Rorγt afkjs 9

Fluorochrome-labeled mAbs including mouse γδTCR (GL3, 1:100), Vγ4 (UC3-10A6, 1:500), Vγ1 (2.11, 1:500), CD24 (M1/69, 1:500), CD27 (LG.3A10, 1:500), CD62L (MEL-14, 1:500), and IL-17A (TC11-18H10.1, 1:500, Biolegend), CD44 (IM7, 1:200), RORγt (AFKJS-9, 1:200), and Ki-67 (SolA15, 1:200, eBioscience), IL-23R (753317, 1:10) and CCR6 (140706, 1:50, R&D system) were used. Anti-mouse Vγ6 (17D1, 1:500) was kindly provided by Dr. Tigelaar (Department of Dermatology, Yale University). For intracellular cytokine staining, cells were first blocked with anti-CD16/32 and then stained with different cell surface Abs. Cells were then fixed, permeabilized and stained intracellularly for IL-17, RORγt or Ki-67. The relevant isotype control mAbs were also used. Samples were harvested with BD FACS Calibur or Canto (Becton Dickinson, San Jose, CA) and analyzed with FlowJo software (TreeStar).

Ova (ova_323–339) (ISQAVHAAHAEINEAGR) and MART-1 (ELAGIGILTV) peptide were purchased from GenScript (Piscataway, NJ). Penicillin, streptomycin, glucose free RPMI-1640, IMDM were purchased from Life Technologies, Grand Island, NY. FBS was procured from BioAbChem Inc., Ladson, SC. All the recombinant cytokines except IL-2 (Shenandoah Biotechnology, Warwick, PA) and fluorochrome conjugated anti-mouse CD4 (GK1.5), CD73 (TY/11.8), CD26 (H194-112), CD44 (IM7), CD62L (MEL-14), IFN-γ (XMG1.2), IL-17a (TC11-18H10.1), IL-22 (Poly5164), IL-2 (JES6-5H4), TNF-α (MP6-XT22), CD25 (PC61) and T-bet (4B10) were purchased from BioLegend, San Diego, CA. Fluorochrome conjugated anti-mouse Vβ5.1,5.2 (MR9-4), IRF-4 (3E4), CD39 (24DMS1) and RORγt (AFKJS-9) were obtained from eBiosciences (San Deigo, CA). Anti-human Vβ12 was from Thermo Scientific (Rockford, IL). Purified anti-CD3, anti-CD28, anti-IFN-γ, anti-IL-4 were obtained from UCSF monoclonal antibody core, UCSF, CA. Anti-mouse pS6 conjugated with Alexa647 was purchased from Cell Signaling Technology (Danvers, MA). Tumor cells tested for antibody production were obtained from our collaborators as follows: 624-MEL (Dr. Michael Nishimura, Loyola University, Chicago), EL4 (Dr. Zihai Li, MUSC), B16-ova (Dr. Mark Rubinstein, MUSC).

Kidneys were minced into ~1 mm³ pieces and then digested with 0.5 mg/mL Collagenase D (Sigma), 0.5 mg/mL DNAse (Sigma) and 1 mg/mL Dispase (Stemcell Technologies) for 45 minutes at 37°C (200 rpm). Thereafter, the solution was filtered through 100 μm mesh, and the supernatant pelleted (1600 rpm, 5 minutes at 4°C), enriched through a percoll gradient (40 to 80%), and kidney leukocytes were collected and analyzed by FACS. Blood obtained from the retro-orbital sinus was immediately placed in heparinized tubes and washed with PBS followed by RBC lysis buffer (10 mM KHCO₃, 16 mM NH₄Cl, pH 7.3) prior to FACS analysis. Fluorophore-conjugated antibodies used for flow cytometry include B220 (RA3-6B2, BioLegend), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD11b (M1/70, BioLegend), CD45 (30-F11, eBioscience), Foxp3 (FJK-16S, eBioscience), IL-17A (eBio17B7, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8, BD Biosciences), Ly6G (RB6-8C5, eBioscience), RORγT (AFKJS-9, eBioscience). For neutrophil depletion, 500 μg of purified anti-Ly6G (1A8, BioXcell) or isotype (Rat IgG2a, BioXcell) antibody was administered by intraperitoneal injection 10 days after C. albicans infection with sustained dosing every three days thereafter.