Modulation of naive CD4+ T-cell responses to an airway antigen during pulmonary mycobacterial infection

Abstract

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.

FIG. 1.

BCG infection causes enrichment and accumulation of OVA-specific T cells after airway OVA challenge. BALB/c mice were infected with BCG, and 4 to 6 weeks later 3 × 10⁶ to 5 × 10⁶ CD4⁺ DO11.10 T cells were transferred into BCG-infected and uninfected mice. Recipient mice were challenged with 500 μg of endotoxin-depleted OVA on days 0 and 2 or with BSA on day 0 and were sacrificed on day 3. Single-cell suspensions were prepared from lungs, MLN, and spleens and stained with anti-CD4 and KJ 1-26 to enumerate the OVA-specific T cells. (A) The percentage of OVA-specific (KJ⁺) T cells among the CD4⁺ T cells present was determined in the MLN, lungs, and spleens of the four different groups. (B) Total KJ⁺ T-cell numbers were calculated by multiplying viable cell counts, determined by trypan blue exclusion, by the percentage of KJ⁺ T cells among viable cells gated from a forward-scatter (FSC) versus side-scatter (SSC) plot during fluorescence-activated cell sorting analysis. This experiment was repeated twice with similar findings. Three mice were included in each group. *, P = 0.02; **, P = 0.01; #, P = 0.004; ##, P = 0.02.

FIG. 2.

BCG infection enhances T-cell activation in the lungs. CD4⁺ CD62L^hi CD44^low FACsorted naive DO11.10 T cells were transferred into BCG-infected and uninfected mice. Mice were challenged with OVA as described in Materials and Methods. Three days later, the mice were sacrificed, and the draining MLN (A) and lungs (B) were stained for TCR, CD25, and CD69. Histograms were gated on KJ⁺ T cells. The baseline levels of CD25 and CD69 on naive KJ⁺ T cells before adoptive transfer are shown in panel A. (C) Percentages of CD25- and CD69-expressing KJ⁺ T cells, above isotype staining, from three mice per group. *, P < 0.01. The data are representative of three separate experiments.

FIG. 3.

Pulmonary infection increases the frequency of antigen-specific T cells responding to an airway antigen. A total of 5 × 10⁶ CFSE-labeled CD4⁺ DO11.10 T cells were transferred into BCG-infected and uninfected recipient mice. Recipient mice were challenged with 500 μg of endotoxin-depleted OVA on days 0 and 2 or with BSA on day 0. Mice were sacrificed on day 3, and single-cell suspensions from tissues were stained and analyzed by flow cytometry to measure T-cell proliferation. (A) Representative dot plots showing KJ⁺ staining and CFSE dye dilution of four individual mice from the four groups. (B) The percentage of OVA-specific T cells that divided in each mouse was calculated as described in Materials and Methods. This experiment was repeated twice with similar results (three mice per group). *, P = 0.02; **, P = 0.03.

FIG. 4.

BCG infection induces OVA-specific T-cell proliferation in the lungs after airway OVA challenge. FACsorted naive (CD62L^hi CD44^low) CD4⁺ T cells from DO11.10 mice were transferred into BCG-infected and uninfected recipient mice. Recipient mice were challenged intranasally with 300 μg of endotoxin-depleted OVA or BSA. At 3 days after challenge, mice were injected intravenously with 2 mg of BrdU. After 1 h of in vivo BrdU pulse, the mice were sacrificed, and single cell suspensions from the lungs were stained with KJ 1-26 and anti-BrdU as described in Materials and Methods. (A) Isotype staining for anti-BrdU and KJ isotype control. (B) Representative dot plots of three mice from the three groups. The numbers represent the percentage of BrdU⁺ cells among the KJ⁺ T cells. (C) Means ± the standard deviation of three mice per group. *, P = 0.01. The experiment was repeated twice with similar findings each time.

FIG. 5.

BCG infection increases the accumulation of effector antigen-specific T cells in the lungs after airway antigen challenge. Flow-sorted CD62L^hi CD44^low naive DO11.10 T cells were labeled with CFSE and transferred into BCG-infected and uninfected recipient mice. Recipient mice were challenged with OVA as described in Materials and Methods. Mice were sacrificed 3 days after OVA challenge, and single cell lung suspensions were prepared. (A) Lung cells were stained with KJ 1-26 and anti-CD62L and analyzed by flow cytometry. Plots were gated on KJ⁺ T cells, and the expression of CD62L and CFSE dilution was analyzed. The numbers represent the percentage of CD62L^low T cells among KJ⁺ T cells that have divided more than twice (CFSE^low). (B) Percentage of CD62L^low T cells among KJ⁺ T cells (three mice per group). *, P = 0.02; **, P = 0.03. The data are representative of two separate experiments.

FIG. 6.

Mycobacterial infection induces naive OVA-specific T cells to differentiate into Th1 effectors in response to airway OVA challenge. Flow-sorted CD62L^hi CD44^low naive DO11.10 T cells were labeled with CFSE and transferred into BCG-infected and uninfected recipient mice. Recipient mice were challenged with OVA as described in Materials and Methods. Mice were sacrificed 3 days after OVA challenge, and single cell lung suspensions were prepared. (A) Lung cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A. Cells were surface labeled with KJ 1-26, stained for intracellular IFN-γ, and analyzed by flow cytometry. Histograms were gated on KJ⁺ T cells. The isotype for anti-IFN-γ is shown. The numbers represent the means ± the standard deviation of triplicate wells for each group. *, P = 0.03. (B) A total of 5 × 10⁵ lung cells were cultured for 48 h at 37°C with or without 2 μM OVA_323-339 peptide. The IL-4 and IFN-γ SFU were quantified by ELISPOT assay. **, P < 0.01. The data are representative of three separate experiments.

FIG. 7.

BCG infection causes upregulation of MHC-II on lung CD11c⁺ cells harboring intranasal Fluos-labeled OVA. BCG-infected and uninfected mice were given 450 μg of Fluos-labeled OVA intranasally. After 18 to 24 h the mice were sacrificed, and single-cell lung suspensions were prepared and stained with anti-CD11c, anti-CD11b, anti-I-Ad, anti-CD80, and anti-CD86. (A) FSC versus SSC plots were gated on live cells, and dot plots of individual mice from three groups are shown examining CD11c and Fluos. The percentages represent live cells (FSC versus SSC gate) harboring Fluos-OVA (three mice per group). (B) The plots were gated on Fluos⁺ CD11c⁺ cells. The percentages represent the distribution of Fluos-OVA to DCs (CD11c^hi CD11b^hi) within the CD11c⁺ population (n = 3). *, P = 0.01. (C) Histogram plots show the surface expression of MHC-II on the gated populations shown in panel A containing lung CD11c⁺ cells that harbor Fluos-OVA. The geometric mean fluorescence intensities of I-A^d staining are 31,000 ± 5,300 and 1,700 ± 450 for the BCG+OVA and OVA groups, respectively (n = 3). P = 0.03. (D) Total numbers of Fluos⁺ CD11c⁺ cells expressing high I-A^d (mean fluorescence intensity > 3,000; n = 3). **, P = 0.04. (E) CD11c⁺ cells were isolated from the lungs of infected and uninfected mice and plated out at different cell densities along with 2 μM OVA peptide and 10⁵ DOBW cells. IL-2 in the culture supernatants was assayed by ELISA. Similar data were obtained in two separate experiments.