Material Supplementary 8.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.090314

Patients with type II lissencephaly, a neuronal migration disorder with ectopic neurons, suffer from severe mental retardation, including learning deficits. There is no effective therapy to prevent or correct the formation of neuronal ectopia, which is presumed to cause cognitive deficits. We hypothesized that learning deficits were not solely caused by neuronal ectopia and that postnatal gene therapy could improve learning without correcting the neuronal ectopia formed during fetal development. To test this hypothesis, we evaluated spatial learning of cerebral cortex-specific protein O-mannosyltransferase 2 (POMT2, an enzyme required for O-mannosyl glycosylation) knockout mice and compared to the knockout mice that were injected with an adeno-associated viral vector (AAV) encoding POMT2 into the postnatal brains with Barnes maze. The data showed that the knockout mice exhibited reduced glycosylation in the cerebral cortex, reduced dendritic spine density on CA1 neurons, and increased latency to the target hole in the Barnes maze, indicating learning deficits. Postnatal gene therapy restored functional glycosylation, rescued dendritic spine defects, and improved performance on the Barnes maze by the knockout mice even though neuronal ectopia was not corrected. These results indicate that postnatal gene therapy improves spatial learning despite the presence of neuronal ectopia.

1335 Poster Board I-357 Alloreactive T cells are crucial for graft-versus-host-disease (GVHD) pathophysiology, and we hypothesized that controlling their trafficking can ameliorate GVHD. P-selectin is a dimeric glycoprotein found on most inflamed endothelium, which interacts with multiple lectin-type molecules on leukocytes, including T cells. We used murine allogenienc BMT models to study GVHD and found that P-selectin−/− recipients exhibited significantly less GVHD mortality and morbidity, as well as decreased GVHD of the skin, liver and small bowels. However, WT and P-selectin−/− allo-BMT recipients had comparable large bowel GVHD. This decrease in target organ and systemic GVHD was associated with diminished infiltration of alloactivated T cells into the Peyer's Patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLO) on day 14 and day 35 post-transplant. However, donor alloreactive T cells in WT and P-selectin−/− allo-BMT recipients had similar alloactivation and apoptosis, and donor alloactivated T cells from WT and P-selectin−/− allo-BMT recipients with GVHD showed similar proliferation in vitro in a mixed leukocyte reaction, suggesting that the inflammatory environment in WT and P-selectin−/− recipients was comparable. Finally, non-transplanted P-selectin−/− mice, and P-selectin−/− mice which had received the allo-BMT conditioning regimen but not a donor graft, had similar cellularity in the majority of tissues examined as corresponding WT controls. This suggests that the differential cellularity of donor alloactivated T cells in WT and P-selectin−/− allo-BMT recipients with GVHD is probably largely dependent on trafficking and tissue infiltration during inflammation. Since P-selectin glycoprotein ligand 1 (PSGL1) is the best-described P-selectin ligand, and all leukocytes constitutively bear high levels of membrane PSGL1, we next hypothesized that PSGL1−/− donor alloreactive T cells would be defective in trafficking into GVHD target organs, and that PSGL1−/− donor T cells would cause decreased target organ damage, systemic GVHD, and mortality. However, allo-BMT recipients of WT and PSGL1−/− donor T cells had comparable survival and clinical GVHD scores, and further analyses on day 14 post-transplant revealed that recipients of WT and PSGL1−/− donor T cells also had similar numbers of donor alloactivated T cells in the spleen, liver, mesenteric and peripheral lymph nodes, and Peyer's Patches. Additionally, WT and PSGL1−/− donor T cells had comparable proliferation as measured by CFSE dilution, and comparable alloactivation in vivo as determined by levels of CD25, CD44, and CD62L, suggesting similar T cell function. As PSGL1−/− and WT donor T cells appeared to have equal functionality and accumulated in GVHD target tissues and lymphoid tissues in a similar fashion, we asked whether PSGL1−/− T cells might display other P-selectin ligands. Flow cytometric analyses of T cells from non-transplanted PSGL1−/− mice, and analyses of PSGL1−/− alloactivated T cells on day 14 after allo-BMT, revealed that these cells displayed substantial levels of cell-surface P-selectin ligands as defined by positive staining with recombinant P-selectin-IgG-Fc fusion protein at levels similar to those found on WT T cells, suggesting that although absence of P-selectin on host tissues may ameliorate GVHD, multiple donor leukocyte P-selectin ligands interact meaningfully with P-selectin. Our studies suggest that P-selectin may be required for trafficking into inflamed tissues but not SLO, and that donor T cells may utilize multiple P-selectin ligands apart from PSGL1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable target for GVHD prophylaxis or treatment. Disclosures No relevant conflicts of interest to declare.

No abstract