Carabin deficiency in B cells increases BCR‐TLR9 costimulation‐induced autoimmunity
2012; Springer Nature; Volume: 4; Issue: 12 Linguagem: Inglês
10.1002/emmm.201201595
ISSN1757-4684
AutoresJean‐Nicolas Schickel, Jean‐Louis Pasquali, Anne Soley, Anne‐Marie Knapp, Marion Décossas, Aurélie Kern, Jean‐Daniel Fauny, Luc Marcellin, Anne‐Sophie Korganow, Thierry Martin, Pauline Soulas‐Sprauel,
Tópico(s)Blood disorders and treatments
ResumoResearch Article29 October 2012Open Access Carabin deficiency in B cells increases BCR-TLR9 costimulation-induced autoimmunity Jean-Nicolas Schickel Jean-Nicolas Schickel CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Jean-Louis Pasquali Jean-Louis Pasquali CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Anne Soley Anne Soley CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Anne-Marie Knapp Anne-Marie Knapp CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Marion Decossas Marion Decossas CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Aurélie Kern Aurélie Kern EA4438, Groupe Borréliose de Lyme, UFR Sciences Pharmaceutiques and UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Jean-Daniel Fauny Jean-Daniel Fauny CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Luc Marcellin Luc Marcellin Department of Anatomopathology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Anne-Sophie Korganow Anne-Sophie Korganow CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Thierry Martin Thierry Martin CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Pauline Soulas-Sprauel Corresponding Author Pauline Soulas-Sprauel [email protected] CNRS UPR9021, IBMC, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France UFR Sciences Pharmaceutiques, Université de Strasbourg, Illkirch, France Search for more papers by this author Jean-Nicolas Schickel Jean-Nicolas Schickel CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Jean-Louis Pasquali Jean-Louis Pasquali CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Anne Soley Anne Soley CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Anne-Marie Knapp Anne-Marie Knapp CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Marion Decossas Marion Decossas CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Aurélie Kern Aurélie Kern EA4438, Groupe Borréliose de Lyme, UFR Sciences Pharmaceutiques and UFR Médecine, Université de Strasbourg, Strasbourg, France Search for more papers by this author Jean-Daniel Fauny Jean-Daniel Fauny CNRS UPR9021, IBMC, Strasbourg, France Search for more papers by this author Luc Marcellin Luc Marcellin Department of Anatomopathology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Anne-Sophie Korganow Anne-Sophie Korganow CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Thierry Martin Thierry Martin CNRS UPR9021, IBMC, Strasbourg, France UFR Médecine, Université de Strasbourg, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France Search for more papers by this author Pauline Soulas-Sprauel Corresponding Author Pauline Soulas-Sprauel [email protected] CNRS UPR9021, IBMC, Strasbourg, France Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France UFR Sciences Pharmaceutiques, Université de Strasbourg, Illkirch, France Search for more papers by this author Author Information Jean-Nicolas Schickel1, Jean-Louis Pasquali1,2,3, Anne Soley1,2, Anne-Marie Knapp1,2, Marion Decossas1, Aurélie Kern4, Jean-Daniel Fauny1, Luc Marcellin5, Anne-Sophie Korganow1,2,3, Thierry Martin1,2,3 and Pauline Soulas-Sprauel *,1,3,6 1CNRS UPR9021, IBMC, Strasbourg, France 2UFR Médecine, Université de Strasbourg, Strasbourg, France 3Department of Clinical Immunology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 4EA4438, Groupe Borréliose de Lyme, UFR Sciences Pharmaceutiques and UFR Médecine, Université de Strasbourg, Strasbourg, France 5Department of Anatomopathology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France 6UFR Sciences Pharmaceutiques, Université de Strasbourg, Illkirch, France *Tel: +33 3 88 41 70 25; Fax: +33 3 88 61 06 80 EMBO Mol Med (2012)4:1261-1275https://doi.org/10.1002/emmm.201201595 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract The 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. The paper explained PROBLEM: SLE is a systemic, frequent and severe autoimmune disease characterized by the production of various pathogenic autoantibodies, which participate in multi-organ damage. The aetiology of SLE is both genetic and environmental. B-lymphocytes play a central role in the disease and carry intrinsic genetic defects, which would make an individual more susceptible to the development of autoimmunity. However, only a few genes have been validated in humans and it is important to identify new gene defects that could be responsible for autoimmunity. RESULTS: We investigated gene expression abnormalities in B cells from SLE patients and from lupic mice and have identified a similar decrease in the expression of Carabin gene. Here, we have studied the role of Carabin in B cell and in the development of autoimmunity using Carabin-deficient cells and mice. We have first shown that Carabin is a new negative regulator of one the most important BCR-dependent signalling pathways, Erk. Its deficiency in B cells accelerates the production of specific antibodies after immunization with classical antigens, and makes the mice more susceptible to the production of autoantibodies and the development of signs of renal damages after immunization with CpG-DNA, mimicking a viral infection by activation of TLR9 receptor (belonging to the TLR family of receptors recognizing pathogen components). We further showed that Carabin deficiency leads to a higher Erk activation after TLR9 and BCR costimulation in B cells. IMPACT: This study identified the deficiency of Carabin expression as a novel gene expression abnormality in SLE B cells. Carabin deficiency could make an individual more susceptible to the development of autoimmunity. These results are important for a better comprehension of biological actors implicated in SLE development. This could give new opportunities into the development of new diagnostic and therapeutic tools. INTRODUCTION Systemic lupus erythematosus (SLE), a prototype of human systemic autoimmune disease, is characterized by a wide variety of multi-organ damage (among which one of the hallmarks is glomerulonephritis), triggered by an autoantibody-mediated inflammation (Croker & Kimberly, 2005). The origin of SLE is generally attributed to a combination of a complex genetic influence (Flesher et al, 2010) and vaguely described environmental factors. In line with this theory, the majority of human SLE occurs in adults and is clinically characterized by a succession of flares interspersed with remission phases. This scenario strongly suggests the influence of flare-inducing external stimuli on a predisposing genetic background. Several lines of evidence indicate that B cells are central to the disease process (Shlomchik & Madaio, 2003): 1) B cells produce the autoantibodies, some of which are clearly pathogenic forming immune complex deposits or destroying their target; 2) (NZB × NZW)F1 and MRL-Faslpr/lpr (murine models of human SLE) mice harbouring the xid mutation, which inactivates Btk and causes a blockade of B-cell development and B-cell responses, no longer develop a lupus phenotype, including autoantibodies and glomerulonephritis (Steinberg et al, 1982; 1983), as do (NZB × NZW)F1 mice having a very restricted IgM transgenic repertoire (Wellmann et al, 2001); 3) the disease can be transferred in mice by B cells since immunodeficient SCID (severe combined immunodeficiency) mice populated with pre-B cells of (NZB × NZW)F1 mice develop many of the characteristics of (NZB × NZW)F1 mice, suggesting that genetic defects responsible for the development of SLE disease in (NZB × NZW)F1 mice are present in their B cells (Reininger et al, 1996). The study of SLE genetics has shown that the disease rarely occurs from a single mutation (except for deficiencies in the early components of complement cascade), but more commonly as a polygenic disease (Moser et al, 2009). On one hand, many polymorphisms of immune and non-immune genes (almost 30) have been described during the last 10 years, owing to large genome-wide association studies (GWAS) (Chung et al, 2011; Graham et al, 2008, 2009; Han et al, 2009; Hom et al, 2008; International Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN) et al, 2008; Kozyrev et al, 2008; Yang et al, 2010, 2011) in lupus patients. They most likely constitute a set of predisposing SLE genes, but the consequences of these polymorphisms, in terms of protein levels or protein function, are generally unknown. Exceptions are BANK1, for which three variants have been associated to SLE and are supposed to lead to an altered B-cell activation threshold (Graham et al, 2009; Moser et al, 2009) and PTPN22, for which Zhang et al have recently developed a knock-in (KI) mouse line expressing the autoimmune disease-associated PTPN22 variant (Pep619W). It is interesting to note that these mice show signs of lymphocyte hyperresponsiveness without developing pathogenic autoantibodies and signs of autoimmunity by their own (Zhang et al, 2011). On the other hand, and in parallel, genetically modified mice have been produced with clear functional consequences like a spontaneous autoimmune phenotype. For example, deficiencies of negative regulators of B lymphocytes induce spontaneous B-cell activation and spontaneous lupus phenotypes (Nitschke, 2005; Pritchard & Smith, 2003): 1) negative regulators of B-cell receptor (BCR) belonging to inhibitory co-receptors pathways [CD22 (O'Keefe et al, 1996; 1999; Otipoby et al, 1996; Poe et al, 2000), 9-O-acetyl sialic acid esterase or Siae (Cariappa et al, 2009), FcγRIIB (Bolland & Ravetch, 2000), PD-1 (Nishimura et al, 1999)], kinases phosphorylating BCR co-receptor ITIM motifs [Lyn (Hibbs et al, 1995)] and phosphatases recruited by the phosphorylated ITIMs [SHP1 (Pao et al, 2007)]; 2) B-cell-negative regulators of BCR-independent pathways like Act1 [TRAF3IP2 (Qian et al, 2004, 2008)] and the ubiquitin-modifying enzyme A20 [TNFAIP3 (Chu et al, 2011; Tavares et al, 2010)]. Although interesting, these data do not fit with the known discrete, not activated B-cell phenotype (normal expression of CD86 and CD40L) of lupus patients during the inactive phases of the disease, which precedes the flares. In addition, among the negative regulators of B cells described above, only two (A20 and FcγRIIB) have been shown to be candidate genes for human SLE (Moser et al, 2009). Altogether, these data suggest that SLE combines many minor susceptibility genes, each representing a single "hit" (for example the Pep619W allele) in a multi-hit and environment-dependent model for the development of SLE and of autoimmunity in general (Behrens, 2011). In order to better undestand this model, it is important to identify gene defects, which can be responsible for an inducible, not a spontaneous, autoimmune disease phenotype, and to provide some mechanistic insights linking these defects to a flare-inducing environmental stimulus. In order to hunt down SLE predisposing gene abnormalities, and considering the important role of B cells in SLE, we started from a transcriptome analysis of B cells purified from SLE patients during latency phases of their disease and of B cells from young (NZW × NZB)F1 mice. We identified a similar deficiency in Carabin gene expression in both human and murine lupus B cells. Carabin, alias TBC1D10C, was recently described as a negative regulator of T-cell function exhibiting a dual inhibitory activity on calcineurin (by its carboxy-terminal domain of interaction with calcineurin) and Ras (by its amino-terminus Ras/GAP domain) pathways. Knockdown of Carabin notably leads to a significant enhancement of IL-2 production by specific T cells after antigen stimulation (Pan et al, 2007). Considering the important molecular similarities of antigen receptor signaling in T and B cells, including the role of Ras and Calcineurin pathways in BCR signaling, we decided to evaluate the role of Carabin in B cells, which is currently unknown, and to look for signs of autoimmunity in Carabin-deficient mice. Using knock-out and B-cell-conditional knock-out murine models, we show that Carabin is a new negative regulator of the Ras/Erk pathway in B cell. The phenotype of Carabin-deficient B cells in non-autoimmune prone mice is subtle: although characterized by an acceleration of early B-cell response after immunization, Carabin knock-out (KO) mice do not present any spontaneous B-cell activation, nor spontaneous production of autoantibodies. However, when Carabin-deficient mice are stimulated with a Toll-like Receptor 9 (TLR9) agonist (CpG-DNA), thereby mimicking a viral infection, we observe the production of anti-dsDNA antibodies and a lupus-like glomerulonephritis with immune deposits in a subgroup of mice. Finally, our in vitro data give some mechanistic insights into these results, proposing Carabin as a new negative regulator of TLR9/BCR costimulation in self-reactive B cells. RESULTS Carabin expression is low in lupus B cells in the quiescent phase of the disease In a transcriptome analysis of purified splenic B cells from 4-month-old (NZB × NZW)F1 mice (8–10 weeks before the occurrence of the disease), Carabin mRNA expression was lower than in control mice (p < 0.05, unpublished observation). A 50% reduction of Carabin expression was confirmed by real-time quantitative RT-PCR in B cells from 4 month-old as well as in 2 month-old (NZB × NZW)F1 mice (43% reduction), a long time before the appearance of autoantibodies, B-cell hyperactivation and disease in this SLE model (Fig 1A). We also performed a pangenomic transcriptome analysis (Affymetrix GeneChip human genome U133 plus 2.0) of purified B cells from 17 patients with SLE in quiescent phase [SLE disease activity score (Selena SLEDAI) less than 4] compared to B cells from age- and sex-matched controls (Garaud et al, 2011). We studied patients with inactive disease and with minimum treatment (less than 10 mg/day of prednisone and no immunosuppressive treatment) in order to avoid background due to non-specific B-cell activation that occurs during flares of the disease. Indeed, the concomitant immunophenotyping of these patients B cells did not show any sign of activation (normal expression of CD86) (Korganow et al, 2010). These data have been deposited in NCBI's Gene Expression Omnibus (Edgar et al, 2002) and are accessible through GEO Series accession number GSE30153 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30153). Most interestingly, Carabin is significantly underexpressed in SLE patients (p = 0.01) (Fig 1B). In addition, we confirmed by real-time quantitative RT-PCR in a new cohort of 10 SLE patients the low level of Carabin expression in purified B cells compared to healthy controls (ranging from 30 to 60% reduction, Fig 1C). Figure 1. Carabin is underexpressed in (NZB × NZW)F1 and in lupus patient B cells. A.. Quantitative real-time RT-PCR analysis of Carabin mRNA expression in splenic mature B cells purified from 2 month-old Balb/c (n = 3) and (NZB × NZW)F1 (n = 4) mice (left), and from 4 month-old Balb/c (n = 4) and (NZB × NZW)F1 (n = 4) mice (right). Each sample was normalized to the endogenous control Hprt1. (errors bars, standard deviation). *p < 0.05, Mann & Whitney test. B.. Carabin mRNA expression levels in transcriptoma analysis of purified B cells from 17 SLE patients, compared to 9 healthy age and sex-matched controls. C.. Quantitative real-time RT-PCR analysis of Carabin mRNA expression in B cells purified from the blood of 10 lupus patients (SLE) compared to 10 healthy individuals. Each sample was normalized to the endogenous control Hprt1. Download figure Download PowerPoint During our transcriptome analysis of SLE B cells, Pan et al. identified Carabin as a negative regulator of T-cell function (Pan et al, 2007). We thus decided to further investigate Carabin function in B cells. Carabin is a new negative regulator of the Erk pathway in B cells Since the role of Carabin in B-cell function was unknown, we analysed the variation of Carabin expression in normal murine B cells after activation and during B-cell development. BCR or LPS activation leads to a fast decrease of Carabin expression (Fig 2A and B). In addition, Carabin expression was tightly regulated during B-cell maturation in normal mice, with a gradual increase from bone marrow pro/preB cells to splenic mature follicular B cells (Fig 2C). In conclusion, the high expression of Carabin in mature B cells could be indicative of a more important role of Carabin in mature B-cell function. Figure 2. Carabin expression is tightly regulated during B-cell maturation and activation. Data correspond to three independent experiments. A,B.. Purified splenic mature B cells from C57/BL6 mice were stimulated with anti-IgM (10 µg/ml), LPS (10 µg/ml), or left unstimulated for the indicated time. Carabin expression was evaluated by (A) quantitative real-time RT-PCR or (B) Western blot. (A, errors bars, standard deviation) C.. Quantitative real-time RT-PCR analysis of Carabin expression in FACS-sorted B-cell subsets from C57/BL6 mice: Pro/PreB (B220+IgM−); Immature (B220medIgM+); recirculating mature (B220highIgM+); T1 (IgM+CD23−CD21−); T2 (IgM+CD23+CD21high); follicular (IgM+CD23+CD21low); marginal zone (IgM+CD23−CD21high). Samples were normalized to the endogenous control Hprt1. (errors bars, standard deviation). Download figure Download PowerPoint To clarify the function of Carabin in B cells, we first analysed the phenotype of Carabin knock-down (KD) in a IgG+, A20 B-cell line transduced with a pTRIP lentivirus allowing for the coexpression of a Carabin-specific shRNA and GFP reporter gene. pTRIP-shCarabin-transduced A20 B cells showed a 70% decrease of Carabin expression compared to pTRIP-control-transduced A20 B cells as assessed by quantitative real-time RT-PCR (Fig 3A). The reduced expression of Carabin was further confirmed by Western Blot analysis (Fig 3B). When compared to control A20 B cells, Carabin KD-A20 B cells displayed a modest increase in the expression of CD86 and CD69 activation markers before and after stimulation with LPS or anti-IgG antibody (Fig 3C). Because Carabin has been shown in T cells to inhibit Ras pathway and Erk1/2 phosphorylation, we evaluated the effects of Carabin KD on the Ras pathway in A20 B cells. Interestingly, Carabin KD accelerated Erk1/2 phosphorylation in BCR-stimulated B cells (Fig 3D). This effect was specific for the BCR pathway, because LPS stimulation did not lead to a faster Erk1/2 phosphorylation in Carabin KD-A20 B cells compared to control B cells. To test the specificity of Carabin for the Ras MAP kinase signaling pathway in B cells, we analysed the activation of another related member of the MAP kinase superfamily, c-Jun N-terminal kinase, which is not targeted by Ras. JNK phosphorylation was not affected by Carabin KD in A20 B cells (Fig 3E). In conclusion, Carabin is a negative regulator of Ras/Erk pathway in B cells, as described for T cells (Pan et al, 2007). Figure 3. Carabin underexpression increases Erk1/2 phosphorylation after stimulation of A20 B cells. Data correspond to three independent experiments. A.. A20 cells were transduced with lentiviral constructs containing no shRNA (pTRIP-control, −) or an shRNA targeting Carabin (pTRIP-shCarabin, +) and GFP+, transduced cells were sorted. Carabin expression was determined by quantitative real-time RT-PCR. Each sample was normalized to the endogenous control 18S. Bars represent the level of Carabin transcript expression in transduced GFP+ A20 cells relative to non-transduced A20 cells. (errors bars, standard deviation). B.. Immunoblot analysis of Carabin expression in A20 B cells after transduction as in A. GAPDH was used as loading control. C.. Flow cytometry analysis of activation markers on GFP+ A20 B cells, transduced and sorted as in A, then stimulated with an anti-IgG antibody (10 µg/ml) or with LPS (10 µg/ml) for 24 h. (errors bars, standard deviation). D.. A20 B cells transduced and sorted as in A, then stimulated with an anti-IgG antibody (10 µg/ml) for 1, 3 or 5 min. Cell lysates were analysed by Western blot using anti-phospho Erk1/2 antibody. Erk1/2 was used as a loading control. The percentages of phospho-Erk1/2 were normalized to the total Erk1/2 proteins in the corresponding lane, and then to unstimulated cells (time 0, 0%). Notation: +, cells stably expressing Carabin specific shRNA; −, control cells. E.. A20 B cells were treated as in D. Cell lysates were analysed by Western blot using anti-phospho JNK antibody. JNK was used as a loading control. Notation: +, cells stably expressing Carabin specific shRNA; −, control cells. Download figure Download PowerPoint Carabin is not involved in B- and T-cell development and in the basal secretion of immunoglobulins In order to fully analyse Carabin function in vivo, we generated Carabin KO and conditional KO mice (see Materials and Methods Section, and Supporting Information Fig 1). Carabin−/− mice were obtained with a Mendelian frequency and developed normally. The development of B and T cells was extensively studied in these mice. Concerning T-cell development, total cellularity of thymus, spleen and lymph nodes and percentages of developing thymocytes and of mature CD4+ and CD8+ T cells were comparable in Carabin−/− and control littermate Carabin +/+ mice (Supporting Information Table 1), thus confirming in a physiological model that Carabin does not influence T-cell maturation as proposed by Pan et al in their Carabin KD hematopoietic stem cell transfer model (Pan et al, 2007). Similarly, there was no statistical difference in the absolute numbers and proportions of the different sub-populations of B cells in primary and secondary lymphoid organs in Carabin−/− and control mice (Supporting Information Table 1). In addition, there was no noticeable difference in the structures of the spleens and lymph nodes. Finally, at baseline, the secretion of serum IgM, IgG and IgG subtypes was not statistically different between the two groups of mice (Supporting Information Fig 2). Thus, Carabin is not involved in B- and T-cell development and in the basal secretion of immunoglobulins. Increased response of Carabin−/− B and T cells in vitro To further study the role of Carabin in B- and T-cell function, we analysed the response of Carabin-deficient B and T cells in vitro and confirmed and completed Pan's description of Carabin deficiency in T cells. Carabin−/− T cells displayed an increased proliferative response (CFSE assay) after stimulation with anti-CD3 or anti-CD3/anti-CD28 antibodies (Fig 4A), an increased spontaneous expression of CD25 and CD44 activation markers at baseline, and an increased expression of CD69 and CD25 after stimulation with anti-CD3 or anti-CD3/anti-CD28 antibodies (Fig 4B and Supporting Information Fig 3A). The phosphorylation of Erk was also enhanced in Carabin−/− T cells before and after stimulation with anti-CD3 or anti-CD3/anti-CD28 antibodies (Supporting Information Figs 3B and 4) in concordance with the data obtained by Pan et al showing that Erk phosphorylation was delayed when N-terminal Ras GAP domain of Carabin was overexpressed in PMA/ionomycin-activated human Jurkat T cells (Pan et al, 2007). On the contrary, considering the B cells of Carabin−/− mice, we made the following observations: 1) the proliferation of B cells (Fig 4C) and the increase of expression of activation markers (CD86, CD69, MHCII) on B cells (Fig 4D and Supporting Information Fig 3C) in response to BCR-dependent (anti-IgM) or BCR-independent (LPS) stimulation is not different in Carabin−/− and in control Carabin+/+ mice; 2) there was no difference in Ig production between Carabin−/− and +/+ mice after stimulation of splenic cells with LPS or LPS plus IL4 in vitro (Supporting Information Fig 5); 3) Carabin−/− B cells showed an increase of Erk phosphorylation compared to Carabin+/+ B cells after BCR activation with anti-IgM antibody (Fig 4E and Supporting Information Fig 3D) confirming the results obtained in Carabin KD-A20 B cells (Fig 3D). This increase or Erk phosphorylation led to a higher induction of Egr1 and TIS11b (Fig 4F), both known to be induced in B cells after BCR stimulation in a Erk-dependent manner (Glynne et al, 2000). Because Carabin has a dual inhibitory activity on Ras and calcineurin pathway in T cells (Pan et al, 2007), we have also analysed the nuclear factor of activated T cells (NFAT) nuclear translocation in Carabin−/− B cells after BCR stimulation and have shown a decrease of cytoplasmic NFAT and an increase of nuclear NFAT in Carabin−/− B cells compared to Carabin+/+ B cells (Fig 4G and H). However, Carabin does not seem to play an inhibitory role on more usptream signals such as calcium influx (Supporting Information Fig 6A and B). To conclude, stimulation-induced increase of Erk activation and NFAT nuclear translocation appears to be a common consequence of Carabin deficiency in T and B cells. But in contrast to T cells, Carabin deficiency in B cells is not associated with a spontaneous activation status. Figure 4. Increased response of Carabin-deficient T and B Cells. A,B.. Flow cytometry analysis of (A) dilution of CFSE-labeled and (B) cell surface expression of CD69, CD44 and CD25 on Carabin+/+ and Carabin−/− CD4+ T cells after stimulation for 72 h with anti-CD3 antibody (2 µg/ml) (dashed line), anti-CD3+anti-CD28 antibodies (2 µg/ml each) (solid line), or medium alone (shaded gray). Data in A correspond to three independent experiments. The corresponding statistical to B analysis is represented in Supporting Information Fig 3A. C,D.. Flow cytometry analysis of (C) dilution of CFSE-labeled and (D) cell surface expression of CD86, CD69 and MHCII on Carabin+/+ and Carabin−/− CD19+ B cells after stimulation for 72 h with LPS (10 µg/ml) (dashed line), anti-IgM antibody (10 µg/ml) (solid line), or medium alone (shaded gray). Data in C correspond to three independent experiments. The corresponding statistical analysis to D is represented in Supporting Information Fig 3C. E.. Flow cytometry analysis of Erk phosphorylation in Carabin+/+ and Carabin−/− B220+ B cells after stimulation for 10 min with anti-IgM antibody (10 µg/ml) (solid line), or with medium alone (shaded gray). Numbers indicate Mean Fluorescence Intensity. The corresponding statistical analysis is represented in Supporting Information Fig 3D. F.. Purified splenic mature B cells were stimulated for 1 h with anti-IgM antibody (0.5 µg/ml), or medium alone. Egr1 and TIS11b expression was determined by quantitative real-time RT-PCR. Each sample was normalized to the endogenous control Hprt1. Bars represent the level of Egr1 and TIS11b transcript expression in Carabin+/+ and Carabin−/− anti-IgM stimulated B cells relative to unstimulated B cells. G,H.. Immunoblot analysis of NFAT nuclear translocation in Carabin+/+ and Carabin−/− splenic purified (CD43-negative) B cells after stimulation for 15 min with anti-IgM antibody (5 µg/ml), ionomycin
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