Abstract
Immunotherapies, including adoptive T cell therapy and immune checkpoint blockade (ICB), have had huge success in cancer treatment; however, they only work in a subset of patients and often fail to induce durable protection. Thus, improving immune-based therapies is of great importance. In recent years, microbial components such as short-chain fatty acids (SCFAs) and lipopolysaccharides (LPS) have been explored as boosters of immune activity. SCFAs have been found to improve the function of CD8+ T cells in infection models and the abundance of bacteria with specific LPS structures have been associated with high immune infiltrates and response to ICB in patients with colorectal cancer. Despite this, SCFAs have not been fully investigated as a booster of CD8+ T cell function in the context of cancer immunotherapy. Similarly, whether the direct administration of bacterial LPS can directly modulate T cell activity and patient response to ICB is also unclear. This thesis explored if short-chain fatty acids (SCFAs) could enhance the function of OT-I CD8+ T cells, that recognise model antigen ovalbumin (OVA) residues 257-264 in the context of H2Kb, and if these cells improve anti-tumour immunity when transferred into tumour-bearing Ptp mice. Flow cytometry was used to characterise the phenotype of activated CD8+ T cells treated with SCFAs in vitro and explore the mechanism behind these. Activated and butyrate-treated CD8+ T cells were adoptively transferred into B16-OVA tumour-bearing mice to determine their tumour protective effects. Finally, flow cytometry and high dimensional analysis were used to phenotype adoptively transferred donor cells within the tumour, and the endogenous host response between treatments.
In vitro, butyrate-treated T cells produced more effector molecules IFN-γ, TNF, Gzmb and IL-2 and expressed higher levels of the TCR and associated molecules CD3, CD8 and CD28, activation markers CD25 and CD44 and mTOR signalling compared to non-butyrate-treated cells. The enhanced production of TNF, IFN-γ and CD25 was also found when CD8+ T cells were treated with Histone Deacetylase (HDAC) inhibitor Mocetinostat, suggesting the HDAC inhibitor activity of butyrate may mediate this effect. When adoptively transferred into B16-OVA tumour-bearing mice, butyrate-treated cells reduced tumour growth compared to PBS-treated mice. Butyrate-treated donor cells were present in higher frequency and number and produced more IFN-γ, IL-2, and GzmB than non-butyrate-treated donor cells. High dimensional analysis revealed that both donor and host cell clusters displaying memory phenotypes were enriched in mice that received butyrate-treated cells. These findings indicate that butyrate has potential as a booster of both the anti-tumour functions of CD8+ T cells and their memory capacity in vivo in mice. Consequently, butyrate could improve adoptive cell therapies for cancer, such as Chimeric Antigen Receptor (CAR)-T cell therapy.
This thesis also explored if intratumoural injection of LPS from Fusobacterium periodontium enhances the anti-tumour effects of anti-PD-1 ICB in CT26 and Colon26 subcutaneous mouse cancer models. Previous studies have shown an enrichment of F. periodontium within colorectal tumours with high immune infiltrates, and colorectal tumours responsive to PD-L1 therapy often have upregulation of genes associated with LPS signalling. Despite this, direct administration of LPS into colorectal tumours has not been explored, nor has its potential ability to boost immunotherapy response. Flow cytometry was used to characterise CD8+ T cell responses within the tumour and spleen of mice after intratumoural injection of LPS, anti-PD-1 ICB, or combined treatments. LPS injection had no additive anti-tumour effects with anti-PD-1 in both models when anti-PD-1 treatment was commenced the same day as LPS and had limited effects on the CD8+ T cell response within the spleen and tumour in both models. When anti-PD-1 treatment was given 48 h after LPS injection, tumour growth was reduced compared to control mice. However, there were still only minor effects on CD8+ T cells. These findings suggest that intratumoural injection of LPS can boost anti-PD-1 therapy in mice; however, the protective mechanism may not be mediated by CD8+ T cells and is sensitive to the timing of anti-PD-1 treatment.
Together, this thesis reveals that both butyrate and F. periodontium-derived LPS have therapeutic potential that should be explored further, with the aim of translating these findings into humans and improving immunotherapy responses in patients.