Phosphorylation of serine-893 in CARD11 suppresses the formation and activity of the CARD11-BCL10-MALT1 complex in T and B cells.

CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/ß-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/ß-dependent CARD11 phosphorylation also suppressed CARD11 functions in T or B cells. Through mass spectrometry-based proteomics analysis, we identified multiple constitutive and inducible CARD11 phosphorylation sites in T cells. We demonstrated that a single TCR- or BCR-inducible phosphorylation on Ser893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton's tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser893 in CARD11 by PKCθ controlled the strength of CARD11 scaffolding by impairing the formation of the CBM complex. Thus, PKCθ simultaneously catalyzes both stimulatory and inhibitory CARD11 phosphorylation events, which shape the strength of CARD11 signaling in lymphocytes.

A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-κB signaling in activated T cells.

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.

TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor­associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Pathological RANK signaling in B cells drives autoimmunity and chronic lymphocytic leukemia.

Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.

MALT1 Phosphorylation Controls Activation of T Lymphocytes and Survival of ABC-DLBCL Tumor Cells.

The CARMA1/CARD11-BCL10-MALT1 (CBM) complex bridges T and B cell antigen receptor (TCR/BCR) ligation to MALT1 protease activation and canonical nuclear factor κB (NF-κB) signaling. Using unbiased mass spectrometry, we discover multiple serine phosphorylation sites in the MALT1 C terminus after T cell activation. Phospho-specific antibodies reveal that CBM-associated MALT1 is transiently hyper-phosphorylated upon TCR/CD28 co-stimulation. We identify a dual role for CK1α as a kinase that is essential for CBM signalosome assembly as well as MALT1 phosphorylation. Although MALT1 phosphorylation is largely dispensable for protease activity, it fosters canonical NF-κB signaling in Jurkat and murine CD4 T cells. Moreover, constitutive MALT1 phosphorylation promotes survival of activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) cells addicted to chronic BCR signaling. Thus, MALT1 phosphorylation triggers optimal NF-κB activation in lymphocytes and survival of lymphoma cells.

Bcl10-controlled Malt1 paracaspase activity is key for the immune suppressive function of regulatory T cells.

Regulatory T cells (Tregs) have crucial functions in the inhibition of immune responses. Their development and suppressive functions are controlled by the T cell receptor (TCR), but the TCR signaling mechanisms that mediate these effects remain ill-defined. Here we show that CARD11-BCL10-MALT1 (CBM) signaling mediates TCR-induced NF-κB activation in Tregs and controls the conversion of resting Tregs to effector Tregs under homeostatic conditions. However, in inflammatory milieus, cytokines can bypass the CBM requirement for this differentiation step. By contrast, CBM signaling, in a MALT1 protease-dependent manner, is essential for mediating the suppressive function of Tregs. In malignant melanoma models, acute genetic blockade of BCL10 signaling selectively in Tregs or pharmacological MALT1 inhibition enhances anti-tumor immune responses. Together, our data uncover a segregation of Treg differentiation and suppressive function at the CBM complex level, and provide a rationale to explore MALT1 inhibitors for cancer immunotherapy.

Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.

Molecular architecture and regulation of BCL10-MALT1 filaments.

The CARD11-BCL10-MALT1 (CBM) complex triggers the adaptive immune response in lymphocytes and lymphoma cells. CARD11/CARMA1 acts as a molecular seed inducing BCL10 filaments, but the integration of MALT1 and the assembly of a functional CBM complex has remained elusive. Using cryo-EM we solved the helical structure of the BCL10-MALT1 filament. The structural model of the filament core solved at 4.9 Å resolution identified the interface between the N-terminal MALT1 DD and the BCL10 caspase recruitment domain. The C-terminal MALT1 Ig and paracaspase domains protrude from this core to orchestrate binding of mediators and substrates at the filament periphery. Mutagenesis studies support the importance of the identified BCL10-MALT1 interface for CBM complex assembly, MALT1 protease activation and NF-κB signaling in Jurkat and primary CD4 T-cells. Collectively, we present a model for the assembly and architecture of the CBM signaling complex and how it functions as a signaling hub in T-lymphocytes.

BCL10 - Bridging CARDs to Immune Activation.

Since the B-cell lymphoma/leukemia 10 (BCL10) protein was first described in 1999, numerous studies have elucidated its key functions in channeling adaptive and innate immune signaling downstream of CARMA/caspase-recruitment domain (CARD) scaffold proteins. While T and B cell antigen receptor (TCR/BCR) signaling induces the recruitment of BCL10 bound to mucosa-associated lymphoid tissue (MALT)1 to the lymphocyte-specific CARMA1/CARD11-BCL10-MALT1 (CBM-1) signalosome, alternative CBM complexes utilize different CARMA/CARD scaffolds in distinct innate or inflammatory pathways. BCL10 constitutes the smallest subunit in all CBM signalosomes, containing a 233 amino acid coding for N-terminal CARD as well as a C-terminal Ser/Thr-rich region. BCL10 forms filaments, thereby aggregating into higher-order clusters that mediate and amplify stimulation-induced signals, ultimately leading to MALT1 protease activation and canonical NF-κB and JNK signaling. BCL10 additionally undergoes extensive post-translational regulation involving phosphorylation, ubiquitination, MALT1-catalyzed cleavage, and degradation. Through these feedback and feed-forward events, BCL10 integrates positive and negative regulatory processes that govern the function as well as the dynamic assembly, disassembly, and destruction of CBM complexes. Thus, BCL10 is a critical regulator for activation as well as termination of immune cell signaling, revealing that its role extends far beyond that of a mere linking factor in CBM complexes.

YOD1/TRAF6 association balances p62-dependent IL-1 signaling to NF-κB.

The ubiquitin ligase TRAF6 is a key regulator of canonical IκB kinase (IKK)/NF-κB signaling in response to interleukin-1 (IL-1) stimulation. Here, we identified the deubiquitinating enzyme YOD1 (OTUD2) as a novel interactor of TRAF6 in human cells. YOD1 binds to the C-terminal TRAF homology domain of TRAF6 that also serves as the interaction surface for the adaptor p62/Sequestosome-1, which is required for IL-1 signaling to NF-κB. We show that YOD1 competes with p62 for TRAF6 association and abolishes the sequestration of TRAF6 to cytosolic p62 aggregates by a non-catalytic mechanism. YOD1 associates with TRAF6 in unstimulated cells but is released upon IL-1ß stimulation, thereby facilitating TRAF6 auto-ubiquitination as well as NEMO/IKKγ substrate ubiquitination. Further, IL-1 triggered IKK/NF-κB signaling and induction of target genes is decreased by YOD1 overexpression and augmented after YOD1 depletion. Hence, our data define that YOD1 antagonizes TRAF6/p62-dependent IL-1 signaling to NF-κB.

Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells.

MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.

89Zr-Labeled Versus ¹²4I-Labeled αHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo.

UNLABELLED: Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide (89)Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide (124)I. METHODS: To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS200) residues was either conjugated with (124)I via an iodination reagent or coupled with deferoxamine (Df) and complexed with (89)Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1(nu) mice bearing HER2-positive human tumor xenografts. RESULTS: (89)Zr⋅Df-Fab-PAS200 and (124)I-Fab-PAS200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt (124)I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for (89)Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. CONCLUSION: Both (89)Zr- and (124)I-labeled versions of αHER2 Fab-PAS200 allowed PET tumor imaging with high contrast. With its residualizing radiometal, the tracer (89)Zr⋅Df-Fab-PAS200 showed better in vivo stability and higher tumor uptake.