In this manuscript, we demonstrate that P-selectin−/− allo-BMT recipients are resistant to the development GVHD, which suggests that P-selectin of recipient origin is an important molecule for GVHD pathophysiology. Furthermore, in cognate experiments, we observe that donor T cells deficient for PSGL1, the most well-described ligand for P-selectin, had a surprisingly intact potential to cause GVHD, suggesting that donor T cells can use multiple ligands, in addition to PSGL1, to mediate their interactions with P-selectin on vessel endothelium during the inflammatory processes associated with acute GVHD.
First, we observed that alloreactive T cells in WT vs. P-selectin−/− allo-BMT recipients demonstrate comparable levels of alloreactivity (activation markers in vivo and T cell proliferation in vitro), apoptosis, and expression of PSGL1. However, upon enumerating donor T cells in mice with GVHD, we noted that compared to the situation in WT recipients, donor alloreactive T cells in P-selectin−/− recipients accumulated in increased numbers in lymphoid tissues, and decreased numbers in GVHD target tissues such as the small intestine. By contrast, non-transplanted WT B6 and P-selectin−/− animals have fairly similar numbers of T cells in lymphoid and non-lymphoid organs.
The observation that P-selectin−/−
recipients had more donor alloactivated T cells in the spleen, SLO, and peripheral blood after allogeneic BMT was initially surprising, in light of the enhanced survival of these mice. Yet while P-selectin has been implicated in the trafficking of T cells into inflamed organs11,22,23
, no data exist regarding its involvement in lymphoid tissue trafficking, which is instead mediated by the peripheral lymph node addressins24
. We therefore believe that P-selectin may be important for the trafficking of alloreactive T cells into non-hematopoietic tissues such as the gut or liver during GVHD, but relatively dispensable for the trafficking of T cells into lymphoid organs.
We were surprised to note that despite a difference in overall mortality, there was no difference in colonic GVHD pathology or degree of lymphocytic or neutrophilic infiltrates in the colons of WT and P-selectin−/−
allo-BMT recipients. However, a number of reports may explain this finding. The first is the observation that in murine models of chronic colitis, T cells require CD18, but not PSGL1, to cause disease, suggesting that in our model systems, there simply may not be a direct requirement for P-selectin for colonic GVHD28
. The second are reports on ulcerative colitis29
and mouse models of dextran sodium sulfuate (DSS) colitis30
, which indicate that P-selectin, as expressed on circulating platelets
, is important for the co-recruitment of leukocytes to the colon. Platelets utilize P-selectin to bind endothelium expressing PSGL1, and leukocyte/platelet aggregates are then subsequent required for leukocyte adhesion in colonic vessels. Indeed, neutralization of platelets reduced leukocyte adhesion in mouse DSS colitis models. Since allo-BMT recipients with GVHD establish full donor chimerism, circulating platelets in our model systems are expected to be of donor origin, and thus P-selectin+/+
; consequently, they would not be expected, according to these reports, to mediate defective leukocyte trafficking to the colon.
Taken together, P-selectin−/− recipients of allo-BMT exhibit diminished systemic, cutaneous, and gastrointestinal GVHD, coupled with increased numbers of donor alloactivated T cells in the spleen and SLO, and decreased numbers of infiltrating donor T cells in the small bowels. This increased cellularity in lymphoid tissues could be due to either a defect in the exit of alloreactive T cells from these SLO, or a defect in their trafficking into GVHD target organs in P-selectin−/− recipients.
Sykes et al. have shown that the sphingosine-1-phosphate receptor agonist FTY720 can sequester T cells in SLO and away from target organs, thus attenuating GVHD5
. Yet the differential accumulation of donor alloactivated T cells in lymphoid vs. non-lymphoid organs in the present study may be due to different requirements in the selectins for entry into these two types of organs. The inability of T cells to enter GVHD target organs without P-selectin, and the requirement for L-selectin/PNAd interactions to enter lymphoid tissues, may explain why P-selectin−/−
allo-BMT recipients had diminished infiltrates into the small bowels coupled with increased numbers of donor T cells in the spleen, peripheral and mesenteric lymph nodes.
In parallel experiments, we assessed the importance of PSGL1, the most well-described leukocyte ligand for P-selectin, for GVHD. Surprisingly, we observed that PSGL1−/− donor T cells caused not only similar GVHD morbidity and mortality as WT T cells, but also that these T cells had similar proliferation, alloactivation, and infiltration into the SLO and liver as WT T cells.
Despite being ablated for PSGL1, PSGL1−/−
T cells appear to still express substantial levels of other P-selectin ligands (Supplemental Figures 1
and ), and these other ligands may interact with P-selectin to compensate for PSGL1 deficiency in the setting of acute GVHD. Indeed, non-transplanted WT and PSGL1−/−
mice displayed similar leukocyte cellularity in lymphoid and non-lymphoid tissues, and numbers of WT and PSGL1−/−
donor T cells were also comparable in lymphoid and non-lymphoid tissues post-transplant in mice with GVHD.
In conclusion, our report suggests although recipient P-selectin is an important molecule for the pathophysiology of GVHD, in strongly inflammatory settings such as acute GVHD, multiple P-selectin ligands on donor T cells may be important for their trafficking and tissue infiltration. Consequently, ablation of PSGL1 alone on donor T cells may not be sufficient to abrogate their interactions with recipient P-selectin. Our results suggest that targeting P-selectin in allo-BMT recipients, or multiple P-selectin ligands on donor T cells and leukocytes, may represent a novel therapeutic strategy for GVHD prophylaxis or treatment.