The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity

Abstract

The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity.

Figure 1.

Generation of Cxcr4 ^f/f Cd19 ^{+ /Cre}mice. (A) Schematic representation of the wild-type Cxcr4 locus, the targeting vector, the targeted allele, the Cxcr4-floxed, and the deleted locus. B, BamHI; C, ClaI; P, PvuII; S, SalI. (B) Southern blot analysis was performed with DNA isolated from wild-type (WT) and targeted (+/T) ES cells, tails of heterozygous mice caring targeted (+/T) or Cxcr4-floxed (+/F) allele, and purified splenic T and B lymphocytes of Cxcr4 ^f/f Cd19 ^{+ /Cre} mice. DNA was digested with BamHI and hybridized with the probe shown in A. Deletion of the Cxcr4 gene did not occur in T cells. About 95% of Cxcr4 alleles were deleted in B cells. (C) Transwell assay was performed to analyze the responsiveness of B cells to CXCL12. Splenic B cells were purified by MACS from Cxcr4 ^f/f Cd19^+/+(open bars) and Cxcr4 ^f/f Cd19 ^{+ /Cre}mice (gray bars). An optimal concentration (500 ng/ml) of CXCL12 was used. Data from two independent experiments are shown.

Figure 2.

Generation of B cell precursors in Cxcr4 ^f/f Cd19 ^{+ /Cre}mice. (A) Expression of CXCR4 on B cells at different developmental stages was analyzed by flow cytometry. Cells isolated from the bone marrow (BM) or spleen (Spl) of mutant and control mice were stained with antibodies against B220, IgM, and CXCR4. B cell precursors (B220^lo) and mature B cells (B220^hi) were gated, and the expression level of CXCR4 on gated B cells is displayed in the histograms. Solid lines represent wild-type B cells and dashed lines indicate CXCR4-deficient B cells. (B) Flow cytometry analysis of CD19⁺ gated populations in the bone marrow (BM), peripheral blood (PBL), and spleen (Spl) of both mutants (n = 7) and wild-type controls (n = 7). B cells at different developmental stages can be defined by the expression of B220 and IgM. B cell precursors including pro– and pre–B cells are B220^lo IgM⁻, immature B cells are B220^lo IgM^hi, and mature B cells are B220^hi IgM^lo. Numbers shown along with standard deviations denote the percentages of cells in the indicated gates.

Figure 3.

Localization of B cell precursors in Cxcr4 ^f/f Cd19 ^{+ /Cre}mice. (A) Immunofluorescent staining of splenic sections with FITC anti-IgM, FITC anti-IgD, and Alexa 568–anti-B220 antibodies (left, a magnification of 10; middle, a magnification of 40). On the right, flow cytometry shows that B cells in the periphery in normal animals express B220 and IgM or IgD, and that B cell precursors in mutants can be detected as they express only B220, but not IgM or IgD. (B) CXCR5 expression in the bone marrow and splenic B cell subsets. Bone marrow cells and splenocytes were isolated from Cxcr4 ^f/f Cd19 ^{+ /Cre}and Cxcr4 ^f/f Cd19 ^+/+ mice and stained with anti-CXCR5, anti-B220, and anti-IgM. B cell subsets of mature B220^hi, immature B220^lo IgM⁺ B cells, and B cell precursors B220^lo IgM⁻ were gated. Histograms show the CXCR5 expression level of these subsets. Solid lines represent CXCR5 level on wild-type B cells and dashed lines denote that on the CXCR4-deficient B cells. The CXCR5 level on wild-type splenic B220^hi cells, shown as the black solid line in both histograms, stands for a positive indicator. Background staining with nonspecific rat IgG on B220⁺ cells is shown as shaded histograms. (C) Splenocytes of Cxcr4 ^f/f Cd19 ^{+ /Cre}mice were analyzed for their CXCL13 responsiveness by a transwell assay with an optimal concentration of 1 μg/ml CXCL13. Input and migrated cells were stained with PE anti-CD19, FITC anti-IgM, and APC anti-B220. The composition of input and migrated cell populations was determined by flow cytometry. The percentages of migration of splenic B cells at different developmental stages are shown. Data are from three experiments performed in duplicates.

Figure 4.

Altered B cell compartments in the peripheral lymphoid organs of Cxcr4 ^f/f Cd19 ^{+ /Cre} mice. (A) Splenocytes were stained with antibodies against B220, CD21, and CD23. The expression of CD21 and CD23 in the B220⁺ gated population is shown. Immature B cells (CD21⁻ CD23⁻), follicular B cells (CD21^lo CD23^hi), and MZ B cells (CD21^hi CD23^lo) are shown in polygons with the mean percentages of each gated population (n = 5). (B) Cell death of splenic B cell subsets was examined by FACS analysis. Bone marrow cells (BM) and splenocytes (Spl) from wild-type controls and Cxcr4 ^f/f Cd19 ^{+ /Cre}mice were stained with anti-B220, anti-IgM, and annexin V. Apoptotic (Annexin V⁺) B cells are boxed and shown with mean percentages (n = 4). (C) Representative sections of small intestine show the structure of Peyer's patches in control (n = 3) and mutant mice (n = 5). B cells were stained by anti-B220 and T cells were labeled with anti-CD3. Arrows indicate enlarged T cell area or ectopic B cell follicles in the mutant mice (a magnification of 10). (D) Peritoneal cells were isolated from 3-wk-old or 6-mo-old mice. Cells within the lymphocyte gate are displayed by staining with anti-B220, anti-IgM, anti-CD5, and anti-CD11b. B2 cells (B220^hi CD5⁻), B1 cells (B220^hi CD5⁺), and T cells (B220⁻ CD5^hi) are shown in marked gates with percentages. (E) Absolute numbers of B2, B1a, and B1b cells in the peritoneal cavity of wild-type (closed bars, n = 5) and mutant mice (open bars, n = 7).

Figure 5.

Basal level of serum Ig and TI responses in CXCR4-conditional knockout mice. (A) Serum concentrations of Ig isotypes in 8-wk-old CXCR4-conditional mutant (○) and control (•) mice. (B) Serum levels of NP-specific Ig. 10-wk-old mice received intraperitoneal injection of NP-Ficoll. Sera from CXCR4-conditional mutant (○) and control (•) mice were collected 8 d after immunization. The relative units (RU) were determined by taking the value of the serum from a naive control littermate as 1 unit. Each symbol represents the value obtained from one mouse. (C) Numbers of NP-binding IgM-secreting cells in the spleen (Spl), bone marrow (BM), and mesenteric lymph nodes (MLN) of control (solid bars) and CXCR4 conditional knockout mice (gray bars) were determined by ELISPOT with cells isolated from animals 9 d after NP-Ficoll immunization.

Figure 6.

CXCR4-deficient B cells are able to elicit normal TD responses. (A) Serum levels of NP-specific Ig isotypes of CXCR4 conditional mutant (○) and control (•) mice at the indicated time points after NP-KLH immunization was measured by ELISA. (B) Relative affinities of IgG1 of CXCR4 conditional mutant (○) and control (•) mice were determined at the indicated time point after antigen challenge. Each symbol represents the value obtained from one mouse. (C) Numbers of NP-specific ASCs in the spleen (Spl) and bone marrow (BM) isolated from control (solid bars) and CXCR4 conditional mutant mice (gray bars) 9 d after first immunization. (D) Frequencies of NP-specific ASCs in the spleen (Spl) and bone marrow (BM) from control (•) and CXCR4 conditional mutant (○) mice 90 d after second immunization, and from 8-mo-old nonimmunized mice (▴). (E) Analysis of CXCR4 expression in plasma cells. Bone marrow cells and splenocytes were isolated from wild-type and mutant mice at day 90 after secondary immunization. Plasma cells are identified as B220⁻ CD138⁺ blast (B220/forward scatter, B220/FSC) with low surface lineage markers (CD3, CD11b, and CD61/FSC). Quadrants that separate CXCR4⁺ from CXCR4⁻ cells are set by the position of CXCR4-deficient B cells in the dot plots. The percentages of cells in the given quadrants are indicated. The level of CXCR4 expression on CD138⁺ cells from the spleen and bone marrow is shown in the histograms along with the isotype control (green shaded histograms).