T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

Antigen-specific CD8 T cells provide an important protective role in response to infection by viruses, intracellular bacteria, and parasites. Pathogen-specific CD8 T cells render this protection by undergoing robust expansion in numbers while gaining the ability to produce cytokines and cytolytic machinery. Following expansion and effector differentiation, pathogen-specific CD8 T cells will contract in number while further differentiating into a highly functional population of memory CD8 T cells. These antigen-experienced cells persist in secondary lymphoid organs and the periphery in order to rapidly respond to repeated infection. Creating optimal CD8 T cell responses to infection can be critical for raising sufficient armament to provide protection against invading intracellular pathogens. Although CD8 T cells have protective value, many vaccine strategies tend to focus on creating productive B cell antibody responses to promote immunological protection. Even though antibody responses can be highly protective, coupling optimal CD8 T cell responses with B cell responses could provide higher orders of protection than either one on their own. Therefore, a deeper understanding of the pathways that ultimately guide the magnitude of CD8 T cell responses is required to achieve this potential therapeutic benefit. My studies evaluate the role of receptor signaling events in guiding the expansion of activated CD8 T cells during primary and secondary responses. Specifically, the first portion of my studies dissect the mechanism by which direct IL-12 and Type I IFN stimulation can substantially bolster primary CD8 T cell responses in vivo. Within this context, I demonstrate that direct IL-12 and Type I IFN signaling increases CD8 T cell accumulation during primary expansion by prolonging division without altering survival. IL-12/Type I IFN signaling promoted prolonged division of activated CD8 T cells by maintaining high-affinity IL-2 receptor subunit (CD25) expression and IL-2 signaling. The other portion of my work was dedicated to understanding the expression and role of the inhibitory FcgR (FcgRIIB) during primary and secondary CD8 T cell responses. FcgRIIB expression could be detected as early as the peak of the CD8 T cell response and marked activated CD8 T cells that were highly sensitive to antigen stimulation. Although FcgRIIB did not appear to play a substantial role in regulating the magnitude of primary CD8 T cell responses, it played an important role in inhibiting the expansion and cytotoxicity of memory CD8 T cells during homologous challenge. Collectively, these data highlight potential avenues that could be exploited by future therapies that aim to achieve appropriately sized CD8 T cell responses.

Vitt snus ökande popularitet har skapat oro för dess påverkan på immunförsvaret. I föreliggande studie undersöks nikotinets påverkan på uttrycket av interleukin-2-receptorn alfa (IL-2R-alfa eller CD25) hos CD4+ T-celler under in vitro-stimulering. Tidigare studier indikerar negativa effekter såsom minskat antingen upptag hos antigenpresenterande celler, cytokinproduktion och uttryck av receptorer som CD28 och CTLA-4, vilka är viktiga för CD4+ T-cellernas aktivering, differentiering samt bromsning av aktiveringen. Andra studier har visat att alfa-4- samt alfa-7-nikotinerg acetylkolinreceptorer uttrycks på CD4+ T-celler, vilket i sin tur har visat sig påverka cytokin produktionen i närvaro av nikotin under stimulering. CD25 är en viktig receptor vid aktiveringen av T-celler och är överuttryckt vid exempelvis akut T-cells leukemi (ATL). Målet med denna studie är att förstå hur nikotin kan påverka CD25-uttrycket och därmed påverka immunförsvaret och effektiviteten av terapier. Med hjälp av flödescytometri och kvantitativ polymeraskedjereaktion (qPCR) kunde en tydlig skillnad detekteras mellan nikotin/PHA-stimulering och enbart PHA-stimulering hos en donator jämfört med de andra donatorerna. Metoderna inkluderar cellseparation, stimulering med fytohemagglutinin (PHA) i närvaro av nikotin, analys av uttrycket CD25 med flödescytometri och analys av genuttryck av CD25 med qPCR. Resultaten visade varierande svar mellan tre donatorer, med en tydlig skillnad mellan nikotin/PHA- och enbart PHA-stimulering för en donator jämfört med de andra donatorerna. Denna studie pekar på potentiella effekter av nikotin på immunförsvaret och betonar behovet av vidare undersökningar för att förstå mekanismerna bakom detta. / The increasing popularity of white snus has raised concerns about its impact on the immune system. The present study investigates the effect of nicotine on the expression of interleukin-2 receptor alpha (IL-2R-alpha or CD25) in CD4+ T cells during in vitro stimulation. Previous studies have indicated negative effects such as decreased uptake by antigen-presenting cells, cytokine production, and expression of receptors such as CD28 and CTLA-4, which are important for the activation, differentiation, and inhibition of CD4+ T cell activation. Other studies have shown that alpha-4 and alpha-7 nicotinic acetylcholine receptors are expressed on CD4+ T cells, which in turn has been shown to affect cytokine production in the presence of nicotine during stimulation. CD25 is an important receptor for T cell activation and is overexpressed in conditions such as acute T cell leukemia (ATL). The aim of this study is to understand how nicotine may affect CD25 expression and thereby influence the immune response and the effectiveness of therapies. Using flow cytometry and quantitative polymerase chain reaction (qPCR), a clear difference was detected between nicotine/PHA stimulation and PHA stimulation alone in one donor compared to the other donors. The methods include cell separation, stimulation with phytohemagglutinin (PHA) in the presence of nicotine, analysis of CD25 expression by flow cytometry and analysis of CD25 gene expression by qPCR. The results showed varying responses among three donors, with a clear difference between nicotine/PHA and PHA stimulation alone for one donor compared to the others. This study highlights potential effects of nicotine on the immune system and underscores the need for further investigations to understand the mechanisms behind this.

Myasthenia Gravis (MG) is an autoimmune disease affecting neuromuscular transmission, in which the thymus is considered pathogenic organ. Earlier ideas suggesting that MG is only the receptors disease have been proven wrong. There are immunopathological changes in both target structures [specific receptors for acetylcholine (AChR] muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein 4 (Lrp4)], the thymus, as well as in peripheral lymphoid organs. Initial findings of the humoral immunity defect with the decisive role of the pathologic autoantibodies, were corrected with findings of the immune dysregulation at the level of T lymphocytes. According to today's knowledge, the development and maintenance of MG involves almost all cell types of immune function in the autoimmune inflammation: helper CD4+ T lymphocytes, cytotoxic CD8+ T lymphocytes, regulatory CD4+CD25+ T lymphocytes, Th17 lymphocytes, B lymphocytes and plasma cells. Thymus plays a dominant immunopathogenetic role in younger patients with MG, while extrathymic mechanisms are applied in older patients. As a result of that, the thymectomy (TE) is generally accepted as part of treatment for MG. However, there is still no data verified by a prospective controlled study, which would demonstrate a useful result of this treatment...

Le polymorphisme et le polygénisme des complexes majeurs d’histocompatibilité (CMH) limitent les succès de la transplantation. En effet, les disparités, tant d’antigènes mineurs que majeurs, exposent le patient transplanté au risque de rejet et imposent l’administration d’un traitement immunosuppresseur. Ce dernier affecte de façon non spécifique l’ensemble des réponses immunitaires et augmente le risque d’infections mortelles et de cancers. En outre, ce traitement ne semble pas prévenir le rejet chronique. <p>Des découvertes récentes ont confirmé l’existence de lymphocytes appelés régulateurs (Tregs) dont le rôle est de garantir l’homéostasie des réponses immunes afin qu’elles ne deviennent incontrôlées et pathologiques. Les Tregs classiquement décrits expriment de manière constitutive l’antigène CD4+, la chaîne alpha du récepteur de l’interleukine (IL)-2 (CD25) et le facteur de transcription Foxp3. Ils représentent 5 à 10% des lymphocytes CD4+ totaux. Les Tregs sont capables de réguler des lymphocytes alloréactifs et ont été décrits comme responsables du maintien de la tolérance d’allogreffe chez la souris. Mais jusqu'alors, les modèles employés pour démontrer l'importance des Tregs en transplantation utilisaient soit un traitement immunosuppresseur transitoire, soit des transferts de cellules T dans des souris lymphopéniques. <p>Toutefois, ces derniers ne permettent pas de distinguer l'effet des Tregs sur la prolifération homéostatique des lymphocytes effecteurs de leur effet sur la réponse allogénique.<p>Dans notre travail, nous montrons que les Tregs jouent un rôle prépondérant dans l’acceptation spontanée d’allogreffes en l’absence d’immunosuppresseur et en dehors d’un contexte lymphopénique chez la souris. En effet, la déplétion des Tregs du receveur par l’administration d’anticorps anti CD25 amplifie les réponses allogéniques de type Th1 et Th2 et, par conséquent, déclenche le rejet d’allogreffe. Les propriétés régulatrices des Tregs ne sont cependant pas illimitées. En effet, dans un second travail, nous décrivons, d’une part, leur incapacité à contrôler la production d’IL-17 par des lymphocytes CD4+CD25pos mémoires et, d’autre, part leur implication directe dans la différenciation de cellules Th17 au départ de lymphocytes CD4+CD25neg alloréactifs.<p>Nous concluons donc que si les Tregs naturellement présents chez le receveur jouent un rôle primordial dans la protection du greffon contre des réponses de type Th1 ou Th2, ils pourraient néanmoins favoriser une voie alterne du rejet d’allogreffe dépendante de l’IL 17.<p>/<p>Major histocompatibility complex (MHC) polymorphism is a major hindrance to transplantation success. Both minor and major antigen disparities between donor and recipient increase the risk of transplant rejection. This is thwarted by the administration of an immunosuppressive therapy that unspecifically affects all immune responses therefore increasing the risk of infections and cancers. Besides, this treatment does not seem to prevent chronic rejection.<p>Recent studies have confirmed the existence of lymphocytes called regulatory T cells (Tregs), whose role is to maintain the general immune homeostasis and to protect the individual from autoimmune diseases.<p>The classically described Tregs express constitutively the CD4 antigen, the alpha chain of the interleukin (IL)-2 receptor (CD25) and the transcription factor Foxp3. They represent 5 to 10% of total CD4+ T cells. Tregs are able to control alloreactive responses and were described to be responsible for the maintenance of allograft tolerance in mice. So far, the tolerogenic capacities of Tregs have been demonstrated either in mice treated with immunomodulatory antibodies (induced Tregs) or by adoptive co-transfer of Tregs and effector cells into lymphopenic mice. However, the latter has the disadvantage of not being able to distinguish the effect of Treg on lymphopenia-induced homeostatic proliferation from their effect on alloreactive responses. <p>Herein, we show that Tregs play a crucial role in spontaneously accepted allografts in the absence of immunosuppressive therapy and in non-lymphopenic condition. Indeed, the depletion of the recipient’s Tregs through the administration of an anti-CD25 antibody enhances type Th1 and type-Th2 allogeneic responses, consequently triggering allograft rejection. However, the regulatory properties of Tregs are not unlimited. Indeed, we found that Tregs are unable to control allogeneic IL-17 production by memory CD4+ T cells and are even necessary for de novo Th17 differentiation. <p>We conclude, therefore, that Tregs naturally present in the recipient play a critical role in protecting the allograft. Nevertheless, despite this context of regulation, IL-17-producing alloreactive T cells, beyond the control of Tregs, could mediate an alternative pathway of allograft rejection. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Myasthenia Gravis (MG) is an autoimmune disease affecting neuromuscular transmission, in which the thymus is considered pathogenic organ. Earlier ideas suggesting that MG is only the receptors disease have been proven wrong. There are immunopathological changes in both target structures [specific receptors for acetylcholine (AChR] muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein 4 (Lrp4)], the thymus, as well as in peripheral lymphoid organs. Initial findings of the humoral immunity defect with the decisive role of the pathologic autoantibodies, were corrected with findings of the immune dysregulation at the level of T lymphocytes. According to today's knowledge, the development and maintenance of MG involves almost all cell types of immune function in the autoimmune inflammation: helper CD4+ T lymphocytes, cytotoxic CD8+ T lymphocytes, regulatory CD4+CD25+ T lymphocytes, Th17 lymphocytes, B lymphocytes and plasma cells. Thymus plays a dominant immunopathogenetic role in younger patients with MG, while extrathymic mechanisms are applied in older patients. As a result of that, the thymectomy (TE) is generally accepted as part of treatment for MG. However, there is still no data verified by a prospective controlled study, which would demonstrate a useful result of this treatment...

Background: Tobacco smoking is the main cause of chronic obstructive pulmonary disease, COPD, but the mechanisms by which cigarette smoke induces COPD are still elusive. T lymphocytes have been implicated in the pathogenesis of the disease, but their role in the airway inflammation in COPD is not fully understood. The aim of this thesis was therefore to address T lymphocyte subsets and their activation in the airways of subjects with COPD, in comparison to smokers with normal lung function (S) and never smokers (NS). Methods: Subjects with moderate to severe COPD were recruited along with controls. They were all non-atopic and clinically stable, without any exacerbation during at least three months prior to inclusion. Only medication with short-acting β2-agonists and/or anti-cholinergic drugs was permitted. All subjects underwent bronchoscopy with endobronchial mucosal biopsy sampling as well as bronchial wash, BW, and bronchoalveolar lavage, BAL, collection. Biopsies were immunohistochemically stained for inflammatory cells and markers. BW and BAL fluids were prepared for differential cell counts. Soluble markers were measured in BW and lymphocyte subsets were determined in BAL using flow cytometry. Results: In biopsies, an increase in epithelial CD3+ and CD8+ cells was found in COPD, compared to NS. In BAL fluid, CD8+ cells were enhanced, whereas CD4+ cells were reduced in subjects with COPD and S, compared to NS. Furthermore, CD4+ and CD8+ cells were more activated both in COPD and S, in terms of increased expression of CD25, CD69 and HLA-DR. NKG2D-expressing CD8+ T cells in BAL fluid were enhanced in both COPD and S. CD4+CD25bright cells were upregulated in COPD and S, suggesting the presence of regulatory T cells. Further analyses of T cell subsets with the more specific markers for regulatory T cells, FoxP3 and CD127, indicated a smoking-induced expansion of non-regulatory T cells, which tended to normalize after smoking cessation in COPD. Currently smoking subjects with COPD still expressed high proportions of activated non-regulatory CD4+ T cells. The data on FoxP3 expression further indicated that the increase in CD25 expression in COPD and S was not only associated with the expansion of regulatory T cells. As CD127 expression is reported to be inversely associated with FoxP3, the data indicate the expansion of a non-regulatory CD25+ population in smokers and patients with stable COPD. The immunohistochemical staining for the NKG2D ligands MICA and MICB on epithelial cells was unchanged. Conclusion: The results of this thesis suggest a role for CD4+ and CD8+ T-cells in clinically stable COPD, indicating that T-cells are of importance in the long-term inflammatory response in COPD. Regardless of current smoking habits, activated CD8+ T lymphocytes were found to be increased in BAL fluid from subjects with COPD, suggesting that changes in CD8+ T cells are associated with a persistent immune response and, thus, of importance in COPD pathogenesis. In contrast, the expansion of non-regulatory CD25+CD4+ cells in BAL fluid seemed to be preferentially smoke-related. In summary, the data indicate that, among airway T cells, changes in CD8+ cells seem to be highly associated with COPD pathogenesis, whereas changes in CD4+ cells appear to be related to cigarette smoke-induced responses. Further, a non regulatory population of helper T cells was identified in BAL fluid of COPD patients, which may contribute to the persistent cytotoxic T cell responses.

Regulatory T cells (Treg) represent a crucial CD4 T cells subset involved in maintenance of immune-tolerance. Since their first description important efforts have been undertaken to better understand their biology, their development and their mechanisms of action. However, little is known about factors controlling Treg peripheral homeostasis. The aim of this thesis work was to better define mechanisms involved in governing Treg homeostasis and to investigate the eventual contribution of perturbation of Treg homeostasis in human disease. In the first part of this thesis work we tried to define in the murine system the role played by IL-7 in governing Treg homeostasis. We showed that Treg surface expression of CD127, the IL-7 receptor alpha chain, is finely regulated as it depends on their activation as well as on their tissue localization. More importantly, we demonstrated that Treg do express functional levels of CD127, identifying these cells as potential target of IL-7. Using both genetically modified murine models of altered IL-7 signaling and adoptive transfer models, we obtained definitive evidence for a direct role of IL-7 in governing Treg cell numbers. Finally, we demonstrated that IL-7 signaling in Treg optimizes their capacity to react to IL-2 an important cytokine regulating Treg homeostasis. In the second part of this work we investigated Treg homeostasis in the context of HIV infection. Employing for the first time in HIV infection a novel consensus Treg identification strategy and applying it to different groups of HIV infected patients, including primary infected patients and HIV controllers, we showed that HIV infection is characterized by an early and long lasting alteration of Treg homeostasis. In particular we demonstrated that effector rather than naive Treg are affected by HIV infection. Moreover, we showed that effector Treg numbers inversely correlated with HIV specific CD8 T cells responses, providing ex vivo evidence of Treg involvement in HIV immunity.