Igg1 isotype control p3.6.2.8.1

Surface marker staining was performed after the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend). Cells were incubated with the Live/Dead Fixable Yellow Dead Cell Stain Kit (Life Technologies). Surface marker-stained cells were analyzed on BD LSRFortessa with FACSDiva software (BD Biosciences). The gating strategy of the FACS analysis was shown in Supplemental Fig. 13. The following antibodies were used for FACS staining: anti-CD45RA-FITC (clone HI100), anti-CD25-PE (BC96), anti-4-1BB-BV421 (4B4-1), anti-CD8-BV510 (RPA-T8), anti-CD103-BV605 (Ber-ACT8), anti-CD4-BV711 (OKT4), anti-Tim-3-APC (F38-2E2), anti-CD3-Alexa Fluor 700 (UCHT1), anti-CD15-BV510 (W6D3), anti-CD11b-BV605 (M1/70), anti-CD45-BV786 (HI30), anti-CD14- Alexa Fluor 700 (HCD14), and IgG1 isotype control (MOPC-21) purchased from Biolegend. Anti-ICOS-PerCP eFluor 710 (ISA-3) and IgG1 isotype control (P3.6.2.8.1) were purchased from eBioscience. Anti-OX-40-PE-CF594 (ACT35), anti-PD-1-PE Cy7 (EH12.1), and anti-CCR3-PE-CF594 (5E8) were purchased from BD Bioscience.

Surface marker staining was performed after the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend). Cells were incubated with the Live/Dead Fixable Yellow Dead Cell Stain Kit (Life Technologies). Surface marker-stained cells were analyzed on BD LSRFortessa with FACSDiva software (BD Biosciences). The following antibodies were used for FACS staining: anti-CD45RA-FITC (clone HI100), anti-CD25-PE (BC96), anti-4-1BB-BV421 (4B4-1), anti-CD8-BV510 (RPA-T8), anti-CD103-BV605 (Ber-ACT8), anti-CD4-BV711 (OKT4), anti-Tim3-APC, (F38-2E2), anti-CD3-Alexa Fluor 700 (UCHT1), anti-CD45-BV786 (HI30), and IgG1 isotype control (MOPC-21) purchased from Biolegend. Anti-ICOS-PerCP eFluor 710 (ISA-3) and IgG1 isotype control (P3.6.2.8.1) were purchased from eBioscience. Anti-OX-40-PE-CF594 (ACT35), anti-PD-1-PE Cy7 (EH12.1), and IgG1 isotype control (X40) were purchased from BD Bioscience.

White blood cells were extracted from peripheral blood by lysis with BD Pharm Lyse (BD Biosciences) red blood cell lysis buffer and then subjected to flow cytometry. Surface marker staining was performed after the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend). Surface marker-stained cells were analyzed on BD LSRFortessa with FACSDiva software (BD Biosciences). The gating strategy of the FACS analysis is shown in Supplementary Fig. S8. The following antibodies were used for FACS staining: anti-CD45RA-FITC (clone HI100), anti-CD25-PE (BC96), anti-4-1BB-BV421 (4B4-1), anti-CD8-BV510 (RPA-T8), anti-CD103-BV605 (Ber-ACT8), anti-CD4-BV711 (OKT4), anti-Tim-3-APC (F38-2E2), anti-CD3-Alexa Fluor 700 (UCHT1), and IgG1 isotype control (MOPC-21) purchased from Biolegend. Anti-ICOS-PerCP-eFluor 710 (ISA-3) and IgG1 isotype control (P3.6.2.8.1) were purchased from eBioscience. Anti-OX-40-PE-CF594 (ACT35), anti-CD27-BV786 (L128), and anti-PD-1-PE Cy7 (EH12.1) were purchased from BD Bioscience. Anti-CD19-APC-eFluor 780 (H1B19) was purchased from Invitrogen.