Anti cd3 anti cd28 beads

To detect the cytokine production by CD4⁺ T cells, 1 × 10⁵ CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of mitochondria at 100 μg/mL in a 96-well plate with a total of 200 μL X-VIVO 15 serum-free culture medium (Lonza, Walkersville, MD, USA) per well. After the treatment for 48 h, the supernatants were collected to examine the level of inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α) using a LEGENDplex™ Human Th Panel (Biolegend, San Diego, CA, USA) by Gallios Flow Cytometer according to the manufacturer’s protocols.

Activated T cells (ATC) were generated from peripheral blood mononuclear cells (PBMCs) by activation with 20 ng/million cells of anti-CD3 monoclonal antibody (OKT3). ATC were expanded by adding 100 IU/million cells of IL-2 every other day for 14 days in RPMI-1640 supplemented with 10% FBS. Harvested ATC were armed with bispecific antibody anti-CD3 x anti-EGFR [EGFRBi] or anti-CD3 x anti-HER2 [HER2Bi] at a pre-optimized concentration of 50 ng/10⁶ ATC. Similar to ATC, co-activated T cells (COATC) were generated from PBMCs by activating T cells with anti-CD3/anti-CD28 beads (Dynabeads, Thermofisher) at the 3:1 bead:cell ratio and transduced with lentivirus (LV) at day 2 (multiplicity of infection, MOI: 5). Beads were removed by magnetic separation at day 6 and cells were expanded till day 14 by adding 100 IU/million cells of IL-2 every other day. Harvested COATC were armed with EGFRBi or HER2Bi at pre optimized concentration of 50 ng/10⁶ COATC.

Purified human CD8+ T cells were labeled with 5 μM cell tracker violet (CTV; ThermoFisher Scientific) and transduced with lentiviral vector coexpressing human ASS1 and GFP (pLenti-GIII-CMV-GFP-2A-Puro, abm). After 24 h cells were stimulated with anti-CD3/anti-CD28 beads (ThermoFisher Scientific), then left for a further 24 h. Cells were PBS-washed then transferred to selection medium (standard growth medium, arginine-free medium and arginine-free medium supplemented with 0.4 mM citrulline). Medium was replenished 48 h later to ensure T cells were not starved of any nutrients except arginine in the appropriate cultures. After 96 h in selection medium, cells were analyzed by flow cytometry for CTV and GFP expression, gating on single, live CTV+ CD8+ T cells.

IL-17 positive Th17 cells were analyzed with accepted methods. Briefly, fresh PBMCs were stimulated for 16 hours with anti-CD3/anti-CD28 beads (Life Technologies) in the presence of Brefeldin A (Sigma-Aldrich). Thereafter, the cells were fixed, permeabilized and stained with anti-CD4, -CD69-APC, and -IL-17A (BD Biosciences), analyzed with BD LSRFortessa flow cytometer (BD Biosciences) and using FlowJo software (BD Biosciences). Th17 CD4+ memory cells were detected from whole blood by a four-color flow cytometry panel with monoclonal antibodies (mAbs) [CD45RA-FITC, CD4-PerCP, CXCR3-APC and CCR6-BV421 (BioLegend)] against surface antigens.

Isolated naïve CD4 T cells were activated with anti‐CD3/anti‐CD28 beads (Life Technologies) at a 1:5 ratio and cultured for 7 days under the following conditions: TH1, 10 ng/ml IL12 and 1 μg/ml neutralizing anti‐IL4 antibody (Ab); TH2, 20 ng/ml IL4 and 10 μg/ml neutralizing anti‐IFNγ Ab; and TH9, 10 ng/ml IL4 and 5 ng/ml TGFβ1. To generate Tregs, naïve CD4 T cells were cultured on plates coated with anti‐CD3 (5 μg/ml) and anti‐CD28 antibody (1 μg/ml) in the presence of IL2 (2 ng/ml) and TGFβ1 (5 ng/ml). All cytokines were purchased from PeproTech. All antibodies were from R&D Systems. TGFβ receptor inhibitor SD‐208 was purchased from Selleck Chemicals.
siRNA for TGFβR3, BATF, IRF4, and plasmids expressing BCL6 or ID3 were transfected into naïve CD4 T cells on day 3 after activation using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). TF expressions were examined on day 5 and the frequencies of cytokine‐producing cells were determined on day 7 after restimulation. siRNA were purchased from Dharmacon Inc. and plasmids were from Genscript.