The relevance of MALT1 protease activity is strongly evidenced for ITAM-containing receptors, which promote activation of lymphoid and NK cells via CARMA1/CARD11 (11–13) and myeloid cells via CARD9 (13, 14). The protease activity of MALT1 also plays a role outside the immune system, in the activation of keratinocytes (15). Stimulation of keratinocytes with zymosan, which triggers the ITAM-containing receptor Dectin-1, leads to CBM complex activation and MALT1 protease activity via CARMA2/CARD14. This suggests that all ITAM containing receptors signal via both the scaffold and protease activity of MALT1 in immune and non-immune cells.

A variety of GPCRs (such as AT1R, Par-1, PAFR, ETA, ETB, CXCR2, CXCR4, LPA1-6) and RTKs (the EGFR and HER2/neu) have been shown to trigger the formation of a CBM complex comprising CARMA3/CARD10 **(Figure 1)** [see (1) and references therein]. Whether the protease function of MALT1 is involved or not in the transmission of the signaling pathway of these receptors is not well understood. For one GPCR (the thrombin receptor PAR1), the protease activity of MALT1 was found to be crucial for the regulation of the integrity of epithelial barriers, whereby stimulation of epithelial cells with thrombin lead to MALT1 proteolytic activity and CYLD cleavage (16), in addition to the previously described formation of a CBM complex comprising CARMA3 (17). These findings suggest the possibility of a broader involvement of the protease activity of MALT1 in CBM-dependent GPCR signaling. In contrast, signaling via the human EGFR2 (HER2), a receptor tyrosine kinase (RTK), has been proposed to depend mainly on the scaffold activity of MALT1 (18). It is possible that MALT1 cleaves a distinct subset of substrates in epithelial cells, or that MALT1 activation requires co-stimulation of the EGFR and another, yet to be identified co-receptor. A need for co-stimulation is observed in T cells, where optimal CBM complex assembly and activation of MALT1 protease activity requires the synergistic stimulation of both, the TCR and the costimulatory receptor CD28 (11, 12, 19, 20). Whether MALT1 protease activity is activated downstream of RTK merits further exploration.

### MULTIPLE ELEMENTS CONTROL THE ASSEMBLY AND ARCHITECTURE OF THE CBM COMPLEX

A common feature of receptors that signal via MALT1 is their capacity to induce the formation of a specific type of CBM signaling complex. In resting cells, the members of the CBM complex reside in an inactive form in the cytoplasm. The catalytically inactive proenzyme MALT1 binds constitutively to BCL10, while the CARMA/CARD scaffold proteins are kept in a monomeric inactive form due to intramolecular inhibitory interactions. The inducible assembly of the CBM complex requires specific protein domains present in each CBM component (**Figure 2**). The CARD proteins CARMA1 (CARD11), CARMA2 (CARD14), and CARMA3 (CARD10)

**Abbreviations:** ABC, activated B-cell; AGTR1, angiotensin II receptor; ATLL, adult T-cell leukemia/lymphoma; BCL10, B-cell lymphoma-10; BCR, B-cell receptor; BENTA, B-cell expansion with NF-κB and T-cell anergy; CARD, caspase recruitment domain; CARMA, CARD-containing membrane-associated guanylate kinase; CBM, CARMA/CARD-BCL10-MALT1; CC, coil-coiled; CDE, constitutive decay element; CLL, chronic lymphocytic leukemia; CTCL, cutaneous T-cell lymphoma; DD death domain; DC, dentritic cells; DLBCL, diffuse large B-cell lymphoma; DUB, deubiquitinating enzyme; EGFR, epidermal growth factor receptor; FcR, Fc receptor; FLIP, Flice-like inhibitory protein; GPCR, G protein-coupled receptor; GUK, guanylate kinase; HER2, human EGFR-2; HOIL1, heme-oxidized IRP2 ubiquitin ligase 1; HOIP, HOIL1-interacting protein; HHV8, human herpes virus-8; Ig, Immunoglobulin; IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked; ITAM, immunoreceptor tyrosinebased activation motif; KSHV, Kaposi's sarcoma-associated herpes virus; LMP2A, latent membrane protein 2A; LUBAC, linear ubiquitin chain assembly complex; Mϕ, macrophage; MAGUK, membrane-associated guanylate kinase; MALT, mucosa-associated lymphoid tissue; MC, myeloid cell; MCL, mantle cell lymphoma; mTORc1, mammalian target of rapamycin complex 1; MZ, marginal zone; nTreg, natural Treg; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor kappa B; NK, natural killer; OSCAR, osteoclast associated receptor; PDZ, PSD95 Dlg1 ZO-1; PEL, primary effusion lymphoma; PI, protease inactive; PKC, protein kinase C; PTCL, peripheral T-cell lymphoma; RTK, receptor tyrosine kinases; TCR, T cell receptor; TREM-1, triggering expression by myeloid cells 1; PTM, posttranslational modification; RA, rheumatoid arthritis; SH3, Src homology 3; SHARPIN, SHANK-associated RH domain interacting protein; UTR, untranslated region.

be addressed are annotated with a red question mark. Deregulated activation of the pathway results in different diseases, as summarized in the lower panel of the figure. BCR, B cell receptor; DC, dendritic cell; EGFR, epidermal growth factor receptor; FcR, Fc receptor; Mϕ, macrophage; MC, myeloid cell; NK, natural killer; OSCAR, osteoclast associated receptor; PKC, protein kinase C; TCR, T cell receptor; TREM-1, triggering expression by myeloid cells 1; RA, rheumatoid arthritis.

are structurally similar. Each contains an N-terminal CARD, followed by a coil-coiled (CC) domain and a C-terminal region that shares features with proteins of the membraneassociated guanylate kinase (MAGUK) family, which contain a PDZ (PSD95, Dlg1, ZO-1), SH3 (Src homology 3), and GUK (guanylate kinase) domain. CARD9 contains a CARD and coiledcoil motif but lacks the C-terminal MAGUK features (21–25). An important structural feature of BCL10 is its N-terminal CARD that allows its inducible association with a CARMA/CARD component (21–25). Additionally, BCL10 contains a C-terminal region with a short sequence of amino acids (107-118) that is necessary for its constitutive interaction with MALT1 (21, 26). MALT1 contains an N-terminal death domain (DD) of unknown function, followed by two immunoglobulin (Ig)-like domains required for BCL10 binding, a caspase-like protease domain and a third Ig domain that plays a regulatory role for the protease function (27, 28) (**Figure 2**).

The inducible assembly of the CBM complexes seems to depend on a common, conserved mechanism that requires the receptor-induced activation of PKC family members (29, 30). These Ser/Thr kinases (and likely other kinases) phosphorylate the CARMA/CARD proteins in the linker region (**Figure 2**, illustrated here for CARMA1 (CARD11)) (29–31). This in turn is thought to weaken an inhibitory intra-molecular interaction between the CARD and linker domain, thereby promoting a CARD-mediated association with BCL10 and the formation of the CBM complex (29–31). Similar mechanisms are thought to control activation of CARMA3 (26), CARMA2 (15), and CARD9 (32), but whether PKC family members directly phosphorylate CARMA2 or CARMA3 is not known. CBM complex formation depends on homotypic CARD-CARD interactions between the CARMA/CARD proteins and BCL10 (21–23, 25, 26) and possibly on an additional interaction of the protease domain of MALT1 with the CC domain of CARD proteins as was demonstrated for CARMA1 (33). The CC region of CARMA1 has been shown to mediate oligomerization that is required for TCR-induced NF-κB activation (30, 34). Structural architecture studies and modeling suggest that the CC domain-mediated oligomerization of CARMA1 nucleates the oligomerization of BCL10 into helical filaments. BCL10 filaments are formed in a cooperative manner and stabilized through homotypic interactions formed between the CARD motifs of adjacent BCL10 monomers in the filament

FIGURE 2 | Structure and function of the CBM complex. In resting conditions, MALT1 and BCL10 interact constitutively, while CARMA1 is dissociated from the other components of the CBM. PKC-mediated phosphorylation triggers a conformational opening and oligomerization of CARMA1, leading to the recruitment of preformed MALT1- BCL10 complexes. The E3 ligase TRAF6 is further recruited and polyubiquitinates MALT1 and BCL10. This favors the recruitment of the IKK and TAB2/3-TAK1 complexes and the downstream activation of NF-κB and AP-1. N-terminal phosphorylation of BCL10 by IKKβ enhances NF-κB activation, while a C-terminal phosphorylation disassembles MALT1 from BCL10. BCL10 can undergo both, linear polyubiquitination and K63-linked polyubiquitination, which are important for IKKγ/NEMO recruitment. Monoubiquitination activates MALT1 protease function. Once activated, MALT1 cleaves several substrates including BCL10 (after R228) and itself (after R149). The CBM signal is thought to be amplified by formation of filamentous BCL10 structures initiated by CARMA1 nucleation. BCL10 filaments are decorated with MALT1, most likely in its dimeric form. Double-headed arrows indicate interactions. Arrows indicate P, phosphorylations; Ub, ubiquitination.

(35). MALT1 is constitutively associated with BCL10, and therefore forms part of these filamentous structures (**Figures 2**,**3**) (35). Although not filamentous in nature, oligomeric signaling complexes containing proteins with CARD motifs, such as the apoptosome, inflammasome, and pyddosome, have been proposed to be key to the activation of proteases of the caspase family (36). Therefore, a prime purpose of the formation of BCL10-MALT1 filaments is likely to allow the oligomerizationdependent activation of MALT1. Crystallographic data from several groups support the idea that active MALT1 forms a dimer that is stabilized by interactions between the protease domain (37, 38). It is tempting to speculate that BCL10 filaments form a scaffold that permits additional interactions between MALT1 dimers leading to its proteolytic activation (**Figure 2**). MALT1 activation strictly requires monoubiquitination in its Ig3 domain that promotes its activation in an intra-or intermolecular manner (39). Exactly how this monoubiquitination contributes to MALT1 activation within the context of the oligomeric BCL10- MALT1 fibers remains unexplored. Although this has not yet been formally addressed, it is likely that similar oligomerizationdependent protein complexes control CBM-driven signaling for all CARMA/CARD proteins.

### MALT1 FUNCTION IS DETERMINED BY ITS MULTIDOMAIN STRUCTURE

The MALT1 gene was originally identified as the target of recurrent translocations in MALT lymphomas (40–42). Later on, in silico studies uncovered that MALT1 shares homology with caspases and metacaspases and therefore falls into a category of caspase-like proteases coined paracaspases (27, 43). The partial structure of the MALT1 caspase-like domain together with the third Ig domain has been recently solved and confirms the expected structural similarities between the protease domains of MALT1 and caspases (37, 38).

Apart from sharing structural homology, several other aspects of MALT1 function are shared with caspases, including cysteine-dependent protease activity (11, 12) a requirement for dimerization (38, 44), and the capacity to auto-process (45, 46). Like caspases, MALT1 contains a protease domain with a catalytic dyad comprising two amino acids that are essential for catalysis, namely C464 and H415 (27). Monomeric MALT1 is inactive and, as for caspases, requires dimerization via the caspase-like protease domain for enzymatic activity (38, 44, 47) (**Figure 3B**). Interestingly, MALT1 has been recently shown to undergo autoprocessing. However, unlike caspases, which autoprocess within the protease domain, MALT1 cleaves itself between the N-terminal death domain and the first Ig domain (45, 46). This does not seem to enhance proteolytic activity but is essential for MALT1-dependent NF-κB activation by a mechanism that is not yet understood (45, 46). In contrast to caspases, the proportion of cellular MALT1 undergoing autoprocessing is very small. It is unclear whether this reflects activation of only a very small proportion of cellular MALT1 or a subtler regulation of its activity, which needs to be investigated in more detail.

Despite the above-mentioned structural similarities between caspases and MALT1, several important differences exist. One major difference between caspases and MALT1 resides in their substrate specificity. While caspases are aspartic acid-specific proteases, MALT1 cleaves its substrates after arginine residues (11, 12, 47). The consensus cleavage site of MALT1 based on

The protease activity of MALT1 controls NF-κB activation, AP-1 activation, mRNA stability, and cellular adhesion. This is mediated by the cleavage of the depicted

known substrates and screening of a tetrapeptide library, is a peptide sequence composed of an arginine at the P1 position, which is in most cases followed by a G or A residue. A hydrophobic residue is usually present at position P4, a rather variable range of amino acids is tolerated at P3 and a preference for serine or proline was found for position P2 (47). The consensus cleavage site could thus be summarized as follows: Φ-X-P/S-**R**'-G/A.

substrates (depicted in blue, also for cleaved BCL10 and autoprocessed MALT1).

A second important difference between caspases and MALT1 concerns the regulation of MALT1 function, which is dependent on posttranslational modification (PTM) by ubiquitination. MALT1 is ubiquitinated, upon antigen receptor triggering, within the Ig3 domain and its C-terminal part by either K63-linked polyubiquitination or monoubquitination. MALT1 polyubiquitination mediated by TRAF6 is crucial for the activation and/or physical recruitment of the IKK complex, which phosphorylates the NF-κB inhibitor IκB to initiate its degradation and thus allows the nuclear translocation of NFκB (4, 6, 7). This K63-linked polyubiquitination of MALT1 likely provides docking sites for the adaptor protein TAB2, which forms a complex with the IKK-activating kinase TAK1 (48). Additionally, the K63-linked ubiquitin chains may serve to physically recruit the IKK complex via its catalytically inactive IKKγ subunit (4, 49). MALT1 mono-ubiquitination on lysine residue 644 (K644), on the other hand, is required for the activation of MALT1 protease activity, most likely by favoring the formation of an active dimeric form of MALT (39).

A third aspect that differentiates MALT1 from caspases is the specific presence of additional protein domains that support its regulation and function. In the N-terminal part preceding the protease domain, MALT1 contains an N-terminal death domain (DD) followed by two Ig-like domains. The C-terminal part following the caspase-like domain contains a third Ig-like domain and a structurally undefined (most likely flexible) Cterminal extension (**Figure 2**). DDs can serve as protein-protein interaction domains in apoptotic signaling pathways. However, and despite its structural similarity with caspases, MALT1 does not induce apoptosis. This suggests that the MALT1 DD recruits another, yet to be identified, DD-containing protein unrelated to apoptosis. It is also possible that this domain plays an inhibitory role, since MALT1 autoprocessing, which removes the DD, is required for optimal NF-κB activation (45, 46). Additional unique features of MALT1 are the presence of three Ig domains that contribute to protein-protein interactions, and the presence of TRAF6 binding motifs. The first two Ig domains of MALT1 are necessary for the binding to BCL10 and are therefore crucial for its ability to induce CBM-mediated NFκB activation upon engagement of the TCR (24). The region between the second Ig domain and the caspase-like domain contains a motif that is important for TRAF6 binding (4, 5), which is lacking in a recently described MALT1 splice variant (see below) (50) (**Figure 2**). Additional TRAF6 binding sites are present in the C-terminal extension, which are important for the downstream signaling event (4). The third, C-terminal Ig domain interacts physically with the protease domain and has a particularly important role in MALT1 protease activity, which is regulated by mono-ubiquitination (**Figure 2**) (39). This and other posttranslational modifications of MALT1 and the CBM complex are important for downstream signaling events that will be discussed below.

### THE CBM COMPLEX FORMS A SCAFFOLDING PLATFORM FOR PROTEIN RECRUITMENT

Once assembled, the CBM complex acts as a signaling platform for the recruitment of multiple signaling complexes, including TRAF6, the TAB1/2-TAK1 complex, the IKK complex, and the linear ubiquitin chain assembly complex (LUBAC) (51). These control various aspects of signal transmission that are essential for NF-κB and AP-1 activation.

The activation of NF-κB requires both the physical recruitment of the IKK complex and its activation by the Ser/Thr kinase TAK1. Recent data suggest an important role for both TRAF6 and the LUBAC complex in promoting IKK recruitment (4, 52). The LUBAC complex is composed of three subunits known as HOIL1 (heme-oxidized IRP2 ubiquitin ligase 1), HOIP (HOIL1-interacting protein; also known as RNF31), and SHARPIN (SHANK-associated RH domain interacting protein). The HOIP subunit has an E3 ligase activity, while HOIP and SHARPIN are important for LUBAC complex regulation, such as assuring its proper localization or stability (52, 53). The CBM complex can associate with the LUBAC complex in a transient manner in stimulated T cells, with kinetics that are similar to the association of LUBAC with the IKK complex (54). The stimulation-induced phosphorylation and conformational opening of CARMA1 is thought to lead to recruitment of both, BCL10 and HOIP (55). BCL10 thereby becomes accessible for HOIP-mediated linear ubiquitination on K17, K31, and K63 residues (55). The linear ubiquitination of BCL10 may promote NF-κB activation by providing docking sites for the physical recruitment of NEMO/IKKγ via its UBAN domain (55). However, alternative mechanisms may contribute to IKK recruitment, since another study showed a role for the LUBAC complex in optimal NF-κB activation that was independent of HOIP's ubiquitin ligase activity (54). Indeed, K31 and K63 on BCL10 can be subject to both linear or K63-linked polyubiquitination (7, 55) and the BCL10 modification by K63-linked ubiquitin chains may also contribute, although with lower affinity, to the physical recruitment of NEMO/IKKγ (7, 49, 56).

The CBM-associated E3 ubiquitin ligase responsible for synthesis of K63-linked ubiquitin chains is TRAF6, which associates with oligomeric BCL10-MALT1 complexes (4) and can be physically incorporated into the BCL10-MALT1 filaments in a cooperative manner (57). It has therefore been proposed that BCL10-MALT1 oligomers serve to activate TRAF6 to promote the ubiquitination-dependent activation of the IKK complex (4) (**Figure 3**). MALT1 is polyubiquitinated by TRAF6 on multiple sites preceding the protease domain and within the Ig3 domain (**Figure 2**). These polyubiquitination events are critical for NF-κB activation, since mutation of key amino acids important for TRAF6 binding to MALT1 impairs TCR-induced NF-κB activation (4, 5, 50). The TRAF6-mediated polyubiquitination of MALT1 and/or itself is thought to recruit the TAB2/3-TAK1 complex (4), since the adaptor proteins TAB2/3 contain ubiquitinbinding motifs that bind to K63 polyubiquitin chains (58). TAK1, which associates with TAB2/3, is thereby brought into proximity of the IKK complex to phosphorylate the IKKβ subunit. IKKβ phosphorylation activates the IKK complex, which phosphorylates the NF-κB inhibitor IκB, inducing its degradation to allow nuclear entry of NF-κB (4). In parallel, TAK1 contributes to AP-1 activation via the MKK7-JNK pathway (**Figure 3**) (59).

Interestingly, a recent study suggests a novel regulatory mechanism for MALT1-dependent TRAF6 recruitment by alternative splicing of MALT1 (50). In primary T cells, two MALT1 isoforms can be expressed, namely MALT1A or MALT1B, the latter of which lacks 11 amino acids encoded by exon 7 (50). These 11 amino acids encode for the first TRAF6 binding site, localized between the second Ig like domain and the paracaspase domain of MALT1 (5, 50) (**Figure 2**). Resting primary T cells express MALT1B, while MALT1A expression is induced upon TCR triggering (50). Therefore, TCR-induced expression of MALT1A likely reinforces the TRAF6-recruiting scaffolding function of the CBM complex (50).

Collectively, these findings support the notion that formation of the CBM complex has a key role in signal transmission as a protein recruitment scaffold.

### THE CBM COMPLEX PROMOTES MALT1 PROTEOLYTIC ACTIVITY

The above-described steps of protein recruitment downstream of the CBM complex are most likely early signaling events that occur within the first minutes of lymphocyte stimulation, since peak IκB phosphorylation is detectable within a few minutes after antigen receptor engagement. The scaffolding function of MALT1 may then be amplified and propagated by its assembly with the BCL10 filamentous structures that are nucleated by CARMA1, as depicted in **Figures 2**, **3**. The activation of the MALT1 protease activity most likely depends on further amplification of the signal through filament formation, since optimal MALT1 activity is detectable later than IKK activation, and peaks roughly 30 min after antigen receptor engagement (11, 12, 19, 20). How exactly filament formation could contribute to MALT1 activation remains unknown. Interestingly, monoubiquitination on K644 within the Ig3 domain of MALT1 is required to license MALT1 protease activity (39). Indeed, a point mutation of K644 preventing monoubiquitination of MALT1 has strongly reduced protease activity and an impaired capacity to sustain IL-2 production in activated T cells (39). The incorporation of MALT1 into filaments might promote MALT1 activation in various ways, for example by allowing recruitment of the (unknown) ubiquitin ligase responsible for MALT1 monoubiquitination and/or assuring physical proximity between MALT1 and its substrates (**Figure 3**).

### MALT1 CLEAVES VARIOUS SUBSTRATES WITH DIVERSE BIOLOGICAL CONSEQUENCES

The last 10 years have witnessed impressive progress in the understanding of MALT1 protease function and its role in NF-κB- dependent and -independent aspects of lymphocyte activation. The best-explored function of MALT1 protease activity is its contribution to NF-κB activation. While MALT1 scaffold-dependent IKK activation promotes rapid but transient NF-κB activation, long-lasting NF-κB activation is assured by the MALT1-dependent cleavage of negative regulators of NF-κB, including RelB (10) and A20 (11), together with MALT1 autoprocessing (45, 46) (**Figure 3**). RelB plays a critical role as an inhibitor of the canonical NF-κB pathway in lymphocytes, by forming transcriptionally inactive complexes with RelA and c-Rel (10). MALT1-dependent RelB cleavage results in RelB proteasomal degradation, thereby favoring RelA-, and c-Reldependent NF-κB activation (10). MALT1-dependent cleavage of the deubiquitinating enzyme (DUB) A20, on the other hand, relieves its inhibitory role in NF-κB activation (11). How exactly A20 cleavage promotes NF-κB activation remains incompletely understood. It has been proposed that A20 negatively regulates the scaffolding function of MALT1 by removing the MALT1 polyubiquitination (60), however this function of A20 does not seem to be affected by its cleavage, since MALT1 inhibitors do not affect IKK-mediated NF-κB activation. A third cleavage event that is important for NF-κB activation is MALT1 autoprocessing (45, 46). MALT1 is autoprocessed after R149, between the Nterminal DD and the first Ig domain. This cleavage is essential for NF-κB activation downstream of its nuclear accumulation by mechanisms that are not yet elucidated (**Figure 2**) (45). Finally, NF-κB can be negatively regulated by the recently identified MALT1 substrate HOIL1, which is part of the LUBAC complex (61, 62). HOIL1 cleavage by MALT1 diminishes linear ubiquitination of cellular proteins, and has thus been proposed to provide negative feedback on the NF-κB pathway (61, 62).

Another important transcriptional pathway that is regulated by MALT1 is the JNK/AP-1 pathway. While the scaffold function of MALT1 is required for JNK activation (63), the protease activity of MALT1 provides an additional layer of control, via the cleavage of the deubiquinating enzyme (DUB) CYLD (64). CYLD cleavage is unlikely to affect JNK activation, since MALT1 knockin mice showed no defect in inducible JNK phosphorylation (13, 14, 65, 66), Thus, CYLD cleavage regulates AP-1 through another mechanism that remains to be explored (**Figure 3**). Despite the described role of CYLD in the negative regulation of NF-κB and the fact that CYLD shares certain substrates with A20 (such as TRAF2, TRAF6, RIP1, and IKKγ) (67), CYLD cleavage is not implicated in TCR-dependent canonical NF-κB activation (64).

Several studies have identified additional NF-κB- and AP-1 independent roles for MALT1. One of these roles is the control of the stability of particular subsets of mRNA transcripts. The MALT1-dependent cleavage of the RNAse Regnase-1 (also known as MCPIP-1 or Zc3h12a) (68) and of the two RNA binding proteins Roquin-1 and Roquin-2 (69) has been shown to stabilize mRNAs by distinct mechanisms. Regnase-1 is an RNAse that cleaves stem loop structures in the 3′ untranslated region (UTR) of mRNAs, including the mRNAs of Regnase-1 itself, c-Rel, ICOS, OX40, IL-2, and IL6 (70). T cell specific Regnase-1 knockout mice developed spontaneous inflammation and premature death, demonstrating the crucial role of Regnase-1 in the regulation of mRNA stability during the adaptive immune response (68). Roquin-1 and Roquin-2 proteins bind to mRNAs via their ROQ domain, which recognizes a secondary RNA structure called constitutive decay element (CDE), and regulate gene expression at the posttranscriptional level (71). Roquin-1 and its paralog Roquin-2 are functionally interchangeable (72). To degrade mRNAs, Roquins recruit an mRNA deadenylase complex via their C-terminal part (71) and regulate the halflife of key cytokines, chemokines, and costimulatory proteins such as ICOS, OX40, and TNF (71, 73, 74). Similar to Regnase-1 deficient mice, Roquin mutant mice (expressing a Roquin mutant that cannot bind CDEs), develop autoimmunity (73). Regnase-1 and Roquins regulate mRNA decay of common and Regnase-1- or Roquin-specific mRNAs. During T cell triggering, the combined mRNAse activity of Regnase-1 and the mRNA decay activity of Roquins are overcome by their MALT1-dependent cleavage and degradation (**Figure 3**) (68, 69).

Finally, the protease activity of MALT1 also regulates transcription-independent cellular functions by the cleavage of BCL10, which promotes lymphocyte adhesion through β1 integrins (11, 12, 19, 20). MALT1-mediated BCL10 cleavage removes 5 amino acids from its C-terminus (11, 12, 19, 20). This cleavage is not required for antigen-dependent NF-κB activation but is important for integrin-mediated T-cell adhesion (11, 12, 19, 20). The molecular mechanisms controlling adhesion via BCL10 remain elusive (**Figures 2**, **3**).

Thus far, 8 different substrates of MALT1 have been functionally described in activated lymphocytes, and two additional substrates (NIK and LIMA1) have been reported as specific substrates of an oncogenic cIAP2-MALT1 fusion protein specifically expressed in MALT lymphoma (75, 76). We can therefore reasonably anticipate that MALT1 has additional substrates with functions that are much wider than anticipated. For example, MALT1 protease function has been suggested to play a key role in metabolic pathways (77). Glutamine uptake is rapidly initiated upon naive T cell activation via the amino acid transporter ASCT2 and this uptake is crucial for activation of the mammalian target of rapamycin complex 1 (mTORc1). Interestingly, both glutamine uptake and mTORc1 activation required proteins of the CBM complex and MALT1 protease activity (77). This suggests a novel role for the MALT1 protease function in the regulation of glutamine uptake via the regulation of mTORc1 and/or ASCT2 (77). Whether this regulation is mediated by one of the already described substrates of MALT1, or whether a yet unidentified MALT1 substrate can modulate the mTORc1 signaling pathway remains to be explored.

### MICE LACKING MALT1 EXPRESSION OR ACTIVITY HAVE DISTINCT IMMUNOLOGICAL PHENOTYPES

The protease activity and most of the substrates of MALT1 have been initially identified and characterized in cell lines. However, recent studies using mouse models have lent strong support to the physiological relevance of MALT1 protease activity in adaptive immunity. MALT1-deficient mice show impaired B- and T-cell responses together with defects in the development of particular lymphocyte subsets such as marginal zone (MZ) and B1 B cells (63, 78). Moreover, these mice are resistant to experimental induction of experimental autoimmune encephalitis, a mouse model of multiple sclerosis (79, 80). Mice expressing a catalytically inactive mutant of MALT1 [C472A knock-in mice, subsequently called MALT1-protease inactive (PI) mice] recapitulate these aspects of the immunodeficiency phenotype of the MALT1 knockout mice to a large extent. Lymphocytes isolated from the MALT1-PI mice also revealed defects in NF-κB activation, AP-1 activation, mRNA stability and lymphocyte adhesion that are all consistent with previous studies from cell lines (13, 14, 65, 66, 81). Despite these impaired responses, the lymphocytes isolated from MALT1-PI mice showed normal antigen receptor-mediated activation of IKK or JNK (63, 78). The impaired cellular responses and the associated immunodeficiency phenotype of the MALT1-PI mice results thus principally from the inactivation of the proteolytic activity of MALT1.

In addition to defects in T- and B-cell responses, MALT1 deficient mice have impaired myeloid and mast cell responses upon Fc-receptor stimulation, reduced NK cell responses and defective innate immunity to yeast infections (63, 78, 82–84). The cellular responses of myeloid and NK cells have also been assessed in MALT1-PI mice and found to be defective.

A surprising finding of all four studies reporting MALT1- PI mice, however, is that these mice develop autoimmune symptoms, in particular a severe autoimmune gastritis of early onset, which progresses with age (13, 65, 66). The observed autoimmunity is likely the result of reduced development of natural Treg (nTreg) cells in MALT1-PI mice (13, 65, 66). Indeed, the autoimmune phenotype of MALT-PI mice can be rescued through transfer of wildtype Tregs into newborn MALT1-PI animals (13, 65). An even stronger reduction of nTregs is found in MALT1-deficient mice (13, 65, 66). However, MALT1-deficient mice show no autoimmune pathology, most likely because their lymphocyte responses are more dramatically affected (13, 14, 63, 65, 66). The reduced Treg numbers are likely caused by the absence of MALT1 function during development, since no spontaneous autoimmunity develops in adult mice treated with small molecule MALT1 inhibitors for up to 17 days (85). Indeed, young MALT1-PI mice do not show autoimmune features and are completely resistant to induction of experimental autoimmune encephalitis (13, 65). While the contribution of individual MALT1 substrate(s) to nTreg development remains largely unknown, a recent study suggests that MALT1 autoprocessing is important for nTreg development (46). Indeed, mice expressing an autoprocessingresistant mutant of MALT1 have a partial Treg deficit that results in enhanced anti-tumor immunity without causing autoimmunity (46).

Mice deficient for the individual CARMA/CARD family members have additional phenotypic features, including a defect in innate immune responses against bacteria, fungi or viruses in CARD9 KO mice (82, 86, 87) and a specific defect in NF-κB signaling upon stimulation of GPCRs in CARMA3-deficient mice (88). Whether these phenotypes depend on MALT1 protease activity remains largely unexplored. In addition, roughly half of CARMA3- and one third of BCL10-deficient mice die prematurely due to a neuronal tube defect, suggesting a common role for CARMA3 and BCL10 in embryonic development (88, 89). This defect is neither present in MALT1-deficient nor in MALT-PI mice and therefore likely depends on MALT1-independent signaling features of CARMA3 and BCL10. CARMA2/CARD14-deficient mice have been recently generated and found to be resistant to psoriasis induction by imiquimod cream or recombinant IL-23 injection (90). The individual contributions of keratinocytes and γδ T cells to the CARMA2/CARD14 dependentinflammation and the extent to which MALT1 protease activity contributes to inflammatory skin reactions remain to be further investigated.

### MALT1 DEREGULATION IS ASSOCIATED WITH IMMUNODEFICIENCY AND LYMPHOMA

A deregulation of MALT1 function is associated with the development of various diseases. Patients with inactivating MALT1 mutations develop immunodeficiency (91–93) or immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX)-like syndrome (94). Patients with an oncogenic activation of the MALT1 signaling pathway, on the other hand, develop lymphoid malignancies or lymphoproliferative disease (**Figures 1**,**4**) (95, 96). An increasing number of T and B cell malignancies are now recognized to be driven by constitutive antigen receptor signaling as a result of foreign or self-antigen recognition (97) and/or gain-of function mutations (**Figures 4A,B**) that trigger CBM complex formation and downstream signaling (95, 98–104). Such mutations can be found in the CBM component CARMA1 itself (98) or in the upstream components of the pathway, including the B-cell receptor-associated CD79 chains (103), the T-cell coreceptor CD28, PLCγ1 and PKCβ (95). Lymphomas with such mutations include the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) (98, 99, 103, 105, 106), in which chronic active BCR signaling is often combined with oncogenic activation of the TLR signaling component MyD88 (107). Constitutive CBM-dependent signaling is also found in mantle cell lymphomas (MCL) (104, 108, 109), chronic lymphocytic leukemia (CLL) (110), acute T-cell leukemia/lymphoma (ATLL) (102), cutaneous T-cell lymphoma (CTCL) and Sézary

upstream regulators or in-frame gene fusions of the T-cell co-receptor CD28 with ICOS or CTLA-4 are thought to promote MALT1 constitutive activation. (B) In the indicated B-cell lymphomas constitutive MALT1 protease activity can be driven by oncogenic mutations in CARMA1, CD79A or CD79B, which may be relevant in combination with self-antigen recognition. A subset of MCL and CLL are also addicted to this pathway by unknown mechanisms. (C) A large proportion of MALT lymphoma harbors a chromosomal translocation leading to formation of an oncogenic MALT1-API2 fusion protein. (D) In primary effusion lymphoma (PEL), NF-κB is activated through viral latency proteins such as K13 and K15, which activate NF-κB directly or via MALT1-BCL10. (A,B), mutations and self-antigen are depicted with a yellow star. (A,D) Open questions are depicted with red question marks. PTCL, peripheral T-cell lymphoma; ATLL adult T-cell leukemia/lymphoma; ABC, activated B-cell subtype; DLBCL, diffuse large B-cell lymphoma; BENTA, B-cell expansion with NF-κB and T-cell anergy; MCL, mantle cell lymphoma; CLL, chronic lymphocytic/leukemia.

syndrome (100, 101), as well as peripheral T-cell lymphomas (PTCL) (111) (**Figures 4A,B**). The relevance of the CBM complex and MALT1 protease activity were formally shown by silencing of the individual CBM components in ABC DLBCL (112, 113) and MCL (104) cell lines, or by inhibition of MALT1 proteolytic function in ABC DLBCL (113, 114), MCL (104), and CLL (110) cell lines. Interestingly, gain of function mutations of CARMA1 were also reported in patients with a congenital lymphoproliferative disease known as BENTA (B-cell expansion with NF-κB and T-cell anergy) syndrome (115–117) (**Figures 4A,B**).

In some types of lymphomas, constitutive MALT1 activation is achieved by mechanisms that bypass the classical CBM signaling events. These include chromosomal translocations involving the MALT1 or BCL10 genes, which are found in MALT lymphomas (1). Such translocations can lead to overexpression of the BCL10 or MALT1 genes or, more frequently, to the generation of an oncogenic API2-MALT1 fusion protein. This fusion protein activates the classical (canonical) and alternative (non-canonical) NF-κB pathways through its scaffold and protease function thereby bypassing the BCR-dependent upstream signaling events (1). The API2- MALT1 fusion protein has constitutive protease activity and cleaves NIK (76) and LIMA1α (75), two non-canonical MALT1 substrates recruited by the cIAP2 moiety (**Figure 4C**). NIK cleavage leads to stabilization of NIK, which promotes activation of the non-canonical NF-κB pathway by phosphorylation and proteolytic maturation of the NF-κB2 precursor p100 into a mature p52 NF-κB2 subunit (76). Cleavage of LIMA1α disrupts the tumor suppressor function of this protein and promotes B cell proliferation and adhesion via the cleavage of its Lim domain, an LMO-like oncogenic protein (75) (**Figure 4C**). The cleavage of NIK and LIMA1α seems to be a specific oncogenic feature of the cIAP2-MALT1 fusion protein since it is not known to be triggered by antigen receptor-mediated MALT1 activation.

Recently, we have uncovered a role for MALT1 protease activity in the development of primary effusion lymphoma (PEL) (118). PEL is a lymphoid tumor triggered by Kaposi's sarcoma herpes virus (KSHV), an oncogenic virus also known as human herpes virus-8 (HHV8). KSHV-derived tumors derive from latently infected B cells that are characterized by constitutive NFκB activation, which suppress the lytic program of the virus. The latency program is controlled by different viral latency genes such as K13 and K15, encoding a viral FLIP (Flicelike inhibitory protein) homolog and a membrane protein homologous to the latent membrane protein 2A (LMP2A), respectively. K13 and K15 induce constitutive activation of NF-κB via the protease and scaffold activity of MALT1 (118) (**Figure 4D**). Pharmacological inhibition or silencing of MALT1 leads to the reactivation of the viral lytic program and induces cell death of PEL cell lines in vitro. MALT1 inhibitors also efficiently block the development of PELs and induce their regression in vivo, in a xenograft model (118). Interestingly, K13 and K15 contribute to MALT1 activation in a CARMA1-independent manner (**Figure 4D**). Whether K13 and K15 mediate MALT1 activation via other CARMA/CARD proteins, or in an entirely distinct manner, remains to be explored.

### MALT1 DEREGULATION IS ASSOCIATED WITH ALLERGY, INFLAMMATORY SKIN DISEASES AND CARCINOMA

Deregulations of MALT1 function are not restricted to the above-described lymphoid pathologies but may also play wider roles in allergies, psoriasis, and various non-lymphoid cancers (**Figure 1**).

Evidence for a potential contribution of MALT1 to allergic asthma comes from two studies showing that CARMA1 is important in mouse models of allergic asthma (119, 120). CARMA3 has also been shown to play an important role in allergic airway inflammation in mice (121). The exact upstream events triggering CBM activation in these models remain undefined.

CARMA2/CARD14-mediated activation of MALT1 has been shown to play a major role in the activation of human keratinocytes upon exposure to the yeast cell wall component zymosan or S. aureus (15). Interestingly, activating mutations in the CARMA2-encoding gene CARD14 have been found to be associated with development of psoriasis, an inflammatory skin disease, in human patients (122, 123). Since then, an increasing number of studies have reported genetic mutations in CARD14 associated to psoriasis diseases (124). Some of the CARD14 mutations found in patients were shown to enhance NF-κB activation and promote secretion of CCL20 and IL-8, two chemokines known to be associated with psoriasis (123, 124). Similar to CARD11 mutations associated with lymphoid malignancies, CARD14 mutations were mostly found in the CC region. This suggests a similar mechanism of activation, via induction of an active CARD14 conformation and oligomerization necessary for CBM complex formation and downstream signaling events. In support of this model, a recent study showed that a single point mutation in CARD14 E138A, which renders CARD14 hyperactive, was sufficient to drive spontaneous psoriasis disease in mice (125). Inhibition of MALT1 protease activity effectively reduces cytokine and chemokine expression induced by overexpression of this hyperactive CARD14 mutant in human primary keratinocytes (126). Therefore, topical application of MALT1 inhibitors may be a future treatment option for psoriasis patients.

Finally, an increasing number of publications support a role for aberrant MALT1 signaling in the development of nonlymphoid cancers, such as glioblastoma (127), breast cancer (18, 128, 129), melanoma (130), lung cancer (18), and various other carcinomas (1, 131, 132). In these cancers, MALT1 seems to be activated by constitutive signaling from GPCRs or the EGFR via CARMA3 and/or by overexpression of MALT1 through various mechanisms.

For breast cancer, two different subtypes, which are HER2 or angiotensin II receptor (AGTR1) positive, have been described to be addicted to CBM signaling via CARMA3 (18, 128, 129). Interestingly, these two subsets show abnormal expressions of AGTR1 or HER2 in a mutually exclusive manner (128). In AGTR1 and HER2 positive breast cancer cell lines, NF-κB is activated upon stimulation with Angiotensin II or the HER2 ligand heregulin, respectively, in a CARMA3-dependent manner (18, 128, 129). Silencing of CBM components abrogates tumor cell growth, migration, and invasion (18, 128, 129) and affects the tumor microenvironment, since endothelial cell chemotaxis and angiogenesis are abolished (128).  It remains possible that other breast cancer subtypes depend on constitutive signaling by other GPCRs that can trigger NFκB signaling via CARMA3 (134). In addition to its relevance in breast cancer, CARMA3 has been shown to play a role in lung cancer (18). In this context, EGFR signaling via CARMA3, BCL10, and MALT1 promotes tumorigenesis through effects on NF-κB activation, cell proliferation, migration and invasion. The relevance of the proteolytic activity of MALT1 was not formally assessed in the above-cited models of breast cancer (128) and lung cancer (18). It would thus be interesting to test the potential effectiveness of pharmacological MALT1 inhibitors in these models.

The relevance of MALT1 as a protooncogene has been recently extended to additional malignancies including melanoma (130) and glioblastoma (127), in which MALT1 can be overexpressed. Indeed, MALT1 is highly expressed in a subset of melanoma associated with poor prognosis (130). MALT1 downregulation or inhibition with the MALT1 inhibitor MI-2 (135), diminishes the growth and the dissemination of melanoma cells in the lung (130). While the proliferation of these melanoma cells is NF-κB dependent, the dissemination is JNK/c-Jun-dependent (130). In glioblastoma, it was shown that a subset of tumors with poor outcome expresses low levels of the microRNA miR181d, leading to high NF-κB activation. One of the targets of miR181d is MALT1, which is highly expressed in the miR181dlow tumors (127). Ectopic expression of miR181d and the consequent downregulation of MALT1 suppresses growth of tumor cells (127). These two studies suggest that, like in a subset of MALT lymphomas (136), MALT1 overexpression per se may play a role in promoting carcinogenesis, potentially by spontaneous aggregation-mediated activation of MALT1. Future studies should address how MALT1 promotes cellular transformation in various cancers, whether and how specific substrates of MALT1 contribute to tumorigenesis and if MALT1 inhibitors might be of therapeutic interest.

### MALT1 INHIBITORS MAY BE OF THERAPEUTIC INTEREST FOR VARIOUS PATHOLOGIES

Several recent studies have reported the development of small molecule MALT1 inhibitors that act as active site or allosteric inhibitors (135, 137–140). These have therapeutic effects in preclinical (mouse) models of diffuse large B-cell lymphoma, (135, 137, 138) and primary effusion lymphoma (118). The idea that MALT1 inhibitors may be useful to dampen inflammatory immune responses has received support from studies using a mouse model of multiple sclerosis, in which paralysis symptoms are alleviated by treatment with a MALT1 inhibitor, or by genetic inactivation of MALT1 (13, 65, 85). The latter also protects mice from experimental induction of colitis (13). Moreover, pharmacological inhibition of MALT1 protects mice from LPS-induced inflammation and lung injury, most likely through inhibitory effects on myeloid cells (141). Nevertheless, the assumption that MALT1 inhibitors will generally dampen immune responses has been challenged by the observation that mice expressing a catalytically inactive form of MALT1 have reduced numbers of natural Treg cells and, as a consequence, progressively develop inflammatory autoimmune disease (13, 14, 65, 66). This raises the interesting possibility that, at least in the context of cancer, MALT1 inhibitors may exert their effects by a dual action on both

### REFERENCES

the inhibition of the growth of cancer cells and boosting antitumor immune responses. Another possible field of application for MALT1 inhibitors are inflammatory skin diseases such as psoriasis, in which inhibitory effects on keratinocytes and possibly on skin T cells may benefit the patients (15, 124–126). As we progress in our knowledge about the molecular and biological function of MALT1, future applications are likely to emerge.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

The Thome laboratory is supported by grants from the Swiss National Science Foundation (310030\_166627), the Swiss Cancer League (KFS-4095-02-2017) and the Emma Muschamp Foundation.

### ACKNOWLEDGMENTS

The authors would like to acknowledge Colin Moore, Laurence Romy, and Julie Vérièpe for comments on the manuscript and Slavica Masina for proofreading.

in lymphocytes and lymphoma cell lines. Proc Natl Acad Sci USA. (2011) 108:14596–601. doi: 10.1073/pnas.1105020108

mRNAs and control follicular helper T cell differentiation. Immunity (2013) 38:655–68. doi: 10.1016/j.immuni.2012.12.004

and -independent signaling pathways. Signal Transduct Target Ther. (2016) 1:16013. doi: 10.1038/sigtrans.2016.13

subtype of diffuse large B-cell lymphoma. J Med Chem. (2015) 58:8491–502. doi: 10.1021/acs.jmedchem.5b01415

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Juilland and Thome. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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

Thomas Seeholzer 1†, Susanne Kurz 1†, Florian Schlauderer <sup>2</sup> , Simone Woods <sup>1</sup> , Torben Gehring<sup>1</sup> , Simon Widmann<sup>1</sup> , Katja Lammens <sup>2</sup> and Daniel Krappmann<sup>1</sup> \*

<sup>1</sup> Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany, <sup>2</sup> Gene Center, Ludwig-Maximilians University, Munich, Germany

#### Edited by:

Andrew L. Snow, Uniformed Services University of the Health Sciences, United States

#### Reviewed by:

Lawrence Kane, University of Pittsburgh, United States Joel L. Pomerantz, Johns Hopkins University, United States Liron David, Boston Children's Hospital, Harvard Medical School, United States

> \*Correspondence: Daniel Krappmann daniel.krappmann@ helmholtz-muenchen.de

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology

Received: 14 August 2018 Accepted: 31 October 2018 Published: 20 November 2018

#### Citation:

Seeholzer T, Kurz S, Schlauderer F, Woods S, Gehring T, Widmann S, Lammens K and Krappmann D (2018) BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation. Front. Immunol. 9:2695. doi: 10.3389/fimmu.2018.02695 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.

Keywords: lymphocyte signaling, CARMA1–BCL10–MALT1 (CBM) signalosome complex, CARD11, NF- kappa B, MALT1 paracaspase

### INTRODUCTION

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signalosome channels T and B cell antigen-receptor (TCR/BCR) ligation to MALT1 protease activation and canonical NF-κB signaling (1, 2). CARD11 phosphorylation, primarily in the central linker region, following antigenic stimulation induces conformational changes that expose the N-terminal CARD (Caspase Recruitment Domain) to recruit pre-assembled BCL10-MALT1 complexes (3, 4). Oncogenic

**49**

CARD11 variants have been identified mainly in the coiledcoil domain, and these activating mutations promote chronic CBM assembly and NF-κB-driven survival in diffuse large B cell lymphomas (DLBCL) in the absence of antigenic stimulation (5, 6).

It is well established that BCL10 associates with the CARDcontaining scaffold protein CARD11 through heterotypic CARD-CARD interactions (7, 8). Overexpression studies indicate that BCL10, via its N-terminal CARD, forms filamentlike clusters in cells, which are required for proper activation of canonical NF-κB signaling (9). Aggregation of BCL10 in foci was also observed following TCR ligation in T cells (10). More recent in vitro structural studies, combined with molecular modeling, have demonstrated that the CARD of CARD11 can function as a seed to nucleate the assembly of BCL10 CARD filaments (11–13). In vitro BCL10 filaments can also form in the absence of CARD11, but CARD11 decreases the lag period of BCL10 polymerization and thus appears to function as an initiator of the process (11). Impaired MALT1 activity and NF-κB signaling upon overexpression of CARD11 or BCL10 mutants, targeting either the heterotypic CARD11-BCL10 or the homotypic BCL10-BCL10 CARD interfaces, highlights the importance of the different CARD surfaces (12, 13). These experiments, however, did not address the contribution of the different interfaces to antigenic activation when expressed at endogenous levels. We have demonstrated that BCL10 oligomerization is also required for its recruitment to CARD11, indicating that both processes are highly interconnected (14). Thus, the cellular relevance of the CARD11 seeding function for BCL10 filament formation, as well as, the order of events after antigenic stimulation, have not been resolved.

Here, we used CRISPR/Cas9 technology to generate CARD11 and BCL10 KO T and B cell lines and stable lentiviral reconstitution, to investigate the cellular necessity of the CARD11 seed and BCL10 filaments in a clean genetic setup under physiological conditions. As noted earlier, we have been unable to definitively determine whether CARD11 nucleates BCL10 filaments or, if BCL10 filament formation happens prior to, or at the same time as CARD11 recruitment in stimulated T cells using missense mutations in the putative CARD11-BCL10 or BCL10- BCL10 interfaces alone (14). Thus, we uncoupled these processes by fusing BCL10 to CARD11 to bypass inducible recruitment and thereby were able to investigate the cellular necessity of CARD11 seeding and BCL10 oligomerization.

### RESULTS

### BCL10 Recruitment to CARD11 and BCL10 Filament Assembly Are Interconnected Processes

In order to predict mutations that would selectively interfere with CARD11 seed function or BCL10 self-assembly, we used structural modeling to fit the CARD11 seed onto the structure of BCL10 CARD filaments (**Figure 1A**). Therefore, the CARD11 CARD domain crystal structure was superimposed on to three BCL10 CARD domains at the bottom of the BCL10 filament cryo EM structure [**Figure 1A**; (13, 14)]. Since the structure of the CARD11-BCL10 interface has not been determined, the CARD11 BCL10 CARD/CARD interaction was modeled in consideration of the surface charge complementarity analysis of the BCL10-MALT1 filament cryo-EM structure and the crystal structure of the CARD11 (**Supplementary Figures 1A–C**). As noted earlier, distinct interfaces between the CARDs are required to mediate heterotypic CARD11-BCL10 interactions or homotypic BCL10-BCL10 interactions (12, 13). On the CARD11 side, R35 serves as a critical contact point to multiple residues in BCL10 including E53 and mediates recruitment of BCL10 to CARD11 [**Figure 1B**; (12)]. On BCL10, R42 contributes to the association at the BCL10-BCL10 interface I that controls BCL10 oligomerization (**Figures 1C,D**). The structure reveals that R42 is not predicted to confer CARD11- BCL10 interaction (11, 13, 14). In vitro, the BCL10 CARD mutation R42E prevents oligomerization of the BCL10-MALT1 complex (14). We expressed BCL10 WT and R42E in adherent U2OS cells to monitor formation of cellular clusters. Indeed, whereas overexpressed BCL10 WT forms cytosolic aggregates in U2OS cells, BCL10 R42E fails to cluster, indicating that oligomerization and filament formation is prevented by the mutation (**Figure 1E**). We performed co-immunoprecipitation (IP) of HA-BCL10 WT, together with Flag-BCL10 WT or R42E in HEK293 cells (**Figure 1F**). Despite the higher expression of BCL10 R42E, only Flag-BCL10 WT co-precipitated with HA-BCL10, validating that the mutation abolishes BCL10 self-association.

To rigorously test the function of CARD11 as a molecular seed and BCL10 as a filament forming CARD11 adaptor in B and T cells, we generated CARD11 and BCL10 KO Jurkat T cells, as well as CARD11 KO BJAB B cells, by CRISPR/Cas9 technology (**Supplementary Figure 2**). CARD11 KO Jurkat T and BJAB B cells were generated using sgRNA targeting Exon3, which induces double stranded breaks and frame shift mutations due to non-homologous end-joining (NHEJ) repair (**Supplementary Figure 2A**). An exon1-intron1 deletion strategy using two sgRNA was employed to knockout BCL10 from Jurkat T cells (**Supplementary Figure 2B**). Using these approaches, we obtained several clones that displayed loss of CARD11 or BCL10 expression as determined by Western Blot analysis (**Supplementary Figures 2C–E**). Destructive frame shift mutations in CARD11 or deletions of BCL10 in both alleles were confirmed by sequencing of the genomic loci in the respective KO cell clones (data not shown). We used PMA/Ionomycin (P/I) stimulation, which bypasses upstream TCR or BCR signaling by directly activating PKCθ or PKCβ in T and B cells and increasing cytosolic calcium levels. In line with the key role of the CBM complex, CARD11 or BCL10 deficiency abolished P/I-induced NF-κB signaling in Jurkat T or BJAB B cells as evident from lack of IκBα degradation and NFκB DNA binding (**Supplementary Figures 2C–E**). In contrast, CBM-independent, TNFα-driven NF-κB signaling as well as ERK activation were not affected by the absence of CARD11 in Jurkat T cells, demonstrating that loss of the CBM complex selectively affects antigenic signaling. Thus, the absence of CARD11 or BCL10 in Jurkat and BJAB cells faithfully mirrors the signaling

defects observed in primary lymphocytes from CARD11−/<sup>−</sup> or BCL10−/<sup>−</sup> mice (15–17).

Flag-BCL10 WT or R42E mutant was determined after Flag-IP.

We then performed rescue experiments in Jurkat KO T cells to study the molecular functions of the CARD11 and BCL10 CARDs, by introducing missense mutations (CARD11 R35A and BCL10 R42E) that are both predicted to selectively interfere with heterotypic CARD11-BCL10 and homotypic BCL10-BCL10 interactions, respectively [**Figures 1**, **2A**; (12, 14)]. Jurkat

s.e.m.; \*\*p ≤ 0.01). (F) HEK 293 cells were transfected with Flag- and HA-tagged BCL10 constructs as indicated, and self-association of HA-BCL10 WT to

and effects on NF-κB signaling were determined by following IκBα degradation by WB and NF-κB activation by EMSA. (C) CARD11 KO Jurkat T cells were reconstituted as in (B), stimulated with P/I (30 min), and MALT1 protease activity was determined via MALT1-ABP PD assay and assessment of substrate cleavage (CYLD and HOIL1) by WB. (D) BCL10 KO Jurkat T cells were reconstituted with BCL10 or BCL10 R42E. Cells were stimulated with P/I (30 min) and NF-κB signaling was analyzed as in (B). MALT1 protease activity was determined by CYLD substrate cleavage by WB. (E) Recruitment of BCL10 or BCL10 R42E to CARD11 after P/I stimulation (15 min) in Jurkat T cells was monitored by ST-PD and subsequent WB. The asterisks indicate an unspecific band in the BCL10 WB.

CARD11 or BCL10 KO T cells were reconstituted by lentiviral transduction and comparable infection rates for epitope-tagged (FS: Flag-StrepTag2) CARD11 and BCL10 constructs were obtained, as determined by co-expression of the surface marker 1CD2 (**Supplementary Figures 3A,B**). Equivalent expression of WT and mutant CARD11 or BCL10 proteins at close to endogenous levels was confirmed by Western Blotting (**Supplementary Figures 3A,B**). Functionally, lack of CARD11 dependent NF-κB activation after P/I stimulation in CARD11 KO cells was rescued upon reconstitution with CARD11 WT, but not with the CARD11 R35A mutant (**Figure 2B**). We also assessed activation of the MALT1 protease by substrate cleavage (CYLD and HOIL-1) (18) and labeling of active MALT1 by a biotinylated MALT1 activity-based probe (ABP) followed by biotin pulldown (PD) to capture active MALT1 [**Figure 2C**; (19)]. Again, CARD11 WT, but not R35A, could rescue P/I stimulated MALT1 protease activation in Jurkat T cells, revealing that this mutation abolishes all CARD11 downstream function. On the side of BCL10, we confirmed that rescue of BCL10 WT, but not BCL10 R42E, was able to trigger MALT1 activation and mediate NF-κB downstream signaling in response to P/I stimulation [**Figure 2D**; (14)]. Since the structural analyses suggested that BCL10 R42E would selectively disrupt the BCL10-BCL10 but not the CARD11-BCL10 interface (13), we asked if BCL10 R42E could still be recruited to CARD11 upon stimulation. However, no stimulation-dependent binding to CARD11 was detected with BCL10 R42E mutant [**Figure 2E**; (14)]. The data demonstrate that CARD11 recruitment and BCL10 filament formation are interconnected processes. Thus, destructive mutations in the CARD11-BCL10 or BCL10-BCL10 interfaces alone are unable to resolve whether CARD11 acts as a seed to induce BCL10 oligomerization or if an initial BCL10 filament assembly may be required for the recruitment of BCL10-MALT1 to CARD11.

### BCL10-CARD11 Fusion Drives Constitutive MALT1 Activation Through BCL10 Oligomerization in Jurkat T Cells

To test the necessity for BCL10 self-association downstream of CARD11, we designed a system that bypasses inducible CARD11-BCL10 association. For this we cloned chimeric proteins that covalently fuse BCL10 through its C-terminus to the N-terminus of CARD11 (**Figure 3A**). We lentivirally transduced CARD11 KO Jurkat T cells with the SF-tagged BCL10-CARD11 construct (herein referred to as B10-C11 fusion) (**Figure 3B**). We detected a faint but distinct band corresponding to the expected size of the B10-C11 fusion using αBCL10 and αCARD11 antibodies (**Figure 3C**). Notably, expression of the B10-C11 protein triggered constitutive cleavage of CYLD and A20, as well as a reduction in endogenous BCL10, which are all MALT1 substrates (18). However, the BCL10-CARD11 fusion itself, which contains a MALT1 cleavage site at R228 in the BCL10 moiety (20), was also prone to processing, giving rise to a fragment the size of endogenous CARD11 (**Figure 3C**). Thus, the data clearly indicate that fusion of BCL10 to CARD11 is sufficient to induce MALT1 activation.

To avoid the indirect reconstitution of CARD11 from the cleaved B10-C11 fusion, we designed all further BCL10-CARD11 fusion constructs so that they contained the MALT1 cleavage resistant BCL10 R228A mutation (**Figure 3A**). The fusion constructs include the destructive mutations R42E in the BCL10 CARD, and R35A in the CARD11 CARD, both alone and in combination. All B10-C11 fusion constructs were transduced into CARD11 KO Jurkat T cells, yielding equivalent infection efficiencies as judged by 1CD2 expression (**Figure 3D**). The chimeric proteins were expressed below the level of endogenous CARD11 and moreover, the fusions containing a functional WT BCL10 CARD were consistently expressed at lower levels compared to the BCL10 mutants R42E that prevent BCL10 oligomerization (**Figure 3E**). StrepTactin pull-downs (ST-PD) demonstrated that the BCL10-CARD11 fusion proteins retained the ability to bind endogenous MALT1, excluding that the fusion or point mutations in the CARDs interfere with MALT1 recruitment (**Figure 3E**).

Using biotin-PD after incubation with bio-MALT1-ABP, we tested MALT1 protease activity in extracts of untreated and P/I stimulated Jurkat T cells (**Figure 3F**). Expression of B10 R228A-C11, containing two functional CARDs, as well as the B10 R228A-C11 R35A fusion, with an inactivation only in the CARD11 CARD, induced strong MALT1 protease activity that was equivalent to the activation obtained in Jurkat T cells after P/I stimulation. Despite robust MALT1 activation, the R228A exchange in BCL10 prevented cleavage of the fusion constructs and thus the appearance of CARD11 (**Figure 3E**). Constitutive MALT1 activation was abrogated in the B10-C11 fusion proteins carrying the oligomerization-defective BCL10 R42E mutation, providing evidence that constitutive MALT1 activation is driven through oligomerization of endogenous BCL10 via the BCL10 CARD (**Figure 3F**). Interestingly, MALT1 activity was further enhanced after stimulation of B10 R228A-C11 expressing cells and this strictly relied on the CARD11 CARD, because the R35A mutation prevented stimulation-dependent induction. In line, even though the B10 R42E/R228A-C11 fusion containing an intact CARD11 CARD was unable to induce constitutive MALT1 activation, it was still able to mediate MALT1 activation in P/I-stimulated Jurkat T cells. Thus, stimulation dependent activation seems to rely on the recruitment of endogenous BCL10 to the CARD11 CARD in the context of the BCL10-CARD11 fusion protein. In fact, the B10 R42E/R228A-C11 fusion acted completely independently of the BCL10 CARD and exactly mirrored the rescue observed when using CARD11 WT (see **Figures 2B,C**). In agreement with these data, the triple mutant B10 R42E/R228A-C11 R35A neither promoted constitutive, nor rescued, stimulus-dependent MALT1 activation.

To confirm these findings on the level of MALT1 substrates, we assessed cleavage of CYLD, HOIL1 and A20 in B10-C11 expressing cells (**Figure 3G**). Cleavage of the three substrates was observed in Jurkat T cells expressing the fusions with an intact BCL10 CARD (B10 R228A-C11 and B10 R228A-C11 R35A), confirming that constitutive MALT1 protease activity relies on BCL10 oligomerization. The intact CARD11 CARD in B10 R42E R228A-C11 still conferred inducible substrate cleavage, which was especially evident for HOIL1 that is also most strongly

FIGURE 3 | A chimeric BCL10-CARD11 fusion protein induces constitutive MALT1 protease activity in Jurkat T cells through the BCL10 oligomerization interface. (A) Schematic representation of BCL10-CARD11 (B10-C11) fusion proteins with the respective CARD mutants. (B) Transduction efficiency of CARD11 KO Jurkat T cells with BCL10-CARD11 (B10-C11) was analyzed by FACS using the surface marker 1CD2. (C) Protein expression of B10-C11 fusion construct and cleavage of MALT1 substrates compared to mock and parental Jurkat T cells was analyzed by WB. (D) Transduction efficiency of CARD11 KO Jurkat T cells with different B10 R228A-C11 fusion constructs as in (B). (E) Expression of the different B10 R228A-C11 fusion proteins compared to mock and parental Jurkat T cells was analyzed by WB and binding to MALT1 assessed by Strep-PD. (F) CARD11 KO Jurkat T cells were reconstituted with B10 R228A-C11 fusion proteins as indicated. Active MALT1 prior or after P/I stimulation (30 min) was detected in the extracts with biotin-labeled MALT1-ABP and MALT1-ABP PD followed by WB. (G) CARD11 KO Jurkat T cells were reconstituted and treated as in (F) and cleavage of MALT1 substrates (CYLD, HOIL1, A20) was detected by WB.

cleaved after P/I stimulation of Jurkat T cells. Again, destruction of both CARDs in B10 R42E/R228A-C11 R35A led to complete loss of constitutive and inducible MALT1 activation. Thus, covalent attachment of BCL10 to the N-terminus of CARD11 is sufficient to induce MALT1 protease activation, which still relies on the oligomerization interface of the fused BCL10 moiety. These data support the concept that, in cells, CARD11 acts as a seed to induce BCL10 filament assembly.

### Transient Expression of BCL10-CARD11 Induces NF-κB Activation in Jurkat T Cells

Interestingly, when we tested activation of NF-κB by B10-C11 fusion constructs in CARD11 or BCL10 KO Jurkat T cells we noticed a severely blunted response in EMSA (**Figure 4A**). There was a weak induction of constitutive NF-κB DNA binding in B10 R228A-C11, but IκBα degradation and NF-κB activation was only mildly triggered after P/I stimulation, revealing that stable expression of the active BCL10-CARD11 fusion may promote a stage of unresponsiveness in Jurkat T cells. Again, missense mutations in both CARDs completely prevented constitutive, as well as, inducible NF-κB activation in the context of the B10-C11 fusion protein. We switched to a transient transfection system and NF-κB reporter assays to investigate if BCL10- CARD11 fusion proteins can activate NF-κB. Indeed, NF-κB was strongly induced by the expression of B10 R228A-C11 fusion activated in parental Jurkat T cells or CARD11 KO Jurkat T cells (**Figures 4B,C**). However, in line with the EMSA results overall NF-κB activation was strongly diminished when the reporter assay was performed in CARD11 KO Jurkat T cells that stably express the B10-C11 fusion constructs (**Figure 4C**). Further, the oligomerization-deficient BCL10 R42E mutant severely reduced NF-κB activation by the BCL10-CARD11 fusion protein. Again, also in transient transfection we observed that BCL10-CARD11 was expressed at much lower levels compared to the B10 R42E/R228A-C11 protein, suggesting that there is a counter-selection against the expression of the active BCL10- CARD11 fusion. NF-κB induction of BCL10-CARD11 fusion protein was comparable to the induction achieved by oncogenic CARD11 L225LI or CARD11 1linker, especially taking into account the much weaker expression of the fusion protein [**Figure 4C**; (6, 21)]. To check if the generated BCL10-CARD11 fusion construct does not trigger unphysiological NF-κB that bypasses the necessity of known regulators, we determined the requirement for MALT1 and for TRAF6 recruitment to MALT1 (**Figure 4D**). NF-κB activation in response to antigenic stimulation is abolished MALT1 KO Jurkat T cells and signaling can be rescued by transduction of MALT1A or MALT1B, but not the respective MALT1 TRAF6 binding mutants (22). As expected, expression of B10 R228A-C11 or CARD11 1linker was unable to activate NF-κB in MALT1 KO Jurkat T cell (**Figure 4D**). While NF-κB activity was recovered by viral complementation with MALT1B WT, the MALT1B E795A mutant that destroys the only functional TRAF6 binding motif on MALT1B failed to rescue reporter gene expression, proving that MALT1 and TRAF6 are utilized by the BCL10-CARD11 fusion protein to activate NF-κB [**Figure 4D**; (22–24)]. Thus, while transient expression of the BCL10-CARD11 fusion protein promotes NF-κB activation, NF-κB responses are dampened in Jurkat T cells after stable expression of the fusion proteins. To corroborate whether the BCL10-CARD11 fusion protein can also compensate for BCL10 deficiency, we transduced BCL10 KO Jurkat T cells with the B10 R228A-C11 fusion protein (**Supplementary Figure 4A**). Indeed, fusion of BCL10 to CARD11 was able to trigger constitutive CYLD, A20, and HOIL1 cleavage and thus to drive MALT1 protease activation in BCL10 deficient cells (**Supplementary Figure 4B**). Again, mutation of BCL10 and CARD11 CARD (B10 R42E/R228A-C11 R35A) prevented constitutive and inducible MALT1 activation, underscoring that dimerization/oligomerization of the fusion proteins is required. Similar to the situation in CARD11 KO cells, NF-κB activation was blunted upon stable expression of B10 R228A-C11 in BCL10 KO Jurkat T cells.

### BCL10-CARD11 Fusion Acts Like Oncogenic CARD11 in BJAB B Cells

To better explore the impact of stable BCL10-CARD11 fusion on NF-κB activation, we switched to GCB DLBCL derived BJAB B cells, which under basal conditions are devoid of NF-κB activity, but overexpression of oncogenic CARD11 mutants induces strong chronic NF-κB activity (5, 6, 25). We generated CARD11 KO BJAB B cells (**Supplementary Figure 2D**) and confirmed that the phenotype of the BJAB KO cells was caused by loss of CARD11. For this we reconstituted the cells with CARD11 WT, or the oncogenic CARD11 coiled-coil (CC) mutant L225LI that induces robust NF-κB and proliferation upon overexpression in B cells [**Figure 5A**; (25, 26)]. After viral transduction, BJAB B cells expressed CARD11 WT and CARD11 L225LI slightly above endogenous levels (**Figures 5B,C**). CARD11 WT was able to recover P/I-inducible NF-κB and MALT1 protease activation, but was not associated with constitutive activation (**Figures 5D,E**). In contrast, transduction of CARD11 L225LI was sufficient to promote constitutive NF-κB activation, as well as cleavage of the MALT1 substrates BCL10, CYLD, A20 and HOIL1. This effect was not further augmented by P/I stimulation (**Figures 5D,E**). Thus, CARD11 KO BJAB B cells represent a valid system to elucidate the impact of BCL10-CARD11 fusions on NF-κB signaling.

Next, we expressed B10 R228A-C11 and B10 R42E/R228A-C11 in CARD11 KO BJAB B cells (**Figure 6A**). In addition, we sought to determine whether an oncogenic mutant of CARD11 also relies on BCL10 oligomerization. We have previously shown that the activating potential of CARD11 L225LI is abolished by the R35A mutation (25). Therefore, we expressed the B10- C11 L225LI fusion constructs in the context of the CARD destructive R35A mutation so that they should rely on the BCL10 CARD (B10 R228A-C11 R35A/L225LI and B10 R42E/R228A-C11 R35A/L225LI) (**Figure 6A**). All constructs were transduced in BJAB B cells to a similar extent, as determined by the surface marker 1CD2, but protein expression was far below endogenous CARD11 (**Figures 6B,C**). As observed in Jurkat T cells, B10 R228A-C11 constructs containing an intact BCL10 CARD were expressed at much lower levels and the variant containing

protein levels visualized on WB (mean ± s.e.m.; n = 3). \*\*p ≤ 0.01, \*\*\*p ≤ 0.001.

the oncogenic mutation (B10 R228A-C11 R35A/L225LI) was hardly detectable (**Figure 6C lane 5**). However, just like in Jurkat T cells, the B10 R228A-C11 fusion induced strong constitutive MALT1 activation, as evident from CYLD, HOIL1 and A20 cleavage, which strictly relied upon the BCL10 CARD interface (**Figure 6D**). Furthermore, the BCL10-CARD11 fusion combined with the oncogenic mutation L225LI (B10 R228A-C11 R35A/L225LI) induced MALT1 activation, despite its very low expression in the BJAB B cells. Again, constitutive MALT1 activation in the oncogenic BCL10-CARD11 fusion required BCL10 oligomerization, but was independent of the CARD11 CARD (**Figure 6D**).

Further, the fusion of BCL10 to CARD11 was sufficient to induce constitutive NF-κB activation in the BJAB B cells that was not further enhanced by P/I treatment (**Figure 6E**). Constitutive NF-κB activation was equivalent to that induced by the oncogenic CARD11 variant L225LI (**Figure 5D**). Despite its very weak expression, the oncogenic fusion construct B10 R228A-C11 R35A/L225LI also induced strong NF-κB activation, suggesting that the combination of fusion with the oncogenic mutant can generate a super-activator that is capable of further boosting CBM signaling. The weak expression of the B10 R228A-C11 R35A/L225LI suggests that there is a strong counterselection against expression of the hyper-active fusion protein. Again, the constitutive activation observed with expression of the fusion constructs was solely driven by the BCL10 filament interface and was severely reduced in cells expressing the double CARD mutant B10 R42E/R228A-C11 R35A/L225LI. Finally, we confirmed that the fusion constructs also induce NF-κB-dependent gene expression by demonstrating that the prototype NF-κB target gene, NFKBIA/IκBα, is upregulated to a similar degree in the BJAB B cells expressing either CARD11 L225LI or the activating B10-C11 fusion constructs (**Figure 6F**). Thus, with respect to signaling, the fusion of BCL10 to CARD11 acts like an oncogenic CARD11 variant in B cells, through oligomerization of the BCL10 CARD interface.

### DISCUSSION

BCL10 CARD filaments are formed in vitro and the molecular architecture of these clusters has been elucidated by cryo-electron microscopy (11, 13, 14). Although it is possible for BCL10 filaments to form in the absence of CARD11, the CARD of CARD11 appears to promote the initiation of BCL10 clustering in a cell free system. Further, CARD11 is solely located at the tip of the BCL10 filaments, lending support to the hypothesis that CARD11 functions as the molecular seed for BCL10 oligomerization (11, 13). Structure-guided missense mutations in the CARDs of CARD11 or BCL10 have been generated to show that destruction of putatively homotypic (BCL10-BCL10) or heterotypic (CARD11-BCL10) CARD-CARD interactions impedes NF-κB signaling and MALT1 protease activation after overexpression in cells (13, 14).

The functional impact of missense mutations in the CARDs of CARD11 or BCL10 on stimulus-dependent CBM complex signaling has not been thoroughly investigated. By reconstituting CARD11 or BCL10 KO Jurkat T cells, we assessed how destruction of putative CARD11-BCL10 or BCL10-BCL10 interfaces would affect signaling when expressed at endogenous levels. As expected, the CARD11 mutation R35A prevented T

cell stimulation due to its inability to recruit BCL10 (12, 25). In addition, the BCL10-BCL10 interface mutant R42E could not rescue NF-κB signaling in BCL10 KO cells, which confirms the critical function of BCL10-BCL10 interface I for filament assembly and signaling (13). Recently, we have been able to solve the architecture of the BCL10-MALT1 filaments using cryo-EM (14). Surprisingly, we found that different mutations in the interface I that lead to loss of BCL10 filament assembly also abolished CARD11 recruitment of BCL10 and thus CBM complex formation (14). Thus, our results question if CARD11 acts as the seed to nucleate BCL10 filaments, or whether an initial oligomerization of BCL10 is required for the recruitment to CARD11 in cells. Alternatively, our data would also be in line with a model in which CARD11-BCL10 association, but not BCL10 oligomerization, is critical to promote signaling in lymphocytes, and that filament formation is only observed with recombinant BCL10 or after high overexpression of BCL10.

To clarify the necessity of the CARD11 seed and BCL10 filaments for T and B cell activation, we fused BCL10 to CARD11 to bypass the initial step of heterotypic CARD-CARD interaction between CARD11 and BCL10. Even though this is an artificial system, it provided the first opportunity to examine the contribution of the individual CARDs in CARD11 and BCL10. Remarkably, stable expression of the BCL10-CARD11 fusion protein promoted strong and chronic MALT1 protease activity in Jurkat T cells. While NF-κB activation was blunted in transduced Jurkat T cells, the transient transfection of BCL10- CARD11 activated NF-κB to a similar extent as an oncogenic CARD11 variant or CARD11 lacking the negative regulatory linker region (CARD11 1linker) (5, 6, 21). Interestingly, the BCL10-CARD11 fusion is not prone to auto-inhibition by the CARD11 linker, which acts as an inhibitory domain (27). In the fusion protein the BCL10 CARD is exposed and most likely not accessible for the CARD11 linker, especially when considering that an additional CARD cannot be bound by the linker in a 1:1 stoichiometry. Moreover, CARD11 and BCL10 CARDs are homologous, but also quite distinct and the BCL10 CARD may complex with the CARD11 linker, which would be critical for auto-inhibition (5, 27). Despite the disrupted auto-inhibition, the BCL10-CARD11 fusion protein signals to NF-κB via MALT1 and the TRAF6 binding motif on MALT1, underscoring that it utilizes the same mechanisms as a physiological CBM complex following T cell activation.

Despite the structural elucidation of BCL10-MALT1 filaments in vitro, the nature and relevance of cellular BCL10 oligomeric structures has not been fully resolved. BCL10 tends to aggregate via its CARD into oligomeric clusters and extended filaments after overexpression in cells (7, 9). Endogenous BCL10 forms oligomeric structures in antigen-stimulated T cells termed POLKADOTS ("punctuated and oligomeric killing or activating domains transducing signals"), which are cellular foci that serve as functional platforms for recruiting NF-κB signaling mediators following TCR stimulation (10, 28). Size exclusion chromatography demonstrated that in stimulated Jurkat T cells or ABC DLBCL tumor cells CARMA1, BCL10, and MALT1 assemble into higher order complexes with an apparent molecular weight >1 Mio. Da and the purified CBM complex displayed a filament-like structure in electron microscopy (13, 29). However, by increasing the concentration of BCL10 during the process of CBM purification, BCL10 filament formation may be initiated in vitro rather than taking place in living cells. Thus, there is good evidence that BCL10 can cluster via the CARD in cells, but the existence of large helical BCL10 filaments under physiological conditions in antigen-stimulated T or B cells has not been formally demonstrated. Since it may be difficult to imagine that the BCL10-CARD11 fusion itself can form long filaments in the absence of endogenous BCL10, activation by the fusion in BCL10 KO cells may indicate that BCL10-dependent dimerization or short oligomerization may be sufficient for lymphocyte activation. As for endogenous CARD11-associated BCL10, monitoring of BCL10 filament formation in the context of BCL10-CARD11 fusion is difficult with current methods, because of low expression levels. High resolution imaging techniques will be necessary to solve the extent of oligomerization and the cellular architecture of the BCL10 clusters. However, so far imaging in lymphocytes has been hampered by the unavailability of high quality antibodies as well as the small and round-shaped T and B cells that contain very little cytoplasm.

An important question that remains is the in vivo necessity for the formation of large, extended BCL10 filaments for signal propagation. The lack of NF-κB and MALT1 activation in BCL10 R42E in the context of the BCL10-CARD11 fusion reflects the need for BCL10 dimerization/oligomerization, but does not prove the requirement for higher order filaments. Alternatively, BCL10-MALT1 recruitment to CARD11 and BCL10 dimerization or short oligomers may be sufficient to facilitate cellular processes such as ubiquitination of CBM complex components. A number of ubiquitin ligases [e.g., TRAF6, LUBAC (linear ubiquitin chain assembly complex) and cIAP2] are recruited to the CBM complex and conjugation of mono-ubiquitin or poly-ubiquitin chains of different topology on all subunits has been implicated in triggering MALT1 activation and/or downstream signaling (1, 2). The complexity of ubiquitindependent regulation is exemplified by BCL10. K48-, K63-, or M1-linked ubiquitin chains are primarily conjugated on K17, K31, and/or K63 in the BCL10 CARD (30–32). Ubiquitination of these lysine residues is required for NF-κB activation in T cells and pro-survival signaling in ABC DLBCL cells (31–33). Deficiency in HOIP/RNF31, the catalytic subunit of LUBAC, suppresses NF-κB signaling in Jurkat T cells whereas HOIP activating mutations enhance NF-κB-dependent pro-survival in ABC DLBCL cells, supporting that M1-linked ubiquitin chains on BCL10 are enhancing CBM complex signaling (32, 34). However, at the same time BCL10 is degraded by lysosomal or proteasomal pathways and these processes are controlled by at least partially overlapping ubiquitination sites (31, 35–37). In addition, K48-linked poly-ubiquitination of CARD11 induces its degradation (38). Thus, the BCL10-CARD11 fusion protein is most likely strongly affected by regulatory ubiquitination and ubiquitin conjugation in each moiety may be responsible for activation, as well as the high turnover of the active BCL10- CARD11 fusions. Due to the multi-layered ubiquitin regulation and the uneven expression levels of active vs. inactive BCL10- CARD11 fusions, this system is not an ideal tool to study the relevance of BCL10 ubiquitination. However, it will be important to clarify in how far BCL10 oligomerization and potential filament formation cooperate with ubiquitination processes to induce downstream signaling or alternatively, if BCL10 ubiquitination may also facilitate CBM complex assembly.

In BJAB B cells stable BCL10-CARD11 expression activates MALT1 and NF-κB as strong as oncogenic variants of CARD11 derived from DLBCL tumor patients (5, 6, 25, 26), clearly showing that the proximity of BCL10 and CARD11 alone is sufficient to activate downstream signaling pathways. Thus, the BCL10-CARD11 fusion already provides compelling evidence that CARD11 acts as a seed in cells to boost lymphocyte activation. By introducing point mutations we were also now able to unravel the contribution of the two different CARD interfaces. Constitutive activity of the BCL10-CARD11 fusion no longer required the presence of an intact CARD11 CARD, but was solely driven by the BCL10 oligomerization interface. In contrast, stimulus-dependent MALT1 activation of the BCL10- CARD11 fusion protein relied on an intact CARD11 CARD, which most likely as in CARD11 WT serves as a platform for recruitment and oligomerization of endogenous BCL10. Interestingly, introduction of the active oncogenic mutation L225LI into the CARD11 coiled-coil (CC) region in the context of the BCL10-CARD11 resulted in a fusion protein that was hyper-active, despite the very low expression level in the BJAB B cells. Indeed, all active BCL10-CARD11 fusion proteins were expressed at a lower level compared to their inactive counterparts. However, the very low expression of the hyperactive fusion constructs indicates that there is a strong counterselection against the expression of these constitutively active variants in T and B cells. Since all fusions constitutively activate the MALT1 paracaspase in the two cell types, it is possible that the sustained MALT1 cleavage of mRNA processing factors Regnase-1 and Roquin1/2 may exert toxic effects that impair T and B cell survival (39, 40). More work is needed to understand under which circumstances chronic activation induces cell survival or toxicity.

While expression of BCL10-CARD11 fusion drives NFκB activation in BJAB B cells, constitutive and inducible NF-κB signaling is severely impaired in Jurkat T cells expressing the fusion protein. Even though NF-κB activation is impaired in Jurkat T cells, MALT1 protease is still strongly activated by BCL10-CARD11 fusions, demonstrating that not all CBM downstream effects are affected. Further, the reduced responsiveness relies on the stable expression of the BCL10-CARD11 fusion protein and it needs to be explored what cell-intrinsic mechanisms operate in Jurkat T cells that counteract constitutive activation of the canonical NF-κB pathway downstream of the CBM complex. Of note, T lymphocytes from patients with germline activating CARD11 mutations are anergic, while the B cells are activated and expanding, causing a phenotype called BENTA (B cell expansion with NF-κB and T cell anergy) (41, 42). It will be interesting to see whether similar negative regulatory mechanisms observed in Jurkat T cells driven by an active BCL10-CARD11 fusion could also operate in primary T cells.

Our data suggest that upon the initial BCL10 recruitment to CARD11, the weak heterotypic interaction of the monomeric CARDs needs to be stabilized by further interactions arising from the oligomerized CARD11 seed and the helical BCL10 filaments. The additional contact points within this multimeric complex are essential for a high affinity binding and the formation of a stable CBM complex that is competent to trigger downstream signaling. Furthermore, the structural rigidity in the core BCL10-MALT1 filament may stabilize the binding, which is in line with the observation that CARD11 binding is also reduced when BCL10 is not complexed with MALT1 (14). In conclusion, we demonstrate that the recruitment of BCL10-MALT1 to CARD11 and BCL10- MALT1 filament formation are highly interconnected processes that cooperate to drive CBM downstream effects in response to physiological or pathological activation of T and B cells.

### MATERIALS AND METHODS

### Cell Lines and Treatments

Cell lines were maintained at 37◦C in a humidified atmosphere at 5% CO2. Jurkat T cells and BJAB B cells were cultured in RPMI 1640 Medium, and U2OS and HEK293T cells in DMEM. Media were supplemented with 10% (Jurkat T cells, U2OS, HEK293T) or 15% (BJAB B cells) fetal calf serum, 100 U/ml penicillin and 100µg/ml streptomycin. U2OS, HEK293T and BJAB B cells were obtained from the DSMZ, Jurkat T cells were authenticated by the Authentication Service of the Leibniz Institute DSMZ. Jurkat T cells were stimulated with Phorbol 12-Myristate 13- Acetate (PMA: 200 ng/ml; Merck) and Ionomycin (300 ng/ml; Calbiochem) for 30 min, except if otherwise stated.

### DNA Constructs and Antibodies

DNA constructs and antibodies used in this study are listed in **Tables 1, 2**, respectively.

### Generation and Reconstitution of Knock-Out Cells

Bicistronic expression vector px458 expressing Cas9 and sgRNA (45, 46) was digested with BbsI and the linearized vector

#### TABLE 1 | DNA constructs.

#### TABLE 2 | Antibodies.

was gel purified. Targeting oligos (CARD11: 5′CTCATCA ATGACCTTACACTGACGCAGGTAGG 3′BCL10: 5′AGT GAGGTCCTCCTCGGTGA 3′ and 5′TTCCGCTTTCGTCTC CCGCT 3′ ) for each targeting site positioned as depicted in **Supplementary Figures 2A,B,** were annealed and ligated to the linearized vector. Jurkat T cells or BJAB B cells (4–8 × 10<sup>6</sup> ) were electroporated (220 V and 1,000 µF) using a Gene pulser X (Biorad) with px458 plasmids expressing sgRNA targeting CARD11 or BCL10, as well as a EGFP expression cassette. Twenty-four to forty-eight hours after electroporation, GFP positive cells were sorted using a MoFlow sorting system. Isolation of clonal cell lines was achieved by serial dilutions and was followed by an appropriate expansion period. KO cell clones were initially identified by detecting CARD11 or BCL10, respectively, by Western Blot. Clones lacking protein expression were genotyped by genomic PCR using intronic primers flanking targeting sides.

For reconstitution, lentivirus was produced in HEK293T cells. 1 × 10<sup>6</sup> HEK293T cells were seeded in 8 ml DMEM medium (10% FCS, 1% Pen/Strep) in 10 cm<sup>2</sup> dishes and grown overnight at 37◦C. The next day, the cells were transfected with 1.5 µg of the packaging vector psPAX2, 1.0 µg of the lentiviral envelope plasmid pMD2.G and 2 µg pHAGE transfer vector using XtremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's protocol. After 3 days the supernatant of the HEK293T cells containing the virus was sterile filtered (0.45µm). For transduction, virus supernatant was transferred to 5 × 10<sup>5</sup> Jurkat T cells or BJAB B cells. For BJAB B cells, supernatant was concentrated with Amicon centrifugal filter units (100 K) prior to transduction. The solution was filled up with RPMI medium (10% FCS, 1% Pen/Strep) to a final volume of 2–2.5 ml and mixed with Polybrene (8µg/ml). To enhance transduction efficiency, BJABs were centrifuged for 1 h at 500 × g. Forty to seventy-two hours later, cells were washed with PBS (without calcium and magnesium) and re-suspended in 1– 2 ml RPMI medium (10% FCS, 1% Pen/Strep). Seven to ten days after transduction infection was analyzed by determining 1CD2 surface expression by FACS and CARD11 or BCL10 protein expression by Western Blot. Only cells yielding a transduction efficiency of >90% as determined by FACS analysis, were used for further analyses.

Generation and reconstitution of MALT1-deficient Jurkat T cells has been described (22).

### Flow Cytometry (FACS)

Surface expression of 1CD2 after lentiviral transduction of BJAB B cells or Jurkat T cells was assessed by incubating 200 µl of the cell culture for 15 min at room temperature with 2 µl anti-CD2- APC (RPA-2.10) antibody. Cells were centrifuged (1,100 rpm, 5 min) and re-suspended in 250 µl PBS before FACS using Attune Acoustic Focusing Flow Cytometer.

### Cell Lysis and Precipitations

For analysis of expression via Western Blot or EMSA, cells (1– 3 × 10<sup>6</sup> ) were harvested (300 × g, 5 min, 4◦C) and washed once with ice cold PBS. The pellet was resuspended in 80–100 µl high salt buffer (20 mM HEPES pH 7.9, 350 mM NaCl, 20% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% NP-40, 1 mM DTT, 10 mM sodium fluoride, 8 mM β-glycerophosphate, 300µM sodium vanadate and Roche protease inhibitor cocktail). For binding studies, cells (1–5 × 10<sup>7</sup> ) were lysed in co-IP buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 0.2% NP-40, 10% glycerol, 1 mM DTT, 10 mM sodium fluoride, 8 mM β-glycerophosphate, 300µM sodium vanadate and protease inhibitor cocktail). Lysate controls were mixed with 4xSDS loading dye and boiled. For StrepTactin pull-downs (ST-PD), 20–40 µl Strep-Tactin Sepharose (1:1 suspension) was used for binding of Strep-tagged BCL10 or BCL10-CARD11 fusion overnight at 4◦C rotating. Sepharose beads were pelleted after incubation (100 × g, 4 min, 4 ◦C), washed 3x with co-IP buffer, and boiled after the addition of 20 µl 2xSDS loading dye (Roti-load). Lysates and ST-PDs were separated by SDS-PAGE and analyzed by Western Blot.

### Western Blot

Proteins were transferred onto PVDF-membranes for immunodetection using an electrophoretic semi-dry transfer system. After transfer, membranes were blocked with 5% BSA for 1 h at RT and incubated with specific primary antibodies (indicated above, diluted 1:1,000 in 2.5% BSA/PBS-T) overnight at 4◦C. Membranes were washed in PBS-T before the addition of HRP-coupled secondary antibodies (indicated above, 1:7,000 in 1.25% BSA in PBS-T; 1 h, RT). HRP was detected by enhanced chemiluminescence (ECL) using the LumiGlo reagent (Cell Signaling) according to manufacturer's instructions.

### Electrophoretic Mobility Shift Assay (EMSA)

For EMSAs, double-stranded NF-κB (H2K: fw: 5′ -GATCCA GGGCTGGGGATTCCCCATCTCCACAGG-3′ , rev: 5′ - GAT CCCTGTGGAGATGGGGAATCCCCAGCCCTG-3′ ), and OCT1 binding sequences (fw: 5′ - GATCTGTCGAATGCA AATCACTAGAA-3′ , rev: 5′ -GATCTTCTAGTGATTTGCATT CGACA-3′ ) were labeled with [α-<sup>32</sup>P] dATP using Klenow Fragment (NEB). To monitor DNA binding, whole cell lysates (3–6 µg) were incubated for 30 min at RT with shift-buffer [20 mM HEPES pH 7.9, 120 mM KCl, 4% Ficoll, 5 mM DTT, 10 µg BSA and 2 µg poly-dI-dC (Roche)] and radioactive double stranded NF-κB or OCT1 probes (10,000–20,000 cpm). Samples were separated on a 5% polyacrylamide gel in TBE buffer, vacuum-dried and exposed to autoradiography.

### Confocal Immunofluorescence Microscopy

The localization and distribution of BCL10 was analyzed by seeding U2OS cells in 96-well plates. Cells were transfected using Lipofectamine 3000 Transfection Reagent (Invitrogene) according to the manufacturer's instructions. To optimize cell attachment, CellCarrier-96 black plates (PerkinElmer) were coated with 100 µl poly-D-lysine at a concentration of 50µg/ml. Twenty-four hours after transfection, cells were washed with PBS and fixed with 60 µl Methanol (−20◦C) for 5–10 min at room temperature and then washed 3x with PBS. For immunostaining, cells were blocked in 2% BSA in PBS. Cells were incubated with primary antibody (anti-StrepTagII) in blocking buffer for 2 h at RT. Cells were washed 3x for 10 min at RT, before incubation with secondary antibody (Alexa Fluor488-donkey anti-mouse) in blocking buffer for 1 h at RT. Cell nuclei were visualized by incubation with Hoechst 33342 dye (Life Technologies) in PBS at a concentration of 0.5µg/ml for 30 min at RT. Afterwards the cells were washed and covered with PBS, sealed with foil and kept at 4◦C in the dark until microscopy. Confocal microscopy was performed with an Operetta high-content imaging system (Perkin-Elmer).

### Labeling and Biotin Pull-Down (PD) of Active MALT1

The biotin-labeled MALT1 activity based probe (MALT1-ABP) has been described previously (19). To investigate MALT1 protease activity, Jurkat T cells (3 x 10<sup>7</sup> ) were washed with PBS, and lysed in 600 µl co-IP buffer without protease inhibitors for 30 min at 4◦C. After clearing the lysates by centrifugation (20,000 × g, 4◦C, 15 min), 30 µl were collected as lysate control, mixed with 4x SDS loading buffer and boiled for 5 min at 95◦C. To 550 µl of the supernatant 12 µl High Capacity Streptavidin Beads (Thermo Fisher) was added and incubated for 1 h at 4◦C for pre-clearing. The beads were pelleted (4,000 rpm, 2 min, 4 ◦C) and 450 µl of supernatant was mixed with MALT1-ABP probe (0.1µM final concentration). After 1 h rotating at room temperature, 15 µl High Capacity Streptavidin Beads was added before 1–2 h incubation at 4◦C (rotating). Beads were collected and washed 3x with co-IP buffer without protease inhibitors. Beads were re-suspended in 22 µl 2x SDS loading buffer and boiled at 95◦C for 7 min before SDS-PAGE and Western Blot analysis.

### NF-κB Reporter Assay

For NF-κB luciferase reporter assays, 8 × 10<sup>6</sup> Jurkat cells were transfected by electroporation with 2 µg NF-κB firefly luciferase reporter plasmid, 1 µg renilla luciferase and 6 µg of CARD11 and BCL10-CARD11 fusion constructs using 220 V and 1,000 µF (Gene Pulser X, BioRad). After cultivation for 72 h cells were lysed in passive lysis buffer and luciferase activity measured using a dual luciferase reporter kit according to the manufacturer's protocol (Promega). All luciferase values were calculated in relation to the Renilla control.

### Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR)

RNA was isolated (QIAGEN RNeasy Kit) and equal amounts of RNA (InviTrap Spin Universal RNA Mini Kit,<PHONE_NUMBER>, Stratec) were transcribed into cDNA using the Verso cDNA synthesis Kit (AB1453B, Thermo Fisher Scientific). Quantitative real-time (qRT) PCR was performed using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) and standard LightCycler protocol on a Roche LightCycler 480. RNA Polymerase II (PolII) served as internal standard. The following primers were used: fw: 5 ′ -CCGCACCTCCACTCCATCC-3′ rev: 5′ -ACATCAGCACCC AAGGACACC-3′ ; RPII fw: 5′ -GCACCACGTCCAATGACA-3′ rev: 5′ -GTGCGGCTGCTTCCATAA-3′ . Results represent the mean and standard error of the mean of three independent experiments.

### Structural Model of CARD11-BCL10 and BCL10-BCL10 Interaction Surfaces

The CARD11-BCL10 CARD/CARD interaction model was prepared under consideration of the surface charge complementarity analysis of the BCL10-MALT1 filament cryo EM structure (EMD-0013, PDB 6GK2) and the crystal structure of the CARD11 domain (PDB 4LWD) (**Supplementary Figure 1**). The CARD11 CARD structure has been superimposed on the BCL10 CARD within the BCL10-MALT1 filament using the align command in PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). Under consideration that the mutation of residue R35 in CARD11 abolishes the interaction to BCL10 (12), we propose that the mainly positively charged top of the CARD11 CARD interacts with the negatively charged bottom of the Bcl10 filament (**Supplementary Figures 1B,C**). The electrostatic surface was calculated with the program APBS Tool 2.1 implemented in PyMOL (47).

### Statistical Analysis

Data for luciferase reporter assay, quantitative RT-PCR and fluorescence microscopy was analyzed for statistical significance using the unpaired Student's t-test (∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). Sample size (n) is specified for each experiment and data are shown as mean ± s.e.m.

### AUTHOR CONTRIBUTIONS

TS and SK conceived and performed most experiments, analyzed, and interpreted the data. FS and KL conceived and performed biophysical experiments and provided structural expertise. SW, TG, and SWi generated, verified and analyzed KO cells. DK conceived the study, experiments, wrote the manuscript, and secured funding. All authors read, acknowledged, and helped with the final version of the manuscript.

### FUNDING

Deutsche Krebshilfe (grant no. 70112622) to DK supported work on oncogenic CARD11 signaling. Deutsche Forschungsgemeinschaft SFB 1054 TP A04 to DK and TP B02 to KL supported structural and mechanistic work on CBM complex signaling in T cells.

### ACKNOWLEDGMENTS

We thank Katrin Demski and Kerstin Kutzner for excellent technical assistance.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.02695/full#supplementary-material

## REFERENCES

in immune cells and lymphomas. Chem Biol. (2015) 22:129–38. doi: 10.1016/j.chembiol.2014.10.021

regulates NF-kappa B signaling. Mol Cell Biol. (2004) 24:3860–73. doi: 10.1128/MCB.24.9.3860-3873.2004

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Seeholzer, Kurz, Schlauderer, Woods, Gehring, Widmann, Lammens and Krappmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Human CARD9: A Critical Molecule of Fungal Immune Surveillance

#### *Rebecca A. Drummond1†, Luis M. Franco2 and Michail S. Lionakis1 \**

*<sup>1</sup> Fungal Pathogenesis Section, Laboratory of Clinical Immunology and Microbiology (LCIM), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States, 2 Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States*

#### *Edited by:*

*Frederic Bornancin, Novartis (Switzerland), Switzerland*

### *Reviewed by:*

*Yuval Itan, Icahn School of Medicine at Mount Sinai, United States Ruben Martinez-Barricarte, Rockefeller University, United States*

#### *\*Correspondence:*

*Michail S. Lionakis<EMAIL_ADDRESS>
#### *†Present address:*

*Rebecca A. Drummond, Institute of Immunology and Immunotherapy, Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom*

#### *Specialty section:*

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

*Received: 01 May 2018 Accepted: 25 July 2018 Published: 06 August 2018*

#### *Citation:*

*Drummond RA, Franco LM and Lionakis MS (2018) Human CARD9: A Critical Molecule of Fungal Immune Surveillance. Front. Immunol. 9:1836. doi: 10.3389/fimmu.2018.01836*

CARD9 is a signaling adaptor protein that is involved in the transduction of signals from a variety of innate pattern recognition receptors, including the C-type lectin receptors and intracellular NOD receptors and nucleic acid sensors. As a result, CARD9 has been shown in animal models to be an important regulator of immunity to bacteria, fungi, and viruses. Studies in humans with autosomal recessive CARD9 deficiency have indicated a highly specific role for this molecule in the activation of antifungal immune responses in the central nervous system, the oral mucosa, and the skin. Moreover, CARD9-dependent functions have recently been indicated to modulate the development of autoimmunity, inflammatory bowel diseases, and cancer. In this mini-review, we highlight the recent studies that have identified several novel functions of CARD9 in various disease contexts, and we summarize the contemporary understanding of the genetics and immunology of human CARD9 deficiency.

Keywords: CARD9, fungi, primary immunodeficiency, C-type lectin receptors, candidiasis, neutrophils

### INTRODUCTION

Innate recognition of microbes by pattern recognition receptors (PRRs) is a critical first step in the defense against infection. To activate antimicrobial immunity, PRRs must initiate intracellular signaling cascades which control cellular responses, such as cytokine production, phagocytosis, and assembly of microbial killing complexes. Many PRRs use signaling molecules and adaptor proteins that are shared with other members of the same PRR family. As a result, deficiency of a shared signaling or adaptor protein often results in immune dysfunctions that are more severe than a deficiency in a single receptor and can have profound consequences for the control of infections (1).

Pathogenic fungi are predominantly recognized by the C-type lectin receptor (CLR) and tolllike receptor (TLR) families, of which many members couple to the signaling adaptor proteins CARD9 and MyD88, respectively, in order to activate their functions and initiate defense against fungal invasion (2). Although both CLRs and TLRs bind to and activate antifungal immune responses, the phenotypic consequences of human deficiencies in either of the two shared signaling adaptors have demonstrated that CARD9 plays a specific, superior role in the control of fungal diseases in humans compared to that of MyD88. Indeed, patients with genetic deficiency in CARD9 exhibit a primary immunodeficiency disorder (PID) which manifests as an extreme susceptibility to fungal infections, but not bacterial, viral, or parasitic infections, and is the only PID described to date that specifically predisposes to fungal diseases without other infectious or non-infectious sequelae (3). In contrast, MyD88 deficiency results in the development of lifethreatening pyogenic bacterial infections without the spontaneous development of fungal disease (4). The highly specific susceptibility to fungi in CARD9-deficient patients further extends to the fungal species and organs affected, indicating that CARD9 is required for organ-specific antifungal immune responses of which we are only now beginning to define. Furthermore, animal models and genome-wide analyses of human singlenucleotide polymorphisms (SNPs) have indicated that CARD9 may also function to promote immunity to other pathogens and contribute toward autoimmune and hyperinflammatory disorders. Thus, CARD9 is a multi-functional signaling protein involved in many aspects of the immune system.

In this review, we discuss recent research that has unveiled the many possible functional roles of CARD9, predominantly defined using animal models. Finally, we discuss the genetics of human CARD9 deficiency and highlight the critical role for this molecule in antifungal immune surveillance in humans.

### THE MULTI-FUNCTIONAL ROLES OF CARD9

### CARD9 Is a Critical Activator of Antifungal Immune Responses

Many of the prototypical PRRs that recognize common components of fungal cell walls are members of the CLR family, and include Dectin-1, Dectin-2, Dectin-3, Mincle, and the Mannose Receptor (CD206). Many of these receptors initiate intracellular signaling cascades that are CARD9-dependent, the best studied of which is the Syk-dependent pathway downstream of Dectin-1 (**Figure 1A**). In brief, ligation of Dectin-1 by β-glucan (the fungal ligand for this receptor) results in recruitment of Syk kinase and the formation of the CBM signalosome, composed of CARD9, BCL10, and MALT1 (5). Activation of CARD9 function requires Vav proteins, of which there are three isoforms (Vav1, Vav2, and Vav3). Deletion of Vav1-3 in mice results in a phenocopy of CARD9-deficiency, indicating that Vav-mediated activation of CARD9 is a critical step in the induction of protective antifungal immunity. In line with this, human *VAV3* SNPs are enriched in patient cohorts with candidemia (6). Activated CARD9 then leads to the production of inflammatory mediators, such as IL-6, IL-12, GM-CSF, TNF, and IL-1β *via* the activation of NFκB and ERK, the latter of which occurs *via* a RASGRF1–H-Ras pathway (7). These CARD9-dependent signaling pathways are regulated by Rubicon, a protein best known for its functions in autophagy (8). Rubicon competitively binds to CARD9 which results in the disassembly of the CBM complex, thus switching off signaling and preventing excessive inflammatory responses. However, modulation of Rubicon expression levels using lentiviral vectors in mice demonstrated that a reduction in Rubicon expression could help promote fungal clearance and survival by enhancing

CARD9-dependent antifungal immune responses, at least in acute infection models (8). Compared to other PRR families, the molecular signaling events that occur upon CLR ligation are poorly defined. Yet, understanding these pathways will be critical to determine how iatrogenic interventions that manipulate these pathways affect vulnerable patients. For example, the recently introduced Syk inhibitors for hematological malignancies and graft-versus-host disease (9, 10), may predispose to the development of dangerous invasive fungal infections in patient cohorts already at risk for these diseases.

Animals with a genetic deletion of Card9 are susceptible to challenge with a variety of fungal species, including *Candida albicans*, *Aspergillus fumigatus, Cryptococcus neoformans*, and some rarer dematiaceous fungi (11–14). In each of these models, *Card9<sup>−</sup>/<sup>−</sup>* mice generally exhibit reduced inflammatory cytokine production, which contributes to the inability to control fungal growth within infected organs. Protective antifungal cytokines that appear critically dependent on CARD9 function include IL-6, TNF, and IL-1β. In humans, CARD9 deficiency predisposes to a smaller range of pathogenic fungi (discussed below) indicating that CARD9-dependent functions in humans may be more context-dependent than in the mouse.

Despite the central position of CARD9 in the CLR signaling pathway, ablation of CARD9 function does not result in complete abrogation of antifungal immune responses (in either mice or human), indicating that CARD9-dependent mechanisms are required only for certain protective processes that may be specific to the cell type in question. Indeed, the dependency on CARD9 for NFκB activation has been shown to vary among different types of myeloid cells (7), including neutrophils, macrophages, and dendritic cells (DCs) (13). Neutrophils are the most important effector cell in the defense against systemic *C. albicans* infections, since neutropenia is a significant independent risk factor for the development of and suffering worse outcome from these infections in humans, and neutrophil depletion significantly reduces survival to *C. albicans* challenge in mice (2, 15). Many intrinsic neutrophil functions, including phagocytosis, production of reactive oxygen species (ROS), and chemotaxis appear largely independent of CARD9 for a variety of fungal species, shown with *ex vivo* studies using human neutrophils (16–19). In contrast, for ROS-independent fungal killing, CARD9 is required for the killing of unopsonized yeast cells as part of a PI3Kγ-dependent pathway, whereas killing of opsonized *C. albicans* yeast largely requires FcRγ and PKC and is independent of CARD9 (20). These two distinct fungal killing pathways utilized by neutrophils helps to explain the observation that neutrophils isolated from CARD9-deficient patients exhibit a selective killing defect toward unopsonized yeast (18, 19). This defect may partly contribute toward the fungal central nervous system (CNS)-tropism observed in CARD9-deficient patients (see below), since opsonization is naturally low in the CNS and thus CARD9-dependent killing of unopsonized cells would be particularly critical at this site. However, human neutrophil killing against the invasive filamentous *Candida* forms observed in tissue appears intact, whether opsonized or unopsonized (19). In addition, we and others have shown that a critical function of CARD9 in antifungal defense is promoting neutrophil recruitment specifically into fungal-infected organs *via* CXC chemokine production in both mice and humans (19, 21), which appears to largely contribute toward the organ-specific manifestation of fungal diseases observed in CARD9-deficient humans.

In experimental *C. albicans* CNS infections in mice, CARD9 promotes neutrophil recruitment in a fungal- and brain-specific manner (19). In this situation, CARD9 is required for the appropriate induction of CXC chemokines in the CNS by resident macrophages (i.e., microglia) and glia cells, as well as recruited neutrophils. Therefore, CARD9 controls local inflammatory chemokine production in addition to a neutrophil-intrinsic positive feedback chemotaxis loop in the brain (19). CARD9 dependent production of neutrophil-attracting chemokines is also evident during pulmonary infection with *A. fumigatus* in mice (14), and we recently reported CARD9-deficient patients who developed extrapulmonary *A. fumigatus* infection that was associated with a lack of neutrophil accumulation at the infected site (21). Moreover, reduced production of CXC chemokines resulting in a lack of neutrophil recruitment has been described in chronically fungal-infected subcutaneous tissue in experimental phaeohyphomycosis models (22). Therefore, CARD9 appears to be a central regulator of neutrophil recruitment to specific organs during invasive fungal infection.

### CARD9 and Anti-Bacterial Immunity

Stimulation of *Card9<sup>−</sup>/<sup>−</sup>* myeloid cells with purified bacterial products has revealed that CARD9 is required for inflammatory cytokine production in response to specific bacterial stimuli. IL-6 production following stimulation with peptidoglycan or muramyl-dipeptide (MDP), predominant components of Grampositive bacterial cell walls, is highly dependent on CARD9 whereas responses to LPS are CARD9-independent (23). Recognition of MDP is largely controlled by the intracellular NOD2 receptor, to which CARD9 couples in order to drive activation of p38 and JNK kinases (**Figure 1B**), thus promoting immunity to intracellular bacterial pathogens including *Listeria monocytogenes* (23) and *Mycobacterium tuberculosis* (24).

In addition to activating innate immune responses to intracellular bacteria, other studies have indicated that CARD9 may be involved in the induction of adaptive immunity to these pathogens. Mouse T-cells specific for flagellin were shown to require Syk-CARD9 signaling for their activation, which was mechanistically linked to TLR5-dependent antigen presentation by CARD9-expressing DCs (25). Polarization to the Th17 lineage also depends on CARD9 in the context of gastrointestinal (GI) bacterial infection (26), similar to what has been described for the generation of Th17 responses in the fungal-infected oral mucosa (27). Moreover, humoral immunity mediated by B-cells has also been shown to be affected by deletion of Card9, since bacterial-specific IgG production during GI infection is significantly reduced in *Card9<sup>−</sup>/<sup>−</sup>* animals (28). Thus, animal models have demonstrated that Card9 can play distinct protective roles in immunity to intracellular bacterial pathogens, however, the relevance of these functions in humans remains to be determined given that CARD9-deficient patients do not appear susceptible to bacterial infections.

### CARD9 and Antiviral Immunity

Studies delineating the molecular pathways controlling IL-1β activation and secretion by myeloid cells in response to viral infection have identified CARD9 as a key regulator of these responses (**Figure 1C**). Following viral infection or transfection with foreign DNA, CARD9 directly interacts with Rad50, a cytosolic DNA sensor, and these CARD9-Rad50 complexes subsequently recruit BCL10 to promote IL-1β secretion. Using a vaccinia virus infection model, *Card9<sup>−</sup>/<sup>−</sup>* animals were shown to produce reduced levels of IL-1β following infection which negatively impacted the ability of these animals to generate an appropriate antiviral CD8<sup>+</sup> T-cell response (29). CARD9 is also required for immunity to retroviruses by transducing RIG-1-dependent signals *via* MAVS and BCL10 to activate NFκB and transcription of pro-IL-1β, which in turn is processed into active IL-1β by ASC-dependent activation of the inflammasome (30). As shown for antifungal immunity, Rubicon can also regulate these CARD9-dependent antiviral functions, since enhancing Rubicon expression levels was found to significantly skew host immunity in favor of pathogen resistance (8).

### CARD9 and Autoimmunity

Recent studies have demonstrated potential novel roles of CARD9 in autoimmune diseases using animal models, providing interesting new insights into how this protein functions in organ-specific inflammation. During experimental uveitis, Mincle-mediated activation of CARD9 is required for the recruitment of pathogenic Th1 and Th17 cells (31), and it was later shown that fungal antigens could exacerbate the development of this disease and this occurred *via* Dectin-2-CARD9 signaling (32). In line with the critical role for CARD9 in neutrophil recruitment, *Card9<sup>−</sup>/<sup>−</sup>* animals showed reduced production of neutrophil-targeted chemokines, such as CXCL1, CXCL2, and CCL3, resulting in decreased accumulation of neutrophils to the arthritic joint (33). As a result, *Card9<sup>−</sup>/<sup>−</sup>* animals are protected from autoantibody-induced arthritis, and this protection was mapped to neutrophils using neutrophil-specific Card9-deficient mice (33). Population-based human studies have indicated that *CARD9* genetic variation may modulate the risk of development of inflammatory bowel diseases (IBD; discussed below), ankylosing spondylitis (34), IgA nephropathy (35), and primary sclerosing cholangitis (36), and more studies are required to determine the role of the CLR/CARD9 axis in organ-specific autoimmune disease development.

### CARD9 and Cancer

CARD9-dependent signaling in tumor development and metastasis has been revealed in several recent studies using animal models and cell lines. Patients with IBD are at risk for the development of colonic cancers. Human SNPs in *CARD9* have been identified as risk factors for IBD by several GWAS-based studies (discussed below), and animal models have shown that CARD9 promotes the production of pro-inflammatory cytokines IL-1β and IL-22 in the gut during active colitis, which in turn drives colonic tumor growth (37). Other work has additionally shown that metastasis of colonic tumor cells to the liver depends on CARD9 signaling. CARD9 is highly expressed by tumorinfiltrating macrophages and has been shown to be a critical modulator of the polarization of these cells toward a highly inflammatory metastatic-inducing phenotype (38).

In addition to colonic cancer, CARD9 signaling has also been implicated in the inappropriate activation of renal cell carcinoma (RCC) cells. Mutation affecting the tumor suppressor gene *VHL* (most often somatic, but occasionally involving germline cells) is a major hallmark of RCC, which in turn causes inappropriate activation of NFκB and c-Jun. CARD9 is a mechanistic link between VHL inactivation and the activation of these inflammatory transcription factors (39, 40). Direct interaction between CARD9 and VHL is required for C-terminal phosphorylation of CARD9 by the kinase CK2, which limits CARD9 activity and NFκB activation. Reducing CARD9 expression with a silencing RNA approach in *VHL−/−* cells lowered NFκB activity to wildtype levels, and significantly reduced the tumorigenic potential of these cells (40). Similarly, the loss of VHL promotes an additional pro-inflammatory CARD9-dependent pathway that results in the activation of JNK signaling and c-Jun activity (39). Collectively, these studies provide the intriguing possibility that inappropriate CARD9 activation contributes to the development of certain cancers and may be a novel target of future therapies for these diseases.

### THE GENETICS AND CLINICAL SPECTRUM OF HUMAN *CARD9* MUTATIONS

Despite the wide range of CARD9-dependent functions identified in animal models and the potential influence of this protein in several disease scenarios, loss-of-function mutations in human *CARD9* have unequivocally demonstrated the importance of CARD9 in antifungal immunity. Human CARD9 deficiency is characterized by the spontaneous development of fungal infections that predominantly localize to the oral mucosa, CNS, bone, and subcutaneous tissues, and often involves specific families of pathogenic fungi including *Candida* species (CNS, bone, and mucosal disease) and dark-walled molds and yeastlike fungi (e.g., *Aspergillus*, *Exophiala*, and *Phialophora*) that localize to the CNS, skin, bone, and abdominal organs (3, 21, 41–43). The underlying mechanisms that cause susceptibility to fungal diseases in CARD9-deficient patients is not well understood, and could be related to the poor production of inflammatory cytokines and chemokines in response to fungal agonists (13, 18, 19). Indeed, a cohort of French-Canadian CARD9 deficient patients were successfully treated with recombinant GM-CSF therapy, which the authors show corrected defective GM-CSF responses and ERK signaling in CARD9-deficient myeloid cells (43), while another study used G-CSF therapy to correct defective IL-17 responses in a CARD9-deficient patient (44). These studies indicate that replacing cytokines that are classically associated with antifungal defense may be an appropriate therapy option for human CARD9 deficiency. However, we recently showed that these approaches may not be effective for all patients which are potentially related to the variety of *CARD9* mutations observed (see below). Our data indicated that although different *CARD9* mutations disrupt GM-CSF production by myeloid cells, the impact of these mutations on ERK activation varied with mutation and appeared to correlate with the efficacy of GM-CSF therapy in these patients (45). Thus, treatment options for human CARD9-deficiency are still limited, and further investigation into the mechanisms that cause fungal susceptibility in these patients is warranted. In addition, the organ- and fungal species-specific nature of CARD9-deficiency disease is also poorly understood. As discussed above, we and others have shown that defects in neutrophil recruitment are likely the major contributing factor toward the organ-specific nature of the disease, however, other contributing roles of tissue-resident macrophages, stromal cells, and/or additional functional deficits in recruited inflammatory cells remain to be fully explored.

Although many CARD9-deficient patients have similar clinical presentations, there is diversity in the genetic mutations underlying the condition [see Ref. (3) for a table of all reported human *CARD9* mutations]. More than 15 missense and nonsense mutations in *CARD9* have now been described in the coiled-coil and CARD domains, as well as the promoter region (3). Inactivation of both alleles appears to be necessary for the occurrence of disease, so the condition follows an autosomal recessive mode of inheritance. *De novo* variants in *CARD9* have also been reported in patients with debilitating fungal infections of the eyes, bone, and skin (46, 47). Interestingly, although some of the reported *CARD9* mutations have been identified in unrelated patients, there are instances where the same mutation does not give rise to a similar clinical phenotype (3). For example, homozygosity for the Q298X mutation has been reported to give rise to *Candida* meningoencephalitis and deep dermatophytosis in different patients (41, 48), while patients with very similar clinical presentations can have mutations in different parts of the *CARD9* gene (47). Therefore, it is currently unclear which mutations predispose to which clinical phenotypes and whether there is any overlap, and this will require more clinical descriptions of CARD9-deficient patients and further investigations into CARD9-dependent immune signaling and functions, especially in humans.

Like many other genes that are associated with Mendelian traits, human *CARD9* is intolerant to sequence variation. To quantify gene-level intolerance to functional genetic variation, Petrovski et al. proposed a Residual Variation Intolerance Score (49). Of the nine CARD genes for which a score was calculated in that study, only three (*CARD9*, *CARD10*, and *CARD11*) had negative scores, suggesting low tolerance to functional genetic variation. In fact, *CARD9* had lower tolerance to variation than 82% of the 16,956 genes for which a score was calculated in the study.

In addition to deleterious mutations which predispose to fungal infection, there have been reports of genetic variants of *CARD9* in humans that associate with autoimmune disease. The CARD9 single-nucleotide polymorphism S12N (rs4077515) was recently shown to be highly enriched in patients with allergic bronchopulmonary aspergillosis, which the authors linked to RelB activation by this CARD9 variant that subsequently activated IL-5 production by alveolar macrophages and drove pathogenic eosinophil recruitment and Th2 responses within the lung (50). Another example is the CARD9 variant in which the C-terminal region is truncated (51, 52). One of these truncated variants of CARD9 (S12NΔ11 or *CARD9*Δ11) is strongly associated with protection against IBD, which is mechanistically linked to the inability of these variants to be activated by TRIM62, a ubiquitin ligase that is required for CARD9 activation and subsequent pro-inflammatory cytokine production. While the lack of TRIM62-mediated activation is protective in the context of inappropriate intestinal inflammation and may represent a target for therapeutic intervention in IBD (53), these mutations would be predicted to negatively affect antifungal immunity, since *Trim62<sup>−</sup>/<sup>−</sup>* animals are unable to control fungal growth and succumb to infection significantly faster compared to their wild-type counterparts (52). Intriguingly, in human cell lines, *CARD9*Δ11 variants act in a dominant-negative fashion (52), raising the possibility of the existence of humans who may be heterozygous for dominantnegative *CARD9* mutations in the C-terminal region and yet may present with functional CARD9-deficiency. This possibility should be considered by clinicians looking for the underlying cause of an extreme fungal infection in an otherwise immunocompetent patient.