Effect of CD62L antibody treatment on chemokine and S1P1 receptor signaling. (A) LN B cells from CD62L antibody-treated mice exhibit reduced chemotaxis. The experiments shown used B cells prepared from the LNs of 10 CD62L antibody–treated mice (24 hours) and 5 untreated ones. Specific migration to various concentrations of CXCL12, CCL19, CXCL13, and S1P are shown (5 μm pore size). (B) Spleen B cells from CD62L-treated mice respond similar to control spleen cells. Specific migration to various concentrations of CXCL13 and CCL19 is shown. Results are from B cells isolated from 3 treated versus 3 untreated spleens. (C) LN B cells from CD62L antibody-treated mice exhibit reduced chemoattractant induced increases in intracellular calcium. Same B cell preparations were used as in panel A. CXCL12 (100 ng/mL), CCL19 (100 ng/mL), and CXCL13 (100 ng/mL) induced changes in [Ca²⁺]_i were monitored over 3 minutes, right panels. The data shown as fluorescent counts and the y-axis labeled as Lm1. Each experimental value is the mean of 3 determinations. (D) CD62L antibody treatment does not affect LN B cell chemokine receptor expression. Results shown are from the same LN cells shown in panels A through C. Flow cytometry used to assess CXCR4, CCR7, and CXCR5 expression. Isotype controls are shown at the left of each profile. The chemotaxis, calcium, and flow cytometry were each replicated using smaller numbers of mice. (E-F) CD62L antibody treatment enhances LN B cell RGS protein mRNA expression. Analysis of RNA extracted from same experiment as other panels analyzed by standard RT-PCR (E) and by quantitative RT-PCR (F). The total LN RNA analyzed was prepared from total lymph node lymphocytes and served as a primer control. The quantitative RT-PCR data are shown as relative expression between PBS-treated and CD62L antibody–treated mice.