The role of WNT and mTOR in human memory T cell formation

Cancer is one of the world's leading causes of death with a rising trend in incidence. These epidemiologic observations underline the need for novel treatment strategies. In this regard, a promising approach takes advantage of the adaptive effector mechanisms of the immune system, using T lymphocytes to specifically target and destroy tumour cells. However, whereas current approaches mainly depend on short-lived, terminally differentiated effector T cells, increasing evidence suggests that long lasting and maximum efficient immune responses are mediated by low differentiated memory T cells. These memory T cells should display characteristics of stem cells, such as longevity, self-renewal capacity and the ability to continuously give rise to further differentiated effectors. These stem celllike memory T (TSCM) cells are thought to be of key therapeutic value as they might not only attack differentiated tumour cells, but also eradicate the root cause of cancer, the cancer stem cells themselves. Thus, efforts are made to characterize TSCM cells and to identify the signalling pathways which mediate their induction. Recently, a human TSCM cell subset was described and the activation of the Wnt-ß-catenin signalling pathway by the drug TWS119 during naive CD8+ T (TN) cell priming was suggested to mediate their induction. However, a precise deciphering of the signalling pathways leading to TSCM cell induction and an in-depth characterization of in vitro induced and in vivo occurring TSCM cells remain to be performed. Here, evidence is presented that the induction of human and mouse CD8+ and CD4+ TSCM cells may be triggered by inhibition of mechanistic/mammalian target of rapamycin (mTOR) complex 1 with simultaneously active mTOR complex 2. This molecular mechanism arrests a fraction of activated TN cells in a stem cell-like differentiation state independently of the Wnt-ß-catenin signalling pathway. Of note, TWS119 was found to also inhibit mTORCl, thereby mediating the induction of TSCM cells. Suggesting an immunostimulatory effect, the acquired data broaden the therapeutic range of mTORCl inhibitors like rapamycin, which are, at present, exclusively used due to their immunosuppressive function. Furthermore, by performing broad metabolic analyses, a well-orchestrated interplay between intracellular signalling pathways and the T cells' metabolic programmes could be identified as important regulator of the T cells' differentiation fate. Moreover, in vitro induced CD4+ TSCM cells possess superior functional capacities and share fate-determining key factors with their naturally occurring counterparts, assessed by a first-time full transcriptome analysis of in vivo occurring CD4+ TN cell, TSCM cells and central memory (TCM) cells and in vitro induced CD4+ TSCM cells. Of interest, a group of 56 genes, with a unique expression profile in TSCM cells could be identified. Thus, a pharmacological mechanism allowing to confer sternness to activated TN cells has been found which might be highly relevant for the design of novel T cell-based cancer immunotherapies.

PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway.

BACKGROUND & AIMS: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. METHODS: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. RESULTS: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. CONCLUSIONS: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen.

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.

The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration.

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

A multicenter phase II trial (SAKK 36/06) of single-agent everolimus (RAD001) in patients with relapsed or refractory mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma accounts for 6% of all B-cell lymphomas and is generally incurable. It is characterized by the translocation t(11;14) leading to cyclin D1 over-expression. Cyclin D1 is downstream of the mammalian target of rapamycin threonine kinase and can be effectively blocked by mammalian target of rapamycin inhibitors. We set out to examine the single agent activity of the orally available mammalian target of rapamycin inhibitor everolimus in a prospective, multicenter trial in patients with relapsed or refractory mantle cell lymphoma (NCT00516412). DESIGN AND METHODS: Eligible patients who had received a maximum of three prior lines of chemotherapy were given everolimus 10 mg for 28 days (one cycle) for a total of six cycles or until disease progression. The primary endpoint was the best objective response. Adverse reactions, progression-free survival and molecular response were secondary endpoints. RESULTS: Thirty-six patients (35 evaluable) were enrolled and treatment was generally well tolerated with Common Terminology Criteria grade ≥ 3 adverse events (>5%) including anemia (11%), thrombocytopenia (11%) and neutropenia (8%). The overall response rate was 20% (95% CI: 8-37%) with two complete remissions and five partial responses; 49% of the patients had stable disease. At a median follow-up of 6 months, the median progression-free survival was 5.5 months (95% CI: 2.8-8.2) overall and 17.0 (6.4-23.3) months for 18 patients who received six or more cycles of treatment. Three patients achieved a lasting complete molecular response, as assessed by polymerase chain reaction analysis of peripheral blood. CONCLUSIONS: Everolimus as a single agent is well tolerated and has anti-lymphoma activity in relapsed or refractory mantle cell lymphoma. Further studies of everolimus in combination with chemotherapy or as a single agent for maintenance treatment are warranted.

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Preparation, maintenance, and use of serum-free aggregating brain cell cultures.

Serum-free aggregating brain cell cultures are free-floating three-dimensional primary cell cultures able to reconstitute spontaneously a histotypic brain architecture to reproduce critical steps of brain development and to reach a high level of structural and functional maturity. This culture system offers, therefore, a unique model for neurotoxicity testing both during the development and at advanced cellular differentiation, and the high number of aggregates available combined with the excellent reproducibility of the cultures facilitates routine test procedures. This chapter presents a detailed description of the preparation, maintenance, and use of these cultures for neurotoxicity studies and a comparison of the developmental characteristics between cultures derived from the telencephalon and cultures derived from the whole brain. For culture preparation, mechanically dissociated embryonic brain tissue is used. The initial cell suspension, composed of neural stem cells, neural progenitor cells, immature postmitotic neurons, glioblasts, and microglial cells, is kept in a serum-free, chemically defined medium under continuous gyratory agitation. Spherical aggregates form spontaneously and are maintained in suspension culture for several weeks. Within the aggregates, the cells rearrange and mature, reproducing critical morphogenic events, such as migration, proliferation, differentiation, synaptogenesis, and myelination. For experimentation, replicate cultures are prepared by the randomization of aggregates from several original flasks. The high yield and reproducibility of the cultures enable multiparametric endpoint analyses, including "omics" approaches.

Molecular signaling in zebrafish development and the vertebrate phylotypic period

During development vertebrate embryos pass through a stage where their morphology is most conserved between species, the phylotypic period (approximately the pharyngula). To explain the resistance to evolutionary changes of this period, one hypothesis suggests that it is characterized by a high level of interactions. Based on this hypothesis, we examined protein-protein interactions, signal transduction cascades and miRNAs over the course of zebrafish development, and the conservation of expression of these genes in mouse development. We also investigated the characteristics of genes highly expressed before or during the presumed phylotypic period. We show that while there is a high diversity of interactions during the phylotypic period (protein-DNA, RNA-RNA, cell-cell, and between tissues), which is well conserved with mouse, there is no clear difference with later, more morphologically divergent, stages. We propose that the phylotypic period may rather be the expression at the morphological level of strong conservation of molecular processes earlier in development.

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Role of the JNK-interacting protein 1/islet brain 1 in cell degeneration in Alzheimer disease and diabetes.

Numerous epidemiological studies and some pharmacological clinical trials show the close connection between Alzheimer disease (AD) and type 2 diabetes (T2D) and thereby, shed more light into the existence of possible similar pathogenic mechanisms between these two diseases. Diabetes increases the risk of developing AD and sensitizers of insulin currently used as diabetes drugs can efficiently slow cognitive decline of the neurological disorder. Deposits of amyloid aggregate and hyperphosphorylation of tau, which are hallmarks of AD, have been also found in degenerating pancreatic islets beta-cells of patients with T2D. These events may have a causal role in the pathogenesis of the two diseases. Increased c-Jun NH(2)-terminal kinase (JNK) activity is found in neurofibrillary tangles (NFT) of AD and promotes programmed cell death of beta-cells exposed to a diabetic environment. The JNK-interacting protein 1 (JIP-1), also called islet brain 1 (IB1) because it is mostly expressed in the brain and islets, is a key regulator of the JNK pathway in neuronal and beta-cells. JNK, hyperphosphorylated tau and IB1/JIP-1 all co-localize with amyloids deposits in NFT and islets of AD and patients with T2D. This review discusses the role of the IB1/JIP-1 and the JNK pathway in the molecular pathogenesis of AD and T2D.