Anti cd8α clone 53 6

T cells were fixed for 10 min in 1.6% PFA on ice and subsequently permeabilized in 90% methanol for at least 30 min at −20 °C. Cells were stained with anti-CD4 (clone RM4–5, Tonbo) and anti-CD8α (clone 53-6.7, Tonbo). Where indicated, T cell IFNγ production was quantified using the mouse IFNγ secretion kit (Miltenyi Biotech). B16-F10 cells were fixed as above and stained with anti-pSTAT1 (58D6: Cell Signaling Technologies). Live B16-F10 cells were stained with anti-MHC-I H2-K^b (AF6-88.5: BioLegend).
Flow cytometric data were collected on an LSR II (BD Biosciences). Analysis was done with FlowJo software (TreeStar) and with custom code written in Python and available on GitHub (https://github.com/oylab/oyFlow).

Tumors (1 day after i.t. injection of PBS, iMSC, or iMSC/CCL19) were snap frozen in Optimal Cutting Temperature (OCT)compound (Sakura Finetek Japan Co.) by using liquid nitrogen. Sections were cut with a cryostat and fixed in cold 4% paraformaldehyde (Wako). Sections were stained with Rat isotype control mAbs (IgG2a: clone eBR2a, IgG2b: clone eB149/10H5, eBioscience), anti-CD3 (clone 17A2, TONBO), anti-CD4 (clone RM4-5, eBioscience), or anti-CD8α (clone 53-6.7, TONBO)+anti-CD8b (clone H35-17.2, eBioscience), and then with ImmPRESS Goat anti-Rat IgG peroxidase polymer detection kit (Vector Laboratories). mAbs were visualized with ImmPACT DAB peroxidase substrate (Vector Laboratories) and sections were counterstained with Hematoxyline QS (Vector Laboratories). For CD11c staining, endogenous biotin blocking with an avidin/biotin blocking kit (Vector Laboratories) was added after fixation. Sections were stained with biotinylated anti-CD11c (clone N418, AbD Serotec) or a Hamster isotype control IgG (clone eBio299Arm, eBioscience), biotinylated goat anti-Hamster IgG (Vector Laboratories), and then VECTASTAIN Elite ABC HRP Kit (Vector Laboratories), and visualized with ImmPACT DAB substrate.