memory ccr6+cd4+ t-cells are selectively imprinted with a transcriptional program favorable to...
TRANSCRIPT
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