Requirement of cognate CD4 + T-cell recognition for the regulation of allospecific ctl by human CD4 +CD127 -CD25 +FOXP3 + cells generated in MLR

Abstract

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4 + Tregs on CD8 + cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4 +CD127 -CD25 +FOXP3 + Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified 51Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4 +CD127 -CD25 +FOXP3 + cells were generated after a 7 day MLR. After immunoselection for CD4 +CD127 -CD25 + cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8 + responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8 + responders or even with CD8 + responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4 + T cells were added to the CD8 + responders. Proliferation of CD8 + cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8 + cells. Therefore, it was concluded that human CD4 +CD127 -CD25 +FOXP3 + MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4 + T cells. CD8 + CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25 + activation epitopes. © 2011 Yu et al.