Introdução: A doação de plaquetas por aférese é um método de coleta que vem aumentando em relevância. Sabe-se que esta técnica apresenta inúmeras vantagens em comparação à doação de sangue total. Observamos que há uma preocupação na qualidade dos hemocomponentes enviados ao paciente, no entanto, não se observam muitas pesquisas em busca do cuidado com o doador. Órgãos como o Food and Drug Administration (FDA) já publicaram normas mais restritivas em relação à doação de plaquetas por aférese, pois pesquisas apontaram uma diminuição de algumas células e proteínas do sistema imunológico em doadores de repetição. Objetivos: Analisar doadores de plaquetas de repetição quanto a parâmetros hematimétricos e quantificação de subpopulações linfocitárias comparando-os com um grupo controle composto por doadores de sangue total que não doam há no mínimo um ano ou doando pela primeira vez e, ainda avaliar se a frequência de doações, o tempo de procedimento e o número de plaquetas doadas influenciam na contagem de leucócitos totais e nas subpopulações de linfócitos. Metodologia: Foram analisados 88 indivíduos em um estudo caso-controle, sendo que o grupo controle (CO) incluído foi de doadores de sangue total que haviam doado pela primeira vez ou haviam doado sangue total há mais de um ano. Os casos (CA) incluídos foram os doadores de repetição de plaquetaférese (quatro ou mais doações no último ano). O pareamento foi feito por sexo e idade. As amostras de sangue periférico foram coletadas em tubos contendo EDTA e analisadas em até 6 horas por citometria de fluxo, através da utilização de anticorpos monoclonais anti-CD3, CD4, CD8, HLADR, CD19 e CD56. Resultados: Foram avaliados 44 pares de doadores (caso vs controle). Destes, 81,8% eram homens, a média de idade dos grupos foi de 46 ±13 anos nos casos e 47 ±11 nos controles. Comparando os dois grupos, observou-se diferença estatisticamente significativa (p<0,05) na média de quantificação de leucócitos absolutos CA= 6476,6/μL vs CO=7115,4/μL (p=0,017), na média de linfócitos absolutos CA= 1862,6/μL vs CO= 2239,2/μL (p=0,007) e nos marcadores: CD3+/CD8+ (absoluto) CA= 437/μL vs CO= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47,3/μL vs CO= 42,77/μL (p=0,007). Conclusões: Neste estudo foi possível observar que há uma diminuição em algumas células linfoides dos doadores de repetição em relação aos doadores convencionais, no entanto essa diferença não tem relevância clínica, demonstrando que os intervalos de doações que estes doadores estão sendo submetidos é adequado. A contagem de plaquetas dos doadores de repetição se mantiveram no decorrer do ano, este dado nos auxilia para mantermos um banco de dados de doadores de repetição com uma quantificação de plaquetas adequada, podendo ser convocado sem risco de ser bloqueado por contagem inferior ao preconizado. / Introduction: The donation of platelets by apheresis as a collection method has lately grown in relevance. This technique presents several advantages when compared to total blood donation. We understand there is a concern about the quality of the hemocomponents that are administered to the patients; however, there are not many researches concerned with caring for the donor. Entities such as the Food and Drug Administration (FDA) have published more restricting regulations regarding the donation of platelets by apheresis, since researches indicate a decrease in some cells and proteins present in the immunological systems of repeat donors. Objectives: To analyze repeat donors of platelets with regards to hematimetric parameters and quantification of lymphocyte sub-populations by comparing them with a control group consisting of total blood donors that have not donated blood for the past year at least or that are donating for the first time. Additionally, to evaluate if the frequency of donations, the duration of the procedure, and the donated platelet counts influence in the total leukocyte counts and in the sub-populations of lymphocytes. Methodology: We analyzed 88 individuals in a control case study. The control group (CG) consisted of total blood donors in their first donation or that had donated for the last time more than a year before. The cases (CA) included were the repeat donors by platelet apheresis (four or more donations in the past year). We matched the individuals by gender and age. Peripheral blood samples were collected in tubes containing EDTA and analyzed up until 6 hours later by flow cytometry, through monoclonal antibodies anti-CD3, CD4, CD8, HLADR, CD19, and CD56. Results: 44 pairs of donor were evaluated (case vs control). Among them, 81.8% were men, the average age of the groups was 46 (±13) years in the cases and 47 (±11) in the controls. When comparing the two groups, we observed a statistically significant difference (p<0,05) in the average of the quantification of absolute leukocytes CA= 6476.6/μL vs CG=7115.4/μL (p=0.017), in the average of absolute lymphocytes CA= 1862.6/μL vs CG= 2239.2/μL (p=0.007), and in the markers: CD3+/CD8+ (absolute) CA= 437/μL vs CG= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47.3/μL vs CG= 42.77/μL (p=0.007). Conclusions: We were able to note in this study that there is a significant decrease in some lymphoid cells of repeat donors when compared to conventional donors. This difference, however, is not clinically relevant, which demonstrates that the donation intervals to which the donors are subject are appropriate. Platelet numbers of repeat donors remained the same throughout the year. This piece of data helps us keep a database of repeat donors with an adequate platelet number. These donors can be called for without risking of their being blocked in the screening for a number lower than the recommended.

Ces dernières années ont été marquées par une progression importante dans la connaissance de la physiologie des cellules B in vivo et de leur différenciation en plasmocytes, grâce aux modèles murins et à l'imagerie intravitale. La transposition à l'Homme des connaissances acquises chez la souris soulève cependant des difficultés, telles que le manque d'outils pour visualiser les évènements qui se déroulent dans les organes lymphoïdes humains. Dans l'optique d'apporter une réponse à cette problématique, nous avons développé au sein du laboratoire un modèle in vitro en deux étapes, permettant la différenciation des lymphocytes B naïfs humains en plasmocytes. Ce modèle est particulièrement adapté pour étudier l'induction, au cours de la différenciation terminale B, des voies de signalisation, des facteurs de transcription et des grands processus cellulaires tels que la prolifération ou la mort programmée. A l'aide de ce modèle, nous avons mené deux études relatives à la différenciation B dans le centre germinatif: (i) La caractérisation sur le plan phénotypique et fonctionnel des populations cellulaires générées in vitro. Par une approche transcriptomique, nous avons comparé le profil d'expression et identifié les gènes et fonctions biologiques spécifiques à chacune de ces populations différenciées. Nous nous sommes concentrés sur l'étude de l'expression et de l'activité de l'enzyme AID, notamment au travers de la mesure des fréquences des hypermutations somatiques à chaque stade de la différenciation. (ii) L'étude des mécanismes cellulaires (cycle cellulaire, apoptose), génétiques et épigénétiques menant à la transition des cellules du centre germinatif vers le stade de plasmablastes. Cette étude a permis de caractériser au plus près une population cellulaire fondatrice à l'origine des cellules différenciées. En outre, nous avons mis en évidence l'importance de la méthylation de l'ADN, en particulier la 5-hydroxyméthylation, dans le contrôle de la différenciation terminale B. / Within the past few years there has been a significant increase in the knowledge of B cell physiology in vivo and their differentiation into plasma cells through the implementation of murine models and intravital imaging. However, the transposition of this knowledge from mouse to man raises difficulties such as the lack of tools available to visualize the ongoing events in human lymphoid organs. In order to overcome this issue, we have developed in our laboratory, a two-step in vitro model allowing naive B cell differentiation into plasma cells. This model is particularly suitable for studying the induction of signaling pathways, transcription factors and major cellular processes such as proliferation or programmed cell death. Using this model, we conducted two studies on B cell differentiation in the germinal center: (i) phenotypic and functional characterization of cell populations generated in vitro. This study involved a transcriptomic approach which identified and compared the expression profile of specific genes and their biological functions within each of these differentiated populations. Specifically, we focused on the expression and activity of the AID enzyme through the measurement of somatic hypermutation frequency at each stage of differentiation. (ii) The study of cellular (cell cycle, apoptosis), genetic and epigenetic mechanisms leading to the transition of the germinal center B cells into plasmablasts. This study allowed us to characterize a founder cell population of the in vitro generated plasmablasts. In addition, we have highlighted the importance of DNA methylation, in particular 5- hydroxymethylation in controlling terminal B cell differentiation.

B and T lymphocytes are critical to the adaptive immune response against invading microorganisms. B and T cells develop in the bone marrow and thymus, respectively, and initiate a series of proliferative responses once they encounter their cognate antigen in the peripheral lymphoid organs. These developmental and functional processes are controlled by different networks of transcriptional regulators that repress and activate gene expression. Identifying proteins that activate or repress specific genes and integrating these proteins into their transcriptional networks is critical to understanding lymphocyte development and function. The study of B lymphopoiesis and CD4+ T cell functional responses has greatly increased our understanding of how transcriptional regulators and other proteins cooperate to specify cell fates and responses. While many of the key components of these protein networks have been defined, several factors have yet to be described. Chemically induced random mutagenesis is a powerful tool for identifying genes that have critical biological functions. Justy mutant mice were generated by injecting wild-type mice with of N-Ethyl-N-Nitrosourea (ENU), a mutagen, which generated a unique point mutation in the mouse Gon4-like (Gon4l) gene. This mutation was found to specifically blunt B cell development and impair the functional responses of CD4+ T cells. Given that the Gon4l protein contains domains implicated in transcriptional regulation and B lymphopoiesis and T cell responses are regulated transcriptionally, the aim of this project was to characterize T and B lymphocyte populations from Justy mice and provide insights into the mechanisms underlying the regulation of gene expression during these biological processes. The work presented in this dissertation demonstrates that the protein encoded by Gon4l is essential for B lymphopoiesis, likely through the repression of alternate lineage genes. This work also shows that in CD4+ T cells, decreased Gon4l protein expression results in reduced levels of proliferation in response to exogenous IL-2 or T cell receptor (TCR) engagement. Additionally, Justy mutant CD4+ T cells display a reduced ability to generate IFNγ-producing cells in response to Th1 polarization in vitro. Collectively, these defects correlate with elevated levels of genes known to specifically inhibit the above developmental and functional processes. Thus, this dissertation proposes that Gon4l acts as a transcriptional repressor within the protein networks controlling B lymphopoiesis and CD4+ T cell responses.

The purpose of this study was to identify the effects of the neurotransmitter, serotonin (SE), on the immune function of peripheral blood mononuclear cells (PBMC) of normal, healthy subjects. This was done as a preliminary investigation to studies on the association of SE with immune changes in autistic subjects. The PBMC isolated from normal male subjects were treated with various concentrations of SE for 48 hrs. Their incubation in SE at a concentration of 10-3 M induced about a 35% decrease in DNA synthesis. However, incubation of the cells in lower concentrations (10-4 to 10-10) of SE produced no significant effect. The ability of natural killer (NK) cells to lyse K562 target cells was also examined after incubation with SE for 48 hrs. The NK activity was almost completely eliminated following incubation in 10-3M of SE, but the activity was not significantly decreased by exposure to lower concentrations of SE. The viability of PBMC was not altered following incubation with SE under identical conditions as those utilized in the NK assay. Preliminary analysis using a fluorescence-activated cell sorter (FACS) of monoclonal antibodies directed against Tll (total T cell), T4 (helper T cell), T8 (suppressor and cytotoxic T cells), B-cell and NK cell markers indicated that the suppressive effect exerted by SE could be attributed to a decrease in the density of these markers or receptors on the cell surface. These findings provide additional evidence for a possible link between neurotransmitters, specifically SE, and immune function.

Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus of the lentivirus subgroup which primarily infects T cells and macrophages, and causes acquired immune deficiency syndrome (AIDS). Since its emergence in the early 1980s, HIV-1 has caused a global pandemic which is still responsible for over one million deaths per year, primarily in sub-Saharan Africa. HIV-1 has been the subject of intense study for over three decades, which has resulted not only in major advances in cell biology, but also in numerous drug treatments that effectively control the infection. However, cessation of treatment always results in reemergence of the infection due to the ability of HIV-1 (and other lentiviruses) to establish a persistent quiescent infection known as latency. The elimination of latently-infected cells is the primary goal of current research towards a cure for HIV-1, alongside efforts to develop vaccines, which have thus far been fruitless. The spread of HIV-1 to susceptible target cells (which express the receptor CD4 and a co-receptor; CXCR4 or CCR5) can take place when antigen-presenting cells, such as dendritic cells, capture virus particles and then pass them on to target cells, without themselves becoming infected. Alternatively, productively infected T cells or macrophages can spread HIV-1 either by shedding virus particles to the milieu, which are then stochastically acquired by target cells, or through transient contacts between infected and uninfected cells known as virological synapses (VSs). VS-mediated cell-to-cell transmission is thought to be highly efficient due to the release of virus directly onto (or very near to) a target cell, and some evidence suggests that the VS is a privileged site which allows the virus to evade neutralizing antibodies and drugs. However, and most importantly, it is of central interest to us because the same transient cell adhesions that facilitate virus transfer can also result in the fusion of the two cells to form a syncytium, due to the presence of the viral fusogen Env and its receptor and co-receptor on either side of the VS. While T cell syncytia can be found in vivo, they remain small, and it appears that the majority of VSs resolve without fusion. The regulation of HIV-1-induced cell-cell fusion and the fate of those syncytia are the focus of the work presented here. A family of host transmembrane proteins, the tetraspanins, which regulate cell-cell fusion in other contexts (e.g. the fusion of myoblasts to form and maintain myotubes), were found to inhibit HIV-1-induced cell-cell fusion. Our investigations have further characterized this regulation, concluding that tetraspanins allow cells to reach the fusion intermediate known as hemifusion before their ability to repress fusion takes effect. In parallel, because syncytia are nevertheless found both in infected individuals and in a humanized mouse model for HIV-1, we also became interested in whether small T cell-based syncytia were able to participate in HIV-1 spread by transmitting virus to target cells. Using a simple three dimensional in vitro culture system which closely recapitulates those in situ observations, we found that small syncytia can contact target cells and transmit virus without fusing with them. Overall, these studies further our understanding of HIV-1-induced syncytia and reveal a previously unrecognized role for these entities as active participants in HIV-1 spread.

Les Lymphocytes T CD4 (LT CD4) sont des cellules du système immunitaire adaptatif extrêmement plastiques qui, en fonction des signaux présents dans le microenvironnement cellulaire, ont la capacité de se différencier en différentes sous-populations de LT CD4 possédant des fonctions distinctes. Ce processus est hautement régulé par l'expression de facteurs de transcription (FT) clés tels que T-Bet, GATA-3, RORgammaT et Foxp3, nécessaires à la mise en place des lignages Th1, Th2, Th17 et Treg respectivement. Néanmoins, ces protéines n'agissent pas seules, et d'autres facteurs de transcription sont nécessaires pour amplifier, soutenir et maintenir ces différents lignages. Chaque lignage permet de lutter efficacement face à différents types de pathogènes ; toutefois, si la réponse immune n'est pas adaptée, ils peuvent également être responsables du développement de maladies auto-immunes. Afin de mettre en évidence les voies de signalisation et les facteurs de transcription impliqués dans la différenciation des LT CD4 pathogènes, nous avons utilisé le modèle de l'Encéphalomyélite Auto-immune Expérimentale (EAE), un modèle murin de Sclérose En Plaques (SEP). Dans ce modèle, nous avons mis en évidence le rôle clef de deux facteurs de transcription, Foxo3 et Eomes, dans la différenciation des LT CD4. En effet, les souris déficientes en Foxo3 développent une EAE moins sévère que les souris WT, et cette moindre sévérité de la maladie est associée à une proportion réduite de cellules productrices d'IFN-gamma et de GM-CSF in vivo, suite à l'immunisation. L'analyse du transcriptôme des souris Foxo3KO et WT a révélé que la déficience en Foxo3 a pour conséquence une diminution drastique de l'expression du FT Eomes. Bien que cette protéine soit nécessaire à la mise en place des réponses cytotoxiques dans les LT CD8 et les NK, son rôle précis dans les LT CD4 reste peu connu. D'un point de vue moléculaire, nous avons pu prouver, par des techniques d'Immuno-Précipitation de la Chromatine (ChIP) et des analyses de gènes rapporteurs, que le FT Eomes est un gène cible direct de Foxo3 dans les LT CD4.[...] / CD4 T cells are extremely plastic, and depending on the cytokines that are present within the microenvironment, they have the ability to differentiate into several subpopulations. This process is finely regulated by the expression of Master Regulator of each lineage such as T-Bet, GATA-3, RORgammaT and Foxp3, that are mandatory for the differentiation of Th1, Th2, Th17 and Treg cells respectively. However, they do not act alone, and several other transcription factors are required to stabilize, amplify and lock CD4 T cell lineages. Each subpopulation of CD4 T cells is highly specialized in the elimination of particular types of pathogen; however, in case of dysregulation of the immune response, they can also be involved in the development of autoimmune diseases. In order to determine how such properties are acquired by pathogenic CD4 T cells, we used the Experimental Autoimmune Encephalomyelitis (EAE) model which mimic Multiple Sclerosis pathology. In this model, we identified two transcription factors, Foxo3 and Eomes, that are critical for the differentiation of a particular and highly pathogenic subset of CD4 T cell. Indeed, Foxo3-deficient mice develop a less severe disease as compared to WT littermate and this decreased disease severity is associated with a decreased proportion of IFN-gamma and GM-CSF producing cells. Transcriptomic analysis of Foxo3KO versus WT CD4 T cells revealed that the most downregulated gene within Foxo3KO CD4 T cells is Eomes, which is essential for/to the acquisition of cytotoxic functions and production of IFN-gamma by NK and CD8 T cells. At the molecular level, using Chromatin Immuno-Precipitation experiments and Luciferase assays, we showed that Eomes is a direct target gene of Foxo3 in CD4 T cells. Then, in order to determine which of the downregulated gene is responsible for the decreased production of IFN-gamma and GM-CSF, we decided to overexpress Eomes in Foxo3KO CD4 T cells. Eomes overexpression restored IFN-gamma and, to a lesser extent, GM-CSF production by CD4 T cells, thus indicating that Eomes is involved in IFN-gamma and GM-CSF regulation in CD4 T cells.[...]

L'athérosclérose est une maladie cardio-vasculaire immuno-inflammatoire se développant sur un terrain de dyslipidémie. De nombreuses composantes de la réponse immunitaire sont capables de moduler le développement des plaques d'athérome. Notamment, les lymphocytes T (LTs) CD4+ dont le rôle dans le processus athérogène dépend de la voie de polarisation. Le mécanisme de polarisation des LTs CD4+ est sous le contrôle des cellules dendritiques conventionnelles (cDCs) CD11b+. Ainsi, moduler ces cDCs et orienter la réponse adaptative vers une polarisation anti-athérogène pourraient représenter une potentielle cible thérapeutique dans la pathologie. Dans cette perspective, nous avons évalué le rôle des cDCs CD11b+ dans le développement de l'athérosclérose qui à ce jour reste totalement inexploré. Nous avons montré que la baisse du nombre des cDCs CD11b+ n'a pas d'impact sur le développement des lésions d'athérome. Cependant, nous montrons que l'absence du facteur de transcription IRF4 nécessaire au développement des cDCs CD11b+ altère le rôle anti-athérogène reconnu de l'adjuvant vaccinal à base d'aluminium (Alum). Nos données suggèrent que les cDCs CD11b+ n'ont pas d'impact direct sur le développement de l'athérosclérose, cependant, elles contrôlent l'effet athéroprotecteur de l'Alum. / Atherosclerosis is a disease characterized by arterial blood vessel thickening due to the accumulation of inflammatory cells in the arterial intima in response to cholesterol deposition. Several components of the immune response are able to modulate the development of atheromatous plaques. In particular, the role of conventional of CD4+ lymphocytes in the atherogenic process depends on their polarization pathway. The polarization mechanism of CD4+ T cells is under the control of conventional CD11b+ dendritic cells (cDCs). Thus, modulating these cDCs and orienting the adaptive response towards anti-atherogenic polarization could represent a potential therapeutic target in pathology. In this context, we evaluated the role of CD11b+ cDCs in the development of atherosclerosis which still remains totally unexplored. We have demonstrated that the decrease of the number of CD11b+ cDCs has no impact on the development of atheroma lesions. However, we show that the deletion of IRF4, the transcription factor necessary for the development of CD11b+ cDCs alters the recognized anti-atherogenic role of the aluminum-based vaccine adjuvant (Alum). However our data suggest that CD11b+ cDCs have no direct impact on the development of atherosclerosis but can control the atheroprotective effect of Alum adjuvant.

Deficiencies in immune responses can lead to increases in the rate of infections and chronic diseases, such as cancer. Critical to the adaptive immune response is the activation of the T helper (Th)/CD4+ cell, the subsequent production of interleukin 2 (IL-2), expression of IL-2 and transferrin receptors (IL-2R, TfR) and transcription of genes resulting in DNA synthesis and T cell clonal expansion. The CD4+ lymphocyte response is impaired with ageing. Recent evidence suggests that moderate, regular aerobic training may increase the responsiveness of CD4+ lymphocytes to antigenic and mitogenic challenge, and thereby improve immune function in the older individual. Large volumes of chronic endurance training, and also high intensity training, may adversely affect the immune response, leading to immunosuppression and increased risk of infections. Impaired immune function and increased rates of URTI are found in athletes who undergo large volumes of training, often at high intensity. Purpose: To investigate if long-term aerobic training improved the immune response in men and women aged 65 to 75 years and, and to investigate if long-term endurance training depressed the immune response in male athletes aged 23 to 36 years. Methods:T helper lymphocyte proliferation was assessed monthly, by inducing the expression of CD25 (IL-2R ) and CD71 (transferrin) receptors with phytohemagglutinin (PHA). Percentage of CD4+ cells positive for the receptors, and the receptor density, were measured using two colour flow cytometry. Concentrations of intracellular calcium (Ca2+) and iron (Fe3+) were also measured monthly to determine the effect of endurance training on intracellular Ca2+ ([Ca2+]i) and Fe3+ ([Fe3+]i) within the CD4+ lymphocyte signal transduction pathway. Results: After twelve months of moderate aerobic training the percentage of CD4+ lymphocytes positive for CD25 increased in males aged 65 to 75 years, but not in females. There was no training effect on the density of CD25 in either gender, nor was there a training-induced increase in [Ca2+]i, total intracellular [Ca2+] from endoplasmic reticulum stores ([Ca2+]t) or [Fe3+] in this age group. Significant month to month variations in leucocyte, erythrocyte and haemoglobin concentration, mean corpuscular haemoglobin concentration, haematocrit, platelets, CD25 expression, CD71 expression, [Ca2+] and [Fe3+] were documented for both trained and untrained male and female groups. Aerobic capacity increased significantly with training for both men and women, with increases in peak, peak power and peak ventilation (p less than 0.05). Twelve months of chronic endurance training produced significantly lower haemoglobin, mean corpuscular haemoglobin and platelet concentration for six ([Hb]) and nine months ([MCHC], platelets) of the year in Ironman-distance triathletes, compared to sedentary males aged 23 to 36 years. There was no evidence of immunosuppression in the trained group, with no significant differences between groups in the percentage of CD4+ cells positive for CD25. The trained group showed a significantly higher density of CD25 receptors in October, January and June, suggesting a better immune response during these months. Endurance training did not effect [Ca2+] or [Fe3+]. The trained group did not show a reduced leucocyte concentration, and reported significantly fewer cases of URTI in twelve months than their sedentary counterparts. The 23 to 36 years age group showed seasonal changes in haematological and immunological indices similar to older individuals, indicating that autumn, late winter and late spring are periods of reduced immuno-competency. Conclusion: Twelve months of moderate intensity training significantly increased functional capacity in older men and women, and the percentage of CD4+ lymphocytes expressing CD25 in older men, thereby improving the lymphoid immune response. Twelve months of endurance training significantly increased CD25 density in CD4+ lymphocytes in Ironman triathletes compared to sedentary young males. The monthly changes in immune variables in young and older subjects suggested that autumn, late winter and late spring might be periods where individuals were more at risk of succumbing to infections due to decreased lymphocyte responsiveness. Summer months appeared to be a period of increased lymphocyte responsiveness and proliferation.

L'immunothérapie cellulaire passive par injection de lymphopcytes T cytotoxiques offre des possibilités thérapeutiques nouvelles dans l'immunité anti-virale et anti-tumorale. Par rapport aux méthodes actuelles de stimulation utilisant des lignées présentatrices d'antigènes, nous essayons de développer une méthode alternative d'amplification des lymphocytes T CD8 à l'aide d'une combinaison de protéines recombinantes immobilisées sur billes.<br />L'interaction 4-1BB/4-1BBL (CD137/CD137L) constitue un des signaux de co-stimulation impliqués dans l'activation des lymphocytes T CD8 effecteurs. Le travail présenté ici décrit pour la première fois la production et la caractérisation d'une forme fonctionnelle de 4-1BBL recombinant soluble. De plus, nous montrons qu'il est possible d'amplifier in vitro des lymphocytes T mémoires anti-CMV et anti-EBV avec des complexes HLA-peptide associés à ce 4-1BBL ou à de l'anticorps anti-CD28. L'intérêt de la co-stimulation du 4-1BB est comparée à celle du CD28 dans les 2 contextes antigéniques étudiés.