Recently, another study used a similar mouse model to demonstrate that Ab responses to Gal do not require CD1 molecules or NKT cells

Recently, another study used a similar mouse model to demonstrate that Ab responses to Gal do not require CD1 molecules or NKT cells.35 Taking these findings into consideration, our results indicate that anti-A Ab production could be specifically inhibited by blocking iTCR-CD1d Morin hydrate interactions using anti-CD1d mAb while maintaining Ab responses to Gal and NeuGc epitopes. in sera. However, these reactions were not observed in mice, which lack NKT cells. Administration of anti-mouse CD1d blocking Morin hydrate monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient Morin hydrate (NOD/SCID/cmice, we investigated whether iNKT cells function to produce anti-A, anti-Gal, anti-NeuGc, or anti-allopeptide Abs. Methods Mice C57BL/6J (B6) (H-2b), BALB/c (H-2d), and nude mice (Balb/c) and F344 rats were purchased from CLEA Japan (Tokyo, Japan). mice on a B6 genetic background and mice on a B6 and Balb/c background, which are established by specific deletion of the J18 and CD1d gene segments, respectively, were used (kindly provided by Dr K. Seino, Laboratory Morin hydrate for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan).18 MHC class II-deficient (C2D) mice around the B6 background were purchased from Jackson Laboratory. mice around the B6 background, which completely lacked Gal expression, were used (kindly provided by Dr M. Sykes, Massachusetts General Hospital, Boston).19 mice around the B6 background, which are completely deficient in NeuGc and completely lacked NeuGc expression, were used (kindly provided by Dr Y. Kozutsumi, Kyoto University or college, Japan).17 Both and mice were crossed with mice to produce double-knockout mice. To generate double-knockout mice, F2 mice (produced by intercrossing F1 mice) were typed for each gene, and the appropriate mice were intercrossed and typed until double-gene knockouts were established (typically 4 generations). Finally, the genotypes were confirmed by fluorescence-activated cell sorting analysis (FACS), genomic Southern blotting, and polymerase chain reaction (PCR). All the mice Rabbit Polyclonal to ENDOGL1 were housed in the animal facility of Hiroshima University or college, Japan, in a pathogen-free, micro-isolated environment and used when they were aged 8C16 weeks. Anti-NeuGc and anti-Gal Ab production was elicited by intraperitoneal immunization of and mice with NeuGc- and Gal-expressing thymocytes obtained from F344 rats 2 times during a 1-week interval (10 106 cells/mouse at each immunization). As indicated, anti-A Ab production was similarly elicited by intraperitoneal immunization of mice with human A-RBCs from blood group A volunteers 2 times during a 1-week interval (5 108 cells/mouse at each immunization). Informed consent was obtained from all human volunteers in accordance with the Declaration of Helsinki. All experiments were approved by the institutional review table of Hiroshima University or college and conducted according to the guidelines of the National Institutes of Health (publication no. 86C23, revised 1996). Conditioning regimen for experimental mice As indicated, each mouse was intraperitoneally injected with 500 g anti-mouse CD1d monoclonal Abs (mAb; 1B1) or with 100 g anti-mouse interleukin (IL)-5 mAb (TRFK5; BD PharMingen, San Diego, CA) diluted in phosphate-buffered saline (PBS) 2 times at 1-week intervals. Mice that received injections of isotype-matched Abs served as the controls. To determine whether iNKT cells enhance Ab responses to specific Ag, we immunized mice with human A-RBCs together with intraperitoneal injection of either GalCer (KRN7000; 4 g/mouse) or PBS (control). Human peripheral blood mononuclear cell-chimeric mouse study Nonobese diabetic/severe combined immunodeficient (NOD/SCID)/cmice were purchased from your Central Institute of Experimental Animals (Kawasaki, Japan). Human peripheral blood mononuclear cells (PBMCs; 20 106 cells/mouse) from type O volunteers were engrafted in NOD/SCID/cmice by intraperitoneal injection after 1 Gy of whole body irradiation. The human PBMC-chimeric mice received intraperitoneal injection of anti-human CD1d mAb (CD1d42) diluted in.