Cxcr3 173

FITC-, PE-, PE-CY7, APC-, APC-CY7 or Percp-labeled antibodies to CD3 (145-2C11), CD45 (30-F11), CD4 (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), B220 (RA36B2), CD25 (PC61), NK1.1 (PK136), PD-L1 (MIH5), FoxP3 (NRRF-30), IFN-γ (XMG1.2), TNF-α (MAb11), CD49 (DX5), and CXCR3 (CXCR3-173) and isotype-matched control antibodies were purchased from Biolegend (antibodies to CD3, CD4, CD8, CD45,CD11b, CD11c, B220, CD25, PD-L1, FoxP3, IFN-γ, TNF-α, CD49, CXCR3, and control antibodies) or BD Biosciences (antibodies to NK1.1 and control antibodies). For identification of cellular phenotypes, disassociated cells from tumors or spleens were suspended in PBS containing 1% bovine serum albumin and incubated with the antibodies on ice for 30 min. Cells were fixed in 1% paraformaldehyde in PBS after washing. For intracellular cytokine staining, cells were stimulated in culture medium for 4 h in the presence of Leukocyte Activation Cocktail, with BD Golgi^Stop (1: 500; BD Biosciences). Viable cells were then fixed and permeabilized with transcription staining buffer set (eBioscience) and stained with respective antibodies to cytokines. FoxP3 staining was performed according to manufacturer’s protocol (eBioscience). Stained cells were analyzed on a FACSCalibur or FACS Canton flow cytometer, and data were analyzed using the flowjo software.

I) For CD4⁺ T cell depletion, intraperitoneal injection of Ultra-LEAF™ Purified anti-mouse CD4 Antibody (10 μg/g, clone GK1.5, BioLegend, San Diego, CA, USA) or corresponding isotype antibody (Ultra-LEAF Purified Rat IgG2b, κ Isotype Ctrl Antibody, clone RTK4530, 10 μg/g, BioLegend) was performed in mice on day 15, 25, and 40 PMI. ii) For CXCR3 blockade, intraperitoneal injection of Ultra-LEAF™ Purified anti-mouse CD183 (CXCR3) Antibody (10 μg/g, clone CXCR3-173, BioLegend) or corresponding isotype antibody (Armenian Hamster IgG, clone: HTK888, BioLegend) was performed in mice every 7 days since day 15 PMI. iii) For systemic IFN-g blocking, mice were injected i.p. with 200 mg of anti-IFN-g antibody (InVivoMAb anti-mouse IFNγ, BioXcell, clone XMG1.2) every 3 days since day 15 PMI. An IgG1 isotype antibody (HRPN, BioXcell) was used to serve as control. iv) To block VCAM-1, a InVivoMAb anti-mouse VCAM-1 (10 mg/kg, BioXcell; corresponding isotype antibody: InVivoMAb rat IgG1 isotype control, clone HRPN, BioXcell) was administrated intravenously in MB-injected mice every 3 days since day 15 PMI. For retinal evaluation in abovementioned in vivo antibody intervention experiments, one retina from each recipient mouse was randomly selected and used for RGC density, while the other was for Iba1 and GFAP staining.

All antibodies were purchased from BD Biosciences or eBioscience (unless otherwise stated): CD4 (L3T4), CD8α (53-6.7), CD11c (HL3), CD45.1 (A20), CD45.2 (104), CD90.2 (30-H12), B220/CD45R (RA3-6B2), NK1.1 (PK136), TCR-β (H57-597), IL-21 (FFA21), CXCR5 (2G8), PD-1 (RMP1-30), ICOS-L (HK5.3), TNF-α (MP6-XT22) and CXCL9 (MIG-2F5.5). αmPDCA-1 mAb (JF05-1C2.4.1) was purchased from Miltenyi Biotec. A CXCR3 specific mAb was obtained from both R&D Systems (220803) and Biolegend (CXCR3-173). αmTNF-α mAb (XT3.11) were purchased from BioXcell for in vivo mTNF-α blocking experiments. Flow cytometry was performed on FACS-Canto with optimal compensation set for six-color staining. The data were analyzed using FlowJo software (Tree Star, Inc.). All cytokine (IL-21 and TNF-α) and chemokine (CXCL9) expressions by dLN-derived cells were measured directly ex vivo without further in vitro re-stimulation.