The Therapeutic Effect of Myeloid-Suppressor Cells Induced By Umbilical Cord Blood CD34+cells in Mice with Aplastic Anemia

Background:Acquired aplastic anemia (AA) is a rare immune-mediated bone marrow failure disease characterized by decreased bone marrow proliferation and decreased whole blood cells. At present, it is believed that abnormal activation of T lymphocytes plays a dominant role in the pathogenesis of AA, and the good effect of immunosuppressive therapy (IST) has been recognized. However, about 30% of patients still have poor response to traditional IST treatment. Myeloid-derived suppressor cells (MDSCs) represents a diverse population of immature myeloid cells. Recent studies have shown that CD34+cells sorted from umbilical cord blood (UCB) can be induced and expanded to UCB-MDSC in vitro. Therefore, inducing UCB-MDSC by sorting CD34+cells in UCB and exploring its application in AA patients may be a potential and effective novel cell therapy strategy. Objective: The aim of this study is to optimize the method of inducing and expanding UCB-MDSC from UCB CD34+cells in vitro. By applying UCB-MDSC to AA mice models, this study explores the immunosuppressive and hematopoietic reconstitution effects of UCB-MDSC on AA mice, aiming to provide new treatment strategies and optimization plans for AA patients. Methods: CD34+cells were sorted from UCB, and were induced and expanded to UCB-MDSC in vitro. Next, the AA mice models were constructed. The mice experiment is mainly divided into two parts. In the first part, we cleared MDSC in AA mice using anti-mouse Gr-1 antibody and compared the severity of bone marrow failure in AA mice before and after MDSC clearance. In the second part, we injected UCB-MDSC into the tail vein of AA mice and compared the improvement of bone marrow failure in AA mice before and after treatment with UCB-MDSC. Subsequently, we performed RNA sequencing (RNA seq) on three groups of bone marrow CD3+T cells from NC mice, AA mice, and AA mice treated with UCB-MDSC (AA+UCB-MDSC mice). We analyzed differentially expressed genes, with a focus on studying the signaling pathways of UCB-MDSC and its impact on biological functions, to explore the mechanism of action of UCB-MDSC on CD3+T cells. Results: Compared with the combination of GM-CSF plus SCF, granulocyte colony-stimulating factor plus SCF, and macrophage colony-stimulating factor plus SCF, the factor combination of GM-CSF, SCF and IL-6 induces higher purity of UCB-MDSC in vitro and stronger immunosuppressive effect on T cells. Subsequently, we cleared the MDSC in AA mice. Compared with AA mice, AA mice that cleared MDSC (anti-Gr-1AA) experienced more severe bone marrow failure, characterized by a significant decrease in bone marrow nuclear count, shortened OS, weight loss and decreased peripheral blood cell counts. Meanwhile, the proportion of lymphocyte subsets in the bone marrow, spleen, and lymph nodes of anti-Gr-1AA mice increased significantly, and CD4+T cells in the bone marrow differentiated more significantly towards Th1, with a lower proportion of Treg. Next, we applied UCB-MDSC to the treatment of AA mice, and the results showed that compared to the AA mice, the bone marrow failure status of AA+UCB-MDSC mice was significantly improved. Sternal HE staining showed an improvement in bone marrow proliferation. Besides, nucleated cell counts in bone marrow, OS and peripheral blood cell counts were also improved. The immune analysis results showed that the proportion of lymphocyte subsets in the bone marrow, spleen, and lymph nodes of AA+UCB-MDSC mice significantly decreased, while the activation level of lymphocytes in the spleen and lymph nodes decreased, and the ability of T cells in lymph nodes to differentiate into Th1 and Tc1 decreased. The RNA seq results showed significant differences in the enrichment of biological functions such as T cell activation, proliferation, and differentiation between the AA+UCB-MDSC mice group and the AA mice group. Compared to AA+UCB-MDSC mice group, the gene set of T cell activation, migration, chemotaxis, and differentiation in the AA mice group was significantly upregulated. Conclusions: We can select CD34+cells from UCB and induce the generation of UCB-MDSC with strong immunosuppressive function in vitro under the factor combination of GM-CSF, SCF and IL-6. Applying UCB-MDSC to AA mice can improve their bone marrow failure status. Therefore, UCB-MDSC is expected to become a new cell therapy for AA patients.