The next day, splenic TCR+ T cells were isolated from na?ve mice and added to the culture (5 105 cells/well). infections have also been reported in travelers and the elderly (26, 27), and there is evidence of colonization AVE 0991 of healthy, nonsymptomatic patients (34). Due to the prevalence of opportunistic microsporidian infections associated with the HIV-AIDS pandemic, recent research has focused on the host’s immune response to these pathogens. Early animal studies showed that cellular immunity was necessary Rabbit Polyclonal to ASAH3L to protect SCID mice from a lethal challenge. Moreover, depletion of CD8+ T cells caused mice to succumb to intraperitoneal (i.p.) infection (21), and previous studies in our laboratory have shown that cytotoxic lymphocytes play a major role in protection against this effect (20, 21). Recent reports from our laboratory have demonstrated that dendritic cells (DC) play an important role in the priming of the immune response against (31, 32). T cells incubated with in response to infection with fungal pathogens (24). However, specific TLR molecules involved in DC activation during infection have not been identified previously. We evaluated the upregulation of specific molecules involved in activation of the DC response after infection. Different TLR molecules were tested, and TLR4 expression was found to be essential for induction of the optimal CD8+ T-cell response by these cells. MATERIALS AND METHODS Mice. Six- to 8-week-old C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). The animals were housed under Institutional Animal Care and Use Committee-approved conditions at the Animal Research Facility at The George Washington University (Washington, DC). Parasites and infection. A rabbit isolate of (genotype II), kindly provided by L. Weiss (Albert Einstein College of Medicine, Bronx, NY), was used throughout this study. The parasites were maintained by continuous passage in rabbit kidney (RK-13) cells, obtained from the American Type Culture Collection (Manassas, VA). The RK-13 cells were AVE 0991 maintained in RPMI 1640 containing 10% fetal calf serum (FCS) (HyClone Laboratories, Logan, UT). Mice were infected via the intraperitoneal (i.p.) route with AVE 0991 2 107 spores/mouse. stimulation was performed using irradiated parasites (220 krads). TLR AVE 0991 expression by dendritic cells. Expression of TLR2, -4, and -9 by dendritic cells was assessed on various days postinfection (p.i.) (2, 4, and 6 p.i.) by performing a phenotypic analysis. Briefly, spleens were harvested, and this was followed by enzymatic (collagenase D and DNase I) and mechanical, disruption, allowing for DC separation. The cell suspension was labeled for CD11c, NK1.1, CD19, and TLR2 (eBioscience, San Diego CA) or TLR4 (BD Bioscience, San Jose CA) expression. Intracellular TLR9 expression was determined after permeabilization and fixation with FoxP3 staining buffer (eBioscience) and intracellular staining with anti-TLR9 antibody (eBioscience). Cells were acquired with a FACSCalibur (BD Biosciences) and were analyzed with FlowJo (Tree Star, Inc., Ashland, OR). TLR2, -4, and -9 messages were detected by real-time PCR according to standard protocol in our laboratory (45). Splenic DC were isolated according to a previously described protocol (45). Briefly, spleens were harvested as described above. A cell suspension system was then tagged with anti-CD11c biotin-conjugated antibodies (eBioscience) and favorably chosen by magnetic purification using the manufacturer’s process (Stem Cell Technology, Vancouver, United kingdom Columbia, Canada). Favorably chosen cells had been tagged after that, and Compact disc11c+ Compact disc19? NK1.1? DC had been purified utilizing a cell sorter (FACSAria; BD Biosciences). RNA was isolated with an RNeasy package (Qiagen, Valencia, CA) based on the manufacturer’s guidelines and change transcribed with Moloney murine leukemia trojan (MMLV) change transcriptase.