Characterising T cell responses to chitosan hydrogel vaccines in an orthotopic mouse model of colorectal cancer

Abstract

Anti-tumour T cell responses have a critical role in determining patient outcome in colorectal cancer (CRC). However, the generation of favourable immune responses in humans is poorly understood. Mouse models of CRC often fail to replicate the manifestation of human CRC by administering tumour cells into environments that do not reflect the colon and/or use genetically altered mice with impaired immune responses. To investigate anti-tumour responses in an orthotopic mouse model of colorectal cancer, intracaecal injection of CT26 colon carcinoma cells was administered. Chitosan hydrogel vaccines, loaded with (Gel + AH1) or without endogenous antigen AH1 (Gel), were used to investigate if anti-tumour T cell responses could be enhanced and lead to a protective immune response against CRC. The aims of this thesis were to 1) design and optimise a 10-parameter flow cytometry panel to identify T cell subsets, 2) to optimise an intracaecal surgery method used in previous studies to inject CT26 cells into the colon, 3) to determine the time point that effector responses were generated to chitosan hydrogels, 4) to investigate the effects of chitosan hydrogel vaccination on T cell populations in mice and finally, 5) to explore if prophylactic and therapeutic chitosan hydrogel vaccination could modulate T cell responses to protect against colorectal cancer, and what T cells are responsible for this protection. The 10-parameter flow panel required manual compensation corrections as software-based corrections were inadequate and often misrepresented populations. The surgical process was optimised to reduce inflammation and animal stress that may impact immune responses; however, the success rate of tumour implantation was lower than previous studies suggesting further optimisation is required. Effector (IFN-γ+) T cell responses were identified after chitosan hydrogel vaccination, peaking at 10 days after vaccination. Initial vaccination experiments revealed that Gel + AH1 vaccinated mice had greater frequencies of effector/effector memory (CD44+CD62L-) and central memory (CD44+CD62L+) CD4+ T cells in the spleen than Gel and PBS vaccinated mice suggesting this vaccine may offer the best protection against tumours. In further experiments, mice were vaccinated prophylactically and challenged with CT26 tumours. Neither Gel nor Gel + AH1 vaccines were able to protect mice from tumour growth. Additionally, the presence of AH1 in the gel led to distinct changes in T cell numbers and frequencies both in the tumour and periphery. In tumours, Gel + AH1 vaccinated mice had a high proportion and number of CD4+CD25+CD127- T regulatory cells and CD4+IFN-γ+ T cells whilst Gel vaccinated mice had a high proportion of CD8+ IFN-γ+ T cells. In the peripheral lymphoid organs Gel + AH1 vaccinated mice had more CD4+CD44+CD62L- effector/effector memory T cells whilst Gel vaccinated mice had more CD8+CD44+CD62L+ central memory T cells. These results suggested that AH1 concentration in the mice may dictate the formation of memory T cell subsets and that high AH1 concentrations may induce T reg development. Despite this, without in vivo monitoring of tumours it was unclear if these responses are protective against tumour growth or promote it and low tumour yield prevented statistical comparison. Overall, this study showed that although Gel vaccines may not be protective against colonic tumour growth, Gel + AH1 vaccines have unique immunomodulatory effects to Gel vaccines without AH1. Furthermore, the use of this model to reflect human CRC requires further investigation including the use of in vivo tumour monitoring, to track growth over a longer period, and also increased sample sizes in order to assess significance of findings.