Modulation of Dendritic Cells Function by H. polygyrus Products

Dendritic cells (DCs) are crucial antigen-presenting cells that can drastically change the development of an immune response by polarising T helper cells toward a Th1 or a Th2 phenotype. Heligmosomoides polygyrus, a murine model of human helminth, has been shown to strongly down-regulate its host immune response. At the DC level, its excretory/secretory products (HES) modulate the cytokine response and co-stimulatory marker expression to bacterial stimulation in favour of an antiinflammatory environment. A regulatory T cell-inducing TGF[beta]-like activity was also discovered in HES. In this work, we further characterised the immunomodulatory properties of HES and heat-inactivated HES both in vitro, using LPS-pulsed GM-CSF-grown bone marrowderived DCs, and in in vivo adoptive transfer of HES-, bacterial extract- or co-pulsed DCs. To determine if the TGF[beta], C-type lectin receptors (CLRs) or toll-like receptors (TLRs) were implicated in this immunomodulation, we treated DCs with blocking antibodies, chemical kinase inhibitors and grew DCs from knockout mice. These studies revealed that HES immunomodulation of DCs was independent of the TGF[beta] receptor, the two CLRs, Dectin-1 and 2, as well as any TLR. Spleen tyrosine kinase (Syk), which is crucial for the signalling of many CLRs, and phosphoinositide 3- kinase seemed not to be needed as well. In an attempt to reduce the number of potential immunomodulators in HES, HES size fractions were tested for their ability to reduce LPS-induced inflammatory cytokine production of DCs. The activity was limited to few fractions, fraction 14 being the most potent. Finally, physical interactions between DCs and biotinylated HES were measured by flow cytometry and revealed that CD24, a molecule required for HES interactions with B cells, was implicated, but not crucial. HES binding to CD24-/- DCs was reduced, but the LPS-induced cytokines and co-stimulatory markers levels were still down-regulated upon HES co-treatment.

Elucidating the mechanisms of paclitaxel-loaded-nanoparticle-induced immune response

Efthymia Vokali

Dentritic cells (DC) are the most potent antigen-presenting cells and play a key role in the induction of effective immune responses, suggesting a novel target for anti-tumor immunotherapy. Paclitaxel (PXL) is a widely used chemotherapeutic, initially characterized as a mitotic inhibitor, that has been shown to enhance maturation and function of DCs as well as up-regulate the production of inflammatory cytokines. Thus, modulation of DC function by the approved-for-human-use PXL could constitute a novel approach for reshaping immune responses against a tumor. For targeted delivery of PXL to DCs in the lymph nodes (LNs), where they are present in high concentrations and apt for antigen uptake, poly(propylene) sulfide core nanopatricles (NPs) developed by our laboratory have been used. PXL loaded-NP (PXL-NP) treatment induces massive immune cell infiltration into the draining LN 24h after intradermal injection of PXL-NPs in mice, suggesting that PXL-NP treatment induces a local and robust immune response (Thomas et al., in preparation). We hypothesized that this effect stems directly from the action of PXL-NPs on LN-resident DCs. We demonstrate that PXL-NPs are able to induce maturation and cytokine production in murine and human DCs in vitro, which may explain the in vivo observations of robust cell recruitment to the draining LN and generation of adaptive immune responses. Furthermore, the striking cell infiltration in the LN upon PXL-NP treatment was abrogated in complement protein 3 (C3) knockout mice in vivo (Thomas et al. in preparation). Interestingly, we show that the presence of C3 regulates IL-12p40 expression and, in turn, plays a crucial role in the activation of mature, functional DCs in response to PXL-NPs. Thus, the absence of functional DCs in case of C3 deficiency might account for the hindered infiltration. Together, this data suggests that PXL-NPs may effectively direct the immunomodulatory action of PXL to LN-resident DCs to alter immune responses.

2011

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

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

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

Iraklis Kourtis

EPFL2012

New immunomodulatory roles of lymphatic endothelium and implications for immunotherapy

Efthymia Vokali

EPFL2016

Elucidating the mechanisms of paclitaxel-loaded-nanoparticle-induced immune response

Efthymia Vokali

2011