Characterization of xenoreactive natural secreting cells in rats

Abstract

Abstract: IgM xenoreactive natural antibodies (XNA) and the activation of complement are thought to be the main components involved in the hyperacute rejection (HAR) of discordant xenografts. Nevertheless few studies have focused on the B cell population responsible for the production of XNA. Using the guinea pig to rat discordant xenograft model we have developed a xenoreactive ELISA‐spot technique allowing the specific detection of XNA secreting cells in rats. In order to detect rat XNA‐secreting cells, guinea pig platelet membrane extracts, which have been shown to share major xenoantigens as recognized by XNA, with endothelial cells, were used as targets for rat XNA. Adult LOU/C rats lymphocytes from spleen, mesenteric lymph nodes, Peyer's patches, bone marrow, and the peritoneal cavity were used as source of XNA secreting cells. The number of IgM, IgG2a, Ig k light chain XNA, and IgM XNA secreting cells was measured using standard and xenoreactive ELISA‐spot techniques respectively. Data presented here show that IgM XNA secreting cells are almost exclusively located in the spleen. Moreover, IgM XNA secreting cells represent around 0.0001% of spleen lymphocytes corresponding to 0.1% of the IgM secreting cells in the spleen. Although circulating IgG2a XNA are detectable, by cellular ELISA, in normal LOU/C rats, IgG2a secreting cells were not found in any of the cell suspensions analyzed. In contrast, Ig k light chain XNA were found in the spleen, mesenteric lymph nodes, and the bone marrow but not in the peritoneal cavity. Taken together these results suggest that extremely low numbers of secreting cells producing isotypes other than IgM and IgG2a might exist in different rat organs but are not detectable using the xenoreactive ELISA‐spot technique. In conclusion, our data show that only 0.0001% of spleen lymphocytes were found to be IgM XNA secreting cells. Moreover this IgM XNA secreting cells population is located almost exclusively in the spleen further illustrating the role of the spleen in the production of XNA.