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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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slide1
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
slide2
Natural cytotoxicity

Killing of virus-infected and tumor cells

Two-signal killing

slide3
In human peripheral blood, 2 primary NK subsets

CD56brightCD16-/dim,CD56dimCD16+

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

Webster & Johnson, Immunology, 2005

natural killer cells in tissues
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
objectives
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
nk cell gating
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

slide7
NK cell heterogeneity

Mucosal

Lymph Nodes

natural killer cell subset distribution in tissues of rhesus macaques
Natural killer cell subset distribution in tissues of rhesus macaques

CD56

“Standard” NK cells nearly absent from mucosae and LN

CD16

slide9
NK cell subset functionality

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

slide10
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

summary and conclusions
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
objectives12
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
slide14
NK cells during HIV/SIV infections

Alter and Altfeld, J Intern Med, 2008

slide15
SIV infection modulates distribution of NK cell subsets

Viral Load Correlation

R = 0.5494

p = 0.0224

slide16
SIV infection increases NK cell activation and cytotoxic markers

SIV infection increases activation and cytotoxic capacity

slide17
SIV perturbs NK trafficking markers

(CD62L)

Mackay, 1999, Nature

summary and conclusions18
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
slide19
View of earliest transmission events

Rhesus macaque model

Hypothesis

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

future directions
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.
acknowledgements
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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