Immune regulation by the ST6Gal sialyltransferase

Abstract

The ST6Gal sialyltransferase controls production of the Siaalpha2-6Galbeta1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.

Figure 1

ST6Gal gene mutagenesis and function. (A) A partial ST6Gal genomic structure (ST6Gal^wt) was cloned and used with pflox in constructing a targeting vector (thick line) shown recombined at the ST6Gal locus (ST6Gal^F[tkneo] locus in ES cell clone 4.8). Following Cre recombinase expression and gancyclovir selection, ES cell subclones B3 and B9, bearing the ST6Gal^F allele or the ST6Gal^Δ allele, respectively, were isolated and used in generating chimeric mice. (B) Genomic Southern blots of ST6Gal alleleic structure in wt ES cells, targeted ES cell clone 4.8 (ST6Gal^F[tkneo]), 4.8 ES cell subclone B3 (ST6Gal^F), and 4.8 ES cell subclone B9 (ST6Gal^Δ) using either a genomic probe outside the targeting vector (Left) or a loxP probe (Center). (Right) A genomic Southern blot analysis of offspring derived from matings of mice heterozygous for the ST6Gal^Δ allele. Genotypes include the presence of mice homozygous for the exon 2 deletion (ST6Gal^Δ allele). (C) Using SNA and CD22-Ig lectins in FACS analyses, splenic CD3⁺ and B220⁺ lymphocytes normally express high levels of Sia6LacNAc in comparison with Gr-1⁺ myeloid and Ter-119⁺ erythroid cells. Lymphocytes from mice homozygous for the B9-derived ST6Gal^Δ allele were deficient in Sia6LacNAc (n = 7). α2-3-linked sialic acids were detected using the MAL II lectin and were found to be expressed at low levels normally and unaltered among cells from mice homozygous for the ST6Gal^Δ allele (n = 7). Mice homozygous for the B3-derived ST6Gal^F allele displayed wt profiles in these lectin-based analyses (data not shown). Similar results were obtained with mesenteric lymph node-derived lymphocytes (data not shown). Fluorescence signal intensity using an anti-human IgG–FITC conjugate is shown (2° and dotted line, Lower Left).

Figure 2

B lymphocyte characterization and serum immunogloblin analyses. (A Left) Bone marrow lymphocytes were analyzed for cell surface expression of IgM, CD24 (HSA), CD43 (S7), and B220 by flow cytometry. No deviations were observed in ST6Gal-deficient mice among the percentages (denoted in parentheses) of pro-B cells (B220^lo CD43(S7)⁺, gate a; ref. 32), pre-B cells (B220^lo HSA^hi, gate b; ref. 33), immature B cells (B220^lo IgM^int, gates b and c; ref. 33), transitional B cells (B220^lo-hi IgM^hi, gate d; ref. 33), and mature B cells (B220^hi IgM^int, gate e, and B220 ^hi HSA^lo, gate f; ref. 33). Percentages shown are the mean of four different analyses with calculated Student’s t test, P > 4 for all genotypic comparisons indicating no significant variations. (A Right) ST6Gal-deficient splenic B cells exhibited reductions in cell surface CD22 and IgM, but not in HSA. Reductions in cell surface IgM levels were found to be at 65 ± 20% of controls (Student’s t test; P < 0.001) and CD22 levels at 38 ± 9% of controls (P < 0.001) as determined by comparisons of peak fluorescence (n = 8). (B) Sia6LacNAc-deficient splenic B cells expressed normal levels of activation markers (CD44, B7.2, and I-A^b) and CD40. Dotted lines represents fluorescence of cells stained using an isotype control antibody (n = 8). (C) Serum Ig levels were measured in 4–6-month-old unimmunized mice of indicated genotypes. The median Ig levels are depicted as horizontal bars. Results revealed a 63% reduction (P < 0.001) in circulating IgM and no statistically significant reduction in IgA or IgG. Genotypes of 4–6-month-old mice are provided on the x-axis. Points represent measurements from individual animals.

Figure 3

Attenuated B cell activation responses in mice deficient in ST6Gal function. (A) B lymphocytes were isolated and stimulated by LPS or by crosslinking IgM or CD40 in the absence and presence of IL-4. Reduced proliferation of Sia6LacNAc-deficient B cells was observed in all experiments in the absence of IL-4. The addition of IL-4 as indicated rescued the defective proliferation response to IgM and CD40 ligation. Proliferation is shown as triplicate measurements of [³H]thymidine incorporation indicating the mean and SD. Results shown are representative of three different experiments (P < 0.005 for IgM crosslinking, P < 0.025 for CD40 crosslinking, and P < 0.001 for LPS stimulation). (B) B lymphocytes were analyzed for Ca²⁺ mobilization in response to IgM crosslinking (arrow). Sia6LacNAc-deficient B cells exhibited a reduction in amount of cytosolic Ca²⁺ mobilization (63 ± 4% of control value, P < 0.01, n = 4) (C) Phosphotyrosine accumulation following IgM crosslinking is altered in Sia6LacNAc-deficient B lymphocytes. Splenic B lymphocytes were isolated and stimulated with anti-IgM antibody. At indicated times, cells were lysed and extracts were subsequently analyzed by SDS/PAGE and immunoblotting with anti-phosphotyrosine antibody (n = 6). Positions of proteins exhibiting continuously altered levels of phosphotyrosine are denoted by asterisks.

Figure 4

Deficient antibody production following immunization of Sia6LacNAc-deficient mice. (A) T-independent antigen immunization (10 μg of DNP–Ficoll) was followed by analysis of anti-DNP antibody levels in sera at indicated times. (B) T-dependent antigen DNP–KLH immunization (10 μg and 100 μg) was accomplished. Anti-DNP antibody levels were measured before and subsequent to a second boost immunization (arrow). Results obtained with 10 μg of DNP–KLH antigen were essentially identical to those depicted with 100 μg (data not shown). Multiple dilutions of sera were assayed to find the linear range of response by OD₄₀₅ measurements (not shown). Sera dilution factors for results depicted are as follows: IgM (1/200), IgG₁ against T-independent antigen (1/200), IgG₃ against T-independent antigen (1/800), IgG₁ against T-dependent antigen (1/1,000), and IgG₃ against T-dependent antigen (1/200). Data are presented as the mean ± SEM from three mice of the indicated genotypes. Use of 0.1 μg of DNP–Ficoll in immunizations resulted in the absence of anti-DNP antibody production specifically in ST6Gal deficient mice, whereas low levels of anti-DNP antibodies were observed in wt mice (data not shown).