Bb515 anti cxcr5

Freshly isolated PBMCs (4 × 10⁶/mL) were cultured in 10 % fetal calf serum RPMI-1640 (Hyclone, Logan, UT, USA) in U-bottom 24-well tissue culture plates (Costar, Lowell, MA, USA), stimulated with or without 50 ng/mL of phorbol myristate acetate (PMA) plus 2 μg/mL of ionomycin (Sigma, St. Louis, MO, USA) for one hour, followed by treatment with Brefeldin A (10 μg/mL, GolgiStop™; BD Biosciences, San Jose, CA, USA) for an additional five hours. Then, these cells were stained in duplicate with BV510-anti-CD3, APC-H7-anti-CD4, BB515-anti-CXCR5, PE-Cy5-anti-CD45RA, PE-CF594-anti-CD279 and BV421-anti-CD278 (Beckton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Subsequently, cells were fixed, permeabilized, and stained with PE-anti-IL-21 (Beckton Dickinson). The frequencies of distinct Tfh cells were analyzed by multicolor flow cytometry (FACSAria™ II, BD Biosciences), and data were processed using FlowJo software (v5.7.2; FlowJo, Ashland, OR, USA).

Blood samples were collected separately from controls and KD patients in different phases. Peripheral blood mononuclear cells (PBMCs) were isolated from individual KD patients and control subjects by density-gradient centrifugation at 800×g for 30 min at 25 °C using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). Freshly isolated PBMCs (4 × 10⁶/mL) were cultured in 10% fetal calf serum RPMI-1640 (Hyclone, Logan, UT, USA) in U-bottom 24-well tissue culture plates (Costar, Lowell, MA, USA) and stimulated for 1 h with or without 50 ng/mL of phorbol myristate acetate (PMA) in the presence of 2 μg/mL of ionomycin (Sigma, St. Louis, MO, USA). The cells were then treated with Brefeldin A (10 μg/mL, GolgiStop™, BD Biosciences, San Jose, CA, USA) for an additional 5 h. For flow cytometric analysis, PBMCs were stained in duplicate with BV510-anti-CD3, APC-H7-anti-CD4, BB515-anti-CXCR5, PE-Cy5-anti-CD45RA, PE-CF594-anti-CD279 and BV421-anti-CD278 (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Subsequently, the cells were fixed, permeabilized, and stained with PE-anti-IL-21 (Becton Dickinson). Multicolor flow cytometry (FACSAria™ II, BD Biosciences) was used to determine the percentages of distinct cTfh cells, and the data were analyzed with FlowJo software (v5.7.2; FlowJo, Ashland, OR, USA).

PBMCs at 4 × 10⁶/ml were analyzed by multicolor flow cytometry (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA). Human PBMCs (10⁶/tube) were stained with BV510-anti-CD3 (clone: UCHT1), allophycocyanin (APC)-H7-anti-CD4 (clone: RPA-T4), BB515-anti-CXCR5 (clone: RF8B2), phycoerythrin (PE)-Cy5-anti-CD45RA (clone: HI100), PE-Cy7-anti-CCR6 (clone: 11A9), or APC-anti-CD183 (clone: IC6/CXCR3) (Becton Dickinson, San Jose, CA, USA) at room temperature for 30 min. Data were processed using FlowJo v.5.7.2 software (Tree Star, Ashland, OR, USA).