Thymus-derived, natural CD4+CD25+ regulatory T cells can educate peripheral CD4+CD25− cells to develop suppressive activity by poorly understood mechanisms. TGF-β has IL-2-dependent costimulatory effects on alloactivated naive, human CD4+ T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4+CD25+ cells are the targets of the costimulatory effects of IL-2 and TGF-β. These cells do not divide, but, instead, greatly increase the numbers of CD4+CD25− cells that become CD25+ cytokine-independent suppressor cells. These CD4+CD25+ regulatory cells, in turn, induce other alloactivated CD4+CD25− cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-β and IL-10. The suppressive effects of these secondary CD4+CD25+ cells depend upon TGF-β and IL-10. Moreover, both the naive CD4+ cells induced by IL-2 and TGF-β to become suppressor cells, and the subsequent CD4+CD25− cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4+CD25+ regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4+ regulatory T cells in vivo.

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-β could not induce CD4+CD25− cells from CTLA-4−/− mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-β to induce FoxP3-expressing mouse suppressor cells. TGF-β accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-β to induce CD4+CD25− cells to express FoxP3 and develop suppressor activity. TGF-β also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4−/− mice, the lack of TGF-β-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 × C57BL/6) F1 mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-β induced both CD4+ and CD8+ cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-β-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-β-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.

Certain CD4+CD25+ T cells can induce and maintain T-cell non-responsiveness to donor alloantigens and have therapeutic potential in solid organ transplantation. Peripheral CD4+CD25- cells alloactivated with IL-2 and transforming growth factor beta (TGF-beta) ex vivo express the transcription factor FoxP3, and become potent antigen-specific CD4+CD25- suppressor cells. Here we report that the transfer of TGF-beta-induced regulatory CD4+ and CD8+ T cells (Tregs) co-incident with transplantation of a histoincompatible heart resulted in extended allograft survival. To account for this result, we injected non-transplanted mice with a single dose of CD4+ and CD8+ Tregs and transferred donor cells every 2 weeks to mimic the continuous stimulation of a transplant. We observed increased splenic CD4+CD25+ cells that were of recipient origin. These cells rendered the animals non-responsive to donor alloantigens by an antigen-specific and cytokine-dependent mechanism of action. Both the increased number of CD4+CD25+ cells and their tolerogenic effect were dependent on continued donor antigen boosting. Thus, Tregs generated ex vivo can act like a vaccine that generates host suppressor cells with the potential to protect MHC-mismatched organ grafts from rejection.

In addition to CD4+CD25+Foxp3+ Tregs, natural and induced CD8+ Tregs have been described. Here we report our progress in generating human CD8+ Tregs ex-vivo. Naïve CD8+ cells were activated with anti-CD3/28 beads with IL-2 ± TGF-β for 1 to 9 days. As with CD4+ cells, TGF-β markedly enhanced expression of Foxp3. TGF-β also enhanced CD122, CD103, and PD-1, but decreased granzymes, HLA-DR, CD80 and CD86 expression. Sustained Foxp3 expression and suppressive function was IL-2 dependent. Secondarily stimulated CD8+ cells proliferated weakly, and suppressed activation, proliferation and cytokine production by CD4+ cells in vitro. In vivo functional activity was assessed in NOD SCID IL-2R cγ chain-/- mice injected with allogeneic human PBMC. While human CD4+Foxp3+ Tregs doubled survival from GVHD, CD8 Regs, remarkably, quadrupled survival. Only previously activated CD8+ cells had suppressive activity and protection was unrelated to Foxp3 expression. Adding retinoic acid or rapamycin to TGF-β did not improve survival. By contrast, these activated CD8+ cells rapidly expressed TNFR2 and PDL-1 within one day, and at all time points studied only cells expressing these markers had suppressive activity. Suppressive activity in vivo was IL-10 dependent and partially TGF-β dependent. Thus, in agreement with functional CD8regs described in lupus, diabetes and multiple sclerosis, protective CD8 regs can be induced ex-vivo, and the therapeutic potential of TNFR2, PDL-1+ cells should be carefully explored.