Michael Engelke

Ca2+ signaling in antigen receptor‐activated B lymphocytes

Summary:  B cells respond to antigen stimulation with mobilization of the Ca2+ second messenger in two phases operated by two distinct sets of effector proteins. First, an antigen receptor‐specific Ca2+ initiation complex is assembled, activated, and targeted to the plasma membrane to trigger the transient release of Ca2+ from intracellular stores of the endoplasmic reticulum. Second, more ubiquitously expressed Ca2+ channels of the plasma membrane are opened to allow for sustained Ca2+ influx from the extracellular medium. Depending on the developmental stage of the B cell, the kinetics and profile of the two phases are adjusted at multiple levels of positive and negative regulation. A molecular basis for the Ca2+ signaling plasticity is provided by cytosolic and transmembrane adapter proteins. They act as signal organizers, which control enzyme/substrate interactions by directing the different signaling modules into specific subcellular compartments. These arrangements orchestrate a graduated activation of Ca2+‐sensitive downstream pathways, which ultimately determine appropriate cellular responses, namely elimination of autoreactive B cells or proliferation and differentiation of immunocompetent B cells into antibody‐secreting plasma cells.

The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

The intramembrane protease SPPL2a cleaves the NTF of invariant chain (CD74), which is essential for normal trafficking of MHC class II–containing endosomes and thus for B cell development and function.

Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells.

Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok‐3 (downstream of kinase‐3) as a key component of negative feedback regulation in Ca2+ signaling from the B‐cell antigen receptor. Dok‐3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok‐3 inhibits antigen receptor‐induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Brutons tyrosine kinase. This leads to diminished activation of phospholipase C‐γ2 and reduced production of soluble inositol trisphosphate. Hence, the Dok‐3/Grb2 module is a membrane‐associated signaling organizer, which orchestrates the interaction efficiency of Ca2+‐mobilizing enzymes.

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85.

Spleen tyrosine kinase Syk and its substrate SLP65 (also called BLNK) are proximal signal transducer elements of the B‐cell antigen receptor (BCR). Yet, our understanding of signal initiation and processing is limited owing to the incomplete list of SLP65 interaction partners and our ignorance of their association kinetics. We have now determined and quantified the in vivo interactomes of SLP65 in resting and stimulated B cells by mass spectrometry. SLP65 orchestrated a complex signal network of about 30 proteins that was predominantly based on dynamic interactions. However, a stimulation‐independent and constant association of SLP65 with the Cbl‐interacting protein of 85 kDa (CIN85) was requisite for SLP65 phosphorylation and its inducible plasma membrane translocation. In the absence of a steady SLP65/CIN85 complex, BCR‐induced Ca2+ and NF‐κB responses were abrogated. Finally, live cell imaging and co‐immunoprecipitation experiments further confirmed that both SLP65 and CIN85 are key components of the BCR‐associated primary transducer module required for the onset and progression phases of BCR signal transduction.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)–kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non–immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

The MHC class I chain‐related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)‐cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA‐129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft‐versus‐host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA‐129Val/Val genotype carriers was improved when treated with anti‐thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA‐129Met isoform was characterized by stronger NKG2D signaling, triggering more NK‐cell cytotoxicity and interferon‐γ release, and faster co‐stimulation of CD8+ T cells. The MICA‐129Met variant also induced a faster and stronger down‐regulation of NKG2D on NK and CD8+ T cells than the MICA‐129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA‐129Met variants appeared to reduce the severity of aGVHD.

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression

OBJECTIVE Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells. MATERIALS AND METHODS A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)-defined NK cells from human leukocyte antigen (HLA)-typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay. RESULTS HLA, but not CD20 expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications. CONCLUSION KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA.

β2 integrin–derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

Complex phosphorylation dynamics control the composition of the Syk interactome in B cells.

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high‐resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full‐length Syk can associate with more than 25 distinct ligands including the 14‐3‐3γ adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor‐proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network.

Conformational plasticity and navigation of signaling proteins in antigen-activated B lymphocytes.

Over the past two decades our view of the B cell antigen receptor (BCR) has fundamentally changed. Being initially regarded as a mute antibody orphan of the B cell surface, the BCR turned out to be a complex multimolecular machine monitoring almost all stages of B cell development, selection, and activation through a plethora of ubiquitously and cell-type-specific effector proteins. A comprehensive understanding of the many BCR signaling facets is still out but a few common biochemical principles outlined in this review operate at the level of receptor activation and orchestrate specific wiring of intracellular transducer cascades. First, initiation and processing of antigen-induced signal transduction relies on transient conformational changes in the signaling proteins to trigger their physical interaction with downstream elements. Second, this dynamic assembly of signalosomes occurs at distinct subcellular locations, most prominently the plasma membrane, which requires dynamic relocalization of one or more of the engaged molecules. For both, precise complex formation and efficient subcellular targeting, B cell signaling components are equipped with a variety of protein interaction domains. Here we provide an overview on how these simple rules are applied by a limited number of transmembrane and cytosolic proteins to convert BCR ligation into Ca(2+) mobilization and Ras activation in an adjustable manner.