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

Lee LN, Baban D, Ronan EO, Ragoussis J, Beverley PC, Tchilian EZ - BMC Med Genomics (2010)

Bottom Line: The gene profiles generated from each condition were compared.It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d.CD8 T cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nuffield Department of Medicine, University of Oxford, The Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK. lian.lee@ndm.ox.ac.uk

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.

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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.
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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.

Mentions: The lung i.n. sample expressed higher levels of the tissue-homing CC chemokines Ccl1, Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl8 and CXC chemokines Xcl1, Cxcl2, Cxcl9 and Cxcl16, which are able to recruit resting and activated T cells as well as other immune cells. Cxcr6, which may aid in localization of activated T cells to non-lymphoid tissue [19-21], was also preferentially up-regulated in the lung i.n. samples. Flow cytometric analysis indicated that this was detectable on the surface of lung i.n. CD8 T cells but was present on very few CD8 T cells from lung i.d. immunized animals, confirming the differential expression detected by microarray analysis (Figure 4A). Similarly, increased expression of Lag3 protein was detected on the surface of a subset of lung i.n. CD8 T cells (Figure 4B). Measurement of chemokine production by ex vivo lung lymphocytes isolated from i.n. and i.d. immunized mice at 3 weeks post-immunization confirmed increased production of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL16 (Table 2).


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

Lee LN, Baban D, Ronan EO, Ragoussis J, Beverley PC, Tchilian EZ - BMC Med Genomics (2010)

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.
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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.
Mentions: The lung i.n. sample expressed higher levels of the tissue-homing CC chemokines Ccl1, Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl8 and CXC chemokines Xcl1, Cxcl2, Cxcl9 and Cxcl16, which are able to recruit resting and activated T cells as well as other immune cells. Cxcr6, which may aid in localization of activated T cells to non-lymphoid tissue [19-21], was also preferentially up-regulated in the lung i.n. samples. Flow cytometric analysis indicated that this was detectable on the surface of lung i.n. CD8 T cells but was present on very few CD8 T cells from lung i.d. immunized animals, confirming the differential expression detected by microarray analysis (Figure 4A). Similarly, increased expression of Lag3 protein was detected on the surface of a subset of lung i.n. CD8 T cells (Figure 4B). Measurement of chemokine production by ex vivo lung lymphocytes isolated from i.n. and i.d. immunized mice at 3 weeks post-immunization confirmed increased production of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL16 (Table 2).

Bottom Line: The gene profiles generated from each condition were compared.It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d.CD8 T cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nuffield Department of Medicine, University of Oxford, The Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK. lian.lee@ndm.ox.ac.uk

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.

Show MeSH
Related in: MedlinePlus