Two subsets of natural and adaptive regulatory T (T reg) cells have been described, but the identity of adaptive type 1 regulatory (Tr1)–like cells in humans is unclear. We analyzed a subset of human blood CD4+ T cells—CD45RA−CD25−interleukin (IL)-7 receptor (R)− cells—that rapidly secreted high levels of IL-10 together with interferon γ, but produced little IL-2. These IL-7R− T cells were rare, anergic, and largely Foxp3−. They expressed low levels of Bcl-2 but high levels of Ki-67 and ICOS, suggesting that they have been recently activated in vivo. Consistently, they responded selectively to persistent foreign and self-antigens under steady-state conditions. Unlike natural CD25+ T reg cells, IL-7R− cells suppressed naive and memory T cell proliferation in an IL-10–dependent fashion, and they required strong T cell receptor stimulation for suppression. To our knowledge, this is the first report that identifies Tr1-like cells in human blood. These IL-10–secreting cells have characteristics of chronically activated Th1 effector cells and are distinct from CD25+ T reg cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 + T-cell subsets, including conventional cytotoxic CD4 + T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 + T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes + GzmK + T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 + Eomes + T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes + Tr1-like cells are effector cells of a unique GzmK-expressing CD4 + T-cell subset.

Interleukin (IL)-10 produced by regulatory T cell subsets is important for the prevention of autoimmunity and immunopathology, but little is known about the phenotype and function of IL-10–producing memory T cells. Human CD4+CCR6+ memory T cells contained comparable numbers of IL-17– and IL-10–producing cells, and CCR6 was induced under both Th17-promoting conditions and upon tolerogenic T cell priming with transforming growth factor (TGF)–β. In normal human spleens, the majority of CCR6+ memory T cells were in the close vicinity of CCR6+ myeloid dendritic cells (mDCs), and strikingly, some of them were secreting IL-10 in situ. Furthermore, CCR6+ memory T cells produced suppressive IL-10 but not IL-2 upon stimulation with autologous immature mDCs ex vivo, and secreted IL-10 efficiently in response to suboptimal T cell receptor (TCR) stimulation with anti-CD3 antibodies. However, optimal TCR stimulation of CCR6+ T cells induced expression of IL-2, interferon-γ, CCL20, and CD40L, and autoreactive CCR6+ T cell lines responded to various recall antigens. Notably, we isolated autoreactive CCR6+ T cell clones with context-dependent behavior that produced IL-10 with autologous mDCs alone, but that secreted IL-2 and proliferated upon stimulation with tetanus toxoid. We propose the novel concept that a population of memory T cells, which is fully equipped to participate in secondary immune responses upon recognition of a relevant recall antigen, contributes to the maintenance of tolerance under steady-state conditions.