Zhao W, Sachsenmeier K, Zhang L, Sult E, Hollingsworth RE, Yang H. expression of PD-L1 through infection was seen in both human and rat intestinal epithelial cell lines. We determined that cellular invasion by the bacteria is necessary for PD-L1 induction, potentially indicating that strains Igfbp4 are delivering mediators from inside the host cell that trigger the increased PD-L1 expression. Using knockout mutants, we determined that this effect largely originates from the pathogenicity island 2. We also show for the first time in any cell type that combined with gamma interferon (IFN-) causes a synergistic induction of PD-L1. Finally, we show that plus IFN- induction of PD-L1 decreased the cytokine production of activated T cells. Understanding immune evasion strategies could generate new therapeutic targets and help to manipulate PD-L1 expression in other diseases. serovar Typhimurium is one such pathogenic bacterium that causes a typhoid-like disease in mice or acute gastroenteritis in humans (10). Although not normally fatal in humans, induces fever, severe diarrhea, and abdominal cramping (11). The epithelial intestinal barrier is MK-0354 crucial in helping to control inflammatory responses and contributes to mucosal tolerance (12). Critical to pathogenicity island 1 (SPI-1) and expressed under the control of the transcription factor (14, 15). Once individual bacteria successfully invade host cells, a shift in pH and limiting nutrients signal to the bacteria the change in environment (16,C18). Consequently, downregulates SPI-1 and induces SPI-2, a T3SS whose gene products facilitate survival in this unique niche. The effectors encoded by SPI-2 facilitate intracellular survival of by preventing the host cell’s lysosome from fusing with the intracellular survival. may have several mechanisms to escape host immune detection, but most MK-0354 recently it has been shown to do so by increasing the PD-L1 expression of infected B cells to limit CD8 T cell responses (22, 23). These findings corroborate previous literature demonstrating that infection of gastric epithelial cells (25), indicating MK-0354 that it is a common and successful immune evasion strategy. Our objective was to determine whether caused an increase of PD-L1 in IECs, and if so, the effects of PD-L1 induction on T cell activation. RESULTS induces PD-L1 in IECs. It is known that induces PD-L1 in cells of the immune system (22,C24, 26). Since this pathogen encounters IECs at an early stage of infection, we sought to determine whether can also induce PD-L1 in this important cell type. In order to investigate changes in expression of PD-L1 on IECs, we used the well-established IEC colorectal adenocarcinoma cell lines, Caco-2 and HT-29. Basal expression of PD-L1 in Caco-2 and HT-29 cells was found to be low (data not shown), making these cell lines excellent models to study PD-L1 production in human IECs, provided the pathway components are expressed. Caco-2 and HT-29 enterocytes are sometimes cultured together to recapitulate intestinal characteristics, including tight-junction formation from Caco-2 cells and mucous secretion from HT-29 cells. IEC-6 cells are cells isolated from rat intestinal epithelium that are also widely used for enterocyte research. Using these IECs, we compared the abilities of several intestinal bacteria to induce PD-L1 expression, as measured with quantitative PCR (qPCR) 24 h after initial exposure (Fig. 1). The Gram-negative and Gram-positive were chosen as representative commensal bacteria that enterocytes MK-0354 regularly encounter. and inoculation elicited no change of basal PD-L1 expression in any cell type. In contrast, the pathogenic bacteria greatly induced PD-L1 mRNA expression. This effect was not unique to human IECs, since similar results were demonstrated in rat IECs (Fig. 1D). increased PD-L1 expression from 5- to 100-fold, depending on the cell type. The largest induction occurred in HT-29 cells (approximately 80-fold compared to nontreated), whereas Caco-2 and IEC-6 cells demonstrated lesser but significant induction ranging from 4- to 12-fold. PD-L1 induction was independent MK-0354 of Gram stain classification, as neither nor had an effect. In order to minimize variability of responses from multiple cell types, we chose to further the investigation of increased PD-L1 mRNA expression in human and rat intestinal epithelial cells. Intestinal epithelial cells were incubated with the commensal bacterium (LaB) or the pathogenic bacterium serovar Typhimurium (ST) for 1 h before bacterial removal and gentamicin addition. Intestinal epithelial cells were cultured for a further 24 h, after which the RNA was isolated, quantified via qPCR, and normalized to GAPDH. PD-L1 expression was measured in a 3:1 mixture of Caco-2:HT-29 cells (A),.