The role of CCR8 in cutaneous vaccination

Our immune system fights microbes, such as bacteria, viruses, and tumours by rapidly activating immune cells, which then kill infected tissue cells and tumour cells in a highly specific manner. In an initial response, effector lymphocytes (T cells) quickly reach the site of infection and tumours where they recognize and kill these targets. Later, an immune memory compartment, composed of lymphocytes sharing the specificity of the primary effector lymphocytes, is established in order to provide long-lasting protection against the same challenges. Similarly, tolerance-inducing memory T (Treg) cells are established to prevent unwanted immunity at discrete tissue locations. Under normal circumstances, memory T cells are known to reside in healthy peripheral tissues, such as skin, lungs and the digestive-urinary tracts (herein referred to as peripheral immune surveillance T [TPS] cells), where they wait for a new attack by the same infectious particles or tumour cells. This cellular immune surveillance system is at the heart of vaccination, a process whereby microbe-specific long-lived memory T and B cells are generated to establish a first-line defense against harmful infections. Smallpox vaccination, for instance, can be considered as one of the most successful examples of skin vaccination that led to world-wide eradication of a deadly infectious disease.

It is clear that TPS cells present in peripheral tissues make up a vast immune memory compartment that protects body surfaces from recurrent infections and autoimmune diseases. Human skin, for instance, harbours twice as many T cells as the combined T cells present in peripheral blood. Although we know much about immune cells present in peripheral blood, our knowledge about peripheral tissue TPS cells is rudimentary. Our lack of understanding is due in part to technical difficulties (access of healthy tissue, cell isolation) when working with TPS cells, which is not the case when working with peripheral blood T cells. Thanks to established collaboration with numerous plastic surgeons, we have been studying TPS cells present in healthy human skin. Our recent work has demonstrated that the majority of human skin TPS cells express the chemokine receptor CCR8, suggesting that CCR8 is involved in the recruitment and/or retention of TPS cells in healthy skin. Preliminary data indicate that T cells in other (skin-unrelated) tissues do not express CCR8, which led us to propose that CCR8 is a selective marker for cutaneous TPS cells. We hypothesize that CCR8+ TPS cells are generated in response to cutaneous vaccination. To test this hypothesis we will turn to mouse models of cutaneous vaccination using the model antigen OVA (ovalbumin) and well defined OVA-specific T cells. Obviously, such studies cannot easily be carried out in humans. In brief, the proposed mouse studies will reveal when in response to vaccination T cells start to express CCR8, which may occur at the early stage of an immune response when short-lived effector T cells predominate or at a late stage when the immune response has resolved and long-lived memory T cells have emerged. We will also find out where (skin tissue or skin-draining lymph nodes) CCR8+ T cells are formed. Ultimately, these studies are essential for understanding the importance of CCR8 and its chemokines in the generation and maintenance of the skin-specific immune surveillance system.

The methodology for the proposed mouse work is up and running in the laboratories of the principal investigator (Moser) and the co-applicant (Gallimore). The second co-applicant (McCully) has substantial experience in mouse models of peritoneal inflammation, which complements well with the expertise of processing skin and other mouse tissues in the Moser laboratroy. Mouse model-specific expertise is provided by our collaborators. In addition, our human studies have prepared us (Moser/McCully) well for carrying out the proposed in vitro studies with T cells recovered from mouse blood/spleen and tissues.
Aim 1 focuses on in vitro studies with mouse T cells that include the measurement of cell surface proteins, including CCR8, on T cells generated under diverse stimulatory conditions. The findings of aim 1 feed directly into aim 2.
Aim 2 studies the kinetics of CCR8 expression in OVA-specific T cells during cutaneous vaccination with OVA/CFA. Core studies will include T cell isolation form various mouse tissues followed by their phenotypic analysis (flow cytometry). These studies will benefit from the CCR8 staining methods defined in aim 1.
Aim 3 addresses the question of the importance of CCR8 (and its chemokine ligands CCL1 and CCL8) in the generation of a OVA-specific memory T cell compartment in mouse skin tissue. The necessary mouse models have been made available to us by our collaborators and the examinations require the same techniques as applied in aim 2.
Aim 4 includes "control" experiments for assessing the degree of skin-specificity of the CCR8 migration system. Again, the relevant mouse models and expertise are provided to us by our collaborators.

The main impact will be felt by colleagues working in similar areas of research. Because of the multifaceted nature of our research topic, we envisage that laboratories working in areas as varied as cellular and molecular immunology, dermatology, ageing, vaccination and immunotherapy will be interested in following our progress. These laboratories will benefit from our research by collaborating with us and/or by including our findings in their own research.
There will be a clear impact on students and young scientists because our project offers the expertise and training of professional skills in a highly competitive area of research. Graduates and young scientists leaving our labs will be well prepared for continuing their research careers in academia or industry.
The proposed project relies heavily on collaborative research between the laboratories of the lead applicant (Moser) and co-applicants (Gallimore) as well as established collaborators in Cardiff (Birchall), Germany (Picher), Madrid (Martinez-A, Manes) and Boston (Luster). Our project will strengthen Infection and Immunity research at Cardiff University School of Medicine through the proposed collaborations between local laboratories with complementing research expertise.
We have considerable experience in translational research as evidenced by numerous consultancies at small biotech and pharmaceutical companies as well as by industrial collaborations. As inventors or co-inventors of several patents we are fully aware of the need to translate the fruits of our fundamental biomedical research into the clinic. In fact, Cardiff University has a strong track record of successful exploitation of intellectual property developed from its research base. A dedicated office of 12 people (www.cf.ac.uk/racdv/) is in place for working on all aspects related to technology transfer and commercial exploitation.
We hope that our research will lead to further funding by the MRC and/or charities allowing us to extend our findings in order to define the importance CCR8+ TPS in cutaneous immunity and skin tissue integrity. In the long run, we envisage that our results will be translated into novel strategies for the benefit of people undergoing vaccination or other forms of immunotherapy aimed at combating skin diseases (chronic infections, allergies, autoimmune diseases and skin cancer).