B16F10 melanoma tumor model and isolation of TILs

To evaluate transgelin-2 functions in T cells for tumor suppression (for Fig. 1b), PBS (none), WT (OTI-T), KO (Tagln2^−/− OTI-T), or transgelin-2-overexpressing T (TG2OE OTI-T) cells were adoptively transferred into tumor-bearing C57BL/6 mice at days 7, 10, and 13 post-tumor implantation. All mice were sacrificed at day 25, and tumors were isolated and weighed.

To evaluate the effect of Tagln2^−/− in mice (for Fig. 1c), OVA⁺B16F10 cells (3 × 10⁵) were s.c. injected into the dorsal flank region of age- and sex-matched WT or Tagln2^−/− mice. To evaluate transgelin-2 functions in DCs for tumor suppression, BMDCs (1 × 10⁷) from WT or Tagln2^−/− were pulsed with 1 µg/mL of pOVA for 2 h and i.v. injected into 8-week-old WT mice. At day 7, OVA⁺B16F10 cells (3 × 10⁵) were s.c. or i.v. injected into the dorsal flank region to induce solid and metastatic tumor models, respectively. Mice were sacrificed at day 8 post-inoculation with tumor cells. At the end of the experiments, tumors were isolated, weighed, and photographed for gross morphology. To analyze TILs, tumor tissues were dissected and mechanically disaggregated before digestion with collagenase D (1 mg/mL, Roche) for 30 min at 37 °C. After digestion, all of the cells were passed through 70-µm filters, and leukocytes were isolated by centrifugation using 38% Percoll for 30 min. Pellets were resuspended with PBS, stained with anti-CD3 and CD8 antibodies, and analyzed by flow cytometry. To analyze cytokine production, isolated TILs were stimulated with PMA/Ionomycin (200 nM/ 1 µM) in the presence of Brefeldin A (1 µg/mL) for 4 h at 37 °C. Cells were subsequently collected and stained for CD8 followed by fixation with IC fixation Buffer (eBioscience) for 20 to 30 min at RT. Then, the cells were washed twice with 1 × Permeabilization Buffer (eBioscience) and stained with IFN-γ antibody. After washing, cells were analyzed by flow cytometry.