Sialic acids on T cells are crucial for their maintenance and survival.

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.

IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

The role of Siglec-G on B cells in autoimmune disease and leukemia.

BACKGROUND: B-cell activation is triggered by the B-cell receptor, but is also controlled by inhibitory receptors, which limit the BCR signaling. CD22 (Siglec-2) and Siglec-G are such inhibitory receptors expressed on B cells. CD22- or Siglec-G deficient mice show enhanced B cell activation. OBJECTIVES: It was the objective of our study to investigate the role of these inhibitory receptors in autoimmune disease and leukemia. RESULTS: Ageing Siglec-G deficient or CD22 x Siglec-G deficient mice develop an SLE-like autoimmune disease with autoantibodies and kidney nephritis. In a mouse model for chronic lymphocytic leukemia (CLL), Siglec-G deficient mice show an earlier and more severe disease. AUTHOR'S CONCLUSIONS: These results show that Siglec-G and CD22 are both involved in preventing autoimmune diseases and leukemia delevopment and could therefore be attractive new targets.

The inhibitory receptor Siglec-G controls the severity of chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eµ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

Epistatic effects of Siglec-G and DNase1 or DNase1l3 deficiencies in the development of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that displays considerable heterogeneity not only in its symptoms, but also in its environmental and genetic causes. Studies in SLE patients have revealed that many genetic variants contribute to disease development. However, often its etiology remains unknown. Existing efforts to determine this etiology have focused on SLE in mouse models revealing not only that mutations in specific genes lead to SLE development, but also that epistatic effects of several gene mutations significantly amplify disease manifestation. Genome-wide association studies for SLE have identified loci involved in the two biological processes of immune complex clearance and lymphocyte signaling. Deficiency in an inhibitory receptor expressed on B lymphocytes, Siglec-G, has been shown to trigger SLE development in aging mice, as have mutations in DNA degrading DNase1 and DNase1l3, that are involved in clearance of DNA-containing immune complexes. Here, we analyze the development of SLE-like symptoms in mice deficient in either Siglecg and DNase1 or Siglecg and DNase1l3 to evaluate potential epistatic effects of these genes. We found that germinal center B cells and follicular helper T cells were increased in aging Siglecg -/- x Dnase1 -/- mice. In contrast, anti-dsDNA antibodies and anti-nuclear antibodies were strongly increased in aging Siglecg-/- x Dnase1l3-/- mice, when compared to single-deficient mice. Histological analysis of the kidneys revealed glomerulonephritis in both Siglecg -/- x Dnase1 -/- and Siglecg-/- x Dnase1l3-/- mice, but with a stronger glomerular damage in the latter. Collectively, these findings underscore the impact of the epistatic effects of Siglecg with DNase1 and Dnase1l3 on disease manifestation and highlight the potential combinatory effects of other gene mutations in SLE.

Targeting Human CD22/Siglec-2 with Dimeric Sialosides as Novel Oligosaccharide Mimetics.

Significant interest in the development of high-affinity ligands for Siglecs exists due to the various therapeutically relevant functions of these proteins. Here, we report a new strategy to develop and design Siglec ligands as disialyl-oligosaccharide mimetics exemplified on Siglec-2 (CD22). We report insights into development of dimeric ligands with high affinity and avidity to cell surface-expressed CD22, assay development, tool compounds, structure activity relationships, and biological data on calcium flux regulation in B-cells. The binding modes of selected ligands have been modeled based on state-of-the-art molecular dynamics simulations on the microsecond timescale, providing detailed views on ligand binding and opening a new perspective on drug design efforts for Siglecs. High-avidity dimeric ligands containing a linker opening the way towards bispecifics are presented as well.

Sialic acids on B cells are crucial for their survival and provide protection against apoptosis.

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

Coordinated changes in glycosylation regulate the germinal center through CD22.

Germinal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC.

The inhibitory coreceptor CD22 restores B cell signaling by developmentally regulating Cd45-/- immunodeficient B cells.

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases. Cd45-/- mice show altered B cell development and a phenotype likely due to reduced steady-state signaling; however, Cd45-/- B cells show relatively normal BCR ligation-induced signaling. In our investigation of how BCR signaling was restored in Cd45-/- cells, we found that the coreceptor CD22 switched from an inhibitory to a stimulatory function in these cells. We disrupted the ability of CD22 to interact with its ligands in Cd45-/- B cells by generating Cd45-/-St6galI-/- mice, which cannot synthesize the glycan ligand of CD22, or by treating Cd45-/- B cells in vitro with the sialoside GSC718, which inhibits ligand binding to CD22. BCR ligation-induced signaling was reduced by ST6GalI deficiency, but not by GSC718 treatment, suggesting that CD22 restored BCR ligation-induced signaling in Cd45-/- mature B cells by altering cellular phenotypes during development. CD22 was required for the increase in the surface amount of IgM-BCR on Cd45-/- B cells, which augmented signaling. Because B cell survival depends on steady-state BCR signaling, IgM-BCR abundance was likely increased by the selective survival of IgM-BCRhi Cd45-/- B cells because of CD22-mediated signaling under conditions of substantially reduced steady-state signaling. Because the amount of surface IgM-BCR is increased on B cells from patients with other BCR signaling deficiencies, including X-linked agammaglobulinemia, our findings suggest that CD22 may contribute to the partial restoration of B cell function in these patients.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Human CD22-Transgenic, Primary Murine Lymphoma Challenges Immunotherapies in Organ-Specific Tumor Microenvironments.

Targeted immunotherapies have greatly changed treatment of patients with B cell malignancies. To further enhance immunotherapies, research increasingly focuses on the tumor microenvironment (TME), which differs considerably by organ site. However, immunocompetent mouse models of disease to study immunotherapies targeting human molecules within organ-specific TME are surprisingly rare. We developed a myc-driven, primary murine lymphoma model expressing a human-mouse chimeric CD22 (h/mCD22). Stable engraftment of three distinct h/mCD22+ lymphoma was established after subcutaneous and systemic injection. However, only systemic lymphoma showed immune infiltration that reflected human disease. In this model, myeloid cells supported lymphoma growth and showed a phenotype of myeloid-derived suppressor cells. The human CD22-targeted immunotoxin Moxetumomab was highly active against h/mCD22+ lymphoma and similarly reduced infiltration of bone marrow and spleen of all three models up to 90-fold while efficacy against lymphoma in lymph nodes varied substantially, highlighting relevance of organ-specific TME. As in human aggressive lymphoma, anti-PD-L1 as monotherapy was not efficient. However, anti-PD-L1 enhanced efficacy of Moxetumomab suggesting potential for future clinical application. The novel model system of h/mCD22+ lymphoma provides a unique platform to test targeted immunotherapies and may be amenable for other human B cell targets such as CD19 and CD20.

Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections.

Siglec-H is a DAP12-associated receptor on plasmacytoid dendritic cells (pDCs) and microglia. Siglec-H inhibits TLR9-induced IFN-α production by pDCs. Previously, it was found that Siglec-H-deficient mice develop a lupus-like severe autoimmune disease after persistent murine cytomegalovirus (mCMV) infection. This was due to enhanced type I interferon responses, including IFN-α. Here we examined, whether other virus infections can also induce autoimmunity in Siglec-H-deficient mice. To this end we infected Siglec-H-deficient mice with influenza virus or with Lymphocytic Choriomeningitis virus (LCMV) clone 13. With both types of viruses we did not observe induction of autoimmune disease in Siglec-H-deficient mice. This can be explained by the fact that both types of viruses are ssRNA viruses that engage TLR7, rather than TLR9. Also, Influenza causes an acute infection that is rapidly cleared and the chronicity of LCMV clone 13 may not be sufficient and may rather suppress pDC functions. Siglec-H inhibited exclusively TLR-9 driven type I interferon responses, but did not affect type II or type III interferon production by pDCs. Siglec-H-deficient pDCs showed impaired Hck expression, which is a Src-family kinase expressed in myeloid cells, and downmodulation of the chemokine receptor CCR9, that has important functions for pDCs. Accordingly, Siglec-H-deficient pDCs showed impaired migration towards the CCR9 ligand CCL25. Furthermore, autoimmune-related genes such as Klk1 and DNase1l3 are downregulated in Siglec-H-deficient pDCs as well. From these findings we conclude that Siglec-H controls TLR-9-dependent, but not TLR-7 dependent inflammatory responses after virus infections and regulates chemokine responsiveness of pDCs.

CD22 Controls Germinal Center B Cell Receptor Signaling, Which Influences Plasma Cell and Memory B Cell Output.

Germinal center reactions are established during a thymus-dependent immune response. Germinal center (GC) B cells are rapidly proliferating and undergo somatic hypermutation in Ab genes. This results in the production of high-affinity Abs and establishment of long-lived memory cells. GC B cells show lower BCR-induced signaling when compared with naive B cells, but the functional relevance is not clear. CD22 is a member of the Siglec family and functions as an inhibitory coreceptor on B cells. Interestingly, GC B cells downregulate sialic acid forms that serve as high-affinity ligands for CD22, indicating a role for CD22 ligand binding during GC responses. We studied the role of CD22 in the GC with mixed bone marrow chimeric mice and found a disadvantage of CD22-/- GC B cells during the GC reaction. Mechanistic investigations ruled out defects in dark zone/light zone distribution and affinity maturation. Rather, an increased rate of apoptosis in CD22-/- GC B cells was responsible for the disadvantage, also leading to a lower GC output in plasma cells and memory B cells. CD22-/- GC B cells showed a clearly increased calcium response upon BCR stimulation, which was almost absent in wild-type GC B cells. We conclude that the differential expression of the low-affinity cis CD22 ligands in the GC normally results in a strong attenuation of BCR signaling in GC B cells, probably due to higher CD22-BCR interactions. Therefore, attenuation of BCR signaling by CD22 is involved in GC output and B cell fate.

Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages.

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.

A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.

The B-cell inhibitory receptor CD22 is a major factor in host resistance to Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major human pathogen, causing pneumonia and sepsis. Genetic components strongly influence host responses to pneumococcal infections, but the responsible loci are unknown. We have previously identified a locus on mouse chromosome 7 from a susceptible mouse strain, CBA/Ca, to be crucial for pneumococcal infection. Here we identify a responsible gene, Cd22, which carries a point mutation in the CBA/Ca strain, leading to loss of CD22 on B cells. CBA/Ca mice and gene-targeted CD22-deficient mice on a C57BL/6 background are both similarly susceptible to pneumococcal infection, as shown by bacterial replication in the lungs, high bacteremia and early death. After bacterial infections, CD22-deficient mice had strongly reduced B cell populations in the lung, including GM-CSF producing, IgM secreting innate response activator B cells, which are crucial for protection. This study provides striking evidence that CD22 is crucial for protection during invasive pneumococcal disease.

Cullin 3 Is Crucial for Pro-B Cell Proliferation, Interacts with CD22, and Controls CD22 Internalization on B Cells.

B lymphocytes are important players of the adaptive immune system. However, not just activation of B cells but also regulation of B cell signaling is important to prevent hyperactivity and dysregulation of the immune response. Different mechanisms and proteins contribute to this balance. One of these is CD22, a member of the Siglec family. It is an inhibitory coreceptor of the BCR and inhibits B cell activation. Upon BCR stimulation, CD22-dependent inhibition of BCR signaling results in a decreased calcium mobilization. Although some CD22 binding partners have already been identified, the knowledge about the CD22 interactome is still incomplete. In this study, quantitative affinity purification-mass spectrometry enabled the delineation of the CD22 interactome in the B cell line DT40. These data will clarify molecular mechanisms and CD22 signaling events after BCR activation and revealed several new CD22-associated proteins. One new identified interaction partner is the E3 ubiquitin ligase cullin 3, which was revealed to regulate CD22 surface expression and clathrin-dependent CD22 internalization after BCR stimulation. Furthermore cullin 3 was identified to be important for B lymphocytes in general. B cell-specific cullin 3-deficient mice show reduced developing B cells in the bone marrow and a severe pro-B cell proliferation defect. Mature B cells in the periphery are also reduced and characterized by increased CD22 expression and additionally by preactivated and apoptotic phenotypes. The findings reveal novel functions of cullin 3 in B lymphocytes, namely regulating CD22 surface expression and internalization after B cell activation, as well as promoting proliferation of pro-B cells.

Critical redundant functions of the adapters Grb2 and Gads in platelet (hem)ITAM signaling in mice.

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets - Grb2 and Grb2-related adapter protein downstream of Shc (Gads) - are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development.