D058 1283

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.

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.