References.
ANDERSON,J., SCHREIER,M.H. & MELCHERS,F. (1980), T-cell-dependent B-cell stimulation is H-2 restricted and antigen-dependent only at the resting B-cell level. Proc. nat. Acad. Sci. (Wash.), 77, 1612. BARTLETT,W.C. & NOELLE,R.J. (1990), In "T cell-dependent and independent B cell activation" (Snow, E.C.) CRC Press, Boca Raton, FL. (in press). CAMBIER,J.C., NEWELL,M.K., JUSTEMENT,L.B., McGUIRE, J.C., LEACH,K.L. & CHEN, Z.Z. (1987), Membrane Ia binding ligands induce increased intracellular cAMP which mediates the transient association of protein kinase C with the nucleus of B lymphocytes. Nature (Lond.), 327, 629. CHARTASH,E.K., IMAI,A., GERSHENGORN,M.C., CROW,M.K. & FRIEDMAN,S.M. (1988), Direct human T helper cell-induced B-cell activation is not mediated by inositol lipid hydrolysis. J. lmmunol., 140, 1974. CROW, M.K., JOVER,J.A. & FRIEDMAN,S.M. (1986), Direct T helper/B cell interaction induce an early B cell activation antigen. J. exp. Med., 164, 1760. CRow, M.K., JUSHNER,B., JOVER,J.A., FRIEDMAN,S.M., I~IECHANIC,S.E. & STOHL,W. (1989),. Induction of B cell BLAST-2 (CD23) expression by anti-CD3-stimulated peripheral blood T h cells. Cell. Immunol., 121, 99. FRIEDMAN,S.M., THOMPSON,G.S. & PRINCIPATO,M.A. (1982), Allospecific human T-cell lines and clones ~hich mediate HLA-DR restricted helper activity. J. lmmunol., 129, 2451. FRIEDMAN,S.M., JOVER,J.A., CHARTASH,E.K. & CROW, M.K. (1986), Antigen-specific, MHC non-restricted T helper cell-induced B-cell activation. J. exp. Med., 164, 1773. GOLDBERG,D.A., GREEN,A., GOTTLIEB,A.B., CRow, M.K., LEWlSON,A. & FRIEDMAN,S.M. (1985), Cloned allospecific human helper T-cell lines induce an MHC-restriced proliferative response by resting B cells. J. lmmunol., 135, 1012. Jt~LIt~S, M.H., VON BOEHMER,H. & SIDMAN,C.L. (1982), Dissociation of two signals required for activation of resting B cells. Proc. nat. Acad. Sci. (Wash.), 79, 1989. JULIUS, M.H. & RAMMENSEE,H-G. (1988), T helper cell-dependent induction of resting B cell differentiation r,eed not require cognate cell interactions. Europ. J. lmmunol., 18, 375. M~XCHISON,N.A. (1971), The carrier effect in the secondary response to hapten-protein conjugates. - - II. Cellular cooperation. Europ. J. Immunol., 1, 18. PRINCIPATO,M.A., THOMPSON,G.A. & FRIEDMAN,S.M. (1983), A cloned major histocoml~atibility complex restricted trinitrophenyl-reactive human helper T cell line whichactivates B cell subsets via two distinct pathways. J. exp. Med., 158, 1444. RIEDEL,C., OWENS,T. & NOSSAL,(.~..;.V. (1988), A significant proportion of normal resting B cells are induced to secrete immunoglobulin through contact with anti-receptor antibody-activated ~helper T cells in cionai cultures. Europ. g-. immunol., i8, 403. SINGER, A. & HODES, R.J. (1983), Mechanisms of T-cell-B cell interactions. Ann. Rev. Immunol., 1, 211.
This work is supported in part by: The Arthritis Foundation Investigator Award, The Irvington House Institute, and NIH Grants no. CA-49283, AI-128367, and P60-AR38520.
T CELLS HELP B CELLS
427
References.
AMOROSO,K. & LIPs~:v, P.E. (1990), Frequency of human B cells differentiating in response to anti-CD3-activated T cells. (Submitted for publication). AMOROSO,K., KELLYP. & LIPSKY,P.E. (1990), Induction of immunoglobulin isotype switching in human B cells stimulated with anti-CD3oactivated T cel~s. Fed. J. (in press). GEPPERT,T.E. & LIPSKY,P.E. (1987), Accessory-cell-independence of the promotion of human B-cell differentiation by IL-6 (BSF-2). J. ImmunoL, 142, 1569-1575. GEPPER'I,T.E. & LiPs~:v, P.E. (1989), Antigen presentation at the inflammatory site. CRC Critical Rev. Immunol., 9, 313-362. HIROHATA,S. • L1PSKY,P.E. (1989), T-cell regulation of human B-cell proliferation and differentiation. Regulatory influences of CD45R ÷ and CD45R- T4-cell subsets. J. Immunol., 142, 2597-2607. HIROHATA,S., JELINEK,D.F. & LIPSKY,P.E. (1988), T-cell-dependent activation of B-cell proliferation and differentiation by immobilized monoclonal antibodies to CD3. J. Immunol., 140, 3726-3744. HIROHATA,S., PATEL,S.S. & LIPSKY,P.E. (1990), Regulation of human B-cell responsiveness by CD8 + T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen. Ceil. Immunol., 127, 35-46. PISETSKY,D.S., JELINEK,D.F., MCANALLY,L.M., REICH,C.F. & LIPSKY,P.E. (1990), In vitro autoantibody production by normal adult and cord blood B ceils. J. Clin. Invest., 85, 899-903. SPL~,WSK~,J.B., JELINE~:,D.F. & Linsey, P.E. (1989), Immunomodulatory role of interleukin 4 on the secretion of immunoglobulin by human B cells. J. ImmunoL, 142, 1569-1575. SPLAWSra, J.B., MCANALLV,L.M. & L~s~Y, P.E. (1990a), IL-2-dependence of the p~omotion of human B-cell differentiation by IL-6 (BSF-2). J. Immunol., 144, 562-569. $OLAWSr~, J.B., JEL~NEK,D.F. & Lmsr:v, P.E. (1990b), Decreased T cell-B cell collaboration by human neonatal lymphocytes reflects functional deficiencies of both T cells and B cells (submitted for publication). TOHMA, S. & Lms~:v, P.E. (1990), Polyclonal activation of human B cells induced by antiCD3-stimulated fixed T cells. Fed. J. (in press). TOHMA,S., HIROHATA,S. & Lli~SKY,P.E. (1990), The role of CDI Ia/CDI8-CD54 interactions in human T-cell-dependent B-cell activation. (Submitted for publication).. VERN~NO, L., MCANALLY, L.M., AMOROSO, K. & LIPsrV, P.E. (1990), Generation of nondividing high-rate immunoglobulin-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells. Fed. J. (in press).
COGNATE T H E L P E R C E L L / B C E L L I N T E R A C T I O N IN MAN M.K. Crow and S.M. Friedman
From the Departments o f Medicine, The Hospital f o r Special Surgery and The New York Hospital-Cotnell University Medical College, New York, N Y 10021 (USA)
Since Mitchison first established the need for " l i n k e d " recognition of hapten-carrier conjugates by T and B cells in order to generate an anti-hapten antibody response (Mitchison, 1971), the nature of the activation signals transmitted from T helper (Th) to B cell have been studied with great interest.
When B-cell populations were depleted of lymphokine-responsive activated cells, the small, resting B cells which remain clearly required close physical contact with Th cells prior to progression to proliferation and differentiation (Julius et aL, 1982; Anderson et aL, 1980; Singer and Hodes, 1983). As the
428
32 nd F O R U M I N I M M U N O I ,
T-B cell contact was MHC-restricted, requiring recognition of antigen/MHC class I! conjugates on the B-cell surface by the TCR of specific T h cells, a logical prediction was that the MHC class II molecule itself might transduce the crucial activation signal when crosslinked by the "cognate" Th/B ceil interaction. Documentation of such a role for B-cell surface MHC class II molecules has been sought in a number of systems, but remains elusive. Indeed, convincing evidence has emerged which shows cell-cell interaction between resting responder B cell and T h cells, preactivated via the TCR, obviates the requirement for MHC restriction (Riedel et al., 1988; Julius and Rammensee, 1988). While ouz ow~ experiments and those from other laboratories strongly support a requirement for intimate physical contact between antigenactivated T h cell and B cell for the generation of proliferating and Igsecreting B cells, the precise events occuring at the Th/B cell interface have yet to be defined. Our own experiments with the human system have utilized T-cell clones specific for allogeneic MHC class II molecules, or for the hapten trinitrophenyl (TNP) in association with an autologous MHC class II molecule. Alternatively, freshly isolated peripheral blood T cells, activated via the TCR-T3 complex with anti-CD3 mAb, have been used. Each of these experimental systems generates a polyclonal B-cell response, readily assayed in at least 4 ways: 1)by the generation of second. messenger molecules, specifically the products of inositol lipid hydrolysis (Chartash et al., 1988); 2) by the induction of B-cell surface CD23, an activation molecule expressed by most sIgM÷ IgD÷ cells after productive T h / B cell interaction (Crow et al., 1986); 3) B-cell proliferation (Goldberg et al., 1985); and 4) B-cell differentiation into Ig-secreting cells (Friedman et aL, 1985; Principato et aL, 1983). As the latter 2 assays require 5-10 days of culture and reflect multiple lymphokinemediated signals, as well as direct T hmediated signals, we have focused our efforts on the assays of second mes-
OG Y
senger molecule generation and CD23 expression. The alloreactive human Th-cell clones were used to show that allospecific Th/resting B cell interaction does not result in inositol lipid hydrolysis, or an increase in intracellular Ca ÷2 within the responder B cells, while antihuman IgM antibody does activate this second messenger cascade (Chartash et al., 1988). As a positive control for these studies, we determined that within 12 h of culture, the target B cells were induced to express cell surface CD23 by the allospecific T h / B cell interaction, but not by sIg cross-linking. These results suggest that antigen and T h cells induce different functional B-cell responses by activating distinct second messenger systems within the B cell. This issue has been addressed in studies by Noelle and Snow, which demonstrate a brief rise in B-cell cAMP after physical interaction with fixed antiCD3-activated T h cells (Bartlett and Noelle, 1990). Moreover, Cambier has shown that anti-MHC class II antibodies stimulate increased B-cell cAMP levels. It appears then that enzymes such as protein kinase A, which generate elevated intracellular cAMP, should be targets of investigation of the biochemical mediators of cognate T-cell help /'C"~ml~,-~ ~,...~,~t~,~
- ,,~. , , ~ ! ~
~g.
,
1~'7~
~ ,~'~.~ 1 ~ .
As IL-4 is known to induce B-cell CD23, we have performed studies in order to distinguish B-cell activation that results from soluble mediators versus physical cell-ceil contact. We have utilised 2-chamber-culture wells and either allospecific T-cell clones or peripheral blood T cells activated with anti-CD3 mAb (Crow et al., 1989). Culture of either group of T cells with unfractionated tonsil or blood non-T cells generates sufficient IL-4 secretion to stimulate CD23 expression on B cells separated by a membrane permeable to lymphokines. However, if the T cells are activated in the presence of resting B cells, strictly depleted of monocytes and low-density B cells, CD23 expression is induced on those resting B cells in physical contact with the activated T h cells, but not on those B cells physically separated. Moreover, IL-4 could not be de-
HOW
tected in supernatants cultures.
T CELLS HELP B CELLS
from those
Gamma interferon has several functions that oppose those of IL-4. Among these activities is gamma interferon's capacity to inhibit IL-4-induced B cell CD23. We found that, while gamma in. terferon inhibited IL-4-induced CD23 expression on resting B cells by about 50 %, it usually resulted in no or slight inhibition of activated Th-cell-induced CD23 expression by resting B cells in close physical proximity. Taken together, the two-chamber and gamma interferon experiments support the requirement for physical Th cell/resting B cell interaction in the induction of an early stage of B-cell activation. While we do not see a need to postulate a second, non-IL-4-mediated mechanism of CD23 induction, it is clear that B-cell activation can be stimulated by unmeasurably low levels of IL-4 delivered at the T-B cell interface, or perhaps by a Tcell surface-bound form of IL-4, present on an activated but not resting T cell. Our studies of CD23 expression induced by human Th cells, when taken together, support a sequence of events in which (1) the T cells are first activated through the TCR, and then (2) efficient cell-mediated B activation is ~.mtatgu, out omy wn~n the ~ and B cells are in close physical proximity. An interesting hapten altered-self reactive human Th-cell clone, Ell, has been useful in approaching these issues (Friedman et al., 1986). Clone Ell proliferates in response to TNP bound to macrophages or B cells of the DR5 haplotype. We found that Eli's requirements for generating help for B-cell activation (CD23 expression) are less stringent than its own requirements for proliferation. Ell will stimulate CD23 expression on TNP-conjugated B cells of any individual, regardless of DR haplotype. Moreover, TNP coupled to BGG or to Sepharose beads can induce B cell CD23 expression when present in cultures of Ell and untreated B cells. Experiments in which Ell cells were pretreated with TNP Sepharose beads for 2 h, fixed C,-,,-,:!:~.,~-~,-I
L..*-
~_1
.....
L
----
~
429
with paraformaldehyde and washed extensively also were successful in stimulating CD23 expression on cocultured B cells. These data suggest that once activated through their TCR, Ell cells undergo a change which facilitates their capacity to provide an activation signal to any B cell in an MHCunrestricted manner. The effect of TNP in this system could include the induction of a confermational change in the TCR, of increased expression or a conformational change .in accessory T-cell ~urface molecules or insertion of lymphokines such as IL-4 into the T-cell membrane. As most investigators have not been successful in stimulating B-cell differentiation using !ymphokines alone (including IL-4), we continue to search for an additional T-cell-mediated signal which permits implementation of the full Bcell program. In recent preliminary studies, we have cultured resting tonsil B cells for 16 h with medium or various combinations of mediators which might mimic the initial B-cell exposure to antigen and activated Th cells. We have used (1) phorbol dibutyrate, the watersolub!~ protein kinase C activator, .~a orovitie a signal analogous to that which might result from antigen interaction with slg; 2) IL-4, the early-acting T-cell Jymphokine; and 3) forskoiin, a cAMP analog, which might mimic a T-cell ~urface-mediated B-cell activation signal. A~ter these analogs were washed from the B cells, the cells were cultured with medium or Staphylococcus aureus Cowan strain I (SAC) for 10 days and IgM secretion measured by ELISA. SAC did not induce any detectable secretion of IgM in B-cell cultures pretreated with any one or two of the stimuli in combination. However, B cells pretreated with the combination of PDB, IL-.4 and forskolin were rendered competent to respond to SAC with active IgM secretion. This data provides further suggestion that B-cell triggering through a molecule that generates cAMP might be responsible for the enigmatic phenomenon of cognate Tcell help.
430
32 'ld F O R U M I N I M M U N O L O G Y
References.
ANDERSON,J., SCHREIER,M.H. & MELCHERS,F. (1980), T-cell-dependent B-cell stimulation is H-2 restricted and antigen-dependent only at the resting B-cell level. Proc. nat. Acad. Sci. (Wash.), 77, 1612. BARTLETT,W.C. & NOELLE,R.J. (1990), In "T cell-dependent and independent B cell activation" (Snow, E.C.) CRC Press, Boca Raton, FL. (in press). CAMBIER,J.C., NEWELL,M.K., JUSTEMENT,L.B., McGUIRE, J.C., LEACH,K.L. & CHEN, Z.Z. (1987), Membrane Ia binding ligands induce increased intracellular cAMP which mediates the transient association of protein kinase C with the nucleus of B lymphocytes. Nature (Lond.), 327, 629. CHARTASH,E.K., IMAI,A., GERSHENGORN,M.C., CROW,M.K. & FRIEDMAN,S.M. (1988), Direct human T helper cell-induced B-cell activation is not mediated by inositol lipid hydrolysis. J. lmmunol., 140, 1974. CROW, M.K., JOVER,J.A. & FRIEDMAN,S.M. (1986), Direct T helper/B cell interaction induce an early B cell activation antigen. J. exp. Med., 164, 1760. CRow, M.K., JUSHNER,B., JOVER,J.A., FRIEDMAN,S.M., I~IECHANIC,S.E. & STOHL,W. (1989),. Induction of B cell BLAST-2 (CD23) expression by anti-CD3-stimulated peripheral blood T h cells. Cell. Immunol., 121, 99. FRIEDMAN,S.M., THOMPSON,G.S. & PRINCIPATO,M.A. (1982), Allospecific human T-cell lines and clones ~hich mediate HLA-DR restricted helper activity. J. lmmunol., 129, 2451. FRIEDMAN,S.M., JOVER,J.A., CHARTASH,E.K. & CROW, M.K. (1986), Antigen-specific, MHC non-restricted T helper cell-induced B-cell activation. J. exp. Med., 164, 1773. GOLDBERG,D.A., GREEN,A., GOTTLIEB,A.B., CRow, M.K., LEWlSON,A. & FRIEDMAN,S.M. (1985), Cloned allospecific human helper T-cell lines induce an MHC-restriced proliferative response by resting B cells. J. lmmunol., 135, 1012. Jt~LIt~S, M.H., VON BOEHMER,H. & SIDMAN,C.L. (1982), Dissociation of two signals required for activation of resting B cells. Proc. nat. Acad. Sci. (Wash.), 79, 1989. JULIUS, M.H. & RAMMENSEE,H-G. (1988), T helper cell-dependent induction of resting B cell differentiation r,eed not require cognate cell interactions. Europ. J. lmmunol., 18, 375. M~XCHISON,N.A. (1971), The carrier effect in the secondary response to hapten-protein conjugates. - - II. Cellular cooperation. Europ. J. Immunol., 1, 18. PRINCIPATO,M.A., THOMPSON,G.A. & FRIEDMAN,S.M. (1983), A cloned major histocoml~atibility complex restricted trinitrophenyl-reactive human helper T cell line whichactivates B cell subsets via two distinct pathways. J. exp. Med., 158, 1444. RIEDEL,C., OWENS,T. & NOSSAL,(.~..;.V. (1988), A significant proportion of normal resting B cells are induced to secrete immunoglobulin through contact with anti-receptor antibody-activated ~helper T cells in cionai cultures. Europ. g-. immunol., i8, 403. SINGER, A. & HODES, R.J. (1983), Mechanisms of T-cell-B cell interactions. Ann. Rev. Immunol., 1, 211.
This work is supported in part by: The Arthritis Foundation Investigator Award, The Irvington House Institute, and NIH Grants no. CA-49283, AI-128367, and P60-AR38520.