Fig. 5. Distribution of SARS-CoV-2-specific effector CD8⁺ and CD4⁺ T cells in mucosal tissues.

(A-B) CD69 and CD103, and antigen-specific T cell responses after ex vivo peptide stimulation from secondary lymphoid organs and non-lymphoid tissues at day 10 post-infection. (A) Representative flow cytometry plots of CD69 and CD103 expression on CD8⁺CD95⁺ or CD4⁺CD95⁺ from BAL, previously hot lung sections, normal lung sections, nasal turbinates, salivary gland (parotid), tonsil, previously hot pulmonary lymph node, normal pulmonary lymph node, peripheral lymph node (axillary), spleen, and blood from ID#DHGF in unstimulated samples at day 10 post-infection. (B) Enumeration of percent CD69⁺ and CD103⁺ of CD8⁺CD95⁺ or CD4⁺CD95⁺ in unstimulated samples. Peripheral lymph node includes axillary, inguinal, and cervical lymph nodes. Previously hot lung sections not done for DGCX and DG3V. Sidak’s multiple comparison test for values on CD8⁺ vs. CD⁺4 T cells for each tissue. (C) Tissue distribution diagram and quantification of the frequency of Ag-specific (IFNγ⁺ and/or TNF⁺) in CD4⁺CD95⁺ (left graph) or CD8⁺CD95⁺ (right graph) in each tissue against peptide pools for Spike (S), Nucleocapsid (N), Membrane (M), and an optimized SARS-CoV-2 peptide megapools for CD4⁺ and CD8⁺ T cells (See Methods for description of peptides). Frequency calculated by subtracting the frequency of IFNγ⁺ and/or TNF⁺ in the unstimulated samples from the frequency in the stimulated samples. Statistics are paired t tests of stimulated vs. unstimulated for each condition. Ag-specific T cells from BAL at day 10, was shown in Fig. 4, with addition of CD8 megapool here. Tissue graphic created with BioRender.com. (D) Representative summary graphs of the immune response to SARS-CoV-2 infection in rhesus macaques. Median genomic and subgenomic viral RNA levels from nasal swabs on a log scale, as in Fig. 1. Mean frequency of myeloid subpopulation 1 and 6 in BAL, as in Fig. 3. Mean frequency of cDC2 (BAL myeloid subpopulation 4) in BAL, as in Fig. 3. Mean frequency of the sum of Ag-specific CD4⁺ and CD8⁺ T cells (S+N+M peptide pools) in BAL, as in Fig. 4. For all statistical analysis p<0.05 for the given test is considered significant: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.