Cd4 rm4

To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro^{30 (link)}. Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).

Cell suspensions were obtained from the spleen, the PP, the MLN and the AA LN (lumbar and renal lymph nodes). Jejunum and colon sections from PBS and LCWE-injected mice were harvested and dissociated into single-cell suspensions with a gentleMACS™ Octo Dissociator (Miltenyi Biotec) and a mouse Lamina Propria Dissociation kit (Miltenyi Biotec) following the complete protocol from the manufacturer. The following antibodies against the respective murine antigens were used: IgA (mA-6E1,Thermo Fisher Scientific), CD19 (eBio1D3, Thermo Fisher Scientific), CD4 (RM4–5, Tonbo Biosciences), CD3 (145–2C11, BioLegend and Tonbo Biosciences), CD45.1 (A20, Thermo Fisher Scientific), CD45.2 (104, BioLegend), CD95 (SA367H8, BioLegend), GL-7 (GL-7, BioLegend). Dead cells were routinely excluded based on the staining of Fixable Viability dye (FVD) eFluor 506 (Thermo Fisher Scientific). Cell numbers were calculated by flow cytometry with the CountBright Absolute Counting Beads (Thermo Fisher Scientific). Stained cells were analyzed on a LSRII (BD Biosciences) and the data were processed using Flowjo (Tree Star Inc.).