The antibody Fc region is critical for the therapeutic potency by virtue of its role in recruiting and activating the cytotoxic pathways of immune cells, complement activation and its role in antibody homeostasis (a process mediated by the pH dependent binding to the neonatal receptor FcRn). Bacterially produced antibodies lack of glycosylation at Asn297 and therefore do not bind to the surface Fc[gamma]Rs on effector innate immune cells, nor can they activate complement. This dissertation describes the engineering of aglycosylated bacterially expressed antibodies for binding to a specific Fc[gamma]R and therefore eliciting therapeutically relevant effector functions. Aglycosylated Fc mutants that bind to desired Fc binding ligands were isolated by a new E. coli homodimeric Fc display system coupled with high throughput flow cytometry. Two amino acids mutation in the CH3 domain (Fc5) conferred selectively high binding affinity of aglycosylated Fc domains to the Fc[gamma]RI receptor. Flow cytometry screening from a randomized Fc5 library resulted in the isolation of Fc mutants exhibiting higher affinity binding to Fc[gamma]RI receptor than the Fc5. Aglycosylated Fc[gamma]RI specific IgG containing the variable regions of the clinically important anti-Her2 antibody trastuzumab elicited dendritic cell-mediated ADCC in sharp contrast to the clinical grade trastuzumab (Herceptin) or the glycosylated coutreparts of the engineered antibodies, neither of which could potentiate target cell lysis with dendritic cells as effectors. In separate studies, a system was developed for the screening of periplasmically anchored E. coli libraries and the isolation of clones expressing antibodies that are specific to insoluble antigens or to cell surface markers. Following three rounds of flow cytometric screening, spheroplasts expressing specific scFvs were enriched 950-fold from a large excess (1,000x) of spheroplasts expressing nonspecific antibodies. / text

Pepper is a popular and important vegetable crop grown and consumed worldwide. However, pepper production is threatened by the gram-negative bacterium Xanthomonas euvesicatoria (Xe) which causes bacterial spot (BS) disease, one of the most common and destructive diseases on pepper. Due to limited genetic resistance resources in host species, a promising strategy for controlling BS disease is to transfer nonhost disease resistance (R) genes from other plant species into pepper plants to confer broad-spectrum and durable resistance. A maize R gene Rxo1 has been functionally transferred to rice plants and confers nonhost resistance to rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc) carrying a type III effector (T3E) AvrRxo1. Most Xe strains carry a T3E Xe4428, a homolog of AvrRxo1. Therefore, Rxo1 could be potentially employed to develop Xe-resistant pepper. In addition, a better understanding of the virulence function of Xe4428 may provide insights into the pathogenesis of Xe and new strategies for crop improvement. In this dissertation, we transformed Rxo1 into the far-related dicot species Nicotiana benthamiana and pepper, and characterized the Rxo1-mediated disease resistance against Xe strains carrying AvrRxo1 or Xe4428. In addition, we explored the virulence function and mechanism of Xe4428. In the Rxo1-transgenic N. benthamiana, we demonstrated that Rxo1 could condition resistance to Xe harboring AvrRxo1 but not Xe4428. We revealed that AvrRxo1 could directly interact with the nucleotide-binding domain of Rxo1 in vivo and in vitro. We further demonstrated that the nucleus localization of AvrRxo1 was required for its avirulence and virulence functions. In addition, the cytosol localization of Rxo1 was also necessary to confer disease resistance. The downstream signaling component NbNDR1 was demonstrated to be involved in Rxo1/AvrRxo1-mediated disease resistance. By RNAseq-based gene expression profiling, we identified six candidate genes of interest up-regulated by the Rxo1-AvrRxo1 recognition. Through virus-induced gene silencing screening, a gene encoding phenylalanine ammonia-lyase 4 was demonstrated to be critical for Rxo1/AvrRxo1-mediated disease resistance in N. benthamiana. Rxo1-transgenic pepper plants were resistant to the Xe strain with the complementary Xoc effector AvrRxo1 but not the wild-type Xe strain that carries Xe4428. A Xe4428 mutant with only one nucleotide substitution could trigger the Rxo1-mediated disease resistance in pepper. Both wild-type and mutant Xe4428 had significant virulence functions that could promote the Xe bacterial proliferation on wild-type pepper plants. In addition, the mutant Xe4428 had a higher expression level than wild-type Xe4428 in Xe bacterial cells, which might explain why the mutant Xe4428 but not wild-type Xe4428, could trigger the Rxo1-mediated disease resistance in pepper. We identified 14 pepper cystatin genes (CaCys), among which two genes (CaCys1 and CaCys13) could be induced, and two genes (CaCys3 and CaCys5) were suppressed by Xe4428. Ectopically expressing one of the induced genes CaCys1 in N. benthamiana increased the stomatal opening and promoted the Xe growth in N. benthamiana plants. Thus, we illuminate one possible mechanism of Xe4428's virulence function is to regulate the stomata apertures in N. benthamiana. Bacterial fruit blotch (BFB) caused by the gram-negative bacterial pathogen Acidovorax citrulli (A. citrulli) is one of the most destructive diseases in cucurbit crops, including melon and watermelon. A better understanding of the virulence and avirulence functions of T3Es in A. citrulli helps breeders engineer crop resistance to BFB. To this end, a clean genetic background of A. citrulli with multiple effector genes deleted is desired. Here, we optimized a marker-exchange-based method for sequential effector deletion and generated an AAC00-1 mutant with five effector genes (Aave2166, Aave3626, Aave1548, Aave2938, Aave2708) deleted (AAC00-15). AAC00-15 was less virulent in watermelon but more virulent in N. benthamiana. Through complementation, we characterized the function of individual effectors and identified a promising R gene, Roq1, that could be used to control BFB disease. / Doctor of Philosophy / As an essential ingredient in almost all cuisines, pepper is grown and consumed worldwide, providing human beings with favorable flavor and nutrients. However, pepper production is threatened by the destructive bacterial spot (BS) disease caused by the bacterial pathogen Xanthomonas euvesicatoria (Xe). Due to limited genetic resistance resources in host species, nonhost resistance (R) genes from other plant species are desired to confer broad-spectrum and durable resistance to the pepper pathogen Xe. Previously, a maize (corn) R gene called Rxo1 was transferred to rice plants. This gene helped these rice plants resist a rice bacterial pathogen that causes leaf streak disease on rice. This rice pathogen has an effector (a virulent protein produced by bacteria to infect plants) that is required for the disease resistance. The pepper pathogen carries a similar effector, so transferring the maize R gene Rxo1 to pepper plants might similarly benefit peppers and help fight against the bacterial spot disease. In this dissertation, we successfully transferred the maize R gene Rxo1 into Nicotiana benthamiana and pepper plants. Our results indicate that this gene can help control disease caused by the pepper pathogen harboring the effector of the rice pathogen but not its native effector. We also illuminate how the disease resistance conferred by this maize gene happens in Nicotiana benthamiana plants. In addition, we explain how the corresponding effector helps infect plants. This research provides insights into the application of R gene transfer between far-related plant species and new tools to improve crop disease resistance.

Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T greffées. / Anti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells.

Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

La majorité des infections par le VIH sont acquises hétérosexuellement surtout chez les femmes en Afrique subsaharienne. Le tractus génital féminin (TGF) est la principale porte d’entrée pour le VIH et joue un rôle important dans la défense de l’organisme. De concert avec les cellules épithéliales, les cellules dendritiques (DC) aident à maintenir une balance immunitaire entre tolérance et inflammation. Dans un groupe de travailleuses du sexe (CSW) à Cotonou, au Bénin, des femmes (CSW ≥ 8 ans) ont été identifiées comme hautement exposées séronégatives (HESN). La fréquence de populations cellulaires myéloïdes de type Monocytes-Derived Dendritic Cells (MoDC) présentant un potentiel antiviral et « tolérogénique/régulateur » est augmentée au niveau du TGF des HESNs et les monocytes pourraient être impliqués dans leur génération. Les résultats de RNA-seq sur les monocytes totaux permettent de constater une augmentation de gènes associés à des fonctions effectrices, de protection/contrôle de l’infection et de régulation chez les HESNs comparé aux contrôles (2,5-5 années CSWs HIV- « early HESN », CSWs HIV+ et des femmes de la population générale Non CSWs HIV-). Les résultats de cytométrie en flux (FACS) démontrent une proportion élevée de non-classiques comparé aux autres sous-populations de monocytes sanguins, exprimant davantage de molécules effectrices et régulatrices, suggérant un lien avec les MoDCs tolérogéniques observées. Cinq individus ont séroconverti et ont présenté des modifications bien avant la séroconversion, soit une diminution de β-chimiokines et des IgG anti-gp41 dans le compartiment sanguin et mucosal du TGF. Un bris du profil « tolérogénique/régulateur » pourrait donc favoriser la séroconversion. / Most HIV infection are acquired through heterosexual intercourse, mostly in women in subsaharian Africa. The female genital tract (FGT) is the principal portal of entry for HIV and plays a critical role in host defense. Together, epithelial cells and dendritic cells (DC) help maintain immunological balance between inflammation and tolerance. In a group of commercial sex worker (CSW) from Cotonou, in Benin, women (CSW 8 ≥ years) have been identified as HIV-1 highly exposed seronegative (HESN). The frequency of myeloid cell populations alike to Monocytes-Derived Dendritic Cells (MoDC) presenting an antiviral potential and a tolerogenic/regulating profile were increased in FGT of HESNs and their monocytes could be implied in their generation. The RNA-seq results on total blood monocytes show an increase expression of genes associated with effector, protection/control of HIV infection and regulation functions in HESNs compared with control groups (2,5-5 years CSWs HIV- « early HESN », CSWs HIV+ and women from general population Non CSWs HIV-). Our flow cytometry (FACS) results show an elevated frequency of non-classical compared with other sub-populations in blood monocytes, expressing more effector and regulator molecules, suggesting a link with observed tolerogenic MoDCs. Moreover, five individuals have seroconverted and presented modifications before seroconversion such as lower levels of β-chemokines and anti-gp41 IgG in blood and mucosal compartments in the FGT. A break of this “tolerogenic/regulating” profile could favor seroconversion.