Développement, implémentation et utilisation pratique d'un score diagnostique [Development, implementation and practical use of a diagnostic score].

In recent years many clinical prediction rules (CPR) have been developed. Before a CPR can be used in clinical practice, different methodical steps are necessary, from the development of the score, the internal and external validation to the impact study. Before using a CPR in daily practice family doctors have to verify how the rules have been developed and whether this has been done in a population similar to the population in which they would use them. The aim of this paper is to describe the development of a CPR, and to discuss advantages and risks related to the use of CPR in order to help family doctors in their choice of scores for use in their daily practice.

HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C.

A variant 35 kb upstream of the HLA-C gene (-35C/T) was previously shown to associate with HLA-C mRNA expression level and steady-state plasma HIV RNA levels. We genotyped this variant in 1,698 patients of European ancestry with HIV. Individuals with known seroconversion dates were used for disease progression analysis and those with longitudinal viral load data were used for viral load analysis. We further tested cell surface expression of HLA-C in normal donors using an HLA-C-specific antibody. We show that the -35C allele is a proxy for high HLA-C cell surface expression, and that individuals with high-expressing HLA-C alleles progress more slowly to AIDS and control viremia significantly better than individuals with low HLA-C expressing alleles. These data strongly implicate high HLA-C expression levels in more effective control of HIV-1, potentially through better antigen presentation to cytotoxic T lymphocytes or recognition and killing of infected cells by natural killer cells.

Development, design and validation of solid reference samples.

We developed a method of sample preparation using epoxy compound, which was validated in two steps. First, we studied the homogeneity within samples by scanning tubes filled with radioactive epoxy. We found within-sample homogeneity better than 2%. Then, we studied the homogeneity between samples during a 4.5 h dispensing time. The homogeneity between samples was found to be better than 2%. This study demonstrates that we have a validated method, which assures the traceability of epoxy samples.

Clinical relevance of CD44 cell-surface expression and N-myc gene amplification in a multicentric analysis of 121 pediatric neuroblastomas.

PURPOSE: In contrast to other human tumors, a repression of the cell-surface glycoprotein CD44 on neuroblastoma is a marker of aggressiveness that usually correlates to N-myc amplification. We thus compared the prognostic value of both markers in the initial staging of 121 children treated for neuroblastoma in collaborative institutions. METHODS: Frozen samples were analyzed by a rapid and well-standardized technique of immunostaining with monoclonal antibodies (MoAbs) against epitopes in the CD44 constant region. RESULTS: In this retrospective series, CD44 was expressed on 102 specimens and strongly correlated with favorable tumor stages and histology, younger age, and normal N-myc copy numbers. In univariate analysis, CD44 expression and normal N-myc were the most powerful markers of favorable clinical outcome (P < 10(-6) and chi 2 = 65.40 and P < 10(-6) and chi 2 = 42.56, respectively), but analysis of CD44 affords significant prognostic discrimination in subgroups of patients with or without N-myc-amplified tumors. In the subgroup of stage IV neuroblastomas, CD44 was the only significant prognostic marker (P < .02, chi 2 = 5.76), whereas N-myc status was not discriminant. In multivariate analysis of five factors, ie, N-myc amplification, CD44 expression, age, tumor stage, and histology, the only independent prognostic factors of event-free survival were CD44 expression and tumor stage. CONCLUSION: The analysis of CD44 cell-surface expression must be recommended as an additional biologic marker in the initial staging of the disease.

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.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

IL-17F co-expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering.

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.

Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Detecting and measuring deprivation in primary care: development, validity and reliability of a self-reported questionnaire - the DiPCare-Q

Background: General practitioners play a central role in taking deprivation into consideration when caring for patients in primary care. Validated questions to identify deprivation in primary-care practices are still lacking. For both clinical and research purposes, this study therefore aims to develop and validate a standardized instrument measuring both material and social deprivation at an individual level. Methods: The Deprivation in Primary Care Questionnaire (DiPCare-Q) was developed using qualitative and quantitative approaches between 2008 and 2011. A systematic review identified 199 questions related to deprivation. Using judgmental item quality, these were reduced to 38 questions. Two focus groups (primary-care physicians, and primary-care researchers), structured interviews (10 laymen), and think aloud interviews (eight cleaning staff) assured face validity. Item response theory analysis was then used to derive the DiPCare-Q index using data obtained from a random sample of 200 patients who were to complete the questionnaire a second time over the phone. For construct and criterion validity, the final 16 questions were administered to a random sample of 1,898 patients attending one of 47 different private primary-care practices in western Switzerland (validation set) along with questions on subjective social status (subjective SES ladder), education, source of income, welfare status, and subjective poverty. Results: Deprivation was defined in three distinct dimensions (table); material deprivation (eight items), social deprivation (five items) and health deprivation (three items). Item consistency was high in both the derivation (KR20 = 0.827) and the validation set (KR20 = 0.778). The DiPCare-Q index was reliable (ICC = 0.847). For construct validity, we showed the DiPCare-Q index to be correlated to patients' estimation of their position on the subjective SES ladder (rs = 0.539). This position was correlated to both material and social deprivation independently suggesting two separate mechanisms enhancing the feeling of deprivation. Conclusion: The DiPCare-Q is a rapid, reliable and validated instrument useful for measuring both material and social deprivation in primary care. Questions from the DiPCare-Q are easy to use when investigating patients' social history and could improve clinicians' ability to detect underlying social distress related to deprivation.

Mécanismes moléculaires de l'homodimérisation de la protéine Scaffold IB1 et son implication dans la sécrétion d'insuline

RÉSUMÉ Les kinases activées par des mitogènes (MAPKs) constituent une importante famille d'enzymes conservée dans l'évolution. Elles forment un réseau de signalisation qui permet à la cellule de réguler spécifiquement divers processus impliqués dans la différenciation, la survie ou l'apoptose. Les kinases formant le module MAPK sont typiquement disposées en cascades de trois partenaires qui s'activent séquentiellement par phosphorylation. Le module minimum est constitué d'une MAPK kinase kinase (MAPKKK), d'une MAPK kinase (MAPKK) et d'une MAPK. Une fois activée, la MAPK phosphoryle différents substrats tels que des facteurs de transcription ou d'autres protéines. Chez les mammifères, trois groupes principaux de MAPKs ont été identifiés. Il s'agit du groupe des kinases régulées par des signaux extracellulaires du type «mitogènes » (ERK), ainsi que des groupes p38 et cJun NH2-terminal kinase (JNK), ou SAPK pour stress activated protein kinase, plutôt activées par des stimuli de type «stress ». De nombreuses études ont impliqué JNK dans la régulation de différents processus physiologiques et pathologiques, comme le diabète, les arthrites rhumatoïdes, l'athérosclérose, l'attaque cérébrale, les maladies de Parkinson et d'Alzheimer. JNK, en particulier joue un rôle dans la mort des cellules sécrétrices d'insuline induite par l'interleukine (IL)-1 β, lors du développement du diabète de type 1. IB1 est une protéine scaffold (échafaud) qui participe à l'organisation du module de JNK. IB1 est fortement exprimée dans les neurones et les cellules β du pancréas. Elle a été impliquée dans la survie des cellules, la régulation de l'expression du transporteur du glucose de type 2 (Glut-2) et dans le processus de sécrétion d'insuline glucose-dépendante. IBl est caractérisée par plusieurs domaines d'interaction protéine-protéine : un domaine de liaison à JNK (JBD), un domaine homologue au domaine 3 de Src (SH3) et un domaine d'interaction avec des tyrosines phosphorylées (PID). Des partenaires d'IB1, incluant les membres de la familles des kinases de lignée mélangée (MLKs), la MAPKK MKK7, la phosphatase 7 des MAPKs (MKP-7) ainsi que la chaîne légère de la kinésine, ont été isolés. Tous ces facteurs, sauf les MLKs et MKK7 interagissent avec le domaine PID ou l'extrême partie C-terminale d'IBl (la chaîne légère de la kinésine). Comme d'autres protéines scaffolds déjà décrites, IBl et un autre membre de la famille, IB2, sont capables d'homo- et d'hétérodimériser. L'interaction a lieu par l'intermédiaire de leur région C-terminale, contenant les domaines SH3 et PID. Mais ni le mécanisme moléculaire, ni la fonction de la dimérisation n'ont été caractérisés. Le domaine SH3 joue un rôle central lors de l'assemblage de complexes de macromolécules impliquées dans la signalisation intracellulaire. Il reconnaît de préférence des ligands contenant un motif riche en proline de type PxxP et s'y lie. Jusqu'à maintenant, tous les ligands isolés se liant à un domaine SH3 sont linéaires. Bien que le domaine SH3 soit un domaine important de la transmission des signaux, aucun partenaire interagissant spécifiquement avec le domaine SH3 d'IB1 n'a été identifié. Nous avons démontré qu'IBl homodimérisait par un nouveau set unique d'interaction domaine SH3 - domaine SH3. Les études de cristallisation ont démontré que l'interface recouvrait une région généralement impliquée dans la reconnaissance classique d'un motif riche en proline de type PxxP, bien que le domaine SH3 d'IB1 ne contienne aucun motif PxxP. L'homodimère d'IB1 semble extrêmement stable. Il peut cependant être déstabilisé par trois mutations ponctuelles dirigées contre des résidus clés impliqués dans la dimérisation. Chaque mutation réduit l'activation basale de JNK dépendante d'IB 1 dans des cellules 293T. La déstabilisation de la dimérisation induite par la sur-expression du domaine SH3, provoque une diminution de la sécrétion d'insuline glucose dépendant. SUMMARY Mitogen activated kinases (MAPK) are an important and conserved enzyme family. They form a signaling network required to specifically regulate process involved in cell differentiation, proliferation or death. A MAPK module is typically organized in a threekinase cascade which are activated by sequential phosphorylation. The MAPK kinase kinase (MAPKKK), the MAPK kinase (MAPKK) and the MAPK constitute the minimal module. Once activated, the MAPK phosphorylates its targets like transcription factors or other proteins. In mammals, three major groups of MAPKs have been identified : the group of extra-cellular regulated kinase (ERK) which is activated by mitogens and the group of p38 and cJun NH2-terminal kinase (JNK) or SAPK for stress activated protein kinase, which are activated by stresses. Many studies implicated JNK in many physiological or pathological process regulations, like diabetes, rheumatoid arthritis, arteriosclerosis, strokes or Parkinson and Alzheimer disease. In particular, JNK plays a crucial role in pancreatic β cell death induced by Interleukin (IL)-1 β in type 1 diabetes. Islet-brain 1 (IB 1) is a scaffold protein that interacts with components of the JNK signal-transduction pathway. IB 1 is expressed at high levels in neurons and in pancreatic β-cells, where it has been implicated in cell survival, in regulating expression of the glucose transporter type 2 (Glut-2) and in glucose-induced insulin secretion. It contains several protein-protein interaction domains, including a JNK-binding domain (JBD), a Src homology 3 domain (SH3) and a phosphotyrosine interaction domain (PID). Proteins that have been shown to associate with IB 1 include members of the Mixed lineage kinase family (MLKs), the MAPKK MKK7, the MAPK phosphatase-7 MKP7, as well as several other ligands including kinesin light chain, LDL receptor related family members and the amyloid precursor protein APP. All these factors, except MLK3 and MKK7 have been shown to interact with the PID domain or the extreme C-terminal part (Kinesin light chain) of IB 1. As some scaffold already described, IB 1 and another member of the family, IB2, have previously been shown to engage in oligomerization through their respective C-terminal regions that include the SH3 and PID domains. But neither the molecular mechanisms nor the function of dimerization have yet been characterized. SH3 domains are central in the assembly of macromolecular complexes involved in many intracellular signaling pathways. SH3 domains are usually characterized by their preferred recognition of and association with canonical PxxP motif. In all these cases, a single linear sequence is sufficient for binding to the SH3 domain. However, although SH3 domains are important elements of signal transduction, no protein that interacts specifically with the SH3 domain of IB 1 has been identified so far. Here, we show that IB 1 homodimerizes through a navel and unique set of SH3-SH3 interactions. X-ray crystallography studies indicate that the dieter interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB 1 SH3 domain lacks this motif. The highly stable IB 1 homodimer can be significantly destabilized in vitro by individual point-mutations directed against key residues involved in dimerization. Each mutation reduces IB 1-dependent basal JNK activity in 293T cells. Impaired dimerization induced by over-expression of the SH3 domain also results in a significant reduction in glucose-dependent insulin secretion in pancreatic β-cells.

CD8+ T cell development: CD4 to the rescue.

How immature CD4+CD8+ thymocytes become committed to either the CD4 (helper) or CD8 (cytotoxic) lineage is controversial. Genetic ablation of a silencer element in the gene encoding CD4 provides new evidence that CD8 lineage commitment occurs via a stochastic, rather than instructive, mechanism.

Role of lymph node fibroblasts in T cell priming

SummarySecondary lymphoid organs, such as lymph nodes or spleen, are the only places in our body where primary adaptive immune responses are efficiently elicited. These organs have distinct Β and Τ cell rich zones and Τ lymphocytes constantly migrate from the bloodstream into Τ zones to scan dendritic cells (DCs) for antigens they present. Specialized fibroblasts, the Τ zone reticular cells (HR.Cs), span the Τ zone in the form a three-dimensional network. lK.Cs guide incoming Τ cells in their migration, both chemically, by the secretion of the chemokines CCL19 and CCL21, and physically, by construction of a road system to which also DCs adhere. In this way TRCs are thought to facilitate encounters of Τ cells with antigen-bearing DCs and thereby accelerate the selection of rare antigen-specific Τ cells. The resulting Τ cell activation, proliferation and differentiation all take place within the TRC network. However, the influence of TRCs on Τ cell activation has so fer not been elucidated with the possible reasons being that TRCs represent a relative rare cell population and that mice devoid of TRCs have not been described.To circumvent these technical limitations, we established TRC clones and lines to have an abundant source to functionally characterize TRCs. Both the clones and lines show a fibroblastic phenotype, express a surface marker profile comparable to ex vivo TRCs and produce extracellular matrix molecules. However, expression of Ccl19, Ccl21 and ZL-7 is lost and could not be restored by cytokine stimulation. When these TRC clones or lines were cultured in a three-dimensional cell culture system, their morphology changed and resembled that of in vivo TRCs as they formed networks. By adding Τ cells and antigen-loaded DCs to these cultures we successfully reconstructed lymphoid Τ zones that allowed antigen-specific Τ cell activation.To characterize the role of TRCs in Τ cell priming, TRCs were co-cultured with antigen-specific Τ cells in the presence antigen-loaded DCs. Surprisingly, the presence of TRC lines and ex vivo TRCs inhibited rather than enhanced CD8+ Τ cell activation, proliferation and effector cell differentiation. TRCs shared this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. TRCs were identified as a strong source of nitric oxide (NO) thereby directly dampening Τ cell expansion as well as reducing the Τ cell priming capacity of DCs. The expression of inducible NO synthase (iNOS) was up- regulated in a subset of TRCs by both DC-signals as well as interferon-γ produced by primed CD8+ Τ cells. Importantly, iNOS expression was induced during viral infection in vivo in both lymph node TRCs and DCs. Consistent with a role for NO as a negative regulator, the primary Τ cell response was exaggerated in iNOS-/- mice. Our findings highlight that in addition to their established positive roles in Τ cell responses TRCs and DCs cooperate in a negative feedback loop to attenuate Τ cell expansion during acute inflammation.RésuméLes organes lymphoïdes secondaires, comme les ganglions lymphoïdes ou la rate, sont les seuls sites dans notre corps où la réponse primaire des lymphocytes Β et Τ est initiée efficacement. Ces organes ont des zones différentes, riches en cellules Β ou T. Des lymphocytes Τ circulent constamment du sang vers les zones T, où ils échantillonent la surface des cellules dendritiques (DCs) pour identifier les antigènes qu'ils présentent. Des fibroblastes spécialisés - nommés Τ zone reticular cells (TRCs)' forment un réseau tridimensionnel dans la zone T. Les TRCs guident la migration des cellules Τ par deux moyens: chimiquement, par la sécrétion des chimiokines CCL19 et CCL21 et physiquement, par la construction d'un réseau routier en trois dimensions, auquel adhèrent aussi des DCs. Dans ce? cas, on pense que la présence des TRCs facilite les rencontres entre les cellules Τ et les DCs chargées de l'antigène et accélère la sélection des rares cellules Τ spécifiques. Ensuite, l'activation de cellules T, ainsi que la prolifération et la différenciation se produisent toutes à l'intérieur du réseau des TRCs. L'influence des TRCs sur l'activation des cellules T n'est que très peu caractérisée, en partie parce que les TRCs représentent une population rare et que les souris déficientes dans les TRCs n'ont pas encore été découvertes.Pour contourner ces limitations techniques, nous avons établi des clones et des lignées cellulaires de TRC pour obtenir une source indéfinie de ces cellules permettant leur caractérisation fonctionnelle. Les clones et lignées établis ont un phénotype de fibroblaste, ils expriment des molécules de surface similaires aux TRCs ex vivo et produisent de la matrice extracellulaire. Mais l'expression de Ccl19, Ccl21 et 11-7 est perdue et ne peut pas être rétablie par stimulation avec différentes cytokines. Les clones TRC ou les lignées cultivées en un système tridimensionnel de culture cellulaire, montrent une morphologie changée, qui ressemble à celle de TRC ex vivo inclus la construction de réseaux tridimensionnels.Pour caractériser le rôle des TRC dans l'activation des cellules T, nous avons cultivé des TRCs avec des cellules T spécifiques et des DCs chargées avec l'antigène. Etonnamment, la présence des TRC (lignées et ex vivo) inhibait plutôt qu'elle améliorait l'activation, la prolifération et la différenciation des lymphocytes T CDS+. Les TRCs partageaient cette fonction avec des fibr-oblastes des organes non lymphoïdes et des cellules souches du type mésenchymateux. Dans ces conditions, les TRCs sont une source importante d'oxyde nitrique (NO) et par ce fait limitent directement l'expansion des cellules T et réduisent aussi la capacité des DCs à activer les cellules T. L'expression de l'enzyme NO synthase inductible (ïNOS) est régulée à la hausse par des signaux dérivés des DCs et par l'interféron-γ produit par des cellules T de type CD8+ activées. Plus important, l'expression d'iNOS est induite pendant une infection virale in vivo, dans les TRCs et dans les DCs. Par conséquent, la réponse primaire de cellules T est exagérée dans des souris iNOS-/-. Nos résultats mettent en évidence qu'en plus de leur rôle positif bien établi dans la réponse immunitaire, les TRCs et les DCs coopèrent dans une boucle de rétroaction négative pour atténuer l'expansion des cellules T pendant l'inflammation aigiie pour protéger l'intégrité et la fonctionnalité des organes lymphoïdes secondaires.

Phorbol 12-myristate 13-acetate induces surface expression of T3 on human immature T cell lines with and without concomitant expression of the T cell antigen receptor complex.

The T3 complex is known to be expressed on the cell surface of mature T cells together with either the alpha-beta heterodimeric T cell receptor (TCR) or the TCR gamma protein. In a number of immature T cell malignancies, however, T3 has been described exclusively in the cytoplasm. We have investigated five such T cell lines with cytoplasmic T3 and could demonstrate by biosynthetic labeling the presence of the alpha and beta chains of the TCR in the cytoplasm of two of them, CEM and Ichikawa. No surface TCR alpha-beta protein could be detected by staining with the WT31 antibody. These observations, therefore, argue against the concept that expression of the TCR alpha chain controls the surface expression of the T3/TCR complex. Interestingly, phorbol 12-myristate 13-acetate (PMA) induced cell surface expression of T3 protein in these two cell lines only. Moreover, on surface-iodinated CEM cells no association of T3 and TCR molecules could be demonstrated after treatment with PMA, and expression of TCR alpha and beta chains was limited to the cytoplasm. In Ichikawa cells, however, PMA induced surface expression of a mature T3/TCR complex. Our findings indicate that separate regulatory mechanisms may exist for the surface expression of the T3 proteins and for the assembly of the T3/TCR complex.