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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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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


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Natural cytotoxicity

Killing of virus-infected and tumor cells

Two-signal killing


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In human peripheral blood, 2 primary NK subsets

CD56brightCD16-/dim,CD56dimCD16+

NK cells in rhesus macaques Poorly defined – CD3-CD16+

Webster & Johnson, Immunology, 2005


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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


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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


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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


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NK cell heterogeneity

Mucosal

Lymph Nodes


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Natural killer cell subset distribution in tissues of rhesus macaques

CD56

“Standard” NK cells nearly absent from mucosae and LN

CD16


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NK cell subset functionality

Up to 4-function polychromatic flow cytometry assay measuring activity against the MHC-devoid cell line 721.221


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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


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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


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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


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NK cells during HIV/SIV infections


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NK cells during HIV/SIV infections

Alter and Altfeld, J Intern Med, 2008


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SIV infection modulates distribution of NK cell subsets

Viral Load Correlation

R = 0.5494

p = 0.0224


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SIV infection increases NK cell activation and cytotoxic markers

SIV infection increases activation and cytotoxic capacity


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SIV perturbs NK trafficking markers

(CD62L)

Mackay, 1999, Nature


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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


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View of earliest transmission events

Rhesus macaque model

Hypothesis

NK cells can interrupt early HIV/SIV replication prior to onset of adaptive responses


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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.


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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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