CD4⁺ T Cell Dysregulation in Long COVID: Insights from an Unvaccinated Cohort

Objective: The pathogenesis of Long COVID (LC) remains poorly understood, with the analysis of immune responses often obscured by variables such as vaccination and reinfection. This study aimed to determine the primary immunological footprint of LC by examining a specific cohort that minimizes these confounding factors. Methods: We analyzed a Brazilian cohort of patients recruited between September 2020 and February 2021, during the first wave of the pandemic (Wuhan-Hu-1 variant), comprising mostly unvaccinated individuals. We assessed humoral responses to SARS-CoV-2 and latent viruses in 104 patients. Additionally, we investigated the immune signatures of CD4+ T cells in a subset of 6 LC and 4 recovered control (RC) unvaccinated patients, recruited 1 to 2 months after symptom onset, utilizing a combination of flow cytometry and single-cell RNA sequencing. Results: Systemic humoral responses to SARS-CoV-2 and reactivated latent viruses were comparable between the LC and RC groups, as were the overall distributions of CD4+ T cell subsets. However, single-cell RNA sequencing revealed distinct immune signatures. Antigen-responsive CD4+ T cells in LC patients demonstrated a signature of acute activation, characterized by the upregulation of genes such as CD38 and interferon-stimulated genes, including IFITM1. Furthermore, gene set enrichment analysis indicated that LC patients exhibit increased expression of interferon-alpha/gamma signatures compared to RC. Analysis of the T cell receptor repertoire presented no evidence of clonal expansion. Discussion: Our findings imply that LC immunopathology is driven by a qualitative T cell dysfunction characterized by CD4+ non-proliferative activation more than one month after symptom onset. This pattern is consistent with persistent antigen stimuli, though the underlying mechanism and its therapeutic implications require further investigation