No description available.

No description available.

La transplantation de sang de cordon ombilical (TSCO) est utilisée pour traiter les enfants atteints de maladies hématologiques en l’absence de donneurs apparentés compatibles. Elle est associée avec des risques plus élevés d’échec de greffe et d’infections opportunistes dans les premiers mois qui suivent la transplantation en comparaison avec une greffe de moelle osseuse. Par contre, la TSCO comporte un risque plus faible de maladie du greffon contre l’hôte et une incidence comparable de rechute de leucémie. Ces quatre complications impliquent directement les lymphocytes T. Dans le but de mieux comprendre le schéma particulier des évènements qui suivent la TSCO et d’améliorer le pronostic des patients, nous avons étudié le potentiel fonctionnel, la persistance et la reconstitution antivirale des lymphocytes T au sein d’un groupe d’enfants transplantés de sang de cordon ombilical (SCO). Étant donné que le SCO contient une majorité de lymphocytes T naïfs, nous avons étudié les lymphocytes T spécifiques au HLA-A2:Melan-A26-35 A27L; seul répertoire naïf et abondant caractérisé chez l’homme. Nous avons observé que les lymphocytes T du SCO se différencient en populations effectrices, s’oligoclonalisent, produisent de l’IFN-γ et lysent spécifiquement leur cible suite à la stimulation. Néanmoins, ces cellules produisent moins d’IFN-γ et sont moins bifonctionnelles que leurs homologues issus du sang périphérique d’adultes. Chez les patients, les lymphocytes T du SCO s’épuisent après la TSCO : ils s’oligoclonalisent dramatiquement, sont principalement en différenciation terminale, et une importante fréquence exprime PD-1 (« programmed death-1 ») dans les 3 à 6 premiers mois post-greffe. Très peu de patients sont capables de développer des réponses antivirales durant cette période et la fréquence de lymphocytes T qui expriment PD-1 semble aussi avoir un impact sur le risque subséquent de faire une rechute de leucémie. La deuxième vague de lymphocytes T émergeant à 6 mois post-TSCO mène à une population fonctionnelle et diversifiée. En conclusion, la fonctionnalité des lymphocytes T présents dans les 3 à 6 premiers mois post-TSCO doit être rétablie pour améliorer les risques d’infections opportunistes et de rechute de leucémie. / Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells to treat a variety of disorders in children. UCB transplantation (UCBT) is associated with a reduced incidence of graft-versus-host disease, a robust graft-versus-leukemia effect, more frequent graft failures and a higher incidence of opportunistic infections (OI) compared to bone marrow transplantation; four processes in which donor-derived T lymphocytes are known to be predominantly involved. UCB T cells are mostly naïve. To examine the differential functionality of UCB T cells, CD8+ T cells specific for the melanoma-associated HLA-A2-restricted Melan-A26-35 A27L peptide were isolated from UCB and UCBT recipients, as it represents an abundant preimmune repertoire in human. Following in vitro stimulation, UCB T cells proliferated, oligoclonalized, acquired cell surface markers reflective of effector/memory differentiation, expressed cytolytic activity and produced IFN-γ. While functional properties of UCB T cells resembled their counterparts in adult peripheral blood, they were more likely to reach terminal differentiation following stimulation, produced less IFN-γ and were less frequently bifunctional (IFN-γ and cytolysis). Following UCBT, T cells became exhausted: they oligoclonalized dramatically, exhibited a terminal differentiation phenotype and a high frequency also expressed PD-1 (“ programmed death 1 ”) in the first 3-6 months post-UCBT. Moreover, very few patients developed an antiviral response during this period. Finally, the frequency of PD-1+ CD8+ T cells was significantly higher in subjects who subsequently experienced leukemic relapse. A second wave of T cells emerging at 6 months post-UCBT was observed and characterized by an increase in the repertoire diversity till 1 year, the development of a naïve population with polyfunctional potential and the progressive reconstitution of antiviral responses. This study reports to the biological properties of UCB-derived CD8+ T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the first wave of CD8+ T cells in the first 3-6 months post-UCBT should be targeted in priority to improve both OI and leukemic relapse risks.

Notre travail montre la prédominance du clone « Lyon » parmi les souches de Staphylococcus aureus responsables d’infections invasives (i.e. bactériémies). Il est caractérisé par la présence du gène codant la «staphylococcal enterotoxin » (SE) A. Afin de mieux comprendre le lien éventuel entre la prédominance de la SEA et la gravité des infections induites, nous avons orienté nos travaux sur le versant immuno‐inflammatoire des SE. L’étude des répertoires Vβ induits par l’ensemble des SE s’est révélée peu discriminante. A l’aide d’approches génomiques et protéiques réalisées sur des lymphocytes et monocytes humains, nous avons observé le puissant potentiel inflammatoire de la SEA en particulier par rapport à la SEG (SE appartenant à l’opéron egc retrouvé dans les infections de faible sévérité) et la prédominance de la réponse lymphocytaire T. Enfin, afin de mieux comprendre la spécificité des effets observés, nous avons conduit des expériences sur lymphocytes purifiés en ciblant les Ly T effecteurs (CD4+CD25‐) et Ly T régulateurs (CD4+CD25+FOXP3+). Nos travaux ont confirmé l’induction par la SEA d’une forte réponse inflammatoire, associant une synthèse de cytokines de type Th1 et Th17 dans les deux lignées lymphocytaires. De plus, nos résultats suggèrent que les SE provoquent une perte des fonctions suppressives des Ly T régulateurs ouvrant de nouvelles perspectives quant à l’implication des SAgs dans de nombreuses situations cliniques en particulier le choc septique, et les infections chroniques / Our work shows the prevalence of the “Lyon” clone among Staphylococcus aureus strains responsible for severe systemic infections (i.e. bacteremia). This clone is characterized by the presence of the staphylococcal enterotoxin (SE) A coding gene. To better understand the possible link between the prevalence of SEA and the severity of infections, we focused our work on the immuno inflammatory responses of SE. By using genomic and protein studies, in human lymphocytes and monocytes, we observed the potent inflammatory property of SEA (especially in comparison with SEG ‐ belonging to the egc cluster mainly found in less severe infections) mainly targeting T cell response over monocytes. Finally, to better understand the specificity of these effects, we performed experiments on purified lymphocytes and targeted effectors (CD4+CD25‐) and regulatory T lymphocytes (CD4+CD25+FOXP3+). Our works confirmed the induction by SEA of a strong inflammatory response, characterized by the release of Th1 and Th17 cytokines in both lymphocyte lineages. Furthermore, our results also showed the loss of the regulatory T cell suppressive functions after SE stimulation. Collectively, these results offer new perspectives for the implication of the SAgs in numerous clinical situations in particular toxic shock and the chronic infections.

Cytotoxic T lymphocytes (CTL) kill virally infected and tumourigenic cells via the regulated secretion of specialised secretory lysosomes. These secretory lysosomes contain cytolytic effector molecules, such as perforin and granzymes, which are able to induce apoptosis in target cells. Secretion occurs at the contact point between the CTL and its target, in a highly structured region termed the immunological synapse (IS). Upon formation of the IS, CTL undergo polarisation of their microtubule cytoskeleton and movement of the microtubule organising centre (MTOC) to the IS. Secretory lysosomes are then able to polarise along microtubules, fuse with the plasma membrane and deliver their effector molecules to the IS. The Adaptor Protein 3 complex (AP-3) sorts transmembrane proteins to lysosomes and deficiency in AP-3 results in missorting of proteins from the lysosomal to plasma membrane. CTL from AP-3 deficient patients, who suffer from Hermansky-Pudlak Syndrome Type 2 (HPS2), show reduced killing of target cells. This thesis describes two new patients with HPS2, both with homozygous mutations in the AP3B1 gene, which codes for the β3A subunit of the AP-3 complex. CTL from the new HPS2 patients show reduced cytotoxicity, which is shown here to be due to impaired secretory lysosome polarisation towards the IS. This impairment is common to HPS2 CTL, but varies between patients. In order to determine differences between HPS2 and wild type CTL, the localisation of a range of lysosomal, cytolytic, transmembrane, inhibitory and activation marker proteins is examined. This shows that in HPS2 CTL, LAMP1, CD63 and CD9 are potential AP-3 cargos. In addition, a possible effect on the key lytic effector perforin is identified. Preliminary experiments to allow proteomic comparison of HPS2 and wild type CTL are also presented. Further investigation of these results will help to shed light on the mechanisms involved in secretory lysosome polarisation in CTL.

L’immunosuppression optimale après greffe d’organe solide est une balance délicate et propre à chaque individu entre le risque de rejet et les risques liés à une surexposition au traitement immunosuppresseur. L’évaluation de la fonction résiduelle des lymphocytes T après stimulation par un mitogène (pharmacodynamie effective) devrait permettre de mesurer l’effet direct des médicaments immunosuppresseurs sur leur cible. Nous avons étudié différents paramètres de pharmacodynamie effective chez 34 receveurs pédiatriques de greffe d’organes solides traités par tacrolimus et mycophénolate. Les tests proposés dans ce travail sont adaptés au milieu pédiatrique et à une réalisation en temps réel. La quantification du CD25 parmi les CD4 activés par l’OKT3 permet de distinguer deux groupes de patients selon leur degré d’immunosuppression. L’âge médian est plus bas et la concentration plasmatique médiane en MPA plus élevée dans le groupe de patients plus fortement immunosupprimés. L’étude des paramètres immunologiques pouvant influencer la réponse (sécrétion des interleukines, proportion des sous-populations lymphocytaires CD4, CD8, T naïfs et Trég) ainsi que l’étude du pouvoir de restauration de la fonction lymphocytaire par l’Il-2, la guanosine ou la xanthosine, ne permettent pas de mieux comprendre les variabilités interindividuelles observées. Ces résultats devront être confirmés sur une cohorte plus grande de patients afin de juger de leur intérêt en pratique clinique. / Optimal immunosuppression following solid organ transplantation is unique to each individual and requires a balance between risks of rejection and overexposure to immunosuppressive therapy. The evaluation of residual function of T lymphocytes after mitogen stimulation (effective pharmacodynamic monitoring) should allow measurement of the direct effect of immunosuppressive drugs on their target. We studied various parameters of effective pharmacodynamic monitoring in 34 paediatric patients receiving solid organ transplants and treated with tacrolimus and mycophenolate (MPA). The tests proposed in this work are adapted to the paediatric setting in real time. Quantification of CD25 among CD4 cells activated by OKT3 can differentiate two groups of patients according to their degree of immunosuppression. Median values for age and MPA plasma concentration are lower and higher, respectively, in the patient group most heavily immunosuppressed. Neither study of the parameters that may influence the response (secretion of interleukins, proportion of lymphocyte subpopulations CD4, CD8, naive and regulatory T cells) nor study of the restoration of basal cell function brought about by Il2, guanosine or xanthosine, helped to explain the observed inter-individual variability. These results should be confirmed in a larger cohort of patients in order to test their relevance in clinical practice.

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.

Une petite population de lymphocytes T exprimant les deux corécepteurs CD4 et CD8 et appelée double positive (DP), a été détectée dans le sang périphérique de donneurs sains et de patients atteints de diverses pathologies dont la sclérose en plaques (SEP). Nous avons émis l’hypothèse qu’il s’agissait de lymphocytes T hautement activés pouvant contribuer à l’inflammation chronique présente dans la SEP. Nous avons comparé les cellules T DP obtenues du sang de donneurs sains et de patients atteints de la SEP et non traités. La fréquence des cellules DP était similaire chez les patients et les donneurs sains. La proportion de lymphocytes T DP qui exprimaient les chaines du récepteur de l’interleukine-15 (IL-15) était plus élevée que pour les autres populations lymphocytaires. Des mesures d’induction de la phosphorylation du STAT5 (signal transducer and activator of transcription) ont démontré que les cellules DP ont répondu à des doses plus faibles et pour de plus longues périodes à l’IL-15 comparativement aux autres lymphocytes T. Le pourcentage de lymphocytes T DP ayant la capacité de produire l’interféron-gamma et des enzymes lytiques était élevé chez les témoins sains mais ces niveaux étaient significativement réduits chez les patients atteints de la SEP. La caractérisation phénotypique de cellules DP a suggéré que ces cellules ont des propriétés similaires aux lymphocytes T activés. Bien qu’il ne s’agisse que d’une caractérisation partielle, il semble que les lymphocytes T DP perdent une partie de leurs propriétés chez les patients atteints de la SEP. / A small population of T lymphocytes expressing both CD4 and CD8 called double positive (DP) T lymphocytes has been detected in the peripheral blood of healthy donors and patients affected by different pathologies such as multiple sclerosis (MS). We hypothesize that these cells represent a highly activated T lymphocyte subset that could contribute to the characteristic inflammation found in MS. Thus, we compared DP T cells from healthy donors to those from untreated MS patients. We found similar frequencies of DP T lymphocytes between both groups. A higher percentage of DP T lymphocytes expressed the IL-15R (interleukin 15 receptor) than other T cell populations. Moreover, IL-15 triggered the phosphorylation of STAT5 in a greater proportion of CD4+CD8+ T lymphocytes compared to other T cells. A greater percentage of CD4+CD8+ T lymphocytes can produce interferon gamma and lytic enzymes compared with other T cell subsets. However, those levels were drastically lower in MS patients. The phenotypic characterization of the DP cells suggests they have similar properties as activated T cells. Even though the characterization process is still in its infancy, it appears that the DP T cells may lose some of their properties in MS patients.

5 Anglický abstrakt Experimental autoimmune encephalomyelitis (EAE) is widely accepted as a murine model of human multiple sclerosis autoimmune disease. Murine EAE is usually actively induced by immunization with a suitable myelin antigen. Following immunization, CD4+ T helper lymphocytes Th1 and Th17 accumulate in the nervous tissue and via the production of cytokines, they mediate an inflammatory reaction and the subsequent destruction of myelin. The main goal of this study was the induction of EAE with clinically observable symptoms and the observation of changes in the counts and phenotypes of cells, mainly NK and T cells. NK cells express a wide range of inhibitory and activation receptors from the C-lectin-like receptor superfamily. The specific ligand of the activating NKR-P1C isoform is still unknown and thus this receptor's involvement in EAE was also observed. Another goal was the use of medication with regard to the disease progress improvement. For the purposes of this study, two inbred murine strains with distinct NKR-P1 surface expression were used - the SJL/J strain (expressing inhibitory NKR-P1B) and C57BL/6 (expression activating NKR-P1C). SJL mice elicited a relapse-remitting of EAE, while C57BL/6 had chronic EAE. Both mouse strains exerted changes in the counts of NK...

INTRODUÇÃO: A hanseníase é uma doença infectocontagiosa granulomatosa crônica, causada por Mycobacterium leprae e representa ainda um problema de saúde pública no Brasil. Em média 30-40% dos doentes tem risco de desenvolver reações hansênicas, que são considerados estados de exacerbação da resposta imunológica do hospedeiro a antígenos M. leprae. Está bem descrita a importância de células T reguladoras (Tregs) em infecções bacterianas crônicas. Estudos anteriores do nosso grupo demonstraram aumento da proporção de Tregs in situ e em sangue periférico de pacientes multibacilares (mas não em paucibacilares), assim como diminuição da proporção de Tregs na reação tipo 2 (porém não na reação tipo 1), sugerindo a participação deste subtipo celular na imunopatogênese da hanseníase. Entretanto, a baixa frequência de Tregs tem dificultado o estudo da sua capacidade funcional. OBJETIVO: Estabelecer protocolo de expansão de Tregs funcionais em pacientes com hanseníase e, posteriormente, avaliar a capacidade funcional das Tregs expandidas. METODOLOGIA: Células mononucleares do sangue periférico (PBMCs) foram separadas e cultivadas, com anti-CD3 e anti-CD28, acrescidos de IL-2 e rapamicina, por 21 dias. Para avaliar a capacidade funcional das Tregs expandidas, realizou-se um ensaio de co-cultivo das Tregs expandidas e PBMCs autólogas (marcadas com CFSE). Utilizou-se diferentes proporções de Tregs:PBMCs (1:1, 1:2 e 1:5). A casuística foi composta por 29 indivíduos diagnosticados com hanseníase, divididos em quatro grupos, de acordo com a classificação de Ridley e Jopling. Além disso, coletou-se material de 6 indivíduos saudáveis, com a finalidade de caracterizar o pool celular do protocolo de expansão. Para isso, culturas de expansão de indivíduos saudáveis foram submetidas a dois protocolos distintos de purificação celular (beads magnéticas e cell sorting), além de imunofenotipagem das moléculas CD39, PD-1 e LAG3. RESULTADOS: Tregs dos pacientes multibacilar sem reação, nas proporções 1:2 e 1:5, e no grupo reacional tipo 2, na proporção 1:5, mostraram maior capacidade supressora, quando comparados aos grupos pacubacilar sem reação e reacional tipo 2, respectivamente. No grupo de indivíduos saudáveis não houve diferenças na capacidade supressora das Tregs obtidas por separação por beads magnéticas e Tregs obtidas por cell sorting, e não houve expressão significativa de células PD-1+ e LAG-3+ nas culturas de expansão. CONCLUSÃO: Em todos os indivíduos testados, independente da classificação clínica da hanseníase, o protocolo proposto resultou em uma expansão exponencial de uma linhagem de Tregs com atividade supressora, porém com sugestão preliminar de maior eficiência supressora das Tregs na forma MB (com ou sem reação), corroborando a correlação entre atividade supressora de Tregs e maior carga bacilar. Além disso, o protocolo com beads magnéticas se mostrou tão eficaz quanto o protocolo de purificação por cell sorting. A imunofenotipagem das Tregs expandidas de indivíduos saudáveis indicou que não havia número significativo de células exaustas ao fim de nosso protocolo, assim como não houve crescimento concomitante de células T reguladoras do tipo 1 na cultura de expansão / INTRODUCTION: Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae and a public health problem in Brazil. On average, 30-40% of patients are at risk of developing leprosy reactions, which are considered as states of exacerbation of the host\'s immune response to M. leprae antigens. The importance of regulatory T cells (Tregs) in chronic bacterial infections has been well described, but their role in leprosy remains unclear. Previous studies from our group have shown increased proportions of Tregs in situ and in peripheral blood of multibacillary patients (but not in paucibacillary patients), and decrease in the proportion of Tregs in type 2 reaction (but not type 1 reaction), reinforcing the role played by these cells in the immunopathogenesis of leprosy. However, the low numbers of Tregs preclude further studies on their functional capabilities. OBJECTIVE: To establish a protocol for expansion of functional Tregs in patients with leprosy and evaluate the functional capacity of the expanded Tregs. METHOD: Peripheral blood mononuclear cells (PBMCs) were separated and cultured under anti-CD3 and anti-CD28 stimulation, plus IL-2 and rapamycin for 21 days. To evaluate the functional capacity of the expanded Tregs, a co-culture assay of the expanded Tregs and autologous PBMCs (labeled with CFSE) was performed. Different ratios of Tregs: PBMCs (1: 1, 1: 2 and 1: 5) were used. The sample consisted of 29 individuals diagnosed with leprosy, divided into four groups, according to the Ridley and Jopling classification. In addition, samples from 6 healthy individuals were collected to characterize better the cellular characteristics of the expansion protocol. For this, cultures of healthy individuals PBMC were submitted to two distinct cell purification protocols (magnetic beads and cell sorting), as well as immunophenotyping of the molecules CD39, PD-1 and LAG3. RESULTS: Expanded Tregs from the multibacillary patients without reaction, at 1:2 and 1:5 ratios, and with type 2 reaction, at 1: 5 ratio, showed greater suppressive capacity that the respective paucibacillary groups. Tregs from healthy individuals obtained through magnetic beads separation and through cell sorting did not show differences in suppressor capacity, and there was no significant expression of PD-1 + and LAG-3 + on cells of the expansion cultures. CONCLUSION: In all individuals tested, irrespective of the leprosy classification, the proposed protocol resulted in an exponential expansion of Tregs with suppressive capacity; however, preliminary evidence indicated stronger suppressor activity of the Tregs from multibacillary patients (with or without reaction), suggesting a direct correlation between Tregs activity and bacillary load in leprosy. In addition, our protocol with magnetic beads proved to be as effective as the cell sorting protocol. Immunophenotyping of expanded Tregs from healthy subjects revealed that there was no significant number of exhausted cells at the end of our protocol, nor there was the concomitant outgrowth of type 1 regulatory T cells in the expansion protocol