Panel A shows an experimental timeline schematic of inoculation, co housing, IVIS imaging, and flow cytometry. Panel B shows flow cytometry plots of CD103 and CD69 expression in nasal cavity, BAL, and lung samples after 30 microliter or 5 microliter inoculation. Panel C shows bar graphs comparing SenNP positive CD103 positive CD69 positive cells across respiratory tissues and spleen. Panel D shows line graphs of viral flux over days post exposure for three inoculation conditions. Panel E shows a bar graph comparing viral flux AUC between uninfected and infected mice. Panel F shows a bar graph of infection probability across inoculation conditions. Panel G shows an experimental timeline testing Ad SenNP 5 microliter with or without FTY720 and anti CD4. Panel H shows line graphs of viral flux over time following isotype or FTY720 plus anti CD4 treatment. Panel I shows a bar graph comparing viral flux AUC between treatment groups. Panel J shows a bar graph comparing infection probability between treatment groups. Panel K shows IVIS images displaying luminescence over days post exposure for two inoculation conditions. Panel L shows pie charts of infection initiation sites for Ad FluNP and Ad SenNP, categorized as nasal first, nasal plus trachea, or trachea first. Panel M shows flow cytometry plots of SenNP and CD69 expression in tracheal samples following 30 microliter or 5 microliter inoculation. Panel N shows a bar graph comparing SenNP positive CD103 positive CD69 positive cells in the trachea between inoculation volumes.
CD8 TRM T cells in the URT alone are sufficient to prevent infection from transmission. (A) Experimental schematic in which immunized contact mice were co-housed with a Sendai-Luc–infected index mouse. (B) Representative flow cytometry plots of CD103 and CD69 expression gated on IV− CD8+ SenNP+ T cells in Ad-SenNP 30 and 5 μl immunized mice. (C) Number of CD103+ CD69+ SenNP+ TRM in the nasal cavity, BAL, lung, and splenic CD8+ SenNP+ cells of Ad-SenNP 30 μl (n = 10) and 5 μl (n = 10) immunized mice. (D) Bioluminescence curves of Ad-FluNP 30 μl (n = 24), Ad-SenNP 30 μl (n = 16), and Ad-SenNP 5 μl (n = 24) immunized contact mice when co-housed with Sendai-Luc–infected index mouse. (E) AUC of bioluminescence in immunized contact mice that became infected following co-housing. Grey circles represent uninfected mice, and triangles represent infected mice. (F) Probability of infection in the respiratory tract for immunized contact mice, calculated as the proportion of contact mice that become infected. (G) Experimental schematic in which contact mice were immunized with 5 μl Ad-SenNP and treated with FTY720 and anti-CD4 or vehicle and isotype controls. (H) Bioluminescence curves of Ad-SenNP 5 μl immunized contact mice treated with FTY720 and anti-CD4 (n = 16) or vehicle and isotype controls (n = 15) when co-housed with Sendai-Luc–infected index mouse. (I) AUC of bioluminescence in immunized contact mice that became infected following co-housing. (J) Probability of infection in the respiratory tract for immunized contact mice. (K) Representative bioluminescence images showing the location of Sendai-Luc replication in Ad-FluNP control mice or Ad-SenNP 5 μl immunized mice that had a breakthrough infection. (L) Proportion of infections that initiated in the nasal region, tracheal region, or nasal and tracheal regions concurrently in Ad-FluNP control (n = 24) and Ad-SenNP 5 μl immunized breakthrough (n = 7) mice. (M and N) Representative flow cytometry plots (M) and quantification (N) of SenNP+ CD69+ CD103+ CD8 T cells in the trachea of Ad-SenNP 30 μl and Ad-SenNP 5 μl immunized mice (n = 9 each) 35 days after immunization. For D and H, solid dark lines represent means, solid pale lines represent individual mice, dashed grey lines represent the limit of detection, and dashed red lines represent the threshold of infection. Error bars (E–I) represent 95% binomial confidence intervals. Data are combined from two (B and C and H–J), or three (D–F) independent replicates. Statistical significance was determined using a two-sided Mann–Whitney test (C, E, F, I, J, and N) or a chi-squared test (L), with ***P < 0.001, ****P < 0.0001, and NS for not significant.