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T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4

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., Graphical abstract, Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening and unpredictable condition of unknown etiology. Ramaswamy et al. use peripheral blood single-cell transcriptomic profiling along with other techniques to define key innate and adaptive signatures that characterize 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.

SummaryA dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5−CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited “B cell help” signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFNγ, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

While inhibition of T cell co-inhibitory receptors has revolutionized cancer therapy, the mechanisms governing their expression on human T cells have not been elucidated. Type 1 interferon (IFN-I) modulates T cell immunity in viral infection, autoimmunity, and cancer, and may facilitate induction of T cell exhaustion in chronic viral infection1,2. Here we show that IFN-I regulates co-inhibitory receptors expression on human T cells, inducing PD-1/TIM-3/LAG-3 while surprisingly inhibiting TIGIT expression. High-temporal-resolution mRNA profiling of IFN-I responses enabled the construction of dynamic transcriptional regulatory networks uncovering three temporal transcriptional waves. Perturbation of key transcription factors on human primary T cells revealed both canonical and non-canonical IFN-I transcriptional regulators, and identified unique regulators that control expression of co-inhibitory receptors. To provide direct in vivo evidence for the role of IFN-I on co-inhibitory receptors, we then performed single cell RNA-sequencing in subjects infected with SARS-CoV-2, where viral load was strongly associated with T cell IFN-I signatures. We found that the dynamic IFN-I response in vitro closely mirrored T cell features with acute IFN-I linked viral infection, with high LAG3 and decreased TIGIT expression. Finally, our gene regulatory network identified SP140 as a key regulator for differential LAG3 and TIGIT expression. The construction of co-inhibitory regulatory networks induced by IFN-I with identification of unique transcription factors controlling their expression may provide targets for enhancement of immunotherapy in cancer, infectious diseases, and autoimmunity.

SUMMARYMultisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV2 infection in otherwise healthy children. Here, we define immune abnormalities in MIS-C compared to adult COVID-19 and pediatric/adult healthy controls using single-cell RNA sequencing, antigen receptor repertoire analysis, unbiased serum proteomics, andin vitroassays. Despite no evidence of active infection, we uncover elevated S100A-family alarmins in myeloid cells and marked enrichment of serum proteins that map to myeloid cells and pathways including cytokines, complement/coagulation, and fluid shear stress in MIS-C patients. Moreover, NK and CD8 T cell cytotoxicity genes are elevated, and plasmablasts harboring IgG1 and IgG3 are expanded. Consistently, we detect elevated binding of serum IgG from severe MIS-C patients to activated human cardiac microvascular endothelial cells in culture. Thus, we define immunopathology features of MIS-C with implications for predicting and managing this SARS-CoV2-induced critical illness in children.

The prominent role of dendritic cells (DC) in promoting tolerance and the development of methods to generate clinical grade products allowed the clinical application of tolerogenic DC (tolDC)-based therapies for controlling unwanted immune responses. We established an efficient method to generate tolerogenic human DC, producing supra-physiological levels of IL-10, by genetically engineering monocyte-derived DC with a bidirectional Lentiviral Vector (bdLV) encoding for IL-10 and a marker gene. DCIL−10 are mature DC, modulate T cell responses, promote T regulatory cells, and are phenotypically and functionally stable upon stimulation. Adoptive transfer of human DCIL−10 in a humanized mouse model dampens allogeneic T cell recall responses, while murine DCIL−10 delays acute graft-vs.-host disease in mice. Our report outlines an efficient method to transduce human myeloid cells with large-size LV and shows that stable over-expression of IL-10 generates an effective cell product for future clinical applications in the contest of allogeneic transplantation.

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.

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.

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule with known immune-modulatory functions. Our group identified a subset of human dendritic cells, named DC-10, that induce adaptive interleukin-10-producing T regulatory type 1 (Tr1) cells via the interleukin-10-dependent HLA-G/ILT4 pathway. In this study we aimed at defining the role of HLA-G in DC-10-mediated Tr1 cell differentiation. We analyzed phenotype, functions, and genetic variations in the 3' untranslated region of the HLA-G locus of in vitro-differentiated DC-10 from 67 healthy donors. We showed that HLA-G expression on DC-10 is donor-dependent. Functional studies demonstrated that DC-10, independently of HLA-G expression, secrete interleukin-10 and negligible levels of interleukin-12. Interestingly, DC-10 with high HLA-G promote allo-specific anergic T cells that contain a significantly higher frequency of Tr1 cells, defined as interleukin-10-producing (P=0.0121) or CD49b(+)LAG-3(+) (P=0.0031) T cells, compared to DC-10 with low HLA-G. We found that the HLA-G expression on DC-10 is genetically imprinted, being associated with specific variations in the 3' untranslated region of the gene, and it may be finely tuned by microRNA-mediated post-transcriptional regulation. These data highlight the important role of HLA-G in boosting DC-10 tolerogenic activity and confirm that interleukin-10 production by DC-10 is necessary but not sufficient to promote Tr1 cells at high frequency. These new insights into the role of HLA-G in DC-10-mediated induction of Tr1 cells provide additional information for clinical use in Tr1- or DC-10-based cell therapy approaches.