Superantigenic TCR Vbeta 21.3 signature in Multisystem Inflammatory Syndrome in Children

ObjectivesMultiple Inflammatory Syndrome in Children (MIS-C) is the most severe pediatric form of COVID-19 and occurs in previously healthy children. MIS-C combines features of Kawasaki disease and Toxic Shock Syndrome (TSS).

MethodsChildren with suspected MIS-C were included within the first week of diagnosis and a large scale immunoassay was performed to determein the immunologic signature of these patients.

ResultsWe characterized the immunological profile of 27 MIS-C cases in comparison with 4 KD and 4 TSS cases. Similarly to TSS, an increase of serum inflammatory cytokines (IL-6, TNF-a, CD25s) was observed in MIS-C contrasting with low expression of HLA-DR monocytes, a feature often associated with immune paralysis. Expansions of T cells expressing the V{beta}21.3 T cell receptor {beta} chain variable region were detected in both CD4 and CD8 subsets in almost 50% of patients and V{beta}21.3-positive T cells expressed high level of HLA-DR highlighting their specific activation. TCR sequencing uncovered the polyclonal nature of the V{beta} 21.3+ population. SARS-CoV2 antigene-specific production of interferon gamma in T cells was not increased in MIS-C T cells compared to COVID-19 patients suggesting the antigen-specific immune response in MIS-C patients is not pivotal to the manifestation.

ConclusionsOur findings argue in favor of a strong activation of the immune system related to a superantigenic immune response in MIS-C with a specific polyclonal V{beta}21.3 T cell expansion.

Key messagesWhat is already known about this subject ?

MIS-C occurs 3-5 weeks after acute SARS-CoV2 infection and overlap features of Toxic Shock syndrome and Kawasaki disease.

MIS-C appears different in term of cytokine and autoantibodies generation from KD with subtle signs of T cells activation

What does this study add?

This study demonstrates that V{beta}21.3+ CD4 and CD8 T cells are highly increased in about 50% of MIS-C and distinctive of the V{beta}2+ expansion observed in toxic shock syndrome in This reflects a specific T cell activation and cytokine release syndrome similar to toxic shock syndrome

How mich this impact on clinical practice or future developments?

V{beta}21.3+ signature can be available on a short term basis by flowcytometry and represents a signature of the MIS-C.

As for TSS, immunomodulating therapies may revert the superantigenic activation and resolve this life threatening pediatric condition.


What does this study add?
This study demonstrates that Vβ21.3+ CD4 and CD8 T cells are highly increased in about 50% of MIS-C and distinctive of the Vβ2+ expansion observed in toxic shock syndrome in This reflects a specific T cell activation and cytokine release syndrome similar to toxic shock syndrome How mich this impact on clinical practice or future developments?
Vβ21.3+ signature can be available on a short term basis by flowcytometry and represents a signature of the MIS-C.
As for TSS, immunomodulating therapies may revert the superantigenic activation and resolve this life threatening pediatric condition.

Introduction
At the end of April 2020, European clinicians warned the Public Health Agencies about an abnormal increase of Kawasaki-like diseases (KLD) and myocarditis requiring critical care support in the context of the ongoing COVID-19 epidemic in children (1)(2)(3). Later on, American clinicians also reported a large outbreak of severe inflammation in children following COVID-19 infection, a condition that is now named Pediatric Inflammatory Multisystemic Syndrome (PIMS) or Multisystem Inflammatory Syndrome in children (MIS-C) (4)(5)(6). The clinical phenotype of this emerging disease is broad and encompasses features of Kawasaki diseases (KD) and toxic shock syndrome (TSS). Many cases require intensive care support, making MIS-C one of the most severe manifestation of COVID-19 in children.
For now, reports on MIS-C have shown slight differences in cytokine profiling and immunophenotype between MIS-C and KD or pediatric COVID-19 (8,9). Analysis of T cells revealed a lower number of T cells in MIS-C with no or subtle signs of activation (10). Multidimensional immune profiling on small numbers of patients showed differences between acute COVID-19 or pre-pandemic Kawasaki disease (8,11). Anti-SARS-CoV2 antibodies were equally produced in pediatric COVID-19 and MIS-C. However, autoantibodies were uniquely produced during MIS-C or KD, which supports a contribution of the humoral response to both diseases (8,11). Finally, a role for genetic factors has been evocated in MIS-C pathogenesis as it seems to occur more frequently in children from Hispanic or African ethnicity (12,13).
Considering the similarities between MIS-C, KD and TSS, it has been hypothesized that SARS-CoV2 may act as a superantigen and induce a hyperinflammatory state in MIS-C (14,15). Superantigens are molecular structures enabling polyclonal T cell response through binding external region of T cell receptor and MHC class II molecules (16). Consequently, superantigens can induce large expansions of T cells expressing one specific T-cell receptor (TCR) beta-chain variable region while classical antigens induce the expansion of T cells bearing different V . For example, TSS is secondary to the effect of Staphylococcal TSS Toxin-1 (TSST-1) and leads to Vβ2+ T cell expansion (17). In KD, previous reports mentioned the expansion of different Vβ including Vβ2 in response to putative TSST1 but this point is controversial and there could be other causes and triggers of KD (14).
Here, we sought to test the hypothesis of a superantigen-type of T cell activation occurring in MIS-C. For this, we explored the cytokine and cellular immune profile in MIS-C and discovered a state of cytokine storm contrasting with a decrease in T cells and a reduction in their HLA-DR expression. We identified by flow cytometry a specific V 21.3 signature in All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251166 doi: medRxiv preprint 12/25 tested patients with an expansion of more than twice the mean normal values, highlighting a superantigenic type of T cell response in MIS-C. TCR sequencing revealed the polyclonal nature of the Vβ21.3+ expansion. No specific HLA bias was identified in patients but we found a specific activation profile within Vβ21.3+ T cells.
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Patients and ethics
Three distinct cohorts were used for the data collection and analyses registred in ClinicalTrial.gov and consent were obtained from parents. Details on IRB and ethical committee are available in supplemental data.
Cytokines and IFN score assessment Whole blood was sampled on EDTA tubes and plasma was frozen at -20°C within 4 hours following blood collection. Plasma concentrations of IL-6, TNF-a, IFN-g, IL-10, MCP-1, IL-1ra and CD25s were measured by Simpleplex technology using ELLA instrument (ProteinSimple), following manufacturer's instructions. Plasma IFN-α concentrations were determined by single-molecule array (Simoa) on a HD-1 Analyzer (Quanterix) using a commercial kit for IFN-α2 quantification (Quanterix). Whole blood was collected on PAXgene blood RNA tubes (BD Biosciences) or on EDTA tubes for IFN signature, RNA extraction was performed with the kit maxwell 16 LEV simply RNA blood associated with the Maxwell extractor (Promega) and quantified by absorbance (Nanovue). IFN score was obtained using nCounter analysis technology (NanoString Technologies) by calculating the mediane of the normalized count of 6 ISGs as previously described (18) T-cell Vβ repertoire analysis and immunophenotyping CD3, CD4 and CD8 T lymphocyte subsets were enumerated on EDTA-anticoagulated peripheral whole blood by single-platform the fully automated volumetric single plateforme technology flow cytometer AQUIOS CL (Beckman-Coulter) as previously described (19).
The phenotypic analysis of T-cell Vβ repertoire was performed on whole blood sample using the IOTest Beta Mark kit (Beckman-Coulter) containing 24 monoclonal antibodies (mAbs) identifying ~ 70% of the T cell repertoire. Details are provided in supplemental data.
Monocyte HLA-DR expression assessment Monocyte HLA-DR expression was determined on EDTA-anticoagulated peripheral whole blood as previously describedde (20).

TCR-sequencing
RNA was extracted from whole blood as reported above. T cell receptor (TCR) alpha/beta libraries were prepared from 300ng of RNA from each sample with SMARTer Human TCR a/b Profiling Kit (Takarabio) following provider protocol as previously described (32). Briefly, the reverse transcription was performed using a mixture of TRBC and TRAC reverse primers and further extended with a template-switching oligonucleotide (SMART-Seq v4). cDNAs were then amplified following two semi-nested PCR: a first PCR with TRBC and TRAC reverse primers as well as a forward primer hybridizing to the SMART-Seq v4 sequence added by template-switching and a second PCR targeting the PCR1 amplicons with reverse and forward primer including Illumina Indexes allowing for sample barcoding. PCR2 are then purified using AMPUre XP beads (Beckman-Coulter). The sequencing was then carried out on a NOVAseq 6000 Illumina sequencer using 2x250bp read length protocol. Paired-end sequences were aligned and annotated using MiXCR 3.0.13 (21) Repertoire analysis All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251166 doi: medRxiv preprint Analysis were performed in R 4.0.3 on the clonotype lists obtained with MiXCR. For each clonotype, read count was recorded. Frequencies for TRBV, TRBJ and clonotypes were calculated based on the total read counts per sample. Chord diagrams were made using the circlize package(22) on TRBVBJ frequencies , CDR3 length barplots were made using ggplot2 (23) on clonotype frequencies.

Statistical analysis
PCA analysis was made in R with stats package and visualized with ggplot2 (23) on Vbeta frequencies obtained by flow cytometry. All rights reserved. No reuse allowed without permission.
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MIS-C presentation overlaps with Toxin Shock Syndrome and KD phenotype
Here, we studied a cohort of 27 children with MISC and compared them with 4 KD diagnosed during the pandemic and 4 retrospective cases of TSS patients. This comparison was motivated by previous descriptions of MIS-C in Europe and in the US that showed a clinical overlap between staphylococcal toxin-mediated TSS and KD in patients with MIS-C. Figure   1 outlines the study flowchart ( Figure 1A) and the clinical and biological parameters ( Figure   1B) that were evaluated in our study. Children samples were not available for all experiments depending on the volume available. In patients with suspicion of MIS-C, we separated KD from MIS-C based on the positivity of the SARS-CoV2 PCR or serology and the absence of SARS-CoV2 exposure history. All three groups of patients were then subjected to a deep immunological analysis combining cytokine profiling, Vβ analysis and for MIS-C in vitro T cell proliferation assays ( Figure 1A). We confirmed the strong clinical overlap between MIS-C, TSS and KD. Indeed, many patients in the MIS-C group fulfilled the major criteria for TSS and KD respectively ( Figure 1B). Considering the clinical parameters, the most frequent features of MIS-C patients in our cohort were fever, cardiac dysfunction, gastrointestinal symptoms, coagulopathy and inflammation (Table 1) Interferons were usually low in MIS-C patients, except for a small subset of patients with high levels of IFN-γ ( Figure 2A). The expression of Interferon-stimulated genes (ISG) in blood cells was accordingly lower in MIS-C patients than in patients with different forms COVID-19 ( Figure 2B). MIS-C patients were rather characterized by high levels of serum TNF, soluble CD25 (rs IL2), IL-1 Ra, IL-10 and IL-6, which was very similar to TSS patients, and different from COVID-19 patients, irrespective of the form. The few KD patients that we could analyse had rather undetectable levels of serum Interferons (Figure 2A) as well as TNFa, IL10 and sCD25 and detectable but low levels of MCP1, IL6 and IL1RA ( Figure 2B). The similarity 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. between MIS-C and TSS cytokines is thus consistent with a cytokine release in MIS-C following superantigenic reaction(28).
To further explore MIS-C immunological profile, we then quantified the number of peripheral lymphocytes of different types, as well as the expression of HLA-DR in patients. T and NK cell counts were on average very low in MISC and KD patients while B cell counts were normal ( Figure 2C). HLA-DR is a marker of T and monocyte activation. We found a decreased expression of HLA-DR in monocytes and normal to low values in T cells in both KD and MIS-C patients compared to controls( Figure 2D). This observation is consistent with immune unresponsiveness, as seen following TSS or septic shock(29-32). Indeed, low HLA-DR expression in monocytes is considered as a very good marker of sepsis-induced immunosuppression(32). (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 February 12, 2021. Interestingly, MIS-C T cells also responded poorly to stimulation with a cocktail of EBV/CMV/Influenza-specific peptides, supporting the notion of T cell anergy in MIS-C, a feature also observed in TSS or septic shock (data not shown).
Finally, we explored whether the HLA subset could be important in the superantigenic activation of patients, and we further sequenced HLA class I and class II genes in 7 patients and analyzed HLA alleles in 75 additional patients from available exome data by genetic inference but did not identify any specific recurrent subset in MIS-C (data not shown). All rights reserved. No reuse allowed without permission.
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The term superantigen (SAg) has been coined by Kappler and Marrack as an operational definition of various T-cell activating substances with specificity for T cell antigen receptors
Vβ subunits regardless of the rearrangement and antigen-specificity (34). TSS is a severe toxic shock secondary to the superantigen TSST-1 that stimulates human T cells expressing TCR Vβ2(35). A superantigenic T cell response has been already suspected in KD but the multiple infectious agents associated with this disease hampered the exploration of this phenomenon (14). Yet, many KD features suggest this disease is indeed caused by an infectious agent(36). In particular, the efficacy of IV immunoglobulins, the occurrence in Altogether, our study supports that MIS-C present a superantigenic responses with a specific Vβ21.3 activated T cell expansion and its place for early diagnosis and/or prognosis has to be further explored. 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.  OT and VB explored HLA in MIS-C patients and JLC, LA supervised genetic inference exploration of HLA. IR and EC provided biobanking and help to generate material for the study TW and AB supervised, designed and funded this study. TW and AB prepared the initial draft. All authors critically reviewed the paper and agreed on the final form.
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(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 February 12, 2021. ; https://doi.org/10.1101/2021.02.11.21251166 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.
(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.  (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.  (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 February 12, 2021.

MIS-C 27
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