key: cord-0781482-y9x1ldjw authors: Alves, I.; Vicente, M. M.; Gaifem, J.; Fernandes, A.; Dias, A. M.; Rodrigues, C. S.; Oliveira, J. C.; Seixas, N.; Malheiro, L.; Abreu, M. A.; Castro, R. S. e.; Pinho, S. S. title: SARS-CoV-2 infection drives a glycan switch of peripheral T cells at diagnosis date: 2021-02-22 journal: nan DOI: 10.1101/2021.02.17.21251918 sha: 5b186ba05148474e36eddde95e613e8ca763cfa6 doc_id: 781482 cord_uid: y9x1ldjw COVID-19 is a highly selective disease in which SARS-CoV-2 infection can result in different clinical manifestations ranging from asymptomatic/mild to severe disease that requires hospitalization. Here, we demonstrated that SARS-CoV-2 infection results in a glycosylation reprogramming of circulating lymphocytes at diagnosis. We identified a specific glycosignature of T cells, defined upon SARS-CoV-2 infection and apparently triggered by a serological factor. This specific glycan switch of T cells is detected at diagnosis being more pronounced in asymptomatic patients. We further demonstrated that asymptomatic patients display an increased expression of a viral-sensing receptor, through the up-regulation of DC-SIGN in monocytes. We showed that higher levels of DC-SIGN in monocytes at diagnosis correlates with better COVID-19 prognosis. These new evidences pave the way to the identification of a novel glycan-based response in T cells that may confer protection against SARS-CoV-2 infection in asymptomatic patients, highlighting a novel prognostic biomarker and potential therapeutic target. 0.5% and 3.5%(1, 2). COVID-19 is a highly selective disease. In fact, only some dysregulation, the so-called "cytokine storm" associated with critical illness(7-10). 68 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in In this study, we discovered that circulating T cells exhibit a glycan switch upon SARS- CoV-2 infection that is detected at COVID-19 diagnosis. This change in the 95 glycosylation profile of T cells appears to be triggered by a serum inflammatory factor 96 present in infected patients. This specific T cell glycan switch is more pronounced in 97 asymptomatic patients, rather than in those who exhibit symptoms. Importantly from a 98 clinical standpoint, we also unveiled that COVID-19 patients with good prognosis 99 exhibit an upregulation of DC-SIGN expression in circulating monocytes. 100 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Cohort description and patient's selection criteria 103 The present study integrates a total of 32 patients diagnosed with SARS-CoV-2 104 from 2 individual Portuguese cohorts (between May 2020 and July 2020), 20 patients perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in In vitro assessment of glycan modulation of T cells 161 PBMCs from healthy and independent from the COVID-19 cohirt human donors 162 were isolated from fresh collected blood, as described above. PBMCs were cultured in 163 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in plasma concentration (25% and 10%) as well as time of culture (4 days, 2 days, 24h and 167 12h). The optimal culture condition (with lower cell death and stronger glycan 168 modulation) was selected (10% human plasma and 24h cell culture). After 24h cell 169 glycoprofile was evaluated using the same antibodies and lectin panel selected above. Figure 1E) , there are no significant differences between the mean age 219 of asymptomatic and symptomatic patients (Supplemental Table 1 ). Curiously, for 220 both glycans, the two COVID-19 convalescent patients seem to recover (or surpass) the 221 levels of expression of β1,6-GlcNAc branched and α2,6-sialic acid glycan structures in 222 all T cell subsets ( Fig. 1A and B, right) . These results showed that CD8 + T cells from perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The differences observed in the glycosylation profile of T cells across severity, were not 226 related to the abundance or activation of T cell populations, since CD3 + , CD4 + , CD8 + 227 and γδ T cells showed similar frequencies as well as similar levels of PD1 and CD69 228 expression between the groups, with the exception of lowered CD3 + population and 229 increased CD69 + within CD8 + T cells, in the mild disease group (Supplemental Figure 230 1A, B and C) . 231 In addition, and since glycosylation is a dynamic process that can change along with 232 disease status, we took advantage of a limited longitudinal follow-up analysis using a Figure 1A and B) . 240 These results showed that SARS-CoV-2 infected individuals led to an en bloc decreased 241 expression of specific glycan structures such as ß1,6-GlcNAc branched N-glycans and 242 α2,6-sialic acid in T cells, predominately in CD8 + T cells and asymptomatic patients. The absence of differences on high mannose structures (GNA binding) or fucose 244 residues on T cells (AAL binding), support the specificity of the glycan switch. In fact, 245 decreased levels of β1,6-branched N-glycans in T cells have been pointed out as a 246 mechanism of TCR threshold regulation, by hampering the TCR complex clustering and 247 lowering the necessary amount of antigen-recognition to signal a cellular response(28). Moreover, surface α2,6-sialylation also regulates T cell immune response through 249 galectins binding modulation(29-31). In order to further clarify the underlying mechanism that imposes the changes in the (Figure 2A) and α2,6-sialylation (Figure 2B) , when compared to T 257 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 268 To gain further insights in the viral-glycan recognition mechanism, we have Supplementary Table 1) . 290 Remarkably, the expression levels of DC-SIGN in monocytes were found to be able to ("Glyco-Immune Alert" mechanism) may constitute a novel mechanism of host-318 response, that contributes to further understand the immunological differences among 319 infected individuals (36) . 320 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251918 doi: medRxiv preprint A 385 dynamic COVID-19 immune signature includes associations with poor prognosis Omer, and 397 A. Iwasaki. 2020. Longitudinal analyses reveal immunological misfiring in severe 398 COVID-19 and 400 U. HLH Across Speciality Collaboration. 2020. COVID-19: consider cytokine storm 401 syndromes and immunosuppression Glycans as Key Checkpoints of 404 T Essentials of Glycobiology Metabolic control of T cell immune response through glycans in 412 All rights reserved preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted Glycans as 417 Immune Checkpoints: Removal of Branched N-glycans Enhances Immune Recognition 418 Preventing Cancer Progression Roles for major histocompatibility complex 420 glycosylation in immune function Negative 422 regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation Genetics and the 427 environment converge to dysregulate N-glycosylation in multiple sclerosis T-cell growth, cell surface 430 organization, and the galectin-glycoprotein lattice Genetic Variants of the MGAT5 Gene Are Functionally Implicated in the Modulation of T Cells Glycosylation and 435 Plasma IgG Glycome Composition in Ulcerative Colitis Bitter-sweet symphony: glycan-lectin interactions in virus biology All rights reserved. No reuse allowed without permission. perpetuity preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted Site-445 specific glycan analysis of the SARS-CoV-2 spike DC-SIGN and DC-SIGNR interact with the 449 glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome 450 coronavirus CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in 454 lung and kidney epithelial and endothelial cells Interleukin-2, Interleukin-7, T 456 cell-mediated autoimmunity, and N-glycosylation Dysregulation of T cell receptor N-glycosylation: a molecular 460 mechanism involved in ulcerative colitis Cell surface sialic acid and the regulation 462 of immune cell interactions: the neuraminidase effect reconsidered Negative 465 regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation Alteration of cell surface sialylation 468 regulates antigen-induced naive CD8+ T cell responses All rights reserved. No reuse allowed without permission. perpetuity doi: medRxiv preprint Kedzierska. 2020. Breadth of concomitant immune responses prior to patient recovery: 472 a case report of non-severe COVID-19 The danger model: a renewed sense of self Clinical and immunological assessment of asymptomatic 477 SARS-CoV-2 infections IL-2 and IFN-γ are biomarkers of 481 SARS-CoV-2 specific cellular response in whole blood stimulation assays Trained Immunity: a Tool for Reducing Susceptibility to and the Severity of SARS-CoV-2 Infection IL-6 and CD8+ T cell 490 counts combined are an early predictor of in-hospital mortality of patients with COVID All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in poor Px in grey) as well as the severity at diagnosis. Each dot represents one patient 519 N=24. Gating strategies for each population are included in Supplemental Figure 1A . 520 Mann-Whitney t-test was performed to evaluate statistically significance differences. * 521 p-value < 0.05, ** < 0.005, or represented. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251918 doi: medRxiv preprint