Human CD4⁺CD25^low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance

Adaptive T regulatory (T_R) cells mediate the suppression of donor-specific, delayed-type hypersensitivity (DTH) in tolerant organ transplant recipients. We hypothesized that cells belonging to the CD4 ⁺CD25⁺ T cell subset but distinct from natural T _R cells may fulfill this role. To test this hypothesis, PBMC and biopsy samples from two tolerant kidney transplant recipients (K1 and K2) were analyzed. When transferred with recipient APC into a SCID mouse footpad, CD4⁺ T cells were hyporesponsive in DTH to donor type HLA-B Ags and derivative allopeptides. However, anti-human TGF-β1 Ab revealed a response to immunodominant allopeptides in both patients, suggesting that CD4⁺ T elector (T_E) cells coexisted with suppressive, TGF-β1-producing CD4⁺ T_R cells. During in vitro culture, allopeptide stimulation induced both IFN-γ-producing and surface TGF-β1⁺ T cells. The relative strength of the latter response in patient K1 was inversely correlated with the level of systemic anti-donor DTH, which varied over a 6-year interval. Allopeptide-induced surface TGF-β1 expression was found primarily in Forkhead box P3 (FoxP3)-negative CD4 ⁺CD25^low T cells, which could adoptively transfer suppression of donor-specific DTH. Biopsy samples contained numerous surface TGF-β1⁺ mononuclear cells that costained for CD4 and, less frequently CD25, but were negative for FoxP3. The CD4⁺TGF- β1⁺ T cells were localized primarily to the tubulointerstitium, whereas TGF-β1^-FoxP3⁺CD25⁺ cells were found mainly in lymphoid aggregates. Thus, adaptive T_R cells suppressing T_E cell responses to donor allopeptides in two tolerant patients appear to be functionally and phenotypically distinct from CD4 ⁺CD25^highFoxP3⁺ T cells.