Immunoglobulin-like transcript 3 (ILT3) and ILT4 belong to a family of inhibitory receptors expressed by human monocytes and dendritic cells. We show here that CD8+CD28(-) alloantigen-specific T suppressor (TS) cells induce the up-regulation of ILT3 and ILT4 on monocytes and dendritic cells, rendering these antigen-presenting cells (APCs) tolerogenic. Tolerogenic APCs show reduced expression of costimulatory molecules and induce antigen-specific unresponsiveness in CD4+ T helper cells. Studies of human heart transplant recipients showed that rejection-free patients have circulating TS cells, which induce the up-regulation of ILT3 and ILT4 in donor APCs. These findings demonstrate an important mechanism of immune regulation.

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Endothelial cells have been shown to activate T cell responses to alloantigens, triggering transplant rejection. However, they may also play a role in tolerance induction. Using RT-PCR we show here that alloantigen specific CD8(+)CD28(-) T suppressor cells generated in vitro are FOXP3 positive and interact with human endothelial cells. This interaction results in the induction of inhibitory receptors and down-regulation of costimulatory and adhesion molecules, thus rendering endothelial cells tolerogenic. In turn, tolerized endothelial cells elicit the differentiation of CD8(+)CD28(-) FOXP3(+) T suppressor cells. Taken together our data demonstrate a functional and phenotypic overlap between tolerogenic dendritic cells and endothelial cells. Furthermore, alloantigen specific CD8(+)CD28(-) FOXP3(+) T cells, which trigger the upregulation of inhibitory receptors in endothelial cells, are present in the circulation of heart allograft recipients in quiescence as demonstrated by flow cytometry, RT-PCR and luciferase transcription assays. Their detection facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppressive therapy, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Modulation of endothelial cells in favor of promoting tolerance may be important for long-term survival of organ allografts.

The role of the indirect allorecognition pathway in acute allograft rejection has been documented both in organ recipients and in experimental models. However, it is unknown whether self-restricted recognition of donor alloantigens also contributes to chronic allograft rejection. The aim of this study was to determine the relationship between allopeptide reactivity, epitope spreading, and chronic rejection. Using synthetic peptides corresponding to the hypervariable region of 32 HLA-DR alleles, we have followed the specificity of self-restricted T cell alloresponses to the donor in a population of 34 heart allograft recipients. T cells from sequential samples of blood collected from the patients up to 36 mo after transplantation were studied in limiting dilution analysis for allopeptide reactivity. The incidence of coronary artery vasculopathy (CAV) was significantly higher in patients who displayed persistent alloreactivity late after transplantation than in patients who showed no alloreactivity after the first 6 mo after transplantation. Both intra- and intermolecular spreading of epitopes was observed with an increased frequency in patients developing CAV in less than 2 yr, compared with patients without CAV; this suggests that diversification of the immune response against the graft contributes to chronic rejection. These data provide a strategy for identifying patients at risk of developing CAV and a rationale for therapeutic intervention aimed to prevent the progression of the rejection process.

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (