(A) Ex-vivo culture induces granzyme B and perforin in SARS-CoV-2-specific T cells. granzyme B and perforin (n=15). (A) Ex-vivo culture induces granzyme B and perforin in SARS-CoV-2-specific T cells. (B) SARS-CoV-2 specific T cells (n=15) were treated with S, Tretinoin M, or N peptide mixtures for at least 18 hours. Stimulated cells were collected for flow cytometric analysis of granzyme-b-producing, and granzyme-b- and perforin-producing cells of each lymphocyte subset. The levels remain unchanged after antigenic re-stimulation. (C) Cell culture supernatants from recovered (n=6, red circles with white bar) and unexposed (n=9, black circles with gray bar) individuals after re-stimulation were collected for granzyme B and perforin measurement. Results represent data from 3 unexposed and 2 recovered individuals. *p .05; **p .01; *p .001. Image_3.tif (72K) GUID:?6B359F94-10A6-4A6E-8490-F71B76E710CB Supplementary Figure?4: Activation markers and related cytokine production of SARS-CoV-2 specific T cells between unexposed and recovered individuals (n=15). (A) HLA?DR+CD38+, CD25+, CD69+ and CD154+ for all lymphocyte subsets were measured prior to, and after 21 days of culture. (B) SARS-CoV-2-specific T cells from recovered (n=6, red circles with white bar) and unexposed (n=9, black circles with gray bar) individuals were treated with S, M, or N peptide mixtures for at least Tretinoin 18 hours, and the supernatant was collected to measure T-cell activation-related cytokine production. Results represent data from 9 unexposed and 6 recovered individuals. Image_4.tif (81K) GUID:?C3E6DA6D-8297-4608-B494-106DEF6CA668 Supplementary Figure?5: (A) The percentages of CD4+CD25highCD127low, CD4+CD25lowCD127high; Tcons, and CD4+CD25highCD127high cells were compared between recovered (n=5) and unexposed (n=9) individuals. (B) Inhibitory markers including PD-1, Tim-3, and LAG-3 were upregulated following cell culture in each lymphocyte subset. Image_5.tif (36K) GUID:?3CF79A5B-9C5B-40FD-B0A5-9A4B9466C5F0 Data Availability StatementThe original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author. Abstract Background Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer Tretinoin of SARS-CoV-2 specific T cells. Methods To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Results Our Tretinoin results show that SARS-CoV-2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Rabbit Polyclonal to DRD4 Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. Discussion In conclusion, our findings show that SARS-CoV-2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients. One Sentence Summary Ex-vivo expanded SARS-CoV-2 antigen specific T cells Tretinoin developed as third-party partial HLA-matching products may be a promising approach for treating severe COVID-19 patients that do not respond to previous treatment options. Culture-Expanded SARS-CoV-2 Specific T Cells Mainly Consisting of Effector Memory Cells We collected either 50 cc of peripheral blood, or one blood volume leukapheresis from both COVID-19 recovered and unexposed donors. Following isolation, PBMCs were stimulated with SARS-CoV-2 S, M, and N peptides along with IFN-. We then induced cell proliferation by addition of IL-2 and expanded the cultures for 21 days ( Figure?1A ). While the expansion rate varied among individuals, overall expansion seemed to be more effective in COVID-19 recovered individuals (range: 1.20C166.50, median: 23.7) compared to unexposed individuals (range: 0.0129C130, median: 7.8; Figures?1B, C ), but the differences were not statistically significant. We investigated the immune cell subsets in the final cell products of COVID-19 recovered and unexposed individuals ( Figure?1D ). The cells showed prominent CD3+ T cell immunophenotype (median: 93.8 and 93.9 in recovered and unexposed, respectively) with the presence of CD3+CD56+ T cells (median: 18.5 and 6.07 in recovered and unexposed, respectively). CD14+ monocytes, CD3-CD56+ natural killer cells, and CD19+ B cells were either absent or less than 5% ( Figure?1C ). There were no significant differences in immunophenotypes of the final cell products between recovered and unexposed individuals. Within CD3+ T cells, we observed slightly higher levels.