Étude de la reprogrammation métabolique des cellules de leucémie lymphoïde chronique dans un modèle ex-vivo de la niche lymphatique

Chronic Lymphocytic Leukemia (CLL), the most prevalent leukemia in adults, is marked by a monoclonal accumulation of tumor and quiescent mature B lymphocytes in the blood. In advanced stages (Binet stage C), marrow invasion and lymphocyte proliferation occurs in secondary lymphoid organs, resulting in adenopathy. The primary complication is the transformation into high-grade lymphoma (Richter's lymphoma), which is detectable by positron emission tomography (PET) and reveals increased binding of a glucose derivative (18FDG) by cancer cells. Our data from the stage C CLL patient cohort at the University Hospital of Nice indicate hyperfixation in lymph node regions, suggesting that the tumor microenvironment promotes proliferation and metabolic changes in CLL cells, which exhibit considerable resistance to current therapies.Developing an ex-vivo tumoroid model that simulates the lymph node microenvironment is crucial for identifying and testing new therapeutic targets because many patients experience relapse or are refractory to existing treatments. Our objectives were: i) to create an ex-vivo cell co-culture model mimicking the lymph node, ii) to metabolically characterize this resistant lymph node, and iii) to assess the efficacy of an antimetabolic therapy along with current treatment (Ibrutinib or Venetoclax).We established an ex-vivo model of the lymphatic niche using a co-culture of peripheral blood mononuclear cells (PBMC) from CLL patients, stimulated with CpG ODN (a TLR9 agonist) and IL2 (activating CD25 on B cell surfaces), and primary human lymph node fibroblasts (HLF). This novel co-culture model replicates the lymphatic niche of CLL patients ex-vivo. Using biochemical and flow cytometry techniques, we validated this model, which demonstrated increased viability, proliferation, and antiapoptotic proteins MCL1, and BCL2 at 48h of co-culture. The model also revealed elevated lactate production, glucose consumption, and metabolic activation detected by the Seahorse analyzer, along with the production of glycolytic enzymes like hexokinase 2 (HK2).HK 2 inhibition disrupts the glycolytic metabolic pathway, which is essential for CLL cell energy supply in co-culture, reducing energy availability for CLL cell survival and heightening vulnerability to treatment. Moreover, glucose flux disruption may affect intracellular signaling pathways and biological processes related to CLL progression.The efficacy of antimetabolic therapy targeting hexokinase 2, combined with current treatments like ibrutinib presents new opportunities to enhance clinical outcomes for patients with relapsed or refractory CLL. The promising results from our ex-vivo lymph node model could serve as a foundation for developing novel targeted therapeutic strategies aimed at disrupting CLL cell energy metabolism and overcoming resistance to existing treatments.La Leucémie Lymphoïde Chronique (LLC), la leucémie la plus courante chez les adultes, est caractérisée par une accumulation monoclonale de lymphocytes B matures tumoraux et quiescents dans le sang. À des stades avancés (stade C selon la classification de Binet), une invasion médullaire et une prolifération de ces lymphocytes sont observées dans les organes lymphoïdes secondaires, entraînant l'apparition d'adénopathies. La complication principale reste la transformation en lymphome de haut grade (lymphome de Richter), décelable par une Tomographie par Émission de Positons (TEP) qui révèle une fixation accrue d'un dérivé du glucose (18FDG) par les cellules cancéreuses. Nos données provenant de la cohorte de patients atteints de LLC stade C à l'Hôpital Universitaire de Nice montrent des indices d'hyperfixations dans les régions ganglionnaires, suggérant que le microenvironnement tumoral de la niche lymphatique favorise la prolifération et un changement métabolique des cellules de LLC qui présentent une résistance significative aux traitements actuels.L'élaboration d'un modèle ex-vivo tumoroïde reproduisant le microenvironnement de la niche ganglionnaire est donc essentiel pour identifier et tester de nouvelles cibles thérapeutiques, car de nombreux patients rechutent ou sont réfractaires aux traitements existants.Nos objectifs étaient de i/ développer un modèle ex-vivo de co-culture cellulaire simulant la niche lymphatique, ii/ caractériser métaboliquement cette niche lymphatique de résistance, et iii/ évaluer l'efficacité d'une thérapie anti-métabolique en association avec le traitement actuel (Ibrutinib ou Venetoclax).Nous avons mis au point un modèle ex-vivo de la niche lymphatique en utilisant une co-culture de cellules mononucléaires du sang périphérique (PBMC) de patients atteints de LLC, stimulées par CpG ODN (un agoniste de TLR9) et IL2 qui active le CD25 à la surface des cellules B, et de fibroblastes ganglionnaires humains primaires (HLF). Ce modèle de co-culture, inédit dans la littérature, reproduit ex-vivo la niche lymphatique des patients LLC. En utilisant des techniques de biochimie et de cytomètrie en flux, nous avons validé ce modèle de co-culture, qui montre une augmentation de la viabilité et de la prolifération associée à une hausse des protéines anti-apoptotiques MCL1 et BCL2 à 48h de co-culture. Ce modèle a également révélé une production accrue de lactate et une consommation de glucose, associées à une activation métabolique détectée par l'automate Seahorse et l‘expression d'enzymes glycolytiques comme l'hexokinase 2 (HK2).L'inhibition de HK2 perturbe la voie métabolique glycolytique, essentielle pour l'approvisionnement énergétique des cellules de LLC en co-culture réduisant l'énergie disponible pour la survie des cellules de LLC et augmentant leur vulnérabilité face aux traitements. Par ailleurs, la perturbation du flux de glucose peut également affecter les voies de signalisation intracellulaire et les processus biologiques liés à la progression de la LLC.L'efficacité d'une thérapie anti-métabolique ciblant HK2, en combinaison avec les traitements actuels tels que l'Ibrutinib, offre de nouvelles perspectives pour améliorer les résultats cliniques des patients atteints de LLC réfractaires ou en rechute. Les résultats prometteurs obtenus à partir de notre modèle ex vivo de la niche lymphatique peuvent constituer une base solide pour le développement de nouvelles stratégies thérapeutiques ciblées. Ces stratégies viseraient à perturber le métabolisme énergétique des cellules de LLC activé dans la niche lymphatique, et à surmonter la résistance aux traitements existants

Inadequate labeling of pork sausages prepared in Corsica causing a trichinellosis outbreak in France

Three cases of human trichinellosis due to Trichinella britovi were reported in 2015 in the Southeast of France resulting from consumption of raw pork sausages (figatelli) prepared in Corsica. Fourteen other people ate figatelli from the same batch but were not infected due to the figatelli being well cooked. This is the first reported human trichinellosis outbreak due to consumption of Corsican sausages prepared from uncontrolled pork. Consumption of raw figatelli is a common tradition in Corsica. As a result, the health recommendation to cook the product well is not always applied. In the present case, the figatelli product label was not sufficiently visible to advise consumers of the risks associated with uncooked pork

Inadequate labeling of pork sausages prepared in Corsica causing a trichinellosis outbreak in France

International audienceThree cases of human trichinellosis due to Trichinella britovi were reported in 2015 in the Southeast of France resulting from consumption of raw pork sausages (figatelli) prepared in Corsica. Fourteen other people ate figatelli from the same batch but were not infected due to the figatelli being well cooked. This is the first reported human trichinellosis outbreak due to consumption of Corsican sausages prepared from uncontrolled pork. Consumption of raw figatelli is a common tradition in Corsica. As a result, the health recommendation to cook the product well is not always applied. In the present case, the figatelli product label was not sufficiently visible to advise consumers of the risks associated with uncooked pork

Th17-Immune Response in Patients With Membranous Nephropathy Is Associated With Thrombosis and Relapses: Th17-Immune Response in Patients With Membranous Nephropathy Is Associated With Thrombosis and Relapses

International audienceExciting times for the field of renal autoimmune diseases have begun. In 2021, for the first time, two new drugs belimumab (1) and voclosporin ( 2) are approved for the treatment of lupus nephritis (LN) (1, 2). Other novel targeted therapies demonstrate clinical efficacy in large, randomized trials, such as avacopan for ANCA-associated vasculitis (AAV) (3), imlifidase for Goodpasture’s disease and iptacopan for IgA nephropathy (IgAN). Pathogenic molecules are specifically targeted by new drugs that help to uncover novel aspects of disease mechanisms leading to glomerulonephritis. Simultaneously, the field is boosted by novel big data technologies on the single cell levels such as high-sensitive multi-color flow cytometry, single-cell genomics (single-cell RNA sequencing - scRNAseq), single cell metabolomics and proteomics. The novel treatment options in renal autoimmune diseases almost simultaneously require new immunomonitoring tools. ‘Immunomonitoring’includes the wide range of approaches to monitoring immune responses by the cellular immune system (e.g. T-cells, B-cells, plasma cells, dendritic cells, neutrophils etc.), or by the humoral immune system (e.g. cytokines, (auto-)antibodies, urinary markers, etc.). Monitoring relevant immune responses in patients with renal autoimmune diseases helps us to better understand a) the underpinning immunological pathophysiology of these diseases; b) the beneficial effects of novel treatments on autoimmunity; and c) can potentially help doctors and patients guide a personalized treatment strategy, adding information on immunological (non-)response to a clinical (non-) response treatment and on disease prognosis. In the present Research Topic, we have been able to collect for you a vast amount of research addressing novel ways and the roleof immunomonitoring in the broad range of renal autoimmune disease including LN, AAV, IgAN, idiopathic membranous nephropathy (iMN) and complement-mediated disease (CMD)

Th17-Immune Response in Patients With Membranous Nephropathy Is Associated With Thrombosis and Relapses

International audienceAlternative strategies targeting dysregulated cytokine balance could be considered for these patients at high risk of relapse

Baseline and early functional immune response is associated with subsequent clinical outcomes of PD-1 inhibition therapy in metastatic melanoma patients

Background Despite significant progress with antiprogrammed cell death protein 1 (PD-1) therapy, a substantial fraction of metastatic melanoma patients show upfront therapy resistance. Biomarkers for outcome are missing and the association of baseline immune function and clinical outcome remains to be determined. We assessed the in vitro nonspecific stimulation of immune response at baseline and during anti-PD-1 therapy for metastatic melanoma.Methods Previously untreated metastatic melanoma patients received nivolumab and radiotherapy as part of the multicentric phase II trial NIRVANA (NCT02799901). The levels of Th1, Th2 and Th17 cytokines on in vitro non-specific stimulation of innate and adaptive immune cells were measured in patient sera before treatment, and at week 2 and week 6 after the beginning of the treatment, and correlated with tumorous response, progression-free survival (PFS) and occurrence of immune-related adverse events (irAEs). The results in melanoma patients were compared with those of a cohort of 9 sex and age-matched healthy donors.Results Seventeen patients were enrolled in this ancillary study. Median follow-up was 16 months (2.2–28.4). The 12-month PFS rate was 67.7%. The incidence of irAEs of any grade was 58.8%. Without in vitro stimulation no differences in cytokines levels were observed between responders and non-responders. On in vitro stimulation, metastatic patients had lower Th1 cytokine levels than healthy donors at baseline for tumor necrosis factor-α and interferon-γ (IFN-γ) (1136 pg/mL vs 5558 pg/mL, p<0.0001; and 3894 pg/mL vs 17 129 pg/mL, p=0.02, respectively). Responders exhibited increasing cytokine levels from baseline to week 6. Non-responders had lower interleukin 17A (IL-17A) levels at baseline than responders (7 pg/mL vs 32 pg/mL, p=0.03), and lower IFN-γ levels at week 6 (3.3 ng/mL vs 14.5 ng/mL, p=0.03). A lower level of IL-17A at week 2 and a lower level of IFN-γ at week 6 correlated with worse PFS (p=0.04 and p=0.04 respectively). At baseline, patients who developed irAEs had higher IL-6 levels (19.3 ng/mL vs 9.2 ng/mL, p=0.03) and higher IL-17A levels (52.5 pg/mL vs 2.5 pg/mL, p=0.009) than those without irAEs.Conclusions Our findings indicate that cytokine levels after in vitro non-specific stimulation could be a promising biomarker to predict the outcome of PD-1 inhibition therapy

Image_2_Low baseline IFN-γ response could predict hospitalization in COVID-19 patients.jpeg

The SARS-CoV-2 infection has spread rapidly around the world causing millions of deaths. Several treatments can reduce mortality and hospitalization. However, their efficacy depends on the choice of the molecule and the precise timing of its administration to ensure viral clearance and avoid a deleterious inflammatory response. Here, we investigated IFN-γ, assessed by a functional immunoassay, as a predictive biomarker for the risk of hospitalization at an early stage of infection or within one month prior to infection. Individuals with IFN-γ levels below 15 IU/mL were 6.57-times more likely to be hospitalized than those with higher values (p65 years, and no vaccination were independently associated with hospitalization. In addition, we found a significant inverse correlation between low IFN-γ response and high level of IL-6 in plasma (Spearman’s rho=-0.38, p=0.003). Early analysis of the IFN-γ response in a contact or recently infected subject with SARS-CoV-2 could predict hospitalization and thus help the clinician to choose the appropriate treatment avoiding severe forms of infection and hospitalization.</p