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It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

It has been shown that innate immune responses can adopt adaptive properties such as memory. Whether T cells utilize innate immune signaling pathways to diversify their repertoire of effector functions is unknown. Gasdermin E (GSDME) is a membrane pore-forming molecule that has been shown to execute pyroptotic cell death and thus to serve as a potential cancer checkpoint. In the present study, we show that human T cells express GSDME and, surprisingly, that this expression is associated with durable viability and repurposed for the release of the alarmin interleukin (IL)-1α. This property was restricted to a subset of human helper type 17 T cells with specificity for Candida albicans and regulated by a T cell-intrinsic NLRP3 inflammasome, and its engagement of a proteolytic cascade of successive caspase-8, caspase-3 and GSDME cleavage after T cell receptor stimulation and calcium-licensed calpain maturation of the pro-IL-1α form. Our results indicate that GSDME pore formation in T cells is a mechanism of unconventional cytokine release. This finding diversifies our understanding of the functional repertoire and mechanistic equipment of T cells and has implications for antifungal immunity.

CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/β-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/β-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 Ser 893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser 893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton’s tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser 893 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.

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-022-04154-z.

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 receptor–associated 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.

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., Ubiquitination may control protein stability or function. Here the authors show that an ubiquitination enzyme, Hectd3, ubiquitinates Stat3 and Malt1 to modulate their function but not degradation in T cells, and thereby promoting the differentiation of pathogenic Th17 cells and susceptibility to a mouse model of multiple sclerosis.

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., The BCL10-MALT1 complex is a central signaling hub in lymphocytes and linked to various human immune pathologies. Here the authors present the cryo-EM structure of the BCL10-MALT1 filament core and verify the identified BCL10/MALT1 interface with mutagenesis studies.

The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.

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-kappa B 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.

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., MALT1 regulates NFκB signalling both as a scaffolding protein and as a protease. Here the authors show that during T cell activation the expression of MALT1 gene switches to an alternatively spliced variant, which increases TCR signal transduction due to enhanced TRAF6 binding.

Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.