Impaired Humoral Immunity and Tolerance in K14-VEGFR-3-Ig Mice That Lack Dermal Lymphatic Drainage

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8(+) T cells and production of IFN-gamma, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity. The Journal of Immunology, 2012, 189: 2181-2190.

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

New immunomodulatory roles of lymphatic endothelium and implications for immunotherapy

Efthymia Vokali

The lymphatic system serves a critical role in fluid homeostasis, lipid metabolism and immune surveillance. The growing appreciation of its implication in various diseases challenges the conventional view of lymphatics as a passive transport system. Traditionally, the lymphatic endothelium has been perceived as a structural scaffold with certain immunological functions but no active involvement in immunomodulation. In the lymph nodes (LNs), the main sites of immune regulation in the periphery, lymphatic endothelial cells (LECs) come to close contact with immune cells, suggesting potential interactions. Indeed, LECs have been recently shown to suppress dendritic cell maturation and present peripheral tissue and tumor antigen for CD8+ T cell deletion. While LECs have only begun to be acknowledged as active regulators of immunity, their function and relative contribution in shaping immune responses is as yet poorly understood. This thesis aimed to elucidate the direct role of LECs in the induction of CD8+ T cell immunity and tolerance. First, we demonstrated that murine LECs can actively scavenge and cross-present exogenous antigen to cognate CD8+ T cells under non-inflamed conditions. By utilizing an in vitro coculture system and the model antigen ovalbumin, we investigated the antigen-specific interactions between LECs and CD8+ T cells. LEC-educated CD8+ T cells proliferated, exhibiting an activated phenotype, however, they displayed early-generation apoptosis and failed to produce effector cytokines. Our findings establish LECs as antigen-presenting cells and suggest that they may assist in the maintenance of peripheral tolerance during homeostasis. The particular differentiation state of LEC-educated CD8+ T cells prompted us to investigate whether they are terminally tolerized or they could escape the dysfunctional state. We demonstrated that LEC-educated CD8+ T cells adopted a distinct phenotype with central memory-like characteristics and shared multiple functional properties with memory cells. Upon antigen re-encounter, LEC-educated CD8+ T cells mounted proliferative responses and generated cytotoxic effector cells. More importantly, they participated in anti-infectious immunity while preserving a secondary-memory persistent population. Our findings reveal a unique differentiation state of antigen-experienced CD8+ T cells, generated under steady-state conditions, which remain inactive but can be functionally reactivated upon antigenic inflammatory challenge. This previously unanticipated feature of LECs triggered questions for their antigen-presenting function in an inflammatory setting. We asked whether the previously observed extensive proliferation of LECs in the LN during inflammation might directly influence the induction of immunity. We employed an anti-VEGFR3 blocking antibody to inhibit LEC proliferation and thus, reduce the number of LECs following vaccine immunization. Alternatively, we generated the Prox1-Cre-DTR mouse model, allowing for specific ablation of LECs following administration of diphtheria toxin in vivo. Our findings advance our perception of the relative contribution of LECs in the establishment of adaptive immunity. This thesis elucidates the multifaceted immunological role of LECs and strongly suggests the importance of harnessing their immunomodulatory function to enhance current vaccines and immunotherapeutic strategies. Looking forward, our work will contribute to future advances in the clinic.

EPFL2016

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

The emergence of recombinant DNA technology has shifted research towards developing subunit vaccines, which use protein or peptide antigens instead of live/attenuated pathogens. Recent research has also shown that the best way to get sustained immunity is to deliver an antigen directly to antigen-presenting cells (APCs), specifically dendritic cells (DCs). However there are two difficulties to overcome in targeting DCs for use in subunit vaccines: (1) DCs are present in a low concentration in peripheral tissue (where typical vaccines are delivered) compared to secondary lymphoid organs such as lymph nodes. Thus delivering antigen to DCs in lymph nodes offers a powerful alternative for vaccine technology. (2) Activating DCs requires co-delivering an adjuvant also sometimes known as a molecular "danger signal" which matures DCs and initiates the subsequent adaptive immune response. Adjuvants are needed since most protein and peptide antigens are insufficiently immunogenic. Current danger signals being explored in experimental vaccine formulations mimic bacterial components (e.g., lipopolysaccharide (LPS), unmethylated CpG DNA) and often signal through a class of pattern recognition receptors knows as Toll-like receptors (TLRs), however limitations exist in that these often cause toxic side effects by inducing high levels pro-inflammatory cytokines. To address the need to target DCs, we used a nanoparticle delivery approach. We investigated the delivery of 20, 45, and 100 nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consisted of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm showed potential for vaccine delivery specifically targeting DCs in lymph nodes. Next we exploited our nanoparticle technology as a vaccine platform. We engineered antigen-bearing nanoparticle vaccines with two novel features: lymph node-targeting and in situ complement activation. Following intradermal injection, ultra-small nanoparticles (25 nm) were readily transported by interstitial flows into lymphatic capillaries, which leads to their accumulation in lymph nodes resident DCs. We further designed the nanoparticle surface chemistry to activate the complement cascade, thus spontaneously generating a danger signal in situ for DC activation. With ovalbumin as a model antigen conjugated to the nanoparticles, we demonstrated humoral and cellular immunity in mice. Finally we exploited nanoparticles as "synthetic pathogens" to isolate the effect of free surface thiols on complement activation and the subsequent immunological response induced. We found that surfaces with both hydroxyl and thiol groups activated complement to significantly higher levels than hydroxl groups alone. We then demonstrated that nanoparticles that induced thiol-associated complement activation were potent danger signals that were able to mature dendritic cells to express high levels of costimulatory molecules and the Th1 promoting cytokine interuleukin-12. Moreover, this dendritic cell maturation was dependent on a Toll-like receptor 4 (TLR4) signaling pathway and independent of the adaptor protein myeloid differentiation protein 88 (MyD88). Thus we uncover a potential direct link between complement and TLR4 signaling that is dependent on the specific activation of complement by thiol-expressing surfaces. In conclusion, this thesis develops a novel vaccine methodology, which is at the interface of nanotechnology and biotechnology, by harnessing lymphatic transport to target DCs and complement activation to activate adaptive immunity; we demonstrated an elegant vaccine technology platform for applications that include global health. Finally we exploit our nanoparticles as synthetic pathogens to discover fundamental mechanisms connecting the complement system and TLR signaling.

EPFL2008