*Purpose: The need to evaluate SARS-CoV-2 immune responses is an important clinical research focus. Detecting neutralizing antibodies is one measure of immune response, but the ability to evaluate CD4 and CD8 T cell responses specific to SARS-CoV-2 is also an important measure of immune status. The role these two arms of the immune system play in protection is currently unknown.

*Methods: We developed a whole blood flow cytometry assay that detects functional CD4 and CD8 responses to stimulation by peptide antigen pools encompassing the spike (S1 – receptor binding domain (RBD) and S2) and nucleocapsid (N) proteins to address this need. Activated T cells were defined as CD4 or CD8 T cells co-expressing the CD69 activation marker with IFN-γ, TNF-α or IL-2. Polyfunctional T cells, expressing multiple cytokines, were also identified. This assay was validated using whole blood from SARS-CoV-2 recovered volunteers and apparently normal donors who self-reported as uninfected by SARS-CoV-2.

*Results: The range in responses varied by donor and was not correlated with the reported severity of their disease. A positive response was defined as a 3-fold increase in the number of activated T cells over the unstimulated control. Of the 32 SARS-CoV-2 recovered donors tested, 25 responded to two or three of the peptide pools and five did not respond to any SARS-CoV-2 peptides. Responses ranged from 0.03% up to 1.23% of the CD4 or CD8 population. The responding T cells were predominantly CD4, but when CD8 T cells responded they tended to recognize the nucleocapsid peptides most strongly. Data from one individual with a robust response are shown below.

For uninfected donors, five of 21 showed a low, but detectable response to stimulation with at least one peptide pool. These responses could be attributed to an asymptomatic infection, or to cross-reactive T cells specific to one of the seasonal respiratory coronaviruses.

*Conclusions: In summary, we developed a sensitive assay to detect SARS-CoV-2 spike- and nucleocapsid-specific T cell responses in whole blood samples. This assay will be valuable in monitoring SARS-CoV-2 T cell response and duration in infected individuals. Measuring the T cell responses to SARS-CoV-2 vaccinated individuals is currently under investigation.

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