Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.

Alloantigen-specific type 1 regulatory T cells suppress through CTLA-4 and PD-1 pathways and persist long-term in patients.

Type 1 regulatory T (Tr1) cells are inducible, interleukin (IL)-10+FOXP3− regulatory T cells that can suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have optimized an in vitro protocol to generate a Tr1-enriched cell product called T-allo10, which is undergoing clinical evaluation in patients with hematological malignancies receiving a human leukocyte antigen (HLA)­mismatched allo-HSCT. Donor-derived T-allo10 cells are specific for host alloantigens, are anergic, and mediate alloantigen-specific suppression. In this study, we determined the mechanism of action of T-allo10 cells and evaluated survival of adoptively transferred Tr1 cells in patients. We showed that Tr1 cells, in contrast to the non-Tr1 population, displayed a restricted T cell receptor (TCR) repertoire, indicating alloantigen-induced clonal expansion. Tr1 cells also had a distinct transcriptome, including high expression of cytotoxic T lymphocyte­associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Blockade of CTLA-4 or PD-1/PD-L1 abrogated T-allo10­mediated suppression, confirming that these proteins, in addition to IL-10, play key roles in Tr1-suppressive function and that Tr1 cells represent the active component of the T-allo10 product. Furthermore, T-allo10­derived Tr1 cells were detectable in the peripheral blood of HSCT patients up to 1 year after T-allo10 transfer. Collectively, we revealed a distinct molecular phenotype, mechanisms of action, and in vivo persistence of alloantigen-specific Tr1 cells. These results further characterize Tr1 cell biology and provide essential knowledge for the design and tracking of Tr1-based cell therapies.