O261* Aims: In previous studies we have demonstrated that allospecific CD8+CD28- FOXP3+ T suppressor (TS) and CD4+CD25+ FOXP3 T regulatory cells (TR) can be generated in vitro by multiple stimulation of human T cells with allogeneic APC. We have now explored the in vivo relevance of TS and TR in 30 recipients of heart allografts. Methods: CD8+CD28- T cells from fresh peripheral blood were obtained using CD8 isolation kit II (Miltenyi Biotech, Auburn, CA) followed by depletion of CD28+ T cells using goat anti-mouse IgD beads coupled with mAb to CD28 (Becton Dickinson). CD4+CD45RO+CD25+ T cells were obtained by depletion of purified CD4+ T cells of CD45RA+ cells followed by selection of CD25+ T cells with magnetic microbeads (Miltenyi). TS and TR were incubated with donor or control APC for 18 hours. The phenotype of TS, TR and of APC was studied by flow cytometry, RT-PCR and luciferase transcription assays. Anti-HLA class I and class II antibodies were monitored in all patients. Results: Serial studies of the phenotype displayed by T cells from transplant recipients demonstrated a progressive increase in the size of the CD8+CD28-CD27+ PRF- FOXP3+ T cell population in rejection-free patients. CD8+CD28- FOXP3+ T cells from these recipients inhibited up-regulation of CD80 and CD86 on CD40-ligated APC from the donor. This inhibitory effect was restricted to the MHC class I antigens expressed by the transplant donor. Patients’ TS also induced the upregulation of the inhibitory receptors ILT3 and ILT4 on donor APC as shown by flow cytometry as well as by trancription assays in which APC transfected with luciferase gene plasmids containing the ILT3 or ILT4 promoter were used. CD8+CD28- FOXP3+ T cells from patients in quiescence induced ILT3 and ILT4 transcription in APC sharing HLA class I antigens with the transplant donor but not in irrelevant control APC. Serial determination of the suppressor activity displayed by patient’s CD4+CD25+ FOXP3+ T cells yielded similar results i.e. they induced transcriptional activation of ILT3 and ILT4 in APC matched to the donor for HLA-DRβ1 antigens. However, while TS could be detected as early as one month post-transplantation CD4+CD25+ TR became detectable at later times (12 months or more). TS and TR were found in 21 out of 30 patients studied. Of the remaining 9 patients who showed no regulatory T cells 7 had a history of early acute rejection episodes while two remained in quiescence. Three of these 7 patients with early rejections showed evidence of CAV two years following transplantation. Taken together our data indicate that the presence of TS and TR in patients’ circulation is negatively associated with acute or chronic rejection and with the development of anti-HLA antibodies (p<0.0001). Conclusions: Serial determination of allospecific TS and TR demonstrated that quiescence is associated with the expansion of regulatory T cell populations. Study of regulatory T cells permits the identification of patients who can benefit from partial withdrawal of immunosuppressive drugs.