Cd8a percp cy5

Bone marrow of Myce and littermate wild‐type control were harvested into single‐cell suspension with red cell lysis and stained with CD19‐Pacific Blue (1D3, BD, NJ, USA), B220‐PE cy7 (RA3‐6B2, BD), CD24‐APC (M1/69, in house), IgM‐FITC (331.12, in house), CD43‐PE (S7, BD). Spleen red cell lysed single cell suspensions were stained with CD19‐BB700 (1D3, BD), B220‐APC (Thermo Fisher, Waltham, USA), CD23‐PE cy7 (B3B4, Thermo Fisher), CD21‐BV421 (7E9, BioLegend, San Diego, USA), CD138‐PE (281–2, BD). The peritoneal cavity wash was stained with CD19‐PE (ID2, in house), B220‐APC (RA3‐6B2, Thermo Fisher), CD23‐PE cy7 (B3B4, Thermo Fisher), CD5‐BB700 (53–7.3, BD), CD11b‐eFlour450 (M1/70, Thermo Fisher). Lymph node and thymus of single cell suspensions were stained with CD19‐BUV395 (1D3, BD), (TCRβ‐PE (H57‐597, BD), CD4‐BV421 (GK1.5, BioLegend) and CD8a‐PerCp/Cy5.5 (53–6.7, eBioscience, San Diego, USA). Flow cytometry analysis was performed on a BD LSRFortessa X‐20 analyzer (BD).

Antibodies used were as follows: CD56 BV421, CD3 PE-Cy7 or APC-Cy7, CD14 APC-Cy7, CD19 APC-Cy7, CD57 Pacific Blue or FITC, CD62L PE-Cy7, CD107a PE-Cy7, CD244 PE-Cy5.5, Perforin Pacific blue, CD160 Alexa Fluor 647 (Biolegend), CD16 eFluor450 or FITC, CD69 FITC, CD94 FITC, CD8a PerCP-Cy5.5 (eBioscience), CD161 PE or APC, CD56 APC, IFNγ FITC, NKp30 APC, NKp46 APC, NKp80 APC (Miltenyi Biotec), CD3 Pacific Orange, CD4 Qdot 605, Granzyme B APC (Invitrogen), NKG2C Alexa Fluor 488 or PE, NKG2D PE, PLZF APC, Granzyme A FITC (R&D Systems), CD56 FITC or PE-Cy7, CD85j FITC, IFNγ Alexa Fluor 700, Ki67 FITC (BD Biosciences), Granzyme K FITC (Immunotools), NKG2A PE, NKp44 PE, CD158e1/e2 PE (Beckman Coulter), Phosphatidylserine Alexa Fluor 488 (Merck Milipore). The viability dye Live/Dead fixable Near-IR (Invitrogen) was used in all experiments. Anti-KLRG1 FITC was kindly provided by H. Pircher. Data were acquired on LSRII (BD Biosciences) and analyzed using FlowJo (Treestar, Inc.).

Lysed cells were acquired and subsequently stained for 30 min at 4 °C with the following fluorescein-conjugated monoclonal antibodies: CD3-PE (Bio- Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CCR7-APC/Cy7 (BioLegend). The T cell subsets were defined as previous study reported [10 (link)]: Naive T cells being CCR7⁺ and CD45RO⁻; central memory T cells as CD45RO⁺ and CCR7⁺; effector memory T cells as CD45RO⁺ and CCR7⁻, and effector memory RA (EMRA) T cells as CD45RO⁻ and CCR7⁻ (Figure S1). A total of 200,000 events were acquired by the BD LSRFortessa™ flow cytometer (BD Bioscience, San Jose, CA, USA). The data analysis was carried out with Flowjo v10.1 Software (Tree Star, Ashland, OR). The absolute number of each T cell subset was calculated as follows: (percentage of each cell population among total lymphocytes) × (total lymphocytes count)/100.

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.

On the day of blood drawing, blood samples mixed with heparin anticoagulant were lysed with red blood cell lysis solution and 0.1 mM EDTA and prepared for flow cytometry analysis with the following fluorescein-conjugated monoclonal antibodies: CD3-PE (Bio- Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and CCR7-APC/Cy7 (BioLegend). The relative expression of CD45RO and CCR7 was used to identify naïve T cell (T_Naïve, CD45RO⁻ CCR7⁺), central memory T cell (T_CM, CD45RO⁺ CCR7⁺), effector memory T cell (T_EM, CD45RO⁺ CCR7⁻), and T effector memory CD45RA cell (T_EMRA, CD45RO⁻ CCR7⁻) subsets of CD4⁺ or CD8⁺ T cells. These markers were selected according to previous studies (7 (link), 17 (link)). The immunophenotyping methods and gating strategy have been elaborated in the supplementary materials (Figure S1).