Definition of a human suppressor T-cell epitope.

The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.

A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders.

It is now well established that cultured human melanocytes are capable of expressing immunologically important cell surface molecules and that they can produce cytokines. Not all cells with the ability to express MHC class II molecules are capable of effective Ag presentation. However, the dendritic nature of melanocytes, their strategic position within the skin, and their phagocytic capacity seem to suggest a role for these cells in processing and presenting Ag. This study demonstrates that cultured normal human skin melanocytes can present peptide Ag, and process and present the mycobacterial HSP65 kDa protein and whole Mycobacterium leprae sonicate to CD4+ cytotoxic proliferative T cell clones in an Ag-specific and HLA-class II-restricted manner. T cell stimulation was dependent on costimulatory signals, i.e., LFA-3/CD2 and LFA-1/ICAM-1. Besides eliciting a T cell proliferative response, our studies further demonstrate that melanocytes can function as target cells for T cell-mediated cytotoxicity. The described Ag-processing and -presenting functions of melanocytes, taken together with in vivo behavior of melanocytes in hypopigmentation, provide new clues for the etiopathogenesis of melanin pigmentary disorders.

Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: heterogeneity in cytotoxic activity and cytokine secretion levels.

Protective immunity against mycobacteria is dependent on antigen-specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium-specific CD4+ CTL are readily detectable among primary peripheral T cells but what proportion of CD4+ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4+ T cells are identical to or distinct from those that produce interferon (IFN)-gamma is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4+ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium-pulsed target cells and to release IFN-gamma and interleukin (IL)-4. Our results show that the vast majority of CD4+ T cell clones are able to lyse mycobacterial antigen-pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN-gamma, but low or nondetectable levels of IL-4. In contrast, control tetanus toxoid-specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL-4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN-gamma release. Interestingly one T cell clone was able to lyse only mycobacterium-pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4+ T helper type 1 (Th1)-like cells that display cytotoxic activity, and release high levels of IFN-gamma but no or little IL-4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL-4.

Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)

HLA class II+ human keratinocytes present Mycobacterium leprae antigens to CD4+ Th1-like cells.

In a variety of inflammatory skin diseases like leprosy, keratinocytes (KC) are induced to express MHC class II molecules and may therefore serve as antigen-presenting cells (APC) for MHC class II restricted T cells infiltrating the lesions. However, KC have been thought to be improper APC for MHC class II restricted T cells and to drive T cells into an anergic rather than into an activation state. We evaluated this issue in relation to leprosy and tested whether HLA-DR+ KC could present M. leprae antigens to well-defined, CD4+, cytotoxic as well as proliferative, Th1-like cell clones. Using a recently developed sensitive assay system which employs intact layers of basal KC as APC we found that most T-cell clones (6/8) lysed HLA-DR+ KC pulsed with M. leprae antigens. KC were only recognized after induction of HLA-DR expression by IFN-gamma, in an antigen-specific and HLA class II restricted manner. All T-cell clones tested also showed significant proliferation and IFN-gamma production in response to M. leprae antigens presented by HLA-DR+ KC, arguing against a KC dependent anergizing effect on T cells. Thus, HLA class II+ KC can function as proper APC for HLA class II restricted CD4+ Th 1-like cells. It seems therefore possible that antigen presentation by KC contributes to the local cell-mediated immune responses in DTH lesions.

Specific killing of cytotoxic T cells and antigen-presenting cells by CD4+ cytotoxic T cell clones. A novel potentially immunoregulatory T-T cell interaction in man.

Mycobacterial antigens not only stimulate Th cells that produce macrophage-activating factors, but also CD4+ and CD8+ CTL that lyse human macrophages. The mycobacterial recombinant 65-kD hsp was previously found to be an important target antigen for polyclonal CD4+ CTL. Because of the major role of 65-kD hsp in the immune response to mycobacterial as well as autoantigens, we have studied CTL activity to this protein at the clonal level. HLA-DR or HLA-DQ restricted, CD4+CD8- T cell clones that recognize different peptides of the M. leprae 65-kD hsp strongly lysed EBV-BLCL pulsed with specific but not irrelevant peptide. No bystander lysis of B cells, T cells, or tumor cells was seen. Target cell lysis could not be triggered by PMA + Ca2+ ionophore alone and depended on active metabolism. Interestingly, these CD4+ CTL also strongly lysed themselves and other HLA-class II compatible CD4+ (TCR-alpha/beta or -gamma/delta) or CD8+ CTL clones in the presence of peptide, suggesting that CTL are not actively protected from CTL-mediated lysis. Cold target competition experiments suggested that EBV-BLCL targets were more efficiently recognized than CD4+ CTL targets. These results demonstrate that hsp65 peptide-specific HLA class II-restricted CD4+ T cell clones display strong peptide-dependent cytolytic activity towards both APCs, and, unexpectedly, CD4+ and CD8+ CTL clones, including themselves. Since, in contrast to murine T cells human T cells express class II, CTL-mediated T cell killing may represent a novel immunoregulatory pathway in man.

A peptidoglycan protein complex purified from M. leprae cell walls contains most or all immunodominant M. leprae T-cell antigens.

The outcome of an infection with Mycobacterium leprae is correlated with the T-cell-mediated immune response developed against this pathogenic agent. The identification of M. leprae antigens that are recognized by T cells is therefore of great importance. In this paper we present the results of in vitro lymphoproliferation assays in which T-cell reactivity was measured against a peptidoglycan-protein complex (PPC) which was purified from the cell wall of M. leprae. Twelve M. leprae-reactive T-cell clones with different antigen specificities from a tuberculoid (TT) leprosy patient showed proliferative responses, but only when PPC was presented by HLA-DR-matched antigen-presenting cells (APCs). Four of these clones were known to react with the recombinant mycobacterial 65-kDa protein. A tetanus-toxoid-reactive T-cell line from a healthy control was not stimulated by this complex, supporting the idea that the stimulation by PPC was antigen specific. Both PPD-reactive and M. leprae-reactive T-cell lines from healthy individuals were stimulated by PPC. However, when this complex was presented to PPD-reactive T-cell lines derived from two lepromatous (LL) leprosy patients, we did not observe any proliferative responses. From these results we conclude that PPC contains most or all of the antigens which stimulate M. leprae-reactive T cells in association with relevant HLA class II molecules, including the 65-kDa protein or at least some immunogenic parts of it.