2014 “towards an hiv cure” symposium melbourne the immune checkpoints pd-1, lag-3 and tigit are...
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2014 “Towards an HIV Cure” symposiumMelbourne
The Immune Checkpoints PD-1, LAG-3 and TIGIT are Biomarkers of HIV Infected Cells During ART
and Identify Distinct Cellular Reservoirs
Remi Fromentin, Wendy Bakeman, Mariam B Lawani, Gabriela Khoury, Elizabeth Sinclair, Frederick M. Hecht, Steven G. Deeks, Sharon R. Lewin, Jean-Pierre Routy, Rafick P. Sékaly,
Nicolas Chomont
Biomarkers of latently infected cells during ART
Identifying biomarkers of latently infected cells is of primary importance to specifically target and eliminate the persistent reservoir
Approach: Combining the “Where” with the “How”
Where: HIV persists in discrete subsets of cells during ART
How: Mechanisms driving the establishment and persistence of the HIV reservoir
Biomarkers of latently infected cells could be:- surrogate markers of higher susceptibility to HIV infection- markers of persistence providing selective advantages (latency maintenance, immune escape, replenishment)
Why immune checkpoints (ICs) could be biomarkers for HIV persistence during ART ?
Chen L, Nat Rev Imm. 2013
• ICs, negative regulators of T cell activation, regulate T cell proliferation and cytokine production.
• Several of these molecules are associated with T cell dysfunction in chronic HIV infection (PD-1, CTLA-4, TIM-3, CD160).(Trautmann et al, NatMed 2006; Kaufmann et al, NatImm 2007;
Jones et al, JEM 2008, Peretz el al, PLoSPath 2012)
• Immune dysregulations persist during ART (residual immune activation, incomplete CD4 T cell restoration, T cell dysfunction).
(Hatano et al, JID 2012; Kelley et al, CID 2009)
Hypothesis
By inhibiting T cell activation, negative regulators (Immune Checkpoints, ICs) may actively maintain viral latency and identify reservoir cells during ART.
ICs silence HIV
Inhibitory signals silencing HIV
APCAPC
Activated/Productivelyinfected CD4 T cell
ICs
Persistent/Latentlyinfected CD4 T cell
+ - + -
ICs
ICs = selective advantage for latently infected cells
A B ART
Association between the expression of ICs and virological markers of HIV persistence
Study population:48 HIV infected subjects virally suppressed for at least 3 years with CD4>350 c/µL
Methods:- Multiparametric
flow cytometry analysis of the expression of 8 ICBs (PD-1, LAG-3, TIGIT, CTLA-4, BTLA, CD160, 2B4, TIM-3) in PBMCs
- ultrasensitive qPCRs to measure the frequency of CD4 T cells harboring virological markers of HIV persistence
The frequency of CD4 T cells expressing PD-1, LAG-3 and TIGIT are positively correlated with the frequency of CD4 T cells harboring integrated HIV DNA during ART.
PD-1 identifies TCM and TTM CD4 T cells enriched in integrated HIV DNA
TCM TTM TEM
The frequency of cells harboring integrated HIV DNA is significantly higher in PD-1 expressing TCM and TTM when compared to their PD-1 negative counterparts.
DifferentiationDifferentiation
A B
LAG-3 identifies TCM and TTM CD4 T cells enriched in integrated HIV DNA
TCM TTM TEM
The frequency of cells harboring integrated HIV DNA is significantly higher in LAG-3 expressing TCM and TTM when compared to their LAG-3 negative counterparts.
DifferentiationDifferentiation
A B
TIGIT identifies TEM CD4 T cells enriched in integrated HIV DNA
TCM TTM TEM
The frequency of cells harboring integrated HIV DNA is significantly higher in TIGIT expressing TEM when compared to their TIGIT negative counterparts.
DifferentiationDifferentiation
A B
Can we further enrich in the reservoir by combining multiple ICs?
Means +/-SD from 5 subjects
A B
Memory CD4 T cells expressing multiple ICs are highly enriched for integrated HIV DNA
Is the virus carried by latently infected cells expressing ICs functional ?
A
B
Principle of “Tat/Rev Induced Limiting Dilution Assay” (TILDA)
Sorted CD4+ T cells
PMA/Ionomycin(12h) 18000 cells/well
9000 cells/well
3000 cells/well
1000 cells/well
Nested RT-PCRFor msHIV RNA(24+40 cycles)
Maximum likelihood method
Frequency of cells with inducible
msHIV RNA
Memory CD4 T cells expressing LAG-3 and/or PD-1 and/or TIGIT are highly enriched for inducible HIV latently infected cells.
Conclusions
Altogether, our data suggest that blocking ICs may reactivate HIV from
latency and paves the way for the development of novel strategies to
cure HIV infection.
BiomarkersCM TM EM
PD-1
LAG-3
TIGIT
1.
2.
Enrichment for HIV infected cellsEnrichment for HIV infected cells
Acknowledgements
• VGTI Florida
Wendy Bakeman
Amanda McNulty
Mariam B. Lawani
Rafick-Pierre Sekaly
Nicolas Chomont
• UCSF
Steven Deeks
Hiroyu Hatano
Rick Hecht
Rebecca Hoh
Elisabeth Sinclair
Lorrie Epling
• Burnet Institute
Gabriela Khoury
Sharon R. Lewin
• McGill University
Jean-Pierre Routy
• Merck
Daria Hazuda
Mike Miller
Richard J.O. Barnard
Dan Gorman
The study participants
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