αcd28

Buffy coats from healthy donors were obtained through the Gulf Coast Regional Blood Center, Houston, TX. Peripheral blood mononuclear cells (PBMCs) were isolated with Lymphoprep density separation (Fresenius Kabi Norge) and activated using 1μg/mL α-CD3 (Miltenyi Biotec) and 1 μg/mL α-CD28 (BD Biosciences) mAb coated plates. Forty eight hr later, T lymphocytes were transduced with retroviral or lentiviral supernatants using retronectin-coated plates (Takara Bio), and expanded in complete medium (45% RPMI-1640 and 45% Click’s medium (Irvine Scientific), 10% FBS (Hyclone), 2mM GlutaMAX, 100 I.U./mL of Penicillin and 100 μg/mL of Streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech) or IL-2 (50 U/ml; R&D) (Zhou et al. 2014 (link)). Four to seven days later, cells were collected for in vitro or in vivo experiments. Lentiviral transduced cells were selected in 1 μg/ml puromycin (Sigma) for 3 – 5 days before T cells were used in functional assays.

Mediastinal lymph nodes associated with healthy donor lungs were dissected digested with 400 U/mL Collagenase Type 4, 0.1% Dispase II and 20 μg/mL DNase I for 30 min at 37°C. Digested samples were mechanically disrupted by pushing through syringe with 18-gauze needle and passage through 100 μm and 40 μm wire meshes. Naïve CD4⁺ T cells were enriched by negative selection using isolation kits (Miltenyi Biotec, 130-094-131). Purified cells (2×10⁶ per well) were cultured in a 12-well plate pre-coated with 2 μg/mL αCD3 (eBioscience, 16-0037-85). RPMI-1640 medium was supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM 2-mercaptoethanol. For Th0 condition, cells were cultured at the presence of 2 μg/mL αCD28 (BD Biosciences, 555725). For Th2 (Th0+IL-4) condition, cells were cultured at the presence of 2 μg/mL αCD28, 5 ng/mL rhIL-2 (R&D Systems, 202-IL-010) and 10 ng/mL rhIL-4 (BioLegend, 204-IL-010).

Murine splenocytes were obtained from NOD spleen after the treatment of either vehicle or MOTS-c for 18 weeks. Human PBMCs were isolated using a Ficoll density gradient (GE Healthcare) of buffy coats from blood of healthy donors or patients. Then, CD4⁺ or CD8⁺ T cells were negatively isolated using T cell isolation kit (MACS, Miltenyi Biotec). Isolated CD4⁺, CD8⁺ T cells, or splenocytes from each group were plated onto Seahorse cell plates coated with Cell-Tak (Corning) to enhance T cell attachment. Cells were analyzed with the Seahorse XFe24/96 Extracellular Flux analyzer (Agilent) to determine real-time oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and Real-time ATP production assay. Real-time ATP production assay measures and quantifies mitoATP and glycoATP, simultaneously, by using oligomycin and Rotenone/Antimycin A. To monitor CD4⁺ and CD8⁺ T cell activation in real-time, αCD3 (4 μg/ml; BD Biosciences) and αCD28 (20 μg/ml; BD Biosciences) were mixed and directly applied onto plated cells via the instrument’s multi-injection ports. Both antibodies were injected 30 minutes after the experiment was initiated for murine T cells, as previously reported (Gubser et al., 2013 (link)). For human T cells, antibodies were introduced 60 minutes after the injection of MOTS-c (10μM) or vehicle.

Stimulation of BDC2.5 T cells with plate-bound α-CD3 (BD Biosciences; 0.5 µg/ml) and α-CD28 (BD Biosciences; 1.0 µg/ml) was performed as described [82 (link), 83 (link)], with the following modifications. BDC2.5 T cells (2×10⁴) were combined with 50 µl conditioned media as indicated in 200 µl supplemented DMEM at 37°C for 72 hr. IFNγ secretion was measured by ELISA.