Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1- CD4+ T cells

Abstract

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4⁺ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4⁺ T cells compared to systemic vaccination. Tissue-resident CD4⁺ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4⁺ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1⁺ KLRG1^- cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4⁺ T cells.

Fig. 1

Mucosal BCG vaccination provides enhanced protection against M. tb infection in the lung. Mice immunised with BCG via IN or ID route were challenged 6 weeks later with M. tb via aerosol. Four weeks post-challenge, CFU were enumerated in lungs and spleen. Individual log₁₀ CFU counts are shown with bars indicating mean ± standard error of the mean (SEM) (n ≥ 7). One-way ANOVA with Tukey’s post-test for significance; ****P < 0.0001; **P < 0.01. Data are representative of one of two independent experiments

Fig. 2

Mucosal BCG recruits CD4⁺ T cells to the lung parenchyma and induces greater frequency of antigen-specific CD4⁺ T cells in the lung parenchyma and BAL. Six weeks after immunisation with BCG via IN or ID route, intravascular staining and ICS identified populations of lung parenchymal and lung vascular antigen- (PPD-T)-specific (cytokine⁺) CD4⁺ or CD8⁺ T cells producing IFN-γ, TNF-α or IL-2 alone or in combination. a Frequency of BCG-induced antigen-specific cytokine⁺ CD4⁺ and CD8⁺ T cells in the lung, spleen and BAL. b Representative flow cytometry plots showing the proportion of lung parenchymal and lung vascular CD4⁺ T cells in the lungs of ID- or IN-immunised mice following intravascular anti-CD45 staining. c Frequency of lung parenchymal and lung vascular CD4⁺ T cells as a % of total CD4⁺ T cells isolated from the lung. d Number of CD4⁺ T cells in the lung parenchymal and lung vascular compartments. e Frequency of BCG-induced antigen-specific cytokine⁺ CD4⁺ T cells. f Number of BCG-induced antigen-specific cytokine⁺ CD4⁺ T cells. For a, c–f bars represent mean ± SEM (n = 6). Two-way ANOVA with Sidak’s post-test (a, c, d) or Tukey’s post-test (e, f); ****P < 0.0001, ***P < 0.001. Data are representative of one of two independent experiments

Fig. 3

Mucosal, but not ID BCG, induces PD-1⁺ KLRG1⁻ CD4⁺ T cells in the lung parenchyma and BAL. Six weeks after immunisation with BCG via IN or ID route, intravascular staining and ICS identified antigen-specific (cytokine⁺) PD-1⁺ KLRG1⁻ and PD-1⁻ KLRG1⁺ CD4⁺ T cells. a Representative plots from IN-immunised mice, pre-gated on cytokine⁺ CD4⁺ T cells, showing surface staining for PD-1, KLRG1 and CXCR3 in the lung parenchyma and lung vasculature. b Frequency of cytokine⁺ CD4⁺ T cells expressing a PD-1⁺ KLRG1⁻ or PD-1⁻ KLRG1⁺ phenotype. c Frequency of cytokine⁺ CD4⁺ T cells expressing CXCR3. d Proportion of lung parenchymal cytokine⁺ CD4⁺ T cells expressing CXCR3 in PD-1/KLRG1 subsets in mice after mucosal BCG vaccination. For b–d bars represent mean ± SEM (n = 6). Two-way ANOVA with Tukey’s post-test (b) or Sidak’s post-test (c); ****P < 0.0001, ***P < 0.001, **P < 0.01. Unpaired t-test (d); **P < 0.01. Data are representative of one of two independent experiments

Fig. 4

Lung parenchymal CD4⁺ T cells have greater proliferative capacity than lung vascular or splenic CD4⁺ T cells following mucosal BCG. Mice were vaccinated with BCG IN 6 weeks prior to intravascular staining. Purified populations of lung parenchymal, lung vascular and splenic CD4⁺ T cells were obtained by sorting. Naïve CD4⁺ lung and spleen T cells were sorted without intravascular staining. Cells were cultured for 3 days with PPD-T before proliferating cells were identified with Ki67 staining. To account for non-specific expression of Ki67, unstimulated values were subtracted from stimulated. Graph shows frequency of Ki67⁺ CD4⁺ T cells in the total lung and spleen of naïve mice and the lung parenchyma, lung vasculature and spleen of BCG-immunised mice. Bars represent mean ± SEM (n = 6). One-way ANOVA with Tukey’s post-test, ****P < 0.0001; **Ρ < 0.01, *Ρ < 0.05. Data are pooled from three independent experiments

Fig. 5

BCG-immunised mice are protected against M. tb infection 26 weeks post-immunisation. IN or ID BCG-immunised mice were challenged 26 weeks later with M. tb via aerosol. Four weeks post-challenge, CFU were enumerated in lung and spleens. Individual log₁₀ CFU values are shown with bars indicating mean ± SEM (n ≥ 5). One-way ANOVA with Tukey’s post-test for significance; ****P < 0.0001; **P < 0.01

Fig. 6

Lung parenchymal PD-1⁺ KLRG1⁻ CD4⁺ T cells decrease with time after mucosal BCG vaccination. Mice were vaccinated with BCG via IN or ID route 3, 6 or 26 weeks prior to intravascular staining and ICS to identify populations of lung parenchymal, lung vascular and spleen antigen-specific (cytokine⁺) CD4⁺ T cells. a Frequency of lung parenchymal and lung vascular CD4⁺ T cells as a % of total CD4⁺ T cells isolated from the lung. Statistical comparison conducted between the same compartments at different time points, all non-significant. b Frequency of BCG-induced antigen-specific cytokine⁺ CD4⁺ T cells. c Number of BCG-induced antigen-specific cytokine⁺ CD4⁺ T cells. d Representative plots from IN-immunised mice, pre-gated on cytokine⁺ CD4⁺ T cells, showing surface staining for PD-1 and KLRG1 in the lung parenchyma. e Frequency of cytokine⁺ CD4⁺ T cells expressing a PD-1⁺ KLRG1⁻ phenotype. For all graphs, bars represent mean ± SEM (n = 6). Two-way ANOVA with Sidak’s post-test, ***Ρ < 0.001, ****Ρ < 0.0001