Cd8α pe

T-GREAT cells were co-cultured with infected BMDMs as described above for T57 CD8 T cells. After 14 to 18 hours of the co-culture, cells were harvested for flow cytometry. With preparations all done on ice, cells were washed with ‘FACS buffer’ [PBS pH 7.4 (Gibco, cat# 10010049), 2% heat-inactivated FBS (Omega Scientific)] and blocked with ‘blocking buffer’ [FACS buffer with 5% normal Syrian hamster serum (Jackson Immunoresearch, cat# 007-000-120), 5% normal mouse serum (Jackson Immunoresearch, cat# 015-000-120), and anti-mouse CD16/CD32 FcBlock (BD Biosciences, clone 2.4G2) at 1:100 dilution)]. Samples were stained at 1:120 dilution with fluorophore-conjugated anti-mouse monoclonal antibodies against CD8α PE (eBioscience) or BV510 (BioLegend) (clone 53–6.7), CD3ϵ APC-eFlour780 (eBioscience, clone 17A2), CD62L eFlour450 (eBioscience, clone MEL-14), and CD69 APC (BioLegend, clone H1.2F3). Samples were washed and then incubated with propidium iodide (PI) at 1:1000 dilution (Sigma, cat# P4170). Flow cytometry was performed on an LSRII (Becton Dickinson) or ZE5 (Bio-Rad) analyzer and processed with FlowJo software (version 10.8.1); PI+ cells were excluded from analysis.

On the 14th day after inoculation, mice were sacrificed and spleens and tumors were removed. Splenocytes were collected and stained with a combination of antibodies specific for CD11c-FITC, CD8α-PE, CD86-APC, CD80-PerCP-Cy5.5 (all purchased from eBioscience, San Diego, CA, USA). Splenocytes were then incubated in the dark at 4°C for 30 minutes and washed twice with FACS buffer (0.1% BSA and 0.05% sodium azide in PBS). Flow cytometry was performed using a BD FACS Arial flow cytometer and the results were analyzed with Flow-Jo software (Tree Star, Inc.).

Spleen, MLN, and small intestine lamina propria cells were resuspended in PBS/1% bovine serum albumin and incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen, San Jose, CA, USA) to block non-specific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE, CD25-AlexaFluor488, CD69-PE-Cy7, CD11c-PerCp-Cy5.5, CD8α-PE, CD11b-PE-Cy7, CD103-APC, CD11b-PE, CD83-FITC, CD86-APC (eBiosciences, San Diego, CA, USA), T1ST2-FITC (MD Biosciences, St. Paul, MN, USA), or CX3CR1-AlexaFluor488 (R&D Systems, Oxon, UK). Viable cells were distinguished by means of a fixable viability dye eFluor^® 780 (eBioscience). For detecting transcription factors, cells were first fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) according to manufacturer’s protocol and then stained with Foxp3-APC and RorγT-PE antibodies (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia).