Anti cd28 cd49d

Splenocytes or lung cells were prepared and counted from each mice in different groups as previously described, respectively [15 (link), 16 (link)]. Cells were plated in triplicate at 5 × 10⁶ cells per well in a 24-well plate and incubated with CMFO (10 μg/mL) and anti-CD28/CD49d (1 μg/mL, eBioscience) overnight. RPMI 1640 medium used is a negative control. Cell responses were monitors through a cell stimulation cocktail (1 μg/mL, eBioscience). Cells were stained for anti-CD4-APC-Cy7 (Cat#552051, BD Biosciences), anti-CD8α-BV510 (Cat#563068, BD Biosciences), anti-CD44-FITC (Cat#561859, BD Biosciences), anti-CD62L-PerCP-Cy5.5 (Cat#560513, BD Biosciences), and intracellular markers anti-IFN-γ-PE (Cat#554412, BD Biosciences) and anti-IL-2-APC (Cat#554429, BD Biosciences). Absolute number of IFN-γ⁺ and IL-2⁺ T cells, central memory T cells (T_CM), and effector memory T cells (T_EM) was determined by an LSRII multicolor flow cytometer (BD Biosciences) and analyzed through FlowJo software. The results were shown as the mean ± SEM per group (n = 6).

Intracellular flow cytometry analysis was performed as previously described (22 (link)). Briefly, 5 × 10⁶ splenocytes were plated in each well of a 24-well plate and incubated with either CMFO (10 µg/mL) or PPD (10 µg/mL) in the presence of 1 µg/mL anti-CD28/CD49d (eBioscience CA, USA). RPMI1640 medium and cell stimulation cocktail (eBioscience, CA, USA) were used as negative and monitoring controls, respectively. Cells were stained with surface markers, including anti-CD4 PE Cy7, anti-CD8a PE, anti-CD44 APC-eFluor^® 780, anti-CD62L FITC mAbs, and intracellular markers anti-IFN-γPerCP-Cy5.5 and anti-IL-2 APC mAbs (all from eBioscience, CA, USA). The stained cells were analyzed by an LSRII multicolor flow cytometer (BD Biosciences, CA, USA). The absolute number of CMFO-specific CD4⁺ or CD8⁺ IFN-γ positive T_EM (effector memory T cells, CD62L^loCD44^hi) and CD4⁺ or CD8⁺ IL-2 positive T_CM (central memory T cells, CD62L^hiCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA). The results are represented as mean ± SD per group (n = 6).

Spleen cells were further seeded in triplicates at 5 × 10⁶ cells/well in a 24-well plate and stimulated with WH121 (10 μg/mL) in the presence of 1 μg/mL anti-CD28/CD49d (eBioscience CA, USA) for 20 h at 37°C under 5% CO₂. For the last 4 h of the stimulation, 3 μg/mL brefeldin A and 2 μM monensin solution (eBioscience CA, USA) were added. The cells were washed in FACS buffer (1% FCS-PBS) and stained for 30 min at RT with anti-CD4-PE-Cy7 (clone GK1.5, eBioscience), anti-CD8α-PE (clone53-6.7, eBioscience), anti-CD62L-FITC (clone MEL-14, BD Pharmingen), and anti-CD44-APC-Cy7 (clone IM7, BD Pharmingen) mAbs. After the cells had been washed and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen CA, USA), the intracellular cytokines were stained with anti-IFN-PerCP-Cy5.5 (clone XMG1.2; eBioscience CA, USA) and anti-IL-2-allophycocyanin (clone JES6-5H4; eBioscience CA, USA) mAbs for 30 min at RT. The cells were washed twice, resuspended in FACS buffer and analyzed using an LSRII multicolor flow cytometer (BD Biosciences CA, USA). The absolute number of IFN-γand/or IL-2 positive CD4⁺ and CD8⁺ T cells, T_CM (central memory T cells, CD62L^hiCD44^hi) and T_EM (effector memory T cells, CD62L^loCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA) as previously described [25 (link)], respectively. The data are represented as the mean ± SD per group (n = 6).