Supplementary Materials1. (200 U/mL), 1% NaPyr, 1% HEPES, 50 M -MeOH, and the designated peptide (2 g/mL). At the time points indicated, cells were stained with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), Live/Dead Ghost dye 780 (Tonbo 13-0865-T100), or corresponding fluorescently labeled IgG controls. Cells were then fixed for 15 min at 4C in cytofix (BD Biosciences, San Jose, CA; 554655), and frozen in FCS + 10% DMSO. After all time points were collected, cells from all times were thawed, rinsed and resuspended in PBS + 3% FCS + 1 mM EDTA and analyzed by flow cytometry. All antibodies used were at 1:100 dilutions and stained for 30 min at 4C in a 1:4 dilution of brilliant stain buffer (BD 563794) in PBS + 3% FCS + 1 mM EDTA. Immunization of HHDII-DR1 mice 6 week-old HHDII-DR1 mice were immunized subcutaneously with 100 g of an individual SSX2-p103 APL in complete Freunds adjuvant (Sigma, F5881). Mice were euthanized seven days later and spleens were processed and analyzed via flow cytometry as described above. For these studies, the following antibodies were used: SSX2-p103 HLA-A2 tetramer-APC (NIH Tetramer Core Facility), CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), PD1-PECF594 (BD 562523), 41BB-PerCPeF710 (eBioscience 46-1371-82), Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls. Types of movement gating are shown in Supplementary Figs. S1CS6. Intracellular cytokine staining Splenocytes were collected from naive OT-1 or immunized HHDII-DR1 mice as described above, cultured with 2 g/mL (unless otherwise indicated) native SSX2-p103, SIINFEKL APL, a non-specific peptide (negative control), or phorbol 12-myristate 13-acetate (40 ng/mL, PMA, Sigma-Aldrich, St. Louis, MO; P8139) and ionomycin (2.6 g/mL, Fisher Scientific, Waltham, MA; ICN15507001) as a positive control. After two hours golgistop (0.67L/mL, BD 554724) was CBB1003 added. Cells were incubated for six additional hours (8 hours total), after which intracellular cytokine staining was performed as per the manufacturers protocol (Cytofix/Cytoperm Kit, BD 554714). Antibodies used CBB1003 for cells surface staining were: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711(BD 563179), PD1-PECF594 (BD 562523), 41BB-PerCPeF710 CBB1003 (eBioscience 46-1371-82). Antibodies used for intracellular staining were: TNF-PECy7 (BD 557644), IL2-APC (eBioscience 17-7021-82), IFN-PE (BD 554412), and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled Rabbit Polyclonal to TALL-2 IgG controls. The number of antigen-specific Th1 cells (expressing IL2 and/or TNF and/or IFN) was determined as a percentage of total CD8 T cells via an OR Boolean gate (FlowJo software v10.1). Adoptive transfer and immunization of wild-type C57BL/6 (B6) mice For adoptive transfer of OT-1 T cells into B6 mice, OT-1 splenocytes were harvested as described above. CD8 T cells were isolated using immunomagnetic negative selection (StemCell, Vancouver, Canada; 19853), rinsed and suspended in PBS, and 2 106 cells were adoptively transferred into 6C10 wk old, female, B6 mice via intraperitoneal injection. The day following transfer, mice were immunized subcutaneously with an individual SIINFEKL APL (100 g) in complete Freunds adjuvant (Sigma, F5881) or vehicle. Mice were euthanized at the times indicated, spleens were collected, processed as described above, and analyzed via flow cytometry using the following antibodies: SIINFEKL H2Kb tetramer-BV421 (NIH Tetramer Core Facility), CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), CD44-BV786 (BD 563736) and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls. Data collected on different days was normalized using rainbow beads (Spherotech, Lake Forest, IL; RFP-30-5A). Microscopy RMA-S cells were loaded with SIINFEKL APL by incubation in full media containing peptide (2 g/mL) for one hour at 37C, stained in full media containing 2M CellTracker Red (Invitrogen,.