Murine Models of B-Cell Lymphomas: Promising Tools for Designing Cancer Therapies

Human B-cell lymphomas, the fourth most common hematologic malignancy, are currently the subject of extensive research. The limited accessibility of biopsies, the heterogeneity among patients, and the subtypes of lymphomas have necessitated the development of animal models to decipher immune escape mechanisms and design new therapies. Here, we summarize the cell lines and murine models used to study lymphomagenesis, the lymphoma microenvironment, and the efficacy of new therapies. These data allow us to understand the role of the immune system in the fight against tumors. Exploring the advantages and limitations of immunocompetent versus immunodeficient models improves our understanding of the molecular and cellular mechanisms of tumor genesis and development as well as the fundamental processes governing the interaction of tumors and their host tissues. We posit that these basic preclinical investigations will open up new and promising approaches to designing better therapies

Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State

Despite a growing interest in CD4+ CD25+ regulatory T cells (Treg) that play a major role in self-tolerance and immunoregulation, fundamental parameters of the biology and homeostasis of these cells are poorly known. Here, we show that this population is composed of two Treg subsets that have distinct phenotypes and homeostasis in normal unmanipulated mice. In the steady state, some Treg remain quiescent and have a long lifespan, in the order of months, whereas the other Treg are dividing extensively and express multiple activation markers. After adoptive transfer, tissue-specific Treg rapidly divide and expand preferentially in lymph nodes draining their target self-antigens. These results reveal the existence of a cycling Treg subset composed of autoreactive Treg that are continuously activated by tissue self-antigens

Th17 Cells Are Involved in the Local Control of Tumor Progression in Primary Intraocular Lymphoma

BACKGROUND: Th17 cells play an important role in the pathogenesis of many autoimmune diseases, but despite some reports of their antitumor properties, too little is known about their presence and role in cancers. Specifically, knowledge is sparse about the relation of Th17 to lymphoma microenvironments and, more particularly, to the microenvironment of primary intraocular B-cell lymphoma (PIOL), an aggressive lymphoma with a poor prognosis. METHODS AND PRINCIPAL FINDINGS: In this work, we investigated the presence of Th17 cells and their related cytokines in a syngeneic model of PIOL, a subtype of non-Hodgkin lymphoma. The very small number of lymphocytes trafficking in normal eyes, which represent a low background as compared to tumor-bearing eyes, allows us to develop the present model to characterize the different lymphocyte subsets present when a tumor is developing. IL-21 mRNA was expressed concomitantly with IL-17 mRNA in tumor-bearing eyes and intracellular expression of IL-17A and IL-21 in infiltrating CD4(+) T lymphocytes. Interestingly, IL-17A production by T cells was negatively correlated with tumor burden. We also showed that IL-21 but not IL-17 inhibits tumor cell proliferation in vitro. CONCLUSIONS: These data demonstrate that IL-17A and IL-21-producing CD4(+) T cells, referred as Th17 cells, infiltrate this tumor locally and suggest that Th17-related cytokines may counteract tumor progression via IL-21 production. Thus, Th17 cells or their related cytokines could be considered to be a new therapeutic approach for non-Hodgkin B-cell lymphomas, particularly those with an ocular localization

Etude du microenvironnement cellulaire et moléculaire des lymphomes B intra-oculaires (mise au point d'un modèle murin et étude du rôle des lymphocytes T régulateurs naturels)

Les lymphomes intraoculaires primitifs (PIOL) constituent une pathologie tumorale intraoculaire maligne mal connue et de pronostic extrêmement sévère. De nombreux facteurs expliquent le pronostic sombre des lymphomes intra-oculaire mais le plus important d entre eux est l absence de modèle animal permettant de comprendre les mécanismes physiopathologique de la maladie. L oeil, de part son anatomie et sa physiologie constitue un site immunologiquement privilégié au sein duquel la réponse anti-tumorale est différente de celle observée en périphérie. Ce travail de thèse à pour but de développer le premier modèle murin de lymphome B intraoculaire chez un hôte immunocompétent, et d étudier l environnement moléculaire et cellulaire de la tumeur. L étude de ce microenvironnement tumoral a permis de mettre en évidence un milieu suppresseur tout à fait particulier, propice au développement tumoral, comprenant de l interleukine-10, ainsi qu un très fort enrichissement en cellules T régulatrices naturelles et induites. Cet environnement tumoral particulier se traduit par une inhibition partielle des lymphocytes T infiltrant la tumeur dont le profil cytokinique Th1 est incomplet. L étude du microenvironnement moléculaire et cellulaire de la tumeur a permis de déterminer les grands axes de futures stratégies thérapeutiques qui devront cibler non seulement la tumeur elle-même, mais également son micro-environnement particulier. Ces conclusions ont abouti au développement d un protocole de recherche clinique dans le but d évaluer l efficacité de l un de ces traitements (anticorps monoclonaux anti-CD20) chez l hommePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude in vitro et in vivo des mécanismes moléculaires d'activation lymphocitaire T et microgliale (identification de nouveaux marqueurs de sous-populations microgliales et de discrimination macrophagique)

La voie costimulatrice B7/CD28, indispensable à l'activation optimale des lymphocytes T (LT) et à la prévention de leur anergie, peut être manipulée pour augmenter ou diminuer les réponses immunitaires et ainsi concevoir des nouvelles approches thérapeutiques pour le traitement respectif des cancers ou des maladies autoimmunes. L'obtention de tels traitements nécessite une meilleure compréhension des mécanismes de transduction associés à la molécule CD28, ainsi que leur relation avec le complexe TCR/CD3. Aussi, l'ADNc de la molécule CD28 humaine a été transfectée dans des thymomes murins exprimant ou non le complexe TCR/CD3 . La caractérisation des transfectants obtenus démontre que la production d'IL2 induite via CD28 nécessite l'expression du complexe TCR/TD3 mais que l'association de la PI3-kinase et la mobilisation calcique qui en découle est indépendante de ce complexe. Au sein du système nerveux central (SNC), les cellules microgliales (principales cellules immunocompétentes résidentes) sont des partenaires privilégiés des LT pour leurs actions effectrices. Dans le but de mieux caractériser leurs mécanismes moléculaires d'activation et de présentation antigénique, les messagers différentiellement exprimés dans 2 clones de cellules microgliales murines activées, potentialisant différemment l'activation lymphocytaire T auxiliaire et cytoxique, ont été isolés par hybridation soustractive (technique RDA). Seize messagers ont ainsi été obtenus dont 15 n'avaient jamais été décrits dans la microglie. Certaines de ces molécules semblent pouvoir servir de marqueurs d'activation et/ou de distinction de certaines ou de toutes les sous-populations microgliales. De plus, trois messagers permettent de discriminer les cellules microgliales des monocytes/macrophages in vitro, ainsi que de distinguer ces cellules dans le modèle animal de la Sclérose en Plaques, l'EAE.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Cellular and Molecular Mechanisms of Anterior Chamber-Associated Immune Deviation (ACAID): What We Have Learned from Knockout Mice

International audienceAnterior chamber-associated immune deviation (ACAID) is a well-known phenomenon that can occur after an antigen is introduced without any danger signal into the anterior chamber of a murine eye. It is reported to lead to an antigen-specific immune deviation throughout the body. Despite the relatively little evidence of this phenomenon in humans, it has been suggested as a potential prophylactic strategy in allograft rejections and in several autoimmune diseases. Cellular and molecular mechanisms of ACAID have been explored in different murine models mainly as proofs of concept, first by direct analyses of immune components in normal immunocompetent settings and by cell transfer experiments. Later, use of knockout (KO) mice has helped considerably to decipher ACAID mechanisms. However, several factors raise questions about the reliability and validity of studies using KO murine models. This mini-review summarizes results obtained with KO mice and discusses their advantages, their potential weaknesses, and their potential methods for further progress

Interelationship between CD3 and CD28 pathways in a murine T cell thymoma

International audienceIt is well known that the CD28 costimulatory signal is important to complement T cell receptor (TCR)/CD3-initiated T cell activation, but the mechanism by which these two distinct signaling pathways are integrated is not clearly understood. In our laboratory, we dispose of a murine T cell hybridoma transfected with human CD28 molecule which is able to produce IL-2 in response to stimulation, suggesting that the signal transduction machinery coupled to the CD28 molecule is capable of triggering effector functions. Nevertheless, the action of three immunosuppressive agents previously shown in our model, suggested an interaction between the CD3 and CD28 pathways. We confirmed here this hypothesis by transfecting the cDNA of the human CD28 molecule in the BW5147 thymoma which lacks CD3 surface expression. Stimulation of the human CD28 did not lead to IL-2 secretion while the restoration of the TCR/CD3 complex re-established the functionality of this costimulatory molecule. These data demonstrate that the IL-2 production induced by the CD28 activation pathway is dependent of the TCR/CD3 complex cell surface expression and suggest the formation of a functional membrane complex between the CD3 and CD28 molecules. The molecular basis of the functional dependence of CD28 signaling on the TCR/CD3 complex is presently unknown. Nonetheless, we showed that some early events induced by CD28 stimulation, such as PI3-kinase association, are independent of the TCR/CD3 complex expression

Immune Responses to Gene Editing by Viral and Non-Viral Delivery Vectors Used in Retinal Gene Therapy

International audienceInherited retinal diseases (IRDs) are a leading cause of blindness in industrialized countries, and gene therapy is quickly becoming a viable option to treat this group of diseases. Gene replacement using a viral vector has been successfully applied and advanced to commercial use for a rare group of diseases. This, and the advances in gene editing, are paving the way for the emergence of a new generation of therapies that use CRISPR-Cas9 to edit mutated genes in situ. These CRISPR-based agents can be delivered to the retina as transgenes in a viral vector, unpackaged transgenes or as proteins or messenger RNA using non-viral vectors. Although the eye is considered to be an immune-privileged organ, studies in animals, as well as evidence from clinics, have concluded that ocular gene therapies elicit an immune response that can under certain circumstances result in inflammation. In this review, we evaluate studies that have reported on pre-existing immunity, and discuss both innate and adaptive immune responses with a specific focus on immune responses to gene editing, both with non-viral and viral delivery in the ocular space. Lastly, we discuss approaches to prevent and manage the immune responses to ensure safe and efficient gene editing in the retina

Les lymphomes primitifs oculo-cérébraux (diagnostic différentiel avec les uvéites et étude de l effet thérapeutique d un anticorps anti-CD20 et du CpG-DNA)

Les lymphomes primitifs du SNC (PCNSL : Primary Central Nervous System Lymphoma) sont génotypiquement associés aux lymphomes diffus à larges cellules B (DLBCL). Ce sont des affectations multifocales pouvant toucher les méninges, la moelle épinière mais plus fréquemment l œil et le cerveau. Le lymphome primaire de l œil (PIOL) et le lymphome primaire du cerveau (PCL) sont des atteintes agressives, avec un pronostic vital sombre, et sans thérapie spécifique. Malgré les avancées considérables découvertes sur la pathogenèse de ces atteintes, leur physiopathologie reste encore mal connue. Le diagnostic est aujourd hui basé sur les techniques modernes d imagerie et sur l étude des marqueurs moléculaires, cependant il reste parfois difficile, notamment pour le PIOL qui peut être considéré comme une pseudo-uvéite . Par ailleurs, en dépit des progrès réalisés dans la thérapie de ces tumeurs (chimiothérapie, radiothérapie), le traitement actuel entraîne une grande toxicité et une rechute est notée en quelques années. En vue de définir de nouveaux biomarqueurs d orientation diagnostique précoce et en perspective d amélioration de la thérapie actuelle des lymphomes oculo-cérébraux, nous avons d abord identifié une nouvelle combinaison cytokinique (les ratios IL-10/IL-6 and IL-10/IFNg) différentielle du PIOL et de l uvéite, à partir de dosages moléculaires sur des échantillons de vitré et d humeur aqueuse. Par la suite, nous avons montré un effet thérapeutique d un nouvel anticorps anti-hCD20 optimisé, testé dans nos modèles murins de PIOL et de PCL, et un effet intéressant du CpG-DNA sur les cellules lymphomateuses B, dépendant de leur microenvironnement moléculaire.Primary central nervous system lymphoma (PCNSL) belongs to the systemic diffuse large B-cell lymphoma family. It can affect meninges as well as cranial, spinal, and peripheral nerves, but most frequently, it develops into the brain and/or eye. Primary cerebral lymphoma (PCL) and primary intraocular lymphoma (PIOL) are highly aggressive malignancies with a dark prognosis. Although the important advances founded from the pathogenesis of extraneural non-Hodgkin's lymphoma, the unique organotropism of PCL/PIOL remains poorly understood. Diagnosis now is facilitated by modern imaging techniques and molecular markers, but remains difficult in particular with PIOL as it is a uveitis masquerade syndrom . Moreover, in spite of remarkable progress through methotrexate-based chemotherapy, the majority of patients experience relapses within a few years. Better diagnostic tools are required for earlier diagnosis and monitoring of treatment response, and a deeper understanding of the pathogenesis of primary nervous system lymphoma may reveal new therapeutic targets. My PhD project was first to find new cytokine combinations as diagnostic and prognostic markers and then to explore new immunotherapeutic strategies on PIOL and PCL preclinical mouse models. Using vitreous and aqueous humor samples from French and Tunisian patients, we define a new highly discriminative combination of the IL-10/IL-6 and IL-10/IFNg ratios between PIOL and uveitis patients. Besides, we show an encouraging therapeutic effect of a novel glycoengineered anti-hCD20 antibody in PCL and PIOL murine lymphomas, and intriguing differences in B-cell lymphoma responsiveness to CpG.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Subretinal Injection of HY Peptides Induces Systemic Antigen-Specific Inhibition of Effector CD4+ and CD8+ T-Cell Responses

International audienceInjection of an antigen into the anterior chamber of the eye induces a peripheral antigen-specific immune modulation mechanism, known as anterior chamber-associated immune deviation (ACAID). Delayed-type hypersensitivity experiments argue that the subretinal space (SR) of the eye displays properties similar to ACAID. However, no investigation was performed regarding the differential impact of a subretinal antigen injection on peripheral CD4+ versus CD8+ T cells, on the potential immune deviation regarding Th profiles, and on the antigen-specificity of the inhibition. A better understanding of these mechanisms is crucial to improve safety and immunomonitoring of ongoing therapeutic approaches targeting the SR. The aim of this study is to characterize the proliferative capacities and cytokine patterns of antigen-specific CD4+ and CD8+ T cells after a subretinal injection of antigen in mice