Sp34 2

Single cell suspensions were prepared from lymph node and gastrointestinal mucosal biopsies as previously described [10] and stored in liquid nitrogen. Cryopreserved samples of PBMC, LNMC and GMMC were thawed at 37 °C in RPMI 1640 containing 10% FBS and benzonase (Millipore) at 50 U/mL. Cells were re-suspended in 1X PBS containing Aqua LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) for 20 min at RT in the dark. Cells were washed and re-suspended in wash buffer containing the following fluorescently conjugated antibodies at the indicated dilutions: CD45 (D058-1283, BD #563861,1:80), CD28 (CD28.2, BD #5622596, 1:200), CD4 (L200, BD #563913, 1:100), CCR7 (150503, BD # 561271, 1:40), CD95 (DX2, BD #555674, 1:100), CD3 (SP34-2, BD # 557917, 1:40), and CD8 (SK1, BD #560179, 1:80), CXCR5 (MU5UBEE, Thermo-Fisher # 25-9185-42, 1:30), and PD-1 (EH12.2H7, Biolegend # 329919, 1:100). Cells were sorted in purity mode, using a BD FACSAria II. Sorted CD4+ T cells were FSC singlets, live, CD45+CD3+CD4+CD8− lymphocytes and subsets were defined as follows. For GMMC: naïve (CD28+CD95−) and memory (CD95+). For LNMC: naive (CD95−CD28+), T follicular helper cells (Tfh, CD28+CD95+CXCR5+PD-1^high), central memory (CD95+CD28+, non-Tfh), effector and effector memory (CD95+CD28−). CXCR5+ events were determined by a CXCR5 FMO antibody stained sample.

Aliquots (50 μl) of EDTA-treated whole blood were stained with monoclonal antibodies to CD3 PerCP (SP34-2, BD Biosciences, 552851), CD4 FITC (OKT-4, Biolegend, 317408), and CD8 PE (RPA-T8, BD Biosciences, 555367). CD4⁺ T-cell counts were determined with BD Trucount tubes according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, United States). PBMCs were isolated using conventional Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Polychromatic flow cytometry was performed to quantitate activated CD4⁺ or CD8⁺ T lymphocytes (Figure 2) and CD4⁺ or CD8⁺ memory T lymphocyte subsets (Figure 3). Activated or memory T lymphocyte subsets (Table 1) from 1 × 10⁶ PBMCs were stained with anti-CD3 BV605 (SP34-2, BD Biosciences, 562994), anti-CD4 BV711 (OKT-4, Biolegend, 317440), anti-CD8 PE (RPA-T8, BD Biosciences, 557086), anti-CCR7 BV421 (G043H7, Biolegend, 352208), anti-CD45RA APC (5H9, BD Biosciences, 561210), anti-CD38 FITC (AT-1, Stemcell, 60131FI), anti-HLA-DR BV510 (G46-6, BD Biosciences, 563083), and anti-PD-1 PerCP-cy5.5 (EH12.2H7, Biolegend, 329914) monoclonal antibodies. Cells were resuspended in 1% paraformaldehyde, subjected to flow cytometry within 24 h on a FACSAriaII (BD Biosciences, San Diego, CA, United States), and analyzed using FlowJo V₁₀ software.

We measured antigen-specific CD8⁺ T-cell responses by flow cytometric analysis detecting gamma interferon (IFN-γ) induction after specific stimulation as described previously [41 (link)]. Autologous herpesvirus papio-immortalized B-lymphoblastoid cell lines (B-LCLs) were pulsed with individual SIVmac239 epitope-coding peptides (at a final concentration of 1–5 μM) or peptide pools (at a final concentration of 1–2 μM for each peptide) using panels of overlapping peptides spanning the entire SIVmac239 Gag, Pol, Vif, Vpx, Vpr, Tat, Rev, Env, and Nef amino acid sequences (Sigma Aldrich Japan). PBMCs were cocultured with these pulsed B-LCLs under GolgiStop (monensin, BD) presence for 6 hours. Intracellular IFN-γ staining was performed with a CytofixCytoperm kit (BD) and fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (M-T477, BD), peridinin chlorophyll protein (PerCP)-conjugated anti-human CD8 (SK1, BD), allophycocyanin (APC)-conjugated anti-human CD3 (SP34-2, BD), and phycoerythrin (PE)-conjugated anti-human IFN-γ monoclonal antibodies (4S.B3, Biolegend). Specific T-cell frequencies were calculated by subtracting non-specific IFN-γ⁺ T-cell frequencies from those after antigen-specific stimulation. Specific CD8⁺ T-cell frequencies lower than 0.02% of CD8⁺ T cells were considered negative.

The following conjugated antibodies were used in these studies: a) from BD Biosciences, D058-1283 (CD45; PE Cy7; cat# 561294), SP34-2 (CD3; Alexa 700; cat# 557917), SP34-2 (CD3; PE; cat# 552127), LP200 (CD4; PerCP-Cy5.5; cat# 552838), RPA-T8 (CD8; PacBlu; cat# 558207), SK1 (CD8; TruRed; cat# 341051) 3GB (CD16; Alexa 700; cat# 560713), 25723.11 (IFN- γ; APC; cat# 502512), 6.7 (TNF-α; PE; cat# 554513), 3A9 (CCR5; APC; cat# 560748), SK1 (CD8; BUV737; cat# 612754), L200 (CD4; BUV395; cat# 564107), FN50 (CD69; PE-Texas Red; cat# 562617), SP34-2 (CD3; Pacific Blue; cat# 558124); b) from BioLegend, OKT4 (CD4; APC-Cy7; cat# 305612), RPA-T4 (CD4; APC; cat# 300537); c) from Beckman Coulter, RMO52 (CD14; PE-Texas Red; cat# IM2707U); d) from Sigma, HP-6025 (IgG4; FITC; cat# F9890); e) from SouthernBiotech, HP6023 (mouse anti-human IgG₄ pFc’; HRP; 1:20,000; cat# 9190–05), f) from NHP Reagent Resource, 1B3 (anti-rhesus IgG1/3; HRP; 1:5000).
The following unconjugated antibodies were used: a) PA-14 (Leronlimab; CytoDyn), conjugated in-house to PacBlu using Pacific Blue Antibody labeling Kit (ThermoFisher) and used at approximately 1:80 depending on the efficacy of conjugation, b) anti-idiotype antibody, PA-22 (CytoDyn). Live/dead Fixable Yellow Dead Cell Stain Kit and Near-IR Dead Cell Stain Kit (ThermoFisher) were amine-reactive dyes used at 1:1000 dilution to assess cell viability.

CD4⁺ T cells and NK cells from fresh or frozen PBMC, pLN and spleen were isolated using,a positive selection with respectively, the anti-human CD4 (CD4 MicroBeads, human Miltenyi Biotec (USA)), or the anti-NKG2a/c monoclonal antibody-PE conjugated (clone Z199 Beckman Coulter (USA)) reveals by anti-PE microbeads (Miltenyi Biotec (USA), the staining and the positive selection of the cells was done according to the supplier’s instructions.
Eight-color panels were used to phenotype, surface stain and sort NK cells from blood and pLNs from chronically infected AGMs. Cells were thawed in 20% FBS-containing media supplemented with benzonase nuclease, and counts and viabilities were performed (Life Technologies). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes). Cells were washed and blocked using normal mouse IgG (Caltag). For the NKG2_a/c^HIGH/lowCD16^+/−phenotyping panel, PBMCs and pLNs were surface stained for CD3 (SP34.2, 1:10 dilution; BD), CD8 (BW135/80, 1:20 dilution; Miltenyi), CD16 (3G8, 1:20 dilution; Beckman Coulter, Inc.), NKG2a/c (Z199, 1:20 dilution; Beckman Coulter, Inc.), CD20 (2H7, 1:20 dilution 1/20; Biolegend), and CD14 (M5E2, 1:25 dilution; BD). Post-staining, cells were washed, filtered, and sorted on a FACS ARIA II (BD). Cells were directly collected in a lysis buffer that contained TCEP. The purity of the cells was >97%.

Single-cell suspensions from rhesus macaque tissues were used as input into the LEGENDScreen assay (Biolegend). Cell suspensions were incubated with Fc block (Human TruStain FcX, Biolegend) prior to labeling with fluorescently conjugated antibodies. To enable assessment of assay markers on cell derived from individual tissues, cells from bone marrow, lymph node, and PBMC were each labeled with anti-NHP CD45 (clone D058-1283, BD Biosciences) conjugated to a unique fluorescent tag to enable sample multiplexing and unmixing during analysis. Following labeling with CD45, samples were washed 2× with FACS buffer before being stained with antibodies targeting CD3 (SP34-2, BD Biosciences), CD20 (2H7, Biolegend), and CD138 (DL101, Biolegend). 1 × 10⁶ cells were used as input into each well of the LEGENDScreen assay. Samples were acquired on a Symphony A5 (BD Biosciences) using FACSDiva (BD Biosciences).