FLT-3 Ligand Enhances Thymopoiesis in Hematopoietic Stem Cell Transplants (HSCT) Involving Aged Donor and Recipient Mice By Increasing Survival and Trafficking of Early Thymocyte Progenitors

Impaired thymopoiesis may contribute to graft-versus-host disease and poor clearance of infections. Early thymic precursor frequency is a point of regulation for thymus reconstitution; however, it is unknown if decreased thymus recovery with age is due to the absence of thymus elements or marrow precursors. To address this, we investigated whether marrow thymus precursor frequency declines with age, contributes to poor aged thymus recovery, and can be enhanced with FMS-like tyrosine kinase 3 (Flt-3), a growth factor for HSC. Total number of HSC (LSK, Lineage- Sca-1+ and cKit+) and Flt-3R+ LSK were equivalent in the marrow of old (> 4 months) and young mice (1 month). By ELISA, marrow concentration of Flt-3L was equivalent between old and young mice. Similarly, there was no difference in splenic LSK numbers. However, the response to exogenous Flt-3L was distinct by age. While young marrow was unaffected after Flt-3L, in aged mice, total LSK and CCR9+ thymus directed LSK increased 4 fold (p < 0.05). The proportion of LSK that expressed Flt-3R decreased in the marrow of older mice in response to Flt3-L and LSK proliferation (Ki67 and BrdU) did not differ in response to Flt3-L in old or young mice, consistent with improved survival rather than proliferation. We then hypothesized that Flt-3L treated cells may also traffic to peripheral spaces, similar to GCSF. Consistent with this theory, in the spleen of old and young mice, the Flt3-R+ LSK number increased in response to Flt3-L (p < 0.05). We then used aged Flt3-L knock-out mice (Flt3-L−/−) to test whether survival and trafficking were part of the mechanism of Flt3-L. Aged Flt3-L −/− mice had significantly lower LSK and Flt3-R+ LSK in the marrow than wild type aged counterparts, consistent with a deficient survival signal. After Flt3-L exposure, although Flt3-R proportion was unchanged in the marrow, LSK numbers increased significantly in the spleen (p < 0.05) with equivalent Ki67, suggesting LSK trafficked from marrow to spleen. Aged donor Flt3-L treated marrow with lupron-treated aged host increased donor thymocytes two-fold within 5 weeks after transplantation (p < 0.05) compared to lupron alone. Collectively, these data suggest that marrow LSK milieu contributes to thymus dysfunction in older donor/host recipient pairs and that Flt-3L may improve thymus recovery by enhancing survival and trafficking of older donor HSC.