Ex Vivo Isolation and Characterization of Cd4+Cd25+ T Cells with Regulatory Properties from Human Blood

Abstract

It has been known for years that rodents harbor a unique population of CD4+CD25+ “professional” regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4+CD25+CD45RO+ T cells (mean 6% of CD4+ T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4+CD25+ T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte–associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4+CD25+ T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4+CD25+ T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4+ and CD8+ T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4+CD25+ T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

“…As T reg have a memory phenotype [8], we additionally studied intracellular levels of T cell receptor excision circles (TREC) in purified T reg derived from 10 MS patients and 10 normal controls. TREC are a marker for recent thymic emigrants and are influenced by both the thymic-dependent T cell generation and the degree of peripheral T cell proliferation [29].…”

“…Within the human CD4 + subset only cells coexpressing high levels of CD25 appear to have potent regulatory function [6,7]. CD4 + CD25 high regulatory T cells (T reg ) are anergic to antigenic stimulation and actively downregulate activation and expansion of conventional T lymphocytes in a cell-contact-dependent and cytokine-independent manner [5, [8][9][10][11][12][13]. Although the molecular interactions and signaling pathways that are critical for their generation and function are nut fully elucidated, T reg require forkhead transcription factor scurfin encoded by the Foxp3 gene, which appears to control their development and regulatory properties [14][15][16].…”

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“…Human CD25 + cells respond to TCR stimulation combined with IL-2, IL-4, and IL-15 although, even under these conditions, their proliferative responses do not approach those of similarly stimulated CD25 − T cells. CD25 + T cells were capable of suppressing the responses of CD25 − cells to stimulation with anti-CD3 or alloantigen 14 15 16 presented by mDCs. Once activated, their suppressor function is nonspecific as was shown in studies of murine CD25 + T cells 7.…”

“…Three papers in this issue 14 15 16 and three papers 17 18 19 appearing elsewhere now describe the properties of human CD4 + CD25 + T cells. Given the obsession of most cellular immunologists with the concept of separating functionally distinct populations of cells based on their differential expression of membrane antigens, it is surprising that 6 yr elapsed between the identification of these cells in the mouse and the confirmation of their existence in man.…”

“…We have been unable to confirm these findings and have recently shown that induction of CD25 + -mediated suppressor function appears to be independent of costimulatory signals mediated by the interaction of either CD28 or CTLA-4 with their ligands, CD80/CD86 (unpublished observations). All of the human studies 14 15 16 17 18 19 fail to demonstrate reversal of suppression when anti–CTLA-4 or its F(ab′) fragment 19 is added. Nevertheless, the in vivo studies of both Takahashi et al 20 and Read et al 21 strongly implicate a role for CTLA-4 at some stage in the induction of CD25 + -mediated suppressor function during the pathogenesis of autoimmunity.…”