Fig 1: T cell responses to SARS-CoV-2, MMR, and Tdap antigens in SARS-CoV-2-infected and uninfected donors(A) Experimental scheme for (B)–(G). Blood was collected from uninfected (UIN) and PCR-confirmed, SARS-CoV-2-infected (INF) donors. Plasma was analyzed for antibodies to SARS-CoV-2 antigens and cytokines. Blood was divided and treated with isotype control (Iso) or AAC for 2 h, and neutrophils were then isolated and cultured in GM-CSF for 2 days. AAC treatment generates nAPCs while isotype-treated controls remain neutrophils. PBMCs were harvested to isolate CD3+ T cells and monocytes, which were cultured in cytokines to generate dendritic cells (moDCs). Neutrophils (isotype), nAPCs (AAC), and moDCs were loaded without (-) or with (+) antigen and co-cultured with autologous T cells on IFN-? ELISpot plates for 18 h. Loaded antigens included spike-S1 subunit (Sp1), spike-S2 subunit (Sp2), receptor binding domain (RBD), nucleocapsid (NC), measles (Measl.), mumps, rubella (Rubel.), diphtheria (Dipth), tetanus (Tetan.), or pertussis (Pertu.). Stimulation of T cells alone with PMA plus ionomycin (PMA+) served as a positive control.(B) nAPCs from uninfected (1–4) and infected (1–9) individuals were co-cultured at a 1:5 ratio with autologous T cells and spike-S1 on IFN?-ELISpot plates, and the number of IFN?+ spots was counted.(C) IgG titers in sera of uninfected and infected donors to SARS-CoV-2 antigens, Sp1, NC, spike-S1 and -S2 (spike), and RBD. Red arrows in (B) and (C) identify samples with IFN-?+ T cells but no detectable SARS-CoV-2 specific IgG titers.(D) nAPCs generated from 8 uninfected and 18 infected donors were loaded with vehicle (-) or indicated individual SARS-CoV-2, MMR, or Tdap antigens were analyzed for T cell responses as in (B). Representative images of wells with IFN-?+ spots are shown (right). *p < 0.05; **p < 0.005 by two-tailed Mann-Whitney test with Bonferroni correction for multiple comparisons.(E) A correlation of spike-S1 or nucleocapsid-derived IFN-?+ spots with indicated vaccine antigens (circles) and percent of nAPCs generated (diamonds), which varies between individuals, in infected donors was conducted using Spearman’s rank correlation coefficients (r).(F) Cytokine levels detected in the supernatants of neutrophils treated with isotype (neut) or AAC (to generate nAPCs) and cultured for 48 h and monocyte-derived dendritic cells (moDCs). **p < 0.005 using two-way analysis of variance and Bonferroni’s multiple comparison test.(G) As in (B), ELISpot assays measuring IFN-? secretion by T cells co-cultured with nAPCs pulsed with combined SARS-CoV-2 (spike-S1, nucleocapsid, RBD), MMR, or TDP (Tdap) antigens were evaluated in the presence of two independent functional blocking anti-IL-15 (top panel), anti-IL-1ß/anti-IL-18, or anti-TNF-a (bottom panel) antibodies. Two-way analysis of variance and Dunnett’s multiple comparison test were used. **p < 0.005.Data in (D), (F), and (G) are average ± SEM, and individual values are plotted. See also Figures S1 and S2.