Anti human cd4

Plasma was separated from EDTA-contained fresh blood samples, aliquoted, and stored at − 80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated over a Ficoll-Paque cushion (GE Healthcare, Wauwatosa, WI). PBMCs were used for annexin V assays. Blood samples were used for all other flow cytometry-based assays except annexin V assays. For surface staining, antibodies were incubated with blood or PBMCs at room temperature for 15 min. After surface staining in blood samples, red cells were lysed, washed, and analyzed by flow cytometry. The fluorochrome-labeled mAbs (BD Pharmingen, San Jose, CA) used for flow cytometry included the following: anti-human CD3 (OKT3), anti-human CD4 (RPA-T4), anti-human CD8 (RPA-T8), anti-human CD19 (HIB19), anti-human CD20 (L27), anti-human CD27 (M-T271), anti-human CD38 (HIT2), anti-human CD45RA (HI100), anti-human HLA-DR (G46-6), anti-human ki67 (B56), anti-human IgD (IA6-2), anti-human IgG (G18-145), isotype control antibodies (BD Pharmingen), and annexin V (BD Pharmingen). Cells were collected in a BD FACSVerse flow cytometer (BD, San Jose, CA), and data was analyzed by FlowJo software (Version 10.0.8).

Monoclonal antibodies used for this study (anti-human CD4, CD25, CD45RO, CCR4, CCR5, CCR6, IL-10, IL-17, FoxP3 and purified antibodies against IL-10 and TGF-β) were obtained from BD Biosciences, CA, USA. Recombinant Soluble proteins of TGF-β, IL-6, IL-17, IL-22 and IL-23 were purchased from eBiosciences, San Diego, CA, USA. The whole cell lysate (WCL) of M. leprae was a kind gift from the Immunology laboratory of National Jalma Institute of Leprosy and Other Mycobacterial Diseases, ICMR, Agra, India.

Cryopreserved bone marrow MNCs were thawed and viable cells were counted. One million viable cells were stained with DAPI viability dye, anti-human CD71 (BD Biosciences, clone L01.1), anti-human CD3 (BD Biosciences, clone SP34–2), anti-human CD4 (BD Biosciences, clone L200), anti-CD8b (Thermo, clone 3B5), anti-CD34 (BD Biosciences, clone 563), anti-human CD20 (BioLegend, clone 2H7), anti-CD14 (Miltenyi, clone TUK4), and anti-human CD11b (BD Biosciences, clone ICRF44) for 20 min at room temperature with Human Fc Block (Miltenyi). Alternatively, 200,000 CD34-enriched viable cryopreserved cells were thawed and stained with anti-human CD38 (Caprico Biotechnologies, clone OKT10), CD45RA (BD Biosciences, clone 5H9), CD90 (BD Biosciences, clone 5E10), and DAPI viability dye. Cells were then passed through a 100-micron cell strainer into FACS sorting buffer, run on a FACSAria flow cytometer (BD Biosciences), and analyzed using FlowJo software (BD Biosciences). Compensation controls were run with UltraComp eBeads (Thermo) during each run and fluorescence minus one (FMO) controls were run for each fluorophore in the panel. MPPs were designated as CD34+CD38-CD90-cells and HSCs were designated as CD34+CD38-CD90+ cells, as published previously in macaques.^{110 (link)}