P-Selectin in Recipients of Allogeneic Bone Marrow Transplantation Regulates Experimental Graft-Versus-Host-Disease.

Abstract 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.