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Cell separation from co-culture system was performed using the CD271+ MicroBeads isolation kit (Miltenyi Biotech) as recommended by the manufacturer. Separation occurs in a MACS Column, which induces a high-gradient magnetic field (∼0.6 Tesla) when placed in an AutoMACS Separator (Miltenyi Biotech). After the automatic sorting, CD271+ MSCs and CRC cells were separated in different falcon tubes for further analysis.

Spleens and lymph nodes from C57BL/6J mice were mechanically disrupted onto 70 μm strainers using the plungers of 3 mL syringes. CD8 T cells were then purified from cell suspensions using CD8a (Ly-2) microbeads (Miltenyi) and autoMACS separator (Miltenyi). Preparing cell suspension with same method, CD4 T cells were purified from spleens and lymph nodes of P25 mice using MojoSort™ CD4 isolation kit and magnet (Biolegend). Purities of cells were determined for each experiment using flow cytometry. 2-5 million of CD8 T cells were transferred into TCRα KO mice with or without distinct congenic marked 10⁵ P25 cells before infecting with Erdman.

CD8 T cells were purified from the lungs of infected mice at indicated time points using CD8a (Ly-2) microbeads (Miltenyi) and an autoMACS separator (Miltenyi). The purity of cells was determined for each experiment and was typically 95%. Purified CD8 T cells were added to infected TG-PMs at a ratio 1:2 (CD8 T: TG-PM) or as indicated. TG-PMs were lysed with 1% Triton X-100 after 4-6 days of co-culture, and CFU was determined by plating serial dilutions of the lysate on 7H10 or 7H11 plates (Hardy Diagnosis). Percent inhibition by CD8 T cells was calculated as: 100 x (Mtb net growth without CD8 T cell – Mtb net growth with CD8 T cell)/ Mtb net growth without CD8 T cell.

For in vitro assays, CD4+ T cells were isolated from either peripheral blood or spleens of mice by negative selection using EasySep Mouse CD4+ T cell Isolation Kit (StemCell Technologies, Canada). Then, CXCR5+PD-1+ Tfh cells were further purified from isolated CD4+ T cells using Streptavidin MicroBeads in an autoMACs Separator (MiltenyiBiotec). In parallel, naïve T cells were purified by negative selection from isolated CD4+ T cells by adding biotinylated anti-CD25 (BD Biosciences, USA) to an EasySep Mouse Memory T cell Depletion Cocktail (StemCell Technologies, Canada). For T cell activation assay, 4×10⁴ naïve T cells, cTfh and spleen Tfh from anti-PD-1-treated and isotype-treated mice were stimulated with Phorbol 12-myristate 13-acetate (PMA)(20 ng/mL) (Merck, Germany) plus ionomycin (1 µM) (Merck, Germany) for 5 hours. Brefeldin A (Merck, Germany) was added for the last 2 hours to allow intracellular cytokine accumulation prior to flow cytometry staining.