Thymic Dendritc Cells Generated Ex Vivo Induce Donor Regulatory T Cell Differentiation and Hasten Immune Reconstitution After Hematopoietic Stem Cell Transplantation

Abstract 1905

Dendritic cells (DCs) are the first committed cells to engraft in the thymus after hematopoietic stem cell transplantation (HSCT). The critical role of thymic DCs in ensuring efficient tolerance and selection has been well demonstrated, however its role in facilitating donor engraftment has not been reported. Here we show DCs accelerates thymic reconstitution by inducing regulatory T cells (T_regs) differentiation and enhancing T cell recovery after HSCT. Lethally irradiated CD45.2 C57BL/6 control group received 10³ CD45.1 lin⁻sca-1⁺c-kit⁺ (LSK) hematopoietic stem cell progenitors while the DCs group received 10³ CD45.1 LSK cells along with 10³ CD45.2 GFP⁺ DCs. DCs were generated ex vivo using bone marrow from CD45.2 GFP⁺ CD57BL/6 mice and cultured for 7 days with GMCSF. At 4 and 7 days after HSCT, the thymus of DC group contained 1.8 and 4.2- fold higher number of thymocytes (p<0.05) and a 3.2 and 7.4-fold, respectively, higher number of donor derived thymoctyes compared to the control group (p<0.05). Moreover, thymuses of the DCs group had GFP⁺ CD11c⁺ cells present in the medulla and 5.6-fold increase in the number of donor derived FoxP3⁺ T_regs compared to control confirmed by immunohistochemistry (IHC). Furthermore, thymic recovery scored by a pathologist blinded to the groups found significant increase in lymphoid regeneration (H&E) and higher number of CD3⁺ lymphoid aggregates (IHC) in the DC group compared to control group that had severe, diffuse lymphoid depletion. Lastly, at 2 and 4 weeks after HSCT, peripheral blood of DCs group contained 2.6 and 4.8-fold, respectively, higher numbers of CD3⁺ cells derived from donor LSK cells compared to the control group (p<0.05). Here, we demonstrate that donor DCs efficiently migrate and home to the thymic medulla and hasten thymic recovery as demonstrated by the higher number of total thymoctyes. Furthermore, DCs facilitate thymic engraftment as shown by increase number of donor derived FoxP3⁺ T_regs and thymocytes. Lastly, recipients of DCs have earlier generation of de-novo donor derived CD3⁺ T cells in the peripheral blood. By using the GFP⁺ cells along with donor LSK cells, we were able to confirm that the facilitation of early thymic recovery was due to the increased engraftment of the donor cells rather than autologous recovery of the host. In conclusion, this study demonstrates that DCs committed prior to thymic entry maintains the ability to home to the medullary region and facilitate thymic recovery by enhancing T_regs differentiation. Thus, ex vivo generation of donor DCs to augment a HSC graft may