Cytotoxic T-Lymphocyte-Associated Protein 4 Haploinsufficiency-Associated Inflammation Can Occur Independently of T-Cell Hyperproliferation

Located contiguously on the long arm of the second chromosome are gene paralogs encoding the immunoglobulin-family co-activation receptors CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4). CD28 and CTLA4 share the same B7 ligands yet each provides opposing proliferative signals to T cells. Herein, we describe for the first time two unrelated subjects with coexisting CD28 and CTLA4 haploinsufficiency due to heterozygous microdeletions of chromosome 2q. Although their clinical phenotype, multi-organ inflammatory disease, is superficially similar to that of CTLA4 haploinsufficient autoimmune lymphoproliferative syndrome type V (ALPS5) patients, we demonstrate our subjects’ underlying immunopathology to be distinct. Unlike ALPS5 T cells which hyperproliferate to T-cell receptor-mediated activation and infiltrate organs, T cells from our subjects are hypoproliferative and do not. Instead of T cell infiltrates, biopsies of affected subject tissues demonstrated infiltrates of lineage negative lymphoid cells. This histologic feature correlated with significant increases in circulating type 3 innate lymphoid cells (ILC3s) and ILC3 cytokines, interleukin 22, and interleukin-17A. CTLA4-Ig monotherapy, which we trialed in one subject, was remarkably effective in controlling inflammatory diseases, normalizing ILC3 frequencies, and reducing ILC3 cytokine concentrations

What contribution for molecular and cellular germinal center components during lupus development?

Le lupus érythémateux disséminé est une maladie auto-immune systémique très invalidante dont les atteintes sont multiples, les plus fréquentes étant cutanées, articulaires et rénales. Dans ce type de maladie, le système immunitaire, hyperactif, ne se limite pas à lutter contre des agents extérieurs mais s'attaque à ses propres cellules, entre autres par le biais d'auto-anticorps. Ces anticorps délétères sont produits par des plasmocytes, cellules issus de la différenciation des lymphocytes B. Ce processus se déroule principalement au sein des centres germinatifs (GC) dans les organes lymphoïdes secondaires, et fait intervenir de nombreux acteurs moléculaires et cellulaires. Mon projet de thèse a porté sur l'étude de la contribution du GC et de ses constituants, tels que les cellules auxiliaires folliculaires (Tfh) et l'IL-21, au cours du lupus. Au cours de ce travail, nous avons mis en évidence une altération à la fois quantitative et qualitative des cellules Tfh chez des patients lupiques et dans un modèle murin, altération entre autres responsable de taux anormalement élevés d'IL-21. Nous avons également observé une sensibilité accrue des cellules B de souris lupiques à cette cytokine, dont la cause est une surexpression de molécules clés telles que STAT3, et dont la conséquence est un surcroit de différenciation plasmocytaire. Tous les éléments sont donc présents pour favoriser l'interaction "Tfh-B" et la réaction du GC, et amplifier la réponse autoimmune. Enfin, la découverte de l'existence de GC ectopiques fonctionnels dans les reins de souris lupiques permet d'envisager l'existence de réponses locales au sein même des organes cibles. Les données obtenues, fondamentales, sont prometteuses et laissent entrevoir de nouvelles perspectives de biothérapies, plus ciblées, pour le traitement de la maladie lupique.Systemic lupus erythematosus is a disabling chronic autoimmune disease characterized by B cell hyperactivity leading to the production of autoantibodies, some of which exerting pathogenic effects. Autoantibodies are produced by plasma cells, which originate from the differentiation of B cells through a process that mainly takes place in germinal centers (GC) in secondary lymphoïd organs and involves many molecular and cellular parameters. The aim of my PhD project was to analyze the individual contribution of GC components, such as follicular helper T cells (Tfh) and IL-21, to lupus development. During this work, we have shown both a quantitative and qualitative impairment of Tfh cells in lupus patients and in a mouse model, leading, among other things, to high IL-21 levels. We also observed that B cells from lupus mice display a specific intrinsic sensitivity to this cytokine, due to over-expression of key molecules such as STAT3, which results in increased plasma cell differentiation. Thus, all elements are gathered that favor "Tfh-B" cell interactions and the GC reaction, and therefore the autoimmune response. Finally, the discovery of functional ectopic GC in the kidneys of lupus mice allows envisaging that local responses occur within the target organs and likely participate to kidney injury. The fundamental data we obtained are promising and anticipate new and better targeted biotherapies for lupus treatment

Biological, physiological, immunological and nutritional assessment of farm-reared Litopenaeus stylirostris shrimp affected or unaffected by vibriosis

Shrimp aquaculture in New Caledonia is subject to seasonal mortalities during grow-out due to highly virulent Vibrio nigripulchritudo (Vn). To understand the mechanisms affecting shrimp resistance and leading to significant mortality, a shrimp ecophysiology and immunology survey was conducted on two farms, the first considered as a "control" farm (HC), the second affected by the disease (DF). Mortality observed during the survey at DF started 50 days after stocking and was typical of this disease. The main observations regarding shrimp were: (a) growth was not affected by the disease and was faster in the DF than in the HC pond; (b) disease did not affect one sex more than the other, or a specific part of the population in terms of weight; (c) the physical condition of shrimp did not specifically allow us to foresee disease outbreak; (d) shrimp at late premolt stage D-2 and early postmolt stage A appeared to be at some points of the mortalities - but not continuously - the most sensitive to disease; (e) physiological, immunological and nutritional parameters of uninfected shrimp in the DF pond were altered, suggesting that environmental stress occurred just before the first mortalities; (f) data suggest that Vn-infected shrimp are more stressed than the presumed healthy shrimp. Combined with pathological and environmental knowledge gained in parallel during this survey, a conceptual model is proposed. Results suggest that an unstable environment induced conditions (i) stressful for the shrimp, increasing their susceptibility to bacterial infections and (ii) favoring the proliferation of the pathogen in the pond. The combination of these two processes could lead to significant mortality

Early Differentiated CD138^highMHCII⁺IgG⁺ Plasma Cells Express CXCR3 and Localize into Inflamed Kidneys of Lupus Mice

<div><p>Humoral responses are central to the development of chronic autoimmune diseases such as systemic lupus erythematosus. Indeed, autoantibody deposition is responsible for tissue damage, the kidneys being one of the main target organs. As the source of pathogenic antibodies, plasma cells are therefore critical players in this harmful scenario, both at systemic and local levels. The aim of the present study was to analyze plasma cells in NZB/W lupus mice and to get a better understanding of the mechanisms underlying their involvement in the renal inflammation process. Using various techniques (i.e. flow cytometry, quantitative PCR, ELISpot), we identified and extensively characterized three plasma cell intermediates, according to their B220/CD138/MHCII expression levels. Each of these cell subsets displays specific proliferation and antibody secretion capacities. Moreover, we evidenced that the inflammation-related CXCR3 chemokine receptor is uniquely expressed by CD138^highMHCII⁺ plasma cells, which encompass both short- and long-lived cells and mostly produce IgG (auto)antibodies. Expression of CXCR3 allows efficient chemotactic responsiveness of these cells to cognate chemokines, which production is up-regulated in the kidneys of diseased NZB/W mice. Finally, using fluorescence and electron microscopy, we demonstrated the presence of CD138⁺CXCR3⁺IgG⁺ cells in inflammatory areas in the kidneys, where they are very likely involved in the injury process. Thus, early differentiated CD138^highMHCII⁺ rather than terminally differentiated CD138^highMHCII^low plasma cells may be involved in the renal inflammatory injury in lupus, due to CXCR3 expression and IgG secretion.</p> </div

Des formations différenciées pour des réussites plurielles : le cas d'écri+ (ANR-17-NCUN-00015)

International audienceDes formations différenciées pour des réussites plurielles : le cas d'écri+ (ANR-17-NCUN-00015

B cell/plasma cell subsets in diseased lupus NZB/W mice.

<p>A) Percentage of CD138-expressing cells among living cells in the blood and spleen of 32–36 week-old NZB/W mice and BALB/c mice (10 and 4 mice per group respectively) as determined by flow cytometry analysis. Results are expressed as mean % +/− SEM. B) Representative FACS figures showing four main populations of cells in the spleen of a sick NZB/W mouse according to surface expression of key B cell and plasma cell markers: (a) B220⁺CD138<b>⁻</b>Ly6C<b>⁻</b>CD79b⁺ cells, (b) B220⁺CD138^intLy6C^intCD79b^int cells, (c) CD138^highMHCII⁺Ly6C⁺CD79b<b>⁻</b> cells and (d) CD138^highMHCII^lowLy6C⁺CD79b<b>⁻</b> cells. This phenotypic analysis was performed in eleven 32–36 week-old proteinuria-positive NZB/W mice with similar results.</p

Characteristic ultrastructural morphology and transcription factor expression in cell subsets identified according to B220/CD138/MHCII expression.

<p>A) The four spleen cell subsets (a to d, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058140#pone-0058140-g001" target="_blank">Figure 1</a>) were sorted by flow cytometry from the spleen of diseased NZB/W mice. Cells were then embedded for electron microscopy observation. Images show one representative cell (out of 20 observed) in each panel. A representative experiment out of two is shown. Scale bars = 500 nm. B) Total RNA was isolated from each FACS-sorted cell subset and the expression of <i>prdm1</i> (Blimp-1; grey bars), <i>xbp1</i> (dark grey bars) and <i>pax5</i> (hatched bars) transcripts was evaluated by quantitative real-time PCR. Results are expressed as the fold induction of gene transcription as compared to the cell subset expressing the lowest transcript amount (B220⁺ cells as for <i>prdm1</i> and <i>xbp1</i>, and CD138^highMHCII⁺ cells as for <i>pax5)</i>. All differences between raised values in each subset are statistically significant except for <i>pax5</i> when comparing the two B220⁺ subsets and the two CD138^high subsets to each other, respectively. Data of one representative experiment out of three are shown.</p

Circulating T_FH Subset Distribution Is Strongly Affected in Lupus Patients with an Active Disease

<div><p>Follicular helper T cells (T_FH) represent a distinct subset of CD4⁺ T cells specialized in providing help to B lymphocytes, which may play a central role in autoimmune diseases having a major B cell component such as systemic lupus erythematosus. Recently, T_FH subsets that share common phenotypic and functional characteristics with T_FH cells from germinal centers, have been described in the peripheral blood from healthy individuals. The aim of this study was to analyze the distribution of such populations in lupus patients. Circulating T_FH cell subsets were defined by multicolor flow cytometry as T_FH17 (CXCR3^-CCR6⁺), T_FH1 (CXCR3 ⁺ CCR6^-) or T_FH2 (CXCR3^-CCR6^-) cells among CXCR5 ⁺ CD45RA^-CD4⁺ T cells in the peripheral blood of 23 SLE patients and 23 sex and age-matched healthy controls. IL-21 receptor expression by B cells was analyzed by flow cytometry and the serum levels of IL-21 and Igs were determined by ELISA tests. We found that the T_FH2 cell subset frequency is strongly and significantly increased in lupus patients with an active disease (SLEDAI score>8), while the T_FH1 cell subset percentage is greatly decreased. The T_FH2 and T_FH1 cell subset frequency alteration is associated with the presence of high Ig levels and autoantibodies in patient’s sera. Moreover, the T_FH2 cell subset enhancement correlates with an increased frequency of double negative memory B cells (CD27^-IgD^-CD19⁺ cells) expressing the IL-21R. Finally, we found that IgE levels in lupus patients’ sera correlate with disease activity and seem to be associated with high T_FH2 cell subset frequency. In conclusion, our study describes for the first time the distribution of circulating T_FH cell subsets in lupus patients. Interestingly, we found an increased frequency of T_FH2 cells, which correlates with disease activity. Our results suggest that this subset might play a key role in lupus pathogenesis.</p> </div