Supplemental Information. Human Circulating PD-1 + CXCR3 CXCR5 + Memory. Tfh Cells Are Highly Functional and Correlate

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1 Immunity, Volume 39 Supplemental Information Human Circulating PD-1 + CXCR3 CXCR5 + Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses Michela Locci, Colin Havenar-Daughton, Elise Landais, Jennifer Wu, Mark A. Kroenke, Cecilia L. Arlehamn, Laura F. Su, Rafael Cubas, Mark M. Davis, Alessandro Sette, Elias K. Haddad, International AIDS Vaccine Initiative Protocol C Principal Investigators, Pascal Poignard, and Shane Crotty 1

2 IAVI Protocol C Principal Investigators: Matt A. Price IAVI New York, NY, USA UCSF Department of Epidemiology and Biostatistics, San Francisco, CA, USA Jill Gilmour IAVI Human Immunology Laboratory, London, UK Pat Fast IAVI New York, NY, USA Anatoli Kamali Medical Research Council/Uganda Virus Research Institute (MRC/UVRI), Uganda Research Unit on AIDS, Masaka & Entebbe, Uganda Eduard J. Sanders Nuffield Department of Medicine, University of Oxford, Headington, UK Centre for Geographic Medicine Research Coast & Kenya Medical Research Institute (CGMRC & KEMRI), Kilifi, Kenya Omu Onzala Kenyan AIDS Vaccine Initiative (KAVI) Nairobi, Kenya Susan Allen Rwanda-Zambia HIV Research Group (RZHRG), Kigali, Rwanda and Lusaka, Zambia Emory University, School of Medicine, Atlanta, GA, USA Eric Hunter Rwanda-Zambia HIV Research Group (RZHRG), Kigali, Rwanda and Lusaka, Zambia Emory University, Emory Vaccine Center, Atlanta, GA, USA Etienne Karita Rwanda-Zambia HIV Research Group (RZHRG), Kigali, Rwanda William Kilembe Rwanda-Zambia HIV Research Group (RZHRG), Lusaka, Zambia Shabir Lakhi Rwanda-Zambia HIV Research Group (RZHRG), Lusaka, Zambia Mubiana Inambao Rwanda-Zambia HIV Research Group (RZHRG), Ndola, Zambia 2

3 Supplemental Experimental Procedures HIV + donors, neutralizing antibody screening, and classification of bnab groups IAVI Protocol C is a large longitudinal sub-sahara African HIV primary infection cohort consisting of 9 clinical research centers. Seronegative individuals were monitored for HIV infection by serum sampling. No pre-infection cell samples were available as individuals were enrolled into Protocol C upon becoming HIV +. Eligibility for enrollment into Protocol C was defined as: male or female at least 18 years of age with documented HIV infection, clinically asymptomatic, and not currently receiving antiretroviral therapy (ART). Blood was drawn at 3-6 month intervals. PBMCs were collected and stored under standard conditions. Serum samples were tested for HIV neutralizing antibodies using a 6 cross-clade pseudovirus panel (Monogram Biosciences); clade A, 94UG13 and 92BR2; clade B, JRCSF; clade C, IAVIC22 and 93IN95; clade AE, 92TH21)(Simek et al., 29). A more extensive explanation of testing for neutralizing antibodies will be published elsewhere (Landais E. and Poignard P, unpublished data). Individuals were categorized as follows: Top neutralizers, 5 or 6 virus neutralized at >4% and at least 4 viruses neutralized at >5% at a 1:1 serum dilution. Low neutralizers were defined as 2 or fewer viruses neutralized at >4% at a 1:1 serum dilution after at least 48 months of follow-up. A total of 25 top and 28 low neutralizers were analyzed in addition to 24 African HIV negative donors (South African blood bank samples). African and USA HIV negative donors gave comparable profiles (Data not shown). PBMC samples from HIV + individuals were tested using the earliest available samples were ~4 months post infection, and samples obtained at the time of bnab development which were ~4 months post infection. For all donors, the ~4 month time point was within 9 months of infection. The ~4 month time points ranged from range months by which time most Top neutralizers had developed neutralizing breadth. All donors were ART-free and had CD4 counts >2 cells/ml at each time point tested. Flow Cytometry and Cell sorting For surface staining, primary staining panels used for phenotypic analysis or for cell sorting in are described in Supplementary Table 1 and 2. For the intranuclear staining: cells were treated with the FoxP3 Fixation/Permeabilization kit (ebioscience) and stained in Permeabilization buffer (ebioscience). The following anti-human/mouse Abs were used: Maf efluor 66 (symf1, ebioscience) and Bcl6 Alexa Fluor 647 (K112-91, BD Bioscience). For cytokine staining: after FACS sorting, cells were let sit overnight at 37 C in AIM V medium supplemented with human recombinant IL-7 (4 ng/ml, Peprotech). The following day, cells were stimulated for 5 hours at 37 C with Phorbol 12-myristate 13-acetate (PMA, 25ng/ml) and Ionomycin (1 g/ml) in the presence of Brefeldin A (5 g/ml). Cells were fixed with PBS 2% FBS, 1% PFA and permeabilized with PBS 2% FBS,.5% Saponin. The following anti-human mabs were used: IL-21 PE (ebio3a3-n2), IFN- efluor 45 (4S.B3), CD154 (CD4L) PerCP-eFluor e71 (24-31) from ebioscience. IL-4 Pe- Cy7 (MPA-25D2) and IL-2 Alexa Fluor 7 (MQ1-17H12) were from Biolegend. Samples were analyzed on a LSR Fortessa Cell Analyzer (BD, Bioscience) or sorted using a FACS Aria (BD, Bioscience). Flow cytometry data were analyzed with FlowJo (Tree Star). For flow cytometry analysis of HIV + and control cohorts: Frozen PBMC were thawed, washed, counted, and resuspended in RPMI supplemented with 1% FBS, 1% Pen/Strep, 1% GlutaMax. 5x1 5 cells were plated and washed with FACS Buffer (PBS, 2% FBS), then stained with antibody mixes (Table S2) for 1 hour at 4 C. Cells were then washed, fixed for 1 minutes with 2% PFA, washed, and resuspended for immediate acquisition. Both top and low neutralizers were included in each batch of samples stained for flow cytometry. Tetramer staining CD4 + T cells were isolated by magnetic beads (CD4 + T cell isolation kit II, Miltenyi Biotec) from 15-5 x1 6 leukopheresed PBMCs of normal donors previously identified as expressing the HLA-DR haplotype HLA-DRB1*41 +. Enriched CD4 + T cells were stained for 1 hour at room 3

4 temperature with CD14, CD16, CD8, CD19 efluor 65NC Abs (ebioscience), PE conjugated HLA-DRB1*41 tetramers (kindly provided by Dr. Mark Davis) loaded with the Tetanus peptides TT (NYSLDKIIVDYNLQSKITLP) and PE-Cy7 conjugated HLA-DRB1*41 tetramers loaded with unrelated peptides. Cells were further enriched by positive selection with anti-pe magnetic beads (Miltenyi Biotec). Following the enrichment, cells were surface stained with the Ab Panel 2 reported in Table S1. Kinetic of CXCR5 and PD-1 maintenance CD4 + T cells were isolated from fresh PBMCs by magnetic bead negative selection using CD4 + T cell isolation kit II (Miltenyi Biotec). For the CXCR5 maintenance study, CD8 - CD19 - CD4 + CD45RA - CXCR5 - or CXCR5 + cells were further isolated by cell sorting. For PD-1 maintenance, cells were FACS purified as CD8 - CD19 - CD4 + CD45RA - CXCR5 + PD-1 + or PD-1 - cells. Isolated cells were cultured in plain AIM V medium (Life Technologies) enriched with human recombinant IL-7 (4 ng/ml, Peprotech). Lentiviral vectors and transduction The vectors and transduction protocol were previously described (Kroenke et al., 212). PD-1 induction kinetic Naïve CD4 + T cells were isolated from PBMCs by magnetic bead negative selection using Naïve CD4 + T cell isolation kit II (Miltenyi Biotec). Samples with purity >95% and lacking PD-1 expression were selected and activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies). 1 5 naïve CD4 + T cells were stimulated with 1 l of Dynabeads in RPMI-164 medium (Corning) supplemented with 1% heat-inactivated FBS, 1% GlutaMax, 1% PenStrep. Human recombinant IL-7 (Peprotech) was added (4ng/ml) to the unstimulated controls. After two days of stimulation, beads were magnetically removed (time point was defined as Day of PD-1 maintenance). PD-1 expression was measured at several time points, until Day 1 or 2. Real time quantitative PCR Total RNA was extracted using RNeasy micro kit (Qiagen) according to manufacture s instruction. Superscript II Reverse Transcriptase (Invitrogen) was used to generate cdna. Quantitative real time PCR of ACTB and POU2AF1 was performed using the following primers: ACTB forward, 5 - ACCTTCTACAATGAGCTGCG-3 ; ACTB reverse, 5 - CCTGGATAGCAACGTACATGG-3 ; POU2AF1 forward, 5 - CCAAACTGTCGGCTTCAAAG-3 ; POU2AF1 reverse, 5 - GTTCCTTCACTGGCTCCTTC-3. Quantitative PCR was set up with BioRad itaq SYBR Green Supermix and transcripts were quantified on LightCycler 48 Real Time PCR system (Roche). CXCL13 Assay Cells were isolated and then stimulated for 2 days with plate-bound anti CD3 (Functional Grade Purified, clone OKT3, ebioscience) and recombinant human ICOS ligand (5 g/ml, B7-H2 Fc Chimera from R&D). CXCL13 released in the supernatants was measured by ELISA (R&D). T:B Co-culture Assay CD4 + T cells were isolated from fresh PBMCs by magnetic bead negative selection using the CD4 + T cell isolation kit II (Miltenyi Biotec). The CD19 - CD14 - CD8 - CD4 + CD45RO + cells were FACS sorted into CXCR5 -, PD1 + CXCR3 - CXCR5 +, PD1 + CXCR3 + CXCR5 +, PD1 - CXCR3 + CXCR5 +, PD1 - CXCR3 - CXCR5 + subpopulations. B cells were isolated from fresh PBMCs by CD19 magnetic bead positive selection (Miltenyi Biotec). Naïve and memory B cells were then sorted as CD3 - CD14 - CD4 - CD19 + IgD + CD27 - CD38 - cells and CD3 - CD14 - CD4 - CD19 + IgD - CD27 + CD38 - cells, respectively. Memory B cells (3x1 4 cells/well) were cocultured with the different T cell populations (3x1 4 cells/well), in the presence of Staphylococcal Enterotoxin, Endotoxin Reduced (SEB, 1ng/ml, Toxin Technology Inc) in AIM-V medium (Life Technologies). Naïve B cells were 4

5 cocultured with T cells in the presence of endotoxin-reduced SEB (1ng/ml) in RPMI-164 medium (Corning) supplemented with 1% heat-inactivated FBS, 1% GlutaMax, 1% PenStrep, sodium pyruvate (11 g/ml, Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Alrich). B cell number, phenotype and Ig concentrations in the supernatants were measured after 7 days. IgM and IgG released in the supernatants were measured by ELISA. IgM ELISA was previously described (Kroenke et al., 212). For IgG ELISA, NUNC MaxiSorp plates (Thermo Scientific) were coated overnight with Ig goat anti human pab (Life Technologies). IgG in the supernatants were detected with HRP-conjugated human IgG Fc (Hybridoma Reagent Laboratory). 5

6 ns 3 25 CXCR5 + of CD4 + ICOS PD1 +++ of CXCR5 CD4 ICOS PD1 +++ of CXCR5 CD4 A B HIV- Tonsil HIV- PBMC HIV+ PBMC PD-1 ICOS 5 HIV- Top Low HIV+ C <.1 D.4 <.1 ns <.1 ns HIV- Top Low HIV- Top Low HIV+ HIV+ E PD-1 ICOS Ki67 Figure S1. Total CXCR5 + CD4 + T Cells in Blood, and Activated ICOS + PD1 +++ CXCR5 + CD4 + T Cells in Blood, Both Fail to Correlate with HIV Broadly Neutralizing Antibody Development (A) At the time of detection of broadly neutralizing antibodies (~4 months post infection), there is not a significant difference between the percentage of CXCR5 + CD4 + T cells and the ability to generate broadly neutralizing antibodies. Each point represents an individual donor. Mean and SEM are plotted. P values were calculated using the two-tailed Mann-Whitney test. (B) Representative flow cytometry plots of HIV - tonsil, HIV - PBMC, and HIV + PBMC donors, gated on CD3 + CD4 + CXCR5 + cells, identifying a population expressing ICOS and very high levels of PD-1 (PD1 +++ ). 6

7 (C) Failure of association between the percentage of activated ICOS + PD1 +++ CXCR5 + CD4 + T cells and the ability to generate bnabs at ~4 months post infection. Each point represents an individual donor. Mean and SEM are plotted. The percentage of activated ICOS + PD1 +++ CXCR5 + CD4 + T cells is significantly increased in HIV + individuals (P <.1) at ~4 months post infection. P values were calculated using the two-tailed Mann-Whitney test. (D) Lack of association between the percentage of activated ICOS + PD1 +++ CXCR5 + CD4 + T cells and the ability to generate bnabs at ~4 months post infection. Each point represents an individual donor. Mean and SEM are plotted. The percentage of activated ICOS + PD1 +++ CXCR5 + CD4 + T cells is significantly increased in HIV + individuals (P <.1) at ~4 months post infection. P values were calculated using the two-tailed Mann-Whitney test. (E)Staining for nuclear Ki67, a proliferation marker, within the ICOS + PD1 +++ CXCR5 + CD4 + T cell population. Related to Figure 1. 7

8 A B C % of CXCR5+ cells % of PD-1+ cells PD CXCR5 Days Day 3 Day Days Not Stimulated Anti CD3+28 Figure S2. Blood PD-1 + CXCR3 - CXCR5 + CD4 + T Cells Display a Resting Phenotype (A) Kinetic of PD-1 expression on activated CD4 T cells. Naïve CD4 + T cells were stimulated with Dynabeads. After two days of stimulation, defined here as Day, the beads were removed and PD-1 expression was assessed until Day 1 after stimuli removal. The graph displays the mean value and SEM of data from three independent experiments. (B) Maintenance of PD-1 expression on sorted blood PD-1 + and PD-1 - CXCR5 + CD45RO + CD4 + T during in vitro culture. (C) CXCR5 expression was evaluated on sorted CD4 + CXCR5 + T cells after 3, 5, 1 and 15 days of in vitro culture. Related to Figure 2. 8

9 MFI 142 PD-1+CXCR Naive 25 2 Bcl Bcl6 5 Naive Figure S3. Bcl6 Expression in the Blood CD4 + T Cell Populations Bcl6 expression was measured by flow cytometry in the four blood CXCR5 + cell populations and in CXCR5 - CD45RO + CD4 + T cells and Naïve (CD4 + CD45RO - ) CD4 + T cells. The mab was validated and optimized in human GC Tfh and GC B cell stains. A representative histogram (left) and the MFI quantitation of 6 donors (right) are shown. Related to Figure 3. 9

10 % Tetanus specific cells Figure S4 A 57 PD PD PD PD B CD PD PD HLA-DR 269 PD PD-1-51 CD4 CXCR5 Ki67 CD PD PD ICOS C CXCR5+ NAIVE D PD-1 2 CXCR3 Figure S4. PD-1 + CXCR3 - CXCR5 + Cells Are Circulating Memory Tfh Cells Blood PD-1 + CXCR3 - CXCR5 + cells display a resting phenotype. Activation markers on blood CXCR5 + CD4 + T cell populations. Expression of CD69, HLA-DR, Ki67, CD25 and ICOS on PD1 + CXCR3 - (blue), PD1 - CXCR3 - (red) and CXCR5 - (grey) CD45RO + CD4 + T cells. Histograms show a representative donor. Numbers are MFI for each cell population. (A) Representative CXCR5 gate strategy on tetanus-specific CD45RO + CD4 T cells (B) Representative PD-1 and CXCR3 gate strategy on CD4 + CD45RO + CXCR5 + cells (CXCR5 + ) and CD4 + CD45RO - cells (NAIVE). (C) Frequency of tetanus-specific cells in all four blood CXCR5 + populations. Related to Figure 4. 1

11 Figure S5 A CXCR5+ PD1+ CXCR3+ PD1+ CXCR3- PD1- CXCR3- PD1- CXCR Unstim PMA/Iono CD4-L IL-4 B IgG B cell number SEB unstim 5 1 ng/ml # cell number 15 IgM Plasmablast number SEB unstim 1 2 ng/ml # cell number 4 5 Figure S5. IL-4 Production Following PMA-lonomycin In Vitro Stimulation and In Vitro T Cell Help to Naive B Cells (A) Representative IL-4 and CD4L expression following stimulation with or without PMA-Ionomicin are depicted in the figure. (B) T-B coculture with naïve B cells. Data are mean and SEM. Experimental setting described in Figure Legend 5D, using naïve instead of memory B cells. Related to Figure 5. 11

12 Figure S6. Highly Functional PD-1 + CXCR3 - CXCR5 + CD4 + T Cells Are Associated with HIV Broadly Neutralizing Antibody Development (A) PD-1 + CXCR3 - CXCR5 + CD4 + T cell frequencies are correlated at the ~4 month and ~4 month time points after HIV infection (R =.561, P =.4). R and p values were calculated using a linear regression model. 12

13 (B)CXCR3 - CXCR5 + CD4 + T cells in blood fail to correlate with HIV broadly neutralizing antibody development at ~4 months post infection (P =.5674). (C) PD-1 + CXCR3 + CXCR5 + CD4 + T cells fail to correlate with HIV broadly neutralizing antibody development at ~4 months post infection (P =.89). Each point represents an individual donor, mean and SEM is plotted; P values were calculated using the two-tailed Mann-Whitney test. (D) PD-1 + CXCR3 + CXCR5 + CD4 + T cells fail to correlate with HIV broadly neutralizing antibody development at the time of broadly neutralizing antibody development (~4 months post infection; P =.3657). There is not a significant correlation between the subset of PD-1 + CXCR3 + CXCR5 + CD4 + T cells and the ability to generate broadly neutralizing antibodies, in contrast to the association for PD-1 + CXCR3 - cells (Fig. 6B and 6C). Each point represents an individual donor, mean and SEM is plotted; P values were calculated using the two-tailed Mann-Whitney test. (E) The association of PD-1 + CXCR3 - CXCR5 + CD4 + T cells with HIV broadly neutralizing antibody development is not dependent on viral load. To control for the effect of viral load, we performed an additional analysis of the ~4 month time point samples, excluding individuals with a low viral load (less than 1 viral copies/ml, dashed line; individuals dropped for this analysis are highlighted in red in panel D: resulting P value of.315). The corresponding frequencies of PD-1 + CXCR3 - CXCR5 + CD4 + T cells is shown in Figure 6E. (F) Viral load of the individuals in the Top and Low neutralizing antibody groups at the ~4 month time point. Viral loads were significantly different between Top and Low neutralizing antibody groups at this later time point. (G) To control for the effect of viral load, we performed an additional analysis of the ~4 month time point samples, excluding individuals with a low viral load (less than 1 viral copies/ml, dashed line; individuals dropped for this analysis are highlighted in red in panel D; resulting P value of.1412). The corresponding frequencies of PD-1 + CXCR3 - CXCR5 + CD4 + T cells is shown in panel H. (H) The percentage of PD-1 + CXCR3 - of CXCR5 + CD4 + T cells within the Top and Low neutralizing antibody groups at the ~4 month time point are shown for individuals with a VL > 1. The association between PD-1 + CXCR3 - CXCR5 + CD4 + T cells and the development of broadly neutralizing antibodies remained significant (P =.75). Two-tailed Mann-Whitney test was used to compare HIV + groups. Related to Figure 6. 13

14 Table S1. Staining Used for Cell Sorting and Phenotypic Analysis panel antibody company clone Sorting/Phenotype CXCR5 biotin BD RF8B2 streptavidin Brilliant Violet 421 TM Biolegend n/a CD8 PE/Cy7 Biolegend HIT8a CD19 PE-Cy7 ebioscience HIB19 CD14 PE-Cy TM 7 BD M5E2 CD4 PerCP-eFluor 71 ebioscience RPA-T4 CD45RO FITC ebioscience UCHL1 PD1 (CD279) PE ebioscience ebioj15 CXCR3 APC BD 1C6/CXCR3 Fixable viability dye efluor 78 ebioscience Panel 2 CD4 V5 BD L2 CD45RA PE-CF594 BD HI1 CXCR5 PerCP-Cy TM 5.5 BD RF8B2 PD1 Brilliant Violet TM 421 BD EH12.1 CXCR3 APC BD 1C6/CXCR3 CD19 efluor 65NC ebioscience HIB19 CD8 efluor 65NC ebioscience RPA-T8 CD16 efluor 65NC ebioscience ebiocb16 CD14 efluor 65NC ebioscience 61D3 Fixable viability dye efluor 78 ebioscience other markers TIGIT PerCP-eFluor 71 ebioscience MBSA43 Ly18 (CD352/NTB-A) PE Biolegend NT-7 CCR7 (CD197) PE-Cy7 BD 3D12 HLA-DR V45 BD G46-6 Ki-67 FITC BD B56 CD2 PE-Cy7 ebioscience 2H7 CD38 APC BD HIT2 CD25 AlexaFluor 488 ebioscience BC96 CD69 PE ebioscience FN5 CD38 PE-Cy7 ebioscience HIT2 14

15 Table S2. Staining Panels for HIV + and Control Individuals Panel 1 Marker Fluorochrome Company Clone CD19 efluor 45 ebioscience HIB19 CD4 FITC ebioscience RPA-T4 PD1 PE ebioscience ebioj15 TIGIT PerCP-eFluor 71 ebioscience MBSA43 CXCR3 PE-Cy7 BD 1C6/CXCR3 CXCR5 Alexa Fluor 647 BD RF8B2 CD3 Alexa Fluor 7 BD SP34-2 CD45RA Brilliant Violet TM 57 Biolegend HI1 Fixable viability dye efluor 78 ebioscience Panel 2 Marker Fluorochrome Company Clone PD1 Brilliant Violet TM 421 BD EH12.1 CD4 FITC ebioscience RPA-T4 IL-6Ra PE BD M5 ICOS PerCP-eFluor 71 ebioscience ISA-3 CD127 PE-Cy7 ebioscience ebiordr5 CXCR5 Alexa Fluor 647 BD RF8B2 CD19 Alexa Fluor 7 Biolegend HIB19 CD45RA Brilliant Violet TM 57 Biolegend HI1 Fixable viability dye efluor 78 ebioscience 15