Figure 6.

Cxcr4 desensitization intrinsically regulates splenic plasmablast homing properties. (A) Representative FACS plot for Cxcr4 surface expression on splenic PBs. The dashed vertical line is placed at the peak of the Cxcr4 staining on WT PBs. (B) Quantification of the geometrical mean of Cxcr4 at the surface of splenic PBs (MFI: geometrical mean fluorescence intensity). (C) Migration index of splenic PBs to Cxcl12 determined by transwell-based assay. Cxcl12 was added to the lower well at the indicated concentrations with or without AMD3100. (D) Schematic diagram describing the transfer experiment protocol. Blimp1^GFP/+ PBs were generated in vitro from splenic B cells and labeled with CTV for Cxcr4^+/1013 cells and with CTY for WT cells. Labeled WT and mutant cells were mixed at a 1:1 ratio and transferred into CD45.1 WT recipients. (E) Representative dot plot of the cell mix (1:1) prior transfer. (F) Representative dot plots and frequency of CTV⁺ (Cxcr4^+/1013 × Blimp1^GFP/+) and CTY⁺ (WT × Blimp1^GFP/+) cells in the BM and the spleen of recipient mice at days 1 and 2 after transfer. (G) Heatmap showing the relative expression of 60 transcripts from sorted BM PBs from both WT and Cxcr4^+/1013 mice determined by Biomark multiplex qPCRs at day 6 after immunization with NP-LPS. Data are presented by applying a column Z score based on (2^−ΔΔCt). (H) Heatmap showing the relative expression of 11 selected transcripts from BM PBs and PCs from both WT and Cxcr4^+/1013 mice determined by Biomark multiplex qPCRs at day 6 after immunization. Data are presented by applying a column Z score based on (2^−ΔΔCt). (I) PCA was performed on BM-PBs and BM-PCs from both groups using a correlation test. (J) Representative histograms for the indicated proteins from BM PBs and PCs from both WT and Cxcr4^+/1013 mice are shown. Results are from 2 to 3 independent experiments (mean ± SEM, n = 4-10). Mann-Whitney U test was used to assess statistical significance (*P ≤ .05, **P < .01, ***P < .001).