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In summary, synthetic peptides corresponding to linear sequences of HLA class I molecules can inhibit T-cell responses in vitro and in vivo. These peptides induce immunologic tolerance by binding to hsp-70 family members, causing an increase in intracellular calcium, and down-regulating the nuclear factor of activated T cells, NF-AT. We suggest that heat shock proteins may function as novel immunophilins (Fig 2). Like cyclophilins and FK 506 binding proteins, heat shock proteins are ubiquitous, are involved in protein folding and trafficking, and bind exogenous drugs. Cyclosporine and FK 506 exert immunosuppressive effects by binding immunophilins, which as a result interrupt the phosphatase activity of calcineurin. Although the precise pathways involved in the synthetic HLA peptide effects are not as well worked out, it seems likely that peptide binding to heat shock protein is disrupting normal events in T-cell activation, giving rise to an apparently permanent state of anergy.

Soluble forms of HLA may be natural immunoregulatory molecules and may explain the "transfusion effect" seen in transplant patients in the precyclosporine era. Synthetic peptides corresponding to short linear sequences of HLA molecules have inhibitory effects on human T lymphocytes in vitro and cause tolerance induction in a rat heterotopic heart transplant model. Recent studies indicate that a subset of these peptides causes an increase in intracellular calcium which appears to account for T cell unresponsiveness and tolerance induction. The peptides bind to members of the heat-shock protein 70 family in a specific manner, depending upon peptide sequence. These studies provide a mechanism to explain the biologic activities of these new immunotherapeutic reagents.

The identification of immunomodulatory approaches that allow the induction of antigen-specific unresponsiveness is required for long-term graft survival without the complications of chronic immunosuppression. Recent novel strategies based upon treatment with synthetic peptides corresponding to linear sequences of MHC class I and II molecules reproducibly induce tolerance to alloantigens. Although the mechanisms involved are still not completely understood, the phenomenology reported makes these approaches promising for evaluation in clinical trials.

CD4 and CD8 are cell surface glycoproteins that serve as co-receptors for Ag with the TCR. Recent studies have shown that both CD4 and CD8 interact with conserved regions of MHC class II and class I, respectively. To investigate further the roles of CD4 and CD8 in the immune response, we prepared synthetic peptides corresponding to the HLA sequences with which CD4 and CD8 are thought to interact. The peptide corresponding to residues 222 to 235 of the HLA class I heavy chain blocked the differentiation of human CTL precursors into active effect cells but affected neither the ability of PBLs to proliferate in response to mitogen nor the cytotoxic activity of established CTLs. In contrast, the peptide corresponding to residues 134 to 152 of the HLA-DR beta-chain inhibited the differentiation of CTL precursors, the proliferative response of freshly isolated PBL, and the proliferation of an established alloreactive CD4+ T cell clone to Ag. The inhibitory effect of the DR.134-152 peptide on CTL differentiation could be overcome by addition of exogenous IL-2 to the limiting dilution cultures, whereas the effect of the HLA-1.222-235 peptide was unaffected by exogenous IL-2. These results directly demonstrate a functional role for these regions of MHC molecules and underscore the central role of both CD4 and CD8 in the effective initiation of a CTL response.

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.

A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis.

The Daudi cell line is a B-lymphoblastoid line derived from a Burkitt lymphoma. Daudi cells lack cell surface expression of class I HLA molecules despite the presence of intracellular class I heavy chains. They have a defect in the gene encoding beta 2-microglobulin (beta 2m), resulting in lack of translatable mRNA for this protein. It has been thought that this deficiency is responsible for the lack of cell surface class I expression. However, data have recently been presented demonstrating that at least one mouse class I heavy chain can be expressed on the cell surface in the absence of beta 2m. These results raised the questions of whether the lack of beta 2m is the only defect in Daudi and whether transfer of this single gene could restore surface class I expression. We found that transfection of the mouse beta 2m gene into Daudi indeed rescued cell surface expression of class I HLA molecules, and that these molecules could be recognized both by monomorphic and allospecific mAbs. CTL clones specific for HLA-B17 or a determinant shared by HLA-B17 and HLA-A2 killed the Daudi cells transfected with the beta 2m gene, but not untransfected Daudi or Daudi transfected with vector alone. Mouse beta 2m on the transfected Daudi cells could exchange with human beta 2m when the cells were incubated in human serum. This exchange did not alter the ability of the cells to be killed by the specific CTLs. These results demonstrate that the lack of beta 2m is the sole reason for lack of surface class I molecules in Daudi cells, and that beta 2m is required for cell surface expression of the specific class I heavy chains of Daudi.

The major histocompatibility complex class I HLA molecules are the primary determinants recognized by allogeneic cytotoxic T lymphocytes (CTL), and serve as restricting elements for CTL recognition of viral, chemical, or minor histocompatibility antigens. HLA-Aw69 is a naturally occurring hybrid class I molecule that we have used to investigate the regions of class I antigens involved in human CTL recognition. HLA-Aw69 appears to have resulted from an exon shuffle between two closely related class I genes: the alpha 1 domain of HLA-Aw69 is identical to that of HLA-Aw68, while the alpha 2 and alpha 3 domains are identical to HLA-A2. The determinants recognized by human allogeneic CTL clones specific for HLA-A2, -Aw68, and/or -Aw69 fall into three patterns: (a) CTL determinants are located on both the alpha 1 and alpha 2 domains; (b) interaction of the alpha 1 and alpha 2 domains results in new combinatorial determinants; (c) interaction of the alpha 1 and alpha 2 domains in the hybrid molecule results in the loss of CTL determinants that are present on both parental molecules. Thus, using human CTL clones, target cells, and HLA molecules, we show that the interaction of the alpha 1 and alpha 2 domains alters CTL determinants in ways not directly predictable from primary structure.