Defining T Cell Immunity to Merkel Cell Polyomavirus

Merkel Cell Carcinoma (MCC) is a rare but aggressive type of skin cancer. It can spread very quickly and so is often fatal. It carries a worse prognosis than melanoma. Recently, a new virus now called Merkel Cell Polyomavirus (MCV) was found to be combined into the DNA of the tumour cells. Although the majority of the population have been exposed to the virus, it usually causes no problems. However, the virus must be important in this cancer as viral proteins expressed by the cancer cells are crucial in allowing the tumour cells grow. Usually, proteins such as these are processed by the cell and portions called 'antigens' are presented on the cell surface. Then immune cells, called T-cells, recognise these abnormal antigens, kill the infected cells that express them and as such control the spread of the virus. One of the major risk factors for developing MCC is immune suppression i.e. having a weakened immune system. This means patients with HIV, blood cancers and auto-immune conditions, for example, are more likely to develop the cancer. This reinforces the idea not only that the MCV is important in MCC, but it may be a problem with T-cell immune response against MCV that allows the virus to become harmful.
In this project we will define exactly how T-cells respond to MCV and the relationship this has with development of cancer.
Firstly, we will assess the dynamics of a normal T-cell response to MCV infection by using healthy donors. Blood from approximately 20 donors will be tested (with full ethical approval and informed consent) against specially manufactured overlapping small lengths of protein ('peptides') that correspond to portions of 2 important antigens expressed by MCV (Large T Antigen (LTA) and Small T Antigen (STA)). We will be able to 'map' which portions of the antigens are recognised by T-cells and we can then focus in on these with smaller peptide lengths until we find the exact target or 'epitope' that is recognised. To allow us to do this we generate T-cell populations from single reactive T-cells. These populations are called 'clones'.
Next, we will use blood and surplus resected tumour from 20 patients with MCC as well as blood from 20 patients with a different skin cancer, basal cell cancer (BCC) (again with ethical approval and consent). These patients will be identified by the Skin Cancer Multi-disciplinary meeting that takes place at the Queen Elizabeth Hospital, Birmingham, with whom the University have close links. We will test T-cell response to epitopes identified in the first set of experiments in these three populations (MCC blood/ MCC tumour/BCC blood) to see if there are any differences between them. BCC patients have been chosen as a control population as this skin cancer affects a similar population group but is not known to be associated with immunosuppression. If T-cells do not react to epitopes, we will do specific experiments to identify potential causes for this. These may include a lack of MCV-specific T-cells, T-cell dysfunction or a generally weakened immune system. If T-cell response is poor, we will investigate mechanisms of enhancing this by use of stimulatory co-factors.
Following this, we will extend our project to see if epitope specific T-cell clones created from our first experiments are able to recognise cells that express them. First this will be done in model cell lines that have been genetically altered to express the peptide in question. It will then be repeated in Merkel cell cancer cell lines and fresh tumour cells themselves. We will also investigate the tumour cells ability to process viral protein and present them as antigens by looking at levels of expression of the important machinery that is involved in this process.

This work is important as by characterising T cell responses to MCV, we will be able to assess if therapies that use the immune system could potentially be used to treat MCC and if so we can make the first initial steps in designing these.

Aim: Defining T cell response to Merkel Cell Virus (MCV)
Objectives:
1. Identify, quantify and characterise MCV-specific T-cell responses to the potentially oncogenic LTA and STA proteins in healthy people, patients with Merkel cell cancer (MCC) and control group with basal cell cancer (BCC).
2. Investigate the immunogenicity of MCC malignant cells and of MCV antigens as targets for T-cell immunity.
First we will map LTA and STA specific epitopes capable of provoking CD4 and CD8 T-cell responses in healthy volunteer donors by using functional assays with synthetic overlapping and then truncated peptides. This is possible in part by establishment of single cell clones from reactive T-cell populations by limited dilution cloning.
We will then use T-cells from both MCC and BCC patients to assess response to these same identified epitopes in both populations.
Next, we will construct epitope specific MHC Class I tetramers to label cells and examine total specific CD8 T-cell frequency of both functional and anergic cells. We will then examine if incubation with co-stimulatory molecules can improve T cell response.
The next stage involves targeting identified epitopes when they are presented on a cell. Firstly, this is done using model cell lines into which we transfect individual peptide that is processed and presented. We will then use epitope specific T-cell clones in functional assays to assess recognition. This will then be expanded to see if clones respond to HLA-matched MCC cell lines (eg.MKL-1) and matched freshly resected tumour cells. We will also investigate the ability of MCC cells to act as antigen presenting cells by using western blots to examine presence of important proteins associated with this such as MHC class I, MHC class II, TAP1 and TAP2.
The major importance of this work is in demonstrating the capability of MCV antigens to act as targets for cytotoxic T-cell responses. This may be the initial step in developing an immunotherapy for MCC.

Although the impact of this work to the general public will be limited during the duration of the fellowship, the long term goals we envisage will have application to a much wider population. These goals will be achievable as a direct result of research to be conducted during this project.
Nevertheless, we will work hard to engage the public during the course of this work, as appropriate. We will aim to highlight the results of our experiments through relevant and accessible formats, such as online and local/national media where appropriate. We are currently developing web content through a programme run by the College and School of Cancer Sciences to increase online visibility of our research. We anticipate being able to discuss the results and relevance of the proposed work during the funding period, but ideally around the time of publication to avoid compromising any IP position. Furthermore, we have excellent established routes through the University Press Office and College's Marketing & Communications Manager that ensures the timely publication of articles relating to scientific innovation, and we aim to involve the MRC Press Office as a major partner in this type of work.

The combination of an emerging research centre alongside a blossoming clinical treatment hub provides us with a fantastic opportunity to become a leading institution in the field of MCC. UHBFT is a member of the Merkel Cell Carcinoma Multi-centre Interest Group Workshop and presentation of research findings from our laboratory will allow the exchange of ideas and information, enhance our reputation as a specialist centre and facilitate the development of clinical partnerships with other groups internationally. This will improve access to clinical trials which will have a direct benefit to local patients by providing therapeutic options that may otherwise have been unavailable. Developing collaborations with other national and international centres will also allow us to lead clinical trials for any immunotherapeutic interventions developed following the finding of suitable molecular targets as a direct result of this project. We expect that if such a target is found, it will be towards the end of this fellowship. The expertise in developing and clinically testing immunotherapies are already present within the T-cell immunology departments. This is further strengthened by the presence of the CR UK Clinical Trials Unit, based at the University which offers an established infrastructure in which developments made in the laboratory can be translated rapidly and effectively into clinical practice.