Anti human cd28 antibody

To provide TCR stimulation to human CD4 T cells, NUNC Maxisorp plates (BioLegend) were coated with purified antihuman CD3 antibodies (BioLegend) diluted to 1.25 µg/ml in sterile PBS for 2 h at 37°C or at 4°C overnight. Control wells were filled with PBS only. Plates were washed two times with PBS and one time with R10 medium before cells were added.
Mononuclear cells were adjusted to either 4 × 10⁶ cells/ml in R10 medium and 100 µl cell suspensions were added to the appropriate well of the coated plates. Purified antihuman CD28 antibodies (BioLegend) and added to all wells previously coated with anti CD3 antibodies (OKT3) to a final concentration of 1 µg/ml. Recombinant human IL-23 (Miltenyi) was diluted to 400 ng/ml in R10 medium and added to the cells to a final concentration of 50 ng/ml.
For stimulation with IL-12 (Miltenyi) and IL-18 (BioLegend), cells were plated out at a concentration of 10⁷ cells/ml in R10 medium on regular 96U plates and mixed with recombinant cytokines (final concentration 50 ng/ml for both IL-12 and IL-18). In all experiments, some cells were left untreated to determine baseline expression of the analyzed effector molecules.
Cells were incubated with different combinations of antibodies or cytokines for 24 h total. Brefeldin A solution (Invitrogen) was added to all well for the last 4 h to prevent cytokine release.

A total of 1 × 10⁶ mononuclear cells from tuberculous pleural effusions or PBMCs were cultured in AIM V^® serum-free medium (Gibco, Life Technologies, Grand Island, NY, USA). The cells were stimulated with M. tuberculosis strain H37Rv sonicated lysates at a concentration of 10 μg/ml overnight at 37 °C, in the presence of 1 μg/ml anti-human CD28 antibody (BioLegend, San Diego, CA, USA). For detection of intracellular cytokines and granzyme B, brefeldin A (BD Biosciences) was added to the cell suspension 4 hours before staining of the cells, as described previously24 (link). The cytokine concentration in the supernatants of cells from pleural effusions and blood PBMCs was measured by Luminex method with Human Cytokine Panel kit (eBioscience, San Diego, CA, USA).

Human PBMC were isolated from fresh buffy coat collected from healthy donors using the density gradient centrifugation method. Briefly, buffy coat was diluted with 2X volume PBS and was gently layered on top of an equal volume Ficoll-Paque PLUS (#17144003, Cytiva, Marlborough, MA), followed by centrifugation at 400 x g for 40 min without break. The PBMC were removed and transferred to a new 50 ml tube, washed in 25 ml DPBS buffer twice, and recovered by centrifugation at 350 x g for 10 min. The cells were counted and cultured in the presence of 1 µg/ml anti-human CD3 antibody (#317302, BioLegend, San Diego, CA) and 2 µg/ml anti-human CD28 antibody (#302902, BioLegend) for 72 h. The cells were then infected with NL4-3 at a MOI as indicated in the presence of 8 µg/ml polybrene by spinoculation at 850 x g, room temperature for 2 h. The cells were recovered by centrifugation, washed with fresh media, and continued to culture in the presence of 100 IU/ml human IL-2 (#21-8029-U050, Tonbo Biosciences, San Diego, CA) and used in subsequent Metformin experiments.

For PBL preparation, donor blood was obtained from healthy volunteers with consent from the Institutional Review Board of the Cancer Institute, Chinese Academy of Medical Sciences, and written informed consent for participation in the study was obtained from participants. After centrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich), PBMCs were plated at 2 × 10⁶ cells/mL in cell culture for 2 h and the non-adherent cells were collected. The cells were then stimulated for 2 d on a non-tissue-culture-treated 24-well plate coated with 1 μg/mL OKT3 (Biolegend) at 1 × 10⁶ cells/mL and in the presence of 1 μg/mL of anti-human CD28 antibody (Biolegend). For retrovirus transduction, a 24-well plate was coated with RetroNectin (Takara) at 4°C overnight, according to the manufacturer’s protocol, and then blocked with 2% BSA at room temperature for 30 min. The plate was then loaded with retrovirus supernatants at 300 μL/well and incubated at 37°C for 6 h. Next, 1 × 10⁶ stimulated PBLs in 1 mL of medium were added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37°C for 2 d. The cells were then transferred to a tissue-culture-treated plate at 1 × 10⁶ cells/mL and cultured in the presence of 100 U/mL of recombinant human IL-2.

Blood samples were collected using Heparin Blood Collection tubes (VACUETTE). After transportation to ICVS, samples were processed in BSL2 laboratories. PBMC were isolated using equal volumes of peripheral blood and Histopaque 1077 (MilliporeSigma, St Louis, Missouri, USA). After centrifugation, plasma samples were collected and stored (−80 °C). PBMC were frozen in Fetal Bovine Serum (FBS, Gibco, Thermo Fisher Scientific) with 10% of DMSO. Samples were defrosted and centrifuged at 250 g for 5 minutes to remove DMSO. Upon suspension in complete RPMI medium (RPMI-1640 culture medium supplemented with 2 mM glutamine, 10% FBS, 10 U/mL penicillin/streptomycin and 10 mM HEPES (Gibco, Thermo Fisher Scientific), PBMC were seeded at a concentration of 1 × 10⁶ cells / mL in a 96-well plate (Corning, NY) and incubated for 24 h with 500 mg/mL of purified anti-human CD28 antibody (ref. 302902, Biolegend, CA, USA) and 500 mg/mL of purified anti-human CD40 antibody (ref. 334302, Biolegend, CA, USA) along with 1 μg/mL of SARS-CoV-2 Spike Glycoprotein (ref. RP30020, Gene Script, USA) or SARS-CoV-2 Nucleoprotein (ref. RP30013, Gene Script, USA). After incubation, supernatant was stored for cytokine quantification and cells were characterised by flow cytometry.

Activation induced marker (AIM) expression assays were conducted using PBMCs as described.³¹ PBMCs were thawed and diluted with complete media (containing 10% fetal bovine serum). Cells were washed and suspended at a density of 2.5 × 10⁶/ml. For each condition, 200 μl complete media containing 0.5 × 10⁶ PBMCs were let to rest in 96‐well plates overnight. After resting, cells were stimulated with spike peptide pools (1 μg/ml, MabTech, Cat#3630‐1) and anti‐human CD28 antibody (2 μg/ml, BioLegend, Cat#302934). Matched control samples for each donor were treated with an equal amount of DMSO. Cells were washed after 24 h stimulation. Cells were surface stained with fluorophore conjugated FACS antibodies against CD3, CD4, CD8, CD45RA, CCR7, CD69, OX40, 4‐1BB, CXCR3, CXCR5, CD95, and CD107a for 20 min. Zombie Red was used for dead cell exclusion.