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...

Primary immunodeficiencies represent a heterogeneous group of hereditary immune system malfunctions with very variable causes and symptoms. Multiparametric flow cytometry has become an important tool in primary immunodeficiency diagnostics and research because it provides detailed information on the phenotype of individual immune cells and their proportions in circulation. We have developed a complex monoclonal antibody panel composed of five eight-color tubes which is designed for immunophenotyping of basic lymphocyte subsets and further analysis of B and T cell subpopulations. We have optimized and standardized the panels so they will identify any changes originating from primary immunodeficiencies and provide comparable data on the level of cooperation between more laboratories. This was achieved by cooperation of six European research facilities which are all parts of the Euroflow consortium. The panels have been validated both on peripheral blood samples from healthy donors and patients with either gentically defined primary immunodeficiency or common variable immunodeficiency. Keywords: T lymphocyte, B lymphocyte, primary immunodeficiency, flow cytometry, immunophenotyping, Euroflow, optimization, standardization

This thesis includes set of published experimental results, which were obtained at the Department of Immunology, Second Faculty of Medicine Charles University within the project focused on the basal and applied research of the Primary immunedeficiencies (PID), particularly Common variable immunodeficiency (CVID). The first, theoretical part, is divided into two sections. The first section is dedicated to the general aspects of Primary antibody deficiencies (PAD). The second section is focused on epidemiology, ethiopathogenesis, classification, clinical and therapeutical aspects of CVID. The main consideration is devoted particularly to non-infectious complications, which significantly contribute to morbidity and mortality of CVID patients. The second part consists of the set of publications describing specific mechanisms of immune system dysregulation and their clinical manifestation, which are briefly commented. The spectrum of issues resolved within the project covers following basic aspects: 1) characteristics of lung complications in CVID from the point of view of bronchial asthma, 2) characteristics of associated malignancies, 3) significance of genetic background for the specific treatment, 4) therapy of CVID focused on aspects of immunoglobulin substitution. The results of the other...

The increased frequencies of two novel B cell populations defined as IgM+ CD19+ CD27- CD21low CD38low CD24+ and IgM+ CD19+ CD27- CD21low CD38low CD24- in peripheral blood of patients with common variable immunodeficiency (CVID) compared to healthy donors were found. The aim was to search for such B cells in patients with rheumatoid arthritis (RA) and their further characterization. The production of immunoglobulin (Ig) mRNA in single B cells was analyzed using flow cytometry, single cell sorting and RT-PCR, IgVH-specific PCR, cycle sequencing and statistical analysis. The study was focused on analysis of variable regions of the heavy chains of Igs and significant differences in the usage of VH, DH and JH gene segments, mutational frequencies, distribution of silent and replacement mutations, length and composition of CDR3 regions, clonal relation and RAG gene expression in above mentioned B cell populations were found. Because of lack of the surface CD27 molecule being regarded as marker of B cells that have undergone antigen-driven germinal reactions, analyzed populations were considered as naive. However, the pattern and type of mutations suggested that these cells could represent a new type of differentiated memory/antigen- experienced B lymphocytes (in CVID less maturated) with the likely role in...