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B LYMPHOCYTE SUBPOPULATIONS INVOLVED IN RAPIDLY-INDUCED, T-CELL INDEPENDENT XENOANTIBODY FORMATION Shengqiao Li, Yehong Yan, Omer Rutgeerts, Caroline Lenaerts, Mark Waer, An D Billiau. Laboratory of experimental transplantation, University of Leuven, Leuven, Belgium. Objectives: While regimens are being developed that aim at inducing T-dependent xenotolerance, T-independent xenoreactivity may still represent a barrier to successful xenografting. It has been shown that splenic CD11bneg B1b cells play a major role in T-independent xenoantibody formation against discordant xenoantigens (Ohdan H, et al. JI 2000,165:5518-5529). For T-independent xenoantibodies directed at concordant xenoantigens, we have previously reported that Marginal Zone B lymphocytes (MZB) may be the major population involved, and that these cells require help from NK cells (Li S et al. Xenotransplantation. 2005, 12: 384). This hypothesis was based on in vivo experiments demonstrating a correlation between the transient disappearance of MZB in T-deficient nude mice after low-dose total body irradiation (2Gy) and the inability of these mice to mount a rapidly-induced IgM anti-hamster xenoantibody response. In order to substantiate this hypothesis, we explored the capacity of various FACS-sorted B lymphocyte subpopulations to enable SCID mice to produce IgM Xab. Materials and Methods. MACS-enriched CD19pos cells from spleens of BALB/c nude mice were purified using FACS-sorting into CD21highCD23low MZB cells and CD21negCD23neg B cells (which include B1 B cells) (purity of sorted populations > 90%). CB17SCID mice were given 2-5x10e6 subset B cells intravenously on day 0, and 200µl whole hamster blood on day 7. Serum IgM xenoantibodies were measured on day 12 using a sensitive FACS assay and hamster red blood cells as targets. Serum IgM xenoantibody levels were expressed as median fluorescence intensity (MFI). Results: As expected, unreconstituted SCID mice were unable to produce IgM anti-hamster xenoantibodies after immunization (MFI 1.3 and 1.8 (n=2)). SCID mice given CD19posCD21negCD23neg B lymphocytes were equally unable to produce a significant IgM Xenoantibody response (mean MFI 1.7 ± SD 0.3 (n=3). In contrast, transfer of CD19posCD21highCD23low MZB cells was very efficient in transferring the ability to produce an anti-hamster IgM xenoantibody response (mean MFI 10 ± SD 2.4 (n=3) (p