Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

Abstract

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Figure 1

Generation of CD-81-deficient mice. (A) Structure of the CD81 gene (Upper) and of the targeting construct (Lower). Exons are represented by bold segments and are numbered. neo, neomycin resistance gene. (B) Southern blot analysis of DNA from ES cell clones. Genomic DNA was digested by XbaI and probed with the KpnI fragment shown in A. The wild-type (WT) allele is represented by the 5-kb band. (C) Southern blot analysis of DNA from tails of littermate mice of brother-sister mating of CD81 heterozygotes (+/−) mice. Analysis was performed as in B. (D) Flow cytometry analysis of surface expression of CD81 on thymocytes from WT and CD81-deficient (KO) mice. Cells were stained with biotinylated anti-CD81 hamster mAb 2F7 (——) or biotinylated hamster IgG as an isotype control (· · · ·) followed by streptavidin-PE.

Figure 2

Flow cytometry analysis of thymocytes (A) and spleen cells (B) from CD81-deficient mice. Single cell suspension from wild-type (WT) and CD81-deficient (KO) littermates were stained for CD4 [anti-CD4-fluorescein isothiocyanate (FITC)] vs. CD8 (anti-CD8-PE). Cells with forward and side light scatter properties of lymphocytes were analyzed by two-color flow cytometry.

Figure 3

Flow cytometry of B lymphocytes in CD81-deficient mice (KO) and wild-type (WT) littermates. (A) Bone marrow cells were stained for IgM (anti-IgM-FITC) vs. B220 (anti-B220-PE); and for B220 (anti-B220-FITC) vs. CD19 (anti-CD19-PE). Cells with forward and side light scatter properties of lymphocytes were analyzed. (B) Spleen cells were stained for CD19 (anti-CD19-FITC) vs. B220 (anti-B220-PE).

Figure 4

Flow cytometry analysis of peritoneal cells. Cells were analyzed with forward (FSC) and side (SSC) light scatter. The gated (R1) lymphocyte population was stained for IgM (anti-IgM-FITC) vs. B20 (anti-b220-PE) and for IgM (anti-IgM-FITC) vs. CD5 (anti-CD5-PE). B-1 (B220^intIgM⁺, B-2 (B220^hiIgM⁺), B-1a (B220^loIgM⁺), and T (CD5^hiIgM⁻) cells are boxed.

Figure 5

Response of CD8-deficient B cells to CD19 ligation. (A) [Ca²⁺]_i response to anti-CD19 mAb in B cells. Splenocytes from wild-type (WT) and CD81-deficient (KO) littermates were loaded with Fluo-3 and stained with anti-B220-PE. Analysis of [Ca²⁺]_i was performed on gated B220-positive cells. The time scale represents 512 s. Cell counts (counts) are represented on the vertical axis. Arrow indicated the addition of anti-CD19 mAb MB19-1 at a concentration of 40 μg/ml. EGTA (10 mM) was added at time ). (B) [Ca²⁺]_i response to anti-IgM in B cells. Arrow indicates the addition of F(ab′)₂ goat anti-mouse IgM 1 μg/ml). (C) Modulation of B cell proliferation in response to anti-IgM by anti-CD19 mAbs. purified splenic B cells (1 × 10⁵ cells per well) from WT (+/+) and CD81-deficient (−/−) littermates were stimulated with F(ab′)₂ fragments of goat anti-mouse IgM (1 and 5 μg/ml) in the presence or absence of anti-CD19 mAb MB19-1 at the indicated concentrations. Results are expressed as percent of the proliferation of cell cultures stimulated with anti-IgM in the absence of anti-CD19 mAb. Each of the experiments shown is representative of four independent experiments.

Figure 6

Antibody responses of wild-type mice (+/+) and CD81-deficient (−/−) mice. (A) Response to the TD antigen TNP-KLH. Mice were immunized i.v. with 100 μg of TNP-KLH conjugate 1:25) in PBS at day 0 and boosted at day 21. (B) response to type I TI antigen TNP-LPS. Mice were immunized i.p. with 10 μg TNP-LPS in PBS at day 0. (C) Response to the type II TI antigen TNP-Ficoll. Mice were immunized i.p. with 10 μg TNP-Ficoll in PBS at day 0. Sera were diluted 1/1,000 and levels of antigen-specific antibody responses of the indicated isotypes were analyzed by TNP-specific ELISA. Mean values ± 2 SD obtained for the four mice per group are shown. ∗, P < 0.005; ∗∗, P < 0.01.

Figure 7

Serum Ig levels in unimmunized wild-type (+/+) and CD81-deficient (−/−) mice. Mean values ± 2 SD obtained for six mice per group are shown. ∗, P < 0.005; ∗∗, P < 0.01.