O vírus linfotrópico das células T humanas tipo 1 (HTLV-1), é responsável por patologias como a mielopatia associada ao HTLV-1 ou paraparesia espástica tropical (HAM/TSP) e a leucemia/linfoma das células T do adulto (ATL) dentre outras. As vias de replicação até hoje demonstradas, não suportam a hipótese de um estado virêmico. Neste estudo, a detecção de partículas virais plasmáticas foi executada, por PCR em Tempo Real e Nested PCR em 190 amostras de pacientes infectados pelo HTLV-1(assintomáticos ou com HAM/TSP), em acompanhamento, no Instituto de Infectologia Emílio Ribas. 12 indivíduos (8%) testados por PCR em tempo real (n=150) e 6 indivíduos (18%) testados por Nested PCR (n=33, dado que sete amostras foram excluídas da análise) apresentaram RNA do HTLV-1 detectável no plasma. Em conclusão, foi possível identificar RNA plasmático do HTLV-1, tanto em pessoas assintomáticas quanto com HAM/TSP. Esta detecção abre novas possibilidades de discussão sobre a replicação do HTLV-1 e das vias de transmissão, sugerindo maiores investigações para elucidar o assunto. / The human T-cell lymphotropic virus type1 (HTLV-1) is responsible for some pathologies such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell Leukemia/ Lymphoma (ATL) among others. Its ways of replication so far presented do not support the hypothesis of a viremic stage. In this study, the detection of the plasmatic viral load was performed by real time PCR and Nested PCR in 190 samples from HTLV-1 infected individuals (Either Asymptomatic or HAM/TSP cases) following up at Instituto de Infectologia Emílio Ribas. 12 individuals (8%) tested by Real time PCR (n= 150) and 6 individuals (18%) tested by Nested PCR (n= 33, given that 7 samples were excluded from the analysis) presented detectable HTLV-1 RNA in the plasma. In conclusion, it was possible to indentify HTLV-1 plasmatic RNA in asymptomatic carriers as well as in HAM/TSP cases. This detection opens new possibilities of discussion about HTLV-1 replication and transmission pathways, suggesting further investigation for clarifying this matter.

A Imunodeficiência Comum Variável (ICV) e a Deficiência Seletiva de Imunoglobulina A (DIgA) são as imunodeficiências primárias humorais de maior freqüência na população mundial. Ambas as doenças são caracterizadas pela ausência ou redução significativa de imunoglobulinas no soro. Embora diversas anormalidades imunológicas tenham sido associadas a estas doenças, nenhuma hipótese unificadora a respeito das bases moleculares das mesmas foi proposta até o presente momento, sendo que o único defeito comum a todos os pacientes é a falha na diferenciação de células B em plasmócitos e conseqüente secreção de anticorpos. Devido à alta incidência de auto-imunidade e alergia em pacientes com ICV e DIgA, no presente trabalho, visamos analisar por citometria de fluxo a população de linfócitos CD4+ com potencial regulador nesses pacientes, para avaliar se possíveis defeitos quantitativos ou funcionais nesta população reguladora poderiam explicar a alta incidência de doenças auto-imunes ou alérgicas associadas a estas imunodeficiências. / Common Variable Immunodeficiency (CVID) and Selective Immunoglobulin A deficiency (IgAD) are the humoral primary immunodeficiencies with the highest incidence in the population. Both diseases are characterized by the absence or significant reduction of serum immunoglobulins. Although several immunological abnormalities have been associated with these diseases, no unifying hypothesis regarding the molecular basis of CVID and IgAD have been proposed to date, and the only defect common to all patients is the failure in differentiation of B cells into plasma cells and consequent secretion of antibodies. Due to the high incidence of autoimmunity and allergy in patients with CVID and IgAD, in the present work we analyzed by flow cytometry the population of CD4+ lymphocytes with regulatory potential in these patients to assess whether possible quantitative or functional defects in this regulatory population could explain the high incidence of autoimmune diseases or allergic reactions associated with these immunodeficiencies.

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

A imunização materna com a vacina acelular para pertussis (dTpa) é uma intervenção adicional que visa fornecer proteção aos recém-nascidos (RN). No entanto, tem sido relatado que altos níveis de anticorpos adquiridos por transferência placentária podem afetar adversamente a resposta imune desses RN após a imunização ativa, devido ao mascaramento antigênico. Neste estudo, avaliamos a aquisição passiva neonatal de anticorpos específicos para pertussis e sua influência na resposta imune celular dos neonatos. A casuística foi composta por gestantes vacinadas com a vacina dTpa (grupo caso, n=66) ou por gestantes que não receberam a vacina (grupo controle, n=101). As concentrações de anticorpos IgG séricos específicos para Bordetella pertussis total (Bp), toxina pertussis (PT), hemaglutinina filamentosa (FHA) e pertactina (PRN) foram quantificadas em soro materno e de cordão umbilical de seu respectivo RN, e as concentrações de anticorpos IgA específicos para Bp e PT foram dosadas nas amostras de colostro por meio de ensaio imunoenzimático. A responsividade dos linfócitos do sangue neonatal foi avaliada após estimulação ex vivo com Bp inativada por citometria de fluxo com o intuito de detectar a proliferação, produção de citocinas e fenótipo de ativação dos linfócitos T em um contexto de altas concentrações de IgG específicas adquiridas após a vacinação materna. As concentrações de anticorpos IgG anti-Bp, PT, FHA e PRN foram maiores nas amostras de soro materno e de cordão umbilical do grupo caso quando comparadas ao grupo controle (p < 0,0001), com índices de correlação positivos em ambos os grupos para todos os antígenos estudados (p < 0,0001). As vacinações realizadas entre 26 e 31 semanas de gestação foram associadas com as melhores taxas de transferência placentária, embora índices significativamente menores foram detectados no grupo caso (p < 0,01). As concentrações de anticorpos IgA anti-Bp e anti-PT no colostro não foram afetadas pelo estado vacinal da parturiente. Os ensaios de cultura celular revelaram que os RN responderam ao estímulo com Bp, com maior expressão de CD40L, CD69 e proliferação de células T CD4, em comparação com células não estimuladas. Também foi observada uma menor resposta Th1, enquanto a resposta Th2 foi preservada, em comparação com os adultos, mas sem diferenças entre os grupos de neonatos em nenhum dos parâmetros estudados. Nossos resultados indicam que níveis mais altos de anticorpos IgG específicos para B. pertussis no soro dos RN após a vacinação materna não afetam a resposta imune neonatal mediada por células / Maternal immunization with pertussis acellular vaccine (Tdap) is an additional intervention that provides protection to newborns. However, it has been reported that high antibody levels acquired via placental transfer may adversely affect the immune response of newborns after active immunization due to epitope masking. In this study, we evaluated neonatal passive acquisition of pertussis-specific antibodies and their influence on the neonatal cell-mediated immune response. The sample consisted of pregnant women vaccinated with the Tdap vaccine (case group, n=66) or pregnant women who received no vaccine (control group n=101). Whole-cell Bordetella pertussis (Bp), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN)-specific serum IgG concentrations were quantified in paired maternal-cord sera, and Bp- and PT-specific IgA concentrations were evaluated in colostrum samples by immunoenzymatic assay. Ex vivo neonatal blood lymphocyte responsiveness after inactivated Bp stimulation was assessed using flow cytometry to detect the proliferation, cytokine production and activation phenotype of T lymphocytes in the context of high specific IgG concentrations acquired after maternal vaccination. Anti-Bp, PT, FHA and PRN IgG antibody concentrations in maternal and cord serum samples from case group were higher than those in control group (p < 0.0001), with positive correlation indexes in both groups for all pertussis antigens (p < 0.0001). Vaccinations performed between 26 and 31 gestation weeks were associated with the best placental transfer ratios, although significantly lower ratios were detected in case group (p < 0.01). Anti-Bp and anti-PT IgA concentrations in colostrum were not affected by vaccine status. Cell culture assays revealed that newborns responded to Bp stimulation with higher expression of CD40L CD69 and CD4+ T cell proliferation compared to unstimulated cells. It was also observed a lower Th1 response, while a preserved Th2 response compared to adults, but there were no differences between neonatal groups for any of the studied parameters. Our results indicate that higher pertussis-specific IgG levels in newborn sera after maternal vaccination do not affect the neonatal cell-mediated immune response

A imunização materna com a vacina acelular para pertussis (dTpa) é uma intervenção adicional que visa fornecer proteção aos recém-nascidos (RN). No entanto, tem sido relatado que altos níveis de anticorpos adquiridos por transferência placentária podem afetar adversamente a resposta imune desses RN após a imunização ativa, devido ao mascaramento antigênico. Neste estudo, avaliamos a aquisição passiva neonatal de anticorpos específicos para pertussis e sua influência na resposta imune celular dos neonatos. A casuística foi composta por gestantes vacinadas com a vacina dTpa (grupo caso, n=66) ou por gestantes que não receberam a vacina (grupo controle, n=101). As concentrações de anticorpos IgG séricos específicos para Bordetella pertussis total (Bp), toxina pertussis (PT), hemaglutinina filamentosa (FHA) e pertactina (PRN) foram quantificadas em soro materno e de cordão umbilical de seu respectivo RN, e as concentrações de anticorpos IgA específicos para Bp e PT foram dosadas nas amostras de colostro por meio de ensaio imunoenzimático. A responsividade dos linfócitos do sangue neonatal foi avaliada após estimulação ex vivo com Bp inativada por citometria de fluxo com o intuito de detectar a proliferação, produção de citocinas e fenótipo de ativação dos linfócitos T em um contexto de altas concentrações de IgG específicas adquiridas após a vacinação materna. As concentrações de anticorpos IgG anti-Bp, PT, FHA e PRN foram maiores nas amostras de soro materno e de cordão umbilical do grupo caso quando comparadas ao grupo controle (p < 0,0001), com índices de correlação positivos em ambos os grupos para todos os antígenos estudados (p < 0,0001). As vacinações realizadas entre 26 e 31 semanas de gestação foram associadas com as melhores taxas de transferência placentária, embora índices significativamente menores foram detectados no grupo caso (p < 0,01). As concentrações de anticorpos IgA anti-Bp e anti-PT no colostro não foram afetadas pelo estado vacinal da parturiente. Os ensaios de cultura celular revelaram que os RN responderam ao estímulo com Bp, com maior expressão de CD40L, CD69 e proliferação de células T CD4, em comparação com células não estimuladas. Também foi observada uma menor resposta Th1, enquanto a resposta Th2 foi preservada, em comparação com os adultos, mas sem diferenças entre os grupos de neonatos em nenhum dos parâmetros estudados. Nossos resultados indicam que níveis mais altos de anticorpos IgG específicos para B. pertussis no soro dos RN após a vacinação materna não afetam a resposta imune neonatal mediada por células / Maternal immunization with pertussis acellular vaccine (Tdap) is an additional intervention that provides protection to newborns. However, it has been reported that high antibody levels acquired via placental transfer may adversely affect the immune response of newborns after active immunization due to epitope masking. In this study, we evaluated neonatal passive acquisition of pertussis-specific antibodies and their influence on the neonatal cell-mediated immune response. The sample consisted of pregnant women vaccinated with the Tdap vaccine (case group, n=66) or pregnant women who received no vaccine (control group n=101). Whole-cell Bordetella pertussis (Bp), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN)-specific serum IgG concentrations were quantified in paired maternal-cord sera, and Bp- and PT-specific IgA concentrations were evaluated in colostrum samples by immunoenzymatic assay. Ex vivo neonatal blood lymphocyte responsiveness after inactivated Bp stimulation was assessed using flow cytometry to detect the proliferation, cytokine production and activation phenotype of T lymphocytes in the context of high specific IgG concentrations acquired after maternal vaccination. Anti-Bp, PT, FHA and PRN IgG antibody concentrations in maternal and cord serum samples from case group were higher than those in control group (p < 0.0001), with positive correlation indexes in both groups for all pertussis antigens (p < 0.0001). Vaccinations performed between 26 and 31 gestation weeks were associated with the best placental transfer ratios, although significantly lower ratios were detected in case group (p < 0.01). Anti-Bp and anti-PT IgA concentrations in colostrum were not affected by vaccine status. Cell culture assays revealed that newborns responded to Bp stimulation with higher expression of CD40L CD69 and CD4+ T cell proliferation compared to unstimulated cells. It was also observed a lower Th1 response, while a preserved Th2 response compared to adults, but there were no differences between neonatal groups for any of the studied parameters. Our results indicate that higher pertussis-specific IgG levels in newborn sera after maternal vaccination do not affect the neonatal cell-mediated immune response

No description available.

L’association entre déficits immunitaires primitifs (DIPs) et manifestations auto-immunes peut sembler paradoxale lorsque l’on aborde les DIPs comme des défauts d’immunité opposés à l’autoimmunité vue comme excès d’immunité adaptative à l’encontre du soi. Néanmoins, loin de se résumer à un simple défaut d’une ou plusieurs composantes du système immunitaire qui prédispose aux infections par divers agents pathogènes, les DIPs sont fréquemment associés à une autoimmunité; parfois révélatrice. Ainsi, les données épidémiologiques issues de registres ou de larges séries de patients atteints de DIPs s’accordent sur une prévalence globale de 25 à 30% de complications auto-immunes (au premier rang desquelles figurent les cytopénies auto-immunes). Différentes hypothèses sont avancées pour rendre compte de l’auto-immunité dans les DIPs. On peut citer : 1°) une perturbation profonde de l’homéostasie lymphocytaire, en particulier dans les déficits immunitaires combinés sévères (CID) avec lymphopénies T et B ; 2°) des défauts intrinsèques des lymphocytes B permettant une rupture de tolérance précoce des LB auto réactifs ; 3°) un comportement aberrant des LT (défaut de maturation, excès d’activation) ; 4°) une absence de lymphocytes T ou de B régulateurs ; 5°) une production inappropriée de certaines cytokines proinflammatoires comme dans les interféronopathies. Ces hypothèses concernent surtout les DIPs pédiatriques sévères. Mon travail de thèse explore la rupture de tolérance immunitaire adaptative au cours des DIPs de l’adulte par différentes approches. Nous nous sommes en particulier attachés au plus fréquent, le DICV (Déficit Immunitaire Commun Variable), déficit immunitaire humoral pas toujours bien défini sur le plan génétique et physiopathologique qui constitue un défi thérapeutique lorsqu’il est compliqué d’une auto-immunité nécessitant un traitement immunosuppresseur. / The association between primary immune deficiency (PID) and autoimmunity may seem paradoxical when PID is considered only as an immune response defect against pathogens and autoimmunity only as an excess of immunity. Nevertheless, far from being simple immune defects increasing the risk of infections, DIPs are frequently associated with autoimmunity. Even more, autoimmunes manifestations can sometimes reveal a PID. Thus, epidemiological data from registers or large series of patients with PIDs agree on an overall prevalence of 25 to 30% of autoimmune complications (with auto-immune cytopenias as first causes). Several hypotheses have been proposed with different underlying mechanisms to explain the tolerance breakdown in PIDs. We can cite : 1°) a severe disturbance of lymphocyte homeostasis, for example in severe combined immunodeficiencies ; 2°) an impaired B-cell developpement with earlystage defects of tolerance ; 3°) a dysregulation of T cells (developpement or activation impairments) ; 4°) a dysfunction of T-reg (or B-reg) ; 5°) an excess of production of proinflammatory cytokines. These hypotheses are especially true for early-onset PIDs (in infancy). In this work (PhD), we explore the mechanisms of tolerance breakdown involved in adults PIDs. We use several approaches to describe the pathways leading to autoimmunity, focusing on the most common PID in adult : CVID (common variable immunodeficiency). This syndrome is not well defined on the genetic and physiopathological level. It is still a therapeutic challenge when complicated by autoimmunity (requiring immunosuppressive therapy).

Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

Το καρκίνωμα εκ πλακώδους επιθηλίου ή πλακώδες καρκίνωμα (ΠΚ) του δέρματος είναι ο δεύτερος πιο συχνός καρκίνος του δέρματος και εμφανίζεται συνήθως σε έδαφος ακτινικής υπερκεράτωσης (ΑΚ). Τα νεοπλασματικά κύτταρα εκφράζουν μια ποικιλία αντιγόνων προσελκύοντας με αυτό τον τρόπο λεμφοκύτταρα, τα διηθούντα τον όγκο λεμφοκύτταρα (Tumor Ιnfiltrating Lymphocytes-TILs) στο μικροπεριβάλλον του όγκου. Με βάση το ανοσοφαινοτυπικό τους προφίλ η πλειοψηφία των TILs εκφράζει το μόριο CD3 και αφορά σε Τ-λεμφοκύτταρα. Αυτά με την σειρά τους διαχωρίζονται σε CD8+ Τ-κυτταροτοξικά λεμφοκύτταρα και CD4+ Τ-λεμφοκύτταρα. Η έκφραση του δείκτη CD25+ επιτρέπει τον διαχωρισμό των CD4+ λεμφοκυττάρων σε δύο επιπλέον υποομάδες, τα T-επικουρικά (CD4+/CD25-) και τα Τ-ρυθμιστικά (CD4+/CD25+) κύτταρα. Ο πιο αξιόπιστος δείκτης για την ανίχνευση των Τ-ρυθμιστικών κυττάρων (Tregs) θεωρείται ο Forkhead box P3 (Foxp3). Ο αυξημένος αριθμός των TILs έχει συσχετισθεί με την πρόγνωση και τη θεραπευτική αντιμετώπιση σε μια ποικιλία νεοπλασμάτων, περιλαμβανομένων και καρκίνων του δέρματος όπως το μελάνωμα. Η μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης, ωστόσο, δεν έχει ακόμα αποσαφηνισθεί και ποικίλλει στα διάφορα είδη νεοπλασμάτων. Στα πλαίσια της παρούσας διπλωματικής εργασίας η ανίχνευση των Τ-λεμφοκυττάρων έγινε με ανοσοϊστοχημική μέθοδο με τη χρήση των κατάλληλων αντισωμάτων (CD3, CD4, CD8, Foxp3). Η παρουσία των κυττάρων αξιολογήθηκε με τη χρήση φωτονικού μικροσκοπίου με δυο διαφορετικά ημιποσοτικά συστήματα βαθμολόγησης για όλους τους μελετηθέντες Τ-κυτταρικούς υποπληθυσμούς καθώς και με ένα τρίτο σύστημα ποσοτικής καταμέτρησης για τα Τregs. Σκοπός ήταν να γίνει σύγκριση των μεθόδων ώστε να βρεθεί η καταλληλότερη μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης στο πλακώδες καρκίνωμα του δέρματος, να υπολογιστεί ο αριθμός των Tregs στις μελετούμενες οντότητες χρησιμοποιώντας το βέλτιστο σύστημα βαθμολόγησης και να αναζητηθούν διαφορές στην πυκνότητα των λεμφοκυτταρικών υποπληθυσμών μεταξύ των οντοτήτων, οι οποίες μπορεί να έχουν παθογενετική ή θεραπευτική κλινική σημασία. / Squamous cell carcinoma (SCC) of the skin is the second most common skin cancer. It usually develops in a background of actinic keratosis (AK). Neoplastic cells express a variety of antigens, attracting lymphocytes in the tumor microenvironment (Tumor Ιnfiltrating Lymphocytes-TILs). Based on the immunophenotypic profile, the majority of TILs expresses the CD3 molecule, so they are T-lymphocytes. These in turn are divided into CD8+ cytotoxic T-lymphocytes and CD4+ T-lymphocytes. The expression of CD25 enables the separation of CD4+ lymphocytes in two further subgroups, the T-helper (CD4+/CD25-) and T-regulatory (CD4+/CD25+) cells. The most reliable marker for the detection of T-regulatory cells (Tregs) is considered to be the Forkhead box P3 (Foxp3). The increased number of TILs is associated with prognosis and treatment in a variety of tumors, including skin cancers, such as melanoma. The evaluation methology of lymphocytic infiltration, however, is not yet clarified and varies in different types of tumors. In the context of this thesis, the detection of T-lymphocytes was performed by immunohistochemistry using the appropriate antibodies (CD3, CD4, CD8, Foxp3). The presence of the cells was evaluated using light microscopy with two different semiquantitative scoring systems, for all the T-lymphocyte subpopulations, as well as with a third system of quantification of Tregs. The purpose was to compare the methods in order to find the most suitable methodology for the evaluation of the lymphocytic infiltration in squamous cell carcinoma of the skin, to evaluate the number of Tregs in the studied entities using the best method and examine for differences in the density of lymphocyte subpopulations.

Le système immunitaire se doit d’être étroitement régulé afin d’éviter que des réponses immunologiques inappropriées ou de trop forte intensité ne surviennent. Ainsi, différents mécanismes permettent de maintenir une tolérance périphérique, mais aussi d’atténuer la réponse lorsque celle-ci n’est plus nécessaire. De tels mécanismes sont cependant aussi exploités par les tumeurs, qui peuvent ainsi échapper à une attaque par le système immunitaire et donc poursuivre leur progression. Ces mécanismes immunosuppresseurs nuisent non seulement à la réponse naturelle contre les cellules tumorales, mais font aussi obstacle aux tentatives de manipulation clinique de l’immunité visant à générer une réponse anti-tumorale par l’immunothérapie. L’un des mécanismes par lesquels les tumeurs s’évadent du système immunitaire est l’expression d’enzymes responsables du métabolisme des acides aminés dont l’une des principales est l’indoleamine 2,3-dioxygénase (IDO). Cette dernière dégrade le tryptophane et diminue ainsi sa disponibilité dans le microenvironnement tumoral, ce qui engendre des effets négatifs sur la prolifération, les fonctions et la survie des lymphocytes T qui y sont présents. Bien que la régulation de l’expression de cette enzyme ait été largement étudiée chez certaines cellules présentatrices d’antigènes, dont les macrophages et les cellules dendritiques, peu est encore connu sur sa régulation dans les cellules tumorales humaines. Nous avons posé l’hypothèse que différents facteurs produits par les cellules immunitaires infiltrant les tumeurs (TIIC) régulent l’expression de l’IDO dans les cellules tumorales. Nous avons effectivement démontré qu’une expression de l’IDO est induite chez les cellules tumorales humaines, suite à une interaction avec des TIIC. Cette induction indépendante du contact cellulaire résulte principalement de l’interféron-gamma (IFN-g) produit par les lymphocytes T activés, mais est régulée à la baisse par l’interleukine (IL)-13. De plus, la fludarabine utilisée comme agent chimiothérapeutique inhibe l’induction de l’IDO chez les cellules tumorales en réponse aux lymphocytes T activés. Cette observation pourrait avoir des conséquences importantes en clinique sachant qu’une forte proportion d’échantillons cliniques provenant de tumeurs humaines exprime l’IDO. Enfin, les lymphocytes B, qui sont retrouvés également dans certaines tumeurs et qui interagissent étroitement avec les lymphocytes T, sont aussi susceptibles à une induction transcriptionnelle et traductionnelle de l’IDO. Cette enzyme est cependant produite sous une forme inactive dans les lymphocytes B, ce qui rend peu probable l’utilisation de l’IDO par les lymphocytes B comme mécanisme pour freiner la réponse immunitaire. Nos travaux apportent des informations importantes quant à la régulation de l’expression de la molécule immunosuppressive IDO dans les cellules cancéreuses. Ils démontrent que l’expression de l’IDO est influencée par la nature des cytokines présentes dans le microenvironnement tumoral. De plus son expression est inhibée par la fludarabine, un agent utilisé pour le traitement de certains cancers. Ces données devraient être prises en considération dans la planification de futurs essais immunothérapeutiques, et pourraient avoir un impact sur les réponses cliniques anti-tumorales. / The immune system is under tight control to avoid inappropriate and excessive immunological responses. Many mechanisms allow the maintenance of peripheral tolerance and mediate attenuation of the immune response after pathogen clearance. Such mechanisms are also exploited by tumors, thereby favoring their escape from assault by the immune system. These immunosuppressive mechanisms hamper host natural immune responses against tumor cells, but also represent an obstacle to the successful clinical manipulation of the immune system in attempts to generate an anti-tumor response through immunotherapy. One immune escape mechanism used by tumors is the production of enzymes responsible for amino acid metabolism, amongst which indoleamine 2,3-dioxygenase (IDO) is of major importance. IDO degrades tryptophan, thus leading to its depletion from intracellular pools and local microenvironments. This culminates in multi-pronged negative effects on T lymphocytes neighboring IDO-expressing cells, notably on proliferation, function and survival. The regulation of IDO expression has been largely studied in antigen-presenting cells such as macrophages and dendritic cells, but its regulation in human tumor cells must still be characterized. We hypothesized that different factors produced by tumor-infiltrating immune cells (TIIC) regulate IDO expression in tumor cells. Accordingly, we have demonstrated that IDO expression is induced in human tumor cells upon interaction with TIIC. This induction is cell contact-independent, and results mainly from interferon-gamma (IFN-g) produced by activated T lymphocytes, while being antagonised by interleukin (IL)-13. Moreover, the chemotherapeutic agent fludarabine inhibits activated T lymphocyte-dependent IDO induction in tumor cells. This observation could have major clinical consequences, considering the large proportion of human cancer clinical samples expressing IDO. Finally, B lymphocytes, which interact closely with T lymphocytes and are found infiltrating human tumors, are also susceptible to transcriptional and translational IDO induction. This enzyme is however produced in an inactive form, suggesting that B lymphocytes do not exploit this mechanism to impede the immune response. In conclusion, our work brings crucial information on the regulation of the immunosuppressive molecule IDO in human tumor cells. We demonstrate that IDO expression is dependent on the nature of cytokines present in the tumor microenvironment. Furthermore, its expression is inhibited by fludarabine, a compound used to treat some types of cancer. These data should be taken into consideration in planning future immunotherapy trials and could impact anti-tumor clinical responses.