Ly6c hk1

Samples were blocked with 2% normal mouse serum or mouse Fc block (2.4G2, BD Biosciences). Fixable yellow (Invitrogen, L34959) was used to stain live/dead cells. Anti-mouse antibodies used were CD45.2 (104, Tonbo Biosciences), CD3 (500A2, BD Bioscience), TCRβ (H57–597, eBioscience), CD8 (53-6.7, BioLegend), CD4 (GK1.5, BioLegend), FoxP3 (FJK-16S, eBioscience), Ly6C (HK1–4, BioLegend), CD11b (M1/70, BioLegend), F4/80 (BM8, eBioscience), MHC II (M5/114.15.2, eBioscience), CD11c (N418, BioLegend), NK1.1 (PK136, BD Biosciences), IFNγ (XMG1.2, Tonbo Biosciences), H-2Db (28-14-8, eBioscience), H-2Kb (AF6–88.5.5.3, eBioscience), PD-1 (29 F.1A12, BioLegend), and PD-L1 (MIH5, eBioscience). Fluorescence was measured on BD LSR Fortessa^TM X-20 or BD FACSymphony^TM flow cytometer (BD Biosciences, North Ryde, New South Wales, Australia) and data analysed using FlowJo, LLC software.

Cells from tumor or spleen were isolated by grinding through 70-μm filters and stained with the following fluorochrome-conjugated antibodies: CD45 (30-F11, BioLegend, 103116); 7-AAD (Tonbo Biosciences, 13-6993-T200); CD3 T cells: CD3e (145-2C11, BioLegend, 100305); CD8 T cells: CD8a (53-6.7, BioLegend, 100708); CD4 T cells (GK1.5, BioLegend,100412); neutrophils and monocytes: Ly6C (HK1.4, BioLegend, 115506) and CD11b (M1/70, BioLegend, 101216); natural killer cells: NK1.1 (PK136, BioLegend, 108722), B cells: CD19 (6D5, BioLegend, 115506). For the intracellular IFNγ staining, cells were incubated for 5 h at 37°C in complete RPMI 1640 containing 2 µM Monensin, 50 ng/mL PMA, 1 µg/mL Ionomycin, followed by incubation in BD Perm buffer for 30 minutes at 4°C, washed by BD wash buffer and stained with the antibody IFNγ (XMG1.2, Biolegend, 505806). The stained cells were acquired on the Invitrogen™ Attune™ NxT acoustic flow cytometer system and the data were analyzed with using FlowJo software (BD Bioscience).

Quadriceps muscles were collected, fixed in 4% paraformaldehyde (PFA), and dehydrated in 30% (wt/vol) sucrose (in PBS). Cryosections that were 14 μm thick were permeabilized in acetone, blocked with 5% bovine serum albumin (BSA) for 1 h and immunolabeled with antibodies against desmin (ab32362; Abcam, Cambridge, UK), CX₃CR1 (ab31331; Abcam, Cambridge, UK), CD68 (MCA1957GA; Bio-Rad, Gladesville, NSW, Australia), CD11b (M1/70; BD Biosciences, San Jose, CA, USA), or Ly6C (HK1.4; Biolegend, San Diego, CA, USA), and detected using Alexa Fluor 488-conjugated anti-rat and Alexa Fluor 568-conjugated anti-rabbit (Thermo Fisher, Australia). Sections were counterstained with Alexa Fluor 647-conjugated phalloidin (Thermo Fisher, Australia), Hoechst 33258 (Sigma-Aldrich USA, Inc.), and mounted with Prolong Gold Antifade (Thermo Fisher, Australia). Images were acquired on an Olympus FV1000 and FV3000 confocal microscope and processed using Imaris 9.2 (Bitplane). Three-dimensional (3D) thresholding analysis of desmin-positive myofibers was performed using Imaris Surfaces function. Desmin-positive and phalloidin-positive myofibers were rendered as surface objects, thresholds were applied using the automated threshold function, and voxel area data were plotted for analysis. Data were generated from ten 30-μm-thick cryosections per group (four mice per group).

For surface markers, single-cell suspensions were stained with relevant fluorochrome-conjugated monoclonal antibodies(mAbs): anti-mouse CD40 (HM40-3), CD80 (16-10A1), CD86 (GL1), and MHCII (M5/114.15.2) from eBioscience, anti-mouse CD11b (M1/70), Gr-1 (RB6-8C5), Ly6G (1A8), and Ly6C (HK1.4) from Biolegend, For intracellular staining, cells were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 µg/ml), monensin (Enzo, 2 µg/ml). After 5 h, cells were stained with antibodies against surface markers, fixed, permeabilized, and stained with anti-IFN-γ mAb (XMG1.2, eBioscience), anti- IL-17 mAb (eBio17B7, eBioscience), or anti-TGF-β mAb (TW7-16B4, eBioscience) according to the Intracellular Staining Kit (Invitrogen) instructions. Flow cytometry was performed using the BD FACSCanto II (Becton Dickinson) and data were analyzed using FlowJo software (Treestar).