Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

Material

Sialic acids are abundant in higher domains of life and lectins recognizing sialosaccharides are heavily involved in the regulation of the human immune system. Modified sialosides are useful tools to explore the functions of those lectins, especially members of the Siglec (sialic acid binding immunoglobulin like lectin) family. Here we report design, synthesis, and affinity evaluation of novel sialoside classes with combined modification at positions 2, 4, and 9 or 2, 3, 4, and 9 of the sialic acid scaffold as human CD22 (human Siglec-2) ligands. They display up to 7.5 × 10(5)-fold increased affinity over αMe Neu5Ac (the minimal Siglec ligand). CD22 is a negative regulating coreceptor of the B-cell receptor (BCR). In vitro experiments with a human B-lymphocyte cell line showed functional blocking of CD22 upon B-cell receptor (BCR) stimulation in the presence of nanomolar concentrations of the novel ligands. The observed increased Ca(2+) response corresponds to enhanced cell activation, providing an opportunity to therapeutically modulate B-lymphocyte responses, e.g., in immune deficiencies and infections.

Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar K values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

CD22 is an inhibitory co-receptor of the B-cell receptor (BCRThe ITIMs are phosphorylated by the Src-family kinase Lyn upon BCR activation, leading to the activation and recruitment of SH2-domain containing protein tyrosin phosphatase 1 (SHP-1) [3,4]. SHP-1 is a tyrosine phosphatase that dephosphorylates signaling molecules, which are critical for initiation and maintenance of the BCR-induced Ca 2+ pathway in B cells [1]. The function of CD22 is regulated by interaction of the first extracellular immunoglobulin (Ig) domain of CD22 to glycan ligands. It recognizes specifically glycans that carry sialic acids attached in α2,6-linkage to an underlying β1, 4-linked galactose residue. The affinity for its native ligand with the sequence Siaα2,6Gal is low * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 2792-2802 Clinical immunology 2793(KD = 0.2 mM) [5] and due to the high concentration of sialic acids on the B-cell surface (25-50 mM), the ligand binding sites of CD22 are masked in cis by self ligands [6,7]. In a structure-aided design, which indicated a hydrophobic pocket within the CD22 ligand-binding site, synthetic glycan ligands were designed [8,9]. To test the affinity of these synthetic glycans to CD22 on the cellular surface, the masking by cis-ligands had to be destroyed by sialidase-treatment. An optimal synthetic ligand targeting CD22 should have a rather high affinity and should be able to overcome the receptor masking without sialidase pretreatment. We had previously determined that the C-9 position of neuraminic acid can be modified with a biphenyl carbonyl-amino (BPC) substituent that increases the affinity for human CD22 about 200 times [8]. Treatment with this synthetic glycan ligands blocks CD22-mediated signal inhibition in human B-cell lines, thereby augmenting BCR signaling [8].CD22 shows a B-cell specific expression from the pre-B to mature B-cell stage but is downregulated on plasma cells. Therefore, it is an attractive target for B-cell targeted immunotherapy and is already used in clinical studies to treat B-cell lymphomas and systemic autoimmune diseases. Several therapeutics based on anti-CD22 antibodies have been developed. Epratuzumab is in phase I/II clinical trials for non-Hodgkin lymphoma (NHL) treatment and systemic lupus erythematosus (SLE) [10,11]. Furthermore, conjugates of single chain anti-CD22 antibodies with RNAses [12] or toxins like Pseudomonas exotoxin A [13] have been tested. CD22 is able to shuttle bound effector molecules inside the cell, since it is endocytosed quickly upon binding of soluble ligands [14,15].The approach we and others use is to replace the anti-CD22 antibodies with high-affinity synthetic glycan ligands as targeting moiety. Until now, this has only been done by attaching synthetic CD22 ligands to liposomes filled with doxorubicin [16] or by attaching high-molecular-weight CD22 ligands to saporin [17]. Here, we show that we can target CD22 by very small, dival...

Background: Dual specificity phosphatases (DUSPs) have emerged as important regulators of MAPK signaling and responses, including immune responses. Results: Dusp16-deficient gene trap mice were generated and show a phenotype of perinatal lethality and altered cytokine responses to TLR stimulation. Conclusion: Dusp16 has non-redundant functions in the innate immune system. Significance: This is the first description of a mouse model of Dusp16 deficiency.

CD22 is a member of the Siglec family. Considerable attention has been drawn to the design and synthesis of new Siglec ligands to explore target biology and innovative therapies. In particular, CD22‐ligand‐targeted nanoparticles with therapeutic functions have proved successful in preclinical settings for blood cancers, autoimmune diseases, and tolerance induction. Here we report the design, synthesis and affinity evaluation of a new class of Siglec ligands: namely sialic acid derivatives with a triazole moiety replacing the natural glycoside oxygen atom. In addition, we describe important and surprising differences in binding to CD22 expressed at the cell surface for compounds with distinct valences. The new class of compounds might serve as a template for the design of ligands for other members of the Siglec family and next‐generation CD22‐ligand‐based targeted therapies.

The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene-targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin μ heavy chain (μHC) mRNA levels and hence lower pre-BCR and BCR levels. Hypomorphic expression of μHC leads to augmented selection processes at all stages of B-cell development, noticeably at the expansion of pre-B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B-lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B-cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre-BCR and BCR receptor levels for the normal development of B-lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.