In the US, the incidence and mortality rates of hepatocellular carcinoma (HCC) are alarmingly increasing since no effective therapy is available for the advanced disease. Activation of IGF signaling is a major oncogenic event in diverse cancers, including HCC. Insulin-like growth factor binding protein-7 (IGFBP7) inhibits IGF signaling by binding to IGF-1 receptor (IGF-1R) and functions as a potential tumor suppressor for hepatocellular carcinoma (HCC). IGFBP7 abrogates tumors by inducing cancer-specific senescence and apoptosis and inhibiting angiogenesis. We now document that Igfbp7 knockout (Igfbp7-/- ) mouse shows constitutive activation of IGF signaling, presents with pro-inflammatory and immunosuppressive microenvironment, and develops spontaneous tumors in lungs and liver and markedly accelerated carcinogen-induced HCC. Loss of Igfbp7 resulted in increased proliferation and decreased senescence in hepatocytes and mouse embryonic fibroblasts that could be blocked by an IGF-1 receptor inhibitor. A significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- livers which was associated with marked inhibition in antigen cross presentation by Igfbp7-/- dendritic cells. IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immune competent mice, which could be abolished by depletion of CD4+ or CD8+ T lymphocytes. Our studies unravel modulation of immune response as a novel component of pleiotropic mechanisms by which IGFBP7 suppresses HCC. Even though HCC has an immunosuppressive milieu, immune targeted therapies are beginning to demonstrate significant objective responses in clinical trials. IGFBP7 might be an effective anti-HCC therapeutic by directly inhibiting cancer cells and stimulating an anti-tumor immune response.

Les lymphocytes T (LT) CD4+ auxiliaires soutiennent l‘action des LT CD8+ cytotoxiques (LTC) au cours des réponses immunitaires anti-tumorales. Des protocoles d‘immunothérapie cellulaire adoptive (ICA) basés sur l‘injection d‘effecteurs T CD4+ ont donc été développés pour traiter les cancers, et ils ont montré une efficacité thérapeutique. Cependant, la difficulté de disposer de cellules présentatrices d‘antigène (CPA) autologues permettant de générer un nombre suffisant de LT CD4+ spécifiques fonctionnels in vitro dans un court délai représente un obstacle majeur au développement de telles approches. Pour contourner cette difficulté, notre groupe a précédemment développé des cellules présentatrices d‘antigène artificielles (CPAA) dérivant de fibroblastes murins NIH/3T3 et exprimant les molécules nécessaires pour activer des LT CD4+ humains : une molécule HLA (Human Leucocyte Antigen) de classe II (ici HLA-DR1), la molécule de costimulation CD80 et les molécules d‘adhérence CD54 et CD58. Dans ce travail, nous avons cherché à optimiser nos CPAA DR1+ (CPAADR1) en permettant une expression endogène et constitutive de l‘antigène d‘intérêt (ici l‘hémagglutinine, HA), sous forme de peptide ou de protéine, au niveau des compartiments cellulaires impliqués dans la présentation des antigènes par les molécules HLA-II. Nous avons montré que les CPAADR1 « endogènes » exprimant le peptide HA au niveau du réticulum endoplasmique (RE) ou la protéine HA à la membrane plasmique, possédaient les meilleures capacités de présentation de l‘antigène. En stimulation primaire, ces deux lignées de CPAADR1 activaient des LT CD4+ spécifiques de HA, mais avec une capacité moindre que des CPA autologues. En revanche, en restimulation, les CPAADR1 exprimant le peptide HA dans le RE étaient même plus efficaces pour amplifier des LT CD4+ spécifiques fonctionnels que des CPAADR1 ou des CPA autologues chargées avec le peptide d‘intérêt. Les LT obtenus étaient des cellules Th1 mémoires exprimant du granzyme B et produisant de l‘IFN-γ et du TNF-α. C‘est la première fois à notre connaissance qu‘un antigène exprimé de façon endogène dans une lignée cellulaire peut-être présenté de façon efficace sur une molécule HLA de classe II. Nos CPAA « endogènes » constituent donc un nouvel outil unique pour générer de façon reproductible et standardisable des réponses T CD4+ spécifiques, et pourraient déboucher sur le développement de nouvelles approches d‘ICA / CD4+ helper T lymphocytes (TLs) sustain CD8+ cytotoxic TL (CTL) activity during anti-tumor immune responses. Adoptive cell immunotherapy (ACI) protocols based on the injection of CD4+ T-effectors have therefore been developed to treat cancers, and they have proven therapeutic efficacy. However, the difficulty of obtaining autologous antigen presenting cells (APCs) for generating a sufficient number of functional specific CD4+ TLs in a short time in vitro is a major obstacle to the development of such approaches. To bypass this difficulty, our group has previously developed artificial antigen presenting cells (AAPCs) derived from NIH/3T3 murine fibroblasts expressing molecules necessary to activate human CD4+ TLs: an HLA (Human Leucocyte Antigen) class II molecule (in this study HLA-DR1), CD80 costimulatory molecule, and CD54 and CD58 adhesion molecules. In this work, we sought to optimize our DR1+ AAPCs (AAPCDR1) by allowing an endogenous and constitutive expression of the antigen of interest (in this study hemagglutinin, HA), as a peptide or a whole protein, in different cell compartments involved in antigen presentation by HLA-II molecules. We have shown that ―endogenous‖ AAPCDR1 expressing HA peptide in the endoplasmic reticulum (ER) or HA protein at the plasma membrane had the best antigen presentation abilities. In a first stimulation, both AAPCDR1cell lines activated HA-specific CD4+ TLs, but to a lower extent than autologous APCs. However, in a second stimulation, AAPCDR1 expressing HA peptide in the ER were even more effective for amplifying functional specific CD4+ TLs than AAPCDR1 or autologous APCs loaded with the peptide of interest. Obtained TLs were memory Th1 cells expressing granzyme B and producing IFN-γ and TNF-α. This is the first time to our knowledge that an antigen expressed endogenously in a cell line can be efficiently presented on an HLA class II molecule. Our ―endogenous‖ AAPCs represent therefore a new and unique tool for reproducible and standardizable generation of specific CD4+ T responses, and could lead to the development of new ACI approaches.

Le dysfonctionnement mitochondrial est associé à de nombreuses maladies neurodégénératives. En effet, plusieurs protéines impliquées dans ces maladies, telles que les protéines PINK1 et Parkin dans la maladie de Parkinson, interviennent dans le recrutement de protéines nécessaires à l’homéostasie mitochondriale. En absence de ces protéines, un nouveau mécanisme se met en place : la formation de vésicules dérivées des mitochondries (MDVs). Notre équipe a démontré que ce mécanisme est responsable de la présentation antigénique mitochondriale (MitAP) et que les protéines PINK1 et Parkin ont un rôle répresseur sur cette voie et que cette nouvelle voie de présentation était capable d’activer des lymphocytes T CD8+ in vivo. Ces découvertes font entrer le système immunitaire comme nouvel acteur des maladies neurodégénératives. Cependant, les protéines impliquées dans MitAP restent à être identifiées. Deux projets ont été initiés afin de pouvoir mieux caractériser MitAP. La première a consisté à mettre au point un protocole d’isolation mitochondrial afin d’identifier de nouveaux partenaires moléculaires à la formation des MDVs au niveau des mitochondries. Le deuxième projet initie l’étude de l’immunopeptidome de cellules présentatrices d’antigène afin d’identifier les peptides mitochondriaux présentés à la surface des cellules. L’identification de protéines par l’isolation des mitochondries et celles générant les peptides mitochondriaux présentés à la surface des cellules sont essentielles pour comprendre le mécanisme des MDVs et le fonctionnement de la MitAP impliquée dans la maladie de Parkinson. Les protéines partenaires de cette voie moléculaire pourraient avoir un rôle dans les maladies neurodégénératives et être des cibles thérapeutiques ou des biomarqueurs. / Mitochondrial dysfunction is associated with many neurodegenerative diseases. Indeed, several proteins involved in these diseases, such as PINK1 and Parkin proteins in Parkinson's disease, are involved in the protein recruitment required for mitochondrial homoeostasis. In the absence of these proteins, a new mechanism is set up: the formation of vesicles derived from mitochondria (MDVs). Our team has demonstrated that this mechanism is responsible for the mitochondrial antigen presentation (MitAP) and that the PINK1 and Parkin proteins play a repressor role on this pathway and that this new presentation pathway is capable of activating CD8 + T cells in vivo. These discoveries bring the immune system as a new player in neurodegenerative diseases. However, the proteins involved in MitAP remain to be identified. Two projects have been initiated to better characterize MitAP. The first was to develop a mitochondrial isolation protocol to identify new molecular partners for MDV formation at the mitochondrial level. The second project initiates the study of the immunopeptidoma of antigen presenting cells to identify the mitochondrial peptides presented on the cell surface. The identification of these proteins is essential to understand the mechanism of MDVs and the functioning of MitAP involved in Parkinson's disease. The protein partners of this molecular pathway may have a role in neurodegenerative diseases and may be therapeutic targets or biomarkers.

Peptides generated from cellular protein degradation via the ubiquitin-proteasome pathway are presented on MHC class I as a means for the immune system to monitor polypeptides being synthesized by cells. For CD8 + T cells to prevent the spread of an incipient infection, it appears essential they should be able to sense foreign polypeptides being synthesized as soon as possible. A prompt detection of viral proteins is of great importance for the success of an adaptive immune response. Defective ribosomal products (DRiPs) have been postulated as a preferential source which would allow for a rapid presentation of peptides derived from the degradation of all newly synthesized proteins. Although this hypothesis is intellectually appealing there is lack of experimental data supporting a mechanism that would prioritize presentation from DRiPs. In this dissertation I describe a series of experiments that probe the DRiPs hypothesis by assessing the contribution to class I presentation of model epitopes derived from DRiPs or from functional proteins. The results show that even at the early stages after mRNA synthesis DRiPs do not account for a significant fraction of the class I presented peptides. These observations suggest that the currently widespread model whereby a mechanism exists which selectively allows for DRiPs to preferentially contribute to class I antigen presentation, is incorrect. Rather, properly folded functional proteins can significantly contribute to class I antigen presentation as they are normally turned over by the ubiquitin-proteasome pathway.

Antigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.

Indiana University-Purdue University Indianapolis (IUPUI) / Globally, protein malnutrition remains problematic, adversely affecting several systems including the immune system. Although poorly understood, protein restriction severely disrupts host immunity and responses to infection. Induction of high-affinity, long-lasting immunity depends upon interactions between B and T lymphocytes. B lymphocytes exploit several pathways including endocytosis, macroautophagy, and chaperone-mediated autophagy to capture and deliver antigens to the endosomal network. Within the endosomal network antigens are processed and loaded onto major histocompatibility complex (MHC) class II molecules for display and recognition by T lymphocytes. To examine the effect of macronutrient malnutrition on MHC class II antigen presentation, we grew B lymphocytes in media containing amino acids, sugars and vitamins but lacking serum, which contains several types of macronutrients. Our studies show macronutrient stress amplified macroautophagy, favoring MHC class II presentation of cytoplasmic antigens targeted to autophagosomes. By contrast, macronutrient stress diminished MHC class II presentation of membrane antigens including the B cell receptor (BCR) and cytoplasmic proteins that utilize the chaperone-mediated autophagy pathway. The BCR plays a critical role in MHC class II antigen presentation, as it captures exogenous antigens leading to internalization and degradation within the endosomal network. While intracellular protease activity increased with macronutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of high molecular mass macronutrients restored endocytosis and antigen presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Cytosolic chaperone HSC70 has been shown to play a role in endocytosis, macroautophagy, chaperone-mediated autophagy and proteolysis by the proteasome, potentially connecting distinct routes of antigen presentation. Here, altering the abundance of HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and antigen presentation suggesting macronutrient deprivation alters the availability of HSC70. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating distinct membrane trafficking pathways during cellular stress. These results offer a new explanation for impaired immune responses in protein malnourished individuals.

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by loss of insulin-producing beta cells resulting in life-long insulin deficiency. Beta cell destruction by autoreactive CD8+ effector T-cells is thought to be the main cause of loss of insulin output. Autoreactive T-cells are similarly to autoantibodies, which have been established as markers of risk and progression of the disease, directed towards autoantigens of T1D. These are most notably, insulin, 65 kDa glutamic acid decarboxylase (GAD65, also known as GAD2), insulinoma-associated protein 2 (IA­2, also known as PTPRN or ICA512) or zinc transporter 8 (ZNT8). Most of the known T1D autoantigens are components of insulin secretory granules (SGs). T1D arises from an interplay of genetic and environmental factors, which are thought to act as triggers in susceptible individuals. Predisposing alleles in genetic loci for human leukocyte antigen (HLA) account for by far the highest contribution to the risk of disease development, followed by an array of polymorphisms thought to play a role in either immune cells or beta cells. Of environmental factors that potentially add to the risk of disease progression, the most evidence-supported are Enteroviruses (EVs). Most notably, their genome and viral proteins, as well as higher expression of cellular proteins involved in viral response were detected more often in blood and pancreata of patients with T1D than in healthy population. In addition, recent evidence from a large long-term observational study has implicated prolonged shedding of specifically species Enterovirus B in the stools of children as a risk factor in development of beta cell autoimmunity in children with high genetic risk of T1D. For these reasons, many researchers have studied the potential mechanisms of EV involvement in T1D pathogenesis. In our laboratory, we have investigated the effects of coxsackievirus B5 (CVB5) infection on murine insulinoma MIN6 cells. Previously, we have reported that glucose-stimulated translation of SG proteins can be carried out in a cap-independent manner and is not shut down as part of the early effects of CVB5 infection on MIN6 cells. We have also observed that mature forms of SG proteins are being degraded during viral infection. As intracellular protein degradation is one of the major pathways to supply peptides for presentation on HLA I for immune recognition, we hypothesized that concomitant production and degradation of SG proteins upon viral infection could lead to altered presentation of mainly peptides derived from insulin SG component proteins and potentially drive the response of autoreactive T-cells. To address this hypothesis, we aimed to identify appropriate conditions to study the impact of EV infection on antigen presentation of ECN90 cells. To that end, we established a panel of markers examined by SDS-PAGE and immunoblotting. Stage of viral infection was assessed based on the detection of the viral protein VP1 and cleavage of cellular factors such as eukaryotic translation initiation factor 4 G (eIF4G), poly(A)-binding protein (PABP1), polypyrimidine tract-binding protein 1 (PTBP1), poly (ADP-ribose) polymerase (PARP) and caspase 3, which is mediated by viral proteases. Furthermore, we assessed the levels of ICA512 and chromogranin A and their pro-forms to estimate the size of insulin SG stores, and the expression of HLA I and β2 microglobulin to confirm sufficient antigen presentation. Peptides presented on both HLA I and II were isolated by immunoaffinity purification and identified by liquid chromatography-tandem mass spectrometry analysis. About 500 unique HLA I-presented peptides were found on average per replicate and condition with purity of 89% (peptides predicted to bind HLA alleles expressed by ECN90 cells). The distribution of unique peptides presented by infected ECN90 cells significantly differed from those presented by control cells as 54 unique peptides were present only in all infected samples and none of uninfected and 13 peptides were only found in uninfected cells. In total, we identified 26 unique peptides from known T1D autoantigens associated with SGs (e.g. insulin, chromogranin A, ICA512) in both conditions. The majority of them were predicted to bind HLA I alleles B*40:01 and A*02:01, while two identified viral peptides were found to bind B*40:01 and A*03:01 alleles. Both of the viral peptides and almost half of the peptides originating from known T1D autoantigens have not been described before. In addition, on average 300 unique HLA II peptides were found per replicate and condition. Similarly to HLA I peptides, the distribution of unique peptides across infected and control cells differed as well, showing that antigen presentation was altered in infected cells. We identified two viral HLA II-eluted peptides and peptides originating from only two known T1D autoantigens, 35 originated from insulin and 157 from chromogranin A. As most of the newly identified HLA I peptides originating from T1D autoantigens and one peptide from viral proteins were restricted by the allele HLA-B*40:01, our further efforts were invested in the development of a recombinant disulfide-stabilized biotinylated peptide-receptive HLA molecule of this allele. This technology has been extensively validated, and will allow us to test the wide array of novel peptides identified by us for the ability to bind this allele, as well as asses frequencies and responses of specific T-cells in subject populations relevant for T1D.

La maladie de Parkinson (MP) est une maladie neurodégénérative qui présente, parmi ces symptômes, une importante inflammation, rapportée tant au niveau du système nerveux central qu’en périphérie. De plus, certains des gènes causant des formes familiales de la MP, soit PINK1 et PRKN, sont impliqués dans la prévention de la présentation des antigènes mitochondriaux (MitAP), un phénomène à risque d’enclencher une réponse auto-immune. Ce travail avait donc pour but d’investiguer la contribution des processus auto-immuns et inflammatoires périphériques dans l’étiologie de la MP, en portant une attention particulière aux phénomènes impliquant la mitochondrie. La quantification des ARNm de PINK1 et PRKN a permis de déterminer que l’expression de ces deux gènes pouvait être inhibée lors de l’activation – par l’exposition au lipopolysaccharide (LPS) ou à l’Escherichia coli entéropathogène (EPEC) – de certaines cellules présentatrices d’antigènes, soit les cellules dendritiques dérivées de monocytes (MDDC). L’expression de PRKN était également inhibée au cours du vieillissement dans les cellules mononucléées du sang périphérique. L’étude des lymphocytes T présents dans la circulation sanguine a permis l’identification d’une population de cellules T CD8+ IL-17+ (Tc17) pouvant être activées par des antigènes mitochondriaux chez les patients avec MP. Les MDDC furent également identifiées comme étant d’importantes régulatrices de l’inflammation, car les cellules des patients avec MP présentaient un profil d’expression spécifique, caractérisé par une surproduction de cytokines pro-inflammatoires. Leur réponse cytokinique était influencée par le vieillissement et correspondait à un profil pouvant faciliter la polarisation des lymphocytes T CD4+ vers le sous-type Th17, lié aux maladies auto-immunes. Nos résultats, dans leur ensemble, supportent l’implication de mécanismes auto-immuns dans le développement de la MP. / Parkinson’s disease (PD) is a neurodegenerative disease which is accompanied by a strong inflammation, present both in the central nervous system and the periphery. Moreover, genes PINK1 and PRKN, which are causative of familial forms of PD, are implicated in the inhibition of mitochondrial antigen presentation (MitAP), a phenomenon which acts as a potential trigger for an autoimmune response. This work’s aim was to investigate the contribution of peripheral autoimmune and inflammatory processes in the etiology of PD, with a particular focus on phenomena implicating the mitochondria. Quantification of PINK1 and PRKN mRNA allowed us to determine that both genes could be inhibited by the activation – through lipopolysaccharide (LPS) or Enteropathogenic Escherichia coli (EPEC) exposition – of specific antigen-presenting cells, the monocyte-derived dendritic cells (MDDC). The expression of PRKN was also inhibited during the aging process in peripheral blood mononuclear cells. Through the investigation of T lymphocytes present in PD patients’ circulation, we identified a population of T CD8+ IL-17+ (Tc17) cells which could be activated by mitochondrial antigens. Furthermore, the MDDC were identified as a major contributor to inflammation since PD patients’ cells presented a specific expression profile characterized by an over-production of pro-inflammatory cytokines. This response was age-associated and corresponded with a pro-Th17 polarization of T CD4+ lymphocytes, a subtype which has been linked with autoimmune diseases. Taken together, our results support an implication of autoimmune mechanisms in the development of PD.

No description available.