*Purpose: COVID-19 has poor outcomes in transplant recipients with reduced antibody responses. However, the exact cellular immune response against SARS-CoV-2 remains largely unclear. We developed novel assays to analyze differential cellular immune responses in individual subjects and groups.

*Methods: We assessed the T cell proliferative responses against spike, membrane, and nuclear proteins of SARS-CoV-2, or a mixture of all these peptides (mix) using ³H-thymidine incorporation and CFSE dilution assays. We have also established a SARS-CoV-2-specific multiplexed cytokine IsoLight at single-cell resolution. This is a very powerful technology that employs IsoPlexis’ IsoCode®chip with 12,000 micro-chambers. Each microchamber is pre-coated with a 32-plex antibody array to capture secreted cytokines. The results were evaluated using IsoSpeak software.

*Results: COVID-19 convalescent subjects (n=3) showed a very strong proliferative response to S/M/N and mix peptides of SARS-CoV-2 when compared to uninfected normal subjects who had only marginal proliferative responses. CFSE dilution assays demonstrated that spike and mix proteins markedly increased the proliferation of CD3 cells comprised of both CD4 and CD8 subsets. In the IsoLight assay, single-cell functional heterogeneity mapping 3D tSNE analysis showed a distinct combinatorial cytokine secretion pattern in stimulated cells compared to unstimulated controls (Fig.1). Polyfunctional activity topography-Principal component analysis (PAT-PCA) revealed that IFN-g, IL2, and MIP-1b drove the polyfunctional heterogeneity. When the percentage of polyfunctional cells (≥2 cytokines/cell), and polyfunctional strength index (PSI) were evaluated, CD8 cells secreted high levels of effector and chemoattractive cytokines while CD4 cells secreted effector and stimulatory cytokines. Most importantly, the depth and breadth of T cell responses, particularly the cytokine polyfunctionality correlated with the severity of the disease the patients had experienced.

*Conclusions: This novel COVID19-specific IsoLight cytokine assay is a powerful technology that can be utilized for in-depth analysis of T cell polyfunctionality at the single-cell level and for further differentiating the anti-SARS-CoV-2 immune capabilities of vulnerable individuals such as transplant patients.

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