Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders causing immune dysfunction that manifest with increased susceptibility to infection. Some PID patients may also have autoimmune and autoinflammatory manifestations. In many cases, PIDs are monogenic disorders that follow Mendelian inheritance and mutations in more than 250 genes have been shown to cause PIDs. However, in the majority of PID patients the causative mutations remain unknown. Here I report a study of a patient from a consanguineous family who presented in infancy with colitis, autoimmune hepatitis, autoimmune anemia and thrombocytopenia. The patient also suffered recurrent respiratory infections leading to bronchiectasis and had several episodes of severe varicella zoster virus (VZV) infections, including pneumonia and meningitis. Immunologically, the patient had increased IgM and IgG levels, absent IgA, low specific antibodies and multiple auto-antibodies, including anti-Interferon- antibodies. Whole blood stimulation assays identified an increased production of the pro-inflammatory cytokine IL-6. Throughout his life the patient received immunosuppressive therapy. Whole exome sequencing of the patient discovered a homozygous frameshift mutation in the ZC3H12A gene that encodes the Regnase-1 protein also known as MCPIP1. Regnase-1 is a regulatory RNase that directly degrades mRNAs of several pro-inflammatory genes, e.g. mRNA of cytokine IL-6, thus curbing the immune activation. The presentation of the patient resembled the phenotype of the Regnase-1-knockout mice that developed spontaneous systemic inflammation, disorganisation of lymphoid organs, severe anaemia and hyperimmunoglobulinemia, with the increased production of IL-6. I studied expression of the mutant Regnase-1 protein using commercial antibodies; also a new custom-made antibody that detects the truncated mutant Regnase-1 protein was developed. Analysis of the patient-derived cells demonstrated absence of the full-length Regnase-1 protein. Cloning and forced expression of the truncated mutant protein showed that it is mislocalized inside the cells and is functionally impaired. Studies of the iPSC-derived macrophages, EBV-transformed B cells and primary fibroblasts of the patient demonstrated increased levels of the IL-6 mRNA in the resting cells. They also showed impaired regulation of the truncated mutant Regnase-1 protein and IL-6 mRNA levels after cell stimulation. Mutations in Regnase-1 have never been associated with human diseases previously. Therefore, this study describes a novel PID caused by the Regnase-1 deficiency.

Ectodermal dysplasia with immunodeficiency (ED-ID) is a rare primary immunodeficiency syndrome characterized by defects in ectodermal tissues (skin, hair and sweat glands), recurrent infections, impaired response to Toll-like receptor ligands, hypogammaglobulinemia and deficient antibody production. It is caused by defective \(NF\kappa B\) signaling. The most common form of ED-ID is X-linked. It is caused by hypomorphic mutations in the \(NF\kappa B\) essential modifier gene NEMO, which is an important regulatory component in the \(NF\kappa B\) signaling pathway. We report the first case of ED-ID caused by insufficient expression of a NEMO protein of normal sequence, due to a mutation in the 5’ untranslated region of the NEMO gene. Autosomal dominant ED-ID, a rare form of ED-ID, has been reported to be caused by a heterozygous S32I mutation in the \(I \kappa B \alpha\). This mutation prevents IκBα phosphorylation and inhibits its degradation. The mutant sequesters \(NF\kappa B\) in the cytoplasm and acts as a dominant negative. We report the first ED-ID patient with a heterozygous mutation (W11X) that causes N-terminal truncation of \(I \kappa B \alpha\) and results in functional haploinsufficiency. We have constructed a knock-in mouse model of ED-ID caused by a heterozygous S32I mutation in \(I \kappa B \alpha\). The mutant mice had ED, increased mortality, complete lack of lymph nodes and Peyer’s patches, and disorganized spleens that lacked follicles, marginal zone B cells and follicular dendritic cells. T cell proliferation and cytokine production was normal in vitro, but in vivo contact hypersensitivity was severely impaired, B cell function in vitro and specific antibody response to antigens were severely reduced. All immune defects, except those that affected B cell function, were absent in \(I \kappa B \alpha\) S32I mutant \(Rag2^{-/- }\) bone marrow chimeras, indicating that defects in non-lymphiod cells play a major role in the immunodeficiency of patients with ED-ID due to mutations in \(I \kappa B \alpha\). This has important clinical implications, as bone marrow transplant may not be able to correct immune function in such patients. The lessons learned in these chapters may be applicable to other mutations that impair \(NF\kappa B\) signaling and have important implications for the treatment of patients who carry these mutations.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Severe combined immunodeficiency (SCID) is a rare group of inherited disorders characterized by defects in both humoral and cellular immune functions. Naturally occurring SCID has been first described in humans in the 1960s and subsequently identified in horses, mice, and dogs, but never before in pigs. Affected animals are characterized by having loss of functional B and T lymphocytes, and in some cases natural killer (NK) cells, but normal numbers of monocytes, granulocytes, and megakaryocytes. As a result, affected animals fail to produce antibodies and succumb to common disease pathogens after circulating maternal antibodies decay. SCID models are extremely valuable for the understanding of molecular mechanisms of immunological processes during viral and bacterial diseases, cancer, and autoimmunity. SCID mice are widely used as the current model; however, the relevance of the murine SCID model to human and veterinary immune research is limited and there is an increasing need for a more representative model of SCID is imperative. We describe the gross, microscopic, and immunophenotypic characteristics of a line of Yorkshire pigs having naturally occurring SCID. Affected pigs lack T and B lymphocytes, but display circulating NK cells, fail to produce antibodies to viral infection, and lack cell-mediated response to tumor xenotransplants. We also describe response of SCID pigs to porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is the most devastating virus in swine industry, causing losses of billions of dollars annually. Understanding the immunopathogenesis of the disease is imperative in order to develop strategies to combat this devastating virus. PRRSV infected-SCID pigs failed to develop lesions of PRRSV infection, demonstrating the significant role of the adaptive immunity to PRRSV infection. Finally, we describe the preliminary results of the adoptive transfer of purified CD3⁺ T lymphocytes to SCID pigs from SLA-II matched wild-type littermates, with the objective of establishing a porcine model for the study of T cell immunopathogenesis with viral diseases.

Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...

Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...

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La sous-unité catalytique p110 de la phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K) est exprimée spécifiquement dans les leucocytes, et possède un rôle central dans la biologie des lymphocytes. Des mutations germinales dominantes gain-de-fonction de PI3KCD (codant p110 ) ont été identifiées récemment chez des patients présentant une immunodéficience combinée caractérisée par une présentation clinique hétérogène incluant des infections récurrentes des voies respiratoires et des lymphoproliférations associées à une virémie EBV et/ou CMV élevée, connue sous le nom de syndromes APDS. L’hétérogénéité des présentations cliniques suggère l’existence de facteurs secondaires, génétiques ou environnementaux. Au cours de ma thèse de doctorat, j’ai rapporté le cas de deux nouveaux patients présentant des anomalies génétiques affectant la voie PI3K. Le premier patient, né dans une famille consanguine, présentait une mutation gain- de-fonction de PIK3CD déjà décrite. Par séquençage de l’exome du patient, nous avons identifié une seconde mutation homozygote non-sens de SEC14L2, un régulateur connu de la voie PI3K. Nous avons analysé le phénotype et la fonctionnalité des lymphocytes du patient, et avons mis en évidence in vitro certains mécanismes de régulation de l’activation lymphocytaire par SEC14L2. Enfin, nous avons réalisé une étude de transcriptome afin d’identifier les voies de régulation dérégulées chez notre patient. Dans l’ensemble, ces résultats ont permis d’identifier le premier co-facteur génétique associé au syndrome APDS. Le second patient est également né dans une famille consanguine et présentait des infections sévères des voies respiratoires et un syndrome d’infection chronique à l’EBV (CAEBV) fatal. Par séquençage de l’exome du patient, nous avons identifié une mutation homozygote rare dans PIK3CD. Une modélisation de la structure de la protéine a montré que l’acide aminé muté se situe dans le domaine catalytique de p110 , dans une boucle phylogénétiquement conservée interagissant avec la sous-unité régulatrice p85↵ de la PI3K, et que la mutation conduisait à la perte de cette interaction. In vitro, la mutation de p110 cause la perte de l’activité PI3K, et les lymphocytes T du patient présentaient une diminution de phosphorylation d’AKT et de p70 S6K, deux cibles de la PI3K. Les lymphocytes T du patient présentaient également une production diminuée d’IFN- et de TNF↵, et un excès de prolifération et de flux calcium suite à la stimulation du TCR. Des lignées cellulaires Jurkat déficientes pour PIK3CD générées par CRISPR/Cas9 ont également présenté un défaut de phosphorylation d’ATK et un excès de prolifération et de flux calcium. L’expression de la version sauvage de p110 dans ces cellules, mais pas de la version mutée, a permis une restauration des réponses normales, prouvant l’implication de la mutation dans les phénotypes cellulaires identifiés. De manière intéressante, nous avons mis en évidence l’existence d’un équilibre entre l’activité PI3K et PLC- 1 lors de l’activation T, qui pourrait être expliqué par une compétition pour l’accès à leur substrat commun, le PIP2. Enfin, nous avons identifié chez notre patient une seconde mutation homozygote délétère dans le gène TNFRSF9. Cette mutation est également retrouvée chez la sœur saine du patient, qui présentait une réplication persistante de l’EBV dans le sang, suggérant que cette mutation pourrait agir comme facteur génétique secondaire. Dans l’ensemble, cette étude à permis l’identification de la première immunodéficience associée à des mutations perte-de-fonction de PIK3CD. / The pathway p110 catalytic subunit of phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K) is selectively expressed in leukocytes and has a central role in lymphocytes biology. Gain-of-function dominant germline mutations of PIK3CD (encoding p110 ) have been recently described in patients presenting a heterogeneous combined immunodeficiency associated with respiratory tract infections, lymphadenopathy and high EBV and/or CMV viremia. The heterogeneous clinical presentation suggests the existence of genetic or environmental modifying factors. In my thesis I report two new patients with different genetic defects causing CID. The first patient, born from a consanguineous family, presented a known gain-of- function mutation of PIK3CD. His clinical presentation was partially compatible with the already described patients. By whole exome sequencing, we identified a homozygous nonsense mutation in SEC14L2, a known regulator of PI3K pathway. We further analyzed the phenotype and functions of patient’s lymphocytes, and performed a transcriptome analysis to better characterize the implication of SEC14L2 mutation in the pathology. The second patient was born from consanguineous family and presented recurrent severe respiratory tract infections and a fatal Chronic Active EBV disease (CAEBV). By Whole Exome Sequencing, we identified a homozygous rare missense mutation in PIK3CD. 3D structure modelization showed that the mutated amino acid is located in p110 catalytic domain, in an evolutionarily conserved loop that interacts with alpha-helix of PI3K regulatory subunit p85a, which interaction is lost in mutated p110 . Phenotyping of patient’s circulating lymphocytes showed increased CD8+ T cells and reduced NK and CD4+ T cells. The mutation in PI3KCD resulted in impaired PI3K activity in vitro and in vivo. Moreover, patient’s T cells exhibited reduced activation-induced phosphorylation of AKT and p70-S6K, two indirect targets of p110 , that we restored by expressing wild type p110 . Patient’s T cells also showed a decreased induction of IFN- and TNF-↵ and an increased proliferation and calcium flux after TCR stimulation. By CRISPR CAS9 technology, we generated Jurkat T- cell lines expressing wild type or mutated PI3K. Jurkat cells expressing mutant PI3K showed decreased AKT phosphorylation and increased calcium flux and proliferation after TCR stimulation, confirming the implication of PIK3CD mutation in patient’s cells phenotype. Interestingly, we highlighted the existence of a balance between PI3K and PLC- 1 activity during T cell activation, that may be due to a competition for access to their shared substrate, the PIP2. Finally, we identified in our patient a second deleterious mutation in TNFRSF9 that is shared by his healthy sister, who also presented persistent EBV replication in blood, suggesting that this additional mutation may act as a modifying genetic factor. Taken together, the results presented in this thesis identified the first loss-of-function mutation in PIK3CD causing CID.

Agamaglobulinemia ligada ao X (XLA) é uma imunodeficiência primária caracterizada por ausência ou número reduzido de células B maduras em sangue periférico, de todos os isotipos de imunoglobulina e um aumento da susceptibilidade a infecções bacterianas e enterovirais graves. XLA é causada por mutações no gene Bruton\'s tirosino quinase, que codifica um membro da proteína da família das tirosino quinases citoplasmáticas que tem papel vital na modulação de muitos processos celulares. Neste estudo foram analisados trinta e três pacientes quanto à presença de mutações de BTK, por SSCP/HA e seqüenciamento. A análise da expressão foi realizada pela técnica por PCR em tempo real. Foram encontradas mutações do tipo stop codons, substituições de aminoácido, defeitos de splicing, pequenas deleções/inserções e frameshift nestes pacientes afetando os domínios PH, SH3, SH2 e o domínio da quinase da proteína. A análise da expressão mostrou níveis baixos nos pacientes com mutação do tipo stop codon, e nas outras mutações, os níveis de expressão foram de aproximadamente 15% e se correlacionaram com os tipos de mutação. / X-linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the absence or decreased numbers of mature B cells in peripheral blood, and by a lack of all immunoglobulin isotypes, leading to an increased susceptibility to severe bacterial and enteroviral infections. XLA is caused by mutations in the gene encoding for Bruton\'s tyrosine kinase (BTK), a protein member of the Tec family of cytoplasmic tyrosine kinases and plays a vital modulation role in many cellular processes. In this study thirty-three patients were analyzed for the presence of BTK mutations by SSCP/HA and sequencing. The expression analysis was carried out by the technique of Real-Time PCR. It was found mutations of the stop codons type, amino acid substitutions, splice defects, small deletions/insertions and frameshift in these patients affecting the PH, SH3, SH2 and tyrosine kinase domains of protein. The expression levels were very low in the patients with stop codon mutations, and in the other mutations, the expression levels were about 15% and were correlated with the mutation types.

A imunodeficiência comum variável (ICV) é uma imunodeficiência primária de origem heterogênea, definida como uma diminuição de pelo menos dois isótipos de imunoglobulinas, a falta de resposta anticórpica a imunizações e a exclusão de outras causas primárias de hipogamaglobulinemia. A ausência de níveis adequados de anticorpos em pacientes com ICV resulta em infecções bacterianas recorrentes, mais freqüentes no trato respiratório e digestivo, que podem levar a seqüelas sinusais e pulmonares. Nos últimos 6 anos iniciou-se a descoberta de genes relacionados à causa de doenças com o fenótipo de ICV, como os genes de TACI, BAFF-R, CD19 e ICOS. Dentre as alterações imunológicas, podemos também relatar deficiência de células B de memória (CD19+IgM-IgD-CD27+), levando a distúrbio de comutação isotípica e redução da secreção de imunoglobulinas. Atualmente tal característica vem sendo utilizada para classificar a ICV. No decorrer do presente trabalho pudemos observar que pacientes com ICV apresentam alterações na expressão de CD27 não somente em células B, mas também em células T, além de resposta linfoproliferativa ao estímulo de PHA reduzida. O CD27 consiste em uma molécula da família TNF presente constitutivamente em células T e após ativação em células B. Sua atuação na resposta imune está relacionada com a proliferação e co-ativação de células T específicas que atuam na interação T-B, na resposta de células B dependente de T. Dessa forma deficiências na via de CD27 podem resultar em defeitos nos mecanismos de comutação isotípica e de diferenciação de células B do centro germinativo, assim como de células de memória. Essas características podem ser observadas em modelos murinos de deficiências de CD27/CD70. Nossos achados permitem que uma nova janela se abra para o estudo da ICV. A avaliação dos distúrbios associados a defeitos de sinalização de CD27/CD70 em humanos pode se tornar uma nova ferramenta para a compreensão de uma deficiência tão pouco esclarecida. Tal enfoque pode eventualmente contribuir para o desenvolvimento de novos tratamentos, atuando diretamente na molécula em questão. Além disso, sugerimos também a utilização da fenotipagem das moléculas CD27 em linfócitos B e T, além da IgM e IgD de membrana para a caracterização da ICV, mais a análise da molécula CD154 para exclusão de outras imunodeficiências / Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder of heterogeneous origin, defined by a decrease of at least two immunoglobulin isotypes, lack of antibody response to immunization and the exclusion of other causes of primary hypogammaglobulinemia. The absence of adequate levels of antibodies in patients with CVID results in recurrent bacterial infections, most frequently in the respiratory and digestive tract, which can lead to sinusal and lung sequels. Over the past six years the discovery of genes related to the phenotype of CVID began, such as the genes of TACI, BAFF-R, CD19 and ICOS. Among the immunological changes, there is impairment of memory B cells (CD19+/IgM-IgD-CD27+), leading to disturbance of isotypic switching and reduced secretion of immunogobulins. Currently this feature has been used to classify CVID. During the present study we observed that patients with CVID present changes in the expression of CD27 not only in B cells, but also in T cells, and reduced lymphoproliferative response to PHA. CD27 molecule is a member of the TNF family present constitutively in T cells, and after activation in B cells. Its importance in the immune response is related to the proliferation and co-activation of specific T cells that act in T-B interaction, in the T cell dependent B cells response. Thus disturbances in the CD27 pathway can result in defects in isotypic switch and differentiation of germinal center B cells, as well as memory cells. These characteristics can be observed in murine models of CD27/CD70 deficiency. Our findings allow a new approach for the study of CVID. The evaluation of defects in CD27/CD70 signaling in humans might become a new tool for understanding an incompletely understood disease. Such an approach may contribute to the development of new treatments, acting directly on the molecule in question. In addition, we also suggest the use of phenotyping of CD27 molecules on B and T lymphocytes, in addition to membrane IgM and IgD to characterize CVID, associated to the analysis of the molecule CD154 to exclude other immunodeficiencies

A imunodeficiência comum variável (ICV) é uma imunodeficiência primária de origem heterogênea, definida como uma diminuição de pelo menos dois isótipos de imunoglobulinas, a falta de resposta anticórpica a imunizações e a exclusão de outras causas primárias de hipogamaglobulinemia. A ausência de níveis adequados de anticorpos em pacientes com ICV resulta em infecções bacterianas recorrentes, mais freqüentes no trato respiratório e digestivo, que podem levar a seqüelas sinusais e pulmonares. Nos últimos 6 anos iniciou-se a descoberta de genes relacionados à causa de doenças com o fenótipo de ICV, como os genes de TACI, BAFF-R, CD19 e ICOS. Dentre as alterações imunológicas, podemos também relatar deficiência de células B de memória (CD19+IgM-IgD-CD27+), levando a distúrbio de comutação isotípica e redução da secreção de imunoglobulinas. Atualmente tal característica vem sendo utilizada para classificar a ICV. No decorrer do presente trabalho pudemos observar que pacientes com ICV apresentam alterações na expressão de CD27 não somente em células B, mas também em células T, além de resposta linfoproliferativa ao estímulo de PHA reduzida. O CD27 consiste em uma molécula da família TNF presente constitutivamente em células T e após ativação em células B. Sua atuação na resposta imune está relacionada com a proliferação e co-ativação de células T específicas que atuam na interação T-B, na resposta de células B dependente de T. Dessa forma deficiências na via de CD27 podem resultar em defeitos nos mecanismos de comutação isotípica e de diferenciação de células B do centro germinativo, assim como de células de memória. Essas características podem ser observadas em modelos murinos de deficiências de CD27/CD70. Nossos achados permitem que uma nova janela se abra para o estudo da ICV. A avaliação dos distúrbios associados a defeitos de sinalização de CD27/CD70 em humanos pode se tornar uma nova ferramenta para a compreensão de uma deficiência tão pouco esclarecida. Tal enfoque pode eventualmente contribuir para o desenvolvimento de novos tratamentos, atuando diretamente na molécula em questão. Além disso, sugerimos também a utilização da fenotipagem das moléculas CD27 em linfócitos B e T, além da IgM e IgD de membrana para a caracterização da ICV, mais a análise da molécula CD154 para exclusão de outras imunodeficiências / Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder of heterogeneous origin, defined by a decrease of at least two immunoglobulin isotypes, lack of antibody response to immunization and the exclusion of other causes of primary hypogammaglobulinemia. The absence of adequate levels of antibodies in patients with CVID results in recurrent bacterial infections, most frequently in the respiratory and digestive tract, which can lead to sinusal and lung sequels. Over the past six years the discovery of genes related to the phenotype of CVID began, such as the genes of TACI, BAFF-R, CD19 and ICOS. Among the immunological changes, there is impairment of memory B cells (CD19+/IgM-IgD-CD27+), leading to disturbance of isotypic switching and reduced secretion of immunogobulins. Currently this feature has been used to classify CVID. During the present study we observed that patients with CVID present changes in the expression of CD27 not only in B cells, but also in T cells, and reduced lymphoproliferative response to PHA. CD27 molecule is a member of the TNF family present constitutively in T cells, and after activation in B cells. Its importance in the immune response is related to the proliferation and co-activation of specific T cells that act in T-B interaction, in the T cell dependent B cells response. Thus disturbances in the CD27 pathway can result in defects in isotypic switch and differentiation of germinal center B cells, as well as memory cells. These characteristics can be observed in murine models of CD27/CD70 deficiency. Our findings allow a new approach for the study of CVID. The evaluation of defects in CD27/CD70 signaling in humans might become a new tool for understanding an incompletely understood disease. Such an approach may contribute to the development of new treatments, acting directly on the molecule in question. In addition, we also suggest the use of phenotyping of CD27 molecules on B and T lymphocytes, in addition to membrane IgM and IgD to characterize CVID, associated to the analysis of the molecule CD154 to exclude other immunodeficiencies