Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Autoinhibition in the Signal Transducer CIN85 Modulates B Cell Activation.

Signal transduction by the ligated B cell antigen receptor (BCR) depends on the preorganization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and the corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85's valency toward SLP65. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction with the adjacent disordered linker is effective in modulating CIN85's valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.

SH2 domain-containing inositol 5-phosphatases support the survival of Burkitt lymphoma cells by promoting energy metabolism.

Burkitt lymphoma cells (BL) exploit antigen-independent tonic signals transduced by the B cell antigen receptor (BCR) for their survival, but the molecular details of the rewired BLspecific BCR signal network remain unclear. A loss of function screen revealed the SH2 domain-containing 5`-inositol phosphatase 2 (SHIP2) as a potential modulator of BL fitness. We characterized the role of SHIP2 in BL survival in several BL cell models and show that perturbing SHIP2 function renders cells more susceptible to apoptosis, while attenuating proliferation in a BCR-dependent manner. Unexpectedly, SHIP2 deficiency did neither affect PI3K survival signals nor MAPK activity, but attenuated ATP production. We found that an efficient energy metabolism in BL cells requires phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2), which is the enzymatic product of SHIP proteins. Consistently, interference with the function of SHIP1 and SHIP2 augments BL cell susceptibility to PI3K inhibition. Notably, we here provide a molecular basis of how tonic BCR signals are connected to energy supply, which is particularly important for such an aggressively growing neoplasia. These findings may help to improve therapies for the treatment of BL by limiting energy metabolism through the inhibition of SHIP proteins, which renders BL cells more susceptible to the targeting of survival signals.

Characterization of testicular macrophage subpopulations in mice.

Testis is an immune privileged site, a feature that prevents germ cells from eliciting an autoimmune response. Macrophages contribute to this state of tolerance by adopting an immunoregulatory phenotype. Here, we further characterized their features in mice by analyzing surface markers, anatomic localization as well as morphology and function. Testicular macrophages (TMΦ) were stained for various surface receptors, and MHCII and CD206 were found to be most suitable to discriminate between two subpopulations. Our immunohistochemical analysis further confirmed a predominant localization of CD206+ cells in the interstitial space. Imaging flow cytometry revealed that both subtypes of TMΦ differed in size and contrast, and to some extent also in their ability to engulf high-molecular dextran. To investigate whether the polarization of the immune system had any influence on the phenotype of TMΦ, we compared C57BL/6 and BALB/c mice. Importantly, our analysis revealed that the abundance of cells expressing either MHCII or any of the scavenger receptors CD206, CD163 and CD71 differed between both mouse strains. In addition, the presence of the glucocorticoid receptor in macrophages affected the ratio between individual subpopulations, which is consistent with a crucial role of glucocorticoids in macrophage polarization. Collectively, our results indicate that TMΦ are composed in a variable ratio of distinct subsets with characteristic features, which may shape the immune privilege of the testis also in humans.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness.

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

Highly selective organ distribution and cellular uptake of inorganic-organic hybrid nanoparticles customized for the targeted delivery of glucocorticoids.

We previously reported that inorganic-organic hybrid nanoparticles (IOH-NPs) containing the synthetic glucocorticoid (GC) betamethasone show efficient anti-inflammatory activity in mice. Here, we employed IOH-NPs with the chemical composition Gd3+2[AMP]2-3 (AMP: adenosine monophosphate) to determine their in vivo distribution by magnetic resonance imaging after intraperitoneal injection. We show that IOH-NPs distribute throughout the peritoneal cavity from where they get rapidly cleared and then localize to abdominal organs. Our findings were confirmed by analyzing individual mouse organs ex vivo following injection of IOH-NPs with the chemical composition [ZrO]2+[(BMP)0.9(FMN)0.1]2- (BMP: betamethasone phosphate, FMN: flavin mononucleotide) or [ZrO]2+[(HPO4)0.9(FMN)0.1]2- using inductively coupled plasma mass spectrometry and flow cytometry. To characterize the mechanism of cellular uptake in vitro, we tested different cell lines for their ability to engulf IOH-NPs by flow cytometric analysis taking advantage of the incorporated fluorescent dye FMN. We found that IOH-NPs were efficiently taken up by macrophages, to a lesser extent by fibroblasts, epithelial cells, and myoblasts, and hardly at all by both T and B lymphocytes. Characterization of the endocytic pathway further suggested that IOH-NPs were internalized by macropinocytosis, and imaging flow cytometry revealed a strong colocalization of the engulfed IOH-NPs with the lysosomal compartment. Intracellular release of the functional anions from IOH-NPs was confirmed by the ability of the GC betamethasone to downregulate the expression of surface receptors on bone marrow-derived macrophages. Taken together, our findings unveil the mechanistic basis of an anti-inflammatory GC therapy with IOH-NPs, which may entail translational approaches in the future.

HSP90 promotes Burkitt lymphoma cell survival by maintaining tonic B-cell receptor signaling.

Burkitt lymphoma (BL) is an aggressive B-cell neoplasm that is currently treated by intensive chemotherapy in combination with anti-CD20 antibodies. Because of their toxicity, current treatment regimens are often not suitable for elderly patients or for patients in developing countries where BL is endemic. Targeted therapies for BL are therefore needed. In this study, we performed a compound screen in 17 BL cell lines to identify small molecule inhibitors affecting cell survival. We found that inhibitors of heat shock protein 90 (HSP90) induced apoptosis in BL cells in vitro at concentrations that did not affect normal B cells. By global proteomic and phosphoproteomic profiling, we show that, in BL, HSP90 inhibition compromises the activity of the pivotal B-cell antigen receptor (BCR)-proximal effector spleen tyrosine kinase (SYK), which we identified as an HSP90 client protein. Consistently, expression of constitutively active TEL-SYK counteracted the apoptotic effect of HSP90 inhibition. Together, our results demonstrate that HSP90 inhibition impairs BL cell survival by interfering with tonic BCR signaling, thus providing a molecular rationale for the use of HSP90 inhibitors in the treatment of BL.

DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk.

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway.

The Dok-3/Grb2 adaptor module promotes inducible association of the lipid phosphatase SHIP with the BCR in a coreceptor-independent manner.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) plays a key role in preventing autoimmune phenomena by limiting antigen-mediated B cell activation. SHIP function is thought to require the dual engagement of the BCR and negative regulatory coreceptors as only the latter appear capable of recruiting SHIP from the cytosol to the plasma membrane by the virtue of phosphorylated immunoreceptor tyrosine-based inhibitory motifs. Here, we demonstrate a coreceptor-independent membrane recruitment and function of SHIP in B cells. In the absence of coreceptor ligation, SHIP translocates to sites of BCR activation through a concerted action of the protein adaptor unit Dok-3/Grb2 and phosphorylated BCR signaling components. Our data reveal auto-inhibitory SHIP activation by the activated BCR and suggest an unexpected negative-regulatory capacity of immunoreceptor tyrosine-based activation motifs in Igα and Igß.

Elucidation of tonic and activated B-cell receptor signaling in Burkitt's lymphoma provides insights into regulation of cell survival.

Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments.

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.

Macromolecular assembly of the adaptor SLP-65 at intracellular vesicles in resting B cells.

The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits. By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain-containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear. We showed that SLP-65 activation required its association at vesicular compartments in resting B cells. A module of ~50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles. Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution but could bind in a structured manner to noncharged lipid components of cellular membranes. Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation.

The intramembrane proteases signal Peptide peptidase-like 2a and 2b have distinct functions in vivo.

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency similar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.

Cutting edge: feed-forward activation of phospholipase Cγ2 via C2 domain-mediated binding to SLP65.

Ag-mediated B cell stimulation relies on phospholipase Cγ2 (PLCγ2) for Ca(2+) mobilization. Enzymatic activity of PLCγ2 is triggered upon Src homology 2 domain-mediated binding to the tyrosine-phosphorylated adaptor SLP65. However, SLP65 phosphorylation outlasts the elevation of cytosolic Ca(2+) concentration suggesting additional levels of PLCγ2 regulation. We show in this article that the functionality of the PLCγ2/SLP65 complex is controlled by the weakly characterized C2 domain of PLCγ2. Usually C2 domains bind membrane lipids, but that of PLCγ2 docks in a Ca(2+)-regulated manner to a distinct phosphotyrosine of SLP65. Hence, early Ca(2+) fluxing provides feed-forward signal amplification by promoting anchoring of the PLCγ2 C2 domain to phospho-SLP65. As the cellular Ca(2+) resources become exhausted, the concomitant decline of Ca(2+) dampens the C2-phosphotyrosine interaction so that PLCγ2 activation terminates despite sustained SLP65 phosphorylation.

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK 4 (BLP).

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam(3)CSK(4) (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.

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.

ß2 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 ß2 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.

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

Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a(-/-) mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.

The Dok-3/Grb2 protein signal module attenuates Lyn kinase-dependent activation of Syk kinase in B cell antigen receptor microclusters.

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5' phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.