GRK2-deficient T cells are resistant to S1PR1 desensitization and have a reduced ability to enter LNs. (A) Flow cytometric analysis of blood and peripheral LN (pLN) CD4 T cells from WT and GRK2^f/− CD4Cre (KO) mice for S1PR1, CCR7 and CXCR4. Shaded histrogram shows background (bg) staining of negative control. (B) Transwell migration of WT and KO CD4 T cells to S1P, CXCL12 or CCL21. Three replicates were run for each group. Bars indicate mean (±SEM). (C) Short term in vivo homing efficiency of GRK2 KO T cells relative to co-transferred WT T cells. Transferred B cells are tracked as an additional control. bld, blood; spln, spleen; mLN, mesenteric LN. Data are from 3 experiments (n=8 mice). Bars indicate mean (±SEM). (D) Short term in vivo homing efficiency of KO T cells relative to co-transferred WT T cells in WT and S1P-deficient recipients. Data are from 3 experiments (n=8 mice). Bars indicate mean (±SEM). (E) Rolling-to-sticking transition frequency and rolling velocity (Vroll) of transferred WT and KO T cells visualized by intravital microscopy in pLN HEVs. Summary of 3 experiments (n=3 mice). Bars indicate mean (±SEM). * p<0.05, ** p<0.005; *** p<0.0005, ns, not significant (p>0.05) by unpaired Student’s t-test.

MZ B cell migration into follicles requires GRK2-dependent S1PR1 desensitization. (A) Flow cytometric analysis of splenic MZ B cells from WT and GRK2^f/− Mb1-cre (KO) mice for S1PR1, CXCR5 and CXCR4. (B) Transwell migration of WT and KO MZ B cells to S1P, CXCL12, CXCL13 and S1PR1 agonist SEW2871. Three replicates were run for each group. Bars indicate mean (±SEM). (C) Flow cytometric detection of 5 min in vivo anti-CD19-PE labeled MZ B cells from WT, GRK2^f/− Mb1-cre (KO) or GRK2^+/− (Het) mice shown as a histogram overlay (left panel) and a summary of data from 4 experiments (WT n=28, KO n=15, Het n=13 mice) (center panel). Right panel shows a summary of data from 3 experiments for WT and GRK2^\f/− Mb1-cre (KO) cells present in mixed BM chimeras (n= 8). Bars indicate mean (±SEM). (D) Flow cytometric detection of 5 min in vivo anti-CD19-PE labeled MZ B cells from WT, GRK2^+/− (GRK2 het), S1PR1^+/− (S1PR1 het) and GRK2^+/− S1PR1^+/− (GRK2/S1PR1 het/het) mice shown as a histogram overlay (left panel) and a summary of data from 5 mice (right panel). Bars indicate mean (±SEM). (E) Immunohistochemical localization of CD1d^hi MZ B cells (blue) with respect to the MAdCAM1+ marginal sinus (brown) in spleen sections from GRK2^+/+ Mb1-cre (WT) and GRK2^f/− Mb1-cre (KO) mice. MZ, marginal zone. FO, follicle.

GRK2-deficient follicular B cells are resistant to S1PR1 desensitization and have a reduced ability to enter LNs. (A) Flow cytometric analysis of blood and peripheral LN follicular B cells from WT and GRK2^f/− Mb1-cre mice (KO) for S1PR1, CXCR5, CXCR4 and CCR7. (B) Transwell migration of WT and KO B cells to S1P, CXCL12 and CXCL13. Three replicates were run for each group. Bars indicate mean (±SEM). (C) Short term homing efficiency of KO B cells relative to co-transferred WT B cells. Transferred T cells are tracked as an additional control. Summary of 3 experiments (n=8 mice). Bars indicate mean (±SEM). (D) Short term homing of GRK2-deficient B cells relative to co-transferred WT B cells in WT and S1P-deficient recipients. Summary of 4 experiments (n=11 mice). Bars indicate mean (±SEM). (E) Flow cytometric detection of 30 min transferred B220+ B cells and CD3+ T cells labeled by 2 min in vivo anti-CD19-PE and anti-CD4/8-PE treatment. (F) Short term homing efficiency of KO B cells relative to cotransferred WT B cells in WT (n=10) and S1P-deficient (n=6) recipients, showing cells that were protected from anti-CD19/4/8-PE exposure (PE−) or blood exposed (PE+), identified as in F. Summary of 3 experiments. Bars indicate mean (±SEM).

MZ B cell shuttling and immune complex delivery into follicles is disrupted by mutation of an S1PR1 desensitization motif. (A) Flow cytometric analysis of S1PR1 expression on MZ B cells from freshly isolated WT and S1PR1^TSS (TSS) mice. (B) Transwell migration of WT and TSS MZ B cells to S1P, CXCL12 and CXCL13. Three replicates were run for each group. Bars indicate mean (±SEM). (C) Flow cytometric detection of 5 min in vivo anti-CD19-PE labeled MZ B cells from the indicated mice shown as a histogram overlay (left panel) and a summary of data from 9 mice. (D) Immunohistochemical localization of CD1d^hi MZ B cells (blue) with respect to the MAdCAM1+ marginal sinus (brown) in spleen sections from WT and TSS mice. (E) Surface S1PR1 internalization and recovery on MZ B cells exposed to 1 μM S1P (internalization) or removed from S1P exposure (recovery) for 10, 30 or 60 min. Shaded histrogram shows background (bg) staining of negative control. (F) Flow cytometric analysis of S1PR1 and CXCR5 expression on 5 min in vivo CD45.2 antibody labeled (‘MZ’) and unlabeled (‘FO’) MZ B cells. Shaded histrogram shows background (bg) staining of negative control. Right panel shows ratio of S1PR1 or CXCR5 mean fluorescence intensities (MFI) of MZ B cells in the two compartments from 3 independent experiments. (G) Immunofluorescence microscopy of spleen sections taken from the indicated mice 18 h after treatment to form systemic PE-ICs (red), stained to detect marginal sinus-associated macrophages (CD169+, green) and FDCs (complement receptor 1 (CR1)+, blue). All data are representative of at least 3 mice of each type.