1. T follicular helper cells in HIV infection
Jan van Lunzen
University Medical Center Hamburg-Eppendorf
Heinrich-Pette-Institut for Experimental Virology & Immunology
Hamburg, Germany

2. 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?

3. Where does HIV replicate in lymph nodes?

4. Lymph Node Pathology HIV - HIV +
Stellbrink & van Lunzen, Curr Opin Infect Dis 2001

5. Immunopathology of LN CD4+Ki-67+ CD8+
Tenner-Racz K, Stellbrink HJ, van Lunzen J et al., J Exp Med 1998

6. Which cells are preferentially infected?

7. Infection rate of CD57+ and CD57- CD4+ T cells in PB and LN
LN CD57+CD4+ T cells
LN CD57–CD4+ T cells
PBL CD57+CD4+ T cells
PBL CD57–CD4+ T cells 10
7,5
Infected cells (%) 5
2,5
Patient no. 1 2 3 5 7
Ratio of the viral loads: CD57+CD4+ T cells [%]/CD57– CD4+ T cells [%]
LN:
PBL: <
Hufert F, van Lunzen J et al.: AIDS 1997

8. What effect/limitations does HAART have?

9. HIV-1 RNA in LN during HAART
Day 0
Day 28
HIV-1 RNA
p24 Ag
Tenner-Racz K, Stellbrink HJ, van Lunzen J et al., J Exp Med 1998
van Lunzen J, Ruiz L et al., AIDS 1998

10. T cell proliferation in LN during HAART
15%
10% 5%
pre-treatment (n = 30)
post-treatment (n = 30)
normal controls (n = 4)
p < 0,0001
p < 0,0001
CD4+Ki-67+
CD8+Ki-67+
van Lunzen J, et al., CROI 1999

11. What is the role of T follicular helper cells in HIV infection?

12. CD4 TFH cells are critical for effective antibody responses
Plasma cell
B cell
CD4 TFH cells
MHC II
TCR
PD-1
CXCR5
Memory B cell
CD40L
CD40
Bcl6
Bcl6
ICOSL
ICOS
IL IFNγ
IL-4
We have known for a long time the importance of CD4 T cell help to B cells for induction of proliferation, somatic hypermutation, class switching and memory formation.
During the last decade these Cd4 T helper cells have been recognized as a new lineage, termed t follicular helper cells. They have been described as reciding in the B cell follicle, due to high expression of CXCR5. They are PD1and ICOS positive, express the transcription factor Bcl6 and secrete IL21.
Given the importance of TFh cells in inducing and regulating B cell responses, it is surprising that nothing is currently known about TFH cells in HIV infection. So the question that we asked ourselves was:
Within the follicle
Express CXCR5, PD1, ICOS
Bcl6+
IL-21 (cardinal cytokine)
B cell proliferation
Antibody maturation (SHM)
Class switching (CSR)
Memory development 12

13. SIV: Relative Accumulation of TFH in LN
SIV acute
SIV chronic (high % of TFH)
SIV-
SIV chronic (low % of TFH)
% of CM CD4 T cells
p=0.0008
p<0.0001
PD-1
CCR7 10 3 4 5 2
ICOS
CD150
Petrovas C. et al. JCI 2012 13

14. Chronic SIV (>2 months) SIV Gag DNA (copies/cell)
Immune activation rather than direct infection with SIV is associated with in vivo TFH accumulation
Chronic SIV (>2 months)
p=0.0013
p=0.0004
p=0.0164
sCD14 (x106 pg/ml)
0.5
1.0
1.5
SIV Gag DNA (copies/cell)
SIV acute
SIV chronic (high % of TFH)
SIV-
SIV chronic (low % of TFH)
PD-1
CCR7 10 3 4 5 2
ICOS
CD150
Petrovas C. et al. JCI 2012 14

15. SIV-specific IgG (titer, x104)
Accumulation of TFH is Associated with Increased Frequency of GC-B cells and titers of SIV-specific Antibodies
SIV acute
SIV chronic (low % TFH)
SIV chronic (high % TFH)
p=0.0286
PNAhighIgGlow
PNAhighIgGhigh 10
7.5
SIV-specific IgG (titer, x104)
p=0.051
p=0.048
p=0.033 5
2.5 ND ND ND ND ND
SIV -
SIV +
low % TFH
SIV +
high % TFH
gp120
gp130
gp140
p27
p55
Petrovas C. et al. JCI 2012 15

16. High frequency of CXCR5+PD-1high CD4 T cell subset in the lymph node
The frequency of this CD4 T cell subset was 100 fold higher in LN compared to in the periphery
we further verified the lineage commitment by staining for Bcl6,
Lindqvist M et al., JCI 2012 16

17. IL-21 preferentially produced by TFH cells
Further, we could show that IL21, which is a cardinal cytokine of Tfh cells and has been shown to regulate B cell diffrentitation, proliferation and subsequently GC responseswas mainly produced by TFH cell subset
With the selective depletion of CD4 T cells in HIV infection, we were interested in looking at the effect of HIV on TFH cells
Lindqvist M et al., JCI 2012 17

18. Expansion of TFH cells correlated with skewing of B cell subsets
GC B cells
Plasma B cells
Late memory B cells
Interestingly, we could detect strong correlations could be between the increase of GC B and PCs with Tfh cell frequency. The loss of memory B cells could also be strongly correlated to the increase of TFh cells.
Indicating a contribution of TFh cells in the skewing of B cell phenotypes in HIV infection
Lindqvist M et al., JCI 2012 18

19. Hypersecretion of IgG was associated with BCL6 expression in TFH cells
Given the significant changes observed in the B cell compartment, we next determined if the expansion of TFH cells is associated with hypergammaglobulinemia
Interestingly,
We could detect a strong correlation between the Bcl6 expression in TFH cells and IgG levels in the sera, this association was mainly driven by IgG1 which constituted 70% of the secreted IgG antibodies in these patients.
Lindqvist M et al., JCI 2012 19

20. Are TFH cells a viral reservoir?

21. PTEs (Gag, Pol, Env)+α-CD28
TFH Cells and CXCR5-PD-1+ CD4 T-Cells Are Enriched in HIV-Specific CD4 T-Cells
PTEs (Gag, Pol, Env)+α-CD28
Brefeldin A
LNMCs
Intracellular staining
Aqua, CD3, CD4, CD8, CD45RA, CXCR5, PD1, IL-21, IL-2, TNF-α, IFN-γ
Perreau M et al JEM 2012

22. 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

23. (>15000 HIV RNA copies/mL)
TFH Cells and CXCR5-PD-1+ CD4 T-Cells Are the Most Efficient in Supporting Production of HIV
Anti-CD3/CD28
Collection of SNs
p24 detection D0 D2 D5
High viremia
(>15000 HIV RNA copies/mL)
Perreau M et al JEM 2012

24. Low viremia (<1000 HIV RNA copies/mL)
HIV Isolation from Patients with Low (<1000 HIV RNA copies/mL of plasma) in Different CD4 T-Cell Populations
Collection of SNs
CD8-depleted PBMCs +
Coated CD3/28
p24 detection D0 D3 D7
Low viremia (<1000 HIV RNA copies/mL)
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

25. What are the consequences of dysregulated TFH function?

27. PD-L1 expression in GC B cells
Cubas RA et al. Nature Med. 2013

28. PD-1 ligand and TFH proliferation, activation and cytokine secretion
Cubas RA et al. Nature Med. 2013

29. PD-1 Triggering affects TFH function
ICOS MFI
P = 0.009
P = 0.025
IL-21 pg/ml
Absolute number ICOS+ Tfh cells
Cubas RA et al. Nature Med. 2013 29

30. Can these functions be restored?

31. Exogenous IL-21 enhances TFH function
P < 0.03
P < 0.03
IgG (ng/ml)
IgG (ng/ml)
-IL IL HIV- LN
HIV+ LN
P < 0.02
P < 0.02
P < 0.02
CD27 MFI (B cells)
CD27 MFI (B cells)
-IL IL21
-IL IL IL21
(10ng/ml) (40ng/ml)
HIV+ LN

32. (Gag-Env stimulation) (Gag-Env stimulation)
Enhancing HIV-specific TFH function by interfering with PD-1/IL-21 axis
Total IgG (ng/ml)
Total IgG (ng/ml)
-IL IL21
Isotype αPD-L1/L2
HIV+ LN
(Gag-Env stimulation)
HIV+ LN
(Gag-Env stimulation)
Cubas RA et al. Nature Med. 2013

33. Hypothesis model Plasma cell Memory B cell Germinal center B cell
HIV viremia
IL-21
Based on our data we propose a model for B cell dysfunction in HIV infection that is not based on lack of CD4 T cell help, but rather that persistent viremia drive an abnormal expansion of TFH cells
We believe that TFH cells stimulate GC B cells, we don’t know today what directs development of GC B cells into memory B cells or plasma cells. The loss of memory B cells could be due to some fucntional loos of the TFH cells. It is also known that IL-21 induce immunoglobulin secreting cells, which might drive antibody secretion
This is however something that needs to be studied further.
Plasma cell
TFH
B cell follicle
Lymph node 33

34. 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
So to summarize:
We were able to detect an increase of CXCR5+,PD1high TFH cells in LN of chronic HIV infected patients
The observed expansion of Tfh cells correlated with an a skewing of the B cell phenotypes towards GC B cells an PCs, with loss of Memory B cells
And we could also see that hypersecretion of IgG and IgG1 was associated with the Bcl6 expression in TFH cells in chronic HIV infection 34

35. 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
So to summarize:
We were able to detect an increase of CXCR5+,PD1high TFH cells in LN of chronic HIV infected patients
The observed expansion of Tfh cells correlated with an a skewing of the B cell phenotypes towards GC B cells an PCs, with loss of Memory B cells
And we could also see that hypersecretion of IgG and IgG1 was associated with the Bcl6 expression in TFH cells in chronic HIV infection 35

36. Pathogenesis Model
Vinuesa CG, JCI 2012

37. O. Degen J. Schulze zur Wiesch S. Hertling I. Toth A. Hüfner
S. Schmiedel
J. Schulze zur Wiesch
I. Toth
P. Hartjen
S. Kummer
Now to the most important part of my talk: First I want to thank my team from the HIV Labor at the university of hamburg, my supervisor Jan van Lunzen and my guardian Julian Schulze zu Wiesch for their financial and intellectual support and their patience with me, for the assistence and emotional support I thank Kristina Colberg, Silke Kummer and Philip Hartjen.
I also thank the team of the department of infectious diseases of the University of Hamburg for their zealous collecting of patients blood. I thank Joachim Hauber from the HPI for his cooperation and support of my work, Dirk Meyer Olson and his PhD student Phillip Keudel from Hannover, who helped me to get enough PBMC of HIV-progressive patients, Gerd Fätkenheuer and Clara Lehmann from cologne for their help with nonprogressive patients. And most of all I thank the patients for their blood donation and you for your attention!

38. Slides and data provided by:
O. Degen
S. Hertling
A. Hüfner
S. Schmiedel
J. Schulze zur Wiesch
I. Toth
P. Hartjen
S. 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)
Now to the most important part of my talk: First I want to thank my team from the HIV Labor at the university of hamburg, my supervisor Jan van Lunzen and my guardian Julian Schulze zu Wiesch for their financial and intellectual support and their patience with me, for the assistence and emotional support I thank Kristina Colberg, Silke Kummer and Philip Hartjen.
I also thank the team of the department of infectious diseases of the University of Hamburg for their zealous collecting of patients blood. I thank Joachim Hauber from the HPI for his cooperation and support of my work, Dirk Meyer Olson and his PhD student Phillip Keudel from Hannover, who helped me to get enough PBMC of HIV-progressive patients, Gerd Fätkenheuer and Clara Lehmann from cologne for their help with nonprogressive patients. And most of all I thank the patients for their blood donation and you for your attention!