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T follicular helper cells in HIV infection. Jan van Lunzen University Medical Center Hamburg-Eppendorf Heinrich-Pette-Institut for Experimental Virology & Immunology Hamburg, Germany. Outline/Questions. Where does HIV replicate in LN? Which cells are preferentially infected? - PowerPoint PPT Presentation
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T follicular helper cells in HIV infection
Jan van LunzenUniversity Medical Center Hamburg-Eppendorf
Heinrich-Pette-Institut for Experimental Virology & ImmunologyHamburg, Germany
Outline/Questions
Where does HIV replicate in LN? Which cells are preferentially infected? What effect/limitations does HAART have? What is the role of T follicular helper cells in HIV
infection? Are TFH cells a viral reservoir? What are the consequences of dysregulated TFH
function? Can these functions be restored?
Where does HIV replicate in lymph nodes?
Lymph Node PathologyHIV - HIV +
Stellbrink & van Lunzen, Curr Opin Infect Dis 2001
Immunopathology of LN
Tenner-Racz K, Stellbrink HJ, van Lunzen J et al., J Exp Med 1998
CD4+Ki-67+
CD8+
Which cells are preferentially infected?
Infection rate of CD57+ and CD57- CD4+ T cells in PB and LN
10
7,5
5
2,5
01 2 3 5 7Patient no.
Infe
cted
cel
ls (%
)
LN CD57+CD4+ T cellsLN CD57–CD4+ T cells
PBL CD57+CD4+ T cellsPBL CD57–CD4+ T cells
Ratio of the viral loads: CD57+CD4+ T cells [%]/CD57– CD4+ T cells [%]
LN: 10.5 3.3 8.0 9.5 3.0PBL: 0.3 2.2 <0.1 2.5 2.5
Hufert F, van Lunzen J et al.: AIDS 1997
What effect/limitations does HAART have?
HIV-1 RNA in LN during HAART
Tenner-Racz K, Stellbrink HJ, van Lunzen J et al., J Exp Med 1998van Lunzen J, Ruiz L et al., AIDS 1998
Day 0 Day 28
HIV-1 RNA p24 Ag
T cell proliferation in LN during HAART
15%
10%
5%
0CD4+Ki-67+ CD8+Ki-67+
p < 0,0001
p < 0,0001pre-treatment (n = 30)post-treatment (n = 30)normal controls (n = 4)
van Lunzen J, et al., CROI 1999
What is the role of T follicular helper cells in HIV infection?
CD4 TFH cells B cell
MHC IITCR
IL-21 IFNγIL-4
• B cell proliferation• Antibody maturation (SHM)• Class switching (CSR)• Memory development
• Within the follicle• Express CXCR5, PD1, ICOS• Bcl6+ • IL-21 (cardinal cytokine)
Plasma cell
Memory B cellCD40L CD40
CD4 TFH cells are critical for effective antibody responses
Bcl6
CXCR5
ICOS ICOSLBcl6
PD-1
SIV: Relative Accumulation of TFH in LN SIV acute SIV chronic (high % of TFH)SIV- SIV chronic (low % of TFH)
% o
f CM
CD
4 T
cells
p=0.0008 p<0.0001
PD-1
CC
R7
0 103 104 1050
102
103
104
105
ICO
S
CD150 Petrovas C. et al. JCI 2012
PD-1
CC
R7
Immune activation rather than direct infection with SIV is associated with in vivo TFH accumulation
0
0.5
1.0
1.5
Chronic SIV (>2 months)
SIV
Gag
DN
A (c
opie
s/ce
ll)
0 103 104 1050
102
103
104
105
ICO
S
CD150
p=0.0013p=0.0004
p=0.0164
sCD
14 (x
106 p
g/m
l)
SIV acute SIV chronic (high % of TFH)SIV- SIV chronic (low % of TFH)
Petrovas C. et al. JCI 2012
Accumulation of TFH is Associated with Increased Frequency of GC-B cells and titers of SIV-specific
Antibodies
PNAhighIgGlow
PNAhighIgGhigh
SIV - SIV +low % TFH
SIV +high % TFH
p=0.0286
SIV-
spec
ific
IgG
(ti
ter,
x104 )
p=0.051
p=0.033p=0.048
ND
ND
ND
ND
ND
0
2.5
5
7.5
10
SIV acuteSIV chronic (low % TFH)SIV chronic (high % TFH)
gp120 gp130 gp140 p27 p55
Petrovas C. et al. JCI 2012
High frequency of CXCR5+PD-1high CD4 T cell subset in the lymph node
Lindqvist M et al., JCI 2012
IL-21 preferentially produced by TFH cells
Lindqvist M et al., JCI 2012
Expansion of TFH cells correlated with skewing of B cell subsets
GC B cells Late memory B cellsPlasma B cells
Lindqvist M et al., JCI 2012
Hypersecretion of IgG was associated with BCL6 expression in TFH cells
Lindqvist M et al., JCI 2012
Are TFH cells a viral reservoir?
TFH Cells and CXCR5-PD-1+ CD4 T-Cells Are Enriched in HIV-Specific CD4 T-Cells
LNMCs Intracellular staining
Aqua, CD3, CD4, CD8, CD45RA, CXCR5, PD1, IL-21, IL-2, TNF-α, IFN-γ
PTEs (Gag, Pol, Env)+α-CD28Brefeldin A
Perreau M et al JEM 2012
TFH Cells and CXCR5-PD-1+ CD4 T-Cells Are Enriched in CD4 T-Cells Containing HIV DNA
DNA extraction Taqman RT-PCR HIV-1 gag gene
Perreau M et al JEM 2012
TFH Cells and CXCR5-PD-1+ CD4 T-Cells Are the Most Efficient in Supporting Production of HIV
p24 detection
D5D0
Anti-CD3/CD28
Collection of SNs
D2
High viremia (>15000 HIV RNA copies/mL)
Perreau M et al JEM 2012
HIV Isolation from Patients with Low (<1000 HIV RNA copies/mL of plasma) in Different CD4
T-Cell Populations
TFH and CXCR5-PD-1+ but not CXCR5-PD-1- and CXCR5+PD-1- CD4 T-cells efficiently support virus isolation and production in patients with low viremia levels
Low viremia (<1000 HIV RNA copies/mL)
p24 detectionCoated CD3/28
CD8-depleted PBMCs+
D7D0
Collection of SNs
D3
What are the consequences of dysregulated TFH function?
PD-L1 expression in GC B cells
Cubas RA et al. Nature Med. 2013
PD-1 ligand and TFH proliferation, activation and cytokine secretion
PD1-L
Cubas RA et al. Nature Med. 2013
IL-21
pg/
ml
Unstim. IgG beads
PD-L1 beads
0
20
40
60
80
100
ICO
S M
FI
P = 0.03P = 0.01
Abso
lute
num
ber
ICO
S+ T
fh ce
lls
P = 0.025P = 0.009
PD-1 Triggering affects TFH function
Cubas RA et al. Nature Med. 2013
Can these functions be restored?
P < 0.03
CD27
MFI
(B ce
lls)
CXCR5hi CXCR5- CD45RA+0
10002000300040005000600070008000 CTRL
IL-21 (10ng/ml)
IgG
(ng/
ml)
-IL-21 +IL21 HIV- LN
-IL-21 +IL21
IgG
(ng/
ml)
P < 0.02
P < 0.03
HIV+ LN
HIV+ LN
Exogenous IL-21 enhances TFH function
CD27
MFI
(B ce
lls)
-IL21 +IL21 +IL21 (10ng/ml) (40ng/ml)
P < 0.02
P < 0.02
Tota
l IgG
(ng/
ml)
Tota
l IgG
(ng/
ml)
-IL-21 +IL21
Isotype αPD-L1/L2
HIV+ LN (Gag-Env stimulation)
HIV+ LN (Gag-Env stimulation)
Enhancing HIV-specific TFH function by interfering with PD-1/IL-21 axis
Cubas RA et al. Nature Med. 2013
Hypothesis model
Lymph nodeB cell follicle
Germinal center
TFH
Plasma cell
Memory B cell
Germinal center B cell
IL-21
HIV viremia
Summary I
• HIV-specific TFH cells are expanded in chronic infection
• Expansion of TFH cells correlated with skewing of B cell subsets in chronic HIV infection
• Hypersecretion of IgG1 was associated with BCL6 expression in TFH cells
• TFH serve as a viral reservoir and promote HIV replication
Summary II
• B memory cell responses are skewed in HIV infection
• PD-L1 is overexpressed on B cells in HIV infection and down regulates TFH function
• TFH and B cell function can be restored by anti-PD-1 and/or
exogenous II-21
• TFH cells may represent the primary obstacle for achieving functional
HIV cure
Pathogenesis Model
Vinuesa CG, JCI 2012
O. DegenS. HertlingA. HüfnerS. Schmiedel
J. Schulze zur WieschI. TothP. HartjenS. Kummer
O. DegenS. HertlingA. HüfnerS. Schmiedel
J. Schulze zur WieschI. TothP. HartjenS. Kummer
Slides and data provided by:C. Petrovas, R. Koup (VRC, NIH, USA)M. Lindqvist, H. Streeck (Ragon Inst., MGH, Boston, USA)M. Perreau, G. Pantaleo (Univ. of Lausanne, Switzerland)R. Cubas, E. Haddad (VGTI, Florida, USA)
