Peptide-specific Allogeneic T-cell Response Induced And Modulated By Peptide/HLA-A2 Complex In Vitro

Alloreactive T cells recognize intact allogeneic MHC molecules expressed on foreign cells and mount vigorous responses as graft rejection in vivo. The role of the bound peptide in alloreactive T cell recognition is controversial, ranging from peptide-independent to peptide-specific. In this study, a single peptide epitope was present to induce alloresponse through mix lymphocyte culture in vitro and prove the pMHC specific character of alloreactive T cells. Meanwhile, novel approaches to induce or inhibit antigen specific allreactive T cells were exploited to aim for tumor tolerance or graft rejection.1. alloreactive T cell recognize allogeneic peptide/MHC complex (pMHC) in peptide dependent way.The aim of this part is to find the evidence that there exist pMHC-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the specific target cell (T2/Tyr) and a higher frequency of Tyr/HLA-A2 specific CD8+ T cell was detected by specific tetramer. Our results indicated the LTMLC was able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs.2. Peptide-specific allogeneic T cell response induced by monocytes-bound HLA-A2 dimerThe allogeneic stimulator cells present a different set of endogenous peptides, leading to potential stimulation of CTLs for the various allogeneic epitopes, which may mount graft-versus-host reaction. In this study, a soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr368-376) to form Tyr/HLA-A2 dimer, which allowed for exploiting autologous monocytes as single peptide epitope presenting cells to generate Tyr/HLA-A2 specific CTLs from HLA-A2 negative (HLA-A2-ve) donors. The studies described here demonstrate that allogeneic divalent peptide/HLA-A2 presented by autologous monocytes can stimulate and expand peptide-specific allo-restricted CTLs in vitro. It provides a novel approach to enrich peptide-specific donor lymphocytes for the treatment of patients with tumor following HLA-mismatched transplantation.3. Peptide-dependent inhibition of alloreactive T-cell response by soluble divalent HLA-A2/IgG molecule in vitroInduction of peripheral tolerance in an Ag-specific manner is currently an unrealized but critical goal of transplant biology. In this study, the soluble divalent HLA-A2/IgG molecule which allowed for specific targeting to Ag-specific alloreactive CTL was used as tolerogen to specific allo-restricted T cells. The Tyr/HLA-A2 dimer suppressed alloreactive T-cell responses by inhibiting its proliferation identified and cytotoxicity against specific target T2/Try in vitro, and it was interesting that the suppression was peptide-specific. The specific Tyr/HLA-A2 tetramer staining results suggested the impaired function of CD8+ T cell was due to deletion of specific alloreactive T cells in 3 subjects. Moreover, the existence of epitope-specific but functional-negative T cells in other two subjects indicated there maight exist other mechanism involved in silencing alloreactive responses by the HLA-A2 dimer. Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression, which may be useful in promoting graft survival.