<p>Splenic CD8α⁺ dendritic cells (DCs) have been described as key antigen presenting cells for the induction of CD8⁺ T cell responses to circulating antigen. This is through a heightened capacity to acquire and present the antigens via the process of cross-presentation, expression of high levels of the co-stimulatory and adhesion molecules required to stimulate CD8⁺ T cells, and the capacity to release high levels of the cytokines required to drive differentiation of CD8⁺ T cells into cytotoxic T lymphocytes (CTLs). However, recent research has indicated that the splenic CD8α⁺ DC population is more heterogeneous than originally thought. A previous study from my own laboratory suggested that a population of CD8α⁺ DCs that express the c-type lectin langerin primarily possess the heightened functions previously attributed to the total CD8α⁺ population. Therefore, the aim of this thesis research was to explore this subset of DCs in more detail, with specific emphasis on gaining mechanistic insight into their ability to elicit CD8⁺ T cell responses to circulating proteins. In the first section of this thesis, the hypothesis that the splenic langerin⁺ CD8α⁺ DCs were the critical subset involved in the induction of strong systemic CD8⁺ T cell responses to circulating antigen was tested in detail. This was examined using a genetically modified mouse model in which langerin-expressing cells could be easily identified and/or specifically depleted. It was first shown that the induction of CD8⁺ T cell responses to the model antigen ovalbumin was dependent on entry into the spleen in the presence of appropriate stimulation, which in these studies was provided by agonists for the toll-like receptors (TLRs) and/or signals from innate-like lymphocytes called natural killer T (NKT) cells. The primary targets for these signals were shown to be splenic langerin⁺ CD8α⁺ DCs, as CD8⁺ T cell responses were significantly reduced in hosts depleted of these cells within the spleen. Furthermore, agonists for TLRs that were not expressed by langerin⁺ CD8α⁺ DCs failed to enhance T cell responses. The langerin⁺ CD8α⁺ DCs were shown to be located in the marginal zone of the spleen, where they could readily screen the blood for antigens, and their function was critical to the induction of CD8⁺ T cell responses within six hours of antigen delivery. Interestingly, other local langerin-negative antigen presenting cells (APCs) were shown to be capable of cross-presentation, but with significantly reduced capacity to prime CD8⁺ T cell responses. Therefore, in the second section of this thesis the hypothesis that the langerin-negative APCs were capable of contributing to CD8⁺ T cell responses with appropriately timed stimuli was investigated. One of the downstream effects of inducing NKT cell activation at the time of priming was shown to be the “pre-conditioning” of langerin-negative DCs, allowing them to respond strongly to subsequent TLR ligation. Using SiglecH-DTR mice, it was shown that plasmacytoid DCs (which are langerin-negative) were pre-conditioned by NKT cell activation, allowing them to respond more actively to the delayed TLR stimulation by producing significantly enhanced levels of IFN-α. This factor was also potentially responsible for “feeding back” to the CD8α⁺ DCs (including langerin-expressing CD8α⁺ DCs), to enhance their function, as indicated by increases in cytokine production. Significantly, the major langerin-negative DC populations, defined as CD8α⁻ DCs, were pre-conditioned to have an enhanced cytokine release response to subsequent stimulation through TLR7, a receptor not expressed by langerin-positive DCs. This enhanced ability to respond to TLR7 ligation permitted these langerin-negative APCs to contribute to increased CD8⁺ T cell accumulation, with enhanced functional activity. Importantly, the CD8⁺ T cell response induced remained significantly dependent on initial cross-priming by langerin⁺ CD8α⁺ DCs, and it was only through pre-conditioning that langerinnegative APCs could contribute to enhancing the T cell response. In the third section of this thesis, the hypothesis that the CD8⁺ T cell responses generated in the presence of langerin⁺ CD8α⁺ DCs were phenotypically and functionally distinct from those responses generated in their absence was tested. No obvious differences were seen in CD8⁺ T cell homing, memory phenotype, restimulatory capacity, and expression of key molecules involved in metabolic function, survival and cytolytic function. However, in vivo cytotoxic function several weeks after priming was comparable, suggesting that this function was not related to initial burst size, providing some evidence of difference in function between CD8⁺ T cells primed in the presence or absence of langerin⁺ CD8α⁺ DCs. In summary, the splenic langerin⁺ CD8α⁺ DCs are the major subset responsible for cross-priming CD8⁺ T cell responses to circulating antigen, and for interpreting multiple stimulatory signals for enhancing the response. However, effective CD8⁺ T cell responses can be generated in their absence, particularly when antigens are provided in the context of appropriately temporally phased stimuli.</p>
Mesothelin-specific CD8+ T Cell Responses Provide Evidence of In Vivo Cross-Priming by Antigen-Presenting Cells in Vaccinated Pancreatic Cancer Patients
