Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8 + T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C.

Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14 + CD16 + CD141 + CD163 + cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4 + T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b + LAG-3 + Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.

ABSTRACTand CD8 low suppressor T cells, 25 interleukin-10 (IL-10)-producing T regulatory type 1 (Tr1) cells, DC-10 are a subset of human tolerogenic DC that are present in vivo [27][28][29] and can be differentiated in vitro by culturing monocytes in the presence of IL-10. DC-10 secrete IL-10, are CD11c + , express CD14, CD16, HLA-G and ILT4 and, although not activated, display a mature phenotype, being CD86+ and HLA-DR + . The secretion of IL-10 and the expression of membrane-bound HLA-G and ILT4 are critical factors involved in DC-10-mediated induction of Tr1 cells. 27 In the present study, we investigated the role of HLA-G in DC-10-mediated Tr1 cell induction and whether polymorphisms present at the 3'UTR of the gene influence the expression of membrane-bound HLA-G on DC-10. MethodsThe methods are described in full in the Online Supplementary Appendix. Peripheral blood was obtained after informed consent in accordance with the Declaration of Helsinki under protocols approved by the ethical committee of the San Raffaele Telethon Institute for Gene Therapy. Dendritic cell differentiation CD14+ monocytes were isolated from peripheral blood mononuclear cells by positive selection using CD14 MicroBeads (Miltenyi Biotech, Germany) according to the manufacturer's instructions. Cells were cultured in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS) (Lonza, Italy) or with 5% human serum (HS) (EuroClone, Italy), 100 U/mL penicillin/streptomycin (Lonza, Italy), 2 mM L-glutamine (Lonza, Italy), (DC medium) at 37°C in the presence of 10 ng/mL recombinant human (rh)IL-4 (R&D Systems, Minneapolis MN, USA) and 100 ng/mL rhGM-CSF (Genzyme, Seattle, WA, USA) with 10 ng/mL of rhIL-10 (BD, Bioscience, CA, USA) for 7 days to differentiate DC-10. Cells cultured with rhIL-4 and rhGM-CSF on day 5 were matured with 1 mg/mL of lipopolysaccharide (Sigma, CA, USA) for 2 more days to generate mature dendritic cells (mDC). At day 7, DC were collected, phenotypically analyzed, and used to stimulate T cells. Statistical analysisSample mean results were compared using the non-parametric Mann-Whitney U test for continuous variables. HLA-G 3'UTR allele and genotype frequencies were obtained by direct counting. Allele and genotype frequencies between populations were compared using the χ 2 test. The correlation between membranebound HLA-G and ILT4 expression was determined using the Spearman correlation test. FBS and DC-10 HS were compared using a paired t-test. All results are presented as mean values ± standard error of mean (SEM). Differences were regarded as statistically significant at *P<0.05, **P<0.01, and ***P<0.001. The results were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA). Results In vitro differentiated DC-10 express variable levels of membrane-bound HLA-GIndependently of the donor, DC-10 differentiated in vitro as described in the Methods section were Figure 1A,B). High variability in the expression of membrane-bound HLA-G (ranging from 3.5% to 97.7%) and of ILT4 (rangin...

The prominent role of tolerogenic dendritic cells (tolDCs) in promoting immune tolerance and the development of efficient methods to generate clinical grade products allow the application of tolDCs as cell-based approach to dampen antigen (Ag)-specific T cell responses in autoimmunity and transplantation. Interleukin (IL)-10 potently modulates the differentiation and functions of myeloid cells. Our group contributed to the identification of IL-10 as key factor in inducing a subset of human tolDCs, named dendritic cell (DC)-10, endowed with the ability to spontaneously release IL-10 and induce Ag-specific T regulatory type 1 (Tr1) cells. We will provide an overview on the role of IL-10 in modulating myeloid cells and in promoting DC-10. Moreover, we will discuss the clinical application of DC-10 as inducers of Ag-specific Tr1 cells for tailoring Tr1-based cell therapy, and as cell product for promoting and restoring tolerance in T-cell-mediated diseases.