Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells.

IL-10-producing CD4(+) type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T-cell responses mainly via cytokine-dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)-dependent mechanism that requires HLA class I recognition, CD54/lymphocyte function-associated antigen (LFA)-1 adhesion, and activation via killer cell Ig-like receptors (KIRs) and CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity. We also showed that high frequency of GZB-expressing CD4(+) T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4(+) T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.

Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy.

BACKGROUND: CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. DESIGN AND METHODS: The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. RESULTS: Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-ß independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. CONCLUSIONS: Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.

Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia.

BACKGROUND: Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood. DESIGN AND METHODS: The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor. RESULTS: Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro. CONCLUSIONS: Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.