Notch1-CD22-Dependent Immune Dysregulation in the SARS-CoV2-Associated Multisystem Inflammatory Syndrome in Children

Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized in association with increased Notch1 expression. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variant impacting inflammation and autoimmunity pathways, including dominant negative mutations in the Notch1 regulators NUMB and NUMBL. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in an mTORC1 dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.


Introduction
COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in massive morbidity and mortality worldwide 1,2 . Acute infection is associated in some subjects with pneumonia and marked hypoxia, leading to acute respiratory distress syndrome as well as other lifethreatening complications 3,4,5 . This in ammation critically involves a dysregulated immune response characterized by intense activation of innate and adaptive immunity associated with features of a cytokine storm 6,7 . While most patients recover from this acute infection, a subset develops persistent symptoms related to different organ system dysfunction including the respiratory, cardiovascular, gastrointestinal, renal, and central nervous systems 8 .
A special case in point is the course of SARS-CoV-2 infection in children. While most children remain asymptomatic or develop from mild infection, some develop a multi-system in ammatory syndrome in children (MIS-C) approximately one month after initial infection 9,10,11,12,13 . These patients exhibit severe immune dysregulation characterized by intense cytokine production and lymphocyte activation associated with fever and end-organ dysfunction including mucocutaneous, cardiovascular, hematologic, and especially gastrointestinal systems 14,15,16,17,18,19,20,21 . In particular, IFNg has been identi ed as a key cytokine in MIS-C with increased levels associated with disease severity and increased organ system involvement 22,23 . There are de ning characteristics of MIS-C that remain perplexing, including the substantial delay between the initial SARS-CoV-2 infection and MIS-C 9, 10, 12 . Unlike children with acute COVID-19 pneumonia, most patients with MIS-C are previously healthy and are able to mount a robust immune response to SARS-CoV-2 with neutralizing antibodies to the virus 11,18,24 . This constellation of features in MIS-C suggests that an evolving hyperin ammatory immune response to SARS-CoV-2 is part of the pathophysiology of this syndrome. Indeed, studies based on relatively small number of patients suggests that a genetic predisposition may contribute to the immune dysregulation in MIS-C 25,26 .
Notch signaling pathways have emerged as important regulators of the immune system by in uencing both Treg and Tconv cells responses 27,28 . In mammals, the Notch family is composed by 4 Notch receptors (Notch1-4) and 5 ligands (Delta-like1, 3, and 4 and Jagged1 and 2) 29 . Recent studies have outlined a prominent role for NOTCH4 in the immune dysregulation in acute COVID19 and related respiratory viral illnesses 30 . Notch4 is upregulated on lung tissue Treg cells in an IL-6-dependent manner to subvert their tissue repair function in favor of an in ammatory response 30,31,32 . The NOTCH4 locus is associated with critical illness in COVID-19 33 . However, the immune dysregulatory mechanisms operative in post-acute COVID19 syndromes including MIS-C remain unclear.
In this study, we demonstrate that while NOTCH4 is also upregulated on circulating Treg cells of children with acute COVID19 as a function of disease severity, the Treg cells in MIS-C additionally upregulate NOTCH1 expression, a pathway previously implicated in Th1 type immune dysregulation, autoimmunity, graft versus host disease and solid organ rejection 34,35 . Gene enrichment using whole genome/exome sequence analysis employing Fischer testing and Monte-Carlo simulation revealed the enrichment in MIS-C patients of rare mutations impacting pathways of in ammation and autoimmunity, many of which contained Notch-related genes. Consistent with these results, a loss of function mutation was identi ed in the negative NOTCH1 regulator NUMB 36 . In mice expressing an active form of Notch1 in Treg cells (Foxp3 EGFPCre R26 N1c ), treatment with Poly I:C to simulate viral infection induced systemic in ammation that recapitulated the phenotype of MIS-C. Notch1 signaling in Treg cells induced the B cell inhibitory receptor CD22 37,38 , which promoted systemic in ammation in association with the expression of the α4β7 gut homing receptor. CD22 destabilized Treg cells and impaired their suppressive function in an mTORC1-dependent manner. Treatment of mice with an anti-CD22 mAb suppressed the development of systemic in ammation following Poly I:C treatment by restoring the Treg cells suppressive function. These ndings point to the mobilization of Treg cell-speci c tissue in ammatory licensing modules involving different Notch receptors that is operative in MIS-C and point to interventions along the Notch1-CD22 axis as therapeutic strategy in MIS-C.

Results
Increased CD4 + T cell activation and Treg cell destabilization in MIS-C. To elucidate the immune dysregulatory mechanisms operative in MIS-C, we studied an international cohort of 45 children with MIS-C and 50 children with COVID-19 from centers in the United States, Italy and Turkey (Table 1 and Patient Cohorts section in Methods). For comparison, 5 children with Kawasaki disease (KD), 12 adults with COVID-19, and 18 pediatric healthy controls were also evaluated. All MIS-C patients met the Centers for Disease Control (CDC) Case De nition for MIS-C while 93% ful lled the WHO case de nition 39,40 . Fever was universal in MIS-C patients and rash (49%), conjunctivitis (58%), and GI symptoms (96%) were also common. Children with MIS-C were highly in amed (median CRP 16.0 mg/dL, IQR 7.8-24.0), lymphopenic (median absolute lymphocyte count 0.91 x10 3 /mL, IQR 0.53-1.35) and coagulopathic (median D-dimer 3.1 mcg/mL, IQR 1.5-6.2). Over 90% of MI S-C patients demonstrated positive SARS-CoV-2 serologies. 18/45 (40%) were considered to have severe MIS-C de ned by admission to the intensive care unit (ICU), need for vasopressor support, and/or development of coronary artery aneurysms. The demographics and key clinical ndings in the respective patient groups are delineated in Supplementary Table 1.
To further delineate the CD4 + T cell dynamics in MIS-C, we carried out single-cell RNA sequencing (scRNAseq) analysis on CD4 + T cells from the peripheral blood of four healthy controls, three MIS-C patients sampled prior to treatment and another ve MIS-C patients sampled post-treatment. To characterize the sub-populations of CD4 + T cells in this high-dimensional analysis, we rst mapped our transcriptomic data to a reference human PBMC dataset using Azimuth 41 , thereby delineating 6 subsets of CD4 + T cells (Fig. 1a,b). To further elucidate the heterogeneity in our dataset, we performed a graph-based clustering analysis using Seurat, which uncovered 16 clusters. Eight of these clusters (Clusters 1 to 8) were enriched in cells annotated as CD4 naïve by Azimuth and expressing genes associated with a naïve CD4 + T cell pro le (e.g., CCR7 and SELL), 5 (Clusters 10 to 14) were enriched in activated CD4 + T cells (CD69), including one with high NF-kB signaling (Cluster 10; NFKB1). The nal 3 clusters encompassed a mix of naïve and activated cells, including one cluster delineated by viral sensing gene transcripts (Cluster 9; IFIT2, IFIT3), one cluster enriched in Treg cell transcripts (Cluster 15; FOXP3) and another with mitotic cells (Cluster 16; TRBC1) (Extended Data Fig. 1a-f). Prior to treatment, MIS-C patients exhibited prominent expansion of cluster 10, enclosing both cells annotated as Tconv and Tregs by Azimuth. Cluster 10 was characterized by increased NFKB1 expression and NF-κB signaling and contracted following immunomodulatory therapy (Extended Data Fig. 1a-f). To further decipher differences in CD4 + T cell transcriptomic programs between patient groups, we also performed pseudobulk differential analysis (DEA) with a focus on both Treg (cells found in Cluster 15 or delineated as Tregs by Azimuth) and Tconv cells (cells found in clusters 9 to 14 and delineated as activated Tconv by Azimuth). For this pseudobulk DEA, we aggregated gene expression data at the patient level for Tregs and activated Tconv, and performed pairwise comparisons of MIS-C pre-treatment, post-treatment and control groups using DESeq2. The DEA were followed by gene set enrichment analyses (GSEA) against the MSigDB Hallmark collection and using the ranked log2 fold changes as input, which reinforced our prior observations of NF-kB pathway activation in pre-treatment MIS-C samples, not only in Tconv but also in Tregs (Fig. 1c-h). Moreover, we also found new pathways that were up regulated in the MIS-C pre-treatment group, including for example MTORC1 and P53 pathway ( Fig. 1c-h). These results indicated that MIS-C is associated with enhanced Tconv activation and Treg dysregulation.
Increased NOTCH1 expression on CD4 + Treg and Tconv cells in MIS-C. Previous studies have demonstrated a key role for Notch signaling-mediated Teg cell dysregulation in licensing tissue in ammation 30,32,34,35 . A case in point is the upregulation of Notch4 on lung tissue Treg cells in viral infections mediated by SARS-CoV2 and in uenza, leading to enahcned tissue in ammation and disease severity 30 . We analyzed the expression of different Notch receptors on CD4 + Treg and Tconv cells in pediatric subjects with mild and severe COVID19 and those with MIS-C. As comparison groups we included healthy children, adults with severe COVID19 and children with KD, some of whose clinical features overlap with those of MIS-C 16, 25, 39, 40 . There was marked increase in Notch1 expression on both Treg and Tconv cells of patients with MIS-C but not on those of other subject groups (Fig. 2a-c). Our previous studies have identi ed Notch4 + Treg cells to emerge in the context of lung in ammation in COVID-19 subjects and related mouse models 30 . Notch4 expression was also selectively increased on the circulating Treg cells of adult and pediatric subjects with severe COVID-19 and with MIS-C but not on their Tconv cells. Notch4 was also not upregulated on Treg cells of patients with mild COVID19 or with KD ( Fig. 2d-f). Expression of Notch1 and Notch4 on Treg cells of MIS-C patients was non-overlapping, suggesting that they may represent distinct Treg cell populations possibly arising in different tissues ( Fig. 2g, lower panel). In contrast, Notch2 expression was increased on Notch1 + Treg and Tconv cells of MIS-C subjects albeit at a lower magnitude than that of Notch1, while there was no difference in the Notch2 single positive Treg and Tconv cell populations between MIS-C and healthy controls ( Fig. 2g higher panel and Extended Data Fig. 2a,b). Also, there was no difference in Notch3 expression between the circulating Treg and Tconv cells of different patient populations and control subjects (Extended Data Fig. 2c,d). Overall, these results identi ed increased Notch1 expression on Treg and Tconv cells as a distinguishing feature of pediatric patients with MIS-C. Fig. 2e-h). Analysis of the serum cytokine pro le of the different patient groups revealed increased expression of IP-10, IL-1β, IL-6 and IFNλ2/3 in MIS-C and to a lesser extent in severe COVID-19 compared to controls (Fig. 2h, Extended Data Fig. 2i). Further analysis showed that Treg and Tconv cells of patients with MIS-C versus severe COVID-19, KD and control subjects had increased IFN-γ production (Extended Data Fig. 2j,k). Notably, IFN-γ expression was selectively increased in Treg cells of MIS-C subjects, while IFN-γ expression in Tconv cells was common to both severe COVID-19 and MIS-C (Extended Data Fig. 2j,k).. We analyzed the capacity of different cytokines found increased in the sera of MIS-C subjects to induce Notch1 expression on cellsorted CD4 + CD25 + CD127 − human Treg cells from control subjects. Results showed that IL-1β and IL-6, and to a lesser extent IFN-γ and IP-10 all induced increased Notch1 expression on human Treg cells ( Fig. 2i). Overall, these results linked the upregulation of Notch1 expression on CD4 + Treg and Tconv cells with the development of MIS-C.

Analysis of T cell phenotype revealed that MIS-C patients present a decrease of naive Tconv and Treg cells associated with an increase of activated T cells (Extended Data
Identi cation of Notch pathway genetic variants in MIS-C. To investigate underlying genetic factors that may predispose to MIS-C versus acute pediatric COVID-19, we performed gene-enrichment tests for rare variants (stop-gain/start-loss, frameshift deletions/insertions and canonical splicing mutations) using 8626 pathways from Gene-Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (8,299 and 327 respectively). We collected genome and exome sequences on 39 MIS-C and 24 acute pediatric COVID-19 subjects, which we compared with 8 different datasets comprising 4,682 exomes collected at the Boston Children's Hospital, including 4 rare disease categories, obesity, myopathy, autism-ADHD and Immune de ciency/dysregulation 42 (see Methods section). All samples were processed using the Variant Explorer (VExP) Pipeline with the same set parameters to avoid bias in the selection of the rare variants 43 . We performed a Fisher test for each group in the different GO and KEGG pathways. Furthermore, we validated these results by Monte-Carlo simulation testing as an unbiased stochastic approach to test for enrichment in genetic variants along individual pathways in the MIS-C group versus the sum total of the clinical comparison groups used for the Fischer tests, as described in the Methods section. Results showed that several in ammation and autoimmunity pathways were signi cantly enriched in rare mutations (≤ 10 in 280,000 chromosomes) in MIS-C versus acute pediatric COVID-19 and the other comparison groups (Fig. 3a,b, Dataset 1). Several of those pathways contained NOTCH related genes. Speci c NOTCH pathway mutations predicted to be damaging and linked to those pathways were identi ed included NOTCH2, NOTCH4 and RPBJL (Dataset 2). Overall, these results supported the presence of an underlying genetic predisposition for MIS-C.
To validate the above ndings from our initial cohort, we screened 88 additional patients with MIS-C from the U.S. multicenter Overcoming COVID-19 Network for mutations in Notch-related genes (see methods in Supplement) 9,44 . Rare damaging mutations were identi ed in NUMB and NUMBL, encoding closely conserved eponymous proteins that negatively regulate Notch receptor signaling and tra cking 36,45,46,47 . We further analyzed three mutation in the phosphotyrosine binding (PTB) domain of NUMB and NUMBL Ser79IIe ; gnomAD = 0) or very rarely so (NUMBL Val88Met ; gnomAD = 4), were predicted to impair NUMB and NUMBL regulatory functions 36,46,47 . This prediction was tested by analyzing the impact of the respective NUMB/NUMBL mutations on NOTCH1 expression and function. Transgenic expression of the respective mutant protein in NUMB/NUMBL-de cient human embryonic kidney 293 (HEK293) cells revealed that their expression was similar to that of wild-type NUMB (NUMB WT ) or NUMBL (NUMBL WT ) protein (Fig. 3d,e). However, whereas transgenic NUMB WT or NUMBL WT decreased the expression of NOTCH1 in HEK293 cells by 32%, the NUMB Leu94Phe , NUMBL Ser79IIe and NUMBL Val88Met mutant completely failed to do so. Similarly, the NUMB Leu94Phe , NUMBL Ser79IIe and NUMBL Val88Met mutant failed to decrease nuclear NOTCH1 cytoplasmic domain (N1c) localization compared to NUMB WT (Fig. 3f, g).
Moreover, Co-transfection of NUMB WT -NUMB Leu94Phe and NUMBL WT -NUMBL Ser79IIe revealed that these mutations behaved as dominant negative mutations (Fig. 3f, g). These results established that the identi ed NUMB/NUMBL mutations were functionally deleterious.
To further delineate the mechanisms by which de cient NUMB activity in Treg cells promotes MIS-C, we employed a mouse model in which a oxed Numb allele was conditionally deleted in Treg cells using a Foxp3 promoter regulated Cre recombinase fused with YFP (Foxp3 YFPCre NUMB D/D ) is) (Fig. 3h).
Treatment of Foxp3 YFPCre NUMB D/D with polyinosinic:polycytidylic acid (poly(I:C)), a proxy model of infection with RNA viruses 30,48,49,50 , resulted in progressive weight loss. In contrast, Poly I:C-treated control Foxp3 YFPCre were minimally affected (Fig. 3i,j). Analysis of CD4 + T cells of Foxp3 YFPCre NUMB D/D mice revealed that their activation phenotype recapitulated that of CD4 + T cells of MIS-C patients, including increased memory markers (CD44 + CD62L − ) and heightened IFNγ production by both Treg and Tconv cells (Fig. 3k,l). Finally, de ciency of NUMB in Treg cell also recapitulate the upregulation of Notch1 and N1c found in MIS-C patients (Fig. 3m,n). Overall, these results indicated that MIS-C subjects may harbor mutations in the Notch pathway that contribute to disease pathogenesis.
Poly I:C-induced multiorgan in ammatory disease in Foxp3 EGFPCre R26 N1c/+ mice. To further delineate the mechanisms by which increased Notch1 signaling in CD4 + T cells promotes MIS-C, and in view of the critical role played by Treg cells in licensing Notch1-dependent immune dysregulation 34,35 , we employed a mouse model in which the intracellular domain of Notch1 is conditionally expressed from the Rosa26 locus (R26 N1c/+ ) in Treg cells using a Foxp3 promoter-regulated Cre recombinase fused with EGFP (Foxp3 EGFPCre ) (Fig. 4a) 34 . Treatment of Foxp3 EGFPCre R26 N1c/+ mice with Poly I:C intraperitoneally resulted in progressive weight loss and multi-organ in ammation. In contrast, Poly I:C-treated control Foxp3 EGFPCre were minimally affected (Fig. 4b-d). Analysis of CD4 + T cells of Foxp3 EGFPCre R26 N1c/+ mice revealed that their activation phenotype recapitulated that of CD4 + T cells of MIS-C patients, including increased memory markers (CD44 + CD62L − ) and heightened IFNγ production by both Treg and Tconv cells ( Fig. 4e-g).
Most MIS-C patients present with gastrointestinal symptoms (Table 1) 14 13 . Notably, the Treg and to a lesser extent the Tconv cells of the Foxp3 EGFPCre R26 N1c/+ mice had increased expression of the gut homing integrin α4β7 (Fig. 4h). Increased expression of integrin α4β7 was also observed on the circulating Treg cells of MIS-C but not acute pediatric COVID-19 subjects, in agreement with a critical role of Notch1 in driving the expression of this marker (Fig. 4i). Consistent with this nding, MIS-C patients exhibited an increase in CD62L − CD38 + mucosally imprinted Treg cells (Extended Data Fig. 3a) 51, 52 . sc-RNA seq analysis demonstrated increased ITGB7 transcripts in the clusters that also present an increase in Notch1 (Fig. 4j and Extended Data Fig. 3b). Expression of integrin α4β7 on Treg cells of MIS-C subjects declined post-treatment, in synchrony with decreased Notch1 expression (Fig. 4k). These results indicated that increased Notch1 activity in Treg cells predisposes to multi organ in ammation in the context of a viral trigger and promotes Treg cell gut homing.
Notch1-mediated CD22 upregulation on Treg cells promotes multi organ in ammation. To delineate the mechanisms by which Notch1 signaling in Treg cells promotes multi organ in ammation in the context of a viral trigger, we analyzed the transcriptome of Notch1c-expressing Treg cells for pathways involved in the immune dysregulation 34 . We found upregulation of CD22, a member of the Siglec family of lectins normally found in B cells, where it acts to regulate B cell receptor signaling 38 . In particular, CD22 directs B cells to the intestinal lymphoid and mucosal tissues by upregulating the expression of the gut homing receptor α4β7 37 . Flow cytometric analysis of Treg cells of Foxp3 EGFPCre R26 N1c/+ mice revealed increased CD22 expression upon treatment of the mice with Poly I:C (Fig. 5a). Expression of CD22 in Treg cells was abrogated upon Treg cell-speci c deletion of Rbpj, the gene encoding the Notch canonical pathway transcriptional co-factor RPBJ (Extended Data Fig. 4a). Analysis of peripheral blood Treg cells of MIS-C subjects revealed increased expression of CD22 that strongly correlated with Notch1 expression on these cells (Fig. 5b). In contrast, CD22 was minimally expressed on Treg cells of control subjects or those of patients with acute COVID-19. CD22 was minimally expressed on CD4 + Tconv cells of control, acute COVID-19 and MIS-C subjects, and it did not correlate with Notch1 expression on these cells, indicating that it uniquely identi ed Treg cells with active Notch1 signaling (Fig. 5b,c).
To determine the role of CD22 expression on Treg cells in the multi organ in ammatory disease triggered by Poly I:C treatment of Foxp3 EGFPCre R26 N1c/+ mice, we examined the impact of therapy with a blocking anti-CD22 mAb on disease outcome in these mice. Anti-CD22 mAb treatment prevented the weight loss and the multi-organ in ammation induced by Poly I:C treatment. It downregulated the activation of splenic CD44 + CD62L − Tconv cells and the expression by Treg and Tconv cells of IFNγ (Fig. 5d-h). Anti-CD22 mAb treatment also downregulated the expression by splenic Treg cells of α4β7 (Fig. 5j). In contrast, B cell depletion with an anti-B cell speci c anti-CD20 mAb failed on its own to protect against disease or to abrogate protection by anti-CD22 mAb treatment (Extended Data Fig. 4b-c). Analysis of gut lamina propria lymphocytes (LPL) revealed increased in ltration with activated (CD44 + CD62L − ) Tconv and Treg cells with increased expression of IFNγ that was similarly downregulated upon treatment with the anti-CD22 mAb (Extended Data Fig. 4d-e). These results indicated that Notch1 canonical pathwayinduced CD22 expression on Treg cells plays a crucial pro-in ammatory role in Poly I:C-treated Foxp3 EGFPCre R26 N1c/+ mice.
To determine the mechanisms by which CD22 subverted Treg cell function, we analyzed the steady state transcriptome of CD22 + Treg cells of Foxp3 EGFPCre R26 N1c/+ mice compared to control Foxp3 EGFPCre Treg cells or CD22 − Treg cells of Foxp3 EGFPCre R26 N1c/+ . KEGG/GO pathway analysis showed increased expression of genes involved in the regulation of the immune response, T cell migration and Notch signaling (Fig. 5a-c). Furthermore, we analyzed the phenotypes of CD22 + colonic and splenic of Treg cells Foxp3 EGFPCre R26 N1c/+ mice isolated at steady state and following Poly I:C-treatment with those of Treg cells of similarly treated Foxp3 EGFPCre mice. The CD22 + Treg cells exhibited decreased expression of Helios and NRP-1 both at steady state and after Poly I:C treatment in the face of similar expression of markers of T cell activation including CD44, suggesting their decreased stability. In agreement with this conclusion, Foxp3 expression also decreased in CD22 + Treg cells following Poly I:C treatment. Treatment with anti-CD22 mAb reversed those defects ( Fig. 6a and Extended DATA Fig. 5). In vitro Treg cell suppression assay revealed profoundly defective suppressive function of CD22 + Treg cells compared to control, which was also corrected upon treatment of the cells with the anti-CD22 mAb (Fig. 6b). Anti-CD22 mAb treatment corrected the decreased cell frequencies and MFI of Foxp3 found at the end of the tissue culture period, indicative of reversal of CD22 + Treg cell instability (Fig. 6c).
CD22 has been described to regulate B cell receptor signaling by forming a molecular scaffold that enables coordinated docking of different downstream signaling pathways 53 . Analysis of CD22 + Treg cells revealed enhanced T cell receptor signaling compared to control Treg cells, with increased phosphorylation of the, extracellular signal-regulated kinases (ERK) 54 and the phospholipase C gamma 1 (pPLCγ1) (Fig. 6d and Extended Fig. 5). Downstream of the PI3-kinase pathway, phosphorylation of the kinase AKT at residue T308, a target of upstream phosphoinositide-dependent kinases, and the mammalian target of rapamycin complex 1 (mTORC1) substrate S6 kinase were also increased (Fig. 6d) with the mTOR inhibitor Rapamycin reversed the regulatory defect in CD22 + Treg cells and the associated loss of Foxp3 expression (Fig. 6e-f). Finally, In vitro Treg cell suppression assay from MIS-C patients also revealed profoundly defective suppressive function compared to Healthy control, which was also corrected upon treatment of the cells with the anti-CD22 mAb or Rapamycin (Fig. 6g). These results indicated that CD22 positively enhanced T cell receptor signaling in Treg cells leading to Treg cell destabilization and loss of regulatory function by an mTORC1-dependent mechanism.

Discussion
In this study, we demonstrate that the evolution of MIS-C entails the mobilization of a Treg cell-speci c pathway involving Notch1-CD22 signaling that promotes immune dysregulation and which can be demonstrated both in human subjects and in proxy mouse models. Patients with MIS-C but not children or adults with acute COVID-19 demonstrated increased Notch1 expression on circulating CD4 + Treg and Tconv cells, which declined precipitously following anti-in ammatory therapy. The pathogenic function of this pathway was con rmed by the identi cation of dominant negative mutations in PTB domains of NUMB and NUMBL in MIS-C subjects that resulted in increased Notch1 expression. It was also supported by the demonstration of a role for Notch pathway-related mutations in MIS-C using Monte Carlo simulation and Fischer Test. Uniquely, MIS-C subjects exhibited increased CD22 expression on Treg but not Tconv cells which could be demonstrated in mice to involve Notch1 signaling via the RBPJ-k canonical pathway. CD22 blockade was su cient to inhibit the immune dysregulation triggered by Our previous studies have identi ed NOTCH4 to be speci cally upregulated on circulating Treg cells in adult subjects with COVID-19; their origin could be traced in mouse models of viral infection to the lung 30 . NOTCH4 was similarly upregulated on circulating Treg cells of pediatric subjects with acute COVID-19, while both NOTCH4 and NOTCH1 were upregulated on those of MIS-C subjects. However, expression of NOTCH1 and NOTCH4 on the Treg cells of the latter group was mutually exclusive, suggesting the respective Treg cell populations were ontogenically distinct. These ndings suggest that NOTCH4 and NOTCH1 regulate distinct checkpoints in the evolution of immune dysregulation following SARS-CoV-2 and other viral infections. Thus, NOTCH4 appears critical for licensing lung in ammation following SARS-CoV2, in uenza and related viral infections 30 . In contrast, NOTCH1 may favor the spread of the initial lung in ammation systemically. Such a division of labor is supported by previous studies showing that the induction Notch1 on Treg cells plays a pathogenic role in mouse models of graft versus host disease and cardiac allograft rejection 34,35 .
Mouse studies revealed that a critical step by which Notch1 signaling in Treg cells promotes systemic in ammation involves its induction of CD22, an inhibitory receptor previously associated with B cells, where it functions as a regulator of B cell receptor signaling, more recently, CD22 has been described to direct the homing of B cells to the gut lymphoid and mucosal tissues by virtue of its upregulation of the gut homing integrin α4β7. Consistent with these ndings, treatment of mice whose Treg cells express a gain of function Notch1 mutant with an anti-CD22 blocking antibody rescued their gut and systemic in ammation following treatment with Poly I: C. CD22 impaired the in vitro Treg cell suppressive function, an effect that was reversed by treatment with the anti-CD22 blocking antibody. Notwithstanding its function as an inhibitory receptor, Treg cells expressing CD22 demonstrated T cell receptor signaling and heightened proliferation.
MIS-C is a rare complication of SARS-CoV-2 infection 57 , suggesting a genetic predisposition to this disorder. In that regard, mutations in a number of immune regulatory genes have been described in MIS-C, including SOCS1, XIAP and CYBB 26 . We found variants in a number of Notch pathway genes in patients with MIS-C. Importantly, dominant negative loss of function mutations in NUMB and NUMBL found in MISC subjects resulted in increased NOTCH1 expression and NOTCH1 signaling, consistent with the pathogenic function of mutations in this pathway in disease pathogenesis.