Human γδ T cells expressing the Vγ2Vδ2 T cell antigen receptor play important roles in immune responses to microbial pathogens by monitoring prenyl pyrophosphate isoprenoid metabolites. Most adult Vγ2Vδ2 cells are memory cytotoxic cells that produce interferon-γ (IFN-γ). Recently, murine γδ T cells were found to be major sources of interleukin (IL)-17A in anti-microbial and autoimmune responses. To determine if primate γδ T cells play similar roles, we characterized IL-17A and IL-22 production by Vγ2Vδ2 T cells. IL-17A-producing memory Vγ2Vδ2 T cells exist at low but significant frequencies in adult humans (1:2,762 T cells) and at even higher frequencies in adult rhesus macaques. Higher levels of Vγ2Vδ2 T cells produce IL-22 (1:1,864 T cells) although few produce both IL-17A and IL-22. Unlike adult humans where many IL-17A+ V#947;2Vδ2 T cells also produce IFN-#947; (T#947;δ1/17), the majority of adult macaques IL-17A+ Vδ2 T cells (T#947;δ17) do not produce IFN-#947;. To define the cytokine requirements for T#947;δ17 cells, we stimulated human neonatal V#947;2Vδ2 T cells with the bacterial antigen, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, and various cytokines and mAbs in vitro. We find that IL-6, IL-1β, and transforming growth factor-β (TGF-β) are required to generate T#947;δ17 cells in neonates whereas T#947;δ1/17 cells additionally required IL-23. In adults, memory T#947;δ1/17 and T#947;δ17 cells required IL-23, IL-1β, and TGF-β but not IL-6. IL-22-producing cells showed similar requirements. Both neonatal and adult IL-17A+ V#947;2Vδ2 T cells expressed elevated levels of retinoid-related orphan receptor-#947;t. Our data suggest that, like Th17 αβ T cells, V#947;2Vδ2 T cells can be polarized into T#947;δ17 and T#947;δ1/17 populations with distinct cytokine requirements for their initial polarization and later maintenance.

The peripheral naive CD4 T cell pool is homeostatically regulated through a balance of thymic production, delivery of survival signals and homeostatic proliferation. CD4 recent thymic emigrants (RTEs) have a high T cell receptor excision circle (TREC) content and express high levels of CD31. We report premature thymic involution in RRMS, initiated by reduced numbers of naive CD4 T cells and various naive CD4 T cell subsets in peripheral blood. Further, CXCR4, a receptor involved in emigration from the thymus, and CD127 and Bcl-2 (survival signals) are upregulated in various naive CD4 T cell subsets in RRMS. As a compensatory process, naive CD4 T cells undergo homeostatic proliferation. This proliferation is a form of peripheral positive selection through self-MHC/self-antigen interaction and thus can contribute to the expansion of autoreactive T cells and predispose to development of RRMS.

According to numerous scientific studies one of the most important points in the area of sustainability in business is related to dialogue with stakeholders. Based on Stakeholder Theory we try to analyze corporate sustainability and the process of preparing a report that a company in the tourism sector in accordance with the guidelines of the guide G3 - Global Reporting Initiative. With the completion of an empirical study seeks to understand the expectations of stakeholders regarding the implementation of the contents of the sustainability report. To achieve the proposed aim we use «The Expertons Method» algorithm that allows the aggregation of opinions of various experts on the subject and represents an important extension of fuzzy subsets for aggregation processes. At the end of our study, we present the results of using this algorithm, the contributions and future research. / De acuerdo con numerosos estudios científicos uno de los puntos más importantes en el ámbito de la sostenibilidad en las empresas radica en el diálogo con los grupos de interés. Tomando como base la teoría de los grupos de interés, trata­remos de analizar la sostenibilidad empresarial y el proceso de elaboración de un informe que una empresa del sector de turismo prepara de acuerdo con las directrices de la guía G3, Global Reporting Initiative (GRI). Con la realización de un estudio empírico se pretende conocer las expectativas de los grupos de interés con respecto al cumplimiento del con­tenido de la memoria de sostenibilidad. Para alcanzar el objetivo propuesto utilizaremos el «método de los expertones», algoritmo que permite la agregación de opiniones de varios expertos sobre el tema y que representa una importante extensión de los subconjuntos borrosos para procesos de agregación. Al final de nuestro estudio, presentaremos los resultados de la utilización de este algoritmo, las aportaciones y futuras líneas de investigación.

Introduction: Sickle cell anemia (SCA) is one of the most common genetic disorders in the world. However, until recently, only the direct consequences of the deoxyHbS polymerization was used to explain the pathophysiology of the disease. Currently, it is known that the clinical repercussions of FA involve complex interactions between the erythrocyte, endothelium and leukocytes: cytokines secreted by inflammatory cells are involved in SCA crisis and in the maintenance of a systemic inflammatory status, suggesting that T cells (Helpers and Cytotoxics) and NK have a fundalmental role in the clinical phenomena of SCA. This study aims to evaluate the profile of T and NK lymphocytes in patients with AF and compare them with the profile of individuals with sickle cell trait and individuals without hemoglobinopathies. Materials and methods: Peripheral blood was collected from 13 individuals; 7 with SS hemoglobinopathy (SS group), 5 with sickle cell trait (AS group) and 5 normal (AA group). The lymphocytes isolation for analysis was performed using Ficoll-Hypaque solution. Immunophenotyping for the determination of lymphocyte subtypes was performed by flow cytometry, using eight-color cytometer and eight BD Biosciences antibodies. Data were analyzed using Flowjo software and tabulated in SPSS IBM 22.0. The results referring to the numerical variables were expressed through measures of central tendency. Results: A lower frequency of T lymphocytes and a greater frequency of NK cells were observed in sickle cell patients.The variation of TCD4 + found between the SCA and the AA group was significant (p = 0.04). Lower frequency tendency in patients of the SS group remained for B lymphocytes. A higher frequency of NK cells was observed in patients with sickle cell anemia (mean: group AA = 15.63%, group AS = 14.82% and group SS = 23.34%) with statistically significant variation. Conclusion: SCA patients present a lower frequency of CD4 + CD8 + T cells and TNK cells when compared with HbAS individuals and individuals without hemoglobinopathies. An increasing and progressive frequency of NK cells is observed between groups AA, AS and SS. We believe that further studies are needed to understand the role of these cells in the genesis of systemic inflammation of SCA. This understanding may contribute to the development of new therapeutic strategies. / INTRODUÇÃO: A anemia falciforme (AF) é uma das desordens genéticas mais comuns do mundo. Entretanto, até pouco tempo atrás, apenas as consequências diretas da polimerização da desoxiHbS explicava a fisiopatogenia da doença. Atualmente, sabe-se que as repercussões clíncas da AF envolvem interações complexas entre o eritrócito, endotélio e leucócitos: citocinas secretadas por células inflamatórias estão envolvidas nas crises e na manutenção de um status inflamatório sistêmico, o que sugere que as células T (auxiliares e citotóxicas) e NK têm papel fundalmental nos fenômenos clínicos da AF. Este estudo visa avaliar o perfil de linfócitos T e NK em portadores de AF e comparar com o perfil de indivíduos com traço falciforme e indivíduos sem hemoglobinopatias. MATERIAIS E MÉTODOS: Foi coletado sangue periférico de 17 indivíduos; 7 com hemoglobinopatia SS (grupo SS), 5 com traço falciforme (grupo AS) e 5 normais (grupo AA). Todos confirmados por eletroforese de hemoglobina. Amostras de pacientes hemotransfundidos 30 dias antes ou em uso de antiinflamatórios 2 dias antes da coleta foram excluídas. O isolamento dos linfócitos para análise foi realizado utilizando solução de Ficoll-Hypaque. A imunofenotipagem para determinação dos subtipos linfocitários foi realizada por citometria de fluxo, utilizando citômetro de oito cores e oito anticorpos da BD Biosciences. Os dados foram analisados com auxílio do software Flowjo e tabulados no SPSS IBM 22.0. Os resultados referentes às variáveis numéricas foram expressos através de medidas de tendência central: média e valores mínimos e máximos. RESULTADOS: Globalmente foi observada menor frequência de linfócitos T e maior frequência de células NK nos pacientes falcêmicos, com média de 31,2% de TCD4+ contra 43,47% do grupo AS e 45,37% do grupo AA. Para os linfócitos TCD8+, o grupo SS obteve média de 12,64% contra 17,1% do AS e 16,42% do AA. A variação de TCD4+ encontrada entre os pacientes AF e o grupo AA foi significativa (p=0,04). Tendência de menor frequência em pacientes do grupo SS se manteve para os linfócitos B. Foi observada maior frequência de células NK em pacientes portadores de anemia falciforme (média do grupo AA=8,83%; AS=11,2% e grupo SS=19,6%), respectivamente, com diferença significativa entre o grupo AA e o grupo SS (p=0,040). CONCLUSÃO: Portadores de AF apresentam tendência de menor frequência de linfócitos T, com diferença significativamente menor comparado ao portador de traço e o paciente portador de AF, para linfócitos T CD4+ verificou-se redução significativa deste subtipo celular entre grupo SS e AA. Neste estudo também foi evidenciado uma frequência crescente e progressiva de células NK entre os grupos AA, AS e SS. Acreditamos que mais estudos são necessários visando compreender o papel destas células na gênese da inflamação sistêmica da AF. Este entendimento possivelmente contribuirá para o desenvolvimento de novas estratégias terapêuticas. / Aracaju, SE

abstract: Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ. To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity. To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

No description available.

The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful. / Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją.

Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją. / The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful.

Identification of recurrent mutations through next-generation sequencing (NGS) has given us a deeper understanding of the molecular mechanisms involved in chronic lymphocytic leukemia (CLL) development and progression and provided novel means for risk assessment in this clinically heterogeneous disease. In paper I, we screened a population-based cohort of CLL patients (n=364) for TP53, NOTCH1, SF3B1, BIRC3 and MYD88 mutations using Sanger sequencing, and confirmed the negative prognostic impact of TP53, SF3B1 or NOTCH1 aberrations, though at lower frequencies compared to previous studies. In paper II, we assessed the feasibility of targeted NGS using a gene panel including 9 CLL-related genes in a large patient cohort (n=188). We could validate 93% (144/155) of mutations with Sanger sequencing; the remaining were at the detection limit of the latter technique, and technical replication showed a high concordance (77/82 mutations, 94%). In paper III, we performed a longitudinal study of CLL patients (n=41) relapsing after fludarabine, cyclophosphamide and rituximab (FCR) therapy using whole-exome sequencing. In addition to known poor-prognostic mutations (NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE), we detected mutations in a ribosomal gene, RPS15, in almost 20% of cases (8/41). In extended patient series, RPS15-mutant cases had a poor survival similar to patients with NOTCH1, SF3B1, or 11q aberrations. In vitro studies revealed that RPS15mut cases displayed reduced p53 stabilization compared to cases wildtype for RPS15. In paper IV, we performed RNA-sequencing in CLL patients (n=50) assigned to 3 clinically and biologically distinct subsets carrying stereotyped B-cell receptors (i.e. subsets #1, #2 and #4) and revealed unique gene expression profiles for each subset. Analysis of SF3B1-mutated versus wildtype subset #2 patients revealed a large number of splice variants (n=187) in genes involved in chromatin remodeling and ribosome biogenesis. Taken together, this thesis confirms the prognostic impact of recurrent mutations and provides data supporting implementation of targeted NGS in clinical routine practice. Moreover, we provide evidence for the involvement of novel players, such as RPS15, in disease progression and present transcriptome data highlighting the potential of global approaches for the identification of molecular mechanisms contributing to CLL development within prognostically relevant subgroups.