LAG-3 is an immune inhibitory marker. These immune inhibitory markers function to reduce the capability of immune cells to elicit a proper immune response and to help maintain tolerance. During an acute infection, these markers assist in controlling the immune system, however, during a chronic infection these markers prevent the immune response from persisting to effectively fight the disease. Contrary to other immune inhibitory markers, LAG-3 is not highly expressed on T cells during chronic viral infections, such as HIV. The majority of information available on LAG-3 has been gained from murine models and cell lines. This thesis uses primary human T cells in order to observe rapid expression of surface LAG-3 from a pre-formed intracellular store and cleavage of these surface molecules into soluble variants by matrix metalloprotease cleavage. LAG-3 and sLAG-3 expression is compared with CD69 and cytokine expression to help understand the early immune response. / October 2015

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB). While approximately one third of the world’s population is infected with this pathogen, only a small minority of these individuals has active TB infection, where these individuals are able to transmit the pathogen to others. In previous microarrays performed in our lab from lung tissue of non-human primates (NHPs), it was noted that animals undergoing the activation of TB showed greatly increased expression of lymphocyte activation gene 3 (LAG-3). This protein performs immunomodulatory roles, which include: increased function of regulatory T cells, decreased function of Th1 effector T cells, and decreased monocyte differentiation. When studied in rhesus macaques infected with Mtb, RNA expression and protein levels of LAG-3 in lung tissue of active TB animals was found to be greatly increased when compared to lung from animals with latent TB. Interestingly enough, there was a bimodal distribution of LAG-3 expression in animals undergoing reactivation of the disease; the animals with greater levels of LAG-3 were the fast reactivators. LAG-3 expression in the lung tissue of animals with Mtb infections was mainly isolated to the outer periphery of the Mtb induced lung granuloma, where predictably, LAG-3 was expressed by lymphocyte populations of immune cells; mainly NK cells and various populations of T cells. To gain a greater understanding of the function of LAG-3, we created a co-culture system where CD4 T cells derived from blood and lung of Mtb infected NHPs were supplemented to Mtb infected differentiated monocytes. With this co-culture model, we utilized short interfering RNA (siRNA) to silence LAG-3. We observed a decreased bacterial burden, as well as decreased frequencies of IL-10 and IFN-γ producing CD4 T cells. This illustrates that the silencing of LAG-3 in CD4 T cells resulted in increased bacterial clearance, not due to up-regulation of IFN-γ. We believe that the bacterial reduction may be due to increased T cell proliferation, along with production of another proinflammatory cytokines. In the near future, we will utilize cytokine assays and microarrays to better understand the mechanism of action through which increased bacterial killing is occurring. / acase@tulane.edu

Etiological agents causing autoimmune diseases largely remain unknown. However, several lines of evidence suggest that environmental factors such as heavy metals (arsenic, lead and mercury) play a crucial role in the development of autoimmune disorders. In our model of mercury-induced autoimmunity, administration of subtoxic doses of HgCl2 to genetically susceptible strains of mice result in an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hypergammaglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of HgCl2 (tolerogenic dose). Previous studies from our lab had demonstrated that CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) control the induction and maintenance of tolerance to mercury. We investigated the therapeutic role of Tregs in our model by utilizing agents that are known to stimulate in vivo expansion of Tregs. We studied two such agents, CD3-specific non-Fc receptor-binding [(Fab’)2 fragment] monoclonal antibody (Anti-CD3) and immune complexes containing recombinant IL-2 and anti-IL-2 monoclonal antibody (IC). In our model, treatment of mice with Anti-CD3 had no effect on Treg population. Administration of Anti-CD3 with the tolerogenic dose prevented induction of tolerance and failed to improve the maintenance period of tolerance. Anti-CD3 in presence of mercury activated the immune-system causing splenomegaly and expansion of B cell population. Overall, in contrast to its protective role in other experimental autoimmune disease models, Anti-CD3 exacerbated mercury-induced autoimmune syndrome. Treatment of mice with IC resulted in selective expansion of Tregs with a modest decrease in IgE levels and autoantibody production. Administration of IC with the tolerogenic dose led to a reduction in autoantibody response, thus IC was able to extend the maintenance period of tolerance to mercury. Lymphocyte Activation Gene-3 (LAG-3) is an inhibitory molecule that maintains lymphocyte homeostatic balance by controlling effector T cell expansion and contributing to the suppressive functions of Tregs. Thus, with the goal to understand the impact of homeostatic balance on Hg-induced autoimmunity, we investigated the role of LAG-3 in our model. Administration of an anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in levels of serum IgE. Additionally, LAG-3-deficient B6.SJL mice exhibited an increased susceptibility to mercury-induced autoimmunity whereas, wild type controls suffered only from a mild disease. Moreover, adoptive transfer of wild-type CD4+ T cells protected LAG-3-deficient mice from mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant. / Microbiology and Immunology

Epithelial ovarian cancer is the sixth most common tumor disease among women and it is the leading cause of death from all types of gynecologic malignancies. The current standart of care consist of debulking surgery followed by platinum-taxane chemotherapy. Althought some patients benefit from the treatment, most eventually experience platinum-resistance and die from this disease. Immunotherapy based on application of immune checkpoint blockers represents a new treatment strategy in different cancer malignancies. However, emerging clinical data show only limited clinical efficacy of these agents in ovarian cancer patients with objective response rates of 10-15%. Therefore there is a strong need to identify a potential biomarkers, which allows to identify the group of patients, who will benefit the most from this costly treatment. The aim of my diploma thesis was to characterize the prognostic and predictive role of the immune checkpoints within the retrospective and prospective cohort of patients with high-grade serous ovarian cancer (HGSOC). Our study follows, that the expression of PD-L1 molecule and high frequencies of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor microenviroment is significantly correlated with a better prognosis of patients with HGSOC. Moreover, PD-L1 and PD-1...