Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy

Abstract The prognosis of follicular lymphoma (FL) patients is suspected to be influenced by tumor-infiltrating regulatory T cells (Treg). The mechanism of Treg enrichment in FL and their impact on malignant FL B cells remains to be elucidated. We analyzed 46 fresh lymph node biopsy samples, including FL (n = 20), diffuse large B-cell lymphoma (n = 10), classical Hodgkin lymphoma (n = 9), and reactive lymphadenitis (n = 7). Using multicolor flow cytometry and cell sorting, we observed an accumulation of CD25highCD127low/neg Tregs in FL tissues. These Tregs comprised activated ICOS+ Tregs that were able to suppress not only conventional T cells, but also FL B cells. These FL B cells were able to express ICOSL in vitro and to generate CD25highFoxP3high Tregs expressing ICOS. Treg generation was associated with ICOS/ICOSL engagement and was abrogated by antagonist anti-ICOS and anti-ICOSL antibodies. Interactions between Tregs and FL B cells resulted in ICOSL downregulation on FL B cells. Our results highlight a key role for Tregs in FL pathogenesis and suggest that targeting the ICOS/ICOSL pathway may be a promising immunotherapy for FL treatment. Cancer Res; 76(16); 4648–60. ©2016 AACR.</jats:p

Caractérisation des fonctions des cellules dentritiques murines déficientes pour l'Asparaginyl Endopeptidase

Mon étude porte sur les cellules dendritiques (DCs), qui patrouillent dans l'organisme à la recherche d'agents pathogènes. Elles disposent pour cela d'une batterie de senseurs , récepteurs membranaires ou cytoplasmiques capables de reconnaître des signatures spécifiques de pathogènes (PAMPs), dont les récepteurs Toll-like (TLRs) font partie. En cas de contact, elles prennent en charge l'élément reconnu et sont alors en mesure de déclencher une réponse immunitaire adaptée. Pour arriver à ce résultat, les DCs ont la capacité d'ingérer l'élément étranger dans des ve sicules intracellulaires, les endosomes ou les phagosomes, de l'y fragmenter et d'en présenter certains résidus sur les molécules du Complexe Majeur d'Histocompatibilité de classe II (CMH II). De par leur localisation et leur activité, les protéases de la voie endocytique sont aptes à jouer un rôle dans la fragmentation des protéines ingérées. Afin d'étudier spécifiquement la contribution de l'une de ces protéases, l'Asparaginyl Endopeptidase (AEP), nous avons utilisé l'approche de la délétion constitutive chez la souris et caractérisé la capacité des DCs déficientes pour l'AEP à dégrader et à présenter l'antigène. Nous avons observé que non seulement l'AEP joue un rôle direct dans la dégradation du fragment C de la toxine tétanique (TTcf), favorisant sa présentation, mais qu'elle est aussi essentielle à la maturation d'autres protéases de la même voie, notamment la Cathepsine L (CatL). La CatL immature montre une augmentation d'activité, résultant en la destruction d'un autre antigène, la Myelin Oligodendrocyte Protein (MOG), entraînant un défaut de présentation de cet antigène chez les souris déficientes pour l'AEP. D'une manière surprenante, nous avons aussi montré que ces DCs ne répondent que faiblement à l'adjonction de CpG, ligand du TLR endosomal TLR9, mais qu'elles maintiennent une réponse normale au LPS, ligand du TLR de surface TLR4. Nous expliquons ce phénomène en montrant que l'activation des TLRs endosomaux nécessite, en plus de la présence de son ligand, un clivage de leur domaine luminal, que peut effectuer l'AEP. Ce travail montre le double rôle de l'AEP dans la fragmentation de l'antigène, directement par son activité protéolytique et indirectement par la régulation de l'activité d'autres protéases. Par cette étude, nous mettons aussi en évidence le rôle jusqu'alors insoupçonné des protéases endosomales, en particulier de l'AEP, dans la régulation de la réponse aux TLRs endosomaux.This is a study on dendritic cells. They are looking all over the body for pathogens that they can recognize thanks to a large number of receptors, on their membrane on cytoplasm. Those receptors, among which are the Toll-like receptors, are able to bind pathogens markers (PAMPs). In case of encounter, DCs can take up the pathogen and to initiate a specific immune response. In this purpose, DCs will internalize the pathogens in intracellular vesicles, called endosomes or phagosomes, to cut it into fragments and to present some of those fragments bound to MHC II on the cell surface. In this mechanism, proteases are localized in the same compartments where the degradation of the pathogens occurs. In order to study the role of one of those proteases, Asparaginyl Endopeptidase, we generate a mouse deleted for the AEP gene. Studying this mouse gave us the opportunity to observe that AEP has a role in the degradation of different antigens : it can degrade TTcf and favors its presentation by DCs; AEP control also the maturation of other proteases, as Cathepsin L, leading to an increase of the activity. Increased CatL leads to the destruction of MOG, and the lack of presentation of this antigen. In an other hand, AEP KO DCs are not able to secrete cytokines when stimulated by CpG, a TLR9 ligand but they respond normally to a TLR4 ligand, a membrane TLR. We show that endosomal TLRs need an intracellular processing to be fully activated by their ligands. This study shows clearly that endosomal proteases are crucial for establishing a normal specific immune response, by controlling the antigen presentation on MHC II by two different ways : controlling their degradation and subsequent presentation and by regulating the activity of TLRs, whose adjuvant role is known for years.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Supplementary figures and tables from Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy

&lt;p&gt;This file contains 3 Supplementary Figures and 1 supplementary table Supplementary Figure S1:ICOSL expression on immune cells in lymphoma and on cell line by flow cytometry Supplementary Figure S2: Antagonist anti-ICOS Abs favor upregulation of ICOSL on FL B cells and on murine B cells Supplementary Figure S3: Inhibition of FL B cells proliferation by Tregs Supplemntary Table S1: Monoclonal antibodies used for flow cytometry&lt;/p&gt;</jats:p

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

International audienceDogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG(+) counterparts do not. Antigen boost modifies the gene expression profile of IgM(+) plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge

Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts

Critical Role for Asparagine Endopeptidase in Endocytic Toll-like Receptor Signaling in Dendritic Cells

SummaryIntracellular Toll-like receptor 3 (TLR3), TLR7, and TLR9 localize in endosomes and recognize single-stranded RNA and nucleotides from viruses and bacteria. This interaction induces their conformational changes resulting in the production of proinflammatory cytokines and upregulation of cell surface molecules. TLR9 requires a proteolytic cleavage for its signaling. Here, we report that myeloid and plasmacytoid dendritic cells (DCs) deficient for the asparagine endopeptidase (AEP), a cysteine lysosomal protease, showed a decrease in the secretion of proinflammatory cytokines in response to TLR9 stimulation in vitro and in vivo. Upon stimulation, full-length TLR9 was cleaved into a 72 kDa fragment and this processing was strongly reduced in DCs lacking AEP. Processed TLR9 coeluted with the adaptor molecule MyD88 and AEP after size exclusion chromatography. When expressed in AEP-deficient DCs, the 72 kDa proteolytic fragment restored TLR9 signaling. Thus, our results identify an endocytic protease playing a critical role in TLR processing and signaling in DCs