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Natural cytotoxicity

Role of Natural Killer Cells in Vaccine-Elicited Control of HIV/SIV Infections R. Keith Reeves, Ph.D. Division of Immunology NEPRC, Harvard Medical School November 19, 2009. Natural cytotoxicity.

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Natural cytotoxicity

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  1. Role of Natural Killer Cells in Vaccine-Elicited Control of HIV/SIV Infections R. Keith Reeves, Ph.D. Division of Immunology NEPRC, Harvard Medical SchoolNovember 19, 2009

  2. Natural cytotoxicity Killing of virus-infected and tumor cells Two-signal killing

  3. In human peripheral blood, 2 primary NK subsets CD56brightCD16-/dim,CD56dimCD16+ NK cells in rhesus macaques Poorly defined – CD3-CD16+ Webster & Johnson, Immunology, 2005

  4. Natural killer cells in tissues • Little is known about the phenotype of NK cells in tissues other than blood of humans and macaques • CD56bright NK cells in human LNs, placenta, and gut mucosa • Transmission and replication of lentiviruses primarily take place at mucosal sites and associated secondary lymphoid tissues

  5. Objectives • To characterize NK cell subsets in blood and tissues of normal rhesus macaques and correlate functionality with discrete cellular phenotypes • To examine NK cell function in blood and tissues of macaques infected with wild-type or live attenuated SIV

  6. NK cell gating DC/B cell exclusion HLA-DR SSC FSC CD45 CD8αα T cell exclusion Macaque NK phenotype in PBMC/tissues: CD45+CD8αα+CD3- NKG2A+ CD20-/dim NKG2A CD8αα CD20 CD3

  7. NK cell heterogeneity Mucosal Lymph Nodes

  8. Natural killer cell subset distribution in tissues of rhesus macaques CD56 “Standard” NK cells nearly absent from mucosae and LN CD16

  9. NK cell subset functionality Up to 4-function polychromatic flow cytometry assay measuring activity against the MHC-devoid cell line 721.221

  10. NK cell subset functionality CD16+ CD56+ DN % responding cells IFN-γ TNF-α MIP-1β CD107a Similar division of labor as seen in human subsets - Unique functionality of undescribed DN subset Underappreciated complexity

  11. Summary and Conclusions • Rigorous definition of macaque NK cells (CD3-CD8+NKG2A+) only achievable by polychromatic flow cytometry • NK subsets vary dramatically in different compartments: • PB: CD16+ • LN: CD56+≈DN • Mucosal tissues: CD56+>DN • Function of NK cell subsets vary significantly • CD16+: primarily cytotoxic effectors • CD56+: primarily cytokine-secreting cells • DN: cytokine secretion and cytotoxic capacity • Standard CD16+ definition of NK cells misses > 80% of those in tissues and in lymph nodes • CD16hiCD56– and CD 56hiCD16– macaque NK subsets are analogous to human NK cells in phenotype, function and tissue distribution

  12. Objectives • To characterize NK cell subsets in blood and tissues of normal rhesus macaques and correlate functionality with discrete cellular phenotypes • To examine NK cell function in blood and tissues of macaques infected with wild-type or live attenuated SIV

  13. NK cells during HIV/SIV infections

  14. NK cells during HIV/SIV infections Alter and Altfeld, J Intern Med, 2008

  15. SIV infection modulates distribution of NK cell subsets Viral Load Correlation R = 0.5494 p = 0.0224

  16. SIV infection increases NK cell activation and cytotoxic markers SIV infection increases activation and cytotoxic capacity

  17. SIV perturbs NK trafficking markers (CD62L) Mackay, 1999, Nature

  18. Summary and Conclusions • Wild-type SIV infection results in: • Expansion of the numbers of circulating CD16+ and DN NK cells • Upregulation of activation and cytotoxic markers during SIV infection • Downregulation of lymph node trafficking molecules during SIV infection

  19. View of earliest transmission events Rhesus macaque model Hypothesis NK cells can interrupt early HIV/SIV replication prior to onset of adaptive responses

  20. Future Directions • Comprehensive phenotypic and functional assessments of NK cells in lymphoid and mucosal tissues: • Study groups: • Normal, WT SIV- and SIV∆nef-infected macaques • Intensive sampling of lymphoid and GALT tissue • Assays: • Cytoxicity assays vs 721 and SIV-infected cells • Viral suppression • Intracellular cytokine staining • Quantitative RT-PCR of NK receptors • Hypotheses to be addressed: • Acute SIV infection induces a rapid influx of NK cells to sites of virus replication (i.e., gut mucosa) that precedes virus-specific adaptive immune responses. • Acute SIV infection results in activation of NK cells associated with increased cytotoxicity and ability to inhibit SIV replication. • NK cell activity is a correlate of control and protection against wild-type and attenuated lentiviruses.

  21. Acknowledgements New England Primate Research Center, Harvard Medical School • Primate Medicine • Angela Carville • Pathology • Kate Hammerman Immunology • Jackie Gillis • Tristan Evans • Michelle Connole • Fay Eng Wong • Yi Yu • Paul Johnson • Miti Kaur FUNDING • Ragon Institute • Galit Alter • Marcus Altfeld • NCI-Frederick • Mike Piatak • Jeff Lifson • U. Minnesota • Ashley Haase • SFBR • Luis Giavedoni

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