- Email: [email protected]
Peptide-mediated anergy in human CD4+ T cells
294
42nd FORUM
IN IMMUNOLOGY
Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J. & Owen, J.J.T. (1989), Antibodies to CD3/Tcell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nurure (Lond.), 337, 181-184. Speiser, D.E., Chvatchko, V., Zinkernagel, R.M. & MacDonald, H.R. (1990), Distinct fates of self-specific T
Peptide-mediated
cells developing in irradiation bone marrow chimeras : clonal deletion, clonal anergy, or in vitro responsivenessto self-MIS- 1a controlled by hematopoietic cells in the thymus. J. exp. Med., 172, 13051314. Webb, S., Morris, C. & Sprent, J. (1990), Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell, 63, 1249-1256.
anergy in human CD4+
T cells
C.R.A. Hewitt and J.R. Lamb Imperial
Department of Immunology, St. Mary’s Hospital Medical School, College of Science, Technology and Medicine, Norfolk Place, London
Introduction The ability to discriminate self from non-self is a fundamental tenet of the peripheral immune system. Tolerance to self requires that the immature repertoire of antigen recognition molecules is purged of self-reactivity before release into the periphery. In order to confer a selective advantage, the antigen receptor repertoire is selected before reproductive maturity. During the development of T cells, the neonatal thymus fulfils this role by directing the deletion of self-reactive T cells whilst retaining antigenreactive MHC-restricted T cells. Whilst central thymic mechanisms of self-tolerance are widely applicable, they fail to account for the control of self-reactive T cells specific for self epitopes expressed exclusively by adults, or at sites remote from the neonatal thymus. It is under these circumstances that T-cell anergy is proposed to “fine-tune” the T-cell repertoire preselected by the thymus. ‘Jr-dike clonal deletion, in which cells with selfreactive T-cell antigen receptors (TcR) are physically removed from the neonatal repertoire (Kappler et al., 1988; MacDonald et al., 1988), antigen-specific clonal T-cell anergy operates by the functional inactivation of mature peripheral self-reactive T cells (Rammensee et al., 1989). Therefore, instead of competing against each other, deletion and anergy cooperate to control self-reactivity in the TcR repertoire. Human
T-cell anergy in vitro
We have investigated non-responsiveness in mature T cells by developing an in vitro model of
W2 IPG
antigen-specific T-cell anergy using human CD4+ T-cell clones. In this model, antigen-specific human T-cell clones are hyperactivated, in the absence of additional antigen-presenting cells (APC), using a concentration of antigen, in the form of a peptide, loto loo-fold greater than that required for maximal proliferation in the presence of APC (Lamb et al., 1983). After rigourous washing, the hyperactivated T cells are rechallenged in the presence of APC with an optimal, immunogenic concentration of antigen. T cells pre-treated, or anergized, with high peptide antigen concentrations then fail to proliferate in response to the immunogenic challenge, but retain responsiveness to exogenously added IL2. The degree of anergy is dependent upon the concentration of peptide used for preincubation; such that T cells preincubated with higher concentrations of peptide make lower responses upon re-challenge than those pretreated with intermediate or low concentrations of peptide. In common with the optimal activation of MHC class-II-restricted T-cell clones, serological inhibition using anti-MHC class II antibodies has demonstrated that hyperactivation, leading to anergy, also requires MHC class II antigens (Lamb and Feldmann, 1984). Since the induction phase of human T cell anergy in vitro takes place in the absence of conventional MHC class-II-bearing APC, peptide-mediated anergy induction is dependent upon MHC class II molecules expressed by the T-cell clone itself (Hewitt and Feldmann, 1989). Although T cell MHC classII-mediated presentation of high concentrations of peptide ultimately renders the T cells anergic to subsequent immunogenic challenge, this route of peptide presentation results in a small but significant
CLONAL
DELETION
AND
ANERGY:
degree of T-cell proliferation. Under these conditions, IL2 and IL4 secretion and mRNA expression are also superinduced (O’Hehir et al., 1991), suggesting that the cells are hyperactivated. Despite this, the lack of accessory cells and the regulatory signals they provide may account for the limited T-cell proliferation observed during anergy induction. Modulation T cells
of cell surface
molecules
in anergic
In an attempt to determine the mechanisms of anergy, T ceils pretreated with anergizing concentrations of antigen have been compared with optimally activated T cells for the modulation of cell surface molecules critical in the induction of anergy and T-cell activation. For up to 36 hours after activation or anergy induction, cell surface expression of the T-cell antigen receptor(TcR)/CD3 complex is downregulated (Zanders et al., 1983). Insufficient antigen recognition due to low TcR expression alone is, however, not responsible for the inability of anergized T cells to respond to immunogenic challenge, since even when the TcR/CD3 level is restored to normal levels, the anergic T cells remain unresponsive to immunogenic challenge (J.R. Lamb and R.E. O’Hehir, unpublished). In common with activated T cells, anergized cells also express high levels of the IL2 receptor (O’Hehir and Lamb, 1990), and accordingly respond well to exogenously added IL2 (Lamb et al., 1983). This confirms that the anergized T cells retain the ability to proliferate to non-TcR-mediated signals, and rules out the potential explanation that anergy might be due to the toxicity of high concentrations of peptide antigen. Thus far, only CD28 appears to be differentially expressed on optimally activated T cells and hyperactivated, anergized T cells. Unlike optimally activated T cells, on which CD28 is strongly induced, hyperactivated anergized T cells express lower levels of CD28 (O’Hehir and Lamb, 1990). This is of particular interest with respect to anergy, as the natural ligand of CD28 thought to be a co-stimulatory molecule, B7/BB 1, is expressed predominantly by B cells (June et al., 1990). Anergy
or activation
and co-stimulation
The importance of co-stimulatory molecules in T-cell activation and anergy has been best demonstrated in a well-characterized in vitro model of anergy using murine T-cell clones (Mueller et al., 1989 ; Schwartz, 1990). These studies suggest that in Thl-type CD4+ T-cell clones, activation occurs when a T cell receives signals from both antigen and other APC-derived co-stimulatory molecules. Aner-
FROM
MODELS
TO REALITY
295
gy, in contrast, occurs when T cells interact with antigen alone in the absence of additional signals from co-stimulatory molecules. This form of anergy, unlike the human model described above, may be induced with concentrations of peptide antigen normally optimal for proliferative responses. Furthermore, murine T-cell anergy is immediate, with unresponsiveness being manifest during the induction phase. This differs from the human model, which requires an additional immunogenic challenge to become’evident. Although finally resulting in anergy, the induction phase of murine anergy does involve transient T-cell activation. In contrast to the human model, however, activation is arrested short of IL2 production and proliferation.
Involvement anergy
of co-stimulatioti
in human
T-cell
Although a variety of differences exist between the human and murine models of clonal T-cell anergy, it has been hypothesized that a lack of costimulation may also account for anergy due to hyperactivation of human T-cell clones (O’Hehir and Lamb, 1990). This hypothesis is suggested by the finding that surface expression of CD28, a costimulatory signal receptor, is downregulated on hyperactivated anergic human T-cell clones compared with CD28 expression on optimally activated T cells. Therefore, rather than involving a defect in the provision of co-stimulatory signals by APC, human T-cell anergy may involve a deficiency in the reception of co-stimulatory signals due to the downregulation of co-stimulatory signal receptors. This is supported by the correlation between anergy induction only at high antigen concentrations and the downregulation of co-stimulatory signal receptors under the same conditions. The hypothesis is also consistent with the proposed mode of action via CD28 (June et al., 1990). When ligated, CD28 appears to be involved in two signal transduction pathways, one of which is dependent upon extensive TcR cross-linking. In combination with TcR-derived signals, ligation of CD28 augments cytokine production. This is due to inhibtion of cytokine mRNA degradation leading to an accumulation of steady mRNA levels, increased levels of translation and protein secretion. We have obtained circumstantial evidence consistent with the mode of action of CD28 from the examination of cytokine mRNA and protein levels during the immunogenic rechallenge of anergic cells. Unlike during optimal activation and anergy induction, the immunogenic challenge of anergized cells does not induce IL2 mRNA in the anergized T cells (O’Hehir et al., 1991). It is, therefore, possible that under circumstances in which CD28 has been down-
296
42nd FORUM
IN IMikWNOLOGY MacDonald, H.R., Schneider, R., Lees, R.K., Howe, R.C., Acha, O.H., Festenstein, H., Zinkernagel, R.M. & Hengartner, H. (1988), T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature (Lond.), 332, 40-45. Mueller, D.L., Jenkins, M.K. & Schwartz, R.H. (1989), Clonal expansion versus functional clonal inactivation : a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy.
regulated by anergy induction, IL2 mRNA is no longer stabilized and is thus degraded; this has the final result of preventing IL2-mediated autocrine proliferation upon immunogenic challenge. An important aspect of this hypothesis is that despite the importance of APC-mediated co-stimulation, this form of anergy is primarily driven by the interaction of high concentrations of antigen with T cells.
Ann.
References Hewitt, C.R. & Feldmann, M. (1989), Human T cell clones present antigen. J. Immunol., 143, 762-769. June, C.H., Ledbetter, J.A., Linsley, P.S. & Thompson, C.B. (1990), Role of the CD28 receptor in T cell activation. Immunol. Today, 11, 211-216. Kappler, J.W., Staerz, U., White, J. & Marrack, P.C. (1988), Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex. Nature (Lond.), 332, 35-40. Lamb, J.R. Jr Feldmann, M. (1984), Essential requirement for major histocompatibility complex recognition in T-cell tolerance induction. Nature (Land.), 308,72-14. Lamb, J.R., Skidmore, B.J., Green, N., Chiller, J.M. & Feldmann, M. (1983), Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J. exp. Med.. 157, 1434-1447.
Tolerance to peripheral non-deletional
Rev.
Immunol.,
7, 445-480.
O’Hehir, R.E. &Lamb, J.R. (1990), Induction of specific clonal anergy in human T lymphocytes by Staphylococcus aureus enterotoxins. Proc. nut. Acad. Sci. (Wash.), 87, 8884-8888. O’Hehir, R.E., Yssel, H., Verma, S., de Vries, J., Spits, H. &Lamb, J.R. (1991), Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy. Int. Immunol., 3, 819-824. Rammensee, H.G., Kroschewski, R. & Frangoulis, B. (1989), Clonal anergy induced in mature V beta 6+ T lymphocytes on immunizing Mls-lb mice with Mls-1’ expressing cells. Nature (Lond.), 339, 541-544. Schwartz, R.H. (1990), A cell culture model for T lymphocyte clonal anergy. Science, 248, 1349-1356. Zanders, E.D., Lamb, J.R., Feldmann, M., Green, N. & Beverly, P.C.L. (1983), Tolerance of T cell clones is associated with membrane antigen changes. Nature (Lond.), 303, 625-627.
antigens must involve mechanisms
D. Lo Department
of Immunology
IMM-4,
The Scripps Research Institute, La Jolla, CA 92037 (USA)
Immunological tolerance to self has always been a topic of major interest to immunologists, but recent years have witnessed a considerable expansion in the number of new experimental tolerance models. As might be expected, different model systems have yielded diverse interpretations of their results. Although it would be natural to seek a unifying explanation for all self-tolerance, it is clear that a single mechanism cannot explain tolerance to all self
10446 North
Torrey Pines Road,
antigens, due to differences in location and amount of antigen. Therefore, in this discussion, we will discuss only T-cell tolerance to peripheral antigens, defined as antigens expressed only on non-lymphoid tissues and not secreted in large amounts into the bloodstream. Such antigens represent the putative targets for a large number of human autoimmune diseases, including type 1 diabetes and multiple sclerosis. We present here our arguments that tolerance to
42nd FORUM
IN IMMUNOLOGY
Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J. & Owen, J.J.T. (1989), Antibodies to CD3/Tcell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nurure (Lond.), 337, 181-184. Speiser, D.E., Chvatchko, V., Zinkernagel, R.M. & MacDonald, H.R. (1990), Distinct fates of self-specific T
Peptide-mediated
cells developing in irradiation bone marrow chimeras : clonal deletion, clonal anergy, or in vitro responsivenessto self-MIS- 1a controlled by hematopoietic cells in the thymus. J. exp. Med., 172, 13051314. Webb, S., Morris, C. & Sprent, J. (1990), Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell, 63, 1249-1256.
anergy in human CD4+
T cells
C.R.A. Hewitt and J.R. Lamb Imperial
Department of Immunology, St. Mary’s Hospital Medical School, College of Science, Technology and Medicine, Norfolk Place, London
Introduction The ability to discriminate self from non-self is a fundamental tenet of the peripheral immune system. Tolerance to self requires that the immature repertoire of antigen recognition molecules is purged of self-reactivity before release into the periphery. In order to confer a selective advantage, the antigen receptor repertoire is selected before reproductive maturity. During the development of T cells, the neonatal thymus fulfils this role by directing the deletion of self-reactive T cells whilst retaining antigenreactive MHC-restricted T cells. Whilst central thymic mechanisms of self-tolerance are widely applicable, they fail to account for the control of self-reactive T cells specific for self epitopes expressed exclusively by adults, or at sites remote from the neonatal thymus. It is under these circumstances that T-cell anergy is proposed to “fine-tune” the T-cell repertoire preselected by the thymus. ‘Jr-dike clonal deletion, in which cells with selfreactive T-cell antigen receptors (TcR) are physically removed from the neonatal repertoire (Kappler et al., 1988; MacDonald et al., 1988), antigen-specific clonal T-cell anergy operates by the functional inactivation of mature peripheral self-reactive T cells (Rammensee et al., 1989). Therefore, instead of competing against each other, deletion and anergy cooperate to control self-reactivity in the TcR repertoire. Human
T-cell anergy in vitro
We have investigated non-responsiveness in mature T cells by developing an in vitro model of
W2 IPG
antigen-specific T-cell anergy using human CD4+ T-cell clones. In this model, antigen-specific human T-cell clones are hyperactivated, in the absence of additional antigen-presenting cells (APC), using a concentration of antigen, in the form of a peptide, loto loo-fold greater than that required for maximal proliferation in the presence of APC (Lamb et al., 1983). After rigourous washing, the hyperactivated T cells are rechallenged in the presence of APC with an optimal, immunogenic concentration of antigen. T cells pre-treated, or anergized, with high peptide antigen concentrations then fail to proliferate in response to the immunogenic challenge, but retain responsiveness to exogenously added IL2. The degree of anergy is dependent upon the concentration of peptide used for preincubation; such that T cells preincubated with higher concentrations of peptide make lower responses upon re-challenge than those pretreated with intermediate or low concentrations of peptide. In common with the optimal activation of MHC class-II-restricted T-cell clones, serological inhibition using anti-MHC class II antibodies has demonstrated that hyperactivation, leading to anergy, also requires MHC class II antigens (Lamb and Feldmann, 1984). Since the induction phase of human T cell anergy in vitro takes place in the absence of conventional MHC class-II-bearing APC, peptide-mediated anergy induction is dependent upon MHC class II molecules expressed by the T-cell clone itself (Hewitt and Feldmann, 1989). Although T cell MHC classII-mediated presentation of high concentrations of peptide ultimately renders the T cells anergic to subsequent immunogenic challenge, this route of peptide presentation results in a small but significant
CLONAL
DELETION
AND
ANERGY:
degree of T-cell proliferation. Under these conditions, IL2 and IL4 secretion and mRNA expression are also superinduced (O’Hehir et al., 1991), suggesting that the cells are hyperactivated. Despite this, the lack of accessory cells and the regulatory signals they provide may account for the limited T-cell proliferation observed during anergy induction. Modulation T cells
of cell surface
molecules
in anergic
In an attempt to determine the mechanisms of anergy, T ceils pretreated with anergizing concentrations of antigen have been compared with optimally activated T cells for the modulation of cell surface molecules critical in the induction of anergy and T-cell activation. For up to 36 hours after activation or anergy induction, cell surface expression of the T-cell antigen receptor(TcR)/CD3 complex is downregulated (Zanders et al., 1983). Insufficient antigen recognition due to low TcR expression alone is, however, not responsible for the inability of anergized T cells to respond to immunogenic challenge, since even when the TcR/CD3 level is restored to normal levels, the anergic T cells remain unresponsive to immunogenic challenge (J.R. Lamb and R.E. O’Hehir, unpublished). In common with activated T cells, anergized cells also express high levels of the IL2 receptor (O’Hehir and Lamb, 1990), and accordingly respond well to exogenously added IL2 (Lamb et al., 1983). This confirms that the anergized T cells retain the ability to proliferate to non-TcR-mediated signals, and rules out the potential explanation that anergy might be due to the toxicity of high concentrations of peptide antigen. Thus far, only CD28 appears to be differentially expressed on optimally activated T cells and hyperactivated, anergized T cells. Unlike optimally activated T cells, on which CD28 is strongly induced, hyperactivated anergized T cells express lower levels of CD28 (O’Hehir and Lamb, 1990). This is of particular interest with respect to anergy, as the natural ligand of CD28 thought to be a co-stimulatory molecule, B7/BB 1, is expressed predominantly by B cells (June et al., 1990). Anergy
or activation
and co-stimulation
The importance of co-stimulatory molecules in T-cell activation and anergy has been best demonstrated in a well-characterized in vitro model of anergy using murine T-cell clones (Mueller et al., 1989 ; Schwartz, 1990). These studies suggest that in Thl-type CD4+ T-cell clones, activation occurs when a T cell receives signals from both antigen and other APC-derived co-stimulatory molecules. Aner-
FROM
MODELS
TO REALITY
295
gy, in contrast, occurs when T cells interact with antigen alone in the absence of additional signals from co-stimulatory molecules. This form of anergy, unlike the human model described above, may be induced with concentrations of peptide antigen normally optimal for proliferative responses. Furthermore, murine T-cell anergy is immediate, with unresponsiveness being manifest during the induction phase. This differs from the human model, which requires an additional immunogenic challenge to become’evident. Although finally resulting in anergy, the induction phase of murine anergy does involve transient T-cell activation. In contrast to the human model, however, activation is arrested short of IL2 production and proliferation.
Involvement anergy
of co-stimulatioti
in human
T-cell
Although a variety of differences exist between the human and murine models of clonal T-cell anergy, it has been hypothesized that a lack of costimulation may also account for anergy due to hyperactivation of human T-cell clones (O’Hehir and Lamb, 1990). This hypothesis is suggested by the finding that surface expression of CD28, a costimulatory signal receptor, is downregulated on hyperactivated anergic human T-cell clones compared with CD28 expression on optimally activated T cells. Therefore, rather than involving a defect in the provision of co-stimulatory signals by APC, human T-cell anergy may involve a deficiency in the reception of co-stimulatory signals due to the downregulation of co-stimulatory signal receptors. This is supported by the correlation between anergy induction only at high antigen concentrations and the downregulation of co-stimulatory signal receptors under the same conditions. The hypothesis is also consistent with the proposed mode of action via CD28 (June et al., 1990). When ligated, CD28 appears to be involved in two signal transduction pathways, one of which is dependent upon extensive TcR cross-linking. In combination with TcR-derived signals, ligation of CD28 augments cytokine production. This is due to inhibtion of cytokine mRNA degradation leading to an accumulation of steady mRNA levels, increased levels of translation and protein secretion. We have obtained circumstantial evidence consistent with the mode of action of CD28 from the examination of cytokine mRNA and protein levels during the immunogenic rechallenge of anergic cells. Unlike during optimal activation and anergy induction, the immunogenic challenge of anergized cells does not induce IL2 mRNA in the anergized T cells (O’Hehir et al., 1991). It is, therefore, possible that under circumstances in which CD28 has been down-
296
42nd FORUM
IN IMikWNOLOGY MacDonald, H.R., Schneider, R., Lees, R.K., Howe, R.C., Acha, O.H., Festenstein, H., Zinkernagel, R.M. & Hengartner, H. (1988), T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature (Lond.), 332, 40-45. Mueller, D.L., Jenkins, M.K. & Schwartz, R.H. (1989), Clonal expansion versus functional clonal inactivation : a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy.
regulated by anergy induction, IL2 mRNA is no longer stabilized and is thus degraded; this has the final result of preventing IL2-mediated autocrine proliferation upon immunogenic challenge. An important aspect of this hypothesis is that despite the importance of APC-mediated co-stimulation, this form of anergy is primarily driven by the interaction of high concentrations of antigen with T cells.
Ann.
References Hewitt, C.R. & Feldmann, M. (1989), Human T cell clones present antigen. J. Immunol., 143, 762-769. June, C.H., Ledbetter, J.A., Linsley, P.S. & Thompson, C.B. (1990), Role of the CD28 receptor in T cell activation. Immunol. Today, 11, 211-216. Kappler, J.W., Staerz, U., White, J. & Marrack, P.C. (1988), Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex. Nature (Lond.), 332, 35-40. Lamb, J.R. Jr Feldmann, M. (1984), Essential requirement for major histocompatibility complex recognition in T-cell tolerance induction. Nature (Land.), 308,72-14. Lamb, J.R., Skidmore, B.J., Green, N., Chiller, J.M. & Feldmann, M. (1983), Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J. exp. Med.. 157, 1434-1447.
Tolerance to peripheral non-deletional
Rev.
Immunol.,
7, 445-480.
O’Hehir, R.E. &Lamb, J.R. (1990), Induction of specific clonal anergy in human T lymphocytes by Staphylococcus aureus enterotoxins. Proc. nut. Acad. Sci. (Wash.), 87, 8884-8888. O’Hehir, R.E., Yssel, H., Verma, S., de Vries, J., Spits, H. &Lamb, J.R. (1991), Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy. Int. Immunol., 3, 819-824. Rammensee, H.G., Kroschewski, R. & Frangoulis, B. (1989), Clonal anergy induced in mature V beta 6+ T lymphocytes on immunizing Mls-lb mice with Mls-1’ expressing cells. Nature (Lond.), 339, 541-544. Schwartz, R.H. (1990), A cell culture model for T lymphocyte clonal anergy. Science, 248, 1349-1356. Zanders, E.D., Lamb, J.R., Feldmann, M., Green, N. & Beverly, P.C.L. (1983), Tolerance of T cell clones is associated with membrane antigen changes. Nature (Lond.), 303, 625-627.
antigens must involve mechanisms
D. Lo Department
of Immunology
IMM-4,
The Scripps Research Institute, La Jolla, CA 92037 (USA)
Immunological tolerance to self has always been a topic of major interest to immunologists, but recent years have witnessed a considerable expansion in the number of new experimental tolerance models. As might be expected, different model systems have yielded diverse interpretations of their results. Although it would be natural to seek a unifying explanation for all self-tolerance, it is clear that a single mechanism cannot explain tolerance to all self
10446 North
Torrey Pines Road,
antigens, due to differences in location and amount of antigen. Therefore, in this discussion, we will discuss only T-cell tolerance to peripheral antigens, defined as antigens expressed only on non-lymphoid tissues and not secreted in large amounts into the bloodstream. Such antigens represent the putative targets for a large number of human autoimmune diseases, including type 1 diabetes and multiple sclerosis. We present here our arguments that tolerance to