Antithymocyte Globulin Enhances Myeloid-Derived Suppressor Cell Expansion and Function in Severe Aplastic Anemia
Authors: Dong Peiyuan , Liu Yilin , Jiang Zhongxing , Sun Huimin , Zhao Yanhong , Wu Linjie , Huo Jiali , Zhang Jing , Ge Meili , Dong Fang , Hao Sha , Cheng Tao 
Published: December 2024

Acquired aplastic anemia (AAA) is the most prevalent bone marrow failure disease, characterized by an autoimmune disorder that damages hematopoietic stem/progenitor cells, which is pivotal in the disease's progression. Nearly 70% of AA patients show hematologic responses to antithymocyte globulin (ATG) and ciclosporin within 6 months. Although massive laboratory data support the immune pathophysiology of AA, the mechanisms underlying abnormal immune response remain unclear. Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells characterized by their immature state and ability to inhibit T cell-mediated immune responses. Our previous studies have shown that the number and inhibitory function of MDSCs are impaired in patients with AA compared to healthy donors (HD). However, the biological alterations of MDSCs from patients with severe AA (SAA) after treatment of ATG are largely unknown.

In this study, a total of 62 HD and 118 SAA patients were included, with a median age of 38 years; 97 were male and 83 were female. Among the 118 SAA patients, 66 were newly diagnosed and 52 had been treated with ATG previously. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation, followed by the evaluation of MDSCs via flow cytometry. The proportion of MDSCs (CD33+CD11b+HLA-DR-), especially polymorphonuclear MDSCs (CD33+CD11b+HLA-DR-CD14-CD15+) and early-stage MDSCs (CD33+CD11b+HLA-DR-Lin-), in circulation was significantly lower in SAA patients than in HD. Following ATG treatment, there was a significant increase in the percentage of MDSCs in PBMCs from SAA patients and HD. Furthermore, MDSC-mediated suppression on T-cell responses correlated with the expression of Arginase 1(Arg1) and interleukin 10 (IL-10). Arg1 and IL-10 expression in circulating MDSCs were analyzed by flow cytometry. Compared to HD, SAA patients had significantly lower Arg1 expression level in MDSCs. After ATG treatment, Arg1 expression was significantly elevated.

PBMCs from SAA patients and HD were cultured with IgG or ATG in vitro and harvested on day 6. ATG could significantly increased the percentage and absolute number of MDSCs in both SAA patients and HD. Furthermore, ATG markedly augmented the expression level of Arg1 and IL-10 in MDSCs from both groups. To further confirm the effect of ATG on MDSCs' immunosuppressive function, MDSCs from both SAA patients and HD were treated with IgG or ATG and then cultured with CFSE-labeled CD3+T cells. The proliferation and activation rate of CD3+T cells and CD4+T cells were lower when cultured with ATG-treated MDSCs, indicating that the immunosuppressive function of MDSCs was significantly enhanced by ATG.

To further investigate the molecular mechanisms by which ATG modulates MDSCs in SAA patients, we performed genome-wide RNA-seq of MDSCs cultured with IgG or ATG. The hierarchical clustering of differentially expressed transcripts showed 1048 upregulated and 1248 downregulated transcripts in ATG-treated MDSCs compared with IgG-treated MDSCs (fold change &gt;2 and P value &lt;0.05). PCA of the transcriptome revealed that MDSCs treated with IgG and ATG clustered separately, representing significant differences in overall gene expression. GO analysis revealed that enriched genes were related to immunoregulation (e.g., inhibitory MHC class I receptor activity) and cellular process (e.g., translation and apoptotic process). Immunologically relevant signaling pathways (T cell receptor signaling pathway, Th17 cell differentiation, and NF-kappa B signaling pathway) and biological processes (ferroptosis and apoptosis) were also enriched, as revealed by KEGG pathway analysis.

In conclusion, our study indicates that MDSCs play a role in the immunopathogenesis of SAA, and that ATG treatment could enhance their expansion and immunosuppressive capabilities.