A unique cytotoxic CD4+ T cells signature defines critical COVID-19

Background and objectives SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T cell response to the resolution or progression of disease is still unclear. Optimal protective immunity may require activation of distinct immune pathways. As such, defining the contribution of individual T cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines. To address this, we performed immunophenotyping of T cell responses in unvaccinated individuals, representing the full spectrum of COVID-19 clinical presentation. Methods Spectral cytometry was performed on peripheral blood mononuclear cell samples from patients with PCR-confirmed SARS-CoV-2 infection. Computational and manual analyses were used to identify T cell populations associated with distinct disease states through unbiased clustering, principal component analysis and discriminant analysis. Results Critical SARS-CoV-2 infection was characterised by an increase in activated and cytotoxic CD4+ (CTL) cells of a T follicular helper (TFH) or effector memory re-expressing CD45RA (TEMRA) phenotype. These CD4+ CTLs were largely absent in those with less severe disease. In contrast, those with asymptomatic or mild disease were associated with high proportions of naïve T cells and reduced expression of activation markers. Conclusion Highly activated and cytotoxic CD4+ T cell responses may contribute to cell-mediated host tissue damage and progression of COVID-19. Potential for induction of these detrimental T cell responses should be considered when developing and implementing effective COVID-19 control strategies. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by MRFF COVID-19 Vaccine Candidate Research Grant 2007221 (J.A.T., C.C., M.S., A.L.F.). F.M.‐W. and T.M.A. are supported by the International Society for the Advancement of Cytometry (ISAC) Marylou Ingram Scholars program. COSIN cohort was supported by Snow Medical Foundation as an investigator-initiated study. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics approval was granted by the Royal Prince Alfred Hospital ethics committee (X20-0117 and 2020/ETH00770), or the Human Research Ethics Committees of the Northern Sydney Local Health District and the University of New South Wales, NSW Australia (ETH00520). Verbal/written consent was given by all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.