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. 2011 Feb 20;410(2):429-436.
doi: 10.1016/j.virol.2010.12.017. Epub 2011 Jan 11.

Phenotypes and functions of persistent Sendai virus-induced antibody forming cells and CD8+ T cells in diffuse nasal-associated lymphoid tissue typify lymphocyte responses of the gut

Rajeev Rudraraju #  1 , Sherri Surman #  1 , Bart Jones  1 , Robert Sealy  1 , David L Woodland  2 , Julia L Hurwitz  1   3
Affiliations

Phenotypes and functions of persistent Sendai virus-induced antibody forming cells and CD8+ T cells in diffuse nasal-associated lymphoid tissue typify lymphocyte responses of the gut

Rajeev Rudraraju et al. Virology. .

Abstract

Lymphocytes of the diffuse nasal-associated lymphoid tissue (d-NALT) are uniquely positioned to tackle respiratory pathogens at their point-of-entry, yet are rarely examined after intranasal (i.n.) vaccinations or infections. Here we evaluate an i.n. inoculation with Sendai virus (SeV) for elicitation of virus-specific antibody forming cells (AFCs) and CD8(+) T cells in the d-NALT. Virus-specific AFCs and CD8(+) T cells each appeared by day 7 after SeV inoculation and persisted for 8 months, explaining the long-sustained protection against respiratory virus challenge conferred by this vaccine. AFCs produced IgM, IgG1, IgG2a, IgG2b and IgA, while CD8+ T cells were cytolytic and produced low levels of cytokines. Phenotypic analyses of virus-specific T cells revealed striking similarities with pathogen-specific immune responses in the intestine, highlighting some key features of adaptive immunity at a mucosal site.

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Figures

1
1. AFCs are rapidly recruited and persistently maintained in the d-NALT following SeV vaccination
AFCs were measured in C57BL/6 mice after a single i.n. inoculation with 250 PFU Sendai virus, on day 7, day 10, month 1, month 3 and month 8. IgM, IgG and IgA AFCs were monitored in the d-NALT, lungs and BAL (panels A, B, C, respectively). At peak activity, d-NALT cells were also assessed for IgG1, IgG2a, IgG2b and IgG3 production (panel A insert). For BAL samplings, due to limited cell numbers, AFCs were examined on day 7 (IgM and IgA only), day 10 (IgM, IgG and IgA), month 1 (IgG and IgA only), and month 3 (IgM, IgG and IgA). Nasal wash IgG and IgA isotypes were measured by ELISA (panel D).
2
2. Rapid and durable appearance of CD8+NP324-332/Kb-specific T cells in the d-NALT after SeV inoculation
The percentages of CD8+ NP324-332/Kb-specific T cells in the d-NALT, lungs and BAL of C57BL/6 mice inoculated with 250 PFU SeV were measured on day 7, day 10, month 1, month 3 and month 8.
3
3. Phenotypic analyses of d-NALT cells
After inoculation of C57BL/6 mice with 250 PFU SeV, d-NALT cells were collected for T cell phenotype analyses. Cells were gated on lymphocytes and on CD8+ cells. NP324-332/Kb-specific T cells were then analyzed for markers CD62L, CD44, CD11a, CD103 and CD69. Test days included day 0, day 10, month 1, month 3 and month 8. Circles highlight the major CD8+ NP324-332/Kb-specific T cell populations at month 1, month 3, and month 8, being of phenotype CD62LLoCD11aHiCD103HiCD69Hi.
4
4. Cells of the d-NALT are cytolytic
Lymphocytes from d-NALT, lungs and BAL were tested for cytotoxicity on day 10. Results from assays with NP324-332-pulsed and unpulsed cells are shown. Effectors were serially diluted (1:2) to test six E:T ratios beginning at 12.5:1.
5
5. Cytokine expression by d-NALT, lung and BAL cells following peptide stimulation
Ten days post-SeV inoculation of C57BL/6 mice, d-NALT, lungs and BAL cells were isolated for measurement of intracellular cytokine production. Results for cytokines IFN-γ and TNF-α are shown, as these were detected at significant levels.
6
6. Free cytokine in the respiratory tract of animals exposed to SeV
Nasal wash and BAL samples were tested for the presence of free cytokines over the course of a 1 month period. Results for cytokines IFN-γ, TNF-α and IL-5 are shown, as these were detected at significant levels.
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