Acceleration and Enhancement of T-Cell Recovery and Immune Competence by Flt3-Ligand (Flt3L) Following BMT with Low Numbers of Progenitor Cells in Immune Deficient Mice.

Deficient thymopoiesis and a retarded or absent recovery of newly developed CD4⁺ T-cells has become one of the most important determinants of impaired immune competence in the later time period after allogeneic transplantation. We previously showed that Interleukin-7 (IL-7) may enhance peripheral T-cell expansion without affecting thymopoiesis after BMT in immunodeficient mice (Broers et al. Blood 2003). In order to improve thymopoiesis, we evaluated whether the cytokine Flt3L alone or combined with IL-7 would affect thymopoiesis and/or the generation of lymphoid progenitors following BMT in immunodeficient RAG-2^−/−γc, ^−/− mice, lacking T-, B- and NK-cells. Following 3 Gy irradiation and transplantation of graded restricted numbers of T-cell depleted (TCD) BM, mice received Flt3L (3 x 20μg/week), Il-7 (35 x 5 μg/week) or the combination of IL-7 and Flt3L until 80 days after BMT. Hematopoietic recovery was evaluated weekly by flowcytometry. While B-cell and NK-cell recovery were moderately enhanced, Flt3L strongly accelerated and enhanced the recovery of T-cells, especially in the setting of BMT with a low dose of 4 x 10⁴ TCD-BM. In contrast, T-cell recovery was still insufficient in control mice treated with PBS (p < 0.01) or mice treated with IL-7 alone by day 80 after BMT with 4 x 10⁴ TCD-BM. The combination of Flt3L and IL-7 did not result in better recovery as compared to Flt3L alone. As early concurrent enhanced T-cell, B-cell, NK-cell and myeloid recovery may suggest an effect on lymphoid progenitors, the number of common lymphoid progenitors (CLP) as characterized by lineage^-, IL-7 Rα⁺, sca-1^low and AA4.1⁺ was assessed. At day 20 post-BMT, 13.2 x 10³ (±10) CLP were harvested from the BM of Flt3L treated mice versus 3.9 x 10³ (±0.6) from control PBS treated mice (p = 0.2). Furthermore, thymic cellularity was increased (18.6 (±8) x 10⁶ thymocytes versus 6.6 (±0.4) x 10⁶, p = 0.1) and especially the number of double positive CD4/CD8 thymocytes were increased in Flt3L treated mice (14.3 x 10⁶ (±7) versus 2.3 x 10⁶ (±1), p = 0.03) at day 20 post-BMT. Next, we studied whether enhanced hematopoietic recovery following Flt3L would result in better immune competence by evaluating survival and clearance of viral load after opportunistic murine cytomegalovirus (mCMV) infection. RAG-2^−/−γc, ^−/− mice were transplanted with 4 x10⁴ TCD-BM and subsequently infected intraperitoneally with 10⁴ PFU mCMV at day 28 post-BMT. All Flt3L treated mice survived and rapidly cleared their viral load as assessed by quantitative real-time Taqman PCR in plasma. T-cell numbers were inversely correlated with viral load (r = −0, 467, p = 0.04), while numbers of NK-cells, B-cells or granulocytes were not associated with viral load. A 100% mortality was observed in control mice developing a viral load > 2 x 10⁵ geq/ml. These results suggest that Flt3L may restore T-cell recovery and immune competence especially in the setting of transplantation with restricted numbers of progenitor cells by promoting thymopoiesis. Enhanced thymopoiesis directly mediated by Flt3L or expansion of lymphoid progenitors by Flt3L may account for the higher numbers of newly developed T-cells.