Tyrosine Kinase Inhibition with Sunitinib Facilitates Thymic Engraftment by Modulating Thymic Niche Accessibility

Abstract

Abstract Stable mixed chimerism is likely to be sufficient for clinical benefit when HSCT is undertaken for benign disease. However, potential short and long term morbidities of current ablative or non-myeloablative conditioning regimens present an unfavorable risk:benefit ratio when HSCT is undertaken in this setting. We sought to develop an entirely non-cytotoxic approach to achieve stable mixed chimerism by modulating accessibility of thymic niches. Previous work identified occupancy of the double negative (DN)3 niche (CD3-4-8-44-25+) as a primary barrier to thymic engraftment based upon evidence that IL-7Rα−/− and γc−/− mice (who have an accessible DN3 niche) are receptive to thymic engraftment, whereas RAG−/− mice (who have a full DN3 niche) are resistant to thymic engraftment. Our results are consistent with this paradigm, since transfer of 5 × 106 TCD BM cells from WT into IL-7Rα−/− mice vs RAG−/− mice showed higher rates of thymic chimerism (mean donor % 83±8 vs 3±3 respectively, p=0.001). In an attempt to “open” the DN3 niche, we pre-treated RAG−/− recipients of WT BM with PC61, an anti-CD25 MoAb that diminished DN3 cell numbers (pre-44±11 × 105, post-14±1 × 105). Anti-CD25 therapy modestly enhanced donor chimerism (20±9%, n=12 vs 12±6%, n=10, p=NS) and significantly increased thymic engraftment (90±20 vs 41±11 x106 thymocytes, p=0.05) in anti-CD25 treated RAG−/− mice vs controls respectively. Importantly however, modulation of the DN3 niche via anti-CD25 treatment in RAG−/− mice did not increase thymic chimerism to the levels achieved in IL-7Rα−/− mice which have defects in the DN1/DN2 niches, implicating a role for DN1/DN2 niche accessibility as well. Indeed, RAG−/−γc−/− mice were receptive to thymic engraftment, with mean donor chimerism of 81.84±14% and a 13-fold increase in thymic cellularity after transplantation of WT BM cells without any prior conditioning. We postulated therefore that the limited efficacy of anti-CD25 alone when RAG−/− recipients were used was due to the fact that donor thymic engraftment is limited by accessibility to the entire range of DN1, DN2 and DN3 niches, while anti-CD25 selectively modulates the DN3 niche. Current models hold that growth factors critical for DN1 and DN2 thymocytes include SCF, FLT3L, and IL-7, among others. To test whether targeted pharmacotherapy could diminish the competitive advantage of resident DN1 and DN2 thymocytes and render the DN1/DN2 niche accessible, we treated RAG−/− recipients with Sunitinib, a receptor tyrosine-kinase (TK) inhibitor that inhibits c-kit and Flt-3, which signal SCF and FLT3L respectively. Sunitinib treatment of donors (60 mg/kg/d on Days -4, -3, -2, -1) followed by transfer of 5 × 106 TCD WT BM cells on Day 0 and 1 enhanced donor chimerism and thymic engraftment (10±4%, n=9 vs 2±0.7%, n=8 donor chimerism, p=0.01; 43±5 vs 13±1 × 106 thymocytes, p<0.0001) in Sunitinib vs. vehicle treated recipients, respectively. Combined anti-CD25 and Sunitinib therapy was superior to Sunitinib alone prior to transplantation of TCD WT BM (15±4%, n=5 vs 7±2%, n=5 donor chimerism, p=NS; 74±14 vs 25±6 × 106 thymocytes, p=0.02) We postulate that Sunitinib plus anti-CD25 renders DN1-DN3 thymic niches accessible to transferred thymic progenitors. This therapy is virtually non-toxic, and could provide new opportunities for HSCT for benign disease. Ongoing studies are underway to clarify effects of this regimen on marrow stem cell engraftment, durability of engraftment, and activity in a minor-mismatched model.