We compared the ability of endogenous uterine CD8+T cells to produce IFN in WT and PD-L1 deficient mice. million people in the United States each year, and the incidence of infection has continued to rise since 2000 (1). Adolescent girls and young women infected withC. trachomatisface reproductive tract damage and increased risk of ectopic pregnancy and infertility (2). During primary infection of mice,C. trachomatisinfection stimulates a 100-fold expansion of CD8+T cells that subsequently contract to form a memory population (3,4). However, upon rechallenge, the response of these cells is significantly smaller in magnitude than the primary response, with fewer cytokine producing CD8+T cells (4). This impaired secondary CD8+T cell response is reminiscent of infections with chronic viral pathogens such as Human Immunodeficiency Virus (HIV) and LCMV clone 13. The memory CD8+T cells that develop after HIV and LCMV Clone 13 infections exhibit an exhausted phenotype defined by low cytokine production, expression of pro-apoptotic genes, and low replicative potential, all of which lead to an extremely deficient secondary CD8+T cell response (5-7). A significant cause of these defective CD8+T responses in chronic viral infections is the engagement of immunoinhibitory pathways (8-11). A well-described immunoinhibitory pathway is made up of the receptor PD-1, which is expressed on CD8+T cells, and its ligand PD-L1, which is expressed on professional antigen presenting cells (pAPC) or on infected target cells. The engagement of the PD-L1/PD-1 pathway can antagonize the T cell signaling mediated by stimulatory molecules, as well as affect downstream signaling pathways that decrease cytokine production and reduce memory potential (12,13). It has not been explored whether PD-L1/PD-1 signaling plays a role in the lack CD8+T cell recall potential resulting fromC. trachomatisinfection. Here we show that the CD8+T cell response to genital infection withC. trachomatis, as with chronic viral infections, is negatively affected by the immunoinhibitory receptor PD-1 and its ligand, PD-L1. We also show that deletion or inhibition of this pathway improves the CD8+T cell response and results in enhanced bacterial clearance. == Materials and Methods == == Mice == C57BL/6, B6.PL-Thy1a (CD90.1/Thy1.1 congenic), and B6.129S7-IFNtm1Agt (IFN/) mice were purchased from The Jackson Labs. PD-L1 (Thy1.2 background) and PD-1(Thy1.1 background) deficient mice have been described previously ZLN024 and were generously provided by Arlene Sharpe. PD-L1 blocking Rabbit Polyclonal to NOX1 antibody (clone 10F.9G2) was generously provided by Gordon Freeman; B7-1 blocking antibody (clone 1G10) was purchased from BioXCell. For transient inhibition experiments, mice were treated with 200ug PD-L1 or B7-1 each day for three days prior to infection and then every other day after infection. All animals were maintained and cared for within the Harvard Medical School Center for Animal Resources and Comparative Medicine (Boston, MA) (14,15). All mice were treated with 2.5 mg medroxyprogesterone subcutaneously 7 days prior to infection withC. trachomatisto synchronize the murine estrous cycle. All experiments were approved by the Institutional Animal Care and Use Committee. == Growth, isolation, and detection of bacteria == C. trachomatisserovar L2 (434/Bu) was propagated within McCoy cell monolayers grown in Eagle’s MEM (Invitrogen, Grand Island, NY) supplemented with 10% FCS, 1.5 g/l sodium bicarbonate, 0.1M nonessential amino acids, and 1 mM sodium pyruvate. Infected monolayers were disassociated from plates using sterile glass beads and were sonicated to disrupt the inclusion. Elementary bodies were purified by density gradient centrifugation, as described previously (16). Aliquots were stored at 80C in medium containing 250 mM sucrose, 10 mM sodium phosphate, and 5 mM L-glutamic acid and were thawed immediately prior to use. To quantify the levels ofC. trachomatis, quantitative PCR (qPCR) with 16S primers specific forC. trachomatiswas performed as has been previously described (16). == Flow cytometry == Tissues were mechanically disaggregated and immediately stained for surface markers or stimulated for 5 h with 50 ng/ml PMA (Alexis Biochemical) and 500 ng/ml ionomycin (Calbiochem) in the presence of brefeldin A (GolgiStop; BD Biosciences) for intracellular cytokine staining. Cells were preincubated with anti-FcRg (Bio ZLN024 X-Cell) before staining with CrpA-APC (National Institute of Health Tetramer Core) or PD-L1-APC, CD4 Q-Dot, CD8-APC-Cy7, and CD90.2-PeCy7 (Biolegend). Cells were also incubated with CD11b-PB, CD11c-PB, B220-PB and CD19-PB to exclude these populations. For activation marker evaluation, we examined Compact disc62L-FITC and Compact disc127-PerCP (BD Biosciences). For intracellular cytokine staining IFN PE (BD Biosciences) was utilized and cells had been permeabilized using the Cytofix/Cytoperm Plus Package based on the manufacturer’s guidelines (BD Biosciences). The overall cellular number in each test was driven ZLN024 using AccuCheck Keeping track of Beads (Invitrogen). Data had been collected with an LSRII (BD Biosciences) and examined using FlowJo (Tree Superstar). == Inhibitory gene transcript appearance == Mice had been transcervically contaminated with 106inclusion developing systems (IFU) as previously defined (17). Five times after infection, tissue were mechanically disaggregated in 2 ml of aliquots and PBS immediately frozen in 20 C. RNA was extracted from 80 ZLN024 ul aliquots by phenol-chloroform precipitation. Quantitative invert transcriptase PCR ZLN024 (qRT-PCR) was performed using 25ng of purified.