Role of c-Myc in homeostasis of memory CD8 T cells

RESUME La mémoire immunologique est essentielle durant la vie et permet aux lymphocytes de répondre plus rapidement et efficacement lors d'une deuxième rencontre avec un antigène connu. Les facteurs contrôlant l'homéostasie des cellules T CD8 mémoires in vivo ne sont pas encore bien définis. Cependant, la prolifération homéostatique de ces cellules dans un hôte déplété en cellules hématopoietiques nécessite l'intéraction du TCR avec les molecules du MHC de class I du soi. De plus, le rôle de cytokines, telles que 1'IL-15 et l'IL-7, est essentiel dans ce mécanisme, aussi bien que dans la maintenance des cellules T CD8 mémoires. Puisque la protéine c-Myc - impliquée dans des mécanismes tells que la division, la prolifération, l'apoptose et la differentiation - a été définie comme étant impliquée dans la réponse à différentes cytokines, nous nous sommes intéressés à l'analyse de l'homéostasie des lymphocytes T CD8 mémoires dans des souris déficientes en c-Myc (c_rnycΔORF/+), qui expriment un niveau réduit de cette protéine. Bien que le développement des cellules T dans le thymus soit normal dans les souris c_rnycΔORF/+, nous avons observé une réduction de 2 à 3 fois dans la population des cellules T CD8 de phenotype mémoire (CD44+) dans les organes lymphoïdes de la périphérie de ces souris. Cette différence ne correspond pas à une réduction de prolifération ou d'expression de protéines de survie telles que Bel-2. Cependant, la prolifération homéostatique de cellules T CD8 c_rnycΔORF/+, mais pas T CD4 c_rnycΔORF/+, est reduite de manière dramatique lorsqu'elles sont transférées dans un hôte irradié. De plus, le transfert adoptif de lymphocytes T dans des souris irradiées déficientes en l'IL-15 nous a permis de montrer que la prolifération homéostatique dépendante de l'IL-15 des cellules T CD8 nécessite l'expression de c-Myc. De plus, contrairement aux cellules T CD8 CD44+ de type sauvage, nous avons observé que l'expansion induite par l'IL-15 des cellules T CD8 CD44+ c_rnycΔORF/+ est altérée aussi bien in vivo (en réponse à une injection de polyI:C) et in vitro. Par conséquent, nos résultats identifient c-Myc comme une nouvelle protéine régulatrice de la signalisation par l'IL-15 impliquée dans l'homéostasie des cellules T CD8 CD44+. SUMMARY Immunological memory is essential throughout life and allows memory lymphocytes to respond faster and more efficiently upon re-encounter of a known antigen. Factors controlling homeostasis of memory CD8 T cells under steady-state conditions in vivo are currently not well defined. However, the homeostatic proliferation of memory CD8 T cells in lymphopenic hosts requires the interaction of the TCR with self MHC class I molecules. In addition, cytokines, such as IL-15 and to a lesser extent IL-7, are essential for both homeostatic proliferation and maintenance of memory CD8 T cells. Since c-Myc, a proto-oncogene involved in cell division, proliferation, apoptosis and differentiation, has been widely implicated in responsiveness to cytokines, we were interested in analyzing homeostasis of memory CD8 T cells in c-myc hypomorph (c_rnycΔORF/+) mice, which express reduced levels of c-Myc. Although T cell development in the thymus was normal in c_rnycΔORF/+ mice, we found a selective 2- to 3-fold reduction in the memory-phenotype CD44high CD8 T cell population in the periphery. Reduced numbers of CD44high CD8 T cells did not correlate with decreased steady-state turnover rate or low expression of survival factors such as Bcl- 2. However, homeostatic proliferation of c_rnycΔORF/+ CD8 T cells, but not c_rnycΔORF/+ CD4 T cells, was dramatically reduced upon transfer into sublethally irradiated wild-type recipients. In addition, upon transfer of c_rnycΔORF/+ and c-myc WT cells into IL-15-/- mice, we observed that IL-15-induced homeostatic proliferation of CD8 T cells requires c-Myc. Moreover, in contrast to c-myc WT CD44high CD8 T cells, IL-15-induced expansion of c_rnycΔORF/+ CD44high CD8 T cells was strongly impaired both in vivo (in response to polyI:C injection) and in vitro. Collectively, our data identify c-Myc as a novel downstream regulator of IL-15 signaling involved in homeostasis of memory CD8 T cells.

Genetic analysis of c-Myc in mouse bone marrow and liver

1. Summary The transcription factor and proto-oncogene c-myc plays an important role in integrating many mitogenic signals within the cell. The consequences are both broad and varied and include the regulation of apoptosis, cellular differentiation, cellular growth and cell cycle progression. It is found to be mis-regulated in over 70% of all cancers, however, our knowledge about c-Myc remains limited and very little is known about its physiological role in mammalian development and in adulthood. We have addressed the physiological role of c-Myc in both the bone marrow and the liver of mice by generating adult c-myc flox/flox mice that lacked c-myc in either the bone marrow or the liver after conversion of the c-myc flox alleles into null alleles by the inducible Mx¬Cre transgene with polyI-polyC. In investigating the role of c-Myc in the haematopoietic system, we concentrated on the aspects of cellular proliferation, cellular differentiation and apoptosis. Mice lacking c-Myc develop anaemia between 3-8 weeks and all more differentiated cell types are severely depleted leading to death. However in addition to its role in driving proliferation in transient amplifying cells, we unexpectedly discovered a new role for c-Myc in controlling haematopoietic stem cell (HSC) differentiation. c-Myc deficient HSCs are able to proliferate normally in vivo. In addition, their differentiation into more committed progenitors is blocked. These cells expressed increased adhesion molecules, which possibly prevent HSCs from being released from the special stem cell supporting stromal niche cells with which they closely associate. Secondly we used the liver as a model system to address the role of c-Myc in cellular growth, meaning the increase in cell size, and also cellular proliferation. Our results revealed c-Myc to play no role in metabolic cellular growth following a period of fasting. Following treatment with the xenobiotic TCPOBOP, c-Myc deficient hepatocytes increased in cell size as control hepatocytes and could surprisingly proliferate albeit at a reduced rate demonstrating a c-Myc independent proliferation pathway to exist in parenchymal cells. However, following partial hepatectomy, in which two-thirds of the liver was removed, mutant livers were severely restricted in their regeneration capacity compared to control livers demonstrating that c-Myc is essential for liver regeneration. Résumé Le facteur de transcription et proto-oncogène c-myc joue un rôle important dans l'intégration de nombreux signaux mitogéniques dans la cellule. Les conséquences de son activation sont étendues et variées et incluent la régulation de l'apoptose, de la différenciation, de la croissance et de la progression du cycle cellulaire. Même si plus de 20% des cancers montrent une dérégulation de c-myc, les connaissances sur ce facteur de transcription restent limitées et ses rôles physiologiques au cours du développement et chez l'adulte sont très peu connus. Nous avons étudié le rôle physiologique de c-Myc dans la molle osseuse et le foie murin en générant des souris adultes c-myc flox/flox. Dans ces souris, les allèles c-myc flox sont convertis en allèles nuls par le transgène Mx-Cre après induction avec du Poly-I.C. Pour notre étude du rôle de c-Myc dans le système hématopoiétique, nous nous sommes concentrés sur les aspects de la prolifération et de la différenciation cellulaire, ainsi que sur l'apoptose. Les souris déficientes pour c-Myc développent une anémie 3 à 8 semaines après la délétion du gène; tous les différents types cellulaires matures sont progressivement épuisés ce qui entraîne la mort des animaux. Néanmoins, outre sa capacité à induire la prolifération des cellules transitoires de la molle osseuse, nous avons inopinément découvert un nouveau rôle pour c-Myc dans le contrôle de la différenciation des cellules souches hématopoiétiques (HSC). Les HSC déficientes pour c-Myc prolifèrent normalement in vivo mais leur différenciation en progéniteurs plus engagés dans une voie de différenciation est bloquée. Ces cellules surexpriment certaines molécules d'adhésion ce qui empêcherait les HSC d'être relachées du stroma spécialisé, ou niche, auquel elles sont étroitement associées. D'autre part, nous avons utilisé le foie comme système modèle pour étudier le rôle de c-Myc dans la prolifération et dans la croissance cellulaire, c'est à dire l'augmentation de taille des cellules. Nos résultats ont révélé que c-Myc ne joue pas de rôle dans le métabolisme cellulaire qui suit une période de jeûne. L'augmentation de la taille cellulaire des hépatocytes déficients pour c-Myc suite au traitement avec l'agent xénobiotique TCPOBOP est identique à celle observée pour les cellules de contrôle. Le taux de prolifération des hépatocytes mutants est par contre réduit, indiquant qu'une voie de différenciation indépendante de c-Myc existe dans les cellules parenchymales. Néanmoins, après hépatectomie partielle, où deux-tiers du foie sont éliminés chirurgicalement, les foies mutants sont sévèrement limités dans leur capacité de régénération par rapport aux foies de contrôle, montrant ainsi que c-Myc est essentiel pour la régénération hépatique.

Essential role for Pbx1 in corneal morphogenesis.

PURPOSE: The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice. METHODS: Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis. RESULTS: Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity. CONCLUSIONS: These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Dlgh1 and Carma1 MAGUK proteins contribute to signal specificity downstream of TCR activation.

Mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), have a key role in T cell receptor (TCR)-induced gene transcription but their precise mechanism of activation is not well understood. The findings of two recent papers provide new insight into the activation of p38 and JNK by the membrane-associated guanylate kinase (MAGUK) family members Dlgh1 and Carma1, respectively, and show how distinct MAGUK proteins control specific aspects of TCR-mediated MAPK activation.

Maf1, a new player in the regulation of human RNA polymerase III transcription.

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

IB1/JIP-1 controls JNK activation and increased during prostatic LNCaP cells neuroendocrine differentiation.

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1.

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.