280 lentiviral mediated gene therapy restores b cell homeostasis and tolerance in wiskott aldrich syndrome patients

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency characterized by micro-thrombocytopenia, eczema and increased risk of infections, autoimmunity and tumors. Allogeneic hematopoietic stem cell (HSC) transplantation is a recognized curative treatment for WAS, but when a matched donor is not available, administration of WAS gene-corrected autologous HSCs represents a valid alternative therapeutic approach. Since alterations of WAS protein (WASp)-deficient B lymphocytes contribute to immunodeficiency and autoimmunity in WAS, we followed the B cell reconstitution in 4 WAS patients treated by lentiviral vector-gene therapy (GT) after a reduced-intensity conditioning regimen combined with anti-CD20 administration. We analyzed the B cell subset distribution in the bone marrow and peripheral blood by flow cytometry and the autoantibody profile by a high-throughput autoantigen microarray platform before and after GT. Lentiviral vector-transduced progenitor cells were able to repopulate the B cell compartment with a normal distribution of transitional, naive and memory B cells. The reduction in the proportion of autoimmune-associated CD21low B cells and in the plasma levels of B cell-activating factor was associated with the decreased autoantibody production in WAS patients after GT. Then, we evaluated the functionality of B cell tolerance checkpoints by testing the reactivity of recombinant antibodies isolated from single B cells. Before GT, we found a decreased frequency of autoreactive new emigrant/transitional B cells in WAS patients, suggesting a hyperfunctional central B cell checkpoint in the absence of WASp. In contrast, high frequency of polyreactive and Hep2 reactive clones were found in mature naive B cells of WAS patients, indicating a defective peripheral B cell checkpoint. Both central and peripheral B cell tolerance checkpoints were restored after GT, further supporting the qualitative efficacy of this treatment. In conclusion, WASp plays an important role in the regulation of B cell homeostasis and in the establishment of B cell tolerance in humans and lentiviral-mediated GT is able to ameliorate the functionality of B cell compartment contributing to the clinical and immunological improvement in WAS patients

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Systemic lupus erythematosus and under expression of Carabon and Sh3kbp1‎ : functional genomic approach

Le lupus érythémateux disséminé (LED) est une maladie autoimmune sévère, caractérisée par la production d’autoanticorps responsables de lésions multiviscérales, et dont l’étiologie est en partie génétique. Les lymphocytes B (LB) jouent un rôle central dans la maladie. Notre analyse du transcriptome des LB de patients lupiques quiescents, en comparaison à des contrôles, a permis d’établir une liste de gènes candidats sous-exprimés ou surexprimés, sur des critères biologiques ou statistiques. Ce projet de thèse propose d’explorer, en utilisant une approche de génomique fonctionnelle, les effets de la sous-expression de deux de ces gènes candidats (Carabin et Sh3kbp1) sur la fonction des LB et le développement d’une autoimmunité chez la souris. Pour cela nous avons produit une lignée de LB et des modèles murins sous-exprimant Carabin ou Sh3kbp1 et étudié leur phénotype. Nos résultats mettent en évidence que Carabin joue le rôle de régulateur négatif des lymphocytes B, et que sa sous-expression (ou son abolition totale) provoque une hypersensibilité du lymphocyte B, qui se caractérise notamment par une accélération de la phosphorylation de Erk après stimulation de la voie du BCR. De plus, Carabin contrôle la cinétique de la réponse lymphocytaire B après immunisation in vivo : en effet, cette réponse est accélérée en absence de Carabin, après immunisation avec un antigène T-dépendant ou T-indépendant. Enfin, Carabin semble jouer un rôle important dans le maintien de la tolérance des lymphocytes B dans le cas d’une stimulation simultanée des voies du BCR et du TLR9. Ceci est illustré par le fait que les souris Carabin KO développent des signes d’autoimmunité après immunisation avec de l’ADN hypométhylé de type CpG. Le deuxième gène étudié est Sh3kbp1. Nous avons montré, dans des cellules B A20 sh3kbp1 knock down, que l’activation du BCR conduit à une accélération des voies Erk et Akt. Ainsi, il semble que les voies de signalisation du BCR soient finement régulées par Sh3kbp1. Ce projet de thèse a permis d’identifier deux gènes dont la sous-expression a une conséquence directe sur la fonction des LB, et peut favoriser l’émergence d’une autoimmunité (dans le cas de Carabin). Ces résultats ouvrent des perspectives très intéressantes quant à l’étude de ces deux gènes en tant que nouveaux gènes de susceptibilité du LED.(Carabin) and SH3KBP1. The aim of this thesis project was to precise the consequences of Carabin and Sh3kbp1 underexpression in B cell function and in the development of autoimmunity. To address those issues, we produced: 1/ Carabin and Sh3kbp1 knock-down (KD) B cells and studied their phenotype; 2) a knock-out (KO) and conditional KO of Carabin in B cells or in mature B or T cells. Our results show that Carabin deficiency leads to an increase in T and B cell activation after TCR and BCR stimulation, respectively. Moreover Carabin KO mice and B cell conditional KO mice shows an accelerated T-dependant and T-independent antigen-specific B cell response in vivo. Finally, Carabin KO mice develop signs of autoimmunity after CpG treatment characterized by sustained production of anti-DNA IgG as well as an important deposition of IgG in renal glomeruli. Altogether these results define a new role for Carabin as a negative regulator of B cell signaling that points out a new defective biological pathway in autoimmunity. For Sh3kbp1, we show an acceleration of Erk and Akt phosphorylation in Sh3kbp1 KD B cells after BCR engagement, showing a role for Sh3kbp1 as a negative regulator of B cell receptor signaling.In conclusion, we have identified two genes that are deregulated in B cells during SLE. A deficiency in one of these two genes speed up the B cells response and predispose for the development of autoimmunity in vivo. Further experiments could potentially identify these two genes as new susceptibility genes in SLE

Lupus érythémateux disséminé et sous-expression de Carabin et Sh3kbp1 (Etude par génomique fonctionnelle)

Le lupus ÈrythÈmateux dissÈminÈ (LED) est une maladie autoimmune sÈvËre, caractÈrisÈe par la production d autoanticorps responsables de lÈsions multiviscÈrales, et dont l Ètiologie est en partie gÈnÈtique. Les lymphocytes B (LB) jouent un rÙle central dans la maladie. Notre analyse du transcriptome des LB de patients lupiques quiescents, en comparaison des contrÙles, a permis d Ètablir une liste de gËnes candidats sous-exprimÈs ou surexprimÈs, sur des critËres biologiques ou statistiques. Ce projet de thËse propose d explorer, en utilisant une approche de gÈnomique fonctionnelle, les effets de la sous-expression de deux de ces gËnes candidats (Carabin et Sh3kbp1) sur la fonction des LB et le dÈveloppement d une autoimmunitÈ chez la souris. Pour cela nous avons produit une lignÈe de LB et des modËles murins sous-exprimant Carabin ou Sh3kbp1 et ÈtudiÈ leur phÈnotype.Nos rÈsultats mettent en Èvidence que Carabin joue le rÙle de rÈgulateur nÈgatif des lymphocytes B, et que sa sous-expression (ou son abolition totale) provoque une hypersensibilitÈ du lymphocyte B, qui se caractÈrise notamment par une accÈlÈration de la phosphorylation de Erk aprËs stimulation de la voie du BCR. De plus, Carabin contrÙle la cinÈtique de la rÈponse lymphocytaire B aprËs immunisation in vivo : en effet, cette rÈponse est accÈlÈrÈe en absence de Carabin, aprËs immunisation avec un antigËne T-dÈpendant ou T-indÈpendant. Enfin, Carabin semble jouer un rÙle important dans le maintien de la tolÈrance des lymphocytes B dans le cas d une stimulation simultanÈe des voies du BCR et du TLR9. Ceci est illustrÈ par le fait que les souris Carabin KO dÈveloppent des signes d autoimmunitÈ aprËs immunisation avec de l ADN hypomÈthylÈ de type CpG. Le deuxiËme gËne ÈtudiÈ est Sh3kbp1. Nous avons montrÈ, dans des cellules B A20 sh3kbp1 knock down, que l activation du BCR conduit une accÈlÈration des voies Erk et Akt. Ainsi, il semble que les voies de signalisation du BCR soient finement rÈgulÈes par Sh3kbp1.Ce projet de thËse a permis d identifier deux gËnes dont la sous-expression a une consÈquence directe sur la fonction des LB, et peut favoriser l Èmergence d une autoimmunitÈ (dans le cas de Carabin). Ces rÈsultats ouvrent des perspectives trËs intÈressantes quant l Ètude de ces deux gËnes en tant que nouveaux gËnes de susceptibilitÈ du LED.(Carabin) and SH3KBP1. The aim of this thesis project was to precise the consequences of Carabin and Sh3kbp1 underexpression in B cell function and in the development of autoimmunity. To address those issues, we produced: 1/ Carabin and Sh3kbp1 knock-down (KD) B cells and studied their phenotype; 2) a knock-out (KO) and conditional KO of Carabin in B cells or in mature B or T cells. Our results show that Carabin deficiency leads to an increase in T and B cell activation after TCR and BCR stimulation, respectively. Moreover Carabin KO mice and B cell conditional KO mice shows an accelerated T-dependant and T-independent antigen-specific B cell response in vivo. Finally, Carabin KO mice develop signs of autoimmunity after CpG treatment characterized by sustained production of anti-DNA IgG as well as an important deposition of IgG in renal glomeruli. Altogether these results define a new role for Carabin as a negative regulator of B cell signaling that points out a new defective biological pathway in autoimmunity. For Sh3kbp1, we show an acceleration of Erk and Akt phosphorylation in Sh3kbp1 KD B cells after BCR engagement, showing a role for Sh3kbp1 as a negative regulator of B cell receptor signaling.In conclusion, we have identified two genes that are deregulated in B cells during SLE. A deficiency in one of these two genes speed up the B cells response and predispose for the development of autoimmunity in vivo. Further experiments could potentially identify these two genes as new susceptibility genes in SLE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

B cell signature during inactive systemic lupus is heterogeneous: toward a biological dissection of lupus.

Systemic lupus erythematosous (SLE) is an autoimmune disease with an important clinical and biological heterogeneity. B lymphocytes appear central to the development of SLE which is characterized by the production of a large variety of autoantibodies and hypergammaglobulinemia. In mice, immature B cells from spontaneous lupus prone animals are able to produce autoantibodies when transferred into immunodeficient mice, strongly suggesting the existence of intrinsic B cell defects during lupus. In order to approach these defects in humans, we compared the peripheral B cell transcriptomas of quiescent lupus patients to normal B cell transcriptomas. When the statistical analysis is performed on the entire group of patients, the differences between patients and controls appear quite weak with only 14 mRNA genes having a false discovery rate ranging between 11 and 17%, with 6 underexpressed genes (PMEPA1, TLR10, TRAF3IP2, LDOC1L, CD1C and EGR1). However, unforced hierarchical clustering of the microarrays reveals a subgroup of lupus patients distinct from both the controls and the other lupus patients. This subgroup has no detectable clinical or immunological phenotypic peculiarity compared to the other patients, but is characterized by 1/an IL-4 signature and 2/the abnormal expression of a large set of genes with an extremely low false discovery rate, mainly pointing to the biological function of the endoplasmic reticulum, and more precisely to genes implicated in the Unfolded Protein Response, suggesting that B cells entered an incomplete BLIMP1 dependent plasmacytic differentiation which was undetectable by immunophenotyping. Thus, this microarray analysis of B cells during quiescent lupus suggests that, despite a similar lupus phenotype, different biological roads can lead to human lupus

TNF receptor superfamily member 13b (TNFRSF13B) hemizygosity reveals transmembrane activator and CAML interactor haploinsufficiency at later stages of B-cell development

Photograph used for a story in the Oklahoma City Times newspaper. Caption: "There's no hang-up, really, in hanging a roof like the one for Oklahoma City's new convention center. It just takes time. Staff photographer Jim Argo was on hand today as workmen continued to hoist in place upright columns for the roof. The columns are topped by horizontal beams from which the roof for Myriad will be hung. Next week, the first of 15 concrete spans forming the roof will go up.

Carabin deficiency in B cells increases BCR-TLR9 costimulation-induced autoimmunity

International audienceThe mechanisms behind flares of human autoimmune diseases in general, and of systemic lupus in particular, are poorly understood. The present scenario proposes that predisposing gene defects favour clinical flares under the influence of external stimuli. Here, we show that Carabin is low in B cells of (NZB × NZW) F1 mice (murine SLE model) long before the disease onset, and is low in B cells of lupus patients during the inactive phases of the disease. Using knock-out and B-cell-conditional knock-out murine models, we identify Carabin as a new negative regulator of B-cell function, whose deficiency in B cells speeds up early B-cell responses and makes the mice more susceptible to anti-dsDNA production and renal lupus flare after stimulation with a Toll-like Receptor 9 agonist, CpG-DNA. Finally, in vitro analysis of NFκB activation and Erk phosphorylation in TLR9- and B-cell receptor (BCR)-stimulated Carabin-deficient B cells strongly suggests how the internal defect synergizes with the external stimulus and proposes Carabin as a natural inhibitor of the potentially dangerous crosstalk between BCR and TLR9 pathways in self-reactive B cells

Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation

Systemic Lupus Erythematosus (SLE) is a severe systemic autoimmune disease, characterized by multi-organ damages, triggered by an autoantibody-mediated inflammation, and with a complex genetic influence. It is today accepted that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility and contributing to a phenotypic trait to the disease. One of the ways to find these gene variations consists in comprehensive analysis of gene expression variation in a precise cell type, which can constitute a good complementary strategy to genome wide association studies. Using this strategy, and considering the central role of B cells in SLE, we analyzed the B cell transcriptome of quiescent SLE patients, and identified an overexpression of FKBP11, coding for a cytoplasmic putative peptidyl-prolyl cis/trans isomerase and chaperone enzyme. To understand the consequences of FKBP11 overexpression on B cell function and on autoimmunity's development, we created lentiviral transgenic mice reproducing this gene expression variation. We showed that high expression of Fkbp11 reproduces by itself two phenotypic traits of SLE in mice: breakdown of B cell tolerance against DNA and initiation of plasma cell differentiation by acting upstream of Pax5 master regulator gene