Cd4 pe cy7 sk3

Prior to stimulation for B10 cells, B cells were negatively isolated from a starting population of 10⁷ PBMCs, using the B cell enrichment kit (Stemcell). Isolated B cells were plated in 96-well flat bottom plates in RPMI + 10% FBS. Cells were stimulated with CpG and rCD40L for 48 h at 37°C in 5% CO₂ incubator. At 43 h, cells were restimulated with PMA and ionomycin. In parallel with the end of the 48-h stimulation, PBMCs from the same patients were enriched for CD4 T cells (Stemcell) and stained with the Violet Proliferation dye (1 µM, BD Bioscience) in PBS and incubated in a 37°C water bath for 15 min. After 48 h, the isolated CD4 cells were combined with B cells at 1:1 and 1:2 ratio of T:B cells along with a CD4 T cell only condition and stimulated with αCD3/αCD28 for 5 days. For experiments using the transwell plates, stimulated B cells were plated in top chamber of 24-well transwell plates while CD4 T cells and αCD3/αCD28 were plated in bottom chamber.
To visualize proliferation of the CD4 T cells, cells were stained with Zombie Violet, CD14 Brilliant Violet 510 (M5E2, Biolegend), CXCR5 AlexaFluor 647 (RF8B2, BDB), CD8 AlexaFluor 700 (SK1, Biolegend), CD3 APC-Cy7 (SK7, Biolegend), CD19 PE (HIB19, Biolegend), and CD4 PE-Cy7 (SK3, Biolegend) conjugate for 25 min at 4°C. Following cell surface staining, cells were fixed with 1% PFA and acquired on a LSRII flow cytometer (BDB).

Frozen PBMC’s were thawed and 5x10⁵ cells were immediately stained with Near-IR live-dead dye (LifeTechnologies, Denmark), blocked and then stained with antibodies to CD4-PE-Cy7 (SK3), CD8⁺-PerCP-Cy5.5 (SK1), CD45RA (HI100), CCR7 (G043H7) CD69-APC (FN50), HLA-DR-PE (G46-6) and CD38-BV605 (HB7) or PD-1 (EH12.1) (all Biolegend except PD-1, CD38, HLA-DR and CD8⁺; BD Bioscience). Only singlet, live cells were included in the data analyses. T cells were gated based upon size and granularity (lymphocyte gate). Within the lymphocyte gate T cells were sub-gated based upon their expression of either CD4 or CD8⁺. Memory subsets within CD4⁺ and CD8⁺ T cells were defined based on CCR7 and CD45RA expression. Activation status was determined based upon CD69 expression or HLA-DR/CD38 co-expression. Gates for activation markers and PD-1 were determined using isotope control antibodies.

Red blood cell-lysed whole blood or cells isolated from mucosal tissues were stained using a combination of multicoloured flow cytometry panels designed to determine Fc-receptor expression on CD14+ monocytic cells, mDC or NK cells. Briefly, for overall cellular phenotyping; CD3 V450 [UCHT1], CD4 PECy7 [SK3] (Biolegend), CD8 Pacific Orange [3B5] (Invitrogen), CD19 BV650 [SJ25C1]. For CD14 and mDC FcR Phenotyping; CD3 V450 [UCHT1], CD14 Qdot 605 [TüK4] (Invitrogen), CD16 Pacific Orange [3G8] (Invitrogen), CD11c A700 [B-ly6], CD123 PECy5 [9F5], CD32 APC [FLI8.26], CD64 APC H7 [10.1], CD89 PE [A59], CD19 FITC [HIB19]. For NK cell FcR phenotyping; CD15 BV650 [W6D3] (Biolegend), CD16 PECy7 [3G8] (Biolegend), CD66b FITC [G10F5] (Biolegend), CD64 APC H7 [10.1], CD56 PECy5 [HCD56] (Biolegend), CD45 A700 [HI30] (Biolegend), CD89 PE [A59], CD3 V450 [UCHT1], CD32 APC [FLI8.26]. Unless otherwise specified, all antibodies were sourced from BD Biosciences. Anti-FcRs were able to detect antibody-occupied FcR as indicated by the manufacture and in house controls. Dead cells were excluded from analysis through staining with Aqua Viability Dye (Invitrogen).