Innate pathogen sensing by local unconventional T cells during microbial infections

The immune system patrols the body constantly in search of "danger" signals derived from microbes or injuries. This response occurs in an innate (inborn) manner where all cells respond in a pre-programmed way, independently of previous infections or immunisations. In many scenarios this early phase of immunity is sufficient to control the infection. In other cases, a second "smarter" wave, the so-called acquired or adaptive immune response, needs to kick in. Certain white blood cells, so-called T cells, carry highly specific receptors, with each individual cell displaying a unique and distinct specificity to microbial molecules such that no two T cells in the body are identical. During infection, those T cells with the strongest potential to mount an effective response will become selected, increase in numbers and develop long-lasting memory against this pathogen. This process is also the mechanism underlying successful immunisations.

A subgroup of T cells behaves in an "unconventional" manner as they appear to detect infections in a pre-programmed fashion and much more rapidly than normal T cells. These unconventional T cells are heavily under-researched and their roles in the immune system very ill-understood, compared to other more "classical" immune cells. This is due to the fact that those unconventional T cells cannot be examined using traditional models and methods; in essence they comprise an entirely unique aspect of immunity that requires researchers to think outside the box.

Research by us and others has shown that unconventional T cells in fact constitute highly specialised cells that play a crucial role in detecting invading germs very early on in infection. This detection affects whether and how other immune cells become involved in the antimicrobial response, which ultimately determines how long the infection may last. We will here study patients with acute infections in their belly, from whom we obtain small samples for the analysis of their immune response. We will collect blood and waste fluid from individuals on home dialysis while they are healthy, and from those unfortunate individuals who may develop an infection during the study period. By comparing the differences between the healthy status and infection we can learn how the immune system responds rapidly to microbial insults. These investigations will be combined with laboratory studies that aim at mimicking the situation at the site of infection as closely as possible. For these experiments in cell culture dishes, we will isolate different types of white blood cells, including unconventional T cells, and study their interaction with each other and the outcome of their responses to bacterial pathogens.

The present proposal provides a detailed characterisation of unconventional T cells during microbial infections at the site of infection (belly), before treatment with antibiotics commences. Such an investigation has never been attempted before but is urgently needed to demonstrate as to how much laboratory-based studies can actually teach us about real infections. These studies not only improve our insight into the complex cell interactions in early infection but will also help identify new markers for future diagnostic tests. Such tests may be able to use unconventional T cells and related parameters to tell the clinician which type of germs actually causes the infection and how best to treat the infection (and avoid having to give unnecessary treatments that are both costly and may cause harm to the body). Finally, our findings will also highlight new avenues for treatments by interfering with the unconventional T cell-induced responses, by improving recovery from infection and by dampening collateral damage on healthy tissues and organs. In a world where people increasingly suffer from conditions related to advanced age (including loss of kidney function), this research will ultimately improve the quality of life of such patients.

This study will
1. Confirm local and systemic pathogen-specific responses of unconventional T cells in PD patients before, during and after acute peritonitis.
2. Demonstrate that local APCs in the peritoneal cavity can present microbial ligands to such unconventional T cells.
3. Demonstrate specific recognition of distinct microorganisms by defined human gamma/delta and MAIT TCR clonotypes.

This work will take advantage of unique access to a cohort of individuals receiving PD as life-saving renal replacement therapy. No other team in the UK has similarly convenient, continuous and non-invasive access to local samples from patients to study infections caused by a range of Gram-negative and Gram-positive organisms. Stable individuals will be evaluated 3-6 monthly, to establish pre-infection baselines and determine whether the composition of peritoneal cells remains constant over time, unless disturbed by infection. Patients who develop peritonitis will be analysed on the day of presentation prior to commencing antibiotic treatment, during the treatment period, and after full recovery.

We will establish for the first time in vivo whether different unconventional T cell populations sense distinct groups of microbes, by defining gamma/delta, MAIT, NKT cells and GEM T cell responses depending on the nature of the causative pathogen, using polychromatic flow cytometry. We will also characterise the potential of peritoneal macrophages and DCs to present non-peptidic ligands to unconventional T cells via the presenting molecules BTN3, MR1, CD1d and CD1b. To achieve further insight into the underlying mechanisms, we will conduct the first TCR clonotyping analysis of locally activated MAIT and Vd2+ T cells and correlate certain clonotypes with distinct pathogens in support of local recruitment/expansion during infection. In addition, we will establish pathogen-specific T cell clones for further characterisation both functionally and, where feasible, structurally.

The immediate impact will be felt by academic beneficiaries. Because of the multifaceted nature of our research topic, we envisage that laboratories working in areas as varied as cellular and molecular immunology, microbiology, nephrology and diagnosis will be interested in following our progress. The proposed project relies heavily on collaborative research between the applicants with basic and clinical research expertise. Successful completion of the proposed studies and dissemination of the findings will enable us to obtain further funding by research councils, charities and/or commercial sponsors, thereby further promoting and strengthening Infection and Immunity research in South Wales.

Novel research findings will be incorporated in the outreach and undergraduate teaching activities of the applicants such as their involvement in the annual Science in Health days and the student-selected component "Basic Concepts in Immunology". There will also be a clear impact on postgraduate students and early career researchers in the Institute as our project offers the expertise and training of laboratory and communication skills in a highly competitive area of research. Transferable skills acquired during this project will include excellent computer literacy, time management and teamworking as well as problem solving and organisational skills. Engagement with the scientific and non-scientific community will be sought via multiple routes through Cardiff University, the British Society for Immunology, the NISCHR Faculty, Involving People, HealthWise Wales, Welsh Kidney Patients Association and other channels, in order to disseminate research findings to the general public, patients, health care professionals, policy makers and funders within Wales, and on a national scale and beyond. This will be achieved through direct presentations, outreach activities and dissemination via press releases, printed news letter and website articles as well as social media.

Chronic kidney disease is a global health problem that is rapidly growing in aging populations. PD is a daily reality for 250,000 patients worldwide, corresponding to about 11% of the total dialysis population. PD is generally associated with a better quality of life but may also be clinically superior compared to haemodialysis, especially in children. However, infection and associated damage on the peritoneal membrane remain a major reason for treatment failure in PD patients. The failure to improve outcomes in dialysis patients indicates that current methods for diagnosis and therapy are neither sufficiently targeted nor effective. Our work addresses the molecular and cellular mechanisms involved in the local recognition of pathogens by the peritoneal immune system. We have considerable experience in translational and applied research as evidenced by productive collaborations with scientists and clinicians in the fields of infection, nephrology/urology, intensive care and oncology as well as industrial partners to pursue commercially and clinically attractive avenues further. In the longer run, we envisage that our results will be translated into novel diagnostic and therapeutic strategies for the benefit of people with local (peritonitis) or systemic infections (sepsis), inflammatory diseases with prominent contributions by unconventional T cells, and cancer. These developments will significantly increase the effectiveness of healthcare services and contribute to improved public health. Work to develop a point-of-care test for gamma/delta T cells is already being undertaken, with seedcorn funding from the Severnside Alliance for Translational Research (SARTRE)/South East Wales Academic Health Science Partnership (SEWAHSP) and the MRC Confidence in Concept Scheme.

The above impact pathways will contribute to raising the profile of South Wales, and the UK as a whole, as being a leader in research, teaching, innovation and engagement in the field of biomedical research.