br Materials and Methods br

Materials and Methods
Results
Discussion Our previous studies in mice with germline or induced ablation of SHIP1 expression suggested that chemical inhibition of SHIP1 in vivo might recapitulate genetic phenotypes that reduce the immune barrier to allogeneic HSCT and or enhance mobilization of HSC (Wang et al., 2002; Ghansah et al., 2004; Wahle et al., 2006; Desponts et al., 2006; Paraiso et al., 2007; Collazo et al., 2009; Hazen et al., 2009). The study presented here reveals that SHIPi does indeed modulate several key components of the immune system (NK flt3 inhibitor were shown to be decreased and hyporesponsive, and MDSC and T regulatory cells were significantly increased) such that we observed considerably improved marrow repopulating activity following transplant of allogeneic BM. Consequently, inhibition of SHIP1 by small molecule therapeutics could have a profound impact on the utility of allogeneic HSCT. Indeed, brief inhibition of SHIP1 failed to display toxicity at doses that allowed for improved engraftment in fully mismatched MHC-I BM graft. By extension of these studies, eventually SHIP1 may prove to be a valuable molecular target that could help facilitate allogeneic grafts particularly for patients who lack an HLA matched donor. Mobilization of HS-PCs by G-CSF administration as it is currently practiced is a very effective and safe procedure. However, since a subset of patients and donors fail to mobilize sufficient numbers of HS-PCs to permit engraftment, other alternatives such as CXCR4 antagonists (AMD-3100) have been developed (Broxmeyer et al., 2005). Clinical trials demonstrated that combining this antagonist, AMD3100-Plerixafor, with G-CSF improved stem cell harvest and reduced the incidence of failure (DiPersio et al., 2009a, 2009b). Nonetheless, even with this combination of mobilizing agents, failure rates of ~7% are still observed (Hosoba and Waller, 2014). New molecular targets are therefore actively sought to further improve HSC mobilization for HSCT and also in the treatment of vascular diseases (e.g. myocardial infarction, peripheral artery disease or coronary artery disease), where periodic HSC mobilization by G-CSF is believed to facilitate vascular repair (Poole et al., 2013; Seiler et al., 2001). Our data show that SHIPi can mobilize significant numbers of HS-PC such that a single blood draw from a donor can provide for radioprotection and long-term reconstitution in the majority of hosts. The SHIPi mobilized graft may therefore represent a new methodology to mobilize patient or donor HSC for HSCT therapies. This could be applied in settings where donors fail G-CSF mobilization, or be used in concert with G-CSF and-or AMD3100, to further increase the yield and efficiency of HS-PC mobilization procedures. Minimally ablative procedures in allogeneic HSCT have seen increased utilization in various clinical settings, but there has also been a concomitant increase in graft failure (Mattsson et al., 2008). The mobilization of HS-PC triggered by SHIPi might also be used in lieu of such cytoablative regimens, or perhaps in combination with minimally ablative chemotherapeutic regimen to decrease the incidence of graft failure. In several diseases, and particularly severe autoimmune diseases, there is a growing effort to use autologous or allogeneic BMT to ‘reboot’ the patient\'s immune system (Li and Sykes, 2012). However, HSCT can be associated with significant morbidity and mortality and thus is only utilized in the most severe cases. As the SHIPi conditioning regimen we describe poses no significant risk to host viability (Brooks et al., 2010), we propose that SHIPi might eventually enable a great proportion of patients to benefit from these autologous BMT procedures, at least in part by creating space in the BM niche for engrafting HSC. In our previous studies, it was observed that germline SHIP1 deficiency caused dramatic alterations in the receptor repertoire such that rejection of MHC-I mismatched BM was severely compromised (Wang et al., 2002; Wahle et al., 2006). More recently, NK cell-specific deletion of SHIP1 has been shown to alter the receptor repertoire and impair induction of INFγ upon receptor crosslinking (Gumbleton et al., in press). Moreover, SHIP1 deficiency in NK cells also enhances engraftment of an MHC-I unmatched BM graft (Gumbleton et al., in press). Interestingly, both receptor specific and general activation of NK cells are compromised by daily, extended treatment with SHIPi. Therefore, alterations of SHIP1 signaling within the NK cell prevent rejection of cells bearing mismatched MHC-I antigens. Furthermore, it is well documented that SHIP-deficiency, whether it be in germline (Ghansah et al., 2004), induced (Paraiso et al., 2007) or triggered by SHIPi (Brooks et al., 2010), causes a dramatic increase in MDSCs that can modulate T-effector functions. Overall, the effects of SHIPi on the both the NK and the myeloid cell compartments are believed to be the important factors in facilitating the observed increase in allogeneic BM engraftment. Finally, germline SHIP-deficiency has been shown to cause increased Treg cell numbers (Collazo et al., 2009; Ghansah et al., 2004; Paraiso et al., 2007), and splenocytes from SHIPi treated mice and SHIPi treated peripheral blood mononuclear cells (PBMC) display reduced priming of allogeneic responses in one-way mixed leukocyte reactions (Brooks et al., 2010). We propose that the significant increase in CD4+FoxP3+ regulatory T-cell frequency observed here also contributed to the immunoregulatory environment, which allowed for better engraftment of allogeneic BM grafts as host Treg cells can also promote MHC-I mismatched BM engraftment (Fujisaki et al. 2011).