Human CD4+CD25+ regulatory T Cells Exhibit Dual Mechanisms of Action in Suppressing in Vitro Alloreactivity

There is extensive evidence from murine models supporting a role for CD4⁺CD25⁺ regulatory T cells in suppressing alloreactivity. However, clinical evidence regarding the role of regulatory T cells in human alloresponses is conflicting and may reflect the difficulty in defining and isolating naturally occurring human CD4⁺CD25⁺ regulatory T cells. Moreover, the mechanism of action of Foxp3⁺ regulatory T cells remains somewhat controversial with a number of proposed modes of suppression described, including cell contact-dependent inhibition, cytokine mediated suppression and cytokine consumption. We have previously used peripheral blood monocyte-derived dendritic cells (DCs) as antigen presenting cells (APCs) in allogeneic mixed lymphocyte reactions (MDCLRs), to assess the in vitro suppressive function of human CD4⁺CD25⁺ T cells. Healthy volunteer donor MACS isolated CD4⁺CD25⁺ regulatory T cells demonstrated anergy and significant dose-dependent suppression of responder cell proliferation, to physiological frequencies, in allogeneic MDCLRs (p<0.005). In this study we investigated the in vitro mechanism of action of human CD4⁺CD25⁺ T cells in suppressing alloreactivity in MDCLRs. Co-culture of CD4⁺CD25⁺ regulatory T cells with autologous CD4⁺ responder cells in allogeneic MDCLRs, at a ratio of 1:4, resulted in suppression of proliferation by 68%. Peak suppression of CD4⁺ responder cell proliferation, observed on day five of co-culture, was accompanied by significant suppression of co-culture supernatant IL-5 concentration (p<0.05), and preceded by inhibition of IL-2. Intracellular cytokine staining confirmed that CD4⁺ responder cell intracellular IL-2 was reduced by 60% on co-culturing with CD4⁺CD25⁺ regulatory T cells in the allogeneic MDCLR. CD4⁺CD25⁺ regulatory T cells mediated suppression of proliferation and cytokine responses across transwell membranes, demonstrating a cell contact-independent mechanism and a potential soluble factor in the mode of action of CD4⁺CD25⁺ regulatory T cells in suppressing human alloresponses. This was confirmed by suppression of responder cell proliferation and cytokine responses by supernatant transfer from DCs co-cultured with allogeneic CD4⁺CD25⁺ regulatory cells. We surveyed possible candidate molecules responsible for mediating the cell contact-independent suppression; in our hands, neither IL-10 nor TGF-β was identified as the soluble factor. Next, we examined the effect of CD4⁺CD25⁺ regulatory T cells on APCs in the allogeneic MDCLR. Immunophenotypic characterization of DCs recovered from MDCLRs in the presence of CD4⁺CD25⁺ regulatory T cells showed down-regulation of HLA-DR, CD83, and co-stimulatory molecules, CD80 and CD86, compared with DCs cultured with CD4⁺CD25⁻ T cells or cytokines, IL-4 and GM-CSF, alone. The suppression of DC activation by CD4⁺CD25⁺ regulatory T cells was not mediated across transwell membranes, demonstrating a cell contact-requirement. Moreover, downregulation of DC activation markers was not accompanied by IL-12 suppression, implying no role for IL-10 in mediating the suppressive effect of the regulatory cells on the APCs. Our results demonstrate both a cell contact-independent mode of action in suppressing CD4⁺ responder cell proliferation, Th1 and Th2 cytokine responses, independent of IL-10 and TGF-β, and a cell contact-requirement in the suppression of allogeneic DC maturation and activation. In summary, our findings suggest that multiple mechanisms of action contribute to the in vitro suppressive effects of human CD4⁺CD25⁺ regulatory T cells on alloreactivity.