Cutting Edge: Influenza-Induced CD11alo Airway CD103+ Tissue Resident Memory T Cells Exhibit Compromised IFN-γ Production after In Vivo TCR Stimulation

Abstract

Although tissue resident memory T cells (TRM) in the lung confer robust protection against secondary influenza infection, their in vivo production of IFN-γ is unknown. In this study, using a mouse model, we evaluated production of IFN-γ by influenza-induced TRM (defined as CD103+) that localize to the airways or lung parenchyma. Airway TRM consist of both CD11ahi and CD11alo populations, with low CD11a expression signifying prolonged airway residence. In vitro, high-dose peptide stimulation evoked IFN-γ from most CD11ahi airway and parenchymal TRM, whereas most CD11alo airway TRM did not produce IFN-γ. In vivo production of IFN-γ was clearly detectable in CD11ahi airway and parenchymal TRM but essentially absent in CD11alo airway TRM, irrespective of airway-instilled peptide concentration or influenza reinfection. The majority of IFN-γ-producing airway TRM in vivo were CD11ahi, suggesting recent airway entry. These results question the contribution of long-term CD11alo airway TRM to influenza immunity and reinforce the importance of defining TRM tissue compartment-specific contributions to protective immunity.

Figure 1.

Airway CD8 T cells do not optimally produce IFN-γ in response to antigen-stimlulation in vitro. Naive P14 (Thy1.1) CD8⁺ T cells were transferred to naive (Thy1.2) C57BL/6 mice 1 d before i.v. injection of DC-GP33. Seven days later the mice were infected i.n. with PR8-GP33 and lungs were harvested day 39–42 post infection. (A) Experimental set-up. (B) Representative flow plots and gating strategy of IN+ vs IN− CD103+ CD8+ T_RM of lung P14s distinguished from PerCPCy5.5-labeled splenocytes. (C) Frequency of IFN-γ within CD103+IN+ or CD103+IN− lung P14s. n = 10 mice per group with data combines from 3 independent experiments. Individual mice are represented by each point. A paired t test was used to determine statistical significance; ** p < 0.01.

Figure 2.

Airway CD11a^lo CD8+ T_RM are most compromised after in vitro stimulation. Experimental setup as in Fig. 1 (A) Representative flow plots of IFN-γ staining in CD11a^lo vs CD11a^hi CD103+ CD8+ T_RM. (B) Frequency of CD11a^lo vs CD11^hi IFN-γ + CD103+ P14s. (C) Naive P14’s were transferred into naive C57BL/6 mice 1 d before i.n. infection with PR8-GP33 and lungs were harvested day 30–40 post infection. P14s were restimulated in vitro with 200 nM GP33, 1 uM GP33 or PMA + ionomycin. n = 4–10 mice per group with data combined from 2–3 independent experiments. Individual mice are represented by each point. A paired t test (B) or one-way ANOVA with Tukey’s multiple comparison test (C) was used to determine statistical significance (B); *** p < 0.001 and **** p < 0.0001.

Figure 3.

CD11a^lo CD8+ airway T_RM fail to make IFN-γ after in vivo peptide administration. Naive P14 CD8⁺ T cells were transferred into naive C57BL/6 mice 1 d before i.n. infection with PR8-GP33 and lungs were harvested day 30–50 post infection. Six hours before lung harvest, mice were i.n. injected with 1–50 ug of GP33 in the presence of brefeldin A. (A) Experimental set-up. (B) Representative flow plots of IFN-γ staining in IN+ vs IN− CD103+ CD8+ T_RM. (C) Frequency of CD11a^lo IN+, CD11^hi IN+ and CD11a^hiIN− IFN-γ + CD103+ P14s. n = 5–7 mice per group with data combines from 3 independent experiments. Individual mice are represented by each point. One-way ANOVA with Tukey’s multiple comparison test was used to determine statistical significance; *** p < 0.001 and **** p < 0.0001.

Figure 4.

CD11a^lo CD8+ airway T_RM are compromised in TCR and bystander activation upon heterologous influenza rechallenge. Naive P14 CD8⁺ T cells were transferred into naive C57BL/6 mice 1 d before i.n. infection with X31-GP33. Immune mice were rechallenged 30–35 days later with a lethal dose of PR8-GP33 or PR8-OVA. Brefeldin A was administered i.n. 48 hr after the rechallenge and lungs were harvested 6 hr later. (A) Experimental set-up. (B) Representative flow plots of IFN-γ and CD25 staining in CD11a^lo vs CD11a^hi CD103+ CD8+ T_RM. (C) Frequency of CD11a^lo IN+, CD11^hi IN+ and CD11a^hiIN− CD25+ or IFN-γ + CD103+ P14s. n = 5–7 mice per group with data combined from 2 independent experiments. Individual mice are represented by each point. An upaired t test was used to determine statistical significance; ** p < 0.01.