Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling

Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super‐resolution microscopy and single‐particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains. The landscape of CD22 and its lateral diffusion were perturbed either in the absence of CD45 or when the CD22 lectin domain was mutated. To understand how a relatively low number of CD22 molecules can keep BCR signalling in check, we generated Brownian dynamic simulations and supported them with ex vivo experiments. This combined approach suggests that the inhibitory function of CD22 is influenced by its nanoscale organization and is ensured by its fast diffusion enabling a “global BCR surveillance” at the plasma membrane.     

Modification of ligand-independent B cell receptor tonic signals activates receptor editing in immature B lymphocytes

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr⁸²² on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr⁸⁰⁷, a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr²⁹² on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.     

Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens

How the B‐cell antigen receptor (BCR) is activated upon interaction with its cognate antigen or with anti‐BCR antibodies is not fully understood. We have recently shown that B‐cell activation is accompanied by the opening of the pre‐organized BCR oligomers, an observation that strengthens the role of receptor reorganization in signalling. We have now analysed the BCR oligomer opening and signalling upon treatment with different monovalent stimuli. Our results indicate that monovalent antigens are able to disturb and open the BCR oligomer, but that this requires the presence and activity of the Src family kinase (SFK) Lyn. We have also shown that monovalent Fab fragments of anti‐BCR antibodies can open the BCR oligomers as long as they directly interact with the antigen‐binding site. We found that monovalent antigen binding opens both the IgM‐BCR and IgD‐BCR, but calcium signalling is only seen in cells expressing IgM‐BCR; this provides a molecular basis for IgM‐ and IgD‐BCR functional segregation.     

Tonic ubiquitylation controls T‐cell receptor:CD3 complex expression during T‐cell development

Expression of the T‐cell receptor (TCR):CD3 complex is tightly regulated during T‐cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ε proline‐rich sequence, Lck, c‐Cbl, and SLAP, which collectively trigger the dynamin‐dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ‐monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T‐cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T‐cell development.     

CD19 amplification of B lymphocyte Ca2+ responses: a role for Lyn sequestration in extinguishing negative regulation

B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca(2+)](i)) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca(2+)](i) mobilization and significantly enhanced BCR-induced [Ca(2+)](i) responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca(2+)](i) responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca(2+)](i) responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca(2+)](i) responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca(2+)](i) responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling.     

CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux

CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation.     

Novel binding site for Src homology 2-containing protein-tyrosine phosphatase-1 in CD22 activated by B lymphocyte stimulation with antigen

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr(843)) and 863 (Tyr(863)), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr(843) and Tyr(863). Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr(843) and Tyr(863) are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr(783) and Tyr(817) fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.     

Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.     

A novel interaction between protein kinase D and TNF receptor-associated factor molecules regulates B cell receptor-CD40 synergy

Signaling by Ag to the B cell Ag receptor (BCR) is enhanced by several cooperating signals, including several provided by B-T cell interactions. One of these, CD40, provides critical signals for B cell differentiation, isotype switching, and B cell memory. The molecular mechanisms by which BCR and CD40 signals synergize are not well understood. Although the BCR and CD40 share certain signaling pathways, we hypothesized that unique signals provided by each could provide mutual enhancement of their signaling pathways. The BCR, but not CD40, activates protein kinase D (PKD), while CD40, but not the BCR, employs the TNFR-associated factor (TRAF) adapter proteins in signaling. In this study, we show that genetic or pharmacologic inhibition of BCR-mediated PKD activation in B lymphocytes abrogated the synergy between the CD40 and the BCR, as measured by activation of Ig and cytokine secretion. Interestingly, the role of PKD was dependent upon the association of CD40 with TRAF2, and was inhibited by the binding of TRAF3, revealing a novel functional link between these two classes of signaling molecules.     

Regulation of B-cell fate by antigen-receptor signals

Recent evidence indicates that B cells are instructed continuously by B-cell receptor (BCR) signals to make crucial cell-fate decisions at several checkpoints during their development. Targeted disruption of BCR signalling components leads to distinct blocks in B-cell maturation, which indicates that key kinases and adaptors fine-tune BCR signalling to direct appropriate cell fates. Recent progress in unravelling the molecular mechanisms of the BCR signalling pathways has helped to clarify how BCR signals regulate the proliferation, survival and apoptosis of developing B cells.     

Cognate B cell signaling via MHC class II: differential regulation of B cell antigen receptor and MHC class II/Ig-alpha beta signaling by CD22

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-alphabeta dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-alphabeta signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.     

Delineating PAS‐HAMP interaction surfaces and signalling‐associated changes in the aerotaxis receptor Aer

The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer‐PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N‐terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase‐off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS‐HAMP surfaces overlapped with a cluster of PAS kinase‐on lesions and with cysteine substitutions that crosslinked the PAS β ‐scaffold to the HAMP AS‐2 helix. A refined Aer PAS‐HAMP interaction model is presented. Compared to the kinase‐off state, the kinase‐on state increased the accessibility of HAMP residues (apparently relaxing PAS‐HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static‐dynamic model in which oxidized Aer‐PAS interacts directly with HAMP AS‐2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase‐off output. When PAS‐FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase‐on output.     

Variant B Cell Receptor Isotype Functions Differ in Hairy Cell Leukemia with Mutated BRAF and IGHV Genes

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD^+ve mult-HCL, IgD mediated persistent Ca²⁺ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD^−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.     

Synthetic glycan ligand excludes CD22 from antigen receptor-containing lipid rafts

CD22/Siglec-2 is a B cell membrane-bound lectin that recognizes glycan ligands containing alpha2,6-linked sialic acid, and negatively regulates signaling through the B cell antigen receptor (BCR). Previous studies demonstrated that synthetic sialosides that bind to CD22 augment BCR signaling by inhibiting CD22-mediated BCR regulation. Here we demonstrate that, after antigen stimulation, CD22 forms a cap together with BCR, and translocates to lipid rafts. Both co-capping of CD22 with BCR and translocation of CD22 to lipid rafts were markedly blocked by a synthetic alpha2,6-linked sialic acid, Neu5Gcalpha2-6GalbetaSE. These results strongly suggest that synthetic glycan ligand excludes CD22 from BCR-containing lipid rafts. Because CD22-mediated signal regulation requires phosphorylation of CD22 by Lyn that localizes in lipid rafts and is activated by BCR, synthetic glycan ligand regulates localization of CD22 crucial for signal regulation.     

Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22

The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.