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. 2009 Apr;10(4):412-9.
doi: 10.1038/ni.1712. Epub 2009 Mar 8.

CD98hc Facilitates B Cell Proliferation and Adaptive Humoral Immunity

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Free PMC article

CD98hc Facilitates B Cell Proliferation and Adaptive Humoral Immunity

Joseph Cantor et al. Nat Immunol. .
Free PMC article

Abstract

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Figures

Figure 1
Figure 1
CD98hc deletion in Slc3a2f/fCD19-Cre+ mice. (a) CD98hc deletion in B cells. BM cells from adult Slc3a2f/fCD19-Cre+ and control mice were isolated and CD98hc expression (filled peaks) measured at different stages of B cell maturation compared to isotype control staining (lines); experiment was repeated once. (b) Specificity of CD98 deletion. Peripheral blood and spleen cells from Slc3a2f/fCD19-Cre+ and control mice were prepared by lysing erythrocytes. CD98hc expression was measured on T cells (CD3+), macrophages (CD11b+B220), and B cells (B220+) by flow cytometry. Data from one representative mouse of each genotype are shown (n=4 per group for each panel); experiment was repeated once.
Figure 2
Figure 2
Impaired antibody responses in Slc3a2f/fCD19-Cre+ mice. (a) T cell-independent antibody response. Adult (8–12 wk old) Slc3a2f/fCD19-Cre+ or control mice were immunized with 50 μg of a T cell-independent antigen, TNP-LPS, in PBS. Mice were bled before immunization (pre-immune) and at one wk after immunization to obtain serum. Concentrations of anti-TNP IgM or anti-TNP IgG3 were measured by direct ELISA. Error bars represent s.e.m. from 5 mice in each group. ** P < 0.025 (b, c) T cell-dependent antibody responses. Slc3a2f/fCD19-Cre+ or control mice were immunized with 100 μg of the T cell-dependent antigen TNP-KLH in Complete Freund’s Adjuvant (CFA) (b) or Incomplete Freund’s Adjuvant (IFA) (c). Anti-TNP IgM and anti-TNP IgG in serum were measured by direct ELISA. Error bars represent s.e.m. 5 mice for each group. (**P <0.025, *P = 0.057). Experiments in (a–c) were repeated once.
Figure 3
Figure 3
Normal B cell distribution and natural antibody concentrations in Slc3a2f/fCD19-Cre+ mice. (a) Enumeration of B cell subsets. Indicated B cell subsets in spleen, BM, and peritoneal cells from adult (8–12 wk old) Slc3a2f/fCD19-Cre+ and control mice were analyzed flow cytometry. Error bars show s.e.m. from 4 mice in each group; experiment was repeated with similar results (b) Analysis of secondary lymphoid architecture. Frozen spleen sections from Slc3a2f/fCD19-Cre+ and littermate control mice were stained with the antibodies specific for the indicated proteins to detect B220+ B cells, metalophillic macrophages that outline the marginal zone (MOMA-1+), T cells (CD5+), and germinal center B cells (PNA+). (c) Natural antibody concentrations. Naïve adult (8–12 wk old) Slc3a2f/fCD19-Cre+ and littermate control mice were bled and serum analyzed by sandwich ELISA for total IgM. Error bars show s.e.m. from 30 mice per group (P = 0.11).
Figure 4
Figure 4
Defective plasma cell formation in Slc3a2f/fCD19-Cre+ mice. (a,b) In vitro plasma cell formation. Resting splenic B cells (CD43CD98hc-deficient) were purified from Slc3a2f/fCD19-Cre+ or littermate control mice, cultured with LPS for 5 days, and stained for surface IgG3 (a) and CD138 (b) (Syndecan-1, a plasma cell marker). Cells were analyzed by flow cytometry. Dot plot is representative data from one mouse; bar graphs summarize IgG3 or CD138 staining on B220lo cells. Error bars show s.e.m. from 3 mice per group. **P < 0.025 Experiment was repeated with similar results. (c) In vitro antibody secretion. Supernatants from resting B cells stimulated for 4 days with LPS were assayed for total IgM and IgG by sandwich ELISA. Error bars show s.e.m. from 4 mice per group. *P <0.05 Experiment was repeated with similar results. (d) In vivo plasma cell formation. Slc3a2f/fCD19-Cre+ and control mice were immunized with TNP-KLH in CFA. One wk later, splenocytes were cultured for 2–3 h on TNP-coated PVDF membrane 96-well plates. After washing to remove cells, and incubation with anti-IgG HRP-conjugated secondary antibody, AEC substrate was used to develop red spots indicating TNP-specific IgG-secreting plasma cells. Error bars indicate s.e.m. from 4 mice for each group (*P = 0.05, **P = 0.08). Experiment was repeated once.
Figure 5
Figure 5
Proliferation of splenic B cells from Slc3a2f/fCD19-Cre+ mice. (a) In vitro proliferation analysis. Resting B cells (CD43) were purified from splenocytes of 8–12 wk-old Slc3a2f/fCD19-Cre+ and littermate control mice and labeled with CFSE. 400,000 CFSE-labeled B cells were cultured per well in a 48-well plate with the indicated stimuli (filled peaks) for 5 days, and proliferation was measured by the dilution of CFSE fluorescence by flow cytometry, as compared with unstimulated resting cells (lines). Each histogram is data from one mouse that represents data from 3 mice of each genotype in one experiment. This experiment was repeated twice with additional mice. (b) CD98hc expression on proliferating cells. CD98 expression was measured on CFSElo (dividing) vs. CFSEhi (non-dividing) cells by antibody staining and 2-color flow cytometry; experiment was repeated once.
Figure 6
Figure 6
Mechanism by which CD98hc enables B cell proliferation. (a) CD98-CD69 chimeric constructs encoded by retroviruses. (b) Rescue of B cell proliferation with CD98hc integrin signaling function. Six to eight wk after transplant of BM cells infected with indicated retroviruses, resting B cells (CD43 mCD98hc) were purified from splenocytes of recipient mice. Cells were labeled with DiD dye. 350,000 DiD-labeled B cells were cultured per well in a 48-well plate with LPS (filled peaks) and proliferation was measured by the dilution of DiD fluorescence by flow cytometry. Dot plots show mouse CD98 expression vs. DiD fluorescence by 2-color flow cytometry; fewer dots in the full-length CD69 or in the integrin-signaling-deficient CD98-CD69 chimera are indicative of the lack of proliferation in these samples. This experiment was repeated twice.
Figure 7
Figure 7
Integrin signaling defects in B cells lacking CD98hc. (a) Late Erk signaling. Resting B cells (CD43CD98) were purified from splenocytes of 8–12 wk-old Slc3a2f/fCD19-Cre+ (n=4) or littermate control mice (n=3) and were stimulated with anti-IgM (30 ug/ml) and IL-4 (15 ng/ml) for 17 h. Cells were then immediatedly washed with ice-cold PBS, lysed, and Erk1/2 phosphorylation was analyzed by immunoblotting. Bands are present, albiet faint, in the Slc3a2f/fCD19-Cre+ lanes. (b) p27 downregulation. Purified B cells from Slc3a2f/fCD19-Cre+ or littermate control mice were stimulated with anti-IgM and IL-4 for 0, 12, or 24 h. Cells were washed and lysed, and expression of p27 was analyzed by immunoblotting (Intervening bands have been omitted for clarity). Bar graph summarizes staining of p27 normalized to total Erk. Error bars represent s.e.m. from n=3 mice per group for (a) and (b). Experiment for (a–b) was repeated once. (c) Integrin-dependent cell spreading. Purified B cells from Slc3a2f/fCD19-Cre+ or littermate control mice were stimulated with anti-CD40 and IL-4 for 24 h, and were plated on anti-LFA-1 for 16 h. Digital pictures show representative fields, and bar graphs summarize area and perimeter of 30 traced cells for each group. Experiment was repeated once.
Figure 8
Figure 8
Selection for CD98hc+ B cells during activation. (a) CD98hc expression on responding B cells in vivo. Cells were isolated from halves of spleens from Slc3a2f/fCD19-Cre+ and control mice immunized with the T cell-dependent antigen TNP-KLH in CFA. CD98hc expression (filled histogram) was measured on indicated B cell subsets by flow cytometry. Isotype control, open histograms. Data from one representative mouse of each genotype are shown (n=3 per group). (b) Selection of CD98hc+ B cells in vivo. Remaining halves of spleens were frozen, sectioned, and stained for B220 and CD98hc; germinal centers were verified with PNA staining. Data from one mouse are shown and are representative of 3 mice per group; experiment for (a-b) was repeated once. (c) Selection of CD98hc+ B cells in vitro. Resting splenic B cells were purified from Slc3a2f/fCD19-Cre+ mice without depletion of CD98hc+ cells or from control mice. Cells were cultured with LPS for 4 days, stained for B220, CD98hc, and CD138 (Syndecan-1, a plasma cell marker), and analyzed by flow cytometry. Representative data (n=3 per group) from one mouse are shown; entire experiment was repeated twice.

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