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

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e são consideradas um problema de saúde pública em vários países, principalmente os em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens adultos. A FR apresenta diversas manifestações, sendo a doença reumática cardíaca (DRC) a seqüela mais grave, caracterizada por lesões cardíacas valvares progressivas e permanentes. O tratamento, frequentemente envolve cirurgia cardíaca para a correção de lesões valvulares, o que acarreta alto custo para o Sistema Único de Saúde no Brasil e em vários países. Em trabalhos anteriores sobre os mecanismos desencadeadores das lesões reumáticas no coração, foi possível identificar o papel do linfócito T como mediador principal da autoimunidade, através da análise do receptor de células T infiltrantes de lesão cardíaca de indivíduos com DRC. Várias expansões oligoclonais com diferentes tamanhos da região que reconhece o antígeno, CDR3 foram encontradas. No presente trabalho, analisou-se a atividade tímica através da quantificação de fragmentos circulares excisados pelo rearranjo do gene do receptor do linfócito T (TREC) em linfócitos T de sangue periférico de indivíduos com FR e DRC. Também foi avaliada a presença de células T naïve e de memória através de citometria de fluxo. Os resultados do presente trabalho mostraram que a quantidade de TREC em amostras de sangue periférico do grupo de pacientes com FR/DRC foi significantemente menor quando comparada a observada em indivíduos saudáveis. Interessantemente, em ambos os grupos a quantidade de TREC apresentou correlação negativa com a idade dos indivíduos estudados. Os resultados indicaram diferenças na atividade tímica em pacientes com FR/DRC, provavelmente decorrente do processo autoimune que envolve linfócitos T / There is a wide spectrum of diseases caused by group A streptococci (GAS), that still being considered a public health problem in developing countries, with about 600 million cases per year. Infections by GAS can cause invasive diseases such as pharyngitis and pyoderma leading to serious autoimmune complications such as rheumatic fever (RF) and glomerulonephritis. RF mainly affects children and young adults, and presents different manifestations. Rheumatic heart disease (RHD) is considered the most serious complication leading to valvular lesions that are characterized by progressive and permanent heart damage, which entails high cost to the Public Health System in Brazil and worldwide. In previous works that focused on the mechanisms leading to rheumatic heart lesions, we identified the role of T lymphocytes as principal mediator of autoimmune reactions. Through the in deep analysis of infiltrating T-cell receptor repertoire of patients with RHD, we identified oligoclonal expansions with different sizes of CDR3 that is the region of antigen recognition. In the present study we analyzed the thymic activity through T cell receptor excision circles (TREC) quantification in T cells from peripheral blood of RF/RHD patients. We also evaluated naïve and memory T cells from peripheral blood by flow cytometry. Our results showed that the amount of TREC in the peripheral blood of patients was significantly lower when compared to the healthy individuals. In addition, both groups showed that the amount of TREC is negatively correlated with age. These results indicated that the thymic activity in RF/RHD patients is altered probably due to the autoimmune process that involves T lymphocytes

Die Effizienz einer Tumortherapie durch einen Transfer von ex vivo aktivierten Tumor-spezifischen zytotoxischen T-Lymphozyten wird durch zahlreiche Faktoren wie geringe Anzahl der isolierten spezifischen T-Zellen, schnelles Abklingen der Aktivität und kurzzeitige Persistenz der transferierten Effektorzellen im Empfängerorganismus stark limitiert. Eine Möglichkeit zur Überwindung dieser Einschränkungen bietet die Entwicklung einer neuen Strategie zur Armierung der zytotoxischen T-Lymphozyten mit Tumor-spezifischen chimären Rezeptoren. Ziel dieser Arbeit war es, die Grundlagen für eine solche immuntherapeutische Strategie zu erarbeiten. Da das Prostatakarzinom die am meisten diagnostizierte maligne Erkrankung und die dritt häufigste Todesursache des Mannes ist, wurde das auf der Oberfläche von Prostatakarzinomzellen exprimierte PSCA (prostataspezifisches Stammzellantigen) als Zielantigen gewählt. Neben der therapierefraktären Spätstadien des Prostatakarzinoms bedürfen die früh entstehenden Mikrometastasen (minimale Resterkrankung) einer neuen adjuvanten Behandlungsoption. Das PSCA ist ein membranständiges Tumor-assoziiertes Antigen, das in mehr als 80 % der primären Prostatakarzinome überexprimiert wird. PSCA wird als besonders aussichtsreiches Zielantigen einer Immuntherapie bei fortgeschrittenen Prostatakarzinomen angesehen, weil sein Expressionsniveau mit der Tumorprogression und der Entwicklung zum androgenunabhängigen Wachstum ansteigt. In der vorgelegten Arbeit wurde zunächst ein neuer monoklonaler PSCA-spezifischer Antikörper generiert, der als Grundlage für die Konstruktion eines Einzelkettenantikörpers (scFv) verwendet wurde. Aus einem Hybridomklon, der sich durch sehr hohe Bindungsstärke auszeichnete, wurden mittels degenerierter Primer die kodierenden Sequenzen für die variablen VH und VL Domänen des Antikörpers amplifiziert. Durch die Verbindung der beiden VH und VL Domänen mittels eines Linkers wurde der PSCA-spezifische Einzelkettenantikörper generiert. Die mit gereinigtem scFv durchgeführten Bindungsanalysen bestätigten die Funktionalität des rekombinanten Proteins und seine Anwendbarkeit zur Chimerisierung eines membranständigen Rezeptors. Nach dem ?Zwei-Signal-Modell? benötigen T-Zellen für eine effiziente Antigen-spezifische Aktivierung neben dem T-Zell-Rezeptorsignal ein zusätzliches kostimulatorisches Signal. Daher wurden chimäre Rezeptoren auf der Basis der Beta-Kette des T-Zell-Rezeptors und des CD28-Moleküls generiert. Bei der Konstruktion des chimären T-Zell-Rezeptors wurde die konstante Domäne der Beta-Kette mit der CD3 &amp;amp;#61562;-Kette fusioniert. Neben einer starken Oberflächenexpression des Rezeptors wurde auch die effiziente Bindung von löslichem PSCA nachgewiesen. Die Bindung des Rezeptors an das PSCA führte zur Phosphorylierung der ITAM-Sequenzen der heterodimeren &amp;amp;#61562;-Kette, was die Funktionalität des chimären Rezeptors bestätigte. Die Stimulation der Zellen über den anti-CD3 Antikörper resultierte ebenfalls in der Phosphorylierung der heterodimeren &amp;amp;#61562;-Kette, was ein Hinweis auf eine mögliche Interaktion der chimären Kette mit dem endogenen CD3-Komplex lieferte. Um die kostimulatorische Wirkung über das selbe Antigen zu erzielen, wurde das CD28 Molekül N-terminal ebenfalls mit dem Einzelkettenantikörper modifiziert. Die durch Bindung des löslichen Proteins induzierte Phosphorylierung der Akt-Kinase bewies die Funktionalität der chimären CD28 Kette als PSCA-spezifischer Rezeptor. Diese Arbeit demonstriert die Generierung eines hochaffinen PSCA-spezifischen Einzelkettenantikörpers als eine Antigen-erkennende Struktur eines chimären Rezeptors. Die Armierung polyklonaler zytotoxischer T-Lymphozyten mit den funktionsfähigen chimären Rezeptoren stellt den ersten Schritt einer neuen Strategie zur Eliminierung hormon-refrektärer und metastasierender Prostatakarzinomzellen dar.

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e são consideradas um problema de saúde pública em vários países, principalmente os em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens adultos. A FR apresenta diversas manifestações, sendo a doença reumática cardíaca (DRC) a seqüela mais grave, caracterizada por lesões cardíacas valvares progressivas e permanentes. O tratamento, frequentemente envolve cirurgia cardíaca para a correção de lesões valvulares, o que acarreta alto custo para o Sistema Único de Saúde no Brasil e em vários países. Em trabalhos anteriores sobre os mecanismos desencadeadores das lesões reumáticas no coração, foi possível identificar o papel do linfócito T como mediador principal da autoimunidade, através da análise do receptor de células T infiltrantes de lesão cardíaca de indivíduos com DRC. Várias expansões oligoclonais com diferentes tamanhos da região que reconhece o antígeno, CDR3 foram encontradas. No presente trabalho, analisou-se a atividade tímica através da quantificação de fragmentos circulares excisados pelo rearranjo do gene do receptor do linfócito T (TREC) em linfócitos T de sangue periférico de indivíduos com FR e DRC. Também foi avaliada a presença de células T naïve e de memória através de citometria de fluxo. Os resultados do presente trabalho mostraram que a quantidade de TREC em amostras de sangue periférico do grupo de pacientes com FR/DRC foi significantemente menor quando comparada a observada em indivíduos saudáveis. Interessantemente, em ambos os grupos a quantidade de TREC apresentou correlação negativa com a idade dos indivíduos estudados. Os resultados indicaram diferenças na atividade tímica em pacientes com FR/DRC, provavelmente decorrente do processo autoimune que envolve linfócitos T / There is a wide spectrum of diseases caused by group A streptococci (GAS), that still being considered a public health problem in developing countries, with about 600 million cases per year. Infections by GAS can cause invasive diseases such as pharyngitis and pyoderma leading to serious autoimmune complications such as rheumatic fever (RF) and glomerulonephritis. RF mainly affects children and young adults, and presents different manifestations. Rheumatic heart disease (RHD) is considered the most serious complication leading to valvular lesions that are characterized by progressive and permanent heart damage, which entails high cost to the Public Health System in Brazil and worldwide. In previous works that focused on the mechanisms leading to rheumatic heart lesions, we identified the role of T lymphocytes as principal mediator of autoimmune reactions. Through the in deep analysis of infiltrating T-cell receptor repertoire of patients with RHD, we identified oligoclonal expansions with different sizes of CDR3 that is the region of antigen recognition. In the present study we analyzed the thymic activity through T cell receptor excision circles (TREC) quantification in T cells from peripheral blood of RF/RHD patients. We also evaluated naïve and memory T cells from peripheral blood by flow cytometry. Our results showed that the amount of TREC in the peripheral blood of patients was significantly lower when compared to the healthy individuals. In addition, both groups showed that the amount of TREC is negatively correlated with age. These results indicated that the thymic activity in RF/RHD patients is altered probably due to the autoimmune process that involves T lymphocytes

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.

Die Effizienz einer Tumortherapie durch einen Transfer von ex vivo aktivierten Tumor-spezifischen zytotoxischen T-Lymphozyten wird durch zahlreiche Faktoren wie geringe Anzahl der isolierten spezifischen T-Zellen, schnelles Abklingen der Aktivität und kurzzeitige Persistenz der transferierten Effektorzellen im Empfängerorganismus stark limitiert. Eine Möglichkeit zur Überwindung dieser Einschränkungen bietet die Entwicklung einer neuen Strategie zur Armierung der zytotoxischen T-Lymphozyten mit Tumor-spezifischen chimären Rezeptoren. Ziel dieser Arbeit war es, die Grundlagen für eine solche immuntherapeutische Strategie zu erarbeiten. Da das Prostatakarzinom die am meisten diagnostizierte maligne Erkrankung und die dritt häufigste Todesursache des Mannes ist, wurde das auf der Oberfläche von Prostatakarzinomzellen exprimierte PSCA (prostataspezifisches Stammzellantigen) als Zielantigen gewählt. Neben der therapierefraktären Spätstadien des Prostatakarzinoms bedürfen die früh entstehenden Mikrometastasen (minimale Resterkrankung) einer neuen adjuvanten Behandlungsoption. Das PSCA ist ein membranständiges Tumor-assoziiertes Antigen, das in mehr als 80 % der primären Prostatakarzinome überexprimiert wird. PSCA wird als besonders aussichtsreiches Zielantigen einer Immuntherapie bei fortgeschrittenen Prostatakarzinomen angesehen, weil sein Expressionsniveau mit der Tumorprogression und der Entwicklung zum androgenunabhängigen Wachstum ansteigt. In der vorgelegten Arbeit wurde zunächst ein neuer monoklonaler PSCA-spezifischer Antikörper generiert, der als Grundlage für die Konstruktion eines Einzelkettenantikörpers (scFv) verwendet wurde. Aus einem Hybridomklon, der sich durch sehr hohe Bindungsstärke auszeichnete, wurden mittels degenerierter Primer die kodierenden Sequenzen für die variablen VH und VL Domänen des Antikörpers amplifiziert. Durch die Verbindung der beiden VH und VL Domänen mittels eines Linkers wurde der PSCA-spezifische Einzelkettenantikörper generiert. Die mit gereinigtem scFv durchgeführten Bindungsanalysen bestätigten die Funktionalität des rekombinanten Proteins und seine Anwendbarkeit zur Chimerisierung eines membranständigen Rezeptors. Nach dem ?Zwei-Signal-Modell? benötigen T-Zellen für eine effiziente Antigen-spezifische Aktivierung neben dem T-Zell-Rezeptorsignal ein zusätzliches kostimulatorisches Signal. Daher wurden chimäre Rezeptoren auf der Basis der Beta-Kette des T-Zell-Rezeptors und des CD28-Moleküls generiert. Bei der Konstruktion des chimären T-Zell-Rezeptors wurde die konstante Domäne der Beta-Kette mit der CD3 &amp;amp;#61562;-Kette fusioniert. Neben einer starken Oberflächenexpression des Rezeptors wurde auch die effiziente Bindung von löslichem PSCA nachgewiesen. Die Bindung des Rezeptors an das PSCA führte zur Phosphorylierung der ITAM-Sequenzen der heterodimeren &amp;amp;#61562;-Kette, was die Funktionalität des chimären Rezeptors bestätigte. Die Stimulation der Zellen über den anti-CD3 Antikörper resultierte ebenfalls in der Phosphorylierung der heterodimeren &amp;amp;#61562;-Kette, was ein Hinweis auf eine mögliche Interaktion der chimären Kette mit dem endogenen CD3-Komplex lieferte. Um die kostimulatorische Wirkung über das selbe Antigen zu erzielen, wurde das CD28 Molekül N-terminal ebenfalls mit dem Einzelkettenantikörper modifiziert. Die durch Bindung des löslichen Proteins induzierte Phosphorylierung der Akt-Kinase bewies die Funktionalität der chimären CD28 Kette als PSCA-spezifischer Rezeptor. Diese Arbeit demonstriert die Generierung eines hochaffinen PSCA-spezifischen Einzelkettenantikörpers als eine Antigen-erkennende Struktur eines chimären Rezeptors. Die Armierung polyklonaler zytotoxischer T-Lymphozyten mit den funktionsfähigen chimären Rezeptoren stellt den ersten Schritt einer neuen Strategie zur Eliminierung hormon-refrektärer und metastasierender Prostatakarzinomzellen dar.

Helper T cells direct the immunological response to foreign pathogens and cancer. To become activated, helper T cells must recognize unique peptides presented on major histocompatibility complex II (pMHCII) by antigen presenting cells (APCs) with their T cell receptor (TCR). While much is known about helper T cell activation signaling cascades and the subsequent roles of helper T cell subsets, the initiation of helper T cell activation by the TCR and other co-receptors is less well understood. Specifically, the affinity of the TCR for its pMHCII can change helper T cell subset fate, proliferation, and alter the risk for activation induced cell death. High affinity TCRs are attractive targets for immunotherapies, but little is known about how helper T cells respond to high affinity TCRs. Here we describe high affinity TCR activation thresholds for both full length TCRs and chimeric antigen receptor TCRs both with and without the presence of the coreceptor CD4 and propose a mechanism whereby CD4 inhibits T cell activation via Lck sequestration and a CD4-independent method. Dysregulated helper T cells play critical roles in the development and perpetuation of systemic lupus erythematosus (SLE), a systemic autoimmune disease that causes widespread inflammation and organ damage throughout the body. Chronic inflammation in SLE affects the immune response to viruses and the risk of developing cancer. However, in SLE patients, it is unclear if viruses initiate the development of cancer directly or if the effects are non-interacting and concomitant. Here we describe the interactions between SLE, viruses, and cancer risk revealing that viruses and SLE do interact to increase the both the overall cancer risk and the risk for hematological malignancies. Due to vaccine efficacy, vaccine preventable diseases (VPDs) are no longer commonly experienced or understood by the public. Vaccines are a victim of their own success and according to the World Health Organization (WHO), vaccine hesitancy (VH) is one of the top threats to global health. VH is the refusal to accept vaccinations and the reasons for VH vary across time, place, and vaccine. Refuting VH is difficult as directly confronting false assumptions can cause individuals to become more entrenched in their position resulting in confirmation bias. Adults with VH attitudes are often motivated by concerns over personal liberty, harm, independence, and body purity. Here we describe the results of a VPD interview- and education-based intervention geared towards promoting positive vaccine attitudes for young adults and demonstrate that education focused on VPDs is more effective than vaccine safety.

Dans le monde, 58 millions de personnes sont chroniquement infectées par le virus de l'hépatite C (VHC). Depuis 2011, l'introduction des antiviraux à action directe a permis la guérison des infections chroniques chez la majorité des sujets traités (~95 %). Toutefois, les traitements sont coûteux et ne protègent pas contre les réinfections, d'où la nécessité de développer un vaccin prophylactique pour freiner efficacement l'épidémie du VHC. Environ 30% des primo-infections sont éliminées spontanément, représentant une occasion unique d'étudier les corrélats de l’immunité protectrice nécessaires pour le développement d’un vaccin efficace. Dans cette thèse, nous avons procédé à la définition des corrélats de l'immunité protectrice au cours des infections par le VHC primaires et subséquentes aux niveaux transcriptomique, clonotypique et fonctionnel à partir d’une cohorte d’utilisateurs de drogues par injection. Le premier objectif était de caractériser le répertoire de récepteurs des cellules T CD8 spécifique de l'épitope immunodominant et cross-réactif NS3 1073-1081 (CINGVCWTV) restreint par HLA-A2 au cours d’une primo-infection aiguë progressant vers une résolution spontanée ou une infection chronique. Nous avons identifié un ensemble de treize clonotypes publics, indépendamment de l'issue de l'infection. Plusieurs clonotypes publics avaient une longue durée de vie après résolution de l’infection et ont proliféré après réinfection par le VHC. En explorant les bases de données publiques, nous avons identifié plusieurs clonotypes partagés avec d'autres épitopes viraux restreints par HLA-A2, mais ils étaient de faible fréquence et de réactivité croisée limitée, suggérant un rôle limité des lymphocytes T CD8 cross-réactifs au cours de l'infection primaire par le VHC. Le deuxième objectif était de caractériser les signatures transcriptomiques longitudinales des cellules mononucléaires du sang périphérique totaux chez huit sujets ayant spontanément résolu deux infections consécutives par le VHC. Nous avons également comparé ces signatures avec un schéma vaccinal composé d'un vecteur à adénovirus de chimpanzé suivi d'un rappel utilisant la vaccine modifiée Ankara, exprimant tout deux les protéines non-structurales du VHC. Nous avons identifié une signature transcriptomique des plasmocytes au cours d'une réinfection aiguë, absente lors de l'infection primaire et après le rappel du vaccin. La résolution spontanée est associée à une expansion rapide des cellules B mémoires spécifiques de la glycoprotéine E2 chez 3 sujets et à une augmentation transitoire des anticorps neutralisants anti- E2 chez 6 sujets. Parallèlement, il y avait une augmentation de l'étendue et de l'ampleur des lymphocytes T spécifiques du VHC chez 7 sujets. En conclusion, nous avons identifié treize clonotypes publics uniques au VHC qui ont proliféré au cours des infections primaire et secondaire. La faible fréquence des clonotypes cross-réactifs suggère qu'ils ne sont pas des déterminants majeurs de l’issue de l’infection. De plus, nous avons observé une augmentation simultanée des réponses des lymphocytes B et T spécifiques du VHC au stade aiguë précoce, suggérant un rôle des deux bras de l’immunité adaptative dans la clairance de la réinfection du VHC. Nos résultats soutiennent l'idée de combiner deux stratégies vaccinales induisant à la fois une immunité à médiation cellulaire et une immunité humorale visant à prévenir les infections chroniques par le VHC. / Worldwide, 58 million individuals are chronically infected with hepatitis C virus (HCV). Since 2011, the introduction of direct acting antivirals enabled the cure of chronic HCV in the majority of treated subjects (~95%). However, direct-acting antivirals treatments are expensive and do not protect against reinfection, urging the need to develop a prophylactic vaccine to efficiently curb the HCV epidemic. Around 30% of acutely infected individuals will spontaneously clear the infection, representing a unique opportunity to study the correlates of immune protection needed to develop a potent vaccine. In this thesis, we proceeded to define the correlates of protective immunity during primary and sub-sequent HCV infections at the transcriptomic, clonotypic and functional levels using longitudinal peripheral blood mononuclear cells samples collected from a cohort of people who inject drugs (PWID). The first aim was to characterize the CD8 T cell receptor repertoire specific to the immunodominant and cross-reactive HLA-A2 restricted NS3 1073-1081 (CINGVCWTV) epitope during acute HCV in PWID progressing to either spontaneous resolution or chronic infection. We identified a set of thirteen public clonotypes in HCV-infected subjects irrespective of infection outcome. Several public clonotypes were long-lived in resolvers and expanded upon reinfection. By mining publicly available data, we identified several TCR clonotypes shared with other HLA-A2 restricted epitopes, but they were of low frequency and limited cross-reactivity, suggesting that they are not major determinants of infectious outcome. The second aim was to characterize longitudinal transcriptomic signatures using total peripheral blood mononuclear cells, as well as T and B cell recall responses in eight subjects who spontaneously resolved two successive episodes of HCV infection. Furthermore, we compared the transcriptomic signatures of primary and secondary resolving HCV infections, with an HCV nonstructural protein vaccine regimen of recombinant chimpanzee adenovirus 3 vector prime followed by modified vaccinia Ankara boost. We identified a plasma cell transcriptomic signature during early acute HCV reinfection that was absent in primary infection and following HCV vaccine boost. Spontaneous resolution of HCV reinfection was associated with rapid expansion of glycoprotein E2-specifc memory B cells in 3 subjects and transient increase in E2-specific neutralizing antibodies in 6 subjects. Concurrently, there was an increase in the breadth and magnitude of HCV-specific T cells in 7 subjects. In conclusion, we identified thirteen new public CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of crossreactive TCRs suggests that they are not major determinants of infectious outcome. Moreover, we observed a concurrent increase of HCV-specific B and T cell responses early during acute HCV reinfection at the transcriptomic and functional levels, suggesting a role for both arms of the adaptive immune response in HCV reinfection clearance. Our results support the combined T and B cell-based vaccine strategy aimed at preventing chronic HCV infections.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.