Identifying Chlamydia trachomatis factors that mediate PD-L1 upregulation

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Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent sexually transmitted infection in the United States. If untreated, infection can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility. Although C. trachomatis infection is treatable with antibiotics, many cases are asymptomatic, and repeat infections are common. Therefore, a vaccine is the best public health solution for managing and preventing infection. To develop a truly effective vaccine, it is critical to understand how C. trachomatis evades adaptive immunity to establish persistent infection during natural infection. CD8 T cells are normally integral for controlling intracellular pathogen infections, but during C. trachomatis infection, the CD8 T cell response is significantly impaired. We have shown that upregulation of the immunoinhibitory ligand PD-L1 during infection contributes to the diminution of the CD8 T cell response. Here we propose to identify C. trachomatis virulence genes that are responsible for upregulating PD-L1, as we hypothesize that these virulence genes are necessary to manipulate CD8 T cell immunity. In our first aim, we propose two complimentary approaches to identify C. trachomatis genes responsible for PD-L1 upregulation during infection. The first is to create and screen a lentiviral library containing individual C. trachomatis genes, testing the ability of individual bacterial genes to upregulate PD-L1. The second approach is based on a fortuitous phenotype in HeLa cells where only C. muridarum and not C. trachomatis is capable of upregulating PD-L1. By using a C. trachomatis/C. muridarum chimera collection developed by collaborators, we will identify portions of the C. muridarum chromosome that are sufficient to upregulate PD-L1. After generating a list of gene candidates from these two approaches, we will create C. trachomatis strains with null mutations in genes responsible for upregulating PD-L1 (CtNP), to validate their necessity for PD-L1 upregulation during infection in vitro and in vivo. In aim 2, we will use the CtNP strains to test if bacterial genes responsible for PD-L1 upregulation are required to manipulate the CD8 T cell response and hinder CD8 T cell-mediated protection. First, we will compare the proliferation and homing of C. trachomatis specific CD8 T cells during the course of infection with CtNP strains or WT C. trachomatis. Secondly, we propose to evaluate the protective capacity of memory CD8 T cells isolated from mice infected with CtNP strains or WT C. trachomatis by performing CD8 T cell transfer experiments into naïve mice. This work will provide the foundation for future studies to interrogate the mechanism by which C. trachomatis upregulates PD-L1. By understanding how C. trachomatis impairs the T cell response, we can develop vaccines that stimulate superior immunity compared to natural infection.