The "ins and outs" of complement-driven immune responses

The complement system represents an evolutionary old and critical component of innate immunity where it forms the first line of defence against invading pathogens. Originally described as a heat-labile fraction of the serum responsible for the opsonisation and subsequent lytic killing of bacteria, work over the last century firmly established complement as a key mediator of the general inflammatory response but also as an acknowledged vital bridge between innate and adaptive immunity. However, recent studies particularly spanning the last decade have provided new insights into the novel modes and locations of complement activation and highlighted unexpected additional biological functions for this ancient system, for example in regulating basic processes of the cell. In this review, we will cover the current knowledge about complement’s established and novel roles in innate and adaptive immunity with a focus on the functional differences between serum-circulating versus intracellularly active complement and will describe and discuss the newly discovered cross-talks of complement with other cell effector systems particularly during T cell induction and contraction

Isoformes membranaires et solubles de HLA-G lors de la différenciation hématopoïétique normale et/ou pathologique (expression et étude fonctionnelle)

La fonction des molécules HLA-Ib HLA-G longtemps restreinte à la tolérance fœto-maternelle semble élargie par leur capacité à inhiber les fonctions des cellules NK/T. Malgré une forte transcription, l'expression de HLA-G reste rare dans les cellules hématopoïétiques normales. Nos résultats montrent que HLA-G est induit dans les macrophages ou cellules dendritiques (CD) dans certaines conditions pathologiques: infection par le cytomégalovirus humain, pathologies pulmonaires inflammatoires ou tumorales. Les lymphopathies montrent une rare expression membranaire et une sécrétion élevée des molécules HLA-G (HLA-Gs)dans la moitié des cas. Le rôle de HLA-Gs sur les CD, cellules clés de la réponse immune, restait à être préciser. Nous avons démontré que les HLA-Gs sont capables d'agir indirectement sur la fonction de présentation des CD via les lymphocytes T. En conclusion, les molécules HLA-G, notamment solubles, pourraient favoriser l'échappement tumoral ou viral au système immunitaire.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9: Key Steps from Design to Genotyping

International audienceThe CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy. While complex genome editing in the mouse since the 1990s has been dominated by the use of embryonic stem (ES) cells, CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes. Yet, this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project. Here, we describe the key steps leading to successful generation of a double knockout (KO) mouse by simultaneously targeting two homolog genes, Tmem176a and Tmem176b, which are located in the same genomic locus. Additionally, we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain. Thus, presented as a detailed case study that should be helpful to the non-specialists, we focus on the genotyping strategy to anticipate the various possibilities

Mouse PRDM9 DNA-Binding Specificity Determines Sites of Histone H3 Lysine 4 Trimethylation for Initiation of Meiotic Recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment

Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules

International audienc

T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

International audienceThe current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs

DataSheet_1_T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations.pdf

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.</p