Anti cd4 percp

In vitro cultured PBMC's were harvested, washed, and aliquoted at 2–4 × 10⁵ cells/well in a round bottom 96 well-plate. The in vitro cultured PBMC's were stimulated for 4 h at 37°C, 5% CO₂ with, for the matrix analysis, each of the 15 column and 15 row mixes (1 μM/peptide), and for the epitope identification with a single peptide (0.8 μM). After 1 h of stimulation 1 μg/mL Brefeldin A (BD Biosciences) was added. The cells were subsequently permeabilized (Becton Dickinson Permeabilizing solution 2) and stained with anti-CD3-Pacific Blue, anti-CD4-PerCp, anti-CD8-APC, anti-CD69-PE, and anti-IFNγ-FITC, according to the “FastImmune” protocol (Becton Dickinson). The cells were subsequently analyzed by flow cytometry using a LSRII (BD Biosciences). The gating strategy is illustrated in Supplementary Figure S4. Staining of more than 0.8% of CD8⁺ or CD4⁺ T cells was considered positive.

For surface staining, 1×10⁶ U937 stable cells were washed twice with PBS and fixed with 4% formaldehyde, followed by staining with either 1 µg/ml anti-CD4-PerCP (Becton Dickinson, Gurgaon, India) or 1 µg/ml anti-tetherin-APC (Abcam, Cambridge, UK). For total staining, the U937 or the LX2 cells were fixed as earlier and permeabilized with 0.4% Triton-X100 followed by staining with either 1 µg/ml anti-TGFβ (R&D Systems) ) or 1∶1000 diluted anti-collagen type I, clone 5D8-G9 (EMD Millipore), respectively and 1∶1500 diluted anti-rabbit Alexa 647 (Life Technologies, Bangalore, India). The cells were acquired using a Dako Cyan flow cytometer (Beckman Coulter, Brea, CA, USA) and the data analyzed with FlowJo (Treestar Inc, Ashland, OR, USA).

Retroviral vector pQCXIP-VpuGFP [18] (link) was a kind gift from Dr. Edward Barker (Rush University Medical Center, Chicago, USA). The pQCXIP-GFP control vector was made by PCR amplification and subcloning of AcGFP. The retroviral constructs containing shRNA against Vpu [19] (link), [20] (link) and control shRNA were purchased from Origene Technologies (Rockville, MD, USA). The 3xAP1pGL3 reporter plasmid was obtained from Dr Alexander Dent through Addgene (#40342) [21] (link). Anti-CD4-PerCP (Becton Dickinson, Gurgaon, India), anti-tetherin-APC (Abcam, Cambridge, UK) and anti-TGFβ (R&D Systems, Minneapolis, MN, USA) were used for flow cytometery; anti-GFP, anti-GAPDH (Santa Cruz Biotechnology, CA, USA) and anti-Vpu antisera [22] (link) (NIH AIDS Reagent Bank, Frederick, MD, USA) were used for western blotting.

Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood and BM mononuclear cells (BMMCs) were obtained from BM in both of OA and RA patients by Ficoll-Paque PLUS (GE Healthcare Biosciences AB, Uppsala, Sweden) density centrifugation. To examine the phenotype of regulatory T cells (Tregs), the following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD45RA-FITC, anti-CD45RO-PE and anti-CD184 (CXCR4)-PE (all from BD Biosciences, San Jose, CA, USA). Next, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set followed by intracellular staining with anti-Foxp3-APC antibody (PCH101; eBioscience, San Diego, CA, USA). Cells were acquired using FACSCalibur (BD Biosciences), and the results were analyzed using CellQuest (BD Biosciences) software. The gating strategy was based on the identification of lymphocytes according to FSC and SSC signal distribution. Then, CD4⁺ lymphocytes were gated, and next, appropriate cell populations were identified. Gates were settled according to the isotype control or fluorescence minus one (FMO) for the desired marker. The dot plots shown are representative for at least six experiments done on different patients. The described gating strategy is shown in Figure 2b and was used though all the analysis of the cell phenotype presented in this paper.

2x10⁶ splenocytes from each sample were plated in a 96-well plate. After centrifugation, cells were resuspended and incubated in culture medium (R10) consisting of RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2mM L-glutamine, 0.01 M HEPES buffer, 100mg/ml gentamicin (Mediatech), and 5×10^-5M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). To test intracellular cytokine, cells were stimulated with 30 mg/ml PMA and 400 ng/ml ionomycin in the presence of GolgiStop (BD Pharmingen) for 4 hours at 37°C.
After incubation and stimulation, cells were surface-stained with anti-CD3-PB (BD), anti-CD4-PerCP (BD), anti-CD8-PO (Biolegend), anti-BTLA-PE, anti-2B4-APC, anti-PD-1-APC-Cy7 (all from eBioscience). Then cells were permeabilized using fixation and permeabilization solution (BD). We used anti-IL-2-FITC (BD), anti-TNF-PE-Cy7 (Biolegend) and anti-IFN-γ-Alexa 700 (BD) for intracellular cytokine staining. In some experiments, cells were stained intracellularly for Ki-67 using the Foxp3 staining kit (BD Pharmingen). Samples were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo software (FlowJo, LLC) and SPADE (Cytobank.org) (see below).

ICS assay was performed according to a standard protocol, as described previously [17 (link)]. Briefly, splenocytes isolated from mice (2 × 10⁶ cells/well) were plated in 24-well plates and stimulated with a mix of VACV-specific peptides (SPYAAGYDL, SPGAAGYDL, VGPSNSPTF, KYGRLFNEI, GFIRSLQTI, and KYMWCYSQV) or with PMA (phorbol myristate acetate, 30 ng/mL) and ionomycin (1 µg/mL). Each peptide was added at a concentration of 20 µg/mL per well, and cells were incubated for 4 h at 37 °C in 5% CO₂, and for additional 16 h with Brefeldin A (5 μg/mL, BD Biosciences, Franklin Lakes, NJ, USA). On the next day, cells were stained with pre-titrated anti-CD3 MCA500SBB700 (Bio-Rad, Hercules, CA, USA), anti-CD8 FITC (BD Pharmingen, San Diego, CA, USA), and anti-CD4 PerCP (BD Pharmingen, San Diego, CA, USA), fixed, and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Cells were then stained for intracellular cytokine detection with anti-IFN-γ APC (BD Pharmingen, San Diego, CA, USA). Samples were analyzed on a ZE5 flow cytometer (Bio-Rad, Hercules, CA, USA). Data were presented as the medians and ranges of variation.