Elucidating the role of regulatory T cells in colorectal cancer progression

Antigens (Ags) expressed by cancer cells can be specifically recognised by T cells contributing to anti-tumour immunity. For this reason, tumour Ag-specific T helper type 1 (Th1) cells can be detected in cancers and their infiltration is predictive of a prolonged disease-free survival. However, their activity is suppressed by T cells with inhibitory functions i.e. regulatory T cells (Tregs). High infiltration of tumours by these cells often correlates with poor survival. It is therefore crucial to understand their activity. Since Th1 and Treg cells are activated after Ag recognition, it is plausible that tumour Ag-specific Tregs inhibit Th1-mediated tumour immunity. Furthermore, vaccination, rather than inducing effector T cells, may be detrimental by activating Tregs. Increased numbers of CD25hi Treg cells are found in cancer patients, including CRC. However, in contrast to many cancers, the phenotype of these cells has not been studied in CRC patients. In this thesis, phenotypes of circulating, colon and tumour infiltrating Tregs are described. A high percentage of intratumoural CD4+CD25hi Tregs was found to express immunosuppressive molecules and these cells were enriched in late-stage CRC tumours. In addition, this thesis describes the measurement of Th1 and Treg cells recognising the oncofetal Ag, 5T4. Four immunogenic regions within the oncofoetal tumour Ag 5T4 and 14 peptides able to bind to most commonly found human HLA-DR alleles were identified. CD4+CD25hi cells mediated the control of tumour Ag 5T4-specific Th1 responses in CRC patients and their suppressive activity was not restricted to Th1 cells of the same Ag specificity. Also, responses to some, but importantly not all, immunogenic helper peptides discovered were influenced by Tregs. Thus Tregs may contribute to CRC progression by influencing tumour Ag- specific responses of Th1 cells. This body of work brings more information regarding the tumour Ag-specificity of Tregs and offers a future rationale to design peptide-based vaccines which exclude Treg peptides

Highly prevalent colorectal cancer-infiltrating LAP+ Foxp3- T cells exhibit more potent immunosuppressive activity than Foxp3+ regulatory T cells

Although elevated CD4+Foxp3+ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4+Foxp3+ and CD4+Foxp3− T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4+Foxp3+ T cells (Tregs) were Helios+ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ~30% of intratumoral CD4+Foxp3− T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and was ~50-fold more suppressive than Foxp3+ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies

Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111–130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR–pHLA-II interactions

The nature of the human T cell response to the cancer antigen 5T4 is determined by the balance of regulatory and inflammatory T cells of the same antigen-specificity: implications for vaccine design

The oncofoetal antigen 5T4 is a promising T cell target in the context of colorectal cancer, as demonstrated by a recent clinical study where 5T4-specific T cell responses, induced by vaccination or cyclophosphamide, were associated with a significantly prolonged survival of patients with metastatic disease. Whilst Th1-type (IFN-γ+) responses specific to 5T4, and other oncofoetal antigens, are often readily detectable in early stage CRC patients and healthy donors, their activity is suppressed as the cancer progresses by CD4+CD25hiFoxp3+ regulatory T cells (Treg) which contribute to the immunosuppressive environment conducive to tumour growth. This study mapped the fine specificity of Th1 and Treg cell responses to the 5T4 protein. Surprisingly, both immunogenic peptides and those recognised by Tregs clustered in the same HLA-DR transcending epitope-rich hotspots within the 5T4 protein. Similarly, regions of low Th1-cell immunogenicity also did not contain peptides capable of stimulating Tregs, further supporting the notion that Treg and Th1 cells recognise the same peptides. Understanding the rules which govern the balance of Th1 and Treg cells responding to a given peptide specificity is, therefore, of fundamental importance to designing strategies for manipulating the balance in favour of Th1 cells, and thus the most effective anti-cancer T cell responses