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Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens world-wide, being responsible for frequent hospital outbreaks and causing sepsis and multi-organ infections with a high mortality rate and frequent hospital outbreaks. The most prevalent and widely disseminated lineage of K. pneumoniae is clonal group 258 (CG258), which includes the highly resistant “high-risk” genotypes ST258 and ST11. Recent studies revealed that very large recombination events have occurred during the recent emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hyper-virulent or resistant clones remains unclear. Here we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100Kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which result to be involved by recombination events with different frequencies. Our results also indicate that certain non-CG258 lineages are more likely to act as donors to CG258 recipients than others. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion.Author SummaryKlebsiella pneumoniae (Kp) is an opportunistic bacterial pathogen, a major cause of deadly infections and outbreaks in hospitals worldwide. This bacterium is able to exchange large genomic portions (up to a fourth of the entire genome) within a single recombination event. Indeed, the most epidemiologically important Kp clone, is actually a hybrid which emerged after a > 1Mb recombination event. In this work, we investigated how recombinations affected the evolution of the most studied Kp Clonal Group, CG258. We found that large recombinations occurred frequently during Kp evolution, and occurred preferentially in a well-delimited genomic region. Furthermore, we found that four epidemiologically important clones emerged after large recombinations. We identified the donors of several large recombinations: despite many Kp lineages acted as donors during CG258 evolution, two of them have been involved more frequently. We hypothesize that the observed pattern of donors-recipients in recombinations, and the presence of a large recombinogenic region in Kp genome, could be related to gene composition. Indeed, genomic analyses showed a pattern compatible with this hypothesis, suggesting that gene content can represent a main factor in the success of a large recombination.

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

International audience; The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high.

International audience; Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

International audience; Reconstructing the lineage of cells is central to understanding development and is now also an important issue in stem cell research. Technological advances in genetically engineered permanent cell labeling, together with a multiplicity of fluorescent markers and sophisticated imaging, open new possibilities for prospective and retrospective clonal analysis.

CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1–lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis -derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1–lipid complexes.

The human CD1a–d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM6. CD1e is formed by the association of β2-microglobulin with an α-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM6 presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.

International audience; BACKGROUND: Kaposi sarcoma (KS) is a complex tumor of uncertain clonality. Studying the viral clonality of the human herpesvirus 8 (HHV-8) in KS to determine clonality of the tumors, a strategy that has been used previously with Epstein-Barr virus and its associated tumors, may elucidate whether multicentric (disseminated) KS lesions correspond to metastatic lesions or to expansions of independent clones. METHODS: A series of 139 KS biopsies (from skin, lymph node, or tonsil) was obtained from 98 patients, with 59 biopsies from 18 patients with disseminated multicentric KS skin lesions. The degree of spindle cell infiltration in biopsies was established by direct observation of hematoxylin-eosin-stained sections, and HHV-8 viral load was quantified by real-time polymerase chain reaction. To determine cellular clonality, the size heterogeneity of the HHV-8-fused terminal repeat (TR) region was determined by probing of electrophoresed restricted genomic DNA from KS biopsies for the HHV-8 TR sequence. RESULTS: HHV-8 clonality analysis was performed on the 62 samples for which sufficient DNA was obtained. Most samples corresponded to histologically nodular lesions with high spindle cell infiltration and high viral load. A clonal HHV-8 pattern was determined for 59 samples; 11 were found to be monoclonal and 48 to be oligoclonal. The informative samples that were from disseminated KS skin lesions (n = 26, from six patients) were either monoclonal or oligoclonal, and the size of HHV-8 episomes varied between these samples. CONCLUSION: Although some tumor KS lesions were monoclonal expansions of HHV-8-infected spindle cells, most advanced lesions were oligoclonal proliferations. Furthermore, individual KS disseminated tumor skin lesions were found to represent distinct expansions of HHV-8-infected spindle cells. Thus, our results suggest that KS lesions, especially in patients with advanced skin tumors, are reactive proliferations rather than true malignancies with metastatic dissemination.

Cells cured of persistent virus infection can be used to investigate cellular pathways of resistance to viral cytopathic effects. Persistent poliovirus (PV) infections were established in human intestinal Caco-2 cells, and spontaneously cured cell cultures were obtained. Two cell clones, cl6 and b13, cured of type 3 PV mutant infection and their parental Caco-2 cells were compared for susceptibility to PV infection, PV receptor CD155 expression, capacity to differentiate into polarized enterocytes, and PV-, staurosporine-, and actinomycin D-induced apoptosis. Our results strongly suggest that cells that are partially resistant to apoptosis can be selected during persistent virus infection.

Lat Y136F knock-in mice harbor a point mutation in Tyr136 of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the LatY136F mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of LatY136F mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In LatY136F mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in LatY136F serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in LatY136F mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.

Follicular lymphoma (FL) is one of the most common B-cell lymphoma, and remains virtually incurable despite its relatively indolent nature. T(14;18)(q32;q21), the genetic hallmark and early initiating event of FL pathogenesis, is also present at low frequency (10−5–10−7) in blood from healthy individuals (HI), indicating that t(14;18) and the ensuing BCL2 overexpression is necessary but not sufficient for malignant transformation. It has long been assumed that in HI, t(14;18) is carried by circulating quiescent naïve B-cells, where its oncogenic potential would be restrained. Yet, several reports, including long-term persistence and immunomodulation of t(14;18)+ cells in lymphoma-free individuals, led us to question this model and investigate the status of circulating t(14;18)+ cells in HI. We first determined if t(14;18)+ cells are naïve B-cells by assessing class-switch recombination (CSR) on the translocated allele. Using 2 long-range PCR assays designed to amplify unswitched BCL2/Sμ and switched BCL2/Sg regions, DNA samples from 6 HI with t(14;18) were tested. Contrary to previous assumptions, our data clearly show that most peripheral t(14;18)+ cells already underwent CSR (n=5/6) and therefore that most t(14;18)+ cells are not naïve B-cells. Are they then memory B cells? Naïve and memory B cell subsets from 9 HI were isolated by cell sorting according to IgD and CD27 markers, and the rate of t(14;18) analyzed in each subset relatively to that of the total B cells. Strikingly, while the level of naïve t(14;18)+ cells remained at baseline for all individuals, memory B-cells tightly accounted for the wide modulation of t(14;18) frequencies observed between individuals. In addition, sequence analysis of t(14;18) clones revealed that this wide modulation was not due to the accumulation of clonally unrelated t(14;18) naïve B-cells, but rather to the clonal expansion of t(14;18)-bearing memory B-cells. To further define the t(14;18)+ cells, we next examined the repartition of the translocation in the IgD−/CD27+ and IgD+/CD27+ memory B-cell subsets. Unexpectedly, we found that the IgD+/CD27+ subset contained significantly higher rates of translocation than the IgD−/CD27+, both in terms of prevalence and frequency. Thus, while CSR is found in the majority of translocated alleles (~75%), most t(14;18)+ memory B cells have not switched their productive allele (~70%) and express an IgM/D. Most importantly, although atypical among physiological peripheral B-cells, this “allelic paradox” is a specific hallmark of FL, and suggests the presence of the same selective pressure in favor of sIgM expression on a B-cell population that is at the same time permanently driven to switch. In line with B-cell hyperplasia in BCL2 transgenic mice slowly progressing to low grade lymphoma, it is likely that “FL-like” cells in HI are rescued by BCL2 from apoptosis, and “frozen” at a differentiation stage in which constitutive AID expression drives continuous somatic hypermutation and CSR activity, two mechanisms conferring a high propensity for genomic instability. Altogether, our findings identify a novel intermediate step in early lymphomagenesis, and strongly impact both on the current understanding of disease progression from potent pre-malignant niches, and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency. It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells. To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated. Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene. The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells. First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing. Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes. In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing. In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.

Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severe invasive infection in humans with a particularly high case-fatality rate. Listeriosis is a major public health concern in all world regions, with an increasing incidence in Europe, especially among elderly persons (1,2). L. monocytogenes is genetically heterogeneous (3–5). To help epidemiologic investigation and to define clones, i.e., groups of genetically similar isolates descending from a common ancestor, a variety of typing methods have been used, including pulsed-field gel electrophoresis (5,6), single nucleotide polymorphism typing (7), and multiple housekeeping and virulence gene sequencing (8,9). Some clones implicated in multiple outbreaks have been defined as epidemic clones (EC) (3,5,9–11). ECI and ECIV have been described in several countries (3,5), but because of the lack of standardization of genotyping, a definition of clones is not widely accepted, and current knowledge on the global distribution of L. monocytogenes clones is virtually absent. Multilocus sequence typing (MLST) is a reference method for global epidemiology and population biology of bacteria, and its application to L. monocytogenes (12) effectively allows isolate comparisons across laboratories (www.pasteur.fr/mlst). The aim of this study was to investigate the global distribution of L. monocytogenes MLST-defined clones.

International audience; In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogenous clonal T cell responses can be accounted for by a single common model of homeostasis.

We treated 5 patients with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome and multifocal bone lesions or diffuse bone marrow plasmacytic infiltration with high-dose therapy (HDT) and autologous blood stem cell transplantation. In all cases, the treatment produced remission of plasma cell proliferation associated with marked improvement in the patients' performance status, neurologic symptoms, and other manifestations of the syndrome. HDT with stem cell support should be investigated further as a therapeutic option in patients with POEMS syndrome and disseminated plasma cell dyscrasia.

International audience; Post-transcriptional regulation plays a central role in cell differentiation and proliferation. Among the regulatory factors involved in this mechanism, Tristetraprolin (ZFP36 or TTP) is the prototype of a family of RNA-binding proteins that bind to adenylate and uridylate (AU)-rich sequences in the 3'UTR of mRNAs, which promotes their physiological decay. Here, we investigated whether TTP correlates with tumor aggressiveness in breast cancer and is a novel prognostic factor for this neoplasia. By immunoblot analysis, we determined the amount of TTP protein in different breast cancer cell lines and found an inverse correlation between aggressiveness and metastatic potential. TTP mRNA levels were very variable among cells lines and did not correlate with protein levels. Interestingly, by sequencing the entire TTP coding region in Hs578T cells that do not express the TTP protein, we identified a synonymous polymorphism (rs3746083) that showed a statistically significant association with a lack of response to Herceptin/Trastuzumab in HER2-positive-breast cancer patients. Even though this genetic change did not modify the corresponding amino acid, we performed functional studies and showed an effect on protein translation associated with the variant allele with respect to the wild-type. These data underline the importance of synonymous variants on gene expression and the potential role of TTP genetic polymorphisms as a prognostic marker for breast cancer.

International audience; Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)$_{206-214}$ epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP$_{206-214}$) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP$_{206-214}$. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.

International audience; Glycosaminoglycans (GAG) are sulfated polysaccharides that play an important role in regulating cell functions. GAG mimetics called RGTAs (for ReGeneraTing Agents) have been shown to stimulate tissue repair. In particular they accelerate myogenesis, in part via their heparin-mimetic property towards growth factors. RGTAs also increase activity of calcium-dependent intracellular protease suggesting an effect on calcium cellular homeostasis. This effect was presently investigated on myoblasts in vitro using one member of the RGTA family molecule named OTR4120. We have shown that OTR4120 or heparin induced transient increases of intracellular calcium concentration ([Ca(2+)]i) in pre-fusing myoblasts from both mouse SolD7 cell line and rat skeletal muscle satellite cells grown in primary culture by mobilising sarcoplasmic reticulum store. This [Ca(2+)]i was not mediated by ryanodine receptors but instead resulted from stimulation of the Inositol-3 phosphate-phospholipase C activation pathway. OTR4120-induced calcium transient was not mediated through an ATP, nor a tyrosine kinase, nor an acetylcholine receptor but principally through serotonin 5-HT2A receptor. This original finding shows that the GAG mimetic can induce calcium signal through serotonin receptors and the IP3 pathway may be relevant to its ability to favour myoblast differentiation. It supports a novel and unexpected function of GAGs in the regulation of calcium homeostasis.

A first indication of the biological role of mucosal associated invariant T (MAIT) cells reveals that this discrete T cell subset is broadly reactive to bacterial infection. In particular MAIT cells recognize Mycobacterium tuberculosis-infected lung airway epithelial cells via the most evolutionarily conserved major histocompatibility molecule., Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Vα7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an “innate” T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection., Author Summary About one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), yet thanks to a robust immune response most infected people remain healthy. CD8 T cells are unique in detecting intracellular infections. Surprisingly, Mtb-reactive CD8 T cells are found in humans with no prior exposure to Mtb. We show that mucosal associated invariant T (MAIT) cells, which have no previously known in vivo function, make up a proportion of these Mtb-reactive CD8 T cells and detect Mtb-infected cells via a specific major histocompatibility molecule called MHC-related molecule 1, which is evolutionarily conserved among mammals. Mtb-reactive MAIT cells are enriched in lung and detect primary Mtb-infected lung epithelial cells from the airway where initial exposure to Mtb occurs. We go on to show that MAIT cells are not specific for Mtb since they can detect cells infected with a variety of other bacteria. Curiously, Mtb-reactive MAIT cells are absent in the blood of individuals with active tuberculosis. We postulate that MAIT cells are innate detectors of bacterial infection poised to play a role in control of intracellular infection.

Helios is a member of the Ikaros family of zinc finger transcription factors. It is expressed mainly in T cells, where it associates with Ikaros-containing complexes and has been proposed to act as a rate-limiting factor for Ikaros function. Overexpression of wild-type or dominant-negative Helios isoforms profoundly alters αβ T cell differentiation and activation, and endogenous Helios is expressed at strikingly high levels in regulatory T cells. Helios has also been implicated as a tumor suppressor in human T cell acute lymphoblastic leukemias. These studies suggest a central role for Helios in T cell development and homeostasis, but whether this protein is physiologically required in T cells is unclear. We report herein that inactivation of the Helios gene by homologous recombination does not impair the differentiation and effector cell function of αβ and γδ T cells, NKT cells, and regulatory T cells. These results suggest that Helios is not essential for T cells, and that its function can be compensated for by other members of the Ikaros family.

International audience; The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.gammac(-/-) immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V-J rearrangements at the TCR-beta chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR-beta chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.gammac(-/-) mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA-restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen-specific immune responses.

Protective T cell responses elicited along chronic human CMV (HCMV) infections are sometimes dominated by CD8 T cell clones bearing highly related or identical public TCR in unrelated individuals. To understand the principles that guide emergence of these public T cell responses, we have performed structural, biophysical, and functional analyses of an immunodominant public TCR (RA14) directed against a major HLA-A*0201-restricted HCMV Ag (pp65495–503) and selected in vivo from a diverse repertoire after chronic stimulations. Unlike the two immunodominant public TCRs crystallized so far, which focused on one peptide hotspot, the HCMV-specific RA14 TCR interacts with the full array of available peptide residues. The conservation of some peptide-MHC complex-contacting amino acids by lower-affinity TCRs suggests a shared TCR-peptide-MHC complex docking mode and supports an Ag-driven selection of optimal TCRs. Therefore, the emergence of a public TCR of an oligoclonal Ag-specific response after repeated viral stimulations is based on a receptor displaying a high structural complementarity with the entire peptide and focusing on three peptide hotspots. This highlights key parameters underlying the selection of a protective T cell response against HCMV infection, which remains a major health issue in patients undergoing bone marrow transplantation.

t(11;14)-positive clones can persist over a long period of time in the peripheral blood of healthy individuals

International audience; HHV-8 belongs to the family Herpesviridae, the subfamily Gammaherpesvirinae and the genus Rhadinovirus. While several viral homologs exist in both old-world and new-world non-human primates, HHV-8 is the only known human rhadinovirus. HHV-8 is considered to be the etiological agent of the four clinical-epidemiological forms of Kaposi's sarcoma (classic, endemic, post-transplant and epidemic/HIV-associated). In several African regions, epidemic KS is the most frequently diagnosed malignancy. In 2002, the estimated annual incidence of KS worldwide was approximately 65,000 cases, representing 1% of all diagnosed cancers. HHV-8 is also associated with primary effusion lymphoma, some cases of multicentric Castleman disease, and other rare lymphomas. All these tumors mainly affect immunodeficient patients. Recent studies indicate that KS tumors are due to mono- or oligoclonal expansion of latently HHV-8-infected spindle cells, Finthermore, advanced multicentric KS lesions are of multiclonal origin. HHV-8 is not a ubiquitous virus. It is mainly endemic in areas where classical or endemic Kaposi's sarcoma is highly endemic, including the Mediterranean basin and East and Central Africa. In the latter areas, the HHV-8 seroprevalence can reach 80% in the adult population. In the homosexual population (mainly in the U.S.A. and Europe), HHV-8 is mainly transmitted during repeated sexual contacts, whereas in Africa it is mainly transmitted from mother to child and among siblings. Saliva seems to play a major role in HH V-8 transmission. Molecular epidemiology studies of the K1 gene (one of the most variable genomic regions) have revealed different molecular subtypes, at least some of which appear to be linked mainly to the geographic origin of the samples rather than to the underlying disease.; L’HHV-8 est un virus de la famille des herpesviridae, de la sous-famille des Gammaherpesvirinae et du genre Rhadinovirus. Alors qu’il existe de nombreux homologues viraux de l’HHV-8 dans plusieurs espèces de primates non-humains, l’HHV-8 est le seul rhadinovirus connu chez l’homme. L’HHV-8 est considéré comme l’agent étiologique de toutes les formes du sarcome de Kaposi (classique, endémique, post-transplant et épidémique ou associée au VIH). Alors que l’incidence de la forme épidémique a fortement diminué ces dernières années dans les pays occidentaux, cette tumeur représente actuellement la prolifération la plus fréquente dans de nombreux pays d’Afrique Centrale et Australe. Le nombre de sarcome de Kaposi annuel est estimé à près de 65 000 cas, soit près de 1 % de tous les cancers diagnostiqués chaque année dans le monde. Cet herpèsvirus oncogène est aussi associé au lymphome des cavités, à certains cas de la maladie de Castleman multicentrique ainsi qu’à d’autres lymphomes rares. Ces dernières tumeurs survenant surtout dans un contexte d’immunodéficience. Des travaux récents ont démontré que le sarcome de Kaposi, dans sa forme tumorale avancée, était une prolifération mono ou oligoclonale de cellules fusiformes infectées par l’HHV-8 et que les lésions multicentriques, très fréquentes dans cette tumeur, étaient d’origine multiclonales. L’HHV-8 n’est pas un virus ubiquitaire. Il est principalement endémique dans les zones où le sarcome de Kaposi est fréquent, c’est-à-dire le pourtour du bassin méditerranéen et surtout l’Afrique Centrale et de l’Est, régions où la séroprévalence virale atteint près de 80 % chez les adultes. Alors que dans la population homosexuelle masculine (principalement aux USA et en Europe), le virus se transmet surtout durant les contacts sexuels, la transmission en zone de forte endémie (Afrique) se fait avant tout de la mère à l’enfant, puis entre enfants. La salive semble jouer un rôle majeur dans la transmission virale. Les études d’épidémiologie moléculaire portant sur le gène K1 (l’une des régions génomiques les plus variables) ont mis en évidence l’existence de plusieurs sous-types viraux qui ne semblent pas liés au type de maladie associée mais dans une grande mesure à l’origine géographique des patients.

International audience; We report on the clinico-biological characteristics of 20 cases of gammadelta T cell large granular lymphocyte (LGL) leukemia. All the data were compared to that of 196 cases with alphabeta T cell subtype, which represents the majority of T cell LGL leukemias. Clinical findings were quite similar in the two groups regarding age, sex ratio, recurrent infections, and association with auto-immune diseases especially rheumatoid arthritis. Gammadelta LGL predominantly expressed a CD3+/CD4-/CD8+/CD16+/CD57+ phenotype, in 50% of cases. Clinical outcome was favorable for these patients with overall survival of 85% at 3 years. Fifty percent of gammadelta patients required treatment and the response to therapy was estimated at 55%. gammadelta and alphabeta T cell LGL leukemia harbor a very similar clinico-biological behavior and represent part of an antigen-driven T cell lymphoproliferation.

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.

Positive selection in the thymus and peripheral T cell survival depend on T cell receptor (TCR)–major histocompatibility complex (MHC) interactions, but it is not yet clear if both events follow exactly the same rules. We studied peripheral T cell survival and clone sizes in conditions of progressive reduction of restricting MHC-bearing cells or progressive ablation of different MHC molecules. Different CD8+ T cell clones/polyclonal populations showed different survival and/or lymphopenia-driven proliferation requirements. We could correlate clone sizes to the capacity of each TCR to interact with different types of MHC complexes. Thus, although repertoire selection in the thymus is mainly conditioned by the affinity of TCR–MHC interactions, peripheral selection is determined by TCR cross-reactivity to environmental ligands.

International audience; Using the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells.

International audience; In L6 skeletal myoblasts induced to differentiate by Arg8-vasopressin treatment, a short-lived lowering of ceramide levels was observed, followed by a long-lasting elevation that was prevented by inhibitors of the de novo synthesis pathway, fumonisin B1 and myriocin. Both inhibitors increased the expression of myogenic differentiation markers and cell fusion rate, whereas short-chain ceramides inhibited these responses. Similar drug effects were observed on primary mouse satellite cell differentiation. Furthermore, bacterial sphingomyelinase overexpression suppressed myogenin nuclear accumulation in L6 cells. These data suggested that endogenous ceramide mediates a negative feedback mechanism limiting myogenic differentiation, and that inhibitors of ceramide synthesis promoted myogenesis by removing this control. Phospholipase D (PLD), a recognized target of ceramide, is required for myogenesis, as shown by the negative effects of PLD1 isoform depletion obtained by siRNA treatment. Fumonisin induced an increase in PLD activity of L6 cells, whereas C6-ceramide decreased it. The expression of PLD1 mRNA transcripts was selectively decreased by C6-ceramide, and increased by ceramide synthesis inhibitors. An early step of myogenic response is the PLD1-dependent formation of actin stress fiber-like structures. C6-ceramide addition or overexpression of sphingomyelinase impaired actin fiber formation. Ceramide might thus regulate myogenesis through downregulation of PLD1 expression and activity.

International audience; Recent results have shown the ability of bone marrow cells to migrate in the brain and to acquire neuronal or glial characteristics. In vitro, bone marrow-derived MSCs can be induced by chemical compounds to express markers of these lineages. In an effort to set up a mouse model of such differentiation, we addressed the neuronal potentiality of mouse MSCs (mMSCs) that we recently purified. These cells expressed nestin, a specific marker of neural progenitors. Under differentiating conditions, mMSCs display a distinct neuronal shape and express neuronal markers NF-L (neurofilament-light, or neurofilament 70 kDa) and class III beta-tubulin. Moreover, differentiated mMSCs acquire neuron-like functions characterized by a cytosolic calcium rise in response to various specific neuronal activators. Finally, we further demonstrated for the first time that clonal mMSCs and their progeny are competent to differentiate along the neuronal pathway, demonstrating that these bone marrow-derived stem cells share characteristics of widely multipotent stem cells unrestricted to mesenchymal differentiation pathways.

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non-cytotoxic CD4(+) T cells are important for the induction and maintenance of anti-tumour immunity exerted by cytotoxic CD8(+) T cells. In contrast, the existence of direct anti-tumour, effector cytotoxic CD4(+) T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B-cell non-Hodgkin lymphomas. This study developed an appropriate, autologous follicular B-cell non-Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA-I) deficient B-cell line, and the generation of three autologous anti-tumour cytotoxic CD4(+) T-cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA-I-deficient B-cell line through a classical HLA-II restricted perforin-mediated pathway, but did not lyse the Epstein-Barr virus-infected autologous normal B lymphocytes. All three CD4(+)clones were T-cell receptor Vbeta17-Dbeta1-Jbeta1.2 and exhibited an identical complementarity-determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4(+) cytotoxic cells in immunotherapeutic strategies.

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure-function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.

International audience; Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.

International audience; In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-alpha. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.

International audience; When and how cells form and pattern the myocardium is a central issue for heart morphogenesis. Many genes are differentially expressed and function in subsets of myocardial cells. However, the lineage relationships between these cells remain poorly understood. To examine this, we have adopted a retrospective approach in the mouse embryo, based on the use of the laacZ reporter gene, targeted to the alpha-cardiac actin locus. This clonal analysis demonstrates the existence of two lineages that segregate early from a common precursor. The primitive left ventricle and the presumptive outflow tract are derived exclusively from a single lineage. Unexpectedly, all other regions of the heart, including the primitive atria, are colonized by both lineages. These results are not consistent with the prespecification of the cardiac tube as a segmented structure. They are discussed in the context of different heart fields and of the evolution of the heart.

International audience; During heart morphogenesis, cardiac chambers arise by differential expansion of regions of the primitive cardiac tube. This process is under the control of specific transcription factors such as Tbx5 and dHAND. To gain insight into the cellular mechanisms that underlie cardiogenesis, we have used a retrospective clonal approach based on the spontaneous recombination of an nlaacZ reporter gene targeted to the murine alpha-cardiac actin locus. We show that clonal growth of myocardial cells is oriented. At embryonic day (E) 10.5, the shape of clones is characteristic of a given cardiac region and reflects its morphology. This is already detectable in the primitive cardiac tube at E8.5, and is maintained after septation at E14.5 with additional modulations. The clonal analysis reveals new subdivisions of the myocardium, including an interventricular boundary region. Our results show that the myocardium, from the time of its formation, is a polarized and regionalized tissue and point to the role of oriented clonal cell growth in cardiac chamber morphogenesis.

The overall degree of complexity of the T cell surface has been unclear, constraining our understanding of its biology. Using global gene expression analysis, we show that 111 of 374 genes encoding well-characterized leukocyte surface antigens are expressed by a resting cytotoxic T cell. Unexpectedly, of 97 stringently defined, T cell-specific transcripts with unknown functions that we identify, none encode proteins with the modular architecture characteristic of 80% of leukocyte surface antigens. Only two encode proteins with membrane topologies found exclusively in cell surface molecules. Our analysis indicates that the cell type-specific composition of the resting CD8+ T cell surface is now largely defined, providing an insight into the overall compositional complexity of the mammalian cell surface and a framework for formulating systematic models of T cell surface-dependent processes, such as T cell receptor triggering.

Human natural killer (NK)-cell receptors are expressed by NK cells and some T cells, primarily TCR+CD8+ cytotoxic T lymphocytes (CTLs). Inhibitory NK cell receptors (iNKRs) can down-regulate antigen-mediated T-cell effector functions, including cytotoxic activity and cytokine release. In the present study we demonstrate that CD3+ T cells that bind tetramers of HLA-E and express its ligand, the NK-cell inhibitory receptor CD94/NKG2A, were significantly decreased in frequency in patients with human T-cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) but not in asymptomatic HTLV-1 carriers. These cells were either alphabeta or gammadelta T cells. T-cell receptor (TCR) Vbeta-specific reverse transcription-polymerase chain reaction and spectratyping analysis revealed that the TCR repertoire in directly isolated HLA-E tetramer-positive cells from peripheral blood mononuclear cells was skewed in both HTLV-1-infected and healthy individuals. However, oligoclonally or monoclonally expanded levels of TCR Vbetawere more frequently detected within HTLV-1-infected individuals than healthy controls. Importantly, HLA-E tetramer-positive or NKG2A+ T cells from HTLV-1 patients do not express Tax and display different TCR usage from the immunodominant Tax11-19-specific CD8+ T cells, suggesting that they do not encounter HTLV-1-infected cells. The expression of NK cell-associated receptors by clonally expanded CD8+ T cells during chronic viral infection suggests that these receptors play a role in regulating CD8+ T cell-mediated antiviral immune responses and that a decrease of this cell subset results in an increased risk of inflammatory diseases such as HAM/TSP.

International audience; Previous experiments with mouse chimeras demonstrated that cellular competition for antigen-specific survival signals plays a crucial role in the maintenance of the naive B cell repertoire. Transgenic (Tg) B cell populations in these chimeras have a shortened lifespan and poor competitive abilities as compared to more diverse non-Tg populations in the same mice. We develop a mathematical model to investigate the mechanism of B cell competition. The model allows for various B cell clones, generated in the bone marrow, to go into the peripheral circulation, where they compete specifically for various ligands providing survival signals. In the model we also find the observed poor competitive abilities of the Tg repertoire. Investigating the nature of the competition in the model, we find that most of the competition is "intraspecific" occurring largely within the clone of truly Tg B cells, and within the repertoire of leaky Tg and non-Tg B cells. This is confirmed by analysing a simplified version of the model, which only allows for intraspecific competition, and resembles a simple ecological model with density-dependent death. The fact that our model accounts for the data, casts doubt on a previous interpretation of the same data arguing that more diverse repertoires outcompete repertoires of lower diversity. Here, we conclude that most of the data can be explained with intraspecific competition, and formulate an experimental prediction that allows one to distinguish between the previous interpretation of inter-specific competition between repertoires, and the current interpretation of intraspecific competition.

Background: Infection with human herpesvirus 8 (HHV8), also termed Kaposi’s sarcoma (KS)-associated herpesvirus, is associated with all forms of KS, with primary effusion lymphoma (PEL), and with some forms of multicentric Castleman’s disease (MCD), but the pathogenic role of HHV8 in these tumors and the clonal nature of KS are still unclear. The purpose of this study was to examine whether the number of terminal repeats (TRs) contained in the fused TR region of HHV8 could be used as a marker of clonality in HHV8-associated tumors. Methods: Pulsed-field gel electrophoresis (PFGE) and multiple-probe Southern blot analysis of the HHV8 TR region were performed on high-molecularweight DNA obtained from tumoral KS, PEL, and MCD lesions. Results: These analysis showed that the fused TR region contains a large but variable number of TR units (ranging from 16 to 75) and that the viral genome is present as extrachromosomal circular DNA in these tumors in vivo, with occasional ladders of heterogeneous linear termini reflecting lytic replication. All PEL tumors and PEL-derived cell lines as well as some KS tumors contained monoclonal or oligoclonal fused TR fragments; however, the TR region appeared polyclonal in MCD tumors and in a few KS lesions. Conclusion: Several KS and PEL lesions are monoclonal expansions of a single infected cell, suggesting that HHV8 infection precedes tumor growth and thus supporting an etiologic role of latent HHV8 in these proliferations. Our finding that nodular KS lesions display all possible patterns of clonality supports the model according to which KS begins as a polyclonal disease with subsequent evolution to a monoclonal process. [J Natl Cancer Inst 2000;92:729‐36]

International audience; Peptidylglycine alpha-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160 +/- 12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive alpha-amidated hormones and/or neuropeptides.

A primary infection by the Plasmodium falciparum Palo Alto O and R antigenic variants induces a variant-specific immunity in the Saimiri sciureus monkey. We have shown that these variants express distinct PfEMP1 antigens and differ in their levels of expression of additional antigens, including two conserved erythrocyte membrane-associated proteins, HRP1 and PfEMP3. To identify the antigens eliciting a variant-specific response, we conducted a differential screening of a λgt11 library with variant-specific sera. We report here the analysis of the 46 anti-R-specific clones. Two specific targets of the anti-R response were identified: (i) PfEMP3, suggesting that immunogenicity of this antigen is modulated by its relative abundance in different variants, and (ii) Asn-rich motifs. Most anti-R-specific clones, derived from so-far-undescribed genes, were detected by a cross-reaction on poly(Asn) stretches, as indicated by elimination of the signal after absorption on Asn-rich sequences. Reverse transcription-PCR (RT-PCR) showed that expression of the gene defined by clone 13 was R specific. Pepscan analysis of clone 13 identified three Asn-rich polypeptides and one unique peptide reacting specifically with antibodies eluted from the R-infected erythrocyte surface. Antisera raised to the unique peptide reacted with an R-specific protein. Attempts to demonstrate that clone 13 was derived from a var gene by using PCRs combining clone 13 and var -derived primers were unsuccessful. The var genes expressed by O and R parasites were identified not by this strategy but by RT-PCR with var -specific primers. This work has provided novel insights into immunity to antigenic variants and has identified a novel gene switched on during antigenic variation.

International audience; In this investigation we compare the repopulation of the CD8+ T cell compartments of bone marrow (BM) chimeras by either normal nontransgenic or T cell receptor (TcR) alpha beta-transgenic (TG) CD8+ T cells, the fate of TG and non-TG CD8+ T cells in different parabionts and the survival of TG and non-TG peripheral CD8+ T cells after transfer into athymic hosts. We found that cellular competition among CD8 T cells occurs at several steps of T cell differentiation including a) during the DN to DP transition, b) positive selection in the thymus, c) export from the thymus and d) in the periphery. Comparison of the results obtained in the BM chimeras and in the parabionts shows that an important step of T cell selection occurs during seeding of peripheral lymphoid tissues. Once established, peripheral T cells resist replacement by recent thymus migrants, i.e. in the periphery, selection of T cell repertoires follows the rule "first come, first served". Peripheral dominance correlates with T cell activation and division. Cell cycling and CD44 expression are more frequent among non-TG CD8 T cells than TG CD8 T cells and within the latter, more frequent among P14 TG CD8 T cells than anti-HYTG CD8 T cells. Thus, in the absence of intentional immunization, the frequencies of CD8+ T cells follow a hierarchy of selection in which non-TG > or = P14 TG > anti-HY TG. We also show that the equilibrium size and the fate of one CD8 T cell population differs according to the presence or absence of other CD8 T cell populations. Under these circumstances, selection of T cell repertoires and T cell survival and memory rely not only on the interactions of each T cell with their respective ligands, but also on the nature and number of other competing cells.

We report the isolation and characterization of HEp-2c cell clones obtained after two successive persistent poliovirus (PV) infections. Once cured, some of the cell clones displayed selective permissivity toward the wild-type Mahoney strain and partial resistance to particular mutants of this strain, including the Sabin 1 strain. Two cell clones, Cl 4 and Cl 10, were studied in greater detail. The cytopathic effects of Mahoney infection were comparable in the cell clones and in HEp-2c cells. The cytopathic effects of infection by Sabin 1 or Mahoney mutants were greatly delayed in Cl 4 and Cl 10. In the genomic region encoding the capsid proteins, determinants involved in the resistance of the cell clones to the Mahoney mutants were localized in the amino-terminal part of VP1 (amino acids 22 and 43), the B-C loop of VP1 (amino acids 94-102), and the loop of VP3 connecting its amino-terminal to β strand B (amino acid 60). These genomic regions are thought to be involved in the early steps of viral infection. Virus adsorption was slower and less efficient on Cl 10 cells than on parental HEp-2c cells. Virus adsorption was faster on Cl 4 than on HEp-2c cells, and at least as efficient, but there was less receptor-induced structural modification of the capsid, a step that is required for decapsidation. Furthermore, infection of Cl 4 by a Mahoney mutant in which the B-C loop of VP1 has been deleted was affected in the later steps of infection. These results indicate that, in cells cured of persistent PV infection, poliovirus multiplication was restricted at several stages and particularly at two steps of virus entry: adsorption and/or the uncoating transitions following adsorption onto the receptor.

International audience; We studied the competitive repopulation by different B cells of irradiated mice reconstituted with bone marrow from either congenic or Ig-transgenic (TG) mice mixed at different ratios. We found that after reconstitution, the number of B cells recovered in the different chimeras is similar and independent of the ratio of injected cells. In chimeras hosting TG and non-TG cells, the relative representation of the donor cell lineages diverges from the ratios present in the inoculum, i.e. at the periphery, non-TG cells are preferentially selected. Selection of non-TG cells only occurs when population growth plateaus, i.e. when resources become limiting and competition starts to operate. Selection of non-TG cells depends on surface Ig expression, and they are selected because they have a longer survival. Finally, the life-expectancy of the same B cell population differs depending upon the second population present. The present results show that the life-span and the population size of each B cell clone can be altered (interfered with) by the presence of a second cell population, demonstrating the existence of cellular competition among B cells. Our findings establish the role of cellular competition in the selection of B cell repertoires and the existence of a hierarchy of B cell selection in the absence of antigenic stimulation. The implications of cellular competition on our understanding of the immune system are discussed.

International audience; We studied the early stages of gene amplification in a Chinese hamster cell line and we show that two distinct mechanisms can operate at a single locus. Both of them rely on an unequal segregation of gene copies at mitosis. We conclude that cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere that lead to further breaks in mitosis, have a key role in the coupling of gene amplification and genome remodeling. Rearrangements are first limited to a single chromosome but can then potentially spread to any additional chromosome. Occasionally, a sequence containing the selected gene can be looped out, generating a "double minute" and thus initiating an independent process of extrachromosomal amplification.

International audience; Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8+ clones were found to be H-2K restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived?) presented by the MHC class I (H-2K or H-2K) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.

Subclones of the murine erythroleukemia cell line DS 19 with different sensitivities towards inhibition by dexamethasone (DEX) of the hexamethylene bisacetamide induced differentiation have been obtained. Scatchard analysis of DEX binding to whole cells and the time course of DEX binding to nuclei show that resistance to inhibition of erythrodifferentiation by DEX is neither associated with a reduction in specific binding of the hormone nor in a lack of activation or translocation to the nuclei of hormone receptor complexes. Hence resistance in these subclones must result from a defect at an event after binding of hormone-receptor complexes to chromatin if inhibition of differentiation is mediated by these complexes.

International audience; A transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed.