Les maladies inflammatoires chroniques sont associées à un maintien de la réponse immunitaire et notamment la présence de cellules T CD4+ mémoires. Les maladies inflammatoires chroniques de l’intestin ont pour conséquence une perte osseuse due à une augmentation du nombre et de l'activité des ostéoclastes, les cellules responsables de la résorption osseuse. Les cellules CD4+ ont été décrites comme participant à la différenciation des ostéoclastes mais la nature exacte de ces cellules reste encore indéterminée. Des études in vivo et in vitro chez la souris et chez l’Homme nous ont permis de décrire une population inflammatoire Th17 TNFα CD4+ présente dans la moelle osseuse participant à la différenciation des ostéoclastes. Ces cellules Th17 participent au recrutement et à la différenciation des monocytes en ostéoclastes en agissant sur les cellules stromales mésenchymateuses. De plus, les maladies inflammatoires chroniques de l’intestin sont associées à une augmentation de la prolifération et de la différenciation des cellules souches hématopoïétiques en cellules myéloïdes. Suite à un stress chimique ou mécanique, les ostéoclastes participent à la mobilisation des cellules souches hématopoïétiques depuis la moelle osseuse. Nous avons montré qu’au cours des maladies inflammatoires de l’intestin, l’augmentation de l’activité des ostéoclastes participe à la prolifération et la différenciation des cellules souches en cellules myéloïdes. Ainsi, les ostéoclastes sont devenus une nouvelle cible thérapeutique intéressante dans la lutte des maladies inflammatoires chroniques de l’intestin / Chronic inflammatory diseases are associated to maintain of memory response and the presence of memory CD4+ T cells. Inflammatory bowel diseases are associated with bone loss due to an increase of the number and activity of osteoclasts, the bone-resorbing cells. CD4+ T cells participated to osteoclasts differentiation, but their nature is still undetermined. In vivo and in vivo studies in mice and human allow us to describe an inflammatory Th17 TNFα CD4+ T cells in the bone marrow that participated to osteoclast differentiation and link inflammation and bone loss. Th17 T cells induce the recruitment and the differentiation of monocytes into osteoclasts acting on mesenchymal stromal cells. Moreover, inflammatory bowel diseases are associated in an increase of hematopoietic stem cell (HSC) mobilization. In stress condition, osteoclasts act on HSC niches and induce their mobilization. We have shown that in inflammatory bowel disease, increase osteoclast activity is involved in the proliferation and differentiation of stem cells into myeloid cells. These myeloid cells are responsible for the intestinal inflammation. Thus, osteoclasts have become an exciting new therapeutic target in the fight of inflammatory bowel diseases

La plasticité cérébrale est une capacité remarquable des cellules du cerveau à adapter leur structure et fonction en réponse à l’expérience et l’environnement. Cette plasticité cérébrale est favorisée par des conditions de vie favorables qui peuvent être modélisées chez le rongeur par le modèle de l’Environnement Enrichi (EE). L’EE consiste à mettre un grand nombre de souris dans de grandes cages comprenant de nombreux objets (nids, tunnels, roues…) qui sont changés régulièrement. L’EE induit une activité physique volontaire accrue, des conditions optimales pour la stimulation des interactions sociales, du comportement exploratoire et des fonctions cognitives. L’EE exerce des effets bénéfiques sur les processus physiologiques au niveau de nombreux systèmes (hormonal, cardiovasculaire, immunitaire…). L’EE réduit les comportements anxio-dépressifs, améliore l'apprentissage et la mémorisation. Ces effets sont sous-tendus par des changements au niveau du cerveau et en particulier de l’hippocampe, où l’on observe en EE plus de neurogenèse et synaptogenèse. De manière intéressante, chez les souris immunodéficientes, les performances mnésiques et la neurogenèse sont très altérées, suggérant une interaction bidirectionnelle entre le système immunitaire et le cerveau. Parmi les cellules du système immunitaire, les lymphocytes T (LT) semblent jouer un rôle particulièrement important dans les mécanismes de plasticité neuronale. Notre objectif a été de caractériser le rôle des LT dans les effets de l’EE sur la plasticité cérébrale et de chercher si ces effets impliquent une modification des LT par l’EE. / Cerebral plasticity is a remarkable ability of brain cells to adapt their structure and function in response to experience and the environment. This cerebral plasticity is enhanced by favorable living conditions that can be modeled in the rodent by the Enriched Environment (EE) model. The EE consists in large number of mice in large cages including numerous objects (nests, tunnels, wheels ...) which are changed frequently. EE induces increased voluntary physical activity, optimal conditions for stimulation of social interactions, exploratory behavior and cognitive functions. EE has beneficial effects on physiological processes in many systems (hormonal, cardiovascular, immune system...). EE reduces anxio-depressive behavior, improves learning and memory. These effects are underpinned by changes in the brain and particularly in the hippocampus, where EE induce more neurogenesis and synaptogenesis. Interestingly, in immunodeficient mice, memory performance and neurogenesis are highly impaired, suggesting a bidirectional interaction between the immune system and the brain. Among the cells of the immune system, T cells appear to play a major role in neuronal plasticity mechanisms. Our objective was to characterize the role of T cells in EE’s effects on cerebral plasticity and to investigate whether these effects imply a modification of T cells by EE.

Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.

CD8+ T cells are essential for host protection against intracellular pathogens and tumors. During antigen-driven responses, CD8+ T cell fate is governed by transcriptional and epigenetic processes that allow naïve CD8+ T cells to develop into a wide range of effector and conventional memory cell subsets. Over the last decades, novel techniques and major efforts led to a better understanding of the origin, nature, and short- and long-term effects of these processes on individual CD8+ T cells. Under certain conditions, naïve CD8+ T cells can acquire memory phenotype and functions in an antigen-independent manner. Although homeostatic cytokines and initial activation pathways that drive the development of these unconventional memory cells had been identified, the ensuing transcriptional profile of these cells and their degree of similarity with conventional memory cells remained ill-defined. The epigenetic events that accompany unconventional memory formation were also not known.Here, we show that innate memory cells, a type of thymic unconventional memory cells, are transcriptionally close to conventional memory cells but only partially epigenetically programmed toward the full memory fate. We also show that the sole overexpression of the transcription factor Eomesodermin (EOMES), a master regulator of effector and conventional memory cells, is able to drive many of the phenotypical, functional, transcriptional, and epigenetic features of innate memory cells, and to induce the recruitment of BRG1, a member of chromatin remodeling complexes, to innate memory gene regulatory regions. We further show that the in vivo interleukine-4-dependent development of innate memory cells is largely dependent on BRG1. We bring to light that, in innate memory cells, EOMES is recruited in many instances to genomic regions previously bound by the transcription factor RUNX3. Overall, we provide insights into the mechanisms that allow memory cell formation and T cell receptor stimulation to be uncoupled. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

No description available.

Regulatory T cells (Treg) represent one of the most important mechanisms of immunoregulation. Treg suppress immune reactions and prevent overactivation of the immune system. There is a lot of ways of Treg action described, here we have focused on Treg interference with macrophages. The suppressor capacity of a highly purified Treg population was demonstrated in proliferation assays. The level of suppression of effector T cell proliferation differs depending on the presence of macrophages in the culture. Treg suppression has been significantly higher in the presence of macrophages. These observations led to hypotesis that Treg affect directly macrophages. However, using flow cytometry, reduction of expression of costimulatory molecules on macrophages after culture with Treg was not observed. Macrophages precultured with Treg showed a comparable functionality as macrophages cultured alone. Neither flow cytometry nor live cell imaging revealed any cytotoxic activity of Treg towards macrophages. Despite the presence of macrophages, Treg did not suppress effector cell proliferation in a model, where stronger activation of effector cells was induced. Therefore, a new hypothesis was presented - initially observed higher suppression in the presence of macrophages was probably caused by a qualitatively or...

Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths. Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus. This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus. We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML. After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia. These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.

The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow). The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates. In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event. Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Chlamydia spp. infect epithelial cells of the respiratory, intestinal, and reproductive tracts. Chlamydial infections in women may lead to pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting infection the female reproductive tract (FRT), Chlamydia also infects the gastrointestinal tract (GIT) of animals and humans. In mice Chlamydia muridarum disseminates from the FRT to the GIT via internal routes and in a stepwise manner. Initially Chlamydia spreads from the FRT to infect the FRT-draining iliac lymph nodes (ILNs), then the spleen, and then the GIT. The first step of this dissemination (FRT to ILN) is mediated by tissue CD11c+ DCs. Chlamydia transport from ILN to the spleen is dependent on cell transport and is mediated by sphingosine 1-phosphate (S1P) signaling. The third step of Chlamydia transport from the spleen to the GIT is significantly hindered in splenectomized mice. However, which cells mediate this transportation of the second and the third step remain unknown. Using mouse-specific C. muridarum as a model pathogen we show that following depletion of CD8+ T cells or monocytes, Chlamydia dissemination to the spleen and the GIT is significantly hindered. Furthermore, this study reveals that Chlamydia may infect various cell types which then mediate its dissemination internally. It remains to be determined what role systemic dissemination may have in Chlamydia pathogenesis.