CD22 regulates adaptive and innate immune responses of B cells

Abstract

B cells sense microenvironments through the B cell receptor (BCR) and Toll-like receptors (TLRs). While signals from BCR and TLRs synergize to distinguish self from nonself, inappropriate regulation can result in development of autoimmune disease. Here we show that CD22, an inhibitory co-receptor of BCR, also negatively regulates TLR signaling in B cells. CD22-deficient (Cd22(-/-)) B cells exhibit hyperactivation in response to ligands of TLRs 3, 4 and 9. Evidence suggests that this results from impaired induction of suppressors of cytokine signaling 1 and 3, well-known suppressors of TLR signaling. Antibody-mediated sequestration of CD22 on wild-type (WT) B cells augments proliferation by TLR ligands. Conversely, expression of CD22 in a Cd22(-/-) B cell line blunts responses to TLR ligands. We also show that lipopolysaccharide-induced transcription by nuclear factor-κB is inhibited by ectopic expression of CD22 in a TLR4 reporter cell line. Taken together, these results suggest that negative regulation of TLR signaling is an intrinsic property of CD22. Since TLRs and BCR activate B cells through different signaling pathways, and are differentially localized in B cells, CD22 exhibits a broader regulation of receptors that mediate adaptive and innate immune responses of B cells than previously recognized.

Fig. 1

CD22 deficiency causes hyperproliferation of B cells in response to TLR ligands. a CFSE-labeled B cells were cultured for 3 days in the absence (black) or presence (gray) of 5.0 μg/ml poly(I:C), 1.5 μg/ml LPS, 15 n M CpG and 3.0 μg/ml anti-IgM. Cells were analyzed by flow cytometry. b Percentage of dividing cells from 2 days' culture subtracted with the value in the absence of stimulation are shown. Statistical analyses were performed by Student's t test. * p < 0.05; * * p < 0.01; * * * p < 0.001. c Living cell number in the wells of 3 days' culture is shown. Before the analysis, 1.0 × 10⁴ of beads was added to the culture well. Cells were then harvested and analyzed by flow cytometry. Living cell number was calculated by the formula: living cell number in the well = the number of living cells acquired × number of beads acquired / 1.0 × 10⁴. Data are representative of 3 independent experiments with similar results.

Fig. 2

Augmented CD86 and class II induction in Cd22^–/– B cells in response to TLR ligands. WT and Cd22^–/– B cells were stimulated for 2 days with 10 μg/ml poly(I:C), 0.15 μg/ml LPS, 7.5 n M CpG and 0.3 μg/ml anti-IgM. Then cells were harvested and stained with anti-CD86 (a) or anti-MHC class II I-A ^b (b) (gray) or isotype-matched control Ab (black). Stained cells were washed and analyzed by flow cytometry. Data are representative of 3 independent experiments with similar results.

Fig. 3

TLR expression in Cd22^–/– B cells is not significantly different from that of WT B cells. a Total RNA was extracted from isolated WT and Cd22^–/– B cells. Expression of TLR3 and 4 was examined by quantitative real-time PCR. Expression levels were normalized to β-actin. Statistical analyses were performed by Student's t test. b Isolated B cells were fixed, permeabilized and then stained with anti-TLR9 Ab followed by R-phycoerythrin-labeled secondary Ab. Stained cells were washed and analyzed by flow cytometry. Geometric mean fluorescence intensity (MFI) of Cd22^–/– B cells relative to WT is shown. Statistical analyses were performed by Student's t test. Data are representative or means of 3 independent experiments with similar results.

Fig. 4

CD22 negatively regulates TLR signaling in B cells. a Expression of CD22 in J2-44 Cd22^–/– B cells suppresses induction of MHC II by TLR ligands. J2-44 cells were retrovirally transduced with mouse CD22 and stimulated with 3.0 μg/ml LPS, 15 n M CpG and 3.0 μg/ml anti-IgM. After 2 days, MHC class II expression was examined by flow cytometry as in figure 2. b Sequestration of CD22 by immobilized anti-CD22 Ab enhances B cell proliferation by TLR ligands. CFSE-labeled WT B cells were stimulated with soluble LPS, CpG and anti-IgM in the presence of immobilized anti-CD22 mAb or isotype-matched control Ab. After 2 days, cells were analyzed by flow cytometry. c Expression of CD22 inhibits NF-κB activation in a TLR4 reporter cell line. mTLR4/ HEK293 cells were transfected with NF-κB-inducible AP and mCD22 or empty vector and stimulated with 10 μg/mL of LPS. The culture supernatant was incubated with the AP substrate at 37°C. Then the enzymatic activity was measured by the absorbance of 405 nm. Background absorbance from culture supernatant only was subtracted. Statistical analyses were performed by Student's t test. Data are representative of 2 or 3 independent experiments with similar results.

Fig. 5

Signaling analysis of Cd22^–/– B cells. a CD22 deficiency results in different spectrum of NF-κB activation. DNA binding activity of the NF-κB subunits, p50, p52, p65 and RelB in the nuclear extracts from WT and Cd22^–/– B cells was assayed by ELISA using oligonucleotides containing the NF-κB-binding site and specific antibodies to each subunit. b Impaired induction of SOCS1 and 3 in Cd22^–/– B cells upon CpG stimulation. Total RNA was extracted from 1.0 × 10⁷ of B cells from WT or Cd22^–/– B cells stimulated with 250 n M CpG for 3 h or unstimulated. Expression of SOCS1 and 3 was examined by quantitative real-time PCR. Expression levels were normalized to β-actin. Statistical analyses were performed by Student's t test. Data are representative of 2 independent experiments with similar results.