Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.

Abstract

Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals. [Display omitted]