Anti ctla 4 apc

For staining of inhibitory receptors, stimulated PBMCs were surface stained with anti-CD4 (V500; BD Biosciences), anti-CD3 (APC-H7; BD Biosciences), anti-CD8 (Alexa Fluor 405; Invitrogen), anti-PD-1 (FITC; BD Bioscience) and anti-human TIM-3 antibody (PE; R&D Systems) for 30 min at 4°C. Cells were washed, fixed, permeabilized (Invitrogen), and intracellularly stained with anti-IFN-γ (PE-Cy7; BD Biosciences), anti-IL-2 (BV605; BD Biosciences), and anti-CTLA-4 (APC; BD Biosciences) for 30 min at 4°C, washed and fixed with 1% formaldehyde. Cells were analyzed using a LSR-II flow cytometer (BD Immunocytometry Systems). One million events were collected. Electronic compensation was performed with Ab capture beads (BD Biosciences) stained separately with individual monoclonal Abs used in the test samples. The data files were analyzed using Diva (BD) and FlowJo Software (Treestar, Co). The expression of TIM-3, PD-1 and CTLA-4 was examined on cytokine-producing cells with frequencies ≥0.03% above the background (media control tube) to ensure an adequate number of events for analysis as previously validated by our laboratory [15 (link)–18 (link)]. Flourescence minus one (FMO) controls were used to set the gates for determining the mean fluorescent intensity (MFI) of TIM-3, PD-1 and CTLA-4 on Gag-responsive T cells expressing IFN-γ or IL-2.

Single cell suspensions of leukocytes from peripheral blood were stained using the following monoclonal antibodies: anti-CD3 APC-H7 (BD, 641397), anti-CD4 Alexa Fluor 700 (BD, 557922), anti-CD8 Alexa Fluor 700 (BD, 557945), anti-CD25 APC (Miltenyi 130-092-858), anti-CD28 BV421(BD, 562613), anti-CD38 BV605 (Biolegend 303532), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-CD39 BV421 (BD 563679), anti-CD45 RO BV510 (BD, 563215), anti-CD56 APC (Biolegend 318310), anti-CCR7 PE (BD 561008), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-Ki-67 FITC (BD 556026), anti-HELIOS (Biolegend, 137214), anti-CTLA-4 APC (BD 560938), anti-OX40 FITC (Biolegend 35006), and anti-PD1 BV510 (Biolegend 329932). Cells were stained in FACs buffer (1 % FBS in PBS with 0.01 % NaN₃) and fixed according to the eBioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were analyzed on a BD X20 Fortessa Flow cytometer. We used Fluorescence Minus One to discriminate between positive and negative cells for each antibody [33 (link)].

PBMCs (2 × 10⁵ cells) were stimulated with M. avium bacilli and M. intracellulare bacilli individually for 48 h and incubated with GolgiPlug (1 μL/mL; BD Pharmingen, San Diego, CA, USA) for the final 5 h of culture. Cells were measured by flow cytometry using a fluorescence activated cell sorter (FACS) (FACSVerse, BD Biosciences, San Jose, CA, USA) and anti-CD3-APC-Cy7, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-4-PE, anti-IL-17A-APC, anti-CD25-PE, anti-Foxp3-APC, anti-T-bet-APC, anti-GATA3-APC, and anti-RORγT-APC antibodies (BD Pharmingen, San Diego, CA, USA). The PD-1, CTLA-4, and TIM-3 were also stained with anti-PD-1-PE, anti-CTLA-4-APC, and anti-TIM-3- APC (BD Biosciences, San Diego, CA, USA). Data were analyzed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). A gate was set on the lymphocytes using characteristic forward scatter and side scatter parameters followed by CD3⁺CD4⁺ T cells (Supplementary Figure S1).