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. 2020 Oct 22;11:566319. doi: 10.3389/fimmu.2020.566319

Figure 6.

Figure 6

Mycobacteria-specific Trm cells accumulate in the lung during BCG immunization. (A) Representative dot plots and gating strategy of tetramer Ag85B binding and CD11a+CD69+ CD4TRM cells in the lungs of mice 3 weeks after i.t. and s.c. BCG immunization. (B) Mice were immunized with BCG s.c. or i.t. and sacrificed 3 weeks later. A group of mice was left untreated. The frequency of CD44+ in gated CD3+ CD4 T cells from lungs and of CD4 TRM ± SEM gated on CD3+ CD44+ cells are shown. The mean % ± SEM of at least 4 animals per group are shown. Differences between groups are significant at *p ≤ 0.05 and **p ≤ 0.001 (Welch's t-test with Holm-Sidak correction for multiple comparisons). (C) The mean numbers ± SEM of tetramer Ag85B binding total and TRM CD4 T cells in the lungs of i.t. and s.c. BCG immunized mice are shown (n ≥ 5 animals per group). Differences between groups are significant at **p ≤ 0.001 and ***p ≤ 0.001 (one-way ANOVA). (D) Mice were immunized i.t. or s.c. with BCG and sacrificed 1 or 3 weeks later. The representative dot plots show the presence of tetramer TB10.4 binding CD44+ CD8 T cells in the lungs of i.t. or s.c. BCG-immunized mice. The CD69+CD103+ CD8 TRM within the tetramer TB10.4 positive or negative cells are also shown. (E–H) The frequency of CD44+ gated on CD3+ CD8 T cells (E), of tetramer TB10.4+ within CD44+ CD8 T cells (F), the numbers of tetramer TB10.4+ CD8 T cells (G) and the frequency of TRM cells within all TB10.4 tetramer binding cells (H) were determined in lungs from mice at 1 and 3 weeks after i.t. or s.c. immunization with BCG. The mean frequencies or log10 transformed cell numbers ± SEM (n = 5 per group) are shown. Differences between groups were statistically significant at *p ≤ 0.05, **p ≤ 0.001, and ***p ≤ 0.001 (Welch's t-test with Holm-Sidak correction for multiple comparisons). (I) The mean frequencies of PD1+ and KRLG1+ cells within the TB10.4 tetramer + or—CD44+ CD8 T cells in the lungs of mice immunized i.t. or s.c. with BCG 3 weeks before sacrifice are shown. Differences between i.t. and s.c. immunized mice (n = 5 per group) were significant at *p ≤ 0.05 and **p ≤ 0.01 (Welch's t-test with Holm-Sidak correction for multiple comparisons).