Mechanisms of tolerance induction by erythrocyte-targeted antigens

Immune-mediated disorders such as autoimmune diseases, graft-versus-host disease or allergies, are a major contemporary burden. Despite significant advances in the field of immunological tolerance, immunosuppressive drugs are still the current hallmark for treatment of transplanted recipients or patients with autoimmune disorders. While these drugs ameliorate the lives of the patients, benefits are unfortunately counterbalanced by the need for life-long treatment and non-negligible adverse side effects. The development of a therapy affecting the immune system in an antigen-specific manner is thus required for these and many other situations. Pursuing this aim, our laboratory has recently developed a novel approach to induce antigen-specific T cell tolerance through targeting antigens to erythrocytes in situ. Apoptotic cells are known to be processed tolerogenically in vivo. The concept of our therapeutic approach is based on the premise that as aged erythrocytes are cleared through an apoptotic-like mechanism, the surface-bound antigen payload will be cleared tolerogenically along with the eryptotic debris. While great strides have been made in showing the potential of the approach in inducing antigen-specific immune tolerance, little is known about the mechanisms driving tolerance. The aim of this thesis is to further understand the mechanisms behind induction of tolerance by erythrocyte-targeted antigens. Aspects such as memory of tolerance, dependence on T cell receptor (TCR) affinity for the peptide major histocompatibility complex (pMHC) and molecular pathways involved in this process will be addressed. Using ovalbumin (OVA) as a model protein antigen, we demonstrated long-term induction of immune tolerance after treatment with erythrocyte-targeted antigens, and requirement of CD25+FOXP3+ regulatory T cells for maintenance of the tolerogenic state. Signaling through programmed death-1/programmed death ligand-1 (PD-1/PD-L1), but not through cytotoxic T lymphocyte antigen 4 (CTLA4), was shown to be required for antigen-specific T cell deletion, anergy and expression of regulatory markers. The recent evidences of a direct implication of low affinity T cells as active contributor of self-destruction in autoimmunity raise questions regarding the ability of peripheral tolerance to deal with T cells of lower affinities. To explore the impact of TCR-pMHC affinity on induction of peripheral tolerance by erythrocyte-targeted antigens, erythrocyte-targeted altered peptide ligands with gradated affinity for the TCR of OTI CD8+ T cells were used. We demonstrated that tolerance induction is dependent on TCR strength as well as activation state of low affinity T cells. Finally, RNA sequencing was performed to determine how genes are regulated in CD8+ T cells and which pathways are activated after treatment with erythrocyte-targeted antigens. Comparison with gene sets characteristic of T cells undergoing anergy or deletional tolerance revealed the similarity between response induced by the natural event of peripheral tolerance and tolerance induced by erythrocyte-targeted antigens. Together, these results pave the way for the continued exploration of erythrocyte-targeting for prophylactic and therapeutic modulation of the immune system.

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

Lymphatic vessels have traditionally been considered as passive conduits for interstitial fluid drainage and for immune cell trafficking to lymph nodes. In the last two decades, many studies have challenged this classical dogma: lymphatic endothelial cells (LECs) were shown to actively interact with immune cells by secreting chemokines and cytokines, and presenting antigens to T cells. These newly described functions pinpoint a multi-faceted role of LECs in orchestrating the immune response through both passive and active mechanisms. Yet, precise mechanisms leading to antigen-specific fine-tuning of the T cell response remain unexplored. Moreover, although lymphatics have been shown to play important roles in cancer, the contributions of LECs in regulating antitumor immunity have been largely overlooked by the biomedical community. This doctoral thesis aimed at filling these gaps. First, in order to investigate the mechanisms and implications of LEC antigen presentation to CD4+ T cells, we engineered a mouse model where LECs lacked the ability to present antigens to CD4+ T cells. In these mice, antigen-specific responses to vaccination were increased when compared to wild-type controls. Moreover, we demonstrated that LECs were poor inducers of CD4+ T cell activation in vitro, and that they could dampen CD4+ T cell activation by dendritic cells. Taken together, these findings suggest that LECs participate to the peripheral immune tolerance of CD4+ T cells. Second, we investigated the immunological consequences of the development of lymphatic vessels, called lymphangiogenesis, in mouse melanoma. Upon induction of tumor lymphangiogenesis, we observed striking tumor enrichment with immune cells, which resulted in a higher sensitivity of those tumors to immunotherapy. We subsequently demonstrated that inducing lymphangiogenesis in a mouse model of cold tumors, which lack immune infiltrates and do not respond to immunotherapies, could restore their responsiveness to immunotherapy treatments. Third, to predict which cancer types were the most likely to replicate the findings we obtained in mouse melanoma, we mined The Cancer Genome Atlas database for gene expression. We identified a set of cancers, such as colon adenocarcinoma and cholangiocarcinoma, where high expression of the lymphangiogenic growth factor was associated with a strong T cell signature. This doctoral thesis unveiled new immunomodulatory functions of LECs by which antigen-specific immune tolerance is induced. Additionally, it opened new avenues to target lymphatics therapeutically in cancer to potentiate immunotherapies. We believe this work contributes to demonstrating the immunological importance of LECs, and hope that lymphatics will be considered as an important immunomodulatory player of the tumor microenvironment when developing novel immunotherapies.

EPFL2018

Optimization of in vitro cord blood hematopoietic stem cell expansion through the identification of secreted factors mediating inter-cellular communication

Aline Roch

Robust and large-scale expansion of umbilical cord blood stem cells in vitro is necessary for widening the usage of transplantation therapies for the treatment of hematological and immune diseases. The lack of understanding of the complex inter-cellular networks regulating stem cell fate in culture explains the low success met so far for the ex vivo expansion of hematopoietic stem cells. The development of a mathematical model of in vitro hematopoiesis coupled with gene expression profiling led to predictions about the secreted factors that play a crucial role in regulating hematopoietic stem cell self-renewal in culture. We tested 18 putative molecules predicted to display effects on primitive progenitor (Long-Term Culture-Initiating Cell; LTC-IC) output, functionally validating three stimulators (VEGF, PDGF, EGF) and three inhibitors (TGFβ, CCL4, CXCL10). Combinatorial studies with the stimulatory molecules showed less-than additive effects, perhaps related to redundant signaling mechanisms. Small molecule-mediated inhibition of the downstream signaling pathways activated by VEGF, PDGF, and EGF led to a decreased expansion of primitive cell compartments, confirming the endogenous activity of these growth factors for the stimulation of blood stem and progenitor cells. Blocking TGFβ-mediated negative feedback signaling in culture enhanced mature cell outputs but had no effect on primitive cell expansion, consistent with the role of TGFβ as a proliferation inhibitor, and underlying the complexity and multi-parametric aspect of the inter-cellular regulatory network. Studies are currently underway to validate the functional activity of VEGF and EGF on in vivo repopulating stem cells (NOD/SCID repopulating cells; SRC) using a clinical-grade, closed-system bioprocess. These results constitute a major step in the functional elucidation of the complex extracellular signaling networks that govern stem cell fate in vitro

2009

Therapeutic Immunomodulation of the Lymphoid-like Tumor Environment Using Nanoparticulate Formulations

Iraklis Kourtis

Cancer is a leading cause of death worldwide. Understanding the underlying mechanisms that lead to tumor escape and evasion is pivotal for the design of new therapeutic applications. This thesis takes an immunobioengineering approach, and navigates through identifying the immunological parameters relevant in the tumor microenvironment, proposing and developing a strategy in response to address these parameters using nanosized drug and antigen carriers. It is widely accepted that changes in the immunological equilibriumof the tumor microenvironment are crucial for the progression, dissemination and metastasis of a developing tumor. Motivated by tumors' natural secretion of CCL21, a chemokine known for dendritic (DC) migration towards the lymphatics, and for attracting DC and T cells in the lymph node (LN), we perturbed the physiological secretion of CCL21 of B16-F10 melanomas, deriving both a knock-down (CCL21low) and an over-expressing (CCL21high) variant. Interestingly, in immunocompetent mice, growth in CCL21-secreting tumors had an advantage over the CCL21low sub-clones and was CCR7 dependent. CCL21low tumors harbored more antigen specific T cells, while CCL21-secreting, more T regulatory cells (Tregs), which also correlated with subsequent biochemical polarization. Moreover, CCL21-secreting tumors formed a reticular fibroblastic (FRC) network (ERTR7+gp38+CCL21+) reminiscent of the lymphoid tissue of the paracortex (T cell zone) within LNs. The lymphoidlike stroma was orchestrated by the recruitment of CCR7+ adult lymphoid tissue inducers (LTis), responsible for LN formation during embryogenesis, present only in the CCL21-secreting tumors. In mice lacking LTis (Rorc(γt)-deficient) control tumors displayed impaired growth, suggesting that tumor growth is stroma dependent. In addition, the stroma of control or CCL21high tumors was infiltrated by more myeloid suppressor cells (MDSCs) than in CCL21low variant. MDSCs are a heterogeneous population of immature myeloid precursor cells that repress T cell activation and antigen presentation in chronic inflammation and cancer, hampering the efficacy of anti-tumoral vaccinations. CCL21's ability to protect allografts was further demonstrated in non-syngeneic recipients and in ectopically CCL21 transduced βTC tumor models. By mimicking secondary lymphoid organs, tumors may hi-jack and modulate the immune response from immunogenic to tolerogenic to allow for growth and metastasis. Intrigued by the excessive stroma infiltration of MDSCs in CCL21-secreting tumors, we performed a detailed spatiotemporal biodistribution analysis of ultrasmall (sub 50 nm) nanoparticles (NPs) in naïve and tumor bearing mice. Intradermally administered NPs rapidly drained to the LNs and spleen and persistently targetedmajor phagocytic populations that play a key role in hosting an immune response in naïve and tumor bearing mice. Furthermore, tumor-induced myeloid compartments, mainlyMDSCs, were highly targeted naturally within the tumormicroenvironment and spleen (tumor macroenvironment) 12 h post administration, without the use of site specific ligand. Following the MDSCs' natural preference towards phagocytosing NPs, and taking advantage of their excessive reductive cytoplasm, we designed a macromolecular pro-drug formulation that bears a cytotoxic payload and can be released in highly reductive microenvironments. Specifically, we engineered a reducible delivery system of 6-tioguanine (TG) in various formulations. Remarkably, bothMDSC compartments were ablated, monocytic ∼20 fold (CD11b+Ly6c+), polymorphonuclear ∼100 fold (CD11b+Ly6g+), for at least 7 d after administration in tumor bearing mice. As the bioidstribution revealed that apart from MDSCs, other antigen presenting cells (APCs) have phagocytosed NPs, we observed only a ∼2 fold decrease in APCs, suggesting that the rest could be used for vaccination. Taking into account that cancer vaccines and cancer immunotherapy rely on the efficient antigen presentation and education of naïve T cells by professional APCs, we explored the ability of NPs to deliver antigens in the appropriate sub-cellular compartment for antigen presentation and activation of T cells. Using the model antigen ovalbumin, we illustrated that exogenous antigen coupled to NPs by reducible bonds were internalized by DCs in vivo and in vitro. The conjugated antigen was preferentially released from the NPs with a reducible formulation, and was successfully presented in the major histocompatibility complex (MHC) I and II, which lead to activation and proliferation of antigen specific CD8+ and CD4+ T cells in vivo, respectively. Taking together, the nanoparticle platformcould be used efficiently for both inducing cytotoxicity and invoking an immune response. From discovery of new suppressive niches to therapeutical applications, we envisioned a two prong strategy that combined I) a modulatory regime to upset the balance of theMDSC populations both within the tumor microenvoronent and systemically with II) a cancer vaccine with the potential to significantly improve anti-tumoral therapeutic applications. This thesis demonstrated that such an approach is both possible and probable.

EPFL2012