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1.
Blood ; 134(1): 9-21, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30940614

RESUMO

Evans syndrome (ES) is a rare severe autoimmune disorder characterized by the combination of autoimmune hemolytic anemia and immune thrombocytopenia. In most cases, the underlying cause is unknown. We sought to identify genetic defects in pediatric ES (pES), based on a hypothesis of strong genetic determinism. In a national, prospective cohort of 203 patients with early-onset ES (median [range] age at last follow-up: 16.3 years ([1.2-41.0 years]) initiated in 2004, 80 nonselected consecutive individuals underwent genetic testing. The clinical data were analyzed as a function of the genetic findings. Fifty-two patients (65%) received a genetic diagnosis (the M+ group): 49 carried germline mutations and 3 carried somatic variants. Thirty-two (40%) had pathogenic mutations in 1 of 9 genes known to be involved in primary immunodeficiencies (TNFRSF6, CTLA4, STAT3, PIK3CD, CBL, ADAR1, LRBA, RAG1, and KRAS), whereas 20 patients (25%) carried probable pathogenic variants in 16 genes that had not previously been reported in the context of autoimmune disease. Lastly, no genetic abnormalities were found in the remaining 28 patients (35%, the M- group). The M+ group displayed more severe disease than the M- group, with a greater frequency of additional immunopathologic manifestations and a greater median number of lines of treatment. Six patients (all from the M+ group) died during the study. In conclusion, pES was potentially genetically determined in at least 65% of cases. Systematic, wide-ranging genetic screening should be offered in pES; the genetic findings have prognostic significance and may guide the choice of a targeted treatment.

2.
J Crohns Colitis ; 2018 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-29788237

RESUMO

Background and Aims: An expanding number of monogenic defects have been identified as causative of severe forms of very early-onset inflammatory bowel diseases (VEO-IBD). The present study aimed at defining how next-generation sequencing (NGS) methods can be used to improve identification of known molecular diagnosis and adapt treatment. Methods: 207 children were recruited in 45 Paediatric centres through an international collaborative network (ESPGHAN GENIUS working group) with a clinical presentation of severe VEO-IBD (n=185) or an anamnesis suggestive of a monogenic disorder (n=22). Patients were divided at inclusion into three phenotypic subsets: predominantly small bowel inflammation, colitis with perianal lesions, and colitis only. Methods to obtain molecular diagnosis included functional tests followed by specific Sanger sequencing, custom-made targeted NGS, and in selected cases whole exome sequencing (WES) of parents-child trios. Genetic findings were validated clinically and/or functionally. Results: Molecular diagnosis was achieved in 66/207 children (32%): 61% with small bowel inflammation, 39% with colitis and perianal lesions and 18% with colitis only. Targeted NGS pinpointed gene mutations causative of atypical presentations and identified large exonic copy number variations previously missed by WES. Conclusions: Our results lead us to propose an optimised diagnostic strategy to identify known monogenic causes of severe IBD.

3.
Front Immunol ; 9: 718, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29686686

RESUMO

Objective: Autoimmune lymphoproliferative syndrome (ALPS) with FAS mutation (ALPS-FAS) is a nonmalignant, noninfectious, lymphoproliferative disease with autoimmunity. Given the central role of natural regulatory T cells (nTregs) in the control of lymphoproliferation and autoimmunity, we assessed nTreg-suppressive function in 16 patients with ALPS-FAS. Results: The proportion of CD25highCD127low Tregs was lower in ALPS-FAS patients than in healthy controls. This subset was correlated with a reduced CD25 expression in CD3+CD4+ T cells from ALPS patients and thus an abnormally low proportion of CD25highFOXP3+ Helios+ T cells. The ALPS patients also displayed a high proportion of naïve Treg (FOXP3lowCD45RA+) and an unusual subpopulation (CD4+CD127lowCD15s+CD45RA+). Despite this abnormal phenotype, the CD25highCD127low Tregs' suppressive function was unaffected. Furthermore, conventional T cells from FAS-mutated patients showed normal levels of sensitivity to Treg suppression. Conclusion: An abnormal Treg phenotype is observed in circulating lymphocytes of ALPS patients. However, these Tregs displayed a normal suppressive function on T effector proliferation in vitro. This is suggesting that lymphoproliferation observed in ALPS patients does not result from Tregs functional defect or T effector cells insensitivity to Tregs suppression.

4.
J Allergy Clin Immunol ; 131(4): 1146-56, 1156.e1-5, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23403044

RESUMO

BACKGROUND: The generation of high-affinity antibodies requires the presence of a population of CD4+ T cells (follicular TH [TFH] cells) in the lymph node follicles. These cells differ from TH1, TH2, and TH17 effector cells in that they strongly express activation markers and the chemokine receptor CXCR5 and secrete large amounts of IL-21 and CXCL13. Small numbers of nonactivated CD4+CD45RO+CXCR5+ T cells are also found in the blood. OBJECTIVE: We sought to obtain in vitro a population close to the TFH cells and to study the presence of this cell population among patients with autosomal dominant hyper-IgE syndrome carrying heterozygous signal transducer and activator of transcription 3 (STAT3) mutations that impair the IL-21 signaling required for B-cell differentiation. METHODS: CD4+CD45RO+CXCR5+ T cells were isolated from blood and activated by CD3/T-cell receptor. RESULTS: We found that CD4+CD45RO+CXCR5+ activated T cells corresponding to circulating bona fide memory TFH cells and that STAT3-deficient patients have abnormally low numbers of "TFH-like" blood T cells. However, STAT3-deficient TFH cells have much the same phenotypic and functional characteristics as TFH cells from healthy control subjects. The ability of STAT3-deficient TFH cells to produce IL-21 on CD28/T-cell receptor activation and to proliferate did not differ from that observed for control TFH cells in vitro. Although the STAT3-deficient TFH cells were also able to help control B cells to produce IgG and IgA, induction of IgG production by naive B cells was impaired. CONCLUSION: Heterozygous mutations in STAT3 lead to reduced numbers of circulating TFH-like cells, a finding that might account (at least in part) for the observed defect in antibody production.


Assuntos
Síndrome de Job/imunologia , Antígenos Comuns de Leucócito/imunologia , Receptores CXCR5/imunologia , Fator de Transcrição STAT3/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular , Criança , Feminino , Expressão Gênica , Heterozigoto , Humanos , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Interleucinas/biossíntese , Interleucinas/imunologia , Síndrome de Job/genética , Síndrome de Job/metabolismo , Síndrome de Job/patologia , Antígenos Comuns de Leucócito/genética , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mutação , Receptores CXCR5/genética , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia
5.
J Immunol ; 188(4): 2023-9, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22250089

RESUMO

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.


Assuntos
Proteínas de Ligação a DNA/deficiência , Switching de Imunoglobulina , Síndromes de Imunodeficiência/genética , Adolescente , Linfócitos B , Sequência de Bases , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Histonas/genética , Humanos , Deficiência de IgG/genética , Imunoglobulina G/sangue , Região Variável de Imunoglobulina/genética , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Hipermutação Somática de Imunoglobulina , Adulto Jovem
6.
Eur J Immunol ; 39(7): 1966-76, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19548248

RESUMO

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8(+) T cells and non-functional CD4(+) T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G-to-A substitution in a non-coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4(+) helper T-cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T-cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.


Assuntos
Síndromes de Imunodeficiência/genética , Mutação Puntual , Proteína-Tirosina Quinase ZAP-70/genética , Sequência de Aminoácidos , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cálcio/metabolismo , Criança , Análise Mutacional de DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Contagem de Linfócitos , Masculino , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Proteína-Tirosina Quinase ZAP-70/metabolismo
7.
Biochem J ; 402(3): 471-81, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17123354

RESUMO

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.


Assuntos
Antígenos CD4/metabolismo , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M3)/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Anticorpos/imunologia , Antígenos CD4/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Proteína Fosfatase 2 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo
8.
Cell Immunol ; 244(1): 33-42, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17408603

RESUMO

We previously showed that CD4 binding induced a down-regulation of LFA-1-dependent-antigen-independent adhesion of T and B lymphocytes in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. We now show in A201-CD4 (+) T cell lines, that anti-CD4 Ab increases activation of phosphoinositide-dependent-protein-kinase 1 (PDK1) or PKC zeta, two main effectors down-stream from PI3K. CD4 binding also increases interactions between PI3K and activated PKCzeta and PDK1. Both events are dependent on CD4/p56Lck association, since they are not detected when p56Lck is unable to bind a truncated form of CD4 in transfected T cell lines. We also show using antisense oligonucleotides that both kinases are necessary for down-regulating LFA-1-dependent adhesion induced by CD4 signalling. We also suggest a role of PDK1 in the recruitment of the phosphatase SHP-2 in a multiprotein complex induced by anti-CD4 Ab. This study thus provides further insights into the mechanism underlying the CD4 triggered regulation of LFA-1-mediated adhesion.


Assuntos
Antígenos CD4/metabolismo , Adesão Celular/fisiologia , Ativação Enzimática/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular , Regulação para Baixo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo
9.
Eur J Immunol ; 34(8): 2168-78, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15259014

RESUMO

We have previously shown that binding of anti-CD4 antibody inhibit LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck) and a PI3-kinase-dependent manner. In this work, we investigated with two different T cell lines (Jurkat and A201) whether CD4 binding could alter interactions of the proteins putatively involved in this adhesion regulatory pathway. Anti-CD4 binding was shown to induce a transient association between PI3-kinase and LFA-1, which took place in different regions of the plasma membrane. It was detected in detergent soluble membrane but also in detergent insoluble membrane consisting in raft microdomains, composed of GM1 and/or GM3 gangliosides. These results show that anti-CD4 Ab could modify the interaction between LFA-1 and signaling molecules, such as PI3-kinase and induce, in part, their recruitment in raft domains. By using specific inhibitors, raft integrity and CD4 association with GM3 were found necessary for observing the CD4-dependent inhibition of LFA-1-mediated adhesion. These results strongly suggest that these molecular rearrangements in the membrane are necessary to induce down-regulation of LFA-1-mediated adhesion.


Assuntos
Linfócitos B/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T/metabolismo , Anticorpos/imunologia , Linfócitos B/imunologia , Antígenos CD4/imunologia , Adesão Celular/fisiologia , Regulação para Baixo , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/imunologia , Microdomínios da Membrana/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Linfócitos T/imunologia
10.
J Biol Chem ; 277(2): 1276-83, 2002 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-11694542

RESUMO

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.


Assuntos
Antígenos CD4/metabolismo , Adesão Celular/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Microscopia Confocal , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Proteico/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Linfócitos T/metabolismo
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