(A) The levels of nucleocapsid antibodies (Units, 0.8-1.4 borderline positive and > 1.4 for positive result) (left) and spike antibodies (BAU/ml, 30 for positive result) (right) were measured in sera from inclusion up to 6-8 months. associated with T cell activation were upregulated at inclusion, and in the case of CD69+ CD4+ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant HLM006474 and long-term alterations in T cell populations and key HLM006474 events associated with COVID-19 pathogenesis. Keywords: SARS-CoV-2, COVID-19, T cell activation, T cell impairment, T cell subsets, neutralizing antibodies, spectral flow cytometry Introduction More than two years since initial reports of the disease, the COVID-19 pandemic has claimed approximately six million lives world-wide, leaving some individuals with long-term health consequences (1, 2). The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gains entry into host cells after first binding to angiotensin-converting enzyme 2 (ACE2) the receptor binding domain (RBD) of the viral spike (S) protein, followed by activation by transmembrane protease, serine 2 (TMPRSS2) or cathepsin L (3C5). While a vast majority of infected individuals remain asymptomatic or pauci-symptomatic, data from early on during the pandemic before the vaccine rollout HLM006474 showed severe and life-threatening disease to occur in 14% and 5-6% of cases, respectively (6, 7). Approximately 20% of all infected individuals, especially the elderly and those with co-morbidities require hospitalization. In fatal cases of COVID-19, death often occurs due to HLM006474 organizing pneumonia (OP), acute respiratory distress syndrome (ARDS) or sepsis-like cytokine storm-associated multi-organ failure (8C10). The hyperinflammation in COVID-19 is caused by the activated inflammatory cells recruited in response to infection and not by the virus per se (11C13). Furthermore, the variations in initial host responses appear to determine the varied disease spectrum observed in COVID-19 (14, 15). Clearance of viral infection involves both cellular and humoral immune responses (16, 17). Virus-specific CD4+ and CD8+ T cells are present in almost all individuals who have had a prior episode of COVID-19 (18, 19), with lower numbers of these cells linked to disease severity (20C22). Furthermore, the rapid appearance of functional SARS-CoV-2-specific T cells is linked to mild disease presentation (17). The humoral immune response also plays a critical role in viral clearance in the host. The SARS-CoV-2-specific antibody responses vary in levels, as well as quality between asymptomatic individuals and those presenting with severe disease. While a few studies have found higher levels and longer duration in more severe cases, others have reported no observable difference (23C29). Follicular helper T cells (TFH) are primarily defined as CXCR5, programmed cell death protein 1 (PD-1), and Bcl-6 expressing CD4+ T cells. The frequency of circulating TFH cells is often low during homeostasis and is believed to mirror their numbers in the germinal centers of peripheral lymphoid organs, where they provide critical support to antigen-specific B cells to facilitate the development of long-term and high-affinity antibodies (30C34). Effects on peripheral TFH cells have been documented in a few studies covering acute (35), mild (26), and severe/fatal SARS-CoV-2 infections (19). However, not many have followed the infected population longitudinally (18, 36). GLURC Peripheral TFH cells have been shown to be linked to neutralizing antibodies (nAb) in diseases such as HIV-1 (37), and now SARS-CoV-2 (25, 28, 38). Furthermore, individuals who have recovered from severe compared to HLM006474 non-severe COVID-19 reportedly have higher nAb titers and CXCR3+ TFH cell frequencies, and faster recovery of lymphocyte levels, a month after leaving the hospital (39). Viral infections lead to the activation of T cells with upregulated expression of markers that define early, i.e., CD69 positive and late, i.e., CD38 and HLA-DR double positive, activation (40, 41). Activated CD4+ and CD8+ T cells exist during active/ongoing COVID-19 and in certain individuals, even after recovery, activated CD8+ T cells persist for over 100 days (42). A case study of mild COVID-19 reported activated CD38+HLA-DR+CD8+ T cells at day 7, which surged to a peak at day 9 but remained high even at day 20. Activated CD4+ T cells were also present, but at lower levels (19). Many studies support the activation of T cells and B cell responses during the onset of COVID-19 disease, but the infection also leads to transient immunosuppression, especially during severe disease (43). The manifestation of bad immune checkpoint molecules on CD8+ and CD4+ T cells examined in 14 individuals with severe COVID-19 revealed an increased abundance.