Cd8α percp cy5

Blood and spleen samples were treated with Red Blood Cell Lysis Solution (Qiagen) for 15 min at 37°C and 3 min at room temperature, respectively, before staining. LIVE/DEAD Aqua fluorescent stain (Invitrogen) was used to discriminate between live and dead cells. For tetramer staining, samples were incubated with phycoerythrin (PE)-conjugated SIINFEKL-H-2k^b multimers (TC Metrix, Switzerland) for 35 min at room temperature. Samples were washed and incubated on ice for 30 min with CD8α-PerCp Cy5.5 (clone 53.6.7–eBioscience), CD3–PE Cy7 (clone 145.2C11–eBioscience), CD4–FITC (clone GK1.5–produced in house, Ludwig Cancer Research). For in vitro binding and chemotaxis assays the following antibodies were used: IgG1–PE (clone A85-1–BD biosciences), B220–Pacific blue (clone RA3-6B2 - LICR), CD8a–PerCp Cy5.5 (clone 53.6.7–eBioscience), CD3–PE Cy7 (clone 145.2C11–eBioscience), CD11c–eFLuor 660 (clone N4/18–eBioscience), CD11b–Alexa700 (clone M1/70–eBioscience), CD103–PE. Data were acquired on a LSRII or LSRII (SORP) and FACS analyses were done with Flow Jo software.

The following antibodies were used in this study: CD3ε-eFlour 450 (145-2C11, eBioscience), CD3ε-APC-eFluor 780 (17A2, eBioscience), CD8α-APC-eFluor 780 (53-6.7, eBioscience), CD8α-BV605 (53-6.7, Biolegend), CD8α-PerCP-Cy5.5 (53-6.7, eBioscience), CD44-PE-Cy7 (IM7, Biolegend), CD62L-APC (MEL-14, eBioscience), CD62L-BV510 (MEL-14, Biolegend), CD69eFluor 450 (H1.2F3, eBioscience) CD69-biotin (H1.2F3, eBioscience), CD122-FITC (TM-B1, BD Biosciences), CD127-BV605 (A7R34, Biolegend), and Streptavidin PerCP-Cy5.5 (BD Biosciences). The MHC class I tetramers H2-D^bGP_33–41 APC and H2-D^bNP_396–404 PE were kind gifts from Dr. Ramon Arens (LUMC, Leiden, The Netherlands). Cells were fixed with Foxp3/Transcription Factor Staining buffer set (eBioscience) and stained with Ki-67 PE or Ki-67 FITC (B56, BD Biosciences). Samples were acquired with the LSR Fortessa (BD) and analyzed with FlowJo software (Tree Star, Inc.).

Flow-cytometry antibodies included CD3e-PE, PD1-BV605, CD45-BV786 (BD-Biosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-APC, CD45.1-PE (eBioscience), CD45.2-Alexa700, CD-8α-Percp-Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas-red (Life Technologies, Carlsbad, CA, USA). Blocking PD-1 antibody (clone RMP1-14, BioXCell, Branford, CT, USA) was administered systemically by intraperitoneal injections of 250 μg [41 (link)]. Blocking anti-CD40L (clone MR1, BioXCell) was administered systemically by intraperitoneal injections of 250 μg [19 (link)].