Memory CCR6+CD4+ T-Cells are Selectively Imprinted with a

Transcriptional Program Favorable to Productive HIV-1 Infection

Patricia Monteiro, Jean-Philippe Goulet, Annie Gosselin, Vanessa Sue Wacleche, Mohamed-Rachid Boulassel, Jean-Pierre Routy, Nathalie

Grandvaux, Elias Haddad, Rafick-Pierre Sekaly, and Petronela Ancuta

Université de Montréal CHUM-Research Center, Saint-Luc Hospital

Montréal, Quebec, Canada

IAS2011, Rome, Italy, July 20, 2011

HIV-1 is a deadly but very selective virus

• Fact:– HIV-1 productively infects and persists in a very

small fraction of memory CD4+ T-cells (<10%)

• Goals:– To identify phenotypic and functional markers of

discrete CD4+ T-cell subsets permissive vs. resistant to HIV infection

– To identify the molecular mechanisms of HIV permissiveness vs. resistance in primary T-cells

Th17: mucosal immunity and HIV infection

CCR6

Th17 cells Mucosal homeostasis

Bridge between innate and adaptive immunity

Immune activation via the production pro-inflammatory cytokines

Sites of HIV replication

Microbial translocation

Chronic immune activation

Preferential HIV-DNA Integration in Memory CCR6+ T-Cells of HIV-Infected Patients

Gosselin/Monteiro et al., J Immunol, 2010

• CCR6:– Marker for Th17

polarization

– Regulates T-cell migration into Peyer’s Patches and other organs (spleen, brain)

• Imprinting of CD4+ T-cells with gut-homing potential: Mucosal dendritic cells → Retinoic acid (RA) production → up-regulation of integrin 47 → Migration into the gut (Mora et al., Immunity, 2004 ; Manicassamy et al., Nat Immunol, 2009)

• Integrin 47 and HIV / SIV:– New binding receptor for HIV gp120 (Arthos et al., Nat

Immunol, 2008)

– Identifies a subset of memory CD4+ T-cells producing IL-17 that is preferentially infected and depleted during acute SIV infection (Kader et al., Mucosal Immunol, 2009; Wang et al., Mucosal Immunol., 2009)

– Peripheral blood 47+ CD4+ T-cells are depleted during primary HIV infection (Krzysiek et al, Blood, 2001)

The Gut-Homing Molecule Integrin 47

• We investigated:–whether memory T-cells co-expressing

CCR6 and integrin 7 are selective HIV targets

–whether RA-induced imprinting for gut-homing selectively increases CCR6+ T-cell permissiveness to infection

Increased Expression of the HIV Coreceptor CCR5 on CCR6+7+ and CCR6+7- T-Cell Subsets

B

CCR5

β7-R6- β7-R6+ β7+R6+β7+R6-

CXCR4

Log Fluorescence Intensity

Cel

l cou

nts

A

Integrin β7

CC

R6

Memory T-cells

CD45RA

CD4+ T-cells

CD4

CD

3

Lymphocytes

CD

4

B

CCR5

β7-R6- β7-R6+ β7+R6+β7+R6-

CXCR4

Log Fluorescence Intensity

Cel

l cou

nts

A

Integrin β7

CC

R6

Memory T-cells

CD45RA

CD4+ T-cells

CD4

CD

3

Lymphocytes

CD

4Integrin β7

CC

R6

Memory T-cells

CD45RA

CD4+ T-cells

CD4

CD

3

Lymphocytes

CD

4

Monteiro et al. J. Immunology 2011

Increased Permissiveness to R5 HIV Replication in CCR6+7+ and CCR6+7- T-Cell Subsets

p<0.0001

p=0.0003

p<0.0001

p=0.0006p=0.0007

p=0.0002

Monteiro et al. J. Immunology 2011

ATRA Upregulates Integrin 47 and CCR5 Expression on CCR6+ and/or CCR6- T-Cells

CCR6- CCR6+

p=0.02

p=0.001Phenotype before FACS sort

Phenotype after FACS sort

CD45RA

FIT

C

CD45RA

FIT

C

CCR6

Cel

lCo

un

t

CCR6

Cel

lCo

un

tTotal CD4+ T-cells

CCR6-

CCR6+

Phenotype before FACS sort

Phenotype after FACS sort

CD45RA

FIT

C

CD45RA

FIT

C

CCR6

Cel

lCo

un

t

CCR6

Cel

lCo

un

tTotal CD4+ T-cells

CCR6-

CCR6+

Monteiro et al. J. Immunology 2011

p=0.04

p=0.02

p=0.04

CCR6- CCR6+CCR6- CCR6+CCR6- CCR6+CCR6- CCR6+

CD3/CD28 Abs±ATRA (10 nM)

ATRA Treatment Selectively Increases CCR6+ T-Cell Permissiveness to HIV at Entry and Post-Entry Levels

p<0.0001

p=0.0001

p<0.0001

p=0.0005

p=0.002

p=0.0003

p<0.0001

p<0.0001

p=0.003

CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+

CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+

p=0.03

p=0.0003

p=0.0023

p=0.0005

p=0.0002

p=0.0002

CCR6- CCR6+CCR6- CCR6+

p=0.028p=0.016

p=0.07

Monteiro et al. J. Immunology 2011

Enhanced TNF- Production and NF-B p65 DNA-Binding Activity in CCR6+ Compared to CCR6- T-Cells

Monteiro et al. J. Immunology 2011

A

CCR6- CCR6+

p=0.07

p=0.0053

p<0.0001

B

CCR6- CCR6+ CCR6- CCR6+ CCR6- CCR6+

p<0.0001

p<0.0001

p=0.0005

p=0.0002

p<0.0001

p<0.0001

A

CCR6- CCR6+CCR6- CCR6+

p=0.07

p=0.0053

p<0.0001

B

CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+

p<0.0001

p<0.0001

p=0.0005

p=0.0002

p<0.0001

p<0.0001

A Systems Biology Approach Toward the Identification of New HIV Permissiveness and Restriction Factors

Ancuta et al., unpublished data

CCR6+ATRA

CCR6+Medium

CCR6-ATRA

CCR6-Medium

• TCR signaling and cell activation (Lck, ZAP-70, PTPN13, MAP3K4, TANK)

• Lineage polarization profiles (IL-22, IL-26, CCL20, IL-5, IL-9)

• Regulation of gene transcription (RORC, RORA, PPARG, ARNTL, KLF2, NLF2, ATF5, E2F2, RUNX1)

• Immunological synapse formation (CXCR6, TNFRSF18)

• CCR6+ memory T-cells expressing or not the gut-homing integrin 7 are highly permissive to HIV replication.

• However, CCR6+ T-cells co-expressing integrin 7 and CCR5 might have an extraordinary ability to disseminate HIV from the portal sites of entry

• ATRA selectively enhances permissiveness to HIV-replication in CCR6+ T-cells via entry (CCR5 upregulation) and yet unidentified post-entry mechanisms

• CCR6 is a marker for memory CD4+ T-cells imprinted with a transcriptional program favorable to HIV replication

• The identification of HIV dependency factors in CCR6+ T-cells will open the path for the design of new therapeutic strategies to limit HIV replication in these cells while maintaining their role in mucosal immunity

Conclusions

Annie Gosselin, MScResearch Assistant

Patricia Monteiro, PhDPostdoctoral fellow

Vanessa Sue WaclechePhD Student

Hanane TouilMSc Student

Ancuta Lab

Petronela Ancuta, PhD

Aurélie Cleret, PhDPostdoctoral fellow

Annie BernierMSc Student

Acknowledgements

CRCHUM and VGTIRafick-Pierre Sekaly, PhDNicolas Chomont, PhDMohamed El-Far, PhDJean-Philippe Goulet, MScElias Haddad, PhD

CRCHUMNathalie Grandvaux, PhD

Flow Cytometry Core Facility – CHUM-Research CenterAnnie GosselinLaurence LejeuneSylvain Gimmig

Primo infection cohort - McGill UniversityJean-Pierre Routy, MDMohamed-Rachid Boulassel, PhD

Slow Progresors Cohort Cécile Tremblay, MD - CRCHUMNicole Bernard, PhD - McGill University

FRSQ-AIDS Infectious Diseases NetworkAnne Vassal and Mario Legault

HIV-infected and uninfected subjects for their gift of leukapheresis and essential contribution to this work

Superior Ki67 Expression in CCR6+ Compared to CCR6- T-Cells ex vivo

Monteiro et al. J. Immunology 2011

Ki67

Ce

ll co

unts

ex vivo

CCR6-

CCR6+

A B

p=0.001

Ki67

Ce

ll co

unts

ex vivo

CCR6-

CCR6+

A B

p=0.001

ATRA does not increase proliferation of CCR6+ T-cells

Monteiro et al. J. Immunology 2011

p=0.02

p=0.003

p=0.007

p=0.007

CCR6- CCR6+ CCR6- CCR6+

p=0.02

p=0.003

p=0.007

p=0.007

CCR6- CCR6+CCR6- CCR6+ CCR6- CCR6+CCR6- CCR6+

CCR6+7- and CCR6+7+ T-Cells are Enriched in Cells with a CCR7-CD27- (EM) and CCR7-CD27+ (TM) Phenotype

Monteiro et al. J. Immunology 2011

A β7-R6- β7-R6+ β7+R6+β7+R6-

CD27

CC

R7

CM

TMEM

CM

TMEM

CM

TMEM

CM

TMEM

p=0.008

p<0.0001p=0.003

B

p=0.02p=0.01

p=0.02

p=0.02

p=0.02

A β7-R6- β7-R6+ β7+R6+β7+R6-

CD27

CC

R7

CM

TMEM

CM

TMEM

CM

TMEM

CM

TMEM

p=0.008

p<0.0001p=0.003

B

p=0.02p=0.01

p=0.02

p=0.02

p=0.02

Decreased Frequency of Circulating CCR6+7- and CCR6+7+ T-Cells in HIV-Infected Subjects

Monteiro et al. J. Immunology 2011

p=0.001

B

A

Integrin β7

CC

R6

Integrin β7C

CR

6

HIV- controls HIV+ subjects

p=0.02p=0.03 p=0.01p=0.001

B

A

Integrin β7

CC

R6

Integrin β7C

CR

6

HIV- controls HIV+ subjects

p=0.02p=0.03 p=0.01

HIV+ (n=14)median CD4: 506 cells/µlmedian VL: 6,500 HIV-RNA copies/mlMedian time of infection: 5.5 monthsART for 1-3 months: 3 of 14 subjects