Epstein-Barr Virus (EBV) is a ubiquitous human virus linked to several diseases, including cancers. CD8 T cells are important for controlling EBV replication. Generation and maintenance of virus-specific CD8 T cells is dependent on specific interaction between MHC-peptide complexes on the infected cell and the CD8 T cell receptor (TCR). Several lines of evidence suggest that the TCR repertoire is an essential component of the CD8 T-cell immune response. The current work focuses on delineating the features of the TCR repertoire that drive the selection of EBV-specific CD8 T cells into the memory phase. We used bulk and single-cell TCRαβ sequencing to analyze the TCR repertoire of human CD8 T cells specific for two immunodominant HLA-A02:01-restricted EBV-derived epitopes: BRLF1109-117 (YVLDHLIVV) and BMLF1280-288 (GLCTLVAML) during the acute and memory phases of primary EBV infection in humans. We showed that persistent EBV-specific clonotypes accounted for only 9% of unique clonotypes but were highly expanded in acute EBV infection and more commonly expressed identifiable features than non-persistent clonotypes. The other 91% of highly diverse unique clonotypes disappeared and were replaced in convalescence by equally diverse “de-novo” clonotypes. We provide evidence suggesting that recognition of BRLF1109-117may be driven by the TCRα. We identified a highly dominant and degenerate BRLF1109-117-specific TCRα sequence, AV8.1-CAVKDTDKLIF-AJ34, that was shared by all donors studied and identified conserved residues within this sequence that were important for antigen recognition. These findings are relevant to current efforts to develop or optimize the efficacy of T cell based therapies or vaccines.

Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function. CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity. Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways. Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation. Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses. Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.

No description available.

[ES] El objetivo principal de este trabajo fue conocer las alteraciones inmunológicas asociadas a la inflamación periférica que desencadenan deterioro cognitivo en los pacientes cirróticos encefalopatía hepática mínima (EHM). Estos cambios pueden ser monitorizados como cascadas de señalización a lo largo de los tipos celulares del sistema inmune. Como estudio preliminar, se analizaron los cambios en la expresión génica (transcriptómica), los metabolitos de plasma (metabolómica) y un panel de citoquinas extracelulares en muestras de sangre de pacientes cirróticos con y sin EHM. Los resultados del análisis transcriptómico apoyaron la hipótesis de alternancias en las poblaciones celulares de linfocitos Th1/Th2 y Th17 como principales impulsores de la EHM. El análisis clúster de las moléculas del suero dio como resultado 6 grupos de compuestos químicamente similares. También se ha realizado un análisis de integración multiómica para detectar las relaciones entre los componentes intra y extracelulares que podrían contribuir a la inducción del deterioro cognitivo. Los resultados de este análisis integrativo sugirieron una relación entre las citocinas CCL20, CX3CL1, CXCL13, IL-15, IL-22 e IL-6 con la alteración de la quimiotaxis, así como un vínculo entre los fosfolípidos insaturados de cadena larga y el aumento del transporte de ácidos grasos y la producción de prostaglandinas. Estudios previos sugieren que un cambio en la inflamación periférica, orquestado principalmente por las células T CD4+, es un factor crítico que desencadena el deterioro cognitivo en EHM. La segunda parte de la tesis se centró en la comprensión de las rutas genéticas y los mecanismos por los que las alteraciones en los linfocitos CD4+ pueden contribuir a la inflamación periférica en EHM. Se analizaron los niveles de expresión de genes, factores de transcripción y miARNs en este subtipo de linfocitos mediante secuenciación de alto rendimiento (RNA-seq y miRNA-seq). El análisis individual de cada grupo de datos mostró diferencias de expresión de ARNm y miARN, así como las vías biológicas alteradas en los linfocitos CD4+ comparando pacientes cirróticos con y sin EHM. Encontramos alteraciones en 167 ARNm y 20 rutas biológicas en los pacientes con EHM, incluyendo los receptores tipo Toll, la señalización de la IL-17 y las vías del metabolismo de histidina y triptófano. Trece miRNAs y 7 factores de transcripción presentaron alteraciones en los pacientes con EHM. Utilizando bases de datos para determinar sus genes diana, encontramos una modulación por el aumento de miR-494-39, miR-656-3p y miR-130b-3p de la expresión de TNFAIP3 (proteína A20) y ZFP36 (proteína TTP) aumentaría los niveles de citoquinas proinflamatorias como IL-17 y TNF¿. Finalmente, estudiamos el repertorio de receptores de células T (TCR) de pacientes control, y de pacientes cirróticos con y sin EHM, a partir del conjunto de datos de RNA-seq procedentes de células T CD4+ aisladas previamente. Dado que los experimentos de RNA-seq contienen genes del TCR en una fracción de los datos, se puede analizar el repertorio sin necesidad de generar datos adicionales. Tras el alineamiento de las lecturas con la base de datos de los genes VDJ realizada por la herramienta MiXCR, recuperamos entre 498-1114 cadenas TCR beta distintas por paciente. Los resultados mostraron un bajo número de clones públicos (convergencia clonal), una alta diversidad (expansión clonal) y una elevada similitud en la arquitectura de la secuencia dentro de los repertorios, independientemente del estado inmunitario de los 3 grupos de pacientes. Además, detectamos una sobrerrepresentación significativa de los TCRs relacionados con la enfermedad celíaca y la enfermedad inflamatoria intestinal en los repertorios de los pacientes con EHM. / [CA] L'objectiu principal d'aquest treball va ser conèixer les alteracions immunològiques associades a la inflamació perifèrica que desencadenen deteriorament cognitiu en els pacients cirròtics amb encefalopatia hepàtica mínima (EHM). Aquests canvis poden ser monitoritzats com cascades de senyalització al llarg dels tipus cel·lulars del sistema immune. Com a estudi preliminar, es van analitzar els canvis en l'expressió gènica (transcriptòmica), els metabòlits de plasma (metabolòmica) i un conjunt de citocines extracel·lulars en mostres de sang de pacients cirròtics amb i sense EHM. Els resultats de l'anàlisi transcriptòmica van recolzar la hipòtesi d'alternances en les poblacions cel·lulars de limfòcits Th1/Th2 i Th17 com a principals impulsors de la EHM. L'anàlisi clúster de les molècules del sèrum va donar com a resultat 6 grups de compostos químicament similars. També s'ha realitzat una anàlisi d'integració multiòmica per detectar les relacions entre els components intra i extracel·lulars que podrien contribuir a la inducció del deteriorament cognitiu. Els resultats d'aquesta anàlisi d'integració van suggerir una relació entre les citocines CCL20, CX3CL1, CXCL13, IL-15, IL-22 i IL-6 amb l'alteració de la quimiotaxis, així com un vincle entre els fosfolípids insaturats de cadena llarga i l'augment del transport d'àcids grassos i la producció de prostaglandines. Estudis previs suggereixen que un canvi en la inflamació perifèrica, orquestrat principalment per les cèl·lules T CD4+, és un factor crític que desencadena el deteriorament cognitiu en EHM. La segona part de la tesi es va centrar en la comprensió de les rutes genètiques i els mecanismes pels quals les alteracions en els limfòcits CD4+ poden contribuir a la inflamació perifèrica en EHM. Es van analitzar els nivells d'expressió de gens, factors de transcripció i miARNs en aquest subtipus de limfòcits mitjançant seqüenciació d'alt rendiment (RNA-seq i miRNA-seq). L'anàlisi individual de cada grup de dades va mostrar les diferències d'expressió d'ARNm i miARN, així com les vies biològiques alterades en els limfòcits CD4+ comparant pacients cirròtics amb i sense EHM. Trobàrem alteracions en 167 ARNm i 20 rutes biològiques en els pacients amb EHM, incloent els receptors tipus Toll, la senyalització de la IL-17 i les vies del metabolisme de la histidina i el triptòfan. Tretze miRNAs i 7 factors de transcripció van presentar alteracions en els pacients amb EHM. Després utilitzàrem bases de dades per determinar els seus gens diana, els quals van resultar ser codificants per proteïnes clau implicades en el canvi immunològic que desencadena la EHM. Per exemple, la modulació per l'augment de miR-494-39, miR-656-3p i miR-130b-3p de l'expressió de TNFAIP3 (proteïna A20) i ZFP36 (proteïna TTP) augmentaria els nivells de citocines proinflamatòries com IL-17 i TNF¿. L'última part de la tesi comprèn un cas pràctic en el qual s'estudia el repertori de receptors de cèl·lules T (TCR) de pacients control, i de pacients cirròtics amb i sense EHM, a partir del conjunt de dades de RNA-seq procedents de cèl·lules T CD4+ aïllades prèviament. Atès que els experiments de RNA-seq contenen gens del TCR en una fracció de les dades, es crea una oportunitat per a l'anàlisi del repertori sense necessitat de generar dades addicionals, el qual redueix la quantitat i els costos de les mostres. Després de l'alineament de les lectures amb la base de dades dels gens VDJ realitzada per l'eina MiXCR, recuperàrem entre 498-1114 cadenes TCR beta diferents per pacient. Els resultats van mostrar un baix nombre de clons públics (convergència clonal), una alta diversitat (expansió clonal) i una elevada similitud en l'arquitectura de la seqüència dins dels repertoris, independentment de l'estat immunitari dels 3 grups de pacients. A més, detectàrem una sobrerepresentació significativa dels TCRs relacionats amb la malaltia celíaca i la malaltia inflamatòria intestinal en els repertoris dels pacients amb EHM. / [EN] The main objective of this work was to understand the immunological alterations associated with the peripheral inflammation that trigger minimal hepatic encephalopathy (MHE) in patients with cirrhosis. These changes can be monitored through the signaling cascades of different immune system cell types. In this work, in a preliminary study, changes in gene expression (transcriptomics), plasma metabolites (metabolomics), and a panel of extracellular cytokines were analyzed in blood samples from patients with cirrhosis with and without MHE. Transcriptomics analysis supported the hypothesis that alternations in the Th1/Th2 and Th17 lymphocyte cell populations are the major drivers of MHE. Cluster analysis of serum molecules highlighted 6 groups of chemically similar compounds. We also developed a multi-omic integration analysis pipeline to detect covariation between intra- and extracellular components that could contribute to the induction of cognitive impairment. Results of this integrative analysis suggested a relationship between cytokines CCL20, CX3CL1, CXCL13, IL-15, IL-22, and IL-6 and altered chemotaxis, as well as a link between long-chain unsaturated phospholipids and increased fatty acid transport and prostaglandin production. A shift in peripheral inflammation in patients with MHE, mainly orchestrated by CD4+ T cells, had been proposed in previous studies as a critical factor that triggers cognitive impairment. The second part of this thesis focused on understanding the pathways and mechanisms by which alterations in CD4+ lymphocytes may contribute to peripheral inflammation in MHE. Thus, the expression levels of genes, transcription factors, and miRNAs were analyzed in this lymphocyte subtype by high throughput sequencing (RNA-seq and miRNA-seq). Separate analysis of each dataset showed mRNA and miRNA expression differences and altered biological pathways in CD4+ lymphocytes when compared to patients with cirrhosis with and without MHE. We found alterations in 167 mRNAs and 20 pathways in patients with MHE, including toll-like receptors, IL-17 signaling, histidine, and tryptophan metabolism pathways. In addition, 13 miRNAs and 7 transcription factors presented alterations in patients with MHE. We used public databases to determine the target genes of these regulatory molecules and found that increased miR-494-39, miR-656-3p, and miR-130b-3p expression may modulate TNFAIP3 (A20) and ZFP36 (TTP) to increase levels of pro-inflammatory cytokines such as IL-17 and TNF¿. Finally, we present a case study of the T-cell receptor (TCR) repertoire profiles of control patients and patients with cirrhosis with and without MHE obtained from the bulk RNA-seq dataset previously generated from isolated CD4+ T cells. Given that RNA-seq experiments contain the TCR genes in a fraction of the data, the receptor repertoire analysis without the need to generate additional data is possible. After read alignment to the VDJ genes was performed with the MiXCR tool, we successfully recovered 498-1,114 distinct TCR beta chains per patient. Results showed fewer public clones (clonal convergence), higher diversity (clonal expansion), and elevated sequence architecture similarity within repertoires, independently of the immune status of the 3 groups of patients. Additionally, we detected significant overrepresentation of celiac disease and inflammatory bowel disease related TCRs in MHE patient repertoires. To the best of our knowledge, this is one of the few studies to have shown a step-by-step pipeline for the analysis of immune repertoires using whole transcriptome RNA-seq reads as source data. In conclusion, our work identified potentially relevant molecular mechanisms of the changes in the immune system associated with the onset of MHE in patients with cirrhosis. Future work with a large sample cohort will be required to validate these results in terms of biomarker determination and the development of new, more effective treatments for MHE. / Rubio Martínez-Abarca, MT. (2022). Multi-omic data integration study of immune system alterations in the development of minimal hepatic encephalopathy in patients with liver cirrhosis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185116 / TESIS

No description available.

Die adoptive T-Zell-Therapie (ATT) ist eine sich schnell entwickelnde Immuntherapie, die bei Patienten, die an verschiedenen Krebsarten leiden, eine positive klinische Reaktion anzeigt. Eine Variante der ATT ist eine T-Zellen-Rezeptor (TCR)-Gentherapie, bei der Patienten-T-Zellen mit krebsspezifischen TCRs ausgestattet werden. Die Herstellung der TCR-erzeugten T-Zellen ist schnell und robust und erfordert eine geringe Anfangsmenge an Patienten-T-Zellen. Der Mangel an verfügbaren krebsspezifischen TCRs, die auf verschiedene Moleküle des menschlichen Leukozytenantigens (HLA) der Klasse I beschränkt sind, schließt jedoch viele Patienten von der Krebsbehandlung aus. Die Generierung einer krebsspezifischen TCR-Bibliothek, die aus gut definierten TCRs besteht, könnte die Zahl der Patienten, die an klinischen Studien teilnehmen, erhöhen. Das Ziel dieser Doktorarbeit war es, Epstein-Barr-Virus (EBV)-spezifische TCRs zu identifizieren und zu isolieren, um eine EBV-spezifische TCR-Bibliothek als ein nützliches Werkzeug der TCR-Gentherapie bei der Behandlung von EBV-bedingten Krebserkrankungen zu generieren. Insgesamt wurden neun EBV-spezifische TCRs von EBV-positiven Spendern isoliert und charakterisiert, die verschiedene pHLA-Komplexe von EBV-Latentmembranproteinen (LMP1, LMP2A) und Kernprotein (EBNA3C) erkannten. Zusätzlich wurde ein neuartiges immunogenes LMP1-Epitop (QQNWWTLLV) entdeckt, das auf HLA-C*15:02 beschränkt ist. Definierte EBV-spezifische TCRs können als Grundlage für die EBV-spezifische TCR-Bibliothek verwendet werden, die eine wertvolle Quelle von TCRs für die schnelle Generierung von EBV-spezifischen T-Zellen zur Behandlung von Krebspatienten mit verschiedenen HLA-Typen darstellt. / Adoptive T cell therapy (ATT) is a fast developing immunotherapy indicating positive clinical response in patients suffering from different type of cancers. One type of the ATT is a T cell receptor (TCR) gene therapy, which involves endowing patient T cells with cancer-specific TCRs. Manufacturing of the TCR-engineered T cells is fast and robust, requiring small initial amount of patient T cells. However, lack of available cancer-specific TCRs restricted to various human leukocyte antigen (HLA) class I molecules eliminates many patients from cancer treatment. Generation of a cancer-specific TCR library consisting of well-defined TCRs could increase the number of patients enrolled in clinical trials. The aim of this PhD thesis was to identify and isolate Epstein-Barr virus (EBV)-specific TCRs in order to generate the EBV-specific TCR library as a useful tool of the TCR gene therapy for treatment of EBV-related malignancies. In total, nine EBV-specific TCRs of EBV-positive donors that recognized various pHLA complexes of EBV latent membrane proteins (LMP1, LMP2A) and nuclear protein (EBNA3C) were isolated and characterized. Additionally, a novel immunogenic LMP1 epitope (QQNWWTLLV) restricted to a HLA-C*15:02 was discovered. Defined EBV-specific TCRs can be used as a basis for the EBV-specific TCR library, which provides a valuable source of TCRs for rapid generation of EBV-specific T cells to treat cancer patients with different HLA types.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.

O timo é um órgão linfoide primário, localizado em região mediastinal, cuja importância funcional é a diferenciação e maturação de todas as subpopulações de linfócitos T provenientes da medula óssea assim como a seleção de células autorreativas. Sua hipoplasia ou aplasia resultam em síndromes de imunodeficiência. Embora de vital importância, o estudo clínico de sua função não é rotineiro na prática clínica, o que pode ser atribuído a sua dificuldade de avaliação em razão de sua localização, necessidade de uso de métodos de imagem não inócuos ao paciente (tomografia computadorizada (TC), PET-SCAN) e complexidade das análises em sangue periférico de subpopulações de células T por citometria de fluxo e, mais recentemente, medição de T cell receptor excision circles (TRECs), por PCR. Um possível método da avaliação do timo sem radiação ionizante ou dor ao paciente seria a elastografia de timo por ultrassom e seu uso na prática clínica poderia substituir a TC, como ocorre na avaliação de lesões hepáticas ou mamárias. Objetivo - Este estudo se propõe a 1. Implantar este método na avaliação da função tímica, 2. Estabelecer valores de referência de TRECs na faixa etária estudada, 3. Investigar se há correlação entre os dois parâmetros. Métodos - Foram incluídas sessenta e quatro crianças de 0-5 anos em acompanhamento no ambulatório de cirurgia infantil sem doença sistêmica ou infecção aguda, e que iriam coletar amostra de sangue para exames pré-operatórios. Quarenta e oito destas coletaram amostra de sangue para avaliação de TRECs, vinte e nove realizaram elastografia num mesmo momento, porém apenas 13 destas apresentaram resultado confiável. A média da idade foi de 36 ± 16meses, predomínio do grupo foi masculino (75%), nascidos a termo (72%) e a principal intervenção cirúrgica foi do tipo urológica de pequeno porte. A elastografia mostrou média de 1,21 ± 0,24m/s, sem diferença significativa quando comparada ano a ano. Observamos uma média de TRECs de 195,6 ± 120,5 cópias/µL, mostrando valores significativamente mais altos quando comparados a adolescentes hígidos da base de dados do laboratório. Os valores de TRECs observados mostram uma ampla variabilidade na faixa etária estudada, sem diferença significativa quando separados por idade ano a ano. Não se encontrou correlação significativa entre a dureza do timo analisada à elastografia e valores de TRECs em sangue periférico. Concluímos que a elastografia é um método que possibilita a avaliação das dimensões e função do timo em crianças a partir de 2 anos de idade, entretanto estudos adicionais são necessários para que se possa recomendar a larga implantação deste método com essa finalidade / The thymus is a primary lymphoid gland responsible for the maturation of T cells as well as the immunological central tolerance. It has been a neglected organ by physicians, despite its relevance in early immunity. Thymic function can be indirectly measured by Computerized Tomography imaging and PET SCAN, T cell subpopulation flow cytometry. More recently, in the beginning of this century, a direct measurement represented by TRECs (T cell receptors excision circles) was developed. Classical thymic imaging has used ionized radiation, which poses a major risk for the pediatric patient and new techniques are needed. Objectives and methods - In this work, we tested the use of elastography ultrasound for the evaluation of the thymus in a group of < 5-year- old healthy children. In parallel, we measured TRECs in peripheral blood and compared the values obtained from both methods. We have reached sixty-four children at the pediatric surgery outpatients ambulatory, scheduled for minor surgeries. A sample of blood was taken during pre operatory and then patients were sent to the imaging service for elastography. Of all, sixty-four had undertaken TRECs and seventeen, elastography. The median age was 36 ±16 months and we had 75% of boys for surgical correction of urologic minor defects. The elastography results showed a median of 1.2 ± 0.24 m/s in all ages, the same stiffness as the liver, as shown in other works. Our median TREC/µL value was 195.6 ± 120.5 copies/µL showing a trend of reduction in older ages, and with statistical significance when compared with healthy teenagers\' values from the lab database. We concluded that elastography may be a good diagnostic tool for thymus evaluation, and additional works are needed for its recommendation in clinical practice. Our TRECs values showed a large variability, as also demonstrated in previous works, and a trend of reduction over age. We could not observe any significant correlation between elastography and TRECs values

CD8+ T Zellen unterstützen die Beseitigung von Pathogenen und sind somit entscheidend bei der Bekämpfung von Infektionen. Neben der Antigendosis und dem inflammatorischen Zytokinemilieu hat auch die Stimulation durch den TZR einen entscheidenden Einfluss auf die CD8+ T Zellantwort. Das transkriptionelle Programm, die finale Größe und Dauer der klonalen Expansion und der Start der Kontraktionsphase werden durch die TZR-Signalstärke bestimmt. Schwache TZR-Stimulation führt zu einer verminderten Expansion und vermittelt eine frühzeitige Kontraktionsphase, die eine Entwicklung von Gedächtniszellen auf den Kosten der Effektorzellen favorisiert. IRF4 wird nach TZR-Ligand-Interaktion in CD8+ T-Zellen hoch reguliert. Seine Expressionskinetik ist stark von der TZR-Signalstärke der Aktivierung abhängig, übersetzt diese und sorgt für die Umsetzung in ein entsprechendes transkriptionelles und differentielles Programm. In dieser Arbeit konnte erstmals gezeigt werden, dass die IRF4-Defizienz in CD8+ T-Zellen zu einem verfrühten Abbruch der Expansion und zu einem vorzeitigen Beginn der Kontraktion führt, die durch den FAS-vermittelten Tod-induzierenden Signalweg initiiert wird. Außerdem präsentieren IRF4-defiziente CD8+ T-Zellen vermehrt Phosphatidylserine an ihrer Oberfläche und Komplementdeposition, beides begünstigt die Erkennung und Aufnahme durch Phagozyten. Diese Ergebnisse weisen zudem stark darauf hin, dass durch die fehlende Expression von IRF4 in CD8+ T Zellen, ein schwaches TZR-Signal übermittelt wird, unabhängig von der tatsächlichen Stärke und Dauer des aktivierenden Signals, dass zu einer Verkürzung der Expansionsphase führt und eine verfrühte Kontraktionsphase der Effektorzellen auslöst. Diese Arbeit erweitert schon bekanntes Wissen um IRF4 als Schlüsselregulator für die Differenzierung und Funktionalität der ag-spezifischen CD8+ T-Zellen, da es den Beginn der Kontraktionsphase diktiert mittels Aktivierung von verschiedenen Apoptose- und Phagocytose Signalwegen. / CD8+ T cells promote pathogen clearance and play a crucial role in controlling infections. Besides antigen dose and inflammatory cytokine milieu, the TCR stimulation contributes to the programming of the CD8+ T cell response. A distinct developmental program, the final magnitude and duration of clonal expansion, as well as the timing of the onset of T cell contraction, are determined by the TCR signaling strength. Weak TCR stimulation results in a diminished magnitude of expansion and accelerates the onset of contraction, as it favors the development of memory cells at the expense of effector cells. IRF4 is a transcription factor, that is upregulated in CD8+ T cells following TCR stimulation. Furthermore, its expression kinetic is highly dependent on the TCR signaling strength, which initiated activation. Therefore, it translates the strength of the activating signal and transmits it into a proper transcriptional and developmental program. This study provides unique evidence that the absence of IRF4 expression in CD8+ T cells leads to a hasted termination of clonal expansion and a premature contraction, initiated by the FAS-mediated cell death pathway. Moreover, IRF4-deficient CD8+ T cells exposed phosphatidylserine on their cell surface and showed complement deposition, both facilitating their recognition and uptake by phagocytes. The findings of this study additionally strongly indicate that IRF4 deficiency mimics weak TCR engagement and in turn transmits every TCR signal, independent of its actually affinity and duration, into a developmental program, that give rise to an early memory formation and results in a premature onset of effector CD8+ T cell contraction. This data extend previous knowledge of IRF4 being essential for the differentiation and functionality of ag-specific effector CD8+ T cells, as it furthermore dictates the onset of CD8+ T cell contraction via the activation of several death and phagocytosis inducing pathways.

Durch den Transfer der Gene des heterodimeren T-Zellrezeptors (TZR) mithilfe viraler Vektoren können T-Zellen programmiert werden, ein ausgewähltes Antigen spezifisch zu erkennen. In klinischen Studien wurden solche T-Zellen bereits mit Erfolg zur Immuntherapie von Krebs und viralen Infektionen eingesetzt. Genmodifizierte T-Zellen unterscheiden sich jedoch von normalen T-Zellen, weil sie neben den beiden zelleigenen auch die zwei übertragenen TZR-Gene exprimieren. Diese Situation erlaubt die Bildung vier verschiedener TZR-Heterodimere: der zelleigene TZR, der übertragene TZR und zwei gemischte TZR, bestehend aus je einer übertragenen und einer zelleigenen TZR-Kette. Gemischte TZR bergen das Risiko von Nebenwirkungen, weil sie durch Zufall gesundes Körpergewebe erkennen und so Autoimmunität auslösen könnten. In dieser Arbeit wurden deshalb virale Vektoren entwickelt, die gleichzeitig mit der Übertragung von neuen TZR-Genen den zelleigenen TZR durch RNA Interferenz (RNAi) unterdrücken. Mikro-RNA (miRNA), die in den Vektor MP71 eingefügt wurden, reduzierten den zelleigenen TZR in Maus-T-Zellen um mehr als 85%. Dies hatte zur Folge, dass beide Ketten des übertragenen P14-TZR in gleicher Menge auf der Zelloberfläche exprimiert wurden und die Bildung von gemischten TZR reduziert wurde. In einem Mausmodell der adoptiven T-Zelltherapie verhinderte die Unterdrückung des zelleigenen TZR die Entstehung von Autoimmunität, die andernfalls durch gemischte TZR verursacht wurde. Im Gegensatz dazu führte die Anwendung von gentechnisch optimierten P14-TZR-Genen weder zur angeglichenen Oberflächenexpression der P14-TZR Ketten noch zu weniger Autoimmunität im Mausmodell. Ein anderes Tierexperiment zeigte, dass die miRNA die Funktion der genmodifizierten T-Zellen nicht beeinträchtigte. Schließlich wurde ein viraler Vektor entwickelt und getestet, der die Expression des zelleigenen TZR in menschlichen T-Zellen effektiv unterdrückte und die Bildung von gemischten TZR reduzieren konnte. / T cells can be genetically modified using viral vectors. The transfer of genes encoding both chains of the heterodimeric T cell receptor (TCR) programs T cells to specifically react towards an antigen of choice. Such TCR gene-modified T cells were already successfully applied in clinical studies to treat cancer and viral infections. However, in contrast to nonmanipulated T cells these cells express the transferred TCR in addition to the endogenous TCR and this situation allows the assembly of four different TCR heterodimers: the endogenous TCR, the transferred TCR, and two mixed TCR dimers, composed of one endogenous and one transferred TCR chain. The formation of mixed TCR dimers represents a safety issue because they may by chance recognize self-antigens and thereby cause autoimmune side effects. To overcome this problem, an RNAi-TCR replacement vector was developed that simultaneously silences the endogenous TCR and expresses an RNAi-resistant therapeutic TCR. The expression of miRNA encoded by a retroviral MP71 vector in transduced mouse T cells reduced the surface levels of the endogenous TCR by more than 85%. The knockdown of the endogenous TCR in turn resulted in equal surface expression levels of both transferred P14 TCR chains and prevented the formation of mixed TCR dimers. Accordingly, the development of lethal mixed TCR dimer-dependent autoimmunity (TI-GVHD) in a mouse model of adoptive T cell therapy was dramatically reduced by the knockdown of the endogenous TCR. In contrast, the usage of genetically optimized TCR genes neither resulted in equal surface levels of both P14 TCR chains nor in reduced autoimmunity. A second mouse model demonstrated that the in vivo functionality of the transduced T cells was not negatively influenced by the expression of the miRNA. Finally, an RNAi-TCR replacement vector for human T cells was developed that effectively reduced the expression of the endogenous TCR and prevented the formation of mixed TCR dimers.