Characterization of Leishmania-Specific T cells in human skin and blood during cutaneous and mucocutaneous leishmaniasis

Parasites belonging to the genus Leishmania are among the most diverse of human pathogens, both in terms of geographical distribution and in the variety of clinical syndromes caused by them. Currently, 12 million people are infected worldwide in 88 tropical/subtropical countries, 2 million new infections are reported annually, and 350 million people are under infection risk. In the past decade, the number of cases in endemic areas has increased sharply. In addition, leishmaniasis is spreading to several non-endemic areas of the world due to coinfections with HIV. Control measures currently available are case detection and treatment with drugs, which are expensive, not always available and cannot be self-administered. The problem is further aggravated by the surge of drug resistance parasites necessitating the development of an anti-Leishmania vaccine or other immunological therapies urgent. Research has demonstrated the importance of the immunity in controlling both cutaneous and mucocotaneous leishmaniasis, however the majority of this work has been performed on blood samples taken from patients and not from the site of infection at the skin. In this context, the current project aims to characterize the immune mechanisms involved in controlling Leishmania in both the blood and the skin. In this proposal, we aim to apply this established approach for the first time in studies with human localized cutaneous (LCL) and mucocutaneous leishmaniasis (MCL) in order to understand the immune mechanisms associated to cell development, function and regulation in the skin and blood. Through this work we will be able to identify in detail components of the cutaneous and systemic immunity that are involved during infection and determine which of these factor can drive lesion development or/and parasite control. The results will also provide important data that will increase the understanding of Leishmania immunity providing explanations about pathological differences observed in cutaneous and mucocotaneous leishmaniasis patients and contribute to the improved design of future vaccines and drug target.

Leishmania (Viannia) brasiliensis causes a wide spectrum of cutaneous disease ranging from self-healing cutaneous leishmaniasis (CL) to tissue destructive mucocutaneous leishmaniasis (MCL) in humans. During infection CD4+ and CD8+ naïve T cells are driven to expand into different subsets including memory compartments which are crucial to infection control and tissue damage observed during CL and MCL. Although Leishmania-reactive memory T cells have been characterised in blood from both CL and MCL patients, little is known about the differentiation/function of these cells in the skin/lesion site. Factors such as persistent antigen stimulation and inflammatory state have been found to play a key role in directing the development of CD4+ and CD8+ differentiation into effector memory (EM) T cells (CD28-CD27-CD45RA-) and also EM T cells that re-express CD45RA (CD28-CD27-CD45RA+; EMRA). These EMRA cell which exhibit terminal differentiation and senescence characteristics contribute to diminished immune responses due to their increased expression of surface inhibitory receptors, DNA damage and reduced replicative capacity.. Mechanisms of memory T cell terminal differentiation during Leishmania infection are still unknown, however may assist the understanding of immune response diversity observed between CL and MCL. In this context, the present study aims to investigate the frequency, differentiation and effector function of CD4+ and CD8+ memory T cell from blood and skin of CL and MCL patients caused by L. brasiliensis.

Identification and reversal of new mechanisms for defective immunity during localized cutaneous and mucocutaneous leishmaniasis. The main aim of this study is to establish a roadmap to understanding both the nature of the defect in antigen specific immunity during leishmaniasis and whether this can be reversed by inhibiting the high level of pro-inflammatory responses in these subjects in vivo. These studies have the potential to identify ways to enhance the Leishmania immunity in humans by manipulating T cell development and function in a highly targeted fashion and also contribute to the improved design of future vaccines, adjuvants and drugs development. We are investigating immune responses to two different clinical form of leishmaniasis, MCL and LCL, which are the two main tegumentary leishmanisis disease in the world and for which treatment strategies are painful. Further, any mechanisms that we may identify will be relevant to the understanding of all different forms of Leishmania immunity and to a wide range of other infectious organisms. This valuable data on immunity in human tissues will be made freely accessible to other scientists, the academic beneficiaries, who will be able to use this information to further extend our knowledge on other aspects of leishmaniasis and also reduce the current spending on patient treatments.

Engagement with industrial collaborators. A second objective and its impact is the potential collaboration with pharmaceutical companies. The work that is proposed in this grant is of considerable interest to Pharmaceutical companies and the data generated will impact directly on strategy for adjuvant design, treatment and vaccination against leishmaniasis. Both PI's involved in this work have experience and successful collaborations with industry generating products currently marketed or in authorization stage.

The reduction of the use of animals in experimentation. As our studies will all be performed in human leukocytes and human samples, there is no need for extra animal testing. In fact this proposal makes use of the availability of unique human tissue samples that will be analysed with state of the art novel technology to investigate immunity during cutaneous and mucocutaneous leishmaniasis.

International dimension of the investigation. This collaboration adds value to the current application and the interchange of ideas, reagents and students between both groups. In addition, the Akbar Lab has hosted visiting scientists from Cuba, Italy, Portugal, Netherlands, USA, Austria and from Spain who have spent between 3 months to 3 years assimilating technology for use in their home institutions. This has established an international network of collaborators that the Akbar Lab is able to draw upon for expertise and reagents in a reciprocal manner. This will offer an opportunity to strengthen the new (started in 2014) collaboration between the Akbar lab and a Brazilian group after a technical visiting period made by Prof. Daniel Gomes.

Developing new expertise. This study involves an exciting multidisciplinary approach using human experimental models for blood and skin biopsies functional responses analyses. UFES has allocated 1 postdoctoral research and 2 PhD students to this project. Members of the Akbar lab, including postdoctoral researchers and PhD students from UCL will also have the opportunity to be involved in this work. The UFES individuals will liases with members of the Akbar group and work on different aspects related to the grant and will be instructed and given access to the above techniques. This will develop a new generation of researchers who will be well versed in the investigation of cellular, molecular and systems biology aspects of human Leishmania immunity.