High-level CXCR5 surface expression on CLL cells and normal CD5+ B cells. The top panel displays overlay histograms that depict the relative red fluorescence intensity after staining of CLL B cells, CXCR5-positive T cells (TFH cells), or neoplastic B cells from a patient with multiple myeloma with PE-labeled anti-CXCR5 mAbs (dark-gray histograms) or PE-conjugated isotype controls (light-gray histograms). CLL cells were identified by gating on the CD19-expressing cells, TFH cells by gating on the CD5+ and CXCR5-positive and CD19− lymphocytes, whereas multiple myeloma cells were gated by costaining with anti-CD38 and anti-CD138 mAbs. The CXCR5 mean fluorescence intensity ratio (MFIR) values are displayed next to each CXCR5 histogram. Compared with other B-cell neoplasias, TFH cells, or CD5− B cells, CLL B cells and normal CD5+ B cells consistently expressed higher levels of surface CXCR5, and multiple myeloma cells did not express surface CXCR5. The right-hand side box in the top panel displays a bar diagram that depicts the mean (± SEM) CXCR5 MFIRs for TFH cells (n = 3), normal CD5− B cells (n = 11), normal CD5+ B cells (n = 3), CLL B cells (n = 49), prolymphocytic leukemia cells (PLL, n = 3), follicular lymphoma cells (FCL, n = 2), mantle cell lymphoma cells (MCL, n = 2), immunocytoma (IC, n = 1), and multiple myeloma (MM, n = 3). The bottom panel displays contour plots that depict the staining and gating of buffy coat cells to determine CXCR5 expression on normal CD5+ B cells. A small proportion of circulating B cells coexpresses CD19 and CD5; this specimen had 10% CD19+ B cells, 0.8% CD19+/CD5+ B cells, and 65% CD5+ T cells, as indicated in the bottom left contour plot. Because of the low frequency of CD5+ B cells, we acquired 500 000 or more CD19+ B cells, which in this case contained 7.6% CD5+ B cells (middle contour plot). This allows us to determine CXCR5 expression levels on a sufficient number of CD19+/CD5+ B cells in gate R1. The right-hand histogram overlay displays the CXCR5 expression on CD5+ B cells in R1 (gray histogram), compared with the respective isotype control (white histogram), and the CXCR5 MFIR is displayed next to the CXCR5 histogram.
