Phorbol 12 myristate 13 acetate pma ionomycin

Fluorescein isothiocyanate, phycoerythrin; allophycocyanin; allophycocyanin-Cy7 and PE/Dazzle labelled CD40, CCR1, IFN-γ, T-bet, IL-17A, IL-22, and TNF-α anti-human monoclonal antibodies; and red blood cell lysing, fixation, and permeabilizing buffers were purchased from BioLegend (San Diego, CA, USA). FcR blocking reagent was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). RORγt, GolgiStop, and acid−citrate−dextrose vacutainer tubes were purchased from BD Biosciences (San Diego, CA, USA). The primers used were purchased from GenScript (Piscataway, NJ, USA). TRIzol reagent was obtained from Life Technologies (Grand Island, NY, USA). The SYBR Green PCR Master Mix and High-Capacity cDNA Reverse Transcription kit was purchased from Applied Biosystems (Paisley, UK). The primary antibodies against CCR1 and secondary anti-human antibodies used for Western blotting were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin, phosphate-buffered saline (PBS), RPMI-1640 medium, Ficoll-Paque, and Hanks’ Balanced Salt Solution (HBSS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitrocellulose membranes were obtained from Bio-Rad Laboratories (Hercules, CA, USA), and a chemiluminescence Western blot detection kit was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA).

The Hev b 5_46–65 peptide (TPEKEEPTAAPAEPEAPAPE), an immunodominant T-cell epitope not associated with a particular MHC II haplotype [25] (link), was synthesized at GenScript (NJ, USA). To induce T-cell differentiation, autologous-naïve T cells were primed with 3×10⁴ Hev b 5_46–65-pulsed DC (T_{Hev b 5-DC}) (10∶1) for 6 days and rested for 4 days with 10 IU/ml IL-2 (Proleukin®, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA) in round-bottomed 96-well plates. Finally, T_{Hev b 5-DC} were harvested after 10 days and re-stimulated for 16 h with Phorbol 12-Myristate 13 Acetate (PMA)/ionomycin (Sigma-Aldrich) to assess IL-10 production by ELISPOT Ready-SET-Go!® (eBioscience) as before.

Antibodies used in this study are listed in online supplemental table S1. Cells were stained according to the manufacturer’s instructions. To detect the expression of the lysosomal-associated membrane protein 1 (LAMP1/CD107a) (as a surrogate marker for degranulation) on the surface of CD8⁺ T-cells, effector cells were incubated for 24 hours with target tumor cells in the presence of monensin (Monensin Solution 1000X, eBioscience) and the CD107a antibody. Phorbol 12-myristate 13-acetate (PMA) + ionomycin (Sigma-Aldrich) were used as control inducers of CD107a expression. The determination of apoptotic cells was performed with a standard Annexin V and propidium iodide (PI) (BD Biosciences) staining according to the manufacturer’s instructions. TCR Vβ repertoire of TCR-engineered T cells in vivo was analyzed by flow cytometry with antibodies from the Beta Mark TCR Vβ Repertoire Kit (Beckman Coulter) directed against 19 individual TCR/Vβ chains. Flow cytometry acquisitions were performed on a FACSCanto II and BD FACSLyric (BD Biosciences), and data were analyzed with FlowJo_V10 software (Tree Star).

The following mAbs were obtained from eBioscience (San Diego, CA, USA) or BioLegend (San Diego, CA, USA) for FACS analysis and other experiments: FITC-labeled anti-CD4 (RMA4-5), CD45 (30-F11), CD80 (16-10-A1), CD86 (GL1), MHC II (M5/114.15.2), CD44 (IM7), CD69 (H1.2F3), CD62L (MEL-14) and CD11b (M1/70); PE-labeled anti-CD8 (53–6.7), F4/80 (BM8), and IFN-γ (XMG1.2), TLR2 (6C2), CD154 (MR1), CD25 (PC61.5); PerCP/Cy5.5-labeled anti-mouse CD11b (M1/70), Ly-6C antibody (HK1.4), and IFN-γ (XMG1.2); PE/Cy7-labeled anti-IL-2 (JES6-5H4); APC-labeled anti-Ly6-G (1A8), IL-12/23p40 (C17.8), iNOS (CXNFT), TNF-α (MP6-XT22), CD206 (MR6F3), and CXCR3 (CXCR3-173). PE-labeled anti-mouse Tmem119 (106–6) was obtained from Abcam (Cambridge, MA, USA). The JEV epitope peptide of CD4⁺ T cells (NS1_132-145 [TFVVDGPETKECPD]; NS3_563-574 [WCFDGPRTNAIL]) or CD8⁺ T cells (NS4B_215-223 [SAVWNSTTA]) was chemically synthesized at Peptron (Daejeon, Korea). Phorbol-12-Myristate-13-Acetate (PMA), ionomycin, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Male BALB/c mice (6–8 weeks old) used for this study were maintained under pathogen-free condition at institutional animal house facility. Anti-mouse antibodies Alexa Fluor 700-CD8 (Clone 53-6.7), APC-cy7-CD19 (Clone 1D3), APC-cy7-CD45R/B220 (Clone RA3-6B2), were procured from BD Biosciences (San Jose, CA, USA), FITC-CD4 (clone GK 1.5), FITC CD278 (7E.17G9), APC-CD278 (Clone-C398.4A), PerCP-Cy 5.5-CD4 (clone RM4-5), PE-Cy7-IFN-γ (clone XMG 1.2), PE-T-bet (clone eBio 4B10), Alexa Fluor 700-CD8 (53-6.7) were from eBioscience (San Diego, CA, USA), Brilliant violet 605 TCR Vb (clone H57-597), APC-CD278 (clone C398.4A), Anti-CD49b (clone DX5) were from BioLegend (San Diego, CA, USA). Purified antibodies Anti-mouse CD278 ICOS (clone 7E.17G9), Anti-Rat IgG2b isotype control, and anti-CD16/32 were procured from BioXcell (West Lebanon, USA). Dynabeads untouched mouse CD4⁺ T cell isolation kit was procured from Life Technologies AS (Oslo, Norway) and CD8⁺ T cell negative selection kit was from Stem cell technologies (Vancouver, BC, Canada). Phorbol 12-myristate 13-acetate (PMA), Ionomycin, Brefeldin A was procured from Sigma-Aldrich (St. Louis, MO, USA). Malarial parasite P. berghei ANKA (MRA-671, MR4, ATCC, Manassas, VA, USA) was obtained from MR4 repository, ATCC, Manassas, VA, USA.