Fig 2: Robust NAb and antigen-specific T cells were responsible for virus control in K18-hACE2 mice. Blood samples were collected on 33 and 42 days (also 4 dpi) after the 2nd vaccination for analysis. (a, b) The mean ± SEM changes of anti-RBD IgG AUC titer (a) and neutralizing IC50 values (b) were determined by anti-RBD IgG ELISA and pseudovirus assay, respectively. (c) Lung CD8 T cells were assayed for IFN-? expression by flow cytometry after re-stimulated with the RBD peptide pool. Tissue-resident memory CD8 T cells (TRM) in lungs were phenotyped for expression CD69 and CD103 (d) and the IFN-? producing TRM were measured by re-stimulated with the RBD peptide pool (e). (f) Viral load (relative gRdRp) in lungs correlated with NAb IC50 titer or percentage of IFN-? producing CD8 T cells. NP+ cells (MFI) in lungs correlated with NAb IC50 titer or percentage of IFN-? producing CD8 T cells. (g) Viral load (relative gRdRp) in NT correlated with NAb IC50 titer or percentage of IFN-? producing CD8 T cells. NP+ cells (MFI) in NT correlated with NAb IC50 titer or percentage of IFN-? producing CD8 T cells. Correlation analysis was performed by linear regression using GraphPad Prism 8.0. Each color represents a vaccination regimen. Each symbol represents an individual mouse. Statistics were generated using the 2-tailed Student's t test. *p<0.05; **p<0.01; ***p<0.001.

Fig 3: AHR signaling promotes TRM differentiation and function in human CD8 T cells(A–C) Human peripheral blood (PBMC) vs. IEL CD8+ T cells were analyzed via flow cytometry. Staining of CD45RA and CD45RO (top), CD103 and CD69 (bottom) (A), as well as T-BET and AHR (B) in tissue fractions as indicated in the figure. Quantification of AHR protein levels (gMFI) in human PBMC, LPL, and IEL CD8+ T cells (C). Data are compiled from three independent experiments and shown as mean ± SEM (n = 4 replicates per group).(D–G) Human naive CD8+ T cells were isolated from PBMCs and then subjected to in vitro TRM-like differentiation culture conditions. The cells were given differentiation cytokines and treated with DMSO (control), FICZ, or CH223191 on day 2. The assay was collected on day 4, and flow cytometry quantification of CD103 expression was performed (D). Data are compiled from three independent experiments and are shown as mean ± SEM (n = 4 replicates per group). RNA was isolated for qRT-PCR expression analysis of AHR (E), AHR direct target gene AHRR (F), and GZMB (G). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of three independent experiments.