It is shown here that antigen-specific, mCD1-restricted lymphocytes can be generated in vivo by immunizing mice with a combination of plasmids encoding chicken ovalbumin, murine CD1d, and costimulatory molecules. isoforms: CD1a, -b, -c, -d, and -e (3). The isoforms are conserved in several mammalian varieties (4) and have been divided into two organizations based on the sequences of their external domains (5). CD1a, -b, -c, and -e comprise group 1, while group 2 consists of CD1d. Although all five isoforms are found in humans, only the group 2 isoforms are conserved from rodents to humans. CD1 molecules share some characteristics with both MHC class I and MHC class II ligands. CD1 proteins carry some resemblance to the classical MHC class I proteins both in overall sequence homology, especially in the 3 website, and by their typical association with 2-microglobulin (2m; referrals 5 and 6). However, unlike MHC class I molecules, CD1 proteins have been reported to be indicated without 2m (7) and don’t require the transporter proteins associated with antigen processing (Faucet) for stable manifestation (8C10). The mechanism for antigen processing for CD1 is definitely more similar to that of MHC class II than class I (11C13). Like MHC class II, human CD1b is definitely localized to endocytic compartments, including the specialized endosomes where MHC class II proteins are believed to bind endocytosed antigens (14C17). The non-MHCC encoded CD1 family of nonpolymorphic glycoproteins is definitely, therefore, much like, yet unique from, additional antigen-presenting molecules in its similarity to MHC class I by sequence, structural homology, and association with 2m, as well as its similarity to MHC class II by its cellular localization and dependence on the endosomal compartment for demonstration of exogenous antigens. Unlike classical MHC, CD1 can present nonpeptide ligands such as mycolic acid (18), lipoarabinomannan (19), and mycobacterial lipid antigens (20) to T cell receptorCbearing lymphocytes. The demonstration of foreign nonpeptide antigens by CD1 has been shown for the human being CD1b and CD1c isoforms from which human CD1d and its related murine isoforms are divergent (5). Casta?o et al. (2) have reported that murine non-MHCC encoded CD1d (mCD1) can bind long peptides with hydrophobic and heavy WYE-125132 (WYE-132) amino WYE-125132 (WYE-132) acids. Immunization of mice with CD1-transfected cells preincubated with peptide generated, CD1-restricted, peptide-specific CTL. These data suggest that mCD1 may have a antigen-presenting function by binding peptides with hydrophobic residues (2). Murine autoreactive, CD1-restricted T cells have been recognized in unimmunized mice (21, 22). To test the biological significance of mCD1 demonstration of foreign protein antigens, we generated an antigen-specific, CD1- restricted response by plasmid DNA immunization. This immunization protocol raised a CD1-restricted, ovalbumin-specific CTL response, demonstrating that protein antigen is definitely identified in the context of mCD1 and elicits a cellular immune response in vivo. Lysis by these cytotoxic lymphocytes are antigen and CD1 dependent, can be partially abrogated by WYE-125132 (WYE-132) anti-CD1 antibodies, and are competitively inhibited by Rabbit polyclonal to CDKN2A an established CD1-binding peptide. Furthermore, these CTLs lyse allogeneic targets in an antigen-specific manner. Materials and Methods Mice. C57BL/6 mice were purchased from your (Bar Harbor, ME) and managed under standard conditions in the University or college of California, San Diego Animal Facility accredited by the American Association of Laboratory Care. Mice of either sex were used at 2C4 mo of age. Preparation of Plasmid DNA. The plasmid pACB-CD1 was constructed by subcloning the BamHICXhoI fragment from your pBluescript vector encoding murine CD1D1 (reference 6; provided by S. Balk, Beth Israel Hospital, Boston, MA) into the BamHICSalI site of the pACB vector (23). The nCMVOVA, nCMVB7-1, and nCMVB7-2 plasmids have been previously explained (24). DNA was prepared using maxiprep kits (Qiagen Inc., Chatsworth, CA), with the modification of adding.