ABSTRACT
Background & Aim: T-cell responses to SARS-CoV-2 have been described in convalescent patients;and adoptive immunotherapy in immunocompromised individuals may be a viable therapeutic strategy to treat or prevent COVID-19 related morbidity and mortality. However, sparse data exists regarding the ideal cytokines to use during viral specific T-cell (VST) expansion in general, and coronavirus specific T-cells (CST) in particular, to maximize specificity and cell count. Here, we demonstrate that polyfunctional coronavirus-specific T cells (CSTs) can be generated rapidly from convalescent donors, and that CSTs can be optimized by varying cytokine expansion cocktails. Methods, Results & Conclusion: PBMCs from SARS-CoV-2 convalescent donors were stimulated with overlapping peptide pools encompassing membrane, spike, envelope and nucleocapsid proteins, and expanded in 96-well plates for 10 days. Cells were expanded either with IL4 (400IU/mL) +IL7 (10ng/mL), IL15 (5ng/mL), IL15+4, IL15+6 (100ng/mL), or IL15+7. After expansion, T-cell phenotype and functionality were assessed using ELISpot and intracellular flow cytometry. Cytokine combinations with the best performance on micro-expansion were retested using GMP-applicable methods. [Figure presented] [Figure presented] CST expansion was maximized using IL15+7 (total CD3+8.2E4±2.6E4 cells) and IL 4+7 (total CD3+ 8.4E4±2.2E4 cells) as compared with IL15 (total CD3+6.1E4±1.0E4 cells), IL4+15 (total CD3+6.6E4 cells±1.4E4 cells), or IL15+6 (total CD3+ 6.1E4±1.7E4 cells). CD4+ specificity was maximized using IL4+7, IL15+6, and IL15+7 (Fig. 1), while CD8+ specificity was maximized using IL15+7, IL15+6, and IL15 (Fig.e 2). T-cell phenotype was similar across cytokine conditions (median 6.9±0.2% naïve, 8.3±0.8% central memory, 79±0.9% effector memory, 4.5±0.2% terminal effector). NK cell expansion was increased in the setting of IL15 and its combinations (median 10.4±12% CD3-/CD56+ versus 1.0±1% without IL-15 p<0.05). Comparison of CSTs grown with IL4+7 and IL15+7 in G-Rex-10 bioreactors showed increased expansion and specificity for spike and membrane peptides with IL15+7 as compared with IL4+7 (Fig. 3). Our study demonstrates that the generation of polyfunctional CSTs targeting SARS-CoV-2 can be optimized using IL15+7 as compared with other cytokine cocktails. We are now translating these techniques to the clinic to examine the safety of adoptive transfer of IL15+7 expanded CSTs for the prevention or early treatment of SARSCoV-2 infection in immunocompromised patients.