Thome Miazza Margot, Associate Professor

Margot Thome studied Biochemistry at the University of Tübingen, Germany, and at the University of Arizona, USA. In 1993 she joined the laboratory of Oreste Acuto at the Pasteur Institute, Paris, where she worked on the role of tyrosine kinases in T-cell activation, and received her PhD from the University of Paris in 1995. Since 1996 she has worked in the Department of Biochemistry at the University of Lausanne. As a postdoctoral fellow she studied viral and cellular regulators of apoptosis in the laboratory of Jürg Tschopp. Since 2004, she holds an SNF Assistant Professorship. Her present research focuses on signaling pathways that control lymphocyte activation and survival. Margot.ThomeMiazza@unil.ch

Lymphocytes play a crucial role in the defense against pathogens and tumor cells. One focus of our research is to understand the molecular mechanisms that control the activation of T-lymphocytes, initiated upon triggering of the T-cell antigen receptor by MHC-bound antigen. This leads to the initiation of multiple signaling pathways that regulate changes in cell shape and gene expression that are critical for efficient T-cell activation, proliferation and survival. Another focus of our research is to understand the molecular mechanisms underlying aberrant lymphocyte proliferation and survival that occurs in certain lymphoid tumors (lymphomas).

By uncovering new molecular players and enzymatic activities relevant to these pathways, we aim at identifying possible targets for therapeutic immuno-modulation or treatment of lymphomas.

T-cell receptor induced changes in cell shape and gene expression

The initiation of the adaptive immune response depends on the recognition of pathogenic substances or tumor-specific molecules (microbial or tumor antigens) by cell surface receptors on lymphocytes. Upon antigen recognition, lymphocytes undergo dramatic changes in cell shape and gene expression, which contribute to the activation, clonal proliferation and survival of the stimulated lymphocytes, which become effector cells capable of eliminating infected or tumor cells.

We are studying a complex of proteins, comprising CARMA1, BCL-10 and MALT1 that play key roles in the initiation of the adaptive immune response (Fig. 1). An important role for these proteins, which we and others have identified over the last few years, is the activation of the transcription factor NF-κB, which in turn controls the expression of genes that are essential for lymphocyte proliferation and survival (Gaide et al., 2002; Thome, 2004). More recently, we have identified another key role for this complex of proteins in the control of cellular adhesion processes that are important for the recognition of target cells by lymphocytes (Rebeaud, Hailfinger et al., 2008). Finally, we have described an essential role for BCL-10, independently of its association with CARMA1 or MALT1, in the control of T-cell- and Fc-receptor mediated actin polymerization, and could show that this is important for the recognition of antigen-presenting target cells by T-lymphocytes, but also for the Fc-receptor-mediated phagocytic uptake of antibody-covered pathogens (Rueda et al., 2007).

 

Figure 1: CARMA1, BCL-10 and MALT1 have multiple functions in T-cell activation. We could show that T-cell receptor (TCR)-induced phosphorylation of BCL-10 is critical for the rapid induction of actin polymerization and actin-dependent changes in cell shape. TCR-induced formation of a CARMA1-BCL-10-MALT1 (CBM) complex is essential for activation of the c-jun kinase (JNK) and NF-κB transcriptional pathways that regulate transcription of genes that control T-cell activation, proliferation and survival. It is thought that in this context, MALT1 contributes to NF-κB activation by recruitment of the ubiquitin ligase TRAF6 and by proteolytic cleavage of proteins with inhibitory function, such as A20. Recently, we could show that CBM complex formation results in the proteolytic cleavage of BCL-10, which is not required for NF-κB activation but essential for integrin-dependent T-cell adhesion.

The proteolytic activity of MALT1 is key to T-cell activation

The transcription factor NF-κB plays a key role in the expression of genes that are essential for lymphocyte activation and the generation of the immune response. In resting lymphocytes, NF-κB family members are present in the cytoplasm in an inactive form, bound to inhibitory κ-B (IκB) proteins. Triggering of the T-cell antigen receptor leads to activation of the IκB kinase (IKK) complex that induces phosphorylation and subsequent degradation of IκB proteins. This allows NF-κB to translocate into the nucleus and to initiate the transcription of genes that control lymphocyte proliferation and survival.

A major issue in the field of T-cell activation has been to understand the molecular mechanisms linking T-cell receptor engagement to the activation of the IKK complex. One of the earliest events following T-cell receptor (TCR) engagement is the activation of tyrosine kinases and the tyrosine phosphorylation of a restricted set of substrates. These in turn control the activation of Ser/Thr kinases of the protein kinase C family. PKCtheta and PKCbeta are T- and B-cell specific PKC family members essential for antigen receptor-induced NF-κB activation, by phosphorylation of CARMA1. We and others have previously identified CARMA1 as an essential signaling component in the antigen-recptor-induced NF-κB pathway, and shown that a caspase-recruitment domain (CARD)-mediated interaction between CARMA1 and BCL-10 is critical for signal transmission to the IKK complex (Gaide et al., 2002; Thome, 2004).

More recently, we could show that the BCL-10- and CARMA1-binding protein MALT1 contributes in a previously unknown manner to NF-κB activation. In particular, we could show that the proteolytic activity of the C-terminal caspase-like domain of MALT1 is critical for optimal NF-κB activation in T cells (Rebeaud, Hailfinger et al., 2008). MALT1 had been identified several years ago as a protein that shares homology with proteases of the caspase family, but despite intensive efforts it had remained enigmatic whether MALT1 has proteolytic activity and whether this might contribute to NF-κB activation. Through the identification of a BCL-10 cleavage product that is present exclusively in activated T- and B-cells (see below), we could recently demonstrate that MALT1 has an unexpected, Arginine-directed proteolytic activity that is transiently induced upon T-cell stimulation and cleaves the C-terminal part of BCL-10 (Fig. 2a). The development of a peptide-based inhibitor of MALT1 (in collaboration with the group of Prof. Nicolas Fasel), has allowed us to show that the proteolytic activity of MALT1 (but not the cleavage of BCL-10) is required for optimal NF-κB activation and cytokine production in human T cells (Fig. 2b). Since over-expression and/or constitutive activity of MALT1 has been associated with lymphomas of the mucosa-associated tissue (MALT lymphomas) and certain forms of diffuse large B-cell lymphomas (ABC-type DLBCL), these findings identify the proteolytic activity of MALT1 as a highly interesting target for the development of immuno-modulatory and anti-lymphoma drugs. Further studies are now targeted at the identification of the NF-κB-relevant MALT1 substrate(s).

Figure 2: T-cell activation induces MALT1 activation and cleavage of BCL-10. (A) Analysis of MALT1 activity and BCL-10 cleavage in T cells activated for the indicated times with PMA and ionomycin to mimic natural T-cell activation. T-cell activation leads to a transient increase in MALT1 activation (but not caspase-3 activation) that peaks at 30 min after stimulation. MALT1 activation correlates with the generation of a BCL-10 cleavage product that is stable for many hours (data not shown). (B) Treatment of human Jurkat T cells with a cell permeable MALT1 inhibitor, z-VRPR-fmk, leads to impaired NF-κB activation upon stimulation with agonistic anti-CD3 and anti-CD28 antibodies and to reduced secretion of the cytokine IL-2 upon stimulation with superantigen (SEE)-presenting Raji cells (Rebeaud, Hailfinger et al., 2008).

BCL-10 controls TCR-induced actin polymerization and adhesion

BCL-10 plays a key role in antigen receptor-induced NF-κB activation, but it also has NF-κB independent functions that we have recently started to characterize (reviewed in Thome, 2004; Thome & Weil, 2007). The starting point for these studies was the observation that, in activated T cells, Bcl10 undergoes characteristic post-translational modifications (Fig. 3a) (Rueda et al., 2007; Rebeaud, Hailfinger et al., 2008). We could show that BCL-10 is rapidly phosphorylated on at least two residues, and have identified Serine 138 as critical for BCL-10 phosphorylation. The study of a non-phosphorylatable Serine138-to-Alanine mutant of BCL-10 has revealed that BCL-10 phosphorylation on this site is not required for NF-κB activation, but rather plays a key role in TCR-induced cell shape changes that are critical for the recognition of the antigen-presenting cells by T cells (Rueda et al., 2007). In collaboration with the group of Dr. Florence Niedergang (Institut Cochin, Paris), we could further show that BCL-10-dependent actin polymerization plays an essential role in the Fc-receptor-induced phagocytosis of antibody-coated particles by macrophages, which is a key event in the elimination of pathogens by the immune system.

More recently, we have identified another posttranslational modification of BCL-10, which is due to the proteolytic removal of five amino acids from the C-terminus of BCL-10 by MALT1 (Fig. 3a) (Rebeaud, Hailfinger et al., 2008). The MALT1-dependent BCL-10 cleavage was generated with kinetics corresponding to MALT1 activation (see Fig. 2a) but remained stable for many hours after activation. Treatment of T cells with a MALT1-inhibitor preventing BCL-10 cleavage or reconstitution of BCL-10-deficient cells with a non-cleavable mutant of BCL-10 led to a clear reduction of TCR-induced adhesion of the T cells to fibronectin, thus demonstrating an impaired capacity of the T-cells to undergo integrin-mediated adhesion. In contrast, BCL-10 cleavage was not required for NF-κB activation.

Together, these results have led to the identification of two previously unsuspected, NF-κB independent functions of BCL-10 in the regulation of signaling pathways that are critical for changes in cell shape and adhesion (Fig. 1 and 3b). Further studies are aimed at the identification of the BCL-10 targets relevant for these pathways, and at the further elucidation of the physiological relevance of these observations.

Figure 3: T-cell activation induces phosphorylation and cleavage of BCL-10 to control various aspects of T-cell activation. (A) Analysis of BCL-10 from unstimulated or activated T cells by 2-dimensional gel electrophoresis, which separates proteins according to size (SDS-PAGE) or isoelectric point (IEF). T-cell activation with PMA and ionomycin induces phosphorylation of BCL-10 on two sites (indicated by open arrowheads) and C-terminal cleavage of BCL-10 after Arginine 228 (red arrowhead and arrow), which leads to acidification of the protein because of removal of the positively charged Arginine residue 232 (lower panel). The position of unmodified BCL-10 is indicated by a black arrowhead. (B) BCL-10 has multiple functions in T cells: binding to CARMA1 (via the CARD motif) and to MALT1 is critical for NF-κB and JNK activation and the control of TCR-induced transcription (Gaide et al., 2002), Ser 138-dependent phosphorylation is required for TCR-induced actin polymerization and actin-dependent changes in cell shape (Rueda et al., 2006), while MALT1-dependent cleavage close to the C-terminus of BCL-10, after Arginine 228, is relevant for beta1-integrin-mediated adhesion of T-cells (Rebeaud, Hailfinger et al., 2008).

Collaborations
Parts of this work were done in collaboration with the laboratories of Nicolas Fasel (Department of Biochemistry of the University of Lausanne), Nathalie Rufer (Multidisciplinary Oncology Center, University Hospital of Lausanne), Jürg Schwaller (formerly at the University Hospital of Geneva), Florence Niedergang (Institut Cochin, Paris) and Jérôme Delon (Institut Cochin, Paris).

Acknowledgments
We gratefully acknowledge the financial support of the Swiss National Science Foundation, the Swiss Cancer League (Oncosuisse) and the Foundations Pierre Mercier, Emma Muschamp and Leenaards.

Recent publications

• Hailfinger, S., Lenz, G., Ngo V., Posevitz-Fejfar A., Rebeaud F., Guzzardi M., Murga Penas, E., Dierlamm J., Chan, W.C., Staudt L.M. and Thome M. Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma. Proc. Natl. Acad. Sci. USA (in press).
• Thurau, M., Marquardt, G., Gonin-Laurent, N., Weinländer, K., Naschberger, E., Jochmann, R., Alkharsah, K.R., Schulz, T.F., Thome, M., Neipel, F. and Stürzl, M. (2009) The Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death. J. Virol. 83, 598-611. PubMed
• Brenner, D., Brechmann, M., Rohling, S., Tapernoux, M., Mock, T., Winter, D. Lehmann, W.D., Kiefer, F., Thome, M., Krammer, P.H., and Arnold, R. (2009) Phosphorylation of CARMA1 by HPK1 is critical for NF-κB activation in T cells. Proc. Natl. Acad. Sci. USA 106, 14508-14513. PubMed
• Rebeaud, F., S. Hailfinger, A. Posevitz-Fejfar, M. Tapernoux, R. Moser, D. Rueda, O. Gaide, M. Guzzardi, E. Iancu, N. Rufer, N. Fasel and M. Thome (2008a) The proteolytic activity of the paracaspase MALT1 is key in T cell activation. Nat Immunol. 9, 272-81. PubMed
• Torgler, R., Bongfen, S.E., Romero, J.C., Tardivel, A., Thome, M., Corradin, G. (2008). Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-kappaB Activation and Inducible NO Synthase Expression. J. Immunol. 180, 3990-3999. PubMed
• Rueda, D., Gaide, O., Ho, L., Lewkowicz, E., Niedergang, F., Hailfinger, S., Rebeaud, F., Guzzardi, M., Conne, B., Thelen, M., Delon, J., Ferch, U., Ruland, J., Mak, T., Schwaller, J., Thome, M. (2007). Bcl10 controls T-cell receptor- and FcgR-induced actin polymerization. J. Immunol. 178, 4373-4384. PubMed
• Loeuillet, C., Martinon, F., Perez, C., Munoz, M., Thome, M., Meylan, P.R. (2006). Mycobacterium tuberculosis subverts innate immunity to evade specific effectors. J. Immunol. 177, 6245-6255. PubMed
• Thurau, M., Everett, H., Tapernoux, M., Tschopp, J., Thome, M. (2006). The TRAF3 binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis. Cell Death Differ. 13, 1577-1585. PubMed
• Teixeiro, M., Daniels, M.A., Hausmann, B., Schrum, A.G., Naeher, D., Luescher, I., Thome, M., Bragado, R., Palmer, E. (2004). T Cell Division and Death Are Segregated by Mutation of TCR Chain Constant Domains. Immunity 21, 515-526. PubMed
• Egawa T., Albrecht B., Favier B., Sunshine M.J., Mirchandani K., O'Brien W., Thome M., Littman D.R. (2003). Requirement for CARMA1 in antigen receptor-induced NF-kappaB activation and lymphocyte proliferation. Curr. Biol. 13, 1252-1258. PubMed

Reviews

• Thome M., Rebeaud, F., Hailfinger, S. Adaptor and enzymatic functions of proteases in T-cell activation. Immunol. Rev. (in press)
• Thome M. (2008). Multifunctional roles for MALT1 in T cell activation. Nat. Rev. Immunol. 8, 495-500. PubMed
• Thome, M., Weil, R. (2007). Posttranslational modifications regulate distinct functions of Carma1 and Bcl10. Trends Immunol. 28, 281-288. PubMed
• Rebeaud, F., Hailfinger, S., Thome, M. (2007). Dlgh1 and Carma1 MAGUK proteins contribute to signal specificity downstream of TCR activation. Trends Immunol. 28, 196-200. PubMed
• Rueda, D., Thome, M. (2005). Phosphorylation of Carma1: the link(er) to NF-kappaB activation. Immunity 23, 551-553. PubMed
• Rueda, D., Thome, M. (2005). Molecule page: Carma1. AfCS Nature Molecule Pages, doi: 10.1038/mp.a003863.01.
• Thome, M. (2004). Carma1, Bcl10 and Malt1 in lymphocyte development and activation. Nature Rev. Immunol. 4, 348-359. PubMed
• Thome M., Tschopp J. (2003). TCR-induced NF-kappaB activation: a crucial role for Carma1, Bcl10 and MALT1. Trends Immunol. 24, 419-424. PubMed
• Thome, M. (2003). Regulation of actin assembly in the immunological synapse: a critical role for PKC theta. Dev. Cell 4, 3-5. PubMed

Book sections

• Thome, M. (2006). Regulation of Fas signaling by FLIP proteins. In Fas signaling, H. Wajant, ed. (New York, Landes Bioscience, Springer Science + Business Media), pp. 38-50.

 

Katrin Cabalzar	Master student
Jean-Enno Charton	Ph.D student
Jessica Gilliard	Trainee
Montserrat Guzzardi	Technician
Stephan Hailfinger	Postdoctoral fellow
Maike Jaworski	Postdoctoral fellow
Christiane Pelzer	Postdoctoral fellow
Mai Perroud	Technician