De nombreux antigènes de tumeur ont été identifiés depuis la découverte du premier antigène de tumeur humain il y a une vingtaine d’années, et plusieurs d’entre eux ont été utilisés comme antigène cible pour l’élaboration de vaccins thérapeutiques anti-Cancer. Cependant, les résultats des essais cliniques visant à évaluer l’efficacité de ces vaccins se sont la plupart du temps révélés décevants. Aussi, il reste indispensable d’identifier de nouveaux antigènes de tumeur cibles capables d’induire des réponses anti-Tumorales fortes et durables. Parmi les antigènes de tumeur considérés comme cibles potentielles pour un vaccin anti-Tumoral se trouve la Cycline B1, une protéine endogène impliquée dans la régulation du cycle cellulaire. Normalement exprimée de façon transitoire dans les cellules saines en division, cette protéine est surexprimée dans diverses tumeurs et est indispensable au développement tumoral. De plus, des réponses immunitaires spontanées spécifiques de cette protéine ont été observées chez des patients atteints de cancer. L’objectif de ma thèse était de caractériser la réponse en lymphocytes T CD4+, qui jouent un rôle capital dans la réponse immunitaire anti-Tumorale, spécifique de la Cycline B1 humaine chez des sujets sains et chez des patients atteints de cancer. Nous avons mis en évidence l’existence, chez des individus sains, de deux populations de lymphocytes T CD4+ préexistants spécifiques de cette protéine, à savoir des lymphocytes T CD4+ naïfs et des lymphocytes T CD4+ mémoires, cette seconde population lymphocytaire se retrouvant également chez des patients atteints de cancer. De multiples épitopes T CD4+ ont été identifiés dans cette protéine, et étaient différemment reconnus par ces deux populations de lymphocytes T CD4+. En outre, des anticorps IgG anti-Cycline B1 ont été détectés chez des patients atteints de cancer comme chez des individus sains, sans différence significative dans les taux d’anticorps entre ces deux catégories de sujets. Ainsi, cette étude montre que la Cycline B1 est un antigène de tumeur caractérisé par un profil singulier de réponses immunitaires, et confirme le potentiel vaccinal de cette protéine pour l’élaboration d’un vaccin anti-Cancer. / Many tumor antigens have been identified since the discovery of the first human antigen about twenty years ago, and some of them have been used as targets for the development of therapeutic cancer vaccines. However, most of the time, the results of clinical trials designed to assess the efficacy of these vaccines proved to be disappointing. Thus, it is still necessary to identify new tumor antigens able to induce strong and long-Lasting anti-Tumor responses that could be used as targets for cancer vaccine. Cyclin B1, an endogenous protein involved in cell cycle regulation, is one of the tumor antigens which are currently considered as potential targets for a cancer vaccine. Usually expressed transiently in healthy dividing cells, this protein is overexpressed in numerous tumors and is necessary for tumor development. Moreover, Cyclin B1 specific spontaneaous immune responses have been observed in cancer patients. My PhD work aimed at characterizing the response of CD4+ T cells, which play a major role in anti-Tumor immune responses, specific to human Cyclin B1 both in healthy individuals and cancer patients. We showed that, in healthy individuals, there exists two pre-Existing Cyclin B1 specific CD4+ T cell populations, namely naive CD4+ T cells and memory CD4+ T cells, the latter lymphocyte population being also found in cancer patients. Multiple CD4+ T cell epitopes have been identified in this protein, and were differently recognized by these two CD4+ T cell populations. Besides, anti-Cyclin B1 IgG antibodies have been detected both in healthy individuals and in cancer patients, without significant differences in antibody levels between these two groups of donors. Therefore, this work shows that Cyclin B1 is a tumor antigen characterized by a singular pattern of immune responses, and confirms the potential of this protein as a target for a cancer vaccine.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Abstract There are approximately 5.5 Million individuals in South Africa infected with HIV-1, predominantly subtype C (HIV-1C). The emergence of drug resistance to the current Antiretroviral (ARV) regimes is of great concern, thus development of novel, effective drugs/vaccines is vital. Certain conserved and thus vulnerable epitopes within the viral envelope (Env) involved in coreceptor binding are usually protected from the immune system in peripheral blood by the variable loops. However, in immune-privileged sites the Env of CD4-independent viruses may exist in a pre-triggered state where these coreceptor binding epitopes are exposed. Targeting the conserved sites could effectively neutralize HIV-1. This study aimed to adapt an HIV-1C primary isolate towards CD4- independence in the Cf2Th cell line through serial in vitro passage. Primary viruses from 20 drug-naïve HIV-1 AIDS patients were isolated and genotypically and phenotypically characterized. The highest percentage (30%) of CXCR4-usage amongst primary isolates from HIV-1C (and CD recombinant) infected AIDS patients worldwide was detected. These data may illustrate the increasing frequency of HIV-1C CXCR4- utilizing (X4) viruses with time and may support the theory that env is capable of evolving. The emergence/evolution of HIV-1C X4 viruses may have profound implications for viral pathogenesis, disease progression and future use of CCR5 antagonists as ARVs. Longitudinal follow-up studies on larger cohorts may confirm this finding. The CXCR4-utilizing isolate 05ZAFV03 was successfully adapted and serially passaged 12 times through Cf2Th cells, whilst gradually decreasing amounts of CD4 expressing cells numbers over time. Viral growth was detected with 10% CD4 expressing cells however, 100% CD4-independence was not reached. Proviral DNA from each stage of the adaptation process was sequenced and analyzed for mutations acquired within env. The only amino acid change noted was an E152K mutation within the V1 region at passage 4. Overall, the extent of env diversity appears to be a complex relationship between isolate-specific and cell-type specific factors. Future attempts to obtain and characterize an HIV-1C CD4-independent isolate will provide potential sites for therapeutic intervention by compounds such as small molecule inhibitors and/or neutralizing antibodies against the most globally prevalent HIV-1 subtype.

Most studies of the induction of cytokine synthesis in monocytes have used an exogenous triggering agent such as Lipolpoysaccharide (LPS). However, during nonseptic chronic inflammatory responses (e.g., rheumatoid arthritis) monocyte activation occurs as a result of T cell generated signals. This report demonstrated that plasma membranes from anti-CD3 activated peripheral CD4$\sp{+}$ T cells (Tm$\sp{\rm A}$) but not from resting CD4$\sp{+}$ cells (Tm$\sp{\rm R}$) induced monocytes to synthesize IL-1 in the absence of costimulatory cytokines. The expression kinetics of the molecule(s) unique to activated T cells which interact with monocyte receptors to induce IL-1 demonstrated that optimal expression occurred at 6h post activation. This matched Lederman's, et al., (1992) previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of IL-1 induction in monocytes. In this work, it was demonstrated that the signal could be reduced up to 85% by addition of 5c8, a monoclonal anti-CD40L antibody. In addition, a monoclonal anti-CD40 IgM (BL-C4) induced resting monocytes to synthesize IL-1. Experiments demonstrated that crosslinking the CD40 molecules on monocytes was critical for IL-1 induction. Tm$\sp{\rm A}$ but not Tm$\sp{\rm R}$ also up-regulated cell surface expression of adhesion/costimulatory molecules on monocytes including CD40, ICAM-1, and LFA-3. Anti-CD40 signaling up-regulated expression of ICAM-1 and LFA-3. Experiments suggested that signaling through CD40 may utilize a protein tyrosine kinase (PTK) mediated pathway but not a protein kinase C mediated pathway and studies using THP-1, a premonocytic cell line, indicated that the transcription factor, NF-$\kappa$B, was activated through anti-CD40 signaling. Since CD40 ligand-transfected cells alone did not induce IL-1 but Tm$\sp{\rm A}$ did, it was considered that an additional costimulatory cell surface molecule was required. Preliminary experiments suggested that CD69 may be required. In summary, these results indicate that contact-dependent T cell-monocyte interactions, alone, can activate inflammatory cytokine production by resting monocytes and that a critical component of this interaction is the CD40-CD40L signaling event.

Cardiac healing after myocardial infarction (MI) represents the cardinal prerequisite for proper replacement of the irreversibly injured myocardium. In contrast to innate immunity, the functional role of adaptive immunity in postinfarction healing has not been systematically addressed. The present study focused on the influence of CD4+ T lymphocytes on wound healing and cardiac remodeling after experimental myocardial infarction in mice. Both conventional and Foxp3+ regulatory CD4+ T cells (Treg cells) became activated in heart draining lymph nodes after MI and accumulated in the infarcted myocardium. T cell activation was strictly antigen-dependant as T cell receptor-transgenic OT-II mice in which CD4+ T cells exhibit a highly limited T cell receptor repertoire did not expand in heart-draining lymph nodes post-MI. Both OT-II and major histocompatibility complex class II-deficient mice lacking a CD4+ T cell compartment showed a fatal clinical postinfarction outcome characterized by disturbed scar tissue construction that resulted in impaired survival due to a prevalence of left-ventricular ruptures. To assess the contribution of anti-inflammatory Treg cells on wound healing after MI, the Treg cell compartment was depleted using DEREG mice that specifically express the human diphtheria toxin receptor in Foxp3-positive cells, resulting in Treg cell ablation after diphtheria toxin administration. In a parallel line of experiments, a second model of anti-CD25 antibody-mediated Treg cell immuno-depletion was used. Treg cell ablation prior to MI resulted in adverse postinfarction left-ventricular dilatation associated with cardiac deterioration. Mechanistically, Treg cell depletion resulted in an increased recruitment of pro-inflammatory neutrophils and Ly-6Chigh monocytes into the healing myocardium. Furthermore, Treg cell-ablated mice exhibited an adverse activation of conventional non-regulatory CD4+ and CD8+ T cells that showed a reinforced infiltration into the infarct zone. Increased synthesis of TNFα and IFNγ by conventional CD4+ and CD8+ T cells in hearts of Treg cell-depleted mice provoked an M1-like macrophage polarization characterized by heightened expression of healing-compromising induced NO synthase, in line with a reduced synthesis of healing-promoting transglutaminase factor XIII (FXIII), osteopontin (OPN) and transforming growth factor beta 1 (TGFβ1). Therapeutic Treg cell activation by a superagonistic anti-CD28 monoclonal antibody stimulated Treg cell accumulation in the infarct zone and led to an increased expression of mediators inducing an M2-like macrophage polarization state, i.e. interleukin-10, interleukin-13 and TGFβ1. M2-like macrophage differentiation in the healing infarct was associated with heightened expression of scar-forming procollagens as well as scar-stabilizing FXIII and OPN, resulting in improved survival due to a reduced incidence of left-ventricular ruptures. Therapeutic Treg cell activation and the induction of a beneficial M2-like macrophage polarization was further achieved by employing a treatment modality of high clinical potential, i.e. by therapeutic administration of IL-2/ anti-IL-2 monoclonal antibody complexes. The findings of the present study suggest that therapeutic Treg cell activation and the resulting improvement of healing may represent a suitable strategy to attenuate adverse infarct expansion, left-ventricular remodeling, or infarct ruptures in patients with MI. / Die kardiale Wundheilung nach einem Herzinfarkt ist unabdingbare Voraussetzung um das unwideruflich beschädigte Myokard zu ersetzen. Im Gegensatz zur Rolle der angeborenen Immunität ist zur Bedeutung der adaptiven Immunität für die kardiale Wundheilung nur wenig bekannt. Im Fokus der Studie stand deshalb die Rolle von CD4+ T-Zellen bei der Wundheilung und kardialem Remodeling nach einem experimentellen Herzinfarkt im Mausmodell. Sowohl konventionelle, als auch Foxp3-positive regulatorische CD4+ T Zellen (Treg-Zellen) wurden im Lymphknoten infarzierter Tiere aktiviert und akkumulierten im Infarktareal. Die Aktivierung von CD4+ T Zellen nach MI setzte die Erkennung von Selbstantigenen voraus, da OT-II Tiere, die ein stark eingeschränktes T-Zell-Rezeptor-Repertoire aufweisen, keine T-Zell-Aktivierung zeigten. Sowohl OT-II Tiere, als auch Haupthistokompatibilitätskomplex Klasse II Knock-out Mäuse, die kein CD4+ T-Zell Kompartment aufweisen, zeigten einen fatalen klinischen Phänotyp, welcher durch eine gestörte Narbenbildung und damit verbunden einem verstärkten Auftreten linksventrikulärer Rupturen verbunden war. Um den Einfluss anti-inflammatorischer Treg-Zellen auf die kardiale Wundheilung nach Herzinfarkt zu untersuchen, wurde das Treg-Zell Kompartment vor MI Induktion depletiert. Hierzu wurden DEREG Mäuse verwendet, in denen Foxp3-positive Zellen den humanen Diphtherietoxin-Rezeptor exprimieren, sodass nach nach Diphtierietoxin-Applikation die Treg-Zellen spezifisch depletiert werden. In einer dazu parallel verlaufenden Versuchsreihe wurden die Treg-Zellen mittels anti-CD25 monoklonaler Antikörper depletiert. Die Depletion des Treg-Zell Kompartments 2 Tage vor MI Induktion bewirkte ein verschlechtertes links-ventrikuläres Remodeling und damit einhergehend eine signifikante Verschlechterung der Herzfunktion. Mechanistisch führte die Treg-Zell Depletion zu einer verstärkten Rekrutierung pro-inflammatorischer Ly-6Chigh Monozyten und neutrophilen Granulozyten ins Infarktareal. Die Depletion von Treg-Zellen war weiterhin mit einer adversen Aktivierung konventioneller CD4+ und CD8+ T-Zellen assoziiert, die eine verstärkte Infiltration ins infarzierte Myokard zeigten. Die erhöhte Synthese von TNFα und IFNγ in konventionellen T-Zellen führte zu einer M1-Makrophagen Polarisierung, welche durch eine verstärkte Expression der induzierbaren NO Synthase charakterisiert war. Weiterhin exprimierten diese M1 Makrophagen signifikant weniger der für eine geordnete Heilung essentiellen Faktoren Osteopontin, Transglutaminase Faktor XIII und transforming growth factor beta 1 (TGFβ1). Im Gegensatz zu den Depletionsversuchen führte die therapeutische Aktivierung der Treg-Zellen durch einen superagonistischen anti-CD28 monoklonalen Antikörper zu einer verstärkten Rekrutierung von Treg-Zellen ins Infarktareal und bewirkte eine erhöhte Synthese von Interleukin (IL)-10, IL-13 sowie TGFβ1. Die damit einhergehende M2 Makrophagenpolarisierung im Infarktareal war mit einer verstärkten Narbenbildung sowie der Synthese von Osteopontin und Transglutaminase Faktor XIII verbunden, welche sich stabilisierend auf die Narbenbildung auswirken. Folgerichtig zeigten die behandelten Tiere ein verbessertes Überleben aufgrund einer signifikant verringerten Inzidenz linksventrikulärer Rupturen. Alternativ zum superagonistischen anti-CD28 monoklonalen Antikörper wurde ein weiterer klinisch relevanter Ansatz zur therapeutischen Aktivierung von Treg-Zellen verfolgt. Durch die Applikation von IL-2/ anti-IL-2 Antikörper-Komplexen konnte ebenfalls eine M2 Makrophagenpolarisierung hervorgerufen werden, die mit einer verstärkten Narbenbildung einherging. Die Ergebnisse der vorliegenden Studie deuten darauf hin, dass eine therapeutische Aktivierung von Treg-Zellen bei Infarktpatienten und die dadurch hervorgerufene Verbesserung der Wundheilung potentiell einen geeigneten Ansatz darstellt, durch welchen adverses linksventrikuläres Remodeling sowie Infarktrupturen verhindert werden können.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: Existen lesiones bucomáxilofaciales que se relacionan con la infección por VIH/SIDA. La asociación de éstas con estados específicos de inmunosupresión a través de su recuento de linfocitos TCD4 tiene gran valor en el proceso diagnóstico, monitorización de progresión y evaluación de terapia en estos pacientes. Material y Métodos: Se realizó un estudio observacional analítico de corte transversal reuniendo una muestra de pacientes del Servicio de Cirugía Máxilofacial del Hospital San Juan de Dios. Se realizó un examen clínico donde se diagnosticaron lesiones bucomáxilofaciales asociadas a VIH/SIDA. Se examinó el recuento de linfocitos TCD4 de estos pacientes, realizado por el Servicio de Infectología del hospital. Se correlacionó la presencia de lesiones con el resultado del examen, categorizado según la clasificación del Centers for Disease Control and Prevention (CDC). Se utilizó el test de Chi2 de Pearson para encontrar asociación entre las variables y se determinó la magnitud del efecto de las variables independientes sobre la variable de resultado. Se determinaron las razones de prevalencia (RP) mediante modelo de regresión de Poisson robusta. Resultados: Se analizó una muestra de 79 pacientes adultos. De estos pacientes, el 83,54% fueron hombres y 16,46% fueron mujeres. El 21,52% de los pacientes tuvo un recuento de linfocitos TCD4 ≥500 células/mm3 de sangre, un 49,37% tuvo un recuento dentro del rango de 499-200 células/mm3 y un 29,11% tenía <200 células/mm3. El 70,89% de los pacientes no presentó lesiones asociadas a VIH/SIDA. El 29,11% restante presentó lesiones. Se estableció una asociación entre presencia de lesiones bucomáxilofaciales y recuento de linfocitos TCD4 (p=0,009). La RP de presentar una lesión en pacientes con un recuento de linfocitos TCD4 de 499-200 células/mm3 unidades respecto a ≥500 células/mm3 es 0,52 (IC 95% 0,18-1,49) mientras que en los pacientes de <200 células/mm3 respecto a ≥500 células/mm3 es 1,77 (IC 95% 0,77-4,11). Conclusiones: Existe una relación estadísticamente significativa entre el recuento de Linfocitos TCD4 y la presencia de lesiones bucomáxilofaciales asociadas a la infección por VIH/SIDA. Existe un mayor riesgo de presentar lesiones asociadas a la infección por VIH/SIDA en pacientes con un recuento de linfocitos TCD4 <200 células/mm3 de sangre. / Adscrito a proyecto PRIO-ODO 14/003

This study is one of the only investigations to examine the complex inter-relationships between immune status, cognitive functioning, and disease progression in school-aged, perinatally infected, HIV+ children on HAART over time and is the first to conduct long-term follow-up assessments beyond one year after initiating HAART. Previous research has shown that HIV+ children on HAART show stability in cognitive functioning for up to one year. The current study investigated cognitive functioning, as measured by the Wechsler Intelligence Scale for Children -III, as a function of immune functioning and disease progression over time in this sample. Overall, results showed that PIQ scores remained stable over the three time points. However, further analyses demonstrated that poorer immune status, as measured by CD4% <25, at the first time point significantly predicted lower Performance IQ (PIQ)scores and PIQ subtest scores at the third time point, even after controlling for covariates. Similarly, additional analyses revealed that PIQ scores significantly declined over time as a function of CD4% category at the first time point. Finally, scores on the PIQ, Verbal IQ (VIQ), Coding, Picture Arrangement, Symbol Search, and Arithmetic at the first time point were all significant predictors of more advanced disease progression, as measured by CDC C classification at follow-up. The clinical relevance of this study and recommendations for future research in this area are discussed

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.

La caracterització genòmica del VHC al 1989, el desenvolupament de tècniques de detecció d’anticossos i, més recentment, la introducció de mètodes moleculars per a la identificació de l’ARN viral han permès pràcticament eliminar el risc de transmissió del VHC associat a la transfusió de productes sanguinis. No obstant, en la pràctica diària, les tècniques de cribatge d’anticossos i inclús els mètodes confirmatoris basats en immunoblots generen resultats indeterminats (presència d’anticossos enfront a un únic antigen viral en absència de virèmia), representant un problema pels Bancs de Sang a l’hora d’informar als donants sobre la causa de l’exclusió definitiva de la donació altruista. Per altra banda, tot i que l’eficàcia del tractament antiviral de la infecció persistent pel VHC ha millorat sensiblement en els últims anys, existeixen grups especials de pacients en els que la teràpia actualment disponible està contraindicada o és francament insuficient, pel que una millor comprensió dels mecanismes de persistència viral o resolució espontània de la infecció poden ser clínicament rellevants. La resposta immune cel·lular CD4+ i CD8+ tipus Th1 potent, multiespecífica i mantinguda és essencial per a la resolució espontània de la infecció, mentre que la infecció persistent es caracteritza per l’absència o la presència de cèl·lules específiques d’antigen funcionalment alterades. Estudis experimentals en ximpanzés han demostrat que l’absència de col·laboració per part dels limfòcits T CD4+ específics d’antigen condiciona la persistència viral, per la qual cosa és possible que la infecció crònica en l’ésser humà sigui deguda primàriament a una disfunció de la resposta CD4+. Les causes d’aquesta disfunció poden ser vàries, incloent la inducció d’anèrgia a través de lligands peptídics alterats tal com s’ha postulat en el cas de la tolerància a cèl·lules tumorals. En base a aquestes troballes, es van plantejar dos objectius principals de la tesi: (1) determinar el significat dels patrons d’immunoblot indeterminat en donants de sang i (2) avaluar la possible presència de limfòcits T CD4+ anèrgics en pacients amb infecció persistent, determinar la especificitat antigènica, i estudiar la possibilitat de restaurar la capacitat funcional de les cèl·lules T CD4+ específiques del VHC ex vivo. Els resultats dels estudis han permès demostrar en primer lloc que aproximadament la meitat dels donants amb patró d’immunoblot indeterminat representen curacions espontànies de la infecció, suggerint la utilitat de la tècnica d’ELISpot-IFN-γ per diferenciar donants falsos positius de les tècniques serològiques de cribatge d’aquells que presenten infeccions resoltes espontàniament. A més, es va determinar que la resposta cel·lular Th1 CD4+ enfront al domini helicasa de la proteïna NS3 és el factor discriminant de la resposta immune en infeccions resoltes. En el segon estudi es va demostrar presència de limfòcits T CD4+ específics de NS3 disfuncionals en la majoria dels pacients amb infecció persistent, independentment del seu perfil funcional, mitjançant l’activació transitòria antigen – específica del lligand del CD40 (CD154). També es va demostrar la possibilitat de revertir l’estat disfuncional de les cèl·lules T CD4+ específiques de NS3 mitjançant l’expansió in vitro en absència de l'estímul inductor d'anèrgia i en presència de citocines homeostàtiques (IL-7 i IL-15), proporcionant un nou instrument per entendre els mecanismes de persistència viral i investigar l'ús d'aquestes cèl·lules per a estratègies d'immunoteràpia adaptativa. / The HCV genome characterization in 1989, the development of antibodies detection techniques and, more recently, the introduction of molecular methods for identification of viral RNA have allowed to reduce the risk of HCV transmission associated with blood products transfusion. However, in current practice, the antibody screening techniques and the confirmatory methods based on immunoblot assays generate indeterminate results (antibodies against a single viral antigen in the absence of viremia), representing a problem for blood banks to inform donors about the cause of the definitive exclusion of altruistic donation. Moreover, although the effectiveness of antiviral treatment for persistent HCV infection has improved considerably in recent years, there are special groups of patients for whom currently available therapy is contraindicated or is frankly inadequate. A better understanding of the mechanisms of viral persistence or spontaneous clearance may be thus clinically relevant. Sustained persistence of powerful and multispecific CD4+ and CD8+ Th1 responses are essential for spontaneous HCV clearance, and persistent infection is characterized by the absence or the functional alteration of antigen-specific T cells. Experimental studies in chimpanzees have shown that the absence of HCV-specific CD4+ T lymphocytes cooperation can generate viral persistence, so it is possible that chronic infection in humans may be primarily due to a dysfunction of the CD4+ immune response. The causes of this dysfunction may be various, including the induction of anergy by altered peptide ligands, as has been postulated in the case of tolerance to tumour cells. Based on these findings, we raised two main objectives for this thesis: (1) to determine the significance of indeterminate immunoblot patterns in blood donors and (2) to evaluate the presence of CD4+ T lymphocytes with anergic phenotype in patients with persistent infection, to determine the antigenic specificity, and whether HCV-specific CD4+ T cells dysfunction can be reversed in vitro. Study results have allowed to demonstrate that approximately half of donors with indeterminate immunoblot pattern have resolved a previous HCV infection and suggested that ELISpot-IFN-γ might be a useful tool to distinguish donors with false positive serological techniques results from those with resolved spontaneous infections. In addition, we showed that the CD4+ Th1 immune response against NS3 helicase domain is the discriminate factor in the immune response in resolved infection. The second study showed that HCV-specific CD4+ T cells, although dysfunctional, are present in the peripheral blood of most patients with persistent HCV infection and can be easily detected, irrespectively of their functional profile, by the transient antigen - specific upregulation of CD40 ligand (CD154). We also demonstrated that it was possible to rescue NS3-specific CD4+ T cell response in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen and presence of homeostatic cytokines (IL-7 and IL-15), providing a new tool to understand the mechanisms of viral persistence and investigate the use of these cells for immunotherapy of adaptive strategies.

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.