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? 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 Pathology HIV - HIV + Stellbrink & van Lunzen, Curr Opin Infect Dis 2001

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

Which cells are preferentially infected?

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: 10.5 3.3 8.0 9.5 3.0 PBL: 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 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

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

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

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-21 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

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

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

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

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

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

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

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

Are TFH cells a viral reservoir?

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

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

(>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

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

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 Cubas RA et al. Nature Med. 2013

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

Can these functions be restored?

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

(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-21 +IL21 Isotype αPD-L1/L2 HIV+ LN (Gag-Env stimulation) HIV+ LN (Gag-Env stimulation) Cubas RA et al. Nature Med. 2013

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

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

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

Pathogenesis Model Vinuesa CG, JCI 2012

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!

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!