PBMCs and nasal cells (1 × 106) were cultured for 24 hours in the presence of 1 μg/mL peptide pools, and 1 μg/mL anti-human CD28 and CD49d mAbs (BD Biosciences). Stimulation was performed using SARS-CoV-2 S OLP pools (PepTivator®, SARS-CoV-2 Prot_S Complete; Miltenyi Biotec) and N OLP pools (PepMix™ SARS-CoV-2 NCAP; JPT Peptide Technologies, Berlin, Germany). CD25+CD137+ cells and OX40+CD137+ cells were considered S-specific AIM+ cells among CD8+ and CD4+ T cells, respectively. Negative controls were treated with DMSO and anti-CD28/anti-CD49d without peptides, and positive controls with PMA/ionomycin.
Finally, we performed cellular indexing of transcriptomes and epitopes (CITE)-seq analysis. SARS-CoV-2 S-reactive T cells were enriched from nasal cells of the vaccinated and BTI groups, by isolating AIM+ (CD137+) T cells following stimulation with S OLPs. Next, these cells were stained with DNA-barcoded antibodies against CD markers, and CITE-seq analysis was performed (Fig. 6a). After initial quality control, we used unsupervised clustering to identify CD8+ T cells from total cells (Figs. 6b, c, Supplementary Fig. 10a). We further gated CD49a+ cells and CD49a− cells among total CD8+ T cells, based on expression levels of CD8 protein and the ITGA1 transcript (which encodes CD49a) (Fig. 6d).
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Analysis of activation-induced markers implicates that a considerable proportion of spike (S)-reactive nasal CD8+ T cells express CD103, a tissue-resident marker. MHC-I multimer staining is performed to analyze the ex vivo phenotype and function of SARS-CoV-2 S-specific CD8+ T cells. We detect multimer+CD8+ T cells with tissue-resident phenotypes in nasal tissue samples from vaccinees without infection as well as vaccinees with BTI. https://immediatespike.info/ Multimer+CD8+ T cells remain present in nasal tissues over one year after the last exposure to S antigen, although the frequency decreases. Upon direct ex vivo stimulation with epitope peptides, nasal multimer+CD8+ T cells–particularly the CD49a+ subset–exhibit immediate effector functions, including IFN-γ production. CITE-seq analysis of S-reactive AIM+CD8+ T cells confirms the enhanced effector function of the CD49a+ subset.
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