Fig 1: Vaccination-induced memory cells have increased functional potential after in vivo recallPBMCs were analyzed pretransfer for the frequency of tetramer+ cells. Cells were divided into two equal fractions and transferred to NSG-HLA:A*02 transgenic mice. On the same day, animals were infected i.p. with 2 × 105 PFU mCMV-COVID or mCMV-FLU. The next day, mice were injected i.p. with human IL-2 (100 ng/mouse). 7 days post-infection, donor cells in peritoneal exudate cells (PECs) were analyzed (n = 25).(A) Experimental setup.(B) Line graph showing expansion of cells of the same donor as fold increase over pretransfer, comparing C19- with FLU-specific CD8+ T cells.(C) Representative FACS plots of CD8+ T cell stained with HLA-A*02 tetramers pretransfer or 7 days post-infection. Non-HLA-A*02 cells are included as negative control. Numbers indicate percentages. Gated is for live hCD45+hCD8+ cells.(D) Fold increase of antigen-specific cells segregated by C19 groups.(E) Correlation between time after last antigen exposure and the expansion of virus-specific cells after mCMV-C19 infection.(F–G) On day 7 post-infection, PECs were re-stimulated in vitro with PMA/IONO for 4 h in the presence of Brefeldin A and analyzed by flow cytometry. Shown are (F) cytokine production of live hCD45+hCD8+tetramer+ cells and (G) representative FACS plots gated for hCD45+hCD8+tetramer+ cells. Indicated are means ± SEM.p values were calculated using paired Student t test (B) and Kruskal-Wallis rank-sum test with Dunn’s post hoc test for multiple comparisons (F). *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S4.
Fig 2: The route of antigen exposure impacts the transcriptional and phenotypic profile of virus-specific memory cellsPBMCs from 3 groups of people were analyzed directly ex vivo (Inf, people with convalescent COVID-19; Inf/Vacc, people with convalescent COVID-19 who received 2 doses of vaccine; Vacc, nonconvalescent people who received 2 doses of COVID-19 vaccine).(A) Frequency of antigen-specific (C19- or FLU-tetramer+) cells. Each dot represents one donor (n = 19 – C19-Inf, n = 15 C19-Inf/Vacc, n = 17 C19-Vacc, n = 51 FLU pooled from all 3 groups).(B) Representative fluorescence-activated cell sorting (FACS) plots of cells stained with HLA A*02 tetramers loaded with the YLQPRTFLL (C19) epitope of SARS-CoV-2 or GILGFVFTL (FLU) epitope of influenza. Gated is for live CD3+CD8+ cells.(C) Quantification of TEM, TCM, and TEMRA cell subsets among C19- and FLU-specific CD8+ T cells (n = 18).(D–F) C19- and FLU-specific CD8+ T cells were sorted and analyzed by RNA sequencing.(D) Principal-component analysis of virus-specific cells based on all differentially expressed genes (n = 17).(E) The 200 most differentially expressed genes between C19-tetramer+ cells from the C19-Inf and C19-Vacc groups were subjected to protein network clustering. Shown is the largest node network for each comparison. Inset shows the largest subcluster.(F) Differential expression of individual genes (n = 17).(G) Geometric mean fluorescence intensity (GeoMean) of selected markers on C19- and FLU-specific CD8+ T cells by flow cytometry (n = 18). Indicated are means ± SEM.Statistical significances at *p < 0.05, **p < 0.01, and ***p < 0.001 using Kruskal-Wallis rank-sum test with Dunn’s post hoc test for multiple comparisons (A) or one-way ANOVA followed with Bonferroni post-testing (C, F, and G).See also Figure S1.
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