CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8(+) T cell than CD4(+) T cell responses. While no specific CD8(+) T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8(+) T cell recruitment to the airways. The recruitment of influenza-specific CD4(+) T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4(+) T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4(+) T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4(+) and CD8(+) T cell expansion during influenza infection.

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

Development of a nano-adjuvant cancer vaccine and its evaluation for melanoma in combination with radiotherapy

Sylvie Hauert

Cancer is the second leading cause of death in the world and melanoma is the deadliest type of skin cancer. Although surgery, radiotherapy and chemotherapy are still standard treatments, the discovery of the role played by the immune system in cancer has allowed the development of new treatments, called immunotherapies. The goal of these treatments is to help the immune system eradicate cancerous cells, for example by increasing the anti-tumor response of the patient. In this particular case, the treatment has to activate cytotoxic CD8+ and CD4+ helper T cells against the cancer cells. This can be done by using subunit vaccines composed of tumor antigens, adjuvants and a delivery system, which will be internalized by dendritic cells (DCs) and induce its activation followed by the migration to the lymph nodes where it will educate T cells against specific antigens. Our laboratory has developed two different delivery systems, nanoparticles (NPs) and polymersomes (PSs) that can enhance the vaccineâs immune response by efficiently entering the lymphatic system, due to their size, and drain to the lymph node. Once they reach that location, they will be internalized by DCs and induce an anti-tumor response. In this thesis, we used these two nanocarriers to create a vaccine composed of different nano-adjuvants capable of activating several toll-like receptors simultaneously and test their efficacy at inducing an anti-tumor response in melanoma. We have developed and characterized the different nano-adjuvants, PS-MPLA, PS-CL075 and NP-CpG-B, and demonstrated, in vitro, synergistic effects on DC activation when PS-MPLA + PS-CL075 and PS-MPLA + NP-CpG-B were combined. In vivo, we showed the enhanced immune response with the different nano-adjuvant combinations, but only PS-MPLA + NP-CpG-B was able to delay tumor growth in melanoma. We then tested this vaccine in combination with radiotherapy, mimicking a clinical situation. We first demonstrated an enhanced immune response when combining a high irradiation dose, 15 Gy, with the nano-adjuvant vaccine draining the tumor-draining lymph node. Moreover, the vaccination of NP-CpG-B + PS-MPLA and radiotherapy increased mice survival by 17 days compared to mice only receiving radiotherapy. â Overall, we have demonstrated that combining different nano-adjuvants, targeting the tumor-draining lymph node, with radiotherapy enhanced significantly the survival in a melanoma model. This approach is particularly promising since ionizing radiation is a standard treatment in cancer and our vaccine can be applied to a multitude of cancer since it does not contain any antigen and its composition can easily be modified.

EPFL2018

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

Miriella Carmela Pasquier, Joseph Rutkowski, Melody Swartz, Susan Thomas

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.