key: cord-0962902-m1wjp99q authors: Diorio, C.; Shraim, R.; Vella, L. A.; Giles, J. R.; Baxter, A. E.; Oldridge, D. A.; Canna, S. W.; Henrickson, S. E.; McNerney, K. O.; Balamuth, F.; Burudpakdee, C.; Lee, J.; Leng, T.; Farrel, A.; Lambert, M. P.; Sullivan, K. E.; Wherry, E. J.; Teachey, D. T.; Bassiri, H.; Behrens, E. M. title: Proteomic Profiling of MIS-C Patients Reveals Heterogeneity Relating to Interferon Gamma Dysregulation and Vascular Endothelial Dysfunction date: 2021-04-20 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.04.13.21255439 sha: 0b7c61e34c82d6a07ccaaf316705e2ec6984fa82 doc_id: 962902 cord_uid: m1wjp99q Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic in pediatric patients. Weeks after an often mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MIS-C present with varying degrees of cardiovascular and hyperinflammatory symptoms. We performed a comprehensive analysis of the plasma proteome of more than 1400 proteins in children with SARS-CoV-2. We hypothesized that the proteome would reflect heterogeneity in hyperinflammation and vascular injury, and further identify pathogenic mediators of disease. Protein signatures demonstrated overlap between MIS-C, and the inflammatory syndromes macrophage activation syndrome (MAS) and thrombotic microangiopathy (TMA). We demonstrate that PLA2G2A is a key marker of MIS-C that associates with TMA. We found that IFN{gamma} responses are dysregulated in MIS-C patients, and that IFN{gamma} levels delineate clinical heterogeneity. press). 16 We have previously demonstrated that infection with SARS-CoV-2 and MIS-C are associated with thrombotic microangiopathy (TMA), and the associated biomarker soluble C5B9 (SC5B9). 17 TMA is a group of diseases characterized by microangiopathic hemolytic anemia, thrombocytopenia and organ dysfunction related to microthrombi. 18 TMA can occur as a primary process or secondary to infection, inflammatory insult or in hematopoietic stem cell transplant (HSCT). 18, 19 The mechanism of TMA in the context of MIS-C is not known, and its association with cytokine dysregulation remains unclear. We interrogated >1400 proteins in the plasma proteome and integrated this information with clinical and high-dimensional flow cytometry data. We hypothesized that distinct dysregulations in proteins and associated pathways that relate to cytokine storm and vascular injury contribute to the underlying pathophysiology of MIS-C. Our goals were to understand the pathophysiology of MIS-C via a thorough examination of the plasma proteome, and to identify candidate biomarkers and predictors of disease severity. Between April 2020 and October 2020, we enrolled 63 hospitalized patients with MIS-C (N=22), Severe COVID-19 ("Severe", N=15) or asymptomatic or mild SARS-CoV-2 infection ("Minimal", N=26). In addition, we included remnant plasma samples from otherwise healthy patients ("Healthy", N=25). Details of the clinical presentation of MIS-C, Severe, and Minimal patients are presented in Supplemental Tables S1 and S2. Similar to previously reported cohorts, a high proportion (N = 17, 77%) of patients with MIS-C were admitted to the PICU. 2, 4 We All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint utilized the Olink Explore 1536/384 protein biomarker platform to interrogate the plasma proteome of all 88 patients. A subset of patients included in this study have been reported previously. 17, [20] [21] [22] [23] Flow cytometry data was available for 20 patients included in this study (MIS-C N=8, Severe N=6, Minimal N=6), reported previously by Vella et al. 20 We first sought to validate the accuracy of the O-link platform. Patients with MIS-C in our sample had marked elevations of brain-type natriuretic peptide (BNP) and troponin measured clinically (Supplemental Table S2 ). The Olink panel measures N-terminal prohormone brain natriuretic peptide (NTproBNP), a protein pre-cursor to BNP associated with cardiac damage. 24 NTproBNP was significantly higher in MIS-C patients (Supplemental Figure S1A ), consistent with other reports. 15 As expected, Olink NTproBNP correlated highly with clinical BNP (R=0.78, p =2.7e-08; Supplemental Figure S1B ) . To further validate the data, we compared cytokines measured in our laboratory with the same cytokines measured by Olink and demonstrated strong correlations (Supplemental Figures S1C). We and others have previously found elevations in IL-10 distinguish patients with MIS-C from those with acute SARS-CoV-2 infection. We confirmed this observation with data from Olink (Supplemental Figure S1D ). 13, 21 Following this validation, we next took an unbiased approach to investigate the proteomic data. The overall architecture of the data is presented in Figure 1 . Patients with MIS-C cluster differently from patients with acute SARS-CoV-2 or healthy patients when visualized with tdistributed stochastic neighbor embedding (tSNE; Figure 1A ). Vella et al. previously All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04. 13.21255439 doi: medRxiv preprint demonstrated that MIS-C is associated with CD8+ T-cell activation and Tbet+ plasmablasts. 20 In the subset of patients on whom flow cytometry data was available, we overlaid flow cytometry markers on tSNE clustering. Interestingly, there was heterogeneity in percent of activated CD8+ T-cells and percent of Tbet+ plasmablasts within the MIS-C clusters ( Figure 1A ). 20 Furthermore, there appears to be an inverse relationship between regions with higher percentage of activated CD8+ cells and a higher percentage Tbet+ plasmablasts within these MIS-C subclusters. In order to look at loading plots of the most prominent drivers of difference between groups, we used principal component analysis (PCA) to visualize clustering between patients. To achieve better separation between groups, we limited the analysis to proteins that were differentially expressed between any of the four groups (N=231). Lists of differentially expressed proteins Figure 1C . Twenty patients with MIS-C received treatment with both steroids and IVIG. One patient was treated with IVIG alone, and one patient did not receive treatment. Convalescent samples were available on 12 patients (54%). To understand how the overall proteome of patients with MIS-C changes over time, we first created a PCA mapping all proteins and all patients ( Figure 1D ). We then PCA transformed data for convalescent samples on that space ( Figure 1E ). Convalescent All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint samples shifted towards the healthy controls, implying that following treatment, the proteome in MIS-C patients returns towards a baseline state. Pre-and post-treatment samples were available on 5 patients. To investigate if the timing of initial collection relative to treatment altered the proteome, we assessed change over time in one of the most differentially expressed proteins, phospholipase A2 (PLA2G2A; Supplemental Figure S1E ). PLA2G2A pre-and post-treatment samples did not significantly differ in acute timepoints suggesting that sampling of plasma shortly after treatment does not immediately affect results. We used unbiased exploration of DEPs to understand pathways of activation between MIS-C, Minimal and Severe patients compared to healthy patients. Volcano plots are presented in Figure 2 . Notably, PLA2G2A is highly differentially expressed between all three groups compared to healthy patients, with the most marked difference occurring in MIS-C patients. In order to identify pathways of relevance to the pathophysiology of MIS-C and pediatric COVID-19 we performed pathway analysis accounting for protein-protein interactions using pathfindR. 25 PathfindR uses an active subnetwork enrichment analysis approach to identify lists of enriched pathways. Ranked pathways and enrichments are presented in Figure 2A -C. Lists of all ranked pathways are available in Supplemental Material S2. A protein to pathway analysis on the pathways of interest was performed to identify interactions between various proteins in the datasets and pathways. Cytokine-cytokine and chemokine-cytokine receptor pathways were dysregulated in all classifications of pediatric SARS-CoV-2 infection. Notably, similar pathways were perturbed in All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure S3A . The majority of DEPs in Severe COVID-19 are also differentially expressed in MIS-C. We directly assessed the DEPs between these groups and surprisingly, there were very few differences (Supplemental Figure S3B ). As this comparison involved a smaller proportion of patients, we used a nominal p-value with a threshold of 0.05. IFNγ has been associated with MIS-C in previous reports. 12, 15 We previously described that IFNγ levels were high in patients with Severe and MIS-C, that levels of IFNγ could not distinguish between these two groups and that IL-10 can distinguish MIS-C from Severe (Supplemental Figure S3B ). 21 In order to better understand these two cytokines, we investigated correlations between IFNγ and IL-10 and their canonical response proteins as well as proteins associated with cellular populations responsible for their production ( Figure 3 ). We examined CXCL9 as the key protein associated with IFNγ response and found that all patients with SARS-CoV-2 infection showed a positive correlation between CXCL9 and IFNγ. Strikingly, MIS-C patients had a disproportionately high CXCL9 response to IFNγ compared to the other groups ( Figure 3A ; p<0.001 by logistical regression modelling). To probe the source of IFNγ production, we correlated IFNγ levels with soluble markers of activated T-cells (IL2RA), NK-cells (NCR1) and macrophages (CD163). IFNγ levels in MIS-C patients associated significantly with IL2RA and NCR1 but not CD163, suggesting that IFNγ levels correlate with T-cells and NK-cells but not macrophages ( Figure 3A ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint Levels of IL-10 have been shown previously, and in this cohort, to be significantly higher in patients with MIS-C than in those with Severe COVID-19 (Supplemental Figure S1D ). 13, 21 Similar to the analysis above, we examined the expression of IL12B, which should be inhibited by IL-10. 26 IL12B was inversely correlated with IL-10 in patients with MIS-C, but not in Minimal, Severe or Healthy patients ( Figure 3B ). This finding implies that the elevated IL-10 in MIS-C patients is bioactive. IL-10 production was significantly associated with markers of activated T-cells and macrophages, but not with NK-cells ( Figure 3B ). We subsequently examined the relationship between IFNγ, CXCL9 and IL-10 and activated CD8+ and CD4+ T-cells from flow cytometry data in order to probe these correlations further ( Figure 3C&D ). We found that CXCL9 was significantly and strongly correlated with both cell types but IFNγ was not, suggesting T-cells are being activated by IFNγ rather than producing IFNγ. IL-10 was significantly associated with activated CD4+ but not activated CD8+ T-cells. The outsized response to IFNγ with excessive CXCL9 expression in patients with MIS-C evoked the cytokine phenotype of macrophage activation syndrome (MAS). 27, 28 MAS is a hyperinflammatory condition that occurs in the setting of rheumatologic diseases, infections or as a primary disorder, characterized by excessive activation of lymphocytes and macrophages in the setting of marked IFNγ production. 29, 30 We therefore investigated whether IFNγ, CXCL9 and other markers associated with MAS could distinguish different SARS-CoV-2 outcomes ( Figure 4A ). 28, 30, 31 Unsupervised hierarchical clustering revealed two distinct groups of MIS-C patients that had elevations in most of the MAS markers. These two clusters primarily differed by their IFNγ expression with an IFNγ-high versus IFNγ-low group evident. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To further interrogate the hypothesis that MAS was associated with MIS-C, we applied a modified version of the Ravelli MAS criteria to all patients on whom ferritin had been measured (N=40; MIS-C N=21, Minimal N=6, Severe N=13). 32 If parameters were missing, they were imputed as negative to bias our score towards the null hypothesis. We compared patients who met criteria for MAS to those who did not (Supplemental Table S3 ). CD163, CXCL9, IFNγ, IL2RA and VSIG4 were significantly elevated in patients who met MAS criteria ( Figure 4B ). We found that maximum ferritin during admission was significantly higher in patients that met criteria for MAS than those that did not (P<0.0001) but did not differ significantly between the disease groups as a whole ( Figure 4C&D ). In MIS-C patients on whom both an acute and a convalescent sample was available (N=12), IFNγ, CXCL9 and IL2RA all significantly decreased over time ( Figure 4E ). For patients with MIS-C, time of sample draw relative to treatment with IVIG or steroids against IFNγ level is presented in Supplemental Figure S1F and shows decay over time. MAS is typically associated with cytopenias, including neutropenia. In contrast, MIS-C is characterized by marked neutrophilia, 4 and in patients who met criteria for MAS high absolute neutrophil counts (ANC) were apparent ( Figure 4F ). ANC was significantly higher in patients with MIS-C than in those with Severe or Minimal ( Figure 4G ). In MIS-C patients, neutrophilia did not correlate with IFNγ or CXCL9 but correlated strongly with CD163, implying neutrophilia in these patients is associated with macrophage activation (R=0.72, p<0.0001; Figure 4H ), but not directly related to IFNγ or CXCL9. Macrophage hyperresponsiveness to IFNγ expression has been linked to TRIM8 dysregulation in MAS. 33 We noted dysregulation of another TRIM protein, the IFNγ signaling suppressive protein TRIM21. 34, 35 Consistent with a All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. derepression of IFNγ signaling, MIS-C patients expressed less TRIM21 compared to healthy controls (Supplemental Figure S3C ). We and others have previously demonstrated that infection with SARS-CoV-2 is associated with clinical TMA and elevations in the TMA biomarker SC5B9. 17, 36 As SC5B9 was not included in the Olink analysis, we correlated SC5B9 levels measured in our laboratory (N=75) to Olink proteins related to vascular or platelet dysfunction to identify surrogate biomarkers. Hierarchical clustering identified that SC5B9 correlated most highly with PLA2G2A, PDGFC, SELE, CALCA, NOS3, VWA1 and TYMP ( Figure 5A ). We then evaluated whether proteins highly correlated with SC5B9 were able to identify disease category ( Figure 5B ). Patients with MIS-C clustered due to very high PLA2G2A expression, and moderate CALCA and TYMP expression. PLA2G2A and CALCA were both significantly higher between MIS-C and Severe patients ( Figure 5C ). PLA2G2A is significantly higher in SARS-CoV-2 infected patients than in healthy patients with the most marked difference in patients with MIS-C ( Figure 5C ). In MIS-C patients, PLA2G2A, SELE, CALCA and VWA1 all improved between the acute and convalescent phases ( Figure 5D ). Notably, levels of PLA2G2A do not return completely to normal during convalescence. For patients who had blood smears available for review (N=34; MIS-C N=15, Minimal N=11, Severe N=8) we applied simple criteria for TMA, as previously published (Supplemental Table S4 ). 17 Meeting criteria for TMA was associated with a significantly higher PLA2G2A and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint CALCA ( Figure 5E ). Patients who met criteria for TMA were more likely to require inotropes during their admission than those who did not meet criteria for TMA (92% vs 23% respectively, N=34, p-value <0.001). Given our hypothesis that PLA2G2A is a marker of TMA and microangiopathic hemolytic anemia, we examined whether levels of PLA2G2A correlated to lowest platelet count and lowest hemoglobin during admission. PLA2G2A levels inversely correlated with platelets and hemoglobin with groupings by disease category evident ( Figure 5F ). Next, we investigated the intersection between the MAS and TMA phenotypes and MIS-C. Among MIS-C patients, TMA and MAS occurred independently (Fisher's exact, p = n.s.; Figure 6A ). Furthermore, when TMA and MAS phenotypes were overlayed on the same tSNE mapping used in Figure 1 , patient clustering by disease state was evident (Supplemental Figure S4A ). To further explore the heterogeneity among MIS-C patients, we compared patients from the IFNγhigh (N=7) and IFNγ-low (N=13) clusters identified in Figure 4A . We looked at DEPs between these two clusters and performed unbiased pathway and clustering analysis ( Figure 6B -D) revealing differences in cytokine signaling including IL-17 pathways. IL-17 is a neutrophil chemotactic factor and may relate to the neutrophilia present in these patients. 37 In Supplemental Figure S4B IFNγ levels are overlayed on the same tSNE map as displayed in Figure 1 , demonstrating heterogenous clustering within MIS-C. We examined occurrence of TMA and MAS among these IFNγ-high and -low MIS-C clusters. There was no increase in TMA among IFNγ-high MIS-C patients (Fisher's exact, p = n.s.; Supplemental Table S5 ). There was a trend towards a significantly increased enrichment in MAS among IFNγ-high MIS-C patients compared to IFNγ-low patients (83% vs. 38%, Fisher's exact All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint p=0.14; Supplemental Table S5 ). In keeping with this trend, IFNγ-high MIS-C patients had significantly higher ferritin (p=0.036; Figure 6E ). IFNγ status did not associate with fibrinogen, d-dimer, platelet, hemoglobin or ANC. In order to understand the relationship between IFNγ and clinical outcomes in MIS-C, we examined surrogate markers of severity. We used requirement of inotropes as a surrogate marker of severity. Surprisingly, patients within the IFNγ-high cluster had a lower likelihood of requiring inotropes than those in the IFNγ-low cluster ( Figure 6F ), implying that higher levels of IFNγ are associated with less severe illness in MIS-C patients. NTproBNP, a marker associated with cardiac damage, showed a similar trend, although the result was not statistically significant, likely due to a lack of power ( Figure 6G ). We performed this analysis on all patients with MIS-C and found that higher levels of NTproBNP were higher in patients requiring inotropes ( Figure 6H ). Thus, it appears higher levels of IFNγ may be associated with less severe illness in patients with MIS-C. Previously, Vella et al. demonstrated that inotrope need was associated with higher frequencies of activation of CX3CR1+ CD8+ T-cells. 20 CX3CR1 is expressed on CD4+ T-cells, monocytes, and effector-like CD8+ T-cells, and allows cells to interact with CX3CL1-expressing vasculature. 38, 39 In MIS-C patients, activation of CX3CR1+ CD8+ T-cells (but not CX3CR1+ CD4+ T-cells; Supplemental Figure S4C ) and IFNγ inversely correlated ( Figure 6I ; N=7, R=-0.83, p=0.02). However, levels of the CX3CR1 ligand, CX3CL1 had no relationship to CX3CR1+ CD8+ T-cell activation ( Figure 6I ). In our unbiased network analysis ( Figure 6D ) we identified CX3CL1 as an enriched protein despite it not reaching the thresholds used for our differential expression analysis ( Figure 6C ). When explicitly tested, we surpassingly observed an All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint association between elevated CX3CL1 and IFNγ-high patients, a group of patients we demonstrated to be at lower risk for inotrope use ( Figure 6J ). The data suggest that free CX3CL1 levels in the plasma are independent of the frequency of CX3CR1+ CD8+ T-cell activation ( Figure 6I ), explaining their differential association with inotrope requirement. Thus, we demonstrate that patients with high IFNγ levels are less likely to require inotrope use and accordingly have lower levels of CX3CR1+ CD8+ T-cell activation. We used proteomic analysis to begin to unravel the complex and heterogeneous pathophysiology associated with MIS-C. We identify PLA2G2A as a candidate biomarker for MIS-C and show that PLA2G2A is associated with clinical features of TMA. We demonstrate that MIS-C patients are characterized by a disproportionately high CXCL9 response to IFNγ, implying a dysregulated response to IFNγ. We found that a subset of patients with MIS-C met modified criteria for the IFNγ-associated syndrome MAS. Patients with MIS-C showed heterogeneity based on IFNγ expression, and surprisingly, patients with higher IFNγ levels were less likely to require inotropes. PLA2G2A is involved in host inflammatory responses and is associated with release of damage associated molecular pattern molecules (DAMPs) that play a key role in activation of the innate immune response. 40, 41 PLA2G2A released from activated platelets is associated with neutrophilia. 40 Expression of PLA2G2A can be driven by IFNγ seceretion. [42] [43] [44] In HSCT associated TMA, IFNγ has been identified as a key causative agent and part of an "interferoncomplement loop" that contributes to vessel injury and TA-TMA pathophysiology. 45 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Overlapping phenotypes of MAS and TMA have been reported by others in other disease settings. 46 We hypothesize that patients with MIS-C may be responding to IFNγ with excessive PLA2G2A production. However, this relationship is likely complex as patients with high IFNγ expression appear to be protected from cardiac toxicity associated with MIS-C. Whether or not there is a causative relationship between PLA2G2A and TMA or MIS-C will need to be determined in future experiments. Understanding the role of PLA2G2A in MIS-C pathophysiology is crucial because PLA2G2A and its associated pathways can be targeted with the common, inexpensive medication indomethacin. 47 Patients with MIS-C produced excessive CXCL9 in response to IFNγ. CXCL9 production has been associated with an increased risk of mortality in adult patients with severe COVID-19 disease and may contribute to cytokine storm in that setting. 48 One potential explanation for the heightened CXCL9 response to IFNγ in MIS-C patients is lower levels of TRIM21, a protein notable for its ability to repress IFNγ signaling through degradation of interferon response factors. 34, 35 TRIM21 is induced by IFNγ, and indeed was one of the upregulated factors in the IFNγ-high MIS-C group, suggesting that this negative feedback loop is at least still partially intact in MIS-C despite the generally lower levels of TRIM21. 49 In keeping with IFNγ dysregulation, found that a subset of patients with MIS-C met modified criteria for MAS. We All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint note that an IFNγ-high and -low subgrouping of MIS-C has been noted by others. 7 While the connection to MAS was also invoked by Esteve-Solie et al., we have clearly shown a connection between IFNγ-high MIS-C patients and objective MAS features in our data. Other features of MAS in patients with MIS-C have been reported in the literature, including overlapping cytokine phenotypes and hemophagocytosis on bone marrow aspirates. 50 Although the evidence suggests that MIS-C does share many things in common with MAS, the clinical presentation of MIS-C patients is not fully consistent with a complete MAS phenotype and may be better understood as an occult MAS. 51, 52 MAS is often treated with IL-1β blockade. 53 Interestingly, IL-1β blockade with anakinra, a recombinant IL-1 receptor antagonist has been reported in many case reports as efficacious in MIS-C, and it is recommended for refractory patients in the American College of Rheumatology guidelines. 54 We further note that IL-1RN, the endogenous IL-1 receptor antagonist is upregulated in the IFNγ high group ( Figure 6B ). It is interesting to speculate whether this confers some of the cardiac protection seen in this group. Curiously, we found that IFNγ-high patients were less likely to require inotropes, a result that is not consistent with other disorders associated with dysregulated IFNγ production. 28 This result was confirmed by an anti-correlation between IFNγ and CD8+ CX3CR1+ T-cells, a cell population which has been previously associated with an increased need for inotropes in MIS-C patients. 20 The novel observation that IFNγ may predict cardiac pathology in MIS-C may prove to be useful clinically as cytokine testing becomes more readily available in hospital settings. Future work should test this prospectively. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Future studies should mechanistically examine the relationships between IFNγ, PLA2G2A, and their drivers in MIS-C initiation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This study was conducted in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board (IRB) at CHOP. Verbal consent for this minimal risk study was obtained from patients or their legally authorized representative. Consent forms were signed by the consenting study team member and a copy was provided to the study participant or legally authorized representative. If appropriate, assent was obtained from children who were 7 years of age or older. For remnant samples obtained from healthy controls, protected health information (PHI) was not recorded. A limited chart review of this cohort was granted by the CHOP IRB to determine that patients met criteria to be considered "healthy". CHOP IRB granted exemption criteria per 45 CFR 46.104(d) 4(ii) and waiver of HIPPA authorization. Patients were eligible to be prospectively enrolled in the Children's Hospital of Philadelphia All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint Patient samples were collected as soon as possible after admission to the hospital. All samples were collected in combination with a clinical blood draw. Blood for protein analysis was collected in a lithium heparin tube and separated into plasma and cell pellets. Components were frozen and stored in liquid nitrogen and -80 freezer respectively. Batch analysis was performed on plasma samples. Blood for flow cytometry was collected in a sodium heparin tube. 20 In patients who remained in hospital, samples were collected weekly. Plasma from otherwise healthy control patients who were being evaluated for symptoms of a bleeding disorder (such as epistaxis) were obtained from discarded plasma from the coagulation lab at CHOP. A limited chart review was performed to confirm these patients had no comorbid medical illnesses. Patients found to have comorbid medical issues other than a possible isolated bleeding disorder were excluded. Clinical and laboratory data were abstracted from electronic patient charts on to standardized case report forms created using the Research Electronic Data Capture database (REDCap; Vanderbilt University, Nashville TN USA). 55 Data were abstracted by a clinician or clinical research assistant. All data elements were validated by a physician. Enrolled patients were prospectively classified in to 3 groups by physicians with expertise pediatric in hematology/oncology (CD, DT), pediatric infectious diseases (HB) or pediatric rheumatology (EB). Patient classification criteria have been described in detail previously. 17 In All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patients were categorized as having thrombotic microangiopathy (TMA) based on criteria reported previously. 17 Only patients on whom a Hematoxylin-Eosin stained peripheral blood smear available were evaluated for TMA (N=34). The criteria used included presence of thrombocytopenia, microangiopathic hemolytic anemia and organ dysfunction. Hemolytic anemia was defined as anemia for age and schistocytes present on a peripheral blood smear. Organ dysfunction was defined as cardiac dysfunction (troponin greater than the upper limit of normal for age or requirement of inotropes), renal dysfunction (based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria) 57 or liver dysfunction (bilirubin greater than twice the upper limit of normal for age, alanine aminotransferase or aspartate aminotransferase greater than three times the upper limit of normal). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In patients on whom a ferritin was available (N=40), a modified version of the Ravelli criteria for MAS were applied. 32 We modified the Ravelli criteria to exclude triglyceride levels because they were not measured in the majority of our patients. We defined a modified Ravelli criteria as ferritin>684ng/mL and any 2 of 1) platelet count<181,000/uL, AST>48U/L, or fibrinogen<360mg/dL. If any data were missing, they were imputed as negative/normal in order to bias our categorization towards the null hypothesis. Plasma protein levels were analyzed using the Olink Explore 1536/384 panel (Olink Proteomics, Uppsala, Sweden). Data were reported in normalized protein expression values (NPX). NPX is an arbitrary unit in a Log2 scale calculated from inverted, normalized Ct values. All assay validation data are available on the Olink website (www.olink.com). sC5b9 levels were determined using enzyme-linked immunosorbent assays (ELISA; Cat. #558315; BD Biosciences, San Jose CA, USA). Assays were performed at two dilutions; all samples were assayed in triplicate, using manufacturer protocols. As previously described, we set the upper limit of normal for our sC5b9 assay at 247 ng/mL. sC5b9 measurements were row normalized to a Z-score and log2 transformed in order to be comparable to NPX scores. Detailed methods for flow cytometry analysis have been described previously. 20 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood collected in sodium All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint heparin tubes. PBMCs were stained with a live/dead stain and then treated with Fc block before staining for chemokine receptors, surface markers and intracellular markers. Samples were acquired on a 5 laser FACS Symphony A5 (BD Biosciences). All analysis was performed in FlowJo (Treestar, version 10.6.2). Flow cytometry data were transformed to a Z-score for all of the values for each gate used in order to be able to compare to the Log2 transformed NPX scores used in the Olink dataset. All statistical analyses were performed in R (version 4.0.4) using RStudio (RStudio, PBC, Boston, MA). 58 Data were assumed to be non-parametric unless normality was demonstrated. Correlations were performed with Spearman's rank correlation coefficient for non-parametric data and Pearson's correlation coefficient for parametric data. Kruskall-Wallis testing was performed to compare three or more groups and Wilcoxcon signed-rank test was performed for paired groups. To test for associations between discrete variables, Fisher's exact test was used if small numbers were available in each cell. Otherwise, Chi-square testing was used. Unless otherwise stated, significance was based on a fold change greater than 2 or less the -2 and a false discovery rate (FDR; using Benjamini-Hochberg correction) value of less than 0.01. The T-distributed Stochastic Neighbor Embedding (tSNE) for R package was used to perform tSNE clustering. 59 Principal component analysis was performed using R version 4.0.4. The factoextra package (https://cran.r-project.org/web/packages/factoextra/index.html) was used to extract and visualize PCA elements. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint We completed a differential expression analysis of all proteins using a log2fold change threshold of 2 and FDR threshold of 0.01. Proteins from the Olink data set were inputted in to PathfindR along with their respective log2fold change and FDR value. 25 Using an active subnetwork enrichment analysis approach, PathfindR outputs a table that represents enriched pathways identified from the protein list inputted into the program. We used the KEGG pathway database (https://www.genome.jp/kegg/pathway.html) which includes a manual collection of pathway maps examining a total of 777,729 molecular pathways, with 544 main pathways included. Only pathways that had a p-value of <=0.01 were considered. The final table produced by PathfindR includes a table of significant pathways with an associated adjusted p-value, a fold enrichment value of the pathway, the lowest and highest p-values generated from each iteration of the pathways analysis, and the upregulated and downregulated proteins from the input protein list for every pathway. 25 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. N=15) patients compared to healthy controls (C). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. T-cells (N=19) and IFNγ, CXCL9, and IL-10. Dots are colored by disease category. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 20, 2021. ; https://doi.org/10.1101/2021.04.13.21255439 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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Boston, MA: PBC; 2020. 59. Van der Maaten L, Hinton G. Visualizing data using t-SNE We gratefully acknowledge the support of the Children's Hospital of Philadelphia Frontiers program and the assistance of Mr. Jansen Weaver, Mr. Daniel Fields and Mr. Kienan O'Brien. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.