Anti mouse cd4 antibody

Cell suspensions were incubated with anti-mouse CD16/32 (eBioscience) for 5 min at RT to block Fc receptors and stained for 20 min at 4 °C with anti-mouse CD4 antibodies (Biolegend). Cells were washed with PBS + 10% FCS and analyzed on a LSRII cytometer (Becton Dickinson).
For intracellular cytokines staining, cells were stained first with anti-mouse CD4 antibodies as described above, fixed for 15 min at RT with fixation buffer (Biolegend), washed twice with permeabilization buffer (BioLegend), and stained with anti-mouse IL-2 or anti-mouse IFN-γ antibodies. After washing with permeabilization buffer, cells were analyzed on a LSRII cytometer.
Cell viability was determined by flow cytometry using Zombie fixable viability kit according to the manufacturer's recommendations (BioLegend).

To assess the killing activity of T cells, degranulation assay was used. Mononuclear cells were isolated from lung tumor tissues as described above, and then cultured alone or with LLC-Luc cells at 1:1 ratio for 4 h at 37°C. Mouse primary peritoneal macrophages and mouse CD4⁺ T cells together co-incubated with LLC-Luc cells at a 1:1:1 ratio with or without different concentrations of YPF at 37°C. After 24h, CD4⁺ T cells were separated from the mixed cells, and cultured alone or with LLC-Luc cells at 1:1 ratio for 4 h at 37°C. Meanwhile, for each assay, anti-mouse CD107α antibody or isotype IgG (BioLegend, USA) was added and incubated for 4h, and then T cells were stained with anti-mouse CD3 antibody, anti-mouse CD4 antibody, and anti-mouse CD8 antibody (BioLegend, USA) for 30 min at 4°C. CD107α expression on the surface of T cells was acquired by BD Accuri C6 (BD Biosciences) instrument. Data was analyzed using the FlowJo software (Ashland, OR).

As previously described [19 (link)], each group's DCs and T cells that were going to be examined in the coculture system were first collected, then washed twice with PBS after being digested with 0.25 percent trypsin. After this, the cells were tested. Following centrifugation, the supernatant was discarded, and the cells were resuspended in Eppendorf (EP) tubes at a concentration of 2 105 cells per 100 μL. Then, l μL of primary antibodies was added to the EP tube, including antimouse CD133 antibody (BioLegend, USA), antimouse CD11c antibody (BioLegend, USA), antimouse CD80 antibody (BioLegend, USA), antimouse CD86 antibody (BioLegend, USA), antimouse MHC-II antibody (BioLegend, USA), antimouse CD4 antibody (BioLegend, USA), antimouse CD8 antibody (BioLegend, USA), and antimouse FOXp antibody (BioLegend, USA). Following a thirty minute incubation period in the dark at a temperature of four degrees Celsius, the cells were resuspended in 0.2 milliliters of PBS before being washed twice with PBS. The phenotypic changes in DCs and T cells were identified with the help of flow cytometry (a BD FACSCalibur), and the analysis of the experiment's results was performed with the FlowJo software tool (FlowJo™ v10.7, http://www.flowjo.com).