Anti tcrβ pe

C57BL/6 (WT) mice were purchased from The Jackson Laboratories (Bar Harbor, ME).
Cd244^-/- mice on the C57BL/6 background [12 (link)], were kindly provided by Dr. Raymond Welsh (University of Massachusetts Medical Center, Worcester, MA). Mice were maintained in the Translational Biomedical Research Center of the Medical College of Wisconsin and both genders were used for experiments in an age (6–12 week) and gender-matched manner. anti-CD45R-PE-Texas Red, anti-CD45R-PE, anti-GL7-FITC and anti-CD5-APC were purchased from BD Biosciences (San Jose, CA). CD21-eFluor 450, anti-CD23-PE-Cy7, anti-CD93-biotin, anti-CD4-APC-eFluor 780, anti-CD8-PE-Cy7, anti-TCRβ-FITC, anti-TCRβ-PE, anti-CD11b-Alexa Fluor 488, anti-CD244, anti-Foxp3-PE, anti-CD19-Alexa 700, anti-CD4-PE, anti-IgG-FITC and Streptavidin PE-Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-CD11b-Brilliant Violet 605, anti-CD11c-PE, anti-NK1.1-APC, anti-IgM-APC Cy7, anti-IgD-Pacific Blue, anti-CD38-Alexa Fluor 647, anti-CD138-APC and rat anti-mouse IgG_2a-biotin were purchased from Biolegend (San Diego, CA). Anti-IgM-FITC and the SBA Clonotyping System-B6/C57J-HRP were purchased from Southern Biotech (Birmingham, AL). 4-Hydroxy-3-nitrophenylacetyl (NP)-ficoll, NP-Chicken Gamma Globulin (CGG), NP(24)-PE and NP-BSA were purchased from Biosearch Technologies (Novato, CA).

To analyze intracellular IFN-γ production by CD8⁺ and CD4⁺ T cells, splenocytes from the mice vaccinated with 1, 3, and 5 doses of cGM-CSF were collected 7 days after the last immunization and stimulated in vitro with 10 µg/mL HPV-16 E7 MHC class I peptide (aa 49–57, RAHYNIVTF) or MHC II (aa 44–60, QAEPDRAHYNIVTFCCK) peptide by incubation at 37 °C with 5% CO₂ for 15 h. After 15 h, the cells were treated with 50 ng/mL phorbol-12-myristate-13-acetate (PMA) and 1 µg/mL of ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop protein transport inhibitor containing monensin (BD Bioscience, San Jose, CA, USA) for 4 h. The cultured cells were analyzed by flow cytometry.
For IKDC analysis, splenic cells were stained with anti-B220-FITC (BD Bioscience, San Diego, CA), anti-NK 1.1-perCP (eBioscience, San Diego, CA, USA), anti-TCRβ-PE (eBioscience, San Diego, CA, USA), anti-CD19-PEcy7 (eBioscience, San Diego, CA, USA), and anti-CD11c-APC antibodies (Biolegend, San Diego, CA, USA) at 4 °C for 20 min followed by flow cytometric analysis.