Fig. 5: Lung resident memory T cells retain intact functionality following pulmonary infection with sAg expressing S. aureus strains.

a DR4-DQ8.F1 mice seeded with 5 × 10⁶ in vitro activated SMARTA.CD90.1 and 5 × 10⁶ in vitro activated gDT-2.CD45.1 cells were intranasally immunised with 30 μg of GP_61-80 and gD_315-327 peptide and adjuvant and were rested for 15 days. a Schematic of experimental set up to lodge SMARTA and gDT-2 CD4⁺ Trm in the lung of mice. Created in BioRender. Wakim, L. (2024) BioRender.com/w79t204. b Representative flow cytometry profiles show the level of expression of the Trm marker CD69 on SMARTA (CD90.1⁺) and gDT-2 (CD45.1⁺) cells in the lung and spleen on day 15 post seeding. Mice generated as described in (a) were intranasally infected with either 10⁸ CFU JKD6159-gD, or 10⁸ CFU JKD6159-gD::seb and spleen and lung were harvested 4 days later. c The absolute number of endogenous CD4⁺Vβ8⁺ and CD4⁺Vβ11⁺ T cells in the lung and spleen. Symbols represent individual mice, and the bars represent the mean ± SEM. Data pooled from 3 experiments. (n = 5–24 mice per group, Two-way ANOVA, Sidak’s multiple comparison). d–h B6 and DR4-DQ8.F1 mice with SMARTA.CD90.1⁺ and gDT-2.CD45.1⁺ lung Trm (generated as described in a) were intranasally infected with either 10⁸ CFU JKD6159-gD, or 10⁸ CFU JKD6159-gD::seb and 4 days later the number of (d) gDT-2.CD45.1⁺ and (e) SMARTA.CD90.1⁺ CD4⁺ T cells in the spleen and lung were measured. Symbols represent individual mice, and the bars represent the mean ± SEM. Data pooled from 5 experiments. (n = 5–24 mice per group, Two-way ANOVA, Sidak’s multiple comparison). The proportion of (f) gDT-2.CD45.1⁺ CD4⁺ T cells (g) SMARTA.CD90.1⁺ CD4⁺ T cells or (h) endogenous CD4⁺Vβ8⁺ T cells in the lung and spleen producing TNFα following a brief in vitro stimulation was measured by flow cytometry. Bars represent the mean ± SEM, and symbols represent individual mice (n = 6–9 mice cohort, Two-way ANOVA, Tukey’s multiple comparison). i DR4-DQ8.F1 mice seeded with 5 × 10⁶ in vitro activated F5.CD45.1⁺ CD8⁺ T cells were intranasally immunised with 30 μg of NP_366-374 peptide and adjuvant and were rested for 15 days. Representative flow cytometry profile showing the proportion of F5.CD45.1⁺ CD8⁺ T cells in the lung expressing Trm markers, CD69 and CD103 on day 15 post seeding. Created in BioRender. Wakim, L. (2024) BioRender.com/w79t204. j Mice generated as described in i were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::sea and spleen, mLN and lung were harvested 4 days later. (j) The absolute number of F5.CD45.1⁺ CD8⁺ T cells in the lung, mLN and spleen. Bars represent the mean ± SEM (n = 2). k–n B6 and DR4-DQ8.F1 mice with F5.CD45.1⁺ CD8⁺ lung Trm generated as described in (i) were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::sea or left uninfected and 21 days later were intranasally challenged with 10⁴ PFU of X31(DAM). k The absolute number of F5.CD45.1⁺ CD8⁺ T cells in the lung and mLN. Symbols represent individual mice, and the bars represent the mean ± SEM. Data pooled from 2 experiments. (n = 3-9, Two-way ANOVA, Sidak’s multiple comparison). The proportion of F5.CD45.1⁺ CD8⁺ T cells in the (l) lung and (m) mLN producing IFNγ following a brief in vitro stimulation was measured by flow cytometry. Bars represent the mean ± SEM, and symbols represent individual mice (n = 4–9 mice cohort, Two-way ANOVA, Tukey’s multiple comparison). n B6 or DR4-DQ8.F1 mice with F5.CD45.1⁺ CD8⁺ lung Trm (generated as described in i) were intranasally infected with either 10⁸ CFU JKD6159, or 10⁸ CFU JKD6159::sea or left uninfected. Fifteen days later, these animals, in addition to a cohort of naïve DR4-DQ8.F1 and B6 mice were infected intranasally with 10⁴ PFU X31(DAM) and viral loads in the lung on day 4 post influenza infection were measured. Bars represent the mean ± SEM, and symbols represent individual mice (n = 4–9 mice cohort, Two-way ANOVA, Tukey’s multiple comparison). *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.