7 aad percp

B cell stimulation with a minor cocktail of stimuli was performed to study the effect of IL-21, co-stimulation, and BCR activation on plasmablast formation. CD19⁺ B cells were isolated via CD43 negative selection with CD43 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (purities ≥85%). B cells were incubated with anti-IL-21R antibody ATR-107 (10 μg/ml, Pfizer) or isotype-matched control (10 μg/ml IgG1-Fc, R&D systems). Next, cells were stimulated with 5 μg/ml soluble anti-CD40 (Bioceros, Utrecht, The Netherlands), 10 μg/ml goat-anti-human IgM (Jackson Immunoresearch, West Grove, PA, USA) and human recombinant IL-21 (100 ng/ml, eBioscience). Subsequently, the presence of plasmablasts on day 0 and the differentiation of memory B cells into plasmablasts on day 8 were determined with flow cytometry. Plasmablasts were defined as CD19^posCD27^highCD38^high cells (16 (link)). The following MoAbs were used: CD19 BV510 (Biolegend), CD27 Pe-Cy7 (eBioscience), IgD APC-Cy7 (Biolegend), and CD38 BV421 (BD Biosciences). In addition, viability staining with 7-AAD PerCP was performed (BD Biosciences).

By use of an AriaII FACS sorter^™ (Becton Dickinson, BD, Franklin Lakes, NJ), pure CD28^POS cells (purity 98% [95–100%]) were isolated. PBMCs were stained with CD3 Brilliant Violet 510 (BioLegend, San Diego, CA), CD4 Pacific Blue (BD, Franklin Lakes, NJ), CD8 APC-Cy7 (BD Pharm, San Diego, CA), CD28 APC (BD), and the viability dye 7-AAD PerCP (BD). Pure memory subsets (≥95% pure) were isolated using CD3 Brilliant Violet 510 (BioLegend), CD45RO PE-Cy7 (BD) and CCR7 PE (BD): naïve (T_N cells: CCR7+CD45RO-), central-memory (T_CM cells: CCR7+, CD45RO+), effector-memory (T_EM cells: CCR7-, CD45RO+), and end-stage terminally-differentiated EMRA (T_EMRA cells: CCR7-CD45RO-) T-cells.

To investigate the killing capacity of CXCR5⁺CD8⁺ T and CXCR5⁻CD8⁺ T cells, we co-cultured PBMCs, purified CXCR5⁺CD8⁺ T cells or CXCR5⁻CD8⁺ T cells with Jurkat cells [HIV-infected CD4⁺ T cells (33 (link))] integration of the HIV cDNA stimulated by overlapping peptides covering the HIV-1 pol, gag, and env antigens (JPT, Berlin, Germany). The total cells were stimulated by the peptide pools (1 µg/mL, 100 µL per sample) and brefeldin A for 8 h at 37°C in the presence of 5% CO₂ before conducting surface and intracellular staining (6 (link)). To evaluate the level of apoptosis, the cells were washed with FACS buffer and stained with CD4-APC (eBioscience, Waltham, MA, USA), 7-AAD-PerCP (BD Biosciences, Franklin Lakes, NJ, USA) and Annexin V-PE (Southern Biotech, Birmingham, AL, USA). Flow cytometric acquisition was performed on a FACSVerse or Caliber.

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA). Isolated PBMCs were stored at −140°C until further use. Total T cells were isolated from the PBMCs by magnetic cell separation on the autoMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the pan T cell protocol using the deplete S settings. Purities were >90% CD3⁺ cells after isolation.
The naive and memory T-cell populations were isolated from the PBMCs using fluorescence-activated cell sorting (FACS) by the BD FACSAria™ II (BD Biosciences, San Jose, CA, USA). The PBMCs were stained with CD3 Brilliant Violet 510 (Biolegend, San Diego, CA, USA), CD4 Pacific Blue (BD Biosciences), CD8 APC-cy7 (BD Biosciences), CD45RO APC (Biolegend), CCR7 PE-cy7 (BD Biosciences), and to exclude non-viable cells the cells were also stained with 7AAD PerCP (BD Biosciences). Naive cells were defined as CCR7⁺CD45RO⁻, central memory cells as CCR7⁺CD45RO⁺, effector memory (EM) as CCR7⁻CD45RO⁺, and the highly differentiated EMRA cells as CCR7⁻CD45RO⁻ (32 (link)). After cell sorting, the purities were >95% for each sorted fraction.

The following anti-human Abs were used in this study: 7-AAD PerCP, CD8 PE, CD16 APC-Cy7, CD21 APC, CD38 PE, CD56 PE-Cy7, DNAM-1 FITC, GM-CSF PerCP/Cy5.5, IFN-γ APC, Ki 67 FITC (all BD Biosciences); CD3 Pacific Blue (Life Technologies); CD4 APC, CD19 PE, (eBioscience); CD20 PE-Cy5, CD27 APC-Cy7, IgD PE-Cy7, NKp44 APC (BioLegend); CD158a, h PE-Cy5.5, CD158b1/b2, j PE-Cy5.5, CD158a, h APC, CD158b1/b2, j APC, CD159a (NKG2A) PE, CD159a (NKG2A) APC (Beckmann Coulter); hIL-12 Rß PerCP (R&D Systems). Live cells were distinguished using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Following endotoxin free recombinant human (rh) cytokines were used: rhIL-12 (R&D Systems), rhIL-2 (PeproTech), and rhIL-15 (Sigma).

PBMCs (5 × 10⁵cells/well) from active TB patients were thawed and rested 6 h before labeling with Carboxyfluorescein succinimidyl ester (CFSE) according to manufacturer’s procedure. In the COX-i treated samples, indomethacin was added 2 h prior to stimulation with ESAT-6 and Ag85. The samples were incubated for 6 days and then washed and stained for surface markers and viability staining. Fluorochromes used in the 6 days proliferation assay; anti-CD3-V450, anti-CD4- APC H7, anti-CD45RA -BV 605, anti-HLA DR- APC, anti-CD25-PE, anti-CD127- PeCy7 and 7AAD- PerCP (all from BD Bioscience) and CellTrace™ CFSE Cell Proliferation (Life technologies).

Immunophenotyping was done using fluorescent-labelled monoclonal antibodies to differentiate T-cell subsets, using CD3 APC-H7 (clone SK7), CD4 FITC (clone RPA-T4) or CD4 PerCP (clone L200), CD8 FITC (clone 3B5), HLA-DR APC (clone TU36), Ki67 FITC (clone B56), CCR7 PE (clone 150503), CD25 PE-Cy7 (clone M-A251), Alpha-4 FITC (clone 9F10), Beta-7 APC (clone FIB504), CD57 APC (clone NK-1), CD27 APC or PE-Cy7 (clone M-T271), CD28 FITC or PE (clone CD28.2), CD31 PE (clone WM59), CD45RA PE-Cy7 (clone HI100) and 7-AAD PerCP (all from BD Biosciences, Gauteng, South Africa) in panels, as outlined below. Data were acquired on a six-colour FACSverse flow cytometer (Becton Dickinson, San Jose, U.S.A.). 100 μL of whole blood was stained, red cells were lysed, then cells were washed and resuspended in 250 μL paraformaldehyde or 10% cell fix before flow cytometric analysis. Intracellular Ki-67 staining was carried out after resuspension in 1X Perm solution (BD) for nuclear membrane permeabilisation. Between 50,000–100,000 lymphocyte events were acquired per tube and fluorescence-minus-one (FMO) controls were used to set all gates. Analysis of markers was carried out using BD FACSuite^TM software v1.0.2.