T cell immunity toward SARS-CoV-2 spike (S-), membrane (M-), and nucleocapsid (N-) proteins may define COVID-19 severity. Therefore, we compare the SARS-CoV-2-reactive T cell responses in moderate, severe, and critical COVID-19 patients and unexposed donors. Overlapping peptide pools of all three proteins induce SARS-CoV-2-reactive T cell response with dominance of CD4 + over CD8 + T cells and demonstrate interindividual immunity against the three proteins. M-protein induces the highest frequencies of CD4 + T cells, suggesting its relevance for diagnosis and vaccination. The T cell response of critical COVID-19 patients is robust and comparable or even superior to non-critical patients. Virus clearance and COVID-19 survival are not associated with either SARS-CoV-2 T cell kinetics or magnitude of T cell responses, respectively. Thus, our data do not support the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. Conversely, it indicates that activation of differentiated memory effector T cells could cause hyperreactivity and immunopathogenesis in critical patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8+ T cell subsets. PD-1-expressing CD8+ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. IMPORTANCE Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients.

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SARS-CoV-2 infection induces a T cell response that most likely contributes to virus control in COVID-19 patients, but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients.Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B, as well as perforin within different effector CD8+ T cell subsets. PD-1 expressing CD8+ T cells also produced cytotoxic molecules during acute infection indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2.Our data provides valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development.ImportanceCytotoxic T cells are responsible for the elimination of infected cells and are key players for the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group in comparison to younger patients.

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Preventing the progression to acute respiratory distress syndrome (ARDS) in COVID-19 is an unsolved challenge. The involvement of T cell immunity in this exacerbation remains unclear. To identify predictive markers of COVID-19 progress and outcome, we analyzed peripheral blood of 10 COVID-19-associated ARDS patients and 35 mild/moderate COVID-19 patients, not requiring intensive care. Using multi-parametric flow cytometry, we compared quantitative, phenotypic and functional characteristics of circulating bulk immune cells, and SARS-CoV-2 S-protein reactive T cell between the two groups. ARDS patients demonstrated significantly higher S-protein reactive CD4 + and CD8 + T cells compared to non-ARDS patients. Of interest, comparison of circulating bulk T cells in ARDS patients to non-ARDS patients demonstrated decreased frequencies of CD4 + and CD8 + T cell subsets with activated memory/effector T cells expressing tissue migration molecule CD11a ++ . Importantly, survival from ARDS (4/10) was accompanied by a recovery of the CD11a ++ T cell subsets in peripheral blood. Conclusively, data on S-protein reactive polyfunctional T cells indicate the ability of ARDS patients to generate antiviral protection. Furthermore, decreased frequencies of activated memory/effector T cells expressing tissue migratory molecule CD11a ++ observed in circulation of ARDS patients might suggest their involvement in ARDS development and propose CD11a-based immune signature as a possible prognostic marker.

Identification of immunogenic targets of SARS-CoV-2 is crucial for monitoring of antiviral immunity and vaccine design. Currently, mainly anti-spike (S)-protein adaptive immunity is investigated. However, also the nucleocapsid (N)-and membrane (M)-proteins should be considered as diagnostic and prophylactic targets.The aim of our study was to explore and compare the immunogenicity of SARS-CoV-2 S-, Mand N-proteins in context of different COVID-19 manifestations. Analyzing a cohort of COVID-19 patients with moderate, severe, and critical disease severity, we show that overlapping peptide pools (OPP) of all three proteins can activate SARS-CoV-2-reactive T-cells with a stronger response of CD4 + compared to CD8 + T-cells. Although interindividual variations for the three proteins were observed, M-protein induced the highest frequencies of CD4 + T-cells, suggesting its relevance as diagnostic and vaccination target. Importantly, patients with critical COVID-19 demonstrated the strongest T-cell response, including the highest frequencies of cytokine-producing bi-and trifunctional T-cells, for all three proteins. Although the higher magnitude and superior functionality of SARS-CoV-2-reactive T-cells in critical patients can also be a result of a stronger immunogenicity provided by severe infection, it disproves the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. To this end, activation of effector T-cells with differentiated memory phenotype found in our study could cause hyper-reactive response in critical cases leading to immunopathogenesis. Conclusively, since the S-, M-, and N-proteins induce T-cell responses with individualdifferences, all three proteins should be evaluated for diagnostics and therapeutic strategies to avoid underestimation of cellular immunity and to deepen our understanding of COVID-19 immunity.