Eomes efluor660

PBMCs were labeled with different combinations of the following antibodies: CD4 BUV737 (BD, clone SK3), CD8 BV786 (BD, clone RPA-T8), CD8 BUV805 (BD, clone SK1), CD3 V500 (BD, clone UCHT1), CD3 eVolve605 (eBioscience clone OKT3), CD27 APC-eFluor780 (eBioscience, clone), CD27 BV510 (Biolegend, clone O323), CD45RA BV650 (BD, clone HI100), CD45RA Qdot655 (Invitrogen, clone MEM-56), CD28 PE-Cy7 (BD, clone 28.2), CX3CR1 APC (eBioscience, clone 2A9-1), CCR7 BV421 (Biolegend, clone G043H7), CD127 BV421 (Biolegend, clone A019D5). Near-IR fixable dye (Invitrogen) was used to exclude dead cells from the analysis. For intracellular staining, the following antibodies were used: Hobit IgM (BD, clone Sanquin-Hobit/1), Eomes eFluor660 (eBioscience, clone WD1928), Tbet BV421 (Biolegend, clone 4B10), Granzyme B AF700 (BD, clone GB11), Perforin FITC (eBioscience, clone dG9), Perforin PE (eBioscience, clone B-D48), Granzyme K PE (Immunotools, clone 24C3), Granzyme K FITC (Immunotools, clone 24C3). To stain for Hobit IgM, a secondary anti-IgM labeled with PE or FITC was used. The cells were labeled according to manufacturer’s instructions. For the intracellular staining, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining kit (eBioscience). The samples were measured in PBS 0.5% FCS with a LSR Fortessa (BD). The analysis was done using FlowJo Version 10 software.

Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).

Frozen PBMC were thawed and stained ex vivo with HLA*0201 or HLA*0101 PE-labelled pentamers (Proimmune Oxford; 20 min in PBS, RT), stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher; 20 min RT), fixed (1% formaldehyde in PBS, 20 mins RT) then stained with: CD3-PO (Pacific Orange; Invitrogen, UCHT1), CD8–PB (Pacific Blue; BD biosciences, RPA-T8), and either stained with CCR7-PeCy7 (CD197; BD biosciences, 3D12), CD45RA-FITC (BD biosciences, HI100), CD38-PerCP-Cy5.5 (Biolegend, HIT2), HLA-DR-AlexaFlour700 (BD biosciences, G46-6) for 30 min RT; or permeabilised (10x permeabilisation buffer; ebiosciences) and stained with PD1-PeCy7 (BD biosciences, EH12.1), Perforin-FITC (BE biosciences, dG9), Granzyme B- Alexa fluor700 (BD biosciences, GB11) and Granzyme A-PerCpCy5.5 (Biolegend, CB9) 30 min RT. For intranuclear staining, PBMC were stained as above for pentamers and Live/Dead dye, and then surface stained with: CD3-Pacific-orange, CD8-PB (30 min RT). PBMC were then fixed (1 h RT) and permeabilised using the Foxp3/Transcription Factor Staining Buffer Set (ebiosciences; 45 min RT) and stained with eomes-eFluor660 (eBiosciences, WD1928) and Tbet-BV605 (Biolegend, 4B10).