- Email: [email protected]
Anti-L3T4 monoclonal antibody inhibits T-Cell activation by Anti-T-Cell receptor antibody through a pathway not involving «associative recognition»
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Acknowledgements. This work was supported by USPHS grants AI-17134 and AI-18785 and American Cancer Society grant IM-49. It was conducted while J. Kappler was funded by a Faculty Research Award from the American Cancer Society.
ANTI-L3T4 MONOCWNAL ANTIBODY INHIBITS T-CELL ACTIVATION BY ANTI-T·CELL RECEPTOR ANTIBODY THROUGH A PATHWAY NOT INVOLVING «ASSOCIATIVE RECOGNITION» by R.L. Moldwin, G.J. Nan, W.L. Havran, D.W. Lancki and F.W. Fitch
The Department of Pathology, Committee on Immunology, and Ben May Laboratory for Cancer Research, the University of Chicago, Chicago, II 60637 (USA) In addition to the T-cell receptor for antigen (TCR), several cell surface structures which participate in T-cell activation have been identified by monoclonal antibodies (mAb). The L3T4 molecule, a 52-Kd protein which appears to be the murine homologue of human CD4, is one such structure [2]. MAb reactive with L3T4 inhibits antigen-induced lymphokine production and proliferation as well as antigenspecific cytolysis by murine T lymphocytes restricted by class II major histocompatability complex (MHC) antigens [2]. Based on these and other observations, it has been suggested that this molecule functions as an «associative recognition» structure, interacting with class II MHC molecules and serving to increase or stabilize the interaction between T cells and antigenpresenting cells [2, 3]. However, other data are difficult to reconcile with this hypothesis. AntiL3T4 mAb can inhibit activation of L3T4+ T cells in the absence of cells which express class II MHC antigens: anti-L3T4 mAb GK1.5 inhibits lectinfacilitated cytolysis of P-815 target cells by an L3T4+ T-cell clone; these target cells do not express class II MHC antigens [2]. Anti-L3T4 mAb also inhibits lectin-induced activation of T-cell clones
[10, 12] and T-cell hybridomas [1,2] in the absence of cells expressing class II MHC antigen. In order to evaluate the effect of anti-L3T4 mAb on our cloned T cells stimulated via the TCR, we studied the activation of cloned helper T lymphocytes (HTL) by anti-TCR mAb alone. When cloned HTL L2 cells are stimulated with appropriate concentrations of immobilized anti-TCR mAb F23.l, they proliferate and secrete lymphokines [9]. However, a biphasic proliferative response is observed (fig. 1). As immobilized anti-TCR mAb concentration is increased up to optimal levels, greater proliferation occurs. When the concentration of anti-TCR mAb is increased further, proliferation decreases. We and others observed similar results when cloned HTL cells were stimulated with increasing concentrations of antigen [5, 7]. Supraoptimallevels of antiTCR mAb inhibit proliferation of HTL in response to exogenous IL-2 as well as to secreted IL-2; this effect also has been observed with cloned cytolytic T lymphocytes exposed to exogenous IL-2 (data not shown). When HTL are stimulated with optimal concentrations of anti-TCR mAb, the addition of anti-L3T4 mAb inhibits proliferation (fig. 1) and lymphokine
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production (data not shown) by
90-100 070. Moreover, anti-L3T4 mAb
produces a shift in the dose-response curve; higher concentrations of antiTCR mAb are required to induce unresponsiveness of HTL to exogenous IL-2 when anti-L3T4 mAb is present than when it is absent (manuscript in preparation). Thus, anti-L3T4 mAb inhibits proliferation and lymphokine secretion of cloned HTL stimulated with optimal concentrations of antiTCR mAb, but anti-L3T4 mAb paradoxically increases proliferation when the cloned HTL are stimulated with higher concentrations of anti-TCR mAb
(fig. 1). This increase in proliferation appears to be due to an inhibition of activation; concentrations of anti-TCR mAb which usually would be supraoptimal become optimal in the presence of anti-L3T4 mAb. Thus, the augmenting effects of anti-L3T4 mAb in this situation can also be attributed to interference with T-cell activation induced by anti-TCR mAb. In order to study the mechanism responsible for inhibition of HTL activation by anti-L3T4 mAb, we exposed the cloned HTL to stimuli which appear to bypass the TCR. When HTL are stimulated with phorbol myristate
80
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lSl
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~
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.0001
.001
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'L mAb
GKI. 5
-'anti-L3T4l
.01
. 1
mRb F23.1 SUPERNRTRNT DILUTION FIG. 1. - Effect of mAb GK1.5 on proliferation of HTL L2 in response to anti- TCR mAb.
Anti-TCR mAb F23.1 was immobilized in microtitre trays by first incubating rat anti-mouse IgG followed by serial3-fold dilutions of mAb F23.1 hybridoma supernatant. Anti-L3T4 mAb GK1.5 was placed in appropriate wells followed by addition of cloned HTL L2 cells (5 x 10 4). Wells were pulsed with 0.5 !J.Cilml [3H]-thymidine for the last 6 h of the 48-h culture period. Counts per minute were corrected to disintegrations per minute (DPM). The data depicted here are representative of more than 10 experiments.
CD4 MOLECULE IN T-CELL ACTIVATION acetate (PMA; to activate protein kinase C) and the calcium ionophore A23187 (to increase intracellular free Ca++), anti-L3T4 mAb does not inhibit proliferation or lymphokine secretion (data not shown). Furthermore, anti-L3T4 does not affect IL-2-induced proliferation of cloned HTL (data not shown). These results indicate that antiL3T4 mAb interferes with a process occurring before the activation of protein kinase C and the increase in intracellular free Ca++ which are induced by stimulation of the TCR [4]. The hypothesis that L3T4 has an associative recognition function has been based mainly on the findings that anti-L3T4 mAb inhibits antigeninduced activation of class II MHC antigen-restricted HTL. Our demonstration that anti-L3T4 mAb inhibits HTL activation in the absence of antigen-presenting cells appears to invalidate the interpretation of much of the data supporting this hypothesis. However, several other observations seem to support an involvement of L3T4 in associative recognition. For example, variant HTL hybridomas which have lost the ability to express L3T4 require higher concentrations of antigen for activation and are more readily inhibited by mAb reactive with class II MHC antigens [6]. These properties would be expected if the loss of L3T4 resulted in decreased avidity of interaction with antigen-presenting cells.
133
Also, anti-L3T4 mAb inhibits conjugate formation between cloned HTL hybridomas and appropriate antigenpresenting cells [12]. In addition, HTL hybridomas can be activated by antigen fragments and planar lipid membranes containing class II MHC antigens [11]. Perhaps, in these situations, L3T4 may be involved in associative recognition, interacting with class II MHC antigens. The associative recognition hypothesis postulates that an interaction of L3T4 with its natural ligand facilitates HTL activation, while our data indicate that anti-L3T4 mAb behaves differently and inhibits HTL activation. It is possible that this difference may relate to the form in which the « ligand» is presented to the T cell, an array for the natural ligand or individual molecules in the case of anti-L3T4 mAb. Such a differential effect has been observed with mAb reactive with the TCR: immobilized anti-TCR or anti-CD3 mAb activates T cells, while soluble forms of these mAb interfere with T-cell activation by antigen [8]. Based on the data presented here, conclusions regarding the role of L3T4 need to be revised. Our data demonstrating that anti-L3T4 mAb inhibits Tcell activation in the absence of cells expressing class II MHC antigens indicate that L3T4 may have a more active function beyond the passive role proposed by the associative recognition hypothesis.
References. [1] BEKOFF, M., KAKIUCHI, T. & GREY, H., Accessory cell function in the ConA response: role of la-positive and la-negative accessory cells. J. Immunol.,
1985, 134, 1337-1342. [2] DIALYNAS, D., WILDE, D., MARRAcK, P., PIERRES, A., WALL, K., HAVRAN, W., OTTEN, G., LOKEN, M., PIERRES, M., KAPPLER, J. & FITCH, F., Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK 1.5: expression of L3T4a by functional T-cell clones appears to correlate primarily with class II MHC antigen reactivity. Immunol. Rev., 1983, 74, 29-56. [3] GREENSTEIN, J., KAPPLER, J., MARRACK, P. & BURAKOFF, S., The role of L3T4 in recognition of la by a cytotoxic, H-2Dd-specific T-cell hybridoma. J. expo Med., 1984, 159, 1213-1224. [4] IMBODEN, J., WEISS, A. & STOBO, J., Transmembrane signalling by the T3-antigen receptor complex. Immunol. Today, 1985, 6, 328-331.
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[5] LANCKI, D., MA, D., HAVRAN, W. & FITCH, F., Cell-surface structures involved in T-cell activation. Immunol. Rev., 1984, 81, 65-94. [6] MARRACK, P., ENDRES, R., SHIMONKEVITZ, R., ZLOTNIK, A., DIALYNAS, D., FITCH, F. & KApPLER, J., The major-histocompatibility-complex-restricted antigen receptor on T cells. - II. Role of the L3T4 product. J. expo Med., 1983, 158, 1077-1091. [7] MATIS, L., GLIMCHER, L., PAUL, W. & SCHWARTZ, R., Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and la molecules. Proc. nat. A cad. Sci. (Wash.), 1983, 80, 6019-6023. [8] MEUER, S., HODGDON, J., HUSSEY, R., PROTENTlS, J., SCHLOSSMAN, S. & REINHERZ, E., Antigen-like effects of monoclonal antibodies directed at receptors on human T-cell clones. J. expo Med., 1983, 158, 988-993. [9] MOLDWIN, R., LANCKI, D., HEROLD, K. & FITCH, F., An antigen-receptor-driven, IL-2-independent pathway for proliferation of murine CTL clones. J. expo Med., 1986, 163, 1566-1582. [10] TITE, J., SLOAN, A. & JANEWAY, C. Jr, The role of L3T4 in T-cell activation: L3T4 may be both an la-binding protein and a receptor which transduces a negative signal. J. mol. Immunol., 1986, 2, 179-190. [11] WATTS, T., BRIAN, A., KAPPLER, J., MARRACK, P. & MCCONNELL, H., Antigen presentation by supported planar membranes containing affinity-purified I-Ad. Proc. nat. A cad. Sci. (Wash.), 1984, 81, 7564-7568. [12] WILDE, D., MARRACK, P., KAPPLER, J., DIALYNAS, D. & FITCH, F., Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GKl.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation release of lymphokines and binding by cloned murine helper T lymphocyte lines. J. Immunol., 1983, 131, 2178-2183. Acknowledgements. This research was supported by grants AI-04I97, AI-I806I and CA-I9266 from the National Institutes of Health. R.L. Moldwin and G.J. Nau were supported by Medical Scientist Training Program Grant 5T32 GM-0728I from the U. S. Public Health Service. W.L. Havran was supported by Training Grant AI-07099 from the U.S. Public Health Service.
THE ROLE OF L3T4 (CD4) IN T-CELL ACTIVATION by J.L. Greenstein and S.J. Burakoff
Department of Pathology, Harvard Medical School and Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 (USA) Recognition of foreign antigen by the T-cell antigen receptor (Ti) occurs in the context of self major histocompatibility complex (MHC) antigens. The expression of the murine T-cell surface molecules L3T4 (CD4) and Lyt2 (CD8) appears to segregate with Ti specifi-
city, i.e. the majority of L3T4+ T cells are restricted to class II MHC and the Lyt2+ T cells are restricted to class I MHC antigens. The possibility that the L3T4 and Lyt2 molecules are receptors for MHC antigens and are involved in T-cell activation was, in part, suggested
131
Acknowledgements. This work was supported by USPHS grants AI-17134 and AI-18785 and American Cancer Society grant IM-49. It was conducted while J. Kappler was funded by a Faculty Research Award from the American Cancer Society.
ANTI-L3T4 MONOCWNAL ANTIBODY INHIBITS T-CELL ACTIVATION BY ANTI-T·CELL RECEPTOR ANTIBODY THROUGH A PATHWAY NOT INVOLVING «ASSOCIATIVE RECOGNITION» by R.L. Moldwin, G.J. Nan, W.L. Havran, D.W. Lancki and F.W. Fitch
The Department of Pathology, Committee on Immunology, and Ben May Laboratory for Cancer Research, the University of Chicago, Chicago, II 60637 (USA) In addition to the T-cell receptor for antigen (TCR), several cell surface structures which participate in T-cell activation have been identified by monoclonal antibodies (mAb). The L3T4 molecule, a 52-Kd protein which appears to be the murine homologue of human CD4, is one such structure [2]. MAb reactive with L3T4 inhibits antigen-induced lymphokine production and proliferation as well as antigenspecific cytolysis by murine T lymphocytes restricted by class II major histocompatability complex (MHC) antigens [2]. Based on these and other observations, it has been suggested that this molecule functions as an «associative recognition» structure, interacting with class II MHC molecules and serving to increase or stabilize the interaction between T cells and antigenpresenting cells [2, 3]. However, other data are difficult to reconcile with this hypothesis. AntiL3T4 mAb can inhibit activation of L3T4+ T cells in the absence of cells which express class II MHC antigens: anti-L3T4 mAb GK1.5 inhibits lectinfacilitated cytolysis of P-815 target cells by an L3T4+ T-cell clone; these target cells do not express class II MHC antigens [2]. Anti-L3T4 mAb also inhibits lectin-induced activation of T-cell clones
[10, 12] and T-cell hybridomas [1,2] in the absence of cells expressing class II MHC antigen. In order to evaluate the effect of anti-L3T4 mAb on our cloned T cells stimulated via the TCR, we studied the activation of cloned helper T lymphocytes (HTL) by anti-TCR mAb alone. When cloned HTL L2 cells are stimulated with appropriate concentrations of immobilized anti-TCR mAb F23.l, they proliferate and secrete lymphokines [9]. However, a biphasic proliferative response is observed (fig. 1). As immobilized anti-TCR mAb concentration is increased up to optimal levels, greater proliferation occurs. When the concentration of anti-TCR mAb is increased further, proliferation decreases. We and others observed similar results when cloned HTL cells were stimulated with increasing concentrations of antigen [5, 7]. Supraoptimallevels of antiTCR mAb inhibit proliferation of HTL in response to exogenous IL-2 as well as to secreted IL-2; this effect also has been observed with cloned cytolytic T lymphocytes exposed to exogenous IL-2 (data not shown). When HTL are stimulated with optimal concentrations of anti-TCR mAb, the addition of anti-L3T4 mAb inhibits proliferation (fig. 1) and lymphokine
16th FORUM IN IMMUNOLOGY
132
production (data not shown) by
90-100 070. Moreover, anti-L3T4 mAb
produces a shift in the dose-response curve; higher concentrations of antiTCR mAb are required to induce unresponsiveness of HTL to exogenous IL-2 when anti-L3T4 mAb is present than when it is absent (manuscript in preparation). Thus, anti-L3T4 mAb inhibits proliferation and lymphokine secretion of cloned HTL stimulated with optimal concentrations of antiTCR mAb, but anti-L3T4 mAb paradoxically increases proliferation when the cloned HTL are stimulated with higher concentrations of anti-TCR mAb
(fig. 1). This increase in proliferation appears to be due to an inhibition of activation; concentrations of anti-TCR mAb which usually would be supraoptimal become optimal in the presence of anti-L3T4 mAb. Thus, the augmenting effects of anti-L3T4 mAb in this situation can also be attributed to interference with T-cell activation induced by anti-TCR mAb. In order to study the mechanism responsible for inhibition of HTL activation by anti-L3T4 mAb, we exposed the cloned HTL to stimuli which appear to bypass the TCR. When HTL are stimulated with phorbol myristate
80
(T)
MEDIA
I
lSl
Q..
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I I I I I ~
X
:L
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~-..
40
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o
.0001
.001
\
'L mAb
GKI. 5
-'anti-L3T4l
.01
. 1
mRb F23.1 SUPERNRTRNT DILUTION FIG. 1. - Effect of mAb GK1.5 on proliferation of HTL L2 in response to anti- TCR mAb.
Anti-TCR mAb F23.1 was immobilized in microtitre trays by first incubating rat anti-mouse IgG followed by serial3-fold dilutions of mAb F23.1 hybridoma supernatant. Anti-L3T4 mAb GK1.5 was placed in appropriate wells followed by addition of cloned HTL L2 cells (5 x 10 4). Wells were pulsed with 0.5 !J.Cilml [3H]-thymidine for the last 6 h of the 48-h culture period. Counts per minute were corrected to disintegrations per minute (DPM). The data depicted here are representative of more than 10 experiments.
CD4 MOLECULE IN T-CELL ACTIVATION acetate (PMA; to activate protein kinase C) and the calcium ionophore A23187 (to increase intracellular free Ca++), anti-L3T4 mAb does not inhibit proliferation or lymphokine secretion (data not shown). Furthermore, anti-L3T4 does not affect IL-2-induced proliferation of cloned HTL (data not shown). These results indicate that antiL3T4 mAb interferes with a process occurring before the activation of protein kinase C and the increase in intracellular free Ca++ which are induced by stimulation of the TCR [4]. The hypothesis that L3T4 has an associative recognition function has been based mainly on the findings that anti-L3T4 mAb inhibits antigeninduced activation of class II MHC antigen-restricted HTL. Our demonstration that anti-L3T4 mAb inhibits HTL activation in the absence of antigen-presenting cells appears to invalidate the interpretation of much of the data supporting this hypothesis. However, several other observations seem to support an involvement of L3T4 in associative recognition. For example, variant HTL hybridomas which have lost the ability to express L3T4 require higher concentrations of antigen for activation and are more readily inhibited by mAb reactive with class II MHC antigens [6]. These properties would be expected if the loss of L3T4 resulted in decreased avidity of interaction with antigen-presenting cells.
133
Also, anti-L3T4 mAb inhibits conjugate formation between cloned HTL hybridomas and appropriate antigenpresenting cells [12]. In addition, HTL hybridomas can be activated by antigen fragments and planar lipid membranes containing class II MHC antigens [11]. Perhaps, in these situations, L3T4 may be involved in associative recognition, interacting with class II MHC antigens. The associative recognition hypothesis postulates that an interaction of L3T4 with its natural ligand facilitates HTL activation, while our data indicate that anti-L3T4 mAb behaves differently and inhibits HTL activation. It is possible that this difference may relate to the form in which the « ligand» is presented to the T cell, an array for the natural ligand or individual molecules in the case of anti-L3T4 mAb. Such a differential effect has been observed with mAb reactive with the TCR: immobilized anti-TCR or anti-CD3 mAb activates T cells, while soluble forms of these mAb interfere with T-cell activation by antigen [8]. Based on the data presented here, conclusions regarding the role of L3T4 need to be revised. Our data demonstrating that anti-L3T4 mAb inhibits Tcell activation in the absence of cells expressing class II MHC antigens indicate that L3T4 may have a more active function beyond the passive role proposed by the associative recognition hypothesis.
References. [1] BEKOFF, M., KAKIUCHI, T. & GREY, H., Accessory cell function in the ConA response: role of la-positive and la-negative accessory cells. J. Immunol.,
1985, 134, 1337-1342. [2] DIALYNAS, D., WILDE, D., MARRAcK, P., PIERRES, A., WALL, K., HAVRAN, W., OTTEN, G., LOKEN, M., PIERRES, M., KAPPLER, J. & FITCH, F., Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK 1.5: expression of L3T4a by functional T-cell clones appears to correlate primarily with class II MHC antigen reactivity. Immunol. Rev., 1983, 74, 29-56. [3] GREENSTEIN, J., KAPPLER, J., MARRACK, P. & BURAKOFF, S., The role of L3T4 in recognition of la by a cytotoxic, H-2Dd-specific T-cell hybridoma. J. expo Med., 1984, 159, 1213-1224. [4] IMBODEN, J., WEISS, A. & STOBO, J., Transmembrane signalling by the T3-antigen receptor complex. Immunol. Today, 1985, 6, 328-331.
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[5] LANCKI, D., MA, D., HAVRAN, W. & FITCH, F., Cell-surface structures involved in T-cell activation. Immunol. Rev., 1984, 81, 65-94. [6] MARRACK, P., ENDRES, R., SHIMONKEVITZ, R., ZLOTNIK, A., DIALYNAS, D., FITCH, F. & KApPLER, J., The major-histocompatibility-complex-restricted antigen receptor on T cells. - II. Role of the L3T4 product. J. expo Med., 1983, 158, 1077-1091. [7] MATIS, L., GLIMCHER, L., PAUL, W. & SCHWARTZ, R., Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and la molecules. Proc. nat. A cad. Sci. (Wash.), 1983, 80, 6019-6023. [8] MEUER, S., HODGDON, J., HUSSEY, R., PROTENTlS, J., SCHLOSSMAN, S. & REINHERZ, E., Antigen-like effects of monoclonal antibodies directed at receptors on human T-cell clones. J. expo Med., 1983, 158, 988-993. [9] MOLDWIN, R., LANCKI, D., HEROLD, K. & FITCH, F., An antigen-receptor-driven, IL-2-independent pathway for proliferation of murine CTL clones. J. expo Med., 1986, 163, 1566-1582. [10] TITE, J., SLOAN, A. & JANEWAY, C. Jr, The role of L3T4 in T-cell activation: L3T4 may be both an la-binding protein and a receptor which transduces a negative signal. J. mol. Immunol., 1986, 2, 179-190. [11] WATTS, T., BRIAN, A., KAPPLER, J., MARRACK, P. & MCCONNELL, H., Antigen presentation by supported planar membranes containing affinity-purified I-Ad. Proc. nat. A cad. Sci. (Wash.), 1984, 81, 7564-7568. [12] WILDE, D., MARRACK, P., KAPPLER, J., DIALYNAS, D. & FITCH, F., Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GKl.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation release of lymphokines and binding by cloned murine helper T lymphocyte lines. J. Immunol., 1983, 131, 2178-2183. Acknowledgements. This research was supported by grants AI-04I97, AI-I806I and CA-I9266 from the National Institutes of Health. R.L. Moldwin and G.J. Nau were supported by Medical Scientist Training Program Grant 5T32 GM-0728I from the U. S. Public Health Service. W.L. Havran was supported by Training Grant AI-07099 from the U.S. Public Health Service.
THE ROLE OF L3T4 (CD4) IN T-CELL ACTIVATION by J.L. Greenstein and S.J. Burakoff
Department of Pathology, Harvard Medical School and Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 (USA) Recognition of foreign antigen by the T-cell antigen receptor (Ti) occurs in the context of self major histocompatibility complex (MHC) antigens. The expression of the murine T-cell surface molecules L3T4 (CD4) and Lyt2 (CD8) appears to segregate with Ti specifi-
city, i.e. the majority of L3T4+ T cells are restricted to class II MHC and the Lyt2+ T cells are restricted to class I MHC antigens. The possibility that the L3T4 and Lyt2 molecules are receptors for MHC antigens and are involved in T-cell activation was, in part, suggested