Siinfekl

Lymphocytes from lung or spleen were incubated in digestion buffer (RPMI medium containing 5% fetal bovine serum, 0.02% Collagenase IV, 0.002% DNase I and 10 U/ml of Heparin) for 40 min before use in phenotypic and functional assays. Primed OT-1 CD8⁺ T cells were isolated and re-stimulated with OVA peptide (SIINFEKL, Sigma, 0.2 μg/ml) in the presence of 1 μl/ml of GolgiPlug™ (BD Bioscience) for 4 h ex vivo. Following incubation, cell surface staining was performed followed by fixation, permeabilization, and intracellular staining. For the in vivo CTL assay, OVA peptide-pulsed or control peptide-pulsed spleen cells (as target cells) from syngeneic mice were labeled with a high dose of CFSE (5 μM) or low dose of CFSE (0.5 μM), mixed at 1:1 (2.5×10⁶ of each) before injection. Target cells were intravenously injected into immunized mice on day 4 after re-challenge with cognate antigen protein. The CTL activity was determined 4 h after target cell transfer. Specific lysis was calculated using the following formulas: ratio=(%CFSE^high/%CFSE^low), % specific lysis=[1−(ratio primed/ratio unprimed)] × 100%.

Lymphocytes were isolated from lymph nodes of OTI or OTII mice and stimulated with SIINFEKL (OVA_257–264; Sigma-Aldrich) or ISQAVHAAHAEINEAGR (OVA_323–339; Sigma-Aldrich) peptide at 5 μM for 24 hours. Cells were then labeled with 5 μM CSFE (Thermo Fisher Scientific) at 37 °C for 10 minutes. After two washes, cells were seeded in 48-well plates (1×10⁶ cells per well) and then treated with the indicated concentrations of GABA. Three days after incubation, stained cells were counted to quantify the proliferation of OTI CD8⁺ T and OTII CD4⁺ T cells using a BD FACS Canto flow-cytometry system.