Abstract
Antigen-presenting cells (APCs), are crucial components of the immune system that are uniquely positioned at the interface between innate and adaptive immunity. In particular, a specialized subset of APCs, dendritic cells (DCs), play a pivotal role in initiating T and B cell responses. This thesis focuses on the initiation of T cell responses, a process that requires DCs to acquire antigens derived from infected or malignant cells and process them for presentation to T cells with appropriate antigen receptors. The DCs also have to receive distinct stimulatory signals in order to increase their expression of chemokines, adhesion molecules, co-stimulatory molecules and cytokines. These phenotypic changes, known as DC activation, facilitate interactions with antigen-specific T cells to promote their proliferation and differentiation into effector cells. Thus, compounds that enhance DC activation can be utilized as vaccine adjuvants; as such, they are highly sought after in the development of novel immunotherapies.
Innate-like T cells express many of the features of conventional T cells but lack diversity in their antigen receptors, and exist poised in a semi-activated state, more like cells of the innate system. Of the different innate-like T cells subsets, CD1d-restricted natural killer T (NKT) cells are known to be potent sources of stimulatory signals that induce activation of DCs. Another innate-like T cell subset, mucosal-associated invariant T (MAIT) cells, have primarily been regarded as anti-bacterial effector T cells because they react to pyrimidines derived from bacterial riboflavin synthesis. However, because they share many phenotypic properties with NKT cells, it is possible that MAIT cells could also play a role in influencing adaptive immune responses by modifying the phenotype of DCs. Indeed, at the start of this thesis, a study was published showing that activated human MAIT cells could induce activation of monocyte-derived DCs in vitro. Importantly, MAIT cells are highly abundant in humans and are enriched at mucosal sites, potentially making them useful cells to exploit in vaccine designs that can be clinically translated. Therefore, this thesis sought to determine if agonists that activate MAIT cells could act as adjuvants by facilitating cellular interactions that modulate the functionality of DCs in vivo in order to augment priming of antigen-specific T cell responses.
The MAIT cell agonist precursor, 5-amino-6-D-ribitylaminouracil (5-A-RU), forms pyrimidine intermediates with cellular by-products of metabolism that bind to the MHC I-related protein 1 (MR1) which engages and activates MAIT cells. For example, when 5-A-RU condenses with methylglyoxal (MG), the potent agonist 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) is formed. One major challenge in MAIT cell research is that 5-OP-RU is an unstable compound. To circumvent this problem, 5-A-RU was used as it is known to condense with endogenous metabolic by-products to form the agonists intracellularly for in vitro or in vivo studies. However, in this thesis, it is shown that 5-A-RU is also unstable, being labile to auto-oxidation resulting in the loss of biological activity. To overcome these challenges, a 5-A-RU prodrug was developed that impeded oxidative degradation and conferred enhanced MAIT cell-activating properties both in vitro and in vivo. Furthermore, this prodrug design also provided a scaffold for chemical conjugation of model antigens to 5-A-RU to facilitate co-localisation of both adjuvant and antigen within the same DC, a mechanism known to enhance T cell priming. In addressing the hypothesis that MAIT cells could help initiate T cells responses, it was initially shown that intravenous administration of 5-A-RU in vivo resulted in DC activation, and when co-administered with soluble antigen, resulted in antigen-specific T cell immunity. Moreover, 5-A-RU-peptide conjugates showed enhanced DC functionality compared to 5-A-RU and drove effective anti-tumour activity. However, further analysis of these compounds revealed the presence of an unexpected, unknown microparticle that activated CD1d-restricted NKT cells, with the NKT cells being the source of the cellular adjuvant activity observed; this observation was shared by colleagues with 5-A-RU manufactured from a different laboratory.
Elimination of the confounding NKT cell-activating microparticle by fractionation or filtration revealed that MAIT cells could still facilitate DC activation, but only when the filtered 5-A-RU was combined with MG to form 5-OP-RU immediately before use. Activation of DCs was only readily detected in the lungs and lung-draining lymph nodes, with MAIT activity potentially driving DC migration from the lung tissue to the lymph nodes. Interestingly, administration of antigen with 5-OP-RU formed from filtered 5-A-RU failed to generate antigen-specific T cells. However, co-administration antigen with 5-OP-RU and limited doses of a toll-like receptor (TLR) agonist provided additional stimulatory signals, enhanced DC activation and resulted in MR1-dependent expansion of antigen-specific T cells.
In summary, this thesis presents evidence that MAIT cells can influence innate and adaptive immune responses. However, the activity of MAIT cells alone is insufficient to initiate T cell responses, as additional signals are required. Nonetheless, this feature of MAIT cell function could be incorporated into vaccine strategies in order to drive antigen-specific T cells to pathogen- or tumour-associated antigens for the development of future immunotherapies.