Cd25 apc clone 2a3

For analysis of suppression of proliferation of bystander T cells by test T cells, 5 × 10⁴ SCFA-pulsed DCs were cocultured with 5 × 10⁵ naive CD4⁺ T cells for 6 days. These T cells (test T cells) were harvested, washed, counted, stained with the cell cycle tracking dye 1 µM Cell Trace Violet dye (Thermo Fisher Scientific) and irradiated (3,000 RAD) to prevent expansion. Bystander target T cells (responder T cells), which were allogeneic memory T cells from the same donor as the test T cells, were labeled with 0.5 µM cell tracking dye 5,6-carboxy fluorescein diacetate succinimidyl ester (CFSE). Subsequently, 5 × 10⁴ test T cells, 2.5 × 10⁴ responder T cells, and 1 × 10³ LPS-stimulated DCs were cocultured for 6 days. Proliferation was determined by flow cytometry, by co-staining with CD4 PE-Cy7 (clone SK3) and CD25 APC (clone 2 A3) (both BD Bioscience). To some cultures, where indicated, 10 µg/mL anti-IL-10 antibody (BioLegend), 10 µM ALK5 (Sigma-Aldrich), 10 µM DEAB, 20 ng/mL recombinant human TGF-β1 (BioLegend) (kind gift from Dr. L. Boon), or control antibody IgG1 (BioLegend) was added during the DC-T cell coculture or during the test-responder T cell coculture.

For analysis of cell surface marker expression, MT-4 cells and bead-selected CD4+ T cells (described above) were incubated with 1 µM of each compound (ING-A, ING-B, ING-C, prostratin, PMA, and bryostatin-1) at 37°C for 48 h. After incubation, cells were washed with PBS and incubated for 20 min at room temperature with antibodies against CD3-Brilliant Violet 510 (clone OKT3 – Biolegend), CD4-Qdot 655 (clone 53.5 - Life Technologies), CXCR4-PE (clone 1265 - BD Biosciences), CCR5-PerCP-Cy5.5 (clone 3A9 - BD Biosciences), CD69-Pacific Blue (clone FN50 – BD Biosciences), CD38-FITC (clone AT-1 – Stem Cell Technologies), CD25-APC (clone 2A3 – BD Biosciences), HLA-DR-Qdot 605 (clone TÜ36 – Life Technologies) and a marker for annexin V-FITC (Life Technologies). Cells (both MT-4 cells and CD4+ T cells) were initially incubated for 2 h with different PKC inhibitors (GÖ6983, GÖ6976 or RO-31-8220). Then, ING-B, prostratin or PMA at 1 µM or TNF-α at 10 ng/mL were added to each condition and incubation was carried out for 24 h at 37°C. Similar protocol was used for the analysis of PKC inhibitors in J-Lat clones 9.2 and 10.6. GFP expression, surface marker expression and activation marker were evaluated on a LSR Fortessa cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).

Three millimeters of blood was added to an anticoagulant-containing (EDTA-K2) tube. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood via Ficoll-Hypaque (Sigma, St. Louis, MO, USA) density gradient centrifugation. Mouse anti-human CD4 monoclonal antibody was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China) CD127 (BD Pharmingen #558598, San Jose, CA) was conjugated to ALEXA FLUOR 488. Goat-anti-mouse FITC IgG was purchased from Kangwei (Beijing, China). CD25 PE-Cy7 (BD, clone M-A251), CD25 APC (clone 2A3), and FoxP3 PE (clone PCH101) were purchased from BD Biosciences (Franklin, NJ, USA). CD4+ T cells were gated on side scatter height vs. CD4. The percentage of CD4⁺CD25⁺ T cells, CD4⁺CD25^high Treg cells, CD4⁺CD25⁺ FoxP3⁺ Treg cells and CD4⁺CD25^highCD127^low Treg cells in PBMCs was measured by using FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA).

Peripheral blood was collected into EDTA. Flow cytometry was performed as previously described.32 (link) Briefly, naïve and IRC CD4+ T cell subsets were identified based on their expression of CD45RB-FITC (clone MEM-55, Serotec, Oxford, UK), CD45RA-PE (clone F8-11-13, Serotec), and CD62L-APC (clone 145/15 Coulter, High Wycombe, UK). Treg were quantified by cell surface staining for CD4-Pacifc blue (clone RPA-T4, Bechton Dickson (BD), Oxford, UK), CD25-APC (clone 2A3, BD) and CD127-PE (R34.34, Beckman coulter), followed by intracellular staining for FOXP3-FITC (clone PCH101 eBioscience, San Diego, California, USA) using the antihuman Foxp3 staining kit (Insight Biotechnology, Wembley, UK). Flow cytometry analysis was performed on a LSRII cytometer (BD), using BD Biosciences FACSDIVA software. Subset frequencies were reported as percentage of gated CD3+/CD4+ T cells.