SUMMARY Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential non-redundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

CD4+ CD25+ Foxp3+ regulatory T (Treg) cells are essential to the balance between pro- and anti-inflammatory responses. There are two major subsets of Treg cells, “natural” Treg (nTreg) cells that develop in the thymus, and “induced” Treg (iTreg) cells that arise in the periphery from CD4+ Foxp3− conventional T cells and can be generated in vitro. Previous work has established that both subsets are required for immunological tolerance. Additionally, in vitro-derived iTreg cells can reestablish tolerance in situations where Treg cells are decreased or defective. This review will focus on iTreg cells, drawing comparisons to nTreg cells when possible. We discuss the molecular mechanisms of iTreg cell induction, both in vivo and in vitro, review the Foxp3-dependent and -independent transcriptional landscape of iTreg cells, and examine the proposed suppressive mechanisms utilized by each Treg cell subset. We also compare the T cell receptor repertoire of the Treg cell subsets, discuss inflammatory conditions where iTreg cells are generated or have been used for treatment, and address the issue of iTreg cell stability.

“Natural” regulatory T (nTreg) cells that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” Treg (iTreg) cells are non-redundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg cell-produced IL-10 and iTreg cell fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naïve CD4+ T cells carrying a non-functional Foxp3 allele. At the onset of weight loss, mice were treated with both iTreg and nTreg cells where one marked subset was selectively IL-10-deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTreg cells were present but IL-10 deficient, iTreg cell-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTreg and ex-iTreg cells, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between Treg subsets that maintains mucosal tolerance. The niche supporting stable iTreg cells is limited and readily saturated, which promotes a large population of ex-iTreg cells with pathogenic potential during immunotherapy.

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1 , OAS2 , or RNASEL in five unrelated children with MIS-C. The cytosolic dsRNA-sensing OAS1 and OAS2 generate 2′-5′-linked oligoadenylates (2-5A) that activate the ssRNA-degrading RNase L. Monocytic cell lines and primary myeloid cells with OAS1 , OAS2 , or RNASEL deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or SARS-CoV-2 stimulation. Exogenous 2-5A suppresses cytokine production in OAS1- but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by MAVS deficiency. Recessive OAS–RNase L deficiencies in these patients unleash the production of SARS-CoV-2–triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.

Summary Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.