BACKGROUND:The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response.RESULTS:In this paper we show that the primary CD8+ T cell response to the LCMV gp33-41 epitope is extremely diverse. Over time while the response remains robust in terms of the number of gp33-tetramer+ T cells, the diversity of the response becomes less so. Strikingly, by 26months after infection the response is dominated by a small number TCRbeta sequences. In addition, it is of note the gp33 specific CD8+ T cells sorted by high and low tetramer binding populations 15 and 22months after infection. High and low tetramer binding cells had equivalent diversity and were dominated by a small number of clones regardless of the time tested. A similar restricted distribution was seen in NP396 specific CD8+ T cells 26months after infection. The identical TCRVbeta sequences were found in both the tetramerhi and tetramerlo binding populations. Finally, we saw no evidence of public clones in the gp33-specific response. No CDR3 sequences were found in more than one mouse.CONCLUSIONS:These data show that following LCMV infection the CD8+ gp33-specific CD8 T cell response becomes highly restricted with enormous narrowing of the diversity. This narrowing of the repertoire could contribute to the progressively ineffective immune response seen in aging.

Des études récentes menées chez l’homme et la souris ont suggéré que la diversité du répertoire TCR pourrait jouer un rôle dans la protection contre des pathogènes à haut pouvoir mutagène. Afin d’étudier le répertoire des lymphocytes T CD4, nous avons utilisé un modèle de souris TCRβ transgéniques exprimant une chaine β spécifique du peptide env122-141 dans le contexte du MHCII. Suite à l’immunisation des souris TCRβ transgéniques avec des cellules dendritiques pulsées avec le peptide env, une rapide prolifération et une restriction du répertoire des lymphocytes T Vα2 CD4 spécifiques est observée. L’analyse de la diversité du répertoire de ces cellules par séquençage à haut débit, a montré l’émergence d’un répertoire plus divers dans des souris déplétées en lymphocytes T régulateurs. Ces résultats suggèrent qu’en plus du rôle des Tregs dans le contrôle de la magnitude de la réponse immunitaire, ces cellules pourraient également contrôler la diversité du répertoire des lymphocytes T suite à une stimulation antigénique. / Recent studies conducted in mice and humans have suggested a role for the TCR repertoire diversity in immune protection against pathogens displaying high antigenic variability. To study the CD4 T cell repertoire, we used a mouse model in which T cells transgenically express the TCRβ chain of a TCR specific to a MHCII-restricted peptide, env122-141. Upon immunization with peptide-pulsed dendritic cells, antigen-specific Vα2+ CD4+ T cells rapidly expand and display a restricted TCRα repertoire. In particular, analysis of receptor diversity by high-throughput TCR sequencing in immunized mice suggests the emergence of a broader CDR3 Vα2 repertoire in Treg-depleted mice. These results suggest that Tregs may play a role in the restriction of the CD4 T cell repertoire during an immune response, raising therefore the possibility that in addition to controlling the magnitude of an immune response, regulatory cells may also control the diversity of TCRs in response to antigen stimulation. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

As doenças falciformes (DF) constituem um grupo de doenças hereditárias monogênicas. São doenças extremamente relevantes no contexto de saúde pública no Brasil, portanto diferentes estratégias terapêuticas devem ser avaliadas. As oclusões vasculares afetam praticamente todos os órgãos, inclusive o baço e a medula óssea, porém não existem dados na literatura se estas comprometem também o tecido tímico. Os pacientes apresentam maior suscetibilidade às infecções cujas causas não são ainda totalmente esclarecidas Embora as infecções observadas nos pacientes sejam atribuídas à disfunção esplênica, o quadro inflamatório crônico e possíveis alterações no timo e na medula óssea, também poderiam causar uma disfunção imunológica. O objetivo deste trabalho foi avaliar a neogênese de células T e B e a diversidade do repertório de células T periféricas em pacientes com anemia falciforme (AF) sem tratamento (N = 15), tratados com hidroxiuréia (N = 20) ou transfusão crônica (N = 21) e em pacientes com DF tratados com transplante de células-tronco hematopoéticas (TCTH) alogênico (N = 29). Pacientes sem tratamento apresentaram menores níveis de sjTREC e ?-TREC, e menor taxa de divisão celular intratímica, demonstrando alterações importantes na neogênese das células T. A produção tímica de novas células T naïve foi reestabelecida em um ano pós-transplante, com normalização dos níveis de sjTREC e ?-TREC. O desenvolvimento de doença do enxerto contra o hospedeiro (DECHa) e reativação de citomegalovírus comprometeu a timopoiese nos primeiros seis meses pós-transplante, com diminuição significativa dos níveis de sjTRECs e ?-TRECs. Análises do repertório da cadeia V? dos receptores de células T (TCRs), pelo método TCRBV CDR3 spectratyping, indicaram que os pacientes com AF apresentaram um repertório menos diverso, composto predominantemente de famílias V? com padrão skewed e picos de CDR3 monoclonais, sendo a família V?3 mais frequente. A composição do repertório de células T foi alterada após o transplante, adquirindo um perfil mais policlonal dos picos de CDR3 ao longo do tempo. A família V?22 foi a mais expressa no período pré-transplante e em todos os seguimentos pós-transplante. Os pacientes com DF apresentaram aumento de linfócitos B naive, demonstrado pelos altos níveis de sjKRECs e pela taxa de proliferação homeostática. As análises multivariadas demonstraram que as alterações esplênicas influenciam diretamente os níveis de sjKREC, indicando que a baixa função esplênica leva ao aumento da produção de células B naive pela medula óssea, sugerindo um mecanismo compensatório. Os resultados desse trabalho demonstraram a existência de um desequilíbrio na neogênese de células T e B e consequentemente nesses compartimentos celulares periféricos, que pode conferir aos pacientes com DF uma maior susceptibilidade a infecções. Entre as diferentes modalidades terapêuticas, o TCTH alogênico sobressaiu-se em relação aos tratamentos convencionais, melhorando a neogênese de células T e B a longo prazo. / Sickle Cell Disease (SCD) are a group of monogenic hereditary diseases. These are extremely relevant diseases in the context of public health in Brazil, thus, different therapeutic strategies must be studied. Vascular occlusions affect practically all organs, including the spleen and bone marrow. However, there are no literature data about the impact of vaso-occlusions on the thymic tissue. Patients are more susceptible to infections whose causes are not fully elucidated. Although the infections observed in these patients are assigned to splenic dysfunction, the chronic inflammatory state and possible alterations of the thymus and bone marrow could also lead to immune dysfunction. The goal of this work was to evaluate the neogenesis of T and B cells and the diversity of peripheral T cell repertoire in patients with sickle cell anemia (SCA) without treatment (N = 15), treated with hydroxyurea (N = 20) or chronic transfusions (N = 21) and in patients with SCD treated with hematopoietic stem cell transplantation (N = 29) allogeneic. Patients without treatment had lower levels of sjTREC and ?-TREC, and lower rate of intrathymic cell division, demonstrating important alterations in the neogenesis of T cells. The thymic production of new naïve T cells was reestablished at one-year post transplantation, with normalization of sjTREC and ?-TREC levels. The development of graft-versus-host disease (aGVHD) and cytomegalovirus activation compromised thymopoiesis in the first six months post transplantation, with a significant decrease of sjTRECs and ?-TRECs levels. Analysis of the TCR V? chain repertoire by TCRBV CDR3 spectratyping indicate that patients with SCA showed a less diverse repertoire, mainly composed by V? families with a skewed pattern and monoclonal CDR3 peaks, being the V?3 family the most frequent one. The composition of the T-cell repertoire was altered after transplantation, changing over time to more polyclonal profile of the CDR3 peaks. The V?22 family was the more expressed at pre-transplantation and at all follow-up periods. Patients with SCD presented increased numbers of naive B cells, demonstrated by higher levels of sjKRECs and homeostatic proliferation. Multivariate analysis demonstrated that splenic function directly influenced sjKREC levels, indicating that compromised splenic function leads to increase of naive B cell output by the bone marrow, suggesting a compensatory mechanism. The results of this study showed the existence of an imbalanced T and B cell neogenesis and, consequently on these peripheral cell compartments, which may confer to patients with SCD an increased susceptibility to infections. Among different therapeutic modalities, allogeneic HSCT stood out in relation to the conventional treatments, improving long-term T and B cell neogenesis.