Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

Abstract

Background: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.

Method: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.

Results: CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.

Conclusions: Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Figure 1

Cytokine responses of T cells to antigen 85A. BALB/c mice were immunized with Ad85A i.d. or i.n.. Lung and splenic lymphocytes were isolated 3 weeks post-immunization and stimulated with the dominant CD4 and dominant and subdominant CD8 peptides. The percentage of cells expressing IFNγ, TNF and IL-2 in (A) splenic CD8, (B) lung CD8, (C) splenic CD4 and (D) lung CD4 T cells as determined by flow cytometry. The values shown are the mean ± SEM from 3 mice per group and are representative of results obtained from at least 2 independent experiments.

Figure 2

Control of mycobacterial growth after aerosol M. tuberculosis challenge of Ad85A immunized mice. BALB/c mice were immunized with Ad85A i.d. or i.n. and challenged 4 weeks later with M. tuberculosis by aerosol. Mice were sacrificed 6 weeks later and mycobacterial burden in the lungs (A) and spleen (B) determined. The results show the log CFU in each mouse and the mean for each group and are representative of at least 2 independent experiments. The data were analyzed using the Kruskal-Wallis test (p = 0.007 comparing all groups), followed by Dunn's multiple comparison test, which returned p-values of < 0.05 for comparisons between naïve vs. i.n. and i.d. vs. i.n. groups. * indicates p < 0.05 compared to naïve or i.d.

Figure 3

Gene Ontology analysis of differentially expressed genes in CD8 T cells. (A) Spleen i.d. vs lung i.n., based on 550 differentially expressed transcripts and (B) lung i.n. vs lung i.d. based on 245 differentially expressed transcripts (>2 fold difference, p < 0.05).

Figure 4

CXCR6 expression on CD8 T cells from lungs of Ad85A immunized mice. BALB/c mice were immunized i.d. or i.n. with Ad85A. At 3 weeks post-immunization, lung lymphocytes were isolated and the percentage of CD8 T cells expressing CXCR6 was determined by flow cytometry. (A) Representative FACS plots of CD8⁺ gated cells showing CD8⁺CXCR6⁺ cells in lungs of i.d. and i.n. immunized mice. The bar chart shows the mean ± SEM from 3 independent experiments with 3 mice per group. (B) Representative FACS plots of CD8⁺ gated cells showing CD8⁺Lag3⁺ cells in lungs of i.d. and i.n. immunized mice 8-12 weeks previously. The bar chart shows the mean ± SEM of 3 i.d. and 5 i.n. immunized mice. (C) A representative histogram showing the expression of Ly6a on lung CD8 T cells. The bar chart shows the mean fluorescence intensity (MFI) ± SEM of 3 i.d. and 5 i.n. immunized mice. * indicates p < 0.05 by Mann-Whitney test.

Figure 5

The overlap of genes differentially expressed between lung i.n. and lung i.d., with genes reported to be related to a common lung inflammatory response. Venn diagram showing the overlap between genes more highly expressed by lung i.n. than i.d. cells, with genes reported as upregulated in lung inflammation [31].