The role of the hematopoietic lineage-restricted minor histocompatibility (H) antigen HA-1 in renal allograft tolerance was explored. We obtained peripheral blood samples from three recipients of histocompatibility leukocyte antigen (HLA)–matched, HA-1–mismatched renal transplants, one of which had discontinued immunosuppression >30 yr ago while sustaining normal kidney function. Peripheral blood mononuclear cells (PBMCs) were injected into the footpads of severe combined immunodeficiency mice to measure human delayed type hypersensitivity (DTH) responses. All three patients manifested regulated DTH responses to HA-1H peptide. By differential tetramer staining intensities, we observed two distinct minor H antigen HA-1–specific CD8+ T cell subsets. The one that stained dimly had the characteristics of a T regulatory (TR) cell and produced interleukin (IL) 10 and/or transforming growth factor (TGF) β. These HA-1–specific TR cells coexisted with bright tetramer-binding CD8+ T effector (TE) cells. The CD8+ TE cells mediated HA-1–specific DTH and produced interferon-γ. Suppression of these TE functions by TR cells was TGFβ, IL-10, and cytotoxic T lymphocyte–associated antigen 4 dependent. In addition, HA-1 microchimerism was detected in two recipients, primarily in the dendritic cell fraction of the PBMCs. This is the first demonstration of coexisting CD8+ memory TR and TE cells, both specific for the same HA-1 antigen, in the context of renal allograft tolerance.

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One hallmark of acquired tolerance is bystander suppression, a process whereby Ag-specific (adaptive) T regulatory cells (TR) inhibit the T effector cell response both to specific Ag and to a colocalized third-party Ag. Using peripheral blood T cells from recipients of HLA-identical kidney transplants as responders in the trans vivo-delayed type hypersensitivity assay, we found that dendritic cells (DC), but not monocyte APCs, could mediate bystander suppression of EBV-specific recall response. When HA-1H peptide was added to mixtures of plasmacytoid DC (pDC) and T cells, bystander suppression of the response to a colocalized recall Ag occurred primarily via indolamine-2,3-dioxygenase (IDO) production. Similarly, addition of HA-1H peptide to cocultures of T cells and pDC, but not myeloid DC (mDC), induced IDO activity in vitro. When mDC presented HA-1H peptide to Ag-specific CD8+ TR, cytokine release (TGF-β, IL-10, or both) was the primary mode of bystander suppression. Bystander suppression via mDC was reversed not only by Ab to TGF-β and its receptor on T cells, but also by Ab to thrombospondin-1. EBV addition did not induce IDO or thrombospondin-1 in T-DC cocultures, suggesting that these DC products are not induced by T effector cells, but only by TR cells. These results shed light upon the mechanism of bystander suppression by donor Ag-specific TR in patients with organ transplant tolerance and underscores the distinct and critical roles of mDC and pDCs in this phenomenon.

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