B cell antigen receptor signal strength and peripheral B cell development are regulated by a 9-O-acetyl sialic acid esterase

Abstract

Generation of Siae ⌬ 2/ ⌬ 2 mice Exon 2 of the Siae gene is unique to Lse . An engineered inframe deletion of exon 2 in a murine Lse complementary DNA resulted in a protein that lacked esterase activity ( Fig. 1 A ). Genomic deletion of exon 2 was achieved as described in the Materials and methods ( Fig. 1 B ). After germline transmission, homozygous mutant mice were generated and were found to be viable. Truncated exon 2 -defi cient Siae mRNA could be detected in KO mice (Fig. S3, available at http://www .jem.org/cgi/content/full/jem.20081399/DC1). Cytosolic esterase mRNA continues to be transcribed in these mutant mice. We refer to these KO animals as Siae ⌬ 2/ ⌬ 2 mice.Enhanced B lymphocyte antigen receptor signaling in Siae ⌬ 2/ ⌬ 2 mice Because Siae has the potential to remove 9-O -acetyl residues from ␣ 2 -6-linked sialic acid containing Siglec ligands, we predicted that B cells from mice lacking this esterase might exhibit enhanced BCR signaling similar to that noted in CD22-null mice ( 13 -16 ). Mice were fi rst bred into the C57BL/6 background for 10 generations. B cells from WT and Siae ⌬ 2/ ⌬ 2 mice were gated on, and the accumulation of cytoplasmic calcium after ligation of the BCR was analyzed using fl ow cytometry. As seen in Fig. 2 , BCR cross-linking resulted in an accelerated and enhanced calcium fl ux. A similar result was seen when purifi ed splenic B cells from mutant and WT mice were analyzed (Fig. S4, available at http://www.jem.org/cgi/content/ full/jem.20081399/DC1). These data suggested that in the absence of functional Siae, BCR signal strength is markedly enhanced and that this alteration in signal strength is an intrinsic property of mutant B lymphocytes.Defective CD22 signaling and hyperacetylation of ␣ 2 -6-linked sialic acid moieties on Siae mutant B cells The defect in Siae could result in the increased acetylation of ␣ 2 -6-linked sialic acid on N -glycans in B cells and, thus, attenuate the ability of glycoproteins on B cells to ligate CD22 and generate inhibitory signals. We sought to examine if there was a defect in CD22 signaling in Siae ⌬ 2/ ⌬ 2 mice. After BCR cross-linking, CD22 was isolated by immunoprecipitation, and immunoprecipitates were examined for CD22 tyrosine phosphorylation and for associated SHP-1 using Western blot assays. As seen in Fig. 3 (left, experiment 1; and right, experiment 2), tyrosine phosphorylation of CD22 after BCR signaling was reduced in Siae ⌬ 2/ ⌬ 2 mice in spite of similar levels of surface CD22 expression in WT and mutant mice ( It remained to be demonstrated whether a defect in Siae would result in enhanced 9-O-acetylation of ␣ 2 -6-linked sialic acid in Siae ⌬ 2/ ⌬ 2 mice. 9-O -acetylation of sialic acid has not

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

“…Mononuclear cells were isolated from spleen cell suspensions by centrifugation through a density gradient on Histopaque-1077 (Sigma-Aldrich), except for B-cell populations. Splenocytes and bone marrow cells for B-cell phenotyping experiments were isolated and processed as previously described (11). Other antibodies used for immunophenotyping included directly conjugated anti-human CD3-APC/Cy7, CD11c-PE, CD14-Pacific Blue, CD27-APC-Cy7, CD34-APC or CD34-PE, CD45-APC, CD45R-FITC (which cross-reacts with mouse and human), CD45RA-PE-Cy5, CD69-PE, CD123-APC, C-C chemokine receptor 7-PE (CCR7-PE), CXCR4-APC, CCR5-PE-Cy7, HLA-DR-FITC, HLA-DR-peridinin chlorophyll protein-Cy5.5 (all from BD Pharmingen), CD16-PE-Cy7 (Biolegend), and programmed death 1-PE (PD-1-PE) (clone EH12; G. Freeman) (14).…”

Impaired B Cell Development in the Absence of Krüppel-like Factor 3

“…Loss of regulators of BCR signaling such as Lyn and CD22 have been reported to lead to an increase in B1 B cells and a reduction of MZ B cells (36,(38)(39)(40)(41)(42)(43)(44). Similarly, positive regulators of BCR signaling such as Btk appear to direct B cell differentiation away from the MZ lineage, while promoting the accumulation of B1 B cells (36,45).…”

Siglec-G Deficiency Leads to More Severe Collagen-Induced Arthritis and Earlier Onset of Lupus-like Symptoms in MRL/lpr Mice

“…This finding indicates functional redundancy of these two Siglecs in the control of B cell tolerance. In addition, mutation in the sialic acid acetyl esterase gene, which codes for an enzyme that modifies CD22 and Siglec-G ligands, leads to autoimmunity in mice and is linked to several autoimmune diseases in humans (11,12). Moreover, the loss of CD22 alone can contribute to autoimmunity, as was demonstrated by crossing Cd22 2/2 mice to the Y chromosome-linked autoimmune accelerator background (13).…”