Induced CD25+CD127dim Γδ Tregs in Acute Myeloid Leukemia Suppress the Activity of Normal Αβ T Cells
Authors: Liang Shuang , Liu Jiangying , Gao Haitao , Liu Ruoyang , Wu Ning , Dong Tianhui , Huang Xiaojun 
Published: November 2020

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in adults with dismal outcomes. γδ T cells have been reported to exert effective anti-tumor activities on various types of solid tumors and hematologic malignancies, and thus become a potential weapon used in the treatment of cancer. However, compared to the striking results demonstrated in vitro and in mouse models, the clinical efficacy of γδ T cells-based immunotherapy is disappointing. Meanwhile, recent studies identified a pro-tumor role of γδ T cells in some types of solid tumors. Whether γδ T cells could be transformed from warriors to foe in the environment of leukemia is unclear. In the current study, we aim to dissect the abnormal changes and functions of intrinsic γδ T cells in the context of AML.
Considering bone marrows (BM) are the primary tumor foci of AML and likely provide more representative immune characteristics during leukemia development, the immunophenotyping analyses of γδ T cells in BM from newly diagnosed AML patients (n = 21) were detected using flow cytometry, compared with healthy individual controls (n = 21). We observed that the proportion of γδ T cells and the composition of Vδ1+ and Vδ2+ subgroups were comparable between AML patients and healthy controls. Interestingly, a dramatically elevated subpopulation in γδ T cells expressing CD25 and dim CD127 was observed in AML patients compared with that in healthy controls (3.2% versus 0.8%, P &lt; 0.001). Since their phenotypes are akin to the conventional CD4+ regulatory T cells (Tregs), these CD25+CD127dim γδ T cells are called γδ Tregs here. Paired comparison was also performed on AML patients at diagnosis (n = 13) and completed remission (CR) after chemotherapy (n = 13). A significant decrease in proportion of γδ Tregs was observed at CR, compared with that at newly diagnoses (3.1% versus 0.4% (P &lt; 0.001). These results suggest that this aberrant γδ T subset, γδ Tregs, is induced in the microenvironment of AML and can be reversed after effective therapy for elimination of leukemia blasts.
To further explore the direct effect of AML blasts on γδ T cells, primary malignant cells were sorted from AML patients, and co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. As expected, γδ Tregs was significantly induced and the frequency was continuously elevated from 0.2% to 4.0% following the increased ratios of AML cells (P &lt; 0.001). These results confirmed that AML cells were capable to trigger the transformation of normal γδ T cells into CD25+CD127dim γδ Tregs.
To determine the function of this special γδ T subset, CD25+CD127dim γδ Tregs were expanded in vitro and co-cultured with AML cell lines, U937 cells. Unlike pamidronate-expanded cytotoxic Vδ2+ T cells, γδ Tregs lost the ability of killing AML cells. Furthermore, we explored the impact of γδ Tregs on the anti-AML activity of normal αβ T cells. γδ T cell-depleted PBMCs (more than 90% are αβ T cells) of healthy donors were co-cultured with U937 cells, with or without autologous γδ Tregs. Flow cytometry analysis showed that 7-AAD-positive fraction in U937 cells was dramatically increased from 0.3% to 7.6% after co-culture with PBMCs and without γδ Tregs. Whereas the mortality of U937 cells was gradually declined (from 7.6% to 4%) in the presence of γδ Tregs at increasing ratios (P = 0.031). During this process, γδ Tregs remarkably downregulated the expressions of HLA-DR and CD38, and productions of TNF-α and IFN-γ in both CD4+ and CD8+ αβ T cells. These findings indicated that γδ Tregs significantly impaired the cytotoxic ability of effector αβ T cells on AML cells, suggesting γδ Tregs play a suppressive role in the anti-leukemia immunity.
In summary, we first reported the significant induction of CD25+CD127dim γδ Tregs in the primary BM of AML patients. These γδ Tregs did not exert anti-leukemia activity and attenuated the cytotoxic effect of normal αβ T cells. Our findings will help advance the current understanding of immune mechanism associated with leukemia cell evasion. It also proposes that boosting the cytotoxic γδ T cells should be coupled with inhibition of the suppressive γδ Tregs, for future improvement of γδ T-cell-based immunotherapy.

Disclosures
No relevant conflicts of interest to declare.