Investigating MAIT cell regulation of lung immune responses during vaccination and infection.

Vaccination remains one of the most effective strategies for reducing the disease burden caused by pathogenic microorganisms. To initiate a pathogen-specific immune response, vaccines must deliver pathogen-derived antigens to antigen presenting cells, particularly dendritic cells (DCs), which if appropriately activated, can then selectively stimulate proliferation and differentiation of antigen-specific B cells and T cells. There is increasing evidence to suggest that pathogen-specific immune responses can be more effective if initially stimulated at the typical site of infection. Mucosal vaccination therefore aims to improve responses to respiratory pathogens by inducing localised immune responses in the lung. However, a major challenge in this approach is providing safe and effective stimulatory signals to DCs, which is typically the role of an immune adjuvant – a compound added to a vaccine that enhances immunity. Immune cells such as T cells are also capable of providing stimulatory signals to DCs through direct and indirect stimuli. This thesis therefore explores the potential of stimulating subsets of T cells that reside in the mucosa to enhance vaccine-induced responses. Specifically, the research explores whether populations of “unconventional” T cells, which share specificity for a limited range of non-peptide compounds, can function as “cellular adjuvants” to enhance lung immune responses. Two subsets of unconventional T cells, Mucosal Associated Invariant T (MAIT) cells and invariant Natural Killer T (iNKT) cells, show promise as cellular adjuvants. Both cell types express T cell receptors (TCR) of limited variability, and exist in a semi-activated state capable of responding rapidly to stimulation. The more well-described iNKT cells respond to specific glycolipids, with alpha-galactosylceramide (α-GalCer) a prototypical agonist. MAIT cells are activated by metabolites of vitamin B, such as 5-(-2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). There is evidence that activation of either population of unconventional T cells can promote DC activation and the subsequent priming of pathogen specific immune responses. However, there is limited data on how they can modulate DC phenotype in the lung. This thesis sought to examine how intranasal administration of agonists for lung MAIT and iNKT cells influences DC phenotype and capacity to stimulate adaptive immune responses in the lung. Initial studies showed that co-administration of either agonist with antigen resulted in enhanced stimulation of antigen-specific T cell and B cell responses; however, activation of iNKT cell promoted a more powerful response. Single cell RNA sequencing of DCs from lung-draining lymph nodes revealed distinct activation states depending on the agonist used. iNKT activation led to a population of DCs that had responded to interferon signalling, a critical regulator of anti-viral immunity and CD8+ T cell responses. In contrast, while MAIT cell activation altered DC phenotype, the stimulus was weaker, and included induction of a small population of regulatory like DCs co-expressing both regulatory and co-stimulatory markers. This suggested a role for MAIT cells in promoting regulatory T cell (Treg) responses. Indeed, coadministration of 5-OP-RU with an influenza antigen resulted in Treg induction alongside conventional T and B cell responses in the lung. This Treg response required MAIT cell activation but did not depend on direct cell-to-cell contact between the MAIT cells and the DCs or Tregs. The direct role of 5-OP-RU activated MAIT cells in modulating the lung immune compartment was further assessed in the context of influenza infection. Intranasal 5-OP-RU treatment, without influenza antigens, protected mice from lethal weight loss during subsequent murine influenza challenge and reduced immune cell infiltration into the lungs, suggesting a regulatory role of the MAIT cells. Single cell RNA sequencing of lung immune cells revealed that administration of 5-OP-RU alone induced MAIT cells to upregulate multiple immunoregulatory markers. Additionally, myeloid cells from 5-OP-RU treated mice exhibited broad downregulation of genes. This downregulation provided additional evidence that 5-OP-RU treatment produced a regulatory signal, most likely originating from the MAIT cells, to limit excessive activation of the surrounding immune cells. These regulatory signatures were long lived, and persisted during subsequent influenza infection, likely contributing to the reduced immunopathology observed. Thus, MAIT cell activation prior to infection can modulate immune responses in a protective manner. Combined, the findings of this thesis suggest that including agonists for MAIT cells may be a useful mucosal vaccination strategy. Not only can this lead to improved adaptive immunity, but can also limit immune mediated lung damage through two distinct mechanisms: by promoting accumulation of regulatory cells, including DCs, Tregs and other myeloid populations, and by directly acting as suppressor cells themselves.