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Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis

View Article: PubMed Central - PubMed

ABSTRACT

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (TRM) cells have been implicated in protective immune responses against viral infections, but the role of TRM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and TRM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4+ T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8+ T cells displayed prototypical TRM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of lung-infiltrating T cells generated by i.t. BCG vaccination (A, B). B6 mice were BCG vaccinated i.t., and BALF T cell subsets were sorted 60 days later by fluorescence-activated cell sorting gated as described in the legend to Fig. 4A. Sorted naive (TCRβ+ CD44lo CD62Lhi) BALF T cells or splenic TEM cells (TCRβ+ CD44hi CD62Llo) from i.t. BCG-vaccinated mice 60 days after immunization were also used as controls. (A) Heat map showing gene expression from sorted BALF T cell populations. Triplicates of 100 BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice were sorted. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm. mRNA concentrations of all sorted T cell populations were normalized to β-actin (NM_007393.4) expression. The color code indicates fold changes (2−ΔΔCT) in transcripts relative to the appropriate internal control as indicated. (B) Fold changes in the expression of selected genes of sorted BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice compared to the appropriate internal control. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm as described for panel A. (C, D) BALF immune cell phenotype measured by flow cytometry 60 days after i.t. BCG vaccination. (C) Representative flow cytometry of intracellular Foxp3 and T-bet expression by sorted CD4+ TRM cells. (D) Representative histograms of selected surface activation markers and IFN-γ expression by CD4+ and CD8+ TEM and TRM cells. Results are presented as pooled individual data points ± the standard error of the mean (B), representative fluorescence-activated cell sorter plots (C), or histograms (D) from two pooled independent experiments (n = 6 to 8 mice per group). The statistical significance of differences from the TRM cell subset (B) is shown. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (B) Analysis of variance with Tukey’s posttest for significance.
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fig5: Phenotypic characterization of lung-infiltrating T cells generated by i.t. BCG vaccination (A, B). B6 mice were BCG vaccinated i.t., and BALF T cell subsets were sorted 60 days later by fluorescence-activated cell sorting gated as described in the legend to Fig. 4A. Sorted naive (TCRβ+ CD44lo CD62Lhi) BALF T cells or splenic TEM cells (TCRβ+ CD44hi CD62Llo) from i.t. BCG-vaccinated mice 60 days after immunization were also used as controls. (A) Heat map showing gene expression from sorted BALF T cell populations. Triplicates of 100 BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice were sorted. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm. mRNA concentrations of all sorted T cell populations were normalized to β-actin (NM_007393.4) expression. The color code indicates fold changes (2−ΔΔCT) in transcripts relative to the appropriate internal control as indicated. (B) Fold changes in the expression of selected genes of sorted BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice compared to the appropriate internal control. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm as described for panel A. (C, D) BALF immune cell phenotype measured by flow cytometry 60 days after i.t. BCG vaccination. (C) Representative flow cytometry of intracellular Foxp3 and T-bet expression by sorted CD4+ TRM cells. (D) Representative histograms of selected surface activation markers and IFN-γ expression by CD4+ and CD8+ TEM and TRM cells. Results are presented as pooled individual data points ± the standard error of the mean (B), representative fluorescence-activated cell sorter plots (C), or histograms (D) from two pooled independent experiments (n = 6 to 8 mice per group). The statistical significance of differences from the TRM cell subset (B) is shown. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (B) Analysis of variance with Tukey’s posttest for significance.

Mentions: TRM cells vary in phenotype and function, depending on the tissue they reside in (25–27). The phenotype of TRM cells in lung airways following mucosal BCG vaccination has not been characterized. Hence, we performed transcriptional gene expression profiling of sorted BALF CD4+ and CD8+ TEM and TRM cell subpopulations induced by i.t. BCG vaccination with a Fluidigm Dynamic Array. The purity of the different sorted cell populations was routinely assessed at 86 to 99% (see Fig. S4). Increased transcription levels of typical markers associated with tissue residency of CD4+ and CD8+ TRM such as Itgae (CD103) and Itga1 (VLA-1) were confirmed (Fig. 5A and B). CD4+ TRM cells displayed a regulatory profile, with high Foxp3 and Il10 mRNA expression (Fig. 5A and B). Additionally, CD4+ TRM cells expressed T-bet, as well as Foxp3, at the protein level (Fig. 5C). Importantly, each marker was expressed by distinct subpopulations, suggesting a heterogeneous population comprising effector and regulatory T cells (28). Therefore, we concluded that CD4+ TRM cells, defined here as CD4+ CD103+ CD69+ cells, comprise a mixture of regulatory and effector T cells rather than solely belonging to the TRM subset. On the other hand, CD8+ TRM cells expressed significantly higher levels of gamma interferon (IFN-γ) (Ifng), tumor necrosis factor alpha (TNF-α) (Tnfa), and Cxcr6 (Fig. 5B) (29) and statistically insignificantly higher levels of perforin (Prf1) and granzyme B (Gzmb) than their CD8+ TEM counterparts (Fig. 5A).


Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis
Phenotypic characterization of lung-infiltrating T cells generated by i.t. BCG vaccination (A, B). B6 mice were BCG vaccinated i.t., and BALF T cell subsets were sorted 60 days later by fluorescence-activated cell sorting gated as described in the legend to Fig. 4A. Sorted naive (TCRβ+ CD44lo CD62Lhi) BALF T cells or splenic TEM cells (TCRβ+ CD44hi CD62Llo) from i.t. BCG-vaccinated mice 60 days after immunization were also used as controls. (A) Heat map showing gene expression from sorted BALF T cell populations. Triplicates of 100 BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice were sorted. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm. mRNA concentrations of all sorted T cell populations were normalized to β-actin (NM_007393.4) expression. The color code indicates fold changes (2−ΔΔCT) in transcripts relative to the appropriate internal control as indicated. (B) Fold changes in the expression of selected genes of sorted BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice compared to the appropriate internal control. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm as described for panel A. (C, D) BALF immune cell phenotype measured by flow cytometry 60 days after i.t. BCG vaccination. (C) Representative flow cytometry of intracellular Foxp3 and T-bet expression by sorted CD4+ TRM cells. (D) Representative histograms of selected surface activation markers and IFN-γ expression by CD4+ and CD8+ TEM and TRM cells. Results are presented as pooled individual data points ± the standard error of the mean (B), representative fluorescence-activated cell sorter plots (C), or histograms (D) from two pooled independent experiments (n = 6 to 8 mice per group). The statistical significance of differences from the TRM cell subset (B) is shown. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (B) Analysis of variance with Tukey’s posttest for significance.
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fig5: Phenotypic characterization of lung-infiltrating T cells generated by i.t. BCG vaccination (A, B). B6 mice were BCG vaccinated i.t., and BALF T cell subsets were sorted 60 days later by fluorescence-activated cell sorting gated as described in the legend to Fig. 4A. Sorted naive (TCRβ+ CD44lo CD62Lhi) BALF T cells or splenic TEM cells (TCRβ+ CD44hi CD62Llo) from i.t. BCG-vaccinated mice 60 days after immunization were also used as controls. (A) Heat map showing gene expression from sorted BALF T cell populations. Triplicates of 100 BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice were sorted. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm. mRNA concentrations of all sorted T cell populations were normalized to β-actin (NM_007393.4) expression. The color code indicates fold changes (2−ΔΔCT) in transcripts relative to the appropriate internal control as indicated. (B) Fold changes in the expression of selected genes of sorted BALF CD4+ and CD8+ TEM and TRM cells from i.t. BCG-vaccinated mice compared to the appropriate internal control. Quantitative PCR was run with the data collection software (36 cycles) from Fluidigm as described for panel A. (C, D) BALF immune cell phenotype measured by flow cytometry 60 days after i.t. BCG vaccination. (C) Representative flow cytometry of intracellular Foxp3 and T-bet expression by sorted CD4+ TRM cells. (D) Representative histograms of selected surface activation markers and IFN-γ expression by CD4+ and CD8+ TEM and TRM cells. Results are presented as pooled individual data points ± the standard error of the mean (B), representative fluorescence-activated cell sorter plots (C), or histograms (D) from two pooled independent experiments (n = 6 to 8 mice per group). The statistical significance of differences from the TRM cell subset (B) is shown. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (B) Analysis of variance with Tukey’s posttest for significance.
Mentions: TRM cells vary in phenotype and function, depending on the tissue they reside in (25–27). The phenotype of TRM cells in lung airways following mucosal BCG vaccination has not been characterized. Hence, we performed transcriptional gene expression profiling of sorted BALF CD4+ and CD8+ TEM and TRM cell subpopulations induced by i.t. BCG vaccination with a Fluidigm Dynamic Array. The purity of the different sorted cell populations was routinely assessed at 86 to 99% (see Fig. S4). Increased transcription levels of typical markers associated with tissue residency of CD4+ and CD8+ TRM such as Itgae (CD103) and Itga1 (VLA-1) were confirmed (Fig. 5A and B). CD4+ TRM cells displayed a regulatory profile, with high Foxp3 and Il10 mRNA expression (Fig. 5A and B). Additionally, CD4+ TRM cells expressed T-bet, as well as Foxp3, at the protein level (Fig. 5C). Importantly, each marker was expressed by distinct subpopulations, suggesting a heterogeneous population comprising effector and regulatory T cells (28). Therefore, we concluded that CD4+ TRM cells, defined here as CD4+ CD103+ CD69+ cells, comprise a mixture of regulatory and effector T cells rather than solely belonging to the TRM subset. On the other hand, CD8+ TRM cells expressed significantly higher levels of gamma interferon (IFN-γ) (Ifng), tumor necrosis factor alpha (TNF-α) (Tnfa), and Cxcr6 (Fig. 5B) (29) and statistically insignificantly higher levels of perforin (Prf1) and granzyme B (Gzmb) than their CD8+ TEM counterparts (Fig. 5A).

View Article: PubMed Central - PubMed

ABSTRACT

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (TRM) cells have been implicated in protective immune responses against viral infections, but the role of TRM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and TRM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4+ T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8+ T cells displayed prototypical TRM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies.

No MeSH data available.


Related in: MedlinePlus