S1P₁ Agonists for Use as Adjunct Mobilizing Agents

Abstract 826

Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P₁ receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB.

Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P₁ expression and an S1P₁ conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models.

Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P₁ conditional knock-out mouse. When mobilised with AMD3100, these S1P₁ knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P₁ agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF.

S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P₁ conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes.