Éric a. cohen laboratory of human rétrovirology ircm

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HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and activate NK cell-mediated killing Éric A. Cohen Laboratory of Human Rétrovirology IRCM 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Cape Town July, 20, 2009

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HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and activate NK cell-mediated killing. Éric A. Cohen Laboratory of Human Rétrovirology IRCM 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Cape Town July, 20, 2009. - PowerPoint PPT Presentation

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Page 1: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and

activate NK cell-mediated killing

Éric A. Cohen Laboratory of Human

RétrovirologyIRCM

5th IAS Conference on HIV Pathogenesis, Treatment

and Prevention,Cape Town

July, 20, 2009

Page 2: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Viral Protein R (Vpr)

• HIV accessory protein of 96 aa (14 kDa)

• Exists in 3 different forms- Virion-associated (~275

molecules/virion)

- Intracellular forms- Extracellular forms:

Plasma and CSF

• Displays cell transduction properties

Page 3: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Vpr Biological Activities

• Facilitates infection of nondividing cells such as terminally differentiated macrophages

• Causes cell-cycle arrest at the G2 phase in CD4+ T cells via activation of the ATR-mediated DNA damage/stress signaling pathway – Conserved among HIV and SIV Vpr

– Abnormal accumulation of infected cells in G2 in HIV- infected subjects

Page 4: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

How Does Vpr Induce a G2 Arrest?

ATR

Chk1

Cdc2

Cyclin B

Cdc2

Cyclin B

?

ATM

DNA stress/damage checkpoint G2 arrest

Belzile et al., PLoS Pathogens, 2007

Page 5: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Functional Relevance of Vpr-induced G2 Arrest

• Vpr-induced G2 arrest has two downstream effects:– Enhances proviral transcription.– Promotes apoptosis of infected cells– Other Effects?

• Activation of the DNA damage/stress checkpoint pathway initiated by ATR or ATM kinase upregulates ligands of the activating NKG2D receptor and enhances susceptibility of target cells to NK cell killing (Gasser et al., 2005)

ATRNKG2D

Page 6: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Control of NK Cell Effector Function

• under control of a sophisticated and tightly regulated network of activating and inhibitory receptors

• NKG2D is a central activating receptor that modulates NK cell function – MHC class 1-related chains A/B

(MIC A/B)– UL16-binding protein (ULBP-1,

-2, -3, -4)

• NKG2D is expressed not only on NK cells, but also on T cells, CD8+T- cells, and a subset of CD4+ T-cells

• Expression of NKG2D ligands is largely confined to virus-infected, tumor and stressed cells.

Page 7: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

RATIONALE

HIV-1 infection of primary CD4+ T-lymphocytes increases cell-surface expression of specific NKG2D ligands both in vitro (Cerboni et al. , 2007;

Ward et al. 2007) and in vivo (Fogli et al., 2008)

GOAL

To investigate whether expression of Vpr could regulate cell-surface expression of NKG2D

ligands and modulate NK cell cytotoxic activity

Page 8: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

HIV-1 upregulates cell-surface expression of ULBP-2 in primary CD4+ T-cells in a

Vpr-dependent manner

PBMCs of healthy

subjects

Infection MOI= 0.5(GFP-marked R5 virus)

WT/ΔVpr-

Cell-surface staining anti-NKG2D ligands

on GFP+ cells

4-5 daysPrimary CD4+ T-lymphocytesactivated with

PHA/IL2

No upregulation of MICA and MICB cell-surface expression

N=5

ULBP-1 ULBP-2 ULBP-3

Rel

ativ

e ce

ll nu

mbe

r

Mock: 2.83

HIVΔVpr: 3.23

HIV WT: 2.96

Mock: 23.91

HIVΔVpr: 23.84

HIV WT: 40.65

Mock: 7.27

HIVΔVpr: 7.89

HIV WT: 7.38

Page 9: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Vpr enhances susceptibility of R5 HIV-1-infected-CD4+ T-lymphocytes to NK cell-mediated killing

Infected cells (GFP+) were exposed to autologous non-activated primary NK cells in a 4h-51Cr release assay

Vpr promotes NK cell-mediated killing at least in part via NKG2D

N=3

Donor 1 Donor 2

Page 10: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Expression of Vpr alone is sufficient to upregulate ULBP2 cell-surface expression

N=2

ULBP2

WPI-Vpr WT 126.9

WPI 45.4

Cel

l rel

ativ

e n

um

ber

Lentiviral vectors expressing GFP

WPI

WPI-Vpr WT

Primary CD4+ T-lymphocytes

Page 11: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Vpr, in the absence of any other HIV proteins, upregulates NKG2D ligands non-selectively

suggest that perhaps other viral protein(s) may limit cell-surface expression of some ligands

Lentiviral vector-transduced (GFP+) CEM.NKR T cells

ULBP-1 ULBP-2 ULBP-3 MICA MICB

Rel

ativ

e ce

ll n

um

ber

WPI-Vpr WT 58.2WPI

25.9

WPI-Vpr WT 97.8WPI

54.5

WPI-Vpr WT 28.6WPI 9.0

WPI-Vpr WT 315.8

WPI 82.8

WPI-Vpr WT 311.6

WPI174.9

APC 41.6

Mock29.4

APC 95.1

Mock52.1

APC 20.3

Mock8.7

APC 427.1Mock

91.7

APC 354.9Mock125.2

Page 12: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Upregulation of NKG2D ligands is dependent on Vpr-mediated G2-checkpoint arrest

R80A

Q65R

CEM.NKR T cells

WPI : 118.7WPI-Vpr R80A : 126.2WPI-Vpr Q65R : 200.1WPI-Vpr WT : 358.1

ULBP-2

Rel

ativ

e ce

ll n

um

ber

A

B

Page 13: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Vpr-mediated upregulation of NKG2D ligands is inhibited by Caffeine

CEM.NKR T cells ULBP-2

Rel

ativ

e ce

ll n

um

ber

WPI-Vpr WT 31.7

WPI 21.1

WPI-Vpr WT 102.5

WPI 35.1

APC 48.4

Mock24.8

APC 327.4

Mock 45.0

+ CaffeineNot treated

A B

Page 14: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Upregulation of NKG2D ligands by Vpr triggers NK cell cytotoxic responses

Transduced cells populations were exposed to non-activated primary NK cells in a 4h-51Cr release assay

CEM.NKR T cells N=2

BA

Page 15: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Delivery of virion-associated Vpr via defective HIV-1 particles upregulates ULBP-2 in non-infected target cells

N=3

Indinavir-treated HIVΔVpr HIVΔVprΔRTΔIN

Donor 1 Donor 2

Indinavir-treated HIVΔVpr

A B C

D

+ Vpr WT46.0

+ Vpr Q65R14.8

+ Vpr WT35.5

+ Vpr Q65R17.8

Page 16: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Conclusions• HIV upregulates selectively cell-surface expression of ULBP-2 in

primary CD4+ T-cells in a Vpr-dependent manner

• Vpr alone is sufficient to upregulate expression of NKG2D ligands, including ULBP-1, -2, -3, MICA and MICB, suggesting that HIV replication and expression of other virus proteins are not required for this activity of Vpr

• The absence of selective NKG2D ligand upregulation when Vpr is expressed alone suggests that another viral factor limits cell-surface expression of some ligands during HIV-1 infection

• Vpr upregulates expression of NKG2D ligands by a process that relies on the recruitment of the DDB1-CUL4A (DCAF1) E3 ligase and on activation of the ATR/ATM-mediated DNA damage/stress checkpoint pathway

Vpr is a key viral determinant responsible for induction of NKG2D ligands during HIV-1 infection

Page 17: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Conclusions

• Vpr expressed alone or during HIV-1 infection promotes NKG2D-dependent NK cell cytotoxic responses via its ability to upregulate NKG2D ligands

• Delivery of virion-associated Vpr via HIV-1 defective particles induces analogous effects in non-infected target cells, suggesting that Vpr may act similarly beyond infected cells

Overall, this study suggests an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced

CD4+ T lymphocyte depletion but may also play a part in HIV-1-induced NK cell dysfunction through sustained

NKG2D receptor activation

Page 18: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Acknowledgements

Dr. D. Trono (EPFL, Lausanne) Dr. H. Göttlinger (U. of Mass.) Dr. Y. Ishizaka (Tokyo)

ReagentsLaboratory of Human Retrovirology

RESEARCH TEAM IN HIV PATHOGENESIS (HPAT)

JP BelzileJ. Richard

S. Sindhu

Flow Cytometry

• Eric Massicote (IRCM)• Martine Dupuis (IRCM)

IRCM Clinic

• Dr P. Larochelle• Mrs M. Gauthier

All the volunteers

Page 19: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

Expression of Vpr WT and Vpr mutants (R80A and Q65R) in CEM.NKR T cells

WPI : 37.9WPI-Vpr R80A : 113.0WPI-Vpr Q65R : 145.5WPI-Vpr WT : 111.4

Rel

ativ

e ce

ll n

um

ber

Vpr

Page 20: Éric A. Cohen Laboratory of Human Rétrovirology IRCM

ULBP-2

Rel

ati

ve

cel

l n

um

ber

GFP + GFP -

WPI-Vpr WT 49.5WPI28.1

WPI-Vpr WT 80.6WPI31.2