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. 2020 Sep 8;9(9):2053.
doi: 10.3390/cells9092053.

Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction

Douâa Moussa Agha  1 , Redouane Rouas  1 , Mehdi Najar  2   3 , Fatima Bouhtit  1   3 , Hussein Fayyad-Kazan  4 , Laurence Lagneaux  5 , Dominique Bron  1 , Nathalie Meuleman  1 , Philippe Lewalle  1 , Makram Merimi  1
Affiliations

Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction

Douâa Moussa Agha et al. Cells. .

Abstract

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy in which antitumor immunity is impaired. The therapeutic management of AML requires understanding the mechanisms involved in the fragility and immune dysfunction of AML T lymphocytes.

Methods: In this study, T lymphocytes from healthy donors (HD) and AML patients were used. Extracellular vesicles (EVs) from leukemic cells were screened for their microRNA content and impact on T lymphocytes. Flow cytometry, transcriptomic as well as lentiviral transduction techniques were used to carry out the research.

Results: We observed increased cell death of T lymphocytes from AML patients. EVs from leukemia myeloid cell lines harbored several miRNAs, including miR-21, and were able to induce T lymphocyte death. Compared to that in HD, miR-21 was overexpressed in both the bone marrow fluid and infiltrating T lymphocytes of AML patients. MiR-21 induces T lymphocyte cell death by upregulating proapoptotic gene expression. It also increases the immunosuppressive profile of T lymphocytes by upregulating the IL13, IL4, IL10, and FoxP3 genes.

Conclusions: Our results demonstrate that miR-21 plays a significant role in AML T lymphocyte dysfunction and apoptosis. Targeting miR-21 may be a novel approach to restore the efficacy of the immune response against AML.

Keywords: AML; T lymphocytes; apoptosis; extracellular vesicles; immunosuppression; miR21.

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Conflict of interest statement

The authors have no conflicts of interest to declare. They confirm that there are no conflicts of interest associated with this publication. The manuscript has been read and approved by all named authors.

Figures

Figure 1
Figure 1
Fragility measurement of AML T lymphocytes. (A) The viability percentage of isolated and activated T lymphocytes (PHA/IL2) was measured in HD and untreated AML patients (=7) within six days post culture using an apoptosis test (cells were labeled with annexin-V-FITC and PI). The level of statistical significance was set at p < 0.01 (**). (B) Relative expression of proapoptotic and antiapoptotic genes in AML T lymphocytes (=27) compared to cells from HDs (=11) in both bone marrow (BM) and peripheral blood (PB) samples as determined using Affymetrix microarray (fold change >1.25 and p < 0.05).
Figure 2
Figure 2
Myeloid leukemia cell line-derived EVs induce cell death in healthy donors (HD) T lymphocytes. (A) Characterization of K562-derived EVs. K562 EVs were characterized by nanoparticle tracking analysis (NTA). They ranged in size from 100 nm to 500 nm, with a mean value of 185 nm. The concentration of K562 EVs reached 28.05 × 109 ± 25.05 × 107 particles/mL. (B) The viability percentage of T lymphocytes (=3) was measured by an apoptosis test after the addition of K562 cell line-derived microvesicles (MVs) at different concentrations. The concentrations used for the MVs derived from HL60 and KG1 cells were similar to those used in the condition of 150 μl for the K562-MVs. MC: medium control. TPE: EV elution buffer. (C) Analysis of miRNA expression in the cargo of EVs purified from the supernatants of K562, KG1 and HL60 cells. The graph shows the expression curves of 385 miRNAs amplified using the TaqMan® Array Human MicroRNA assay. The levels of statistical significance were set at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).
Figure 3
Figure 3
Relative expression level of miR-21 in bone marrow. (A) Bone marrow-circulating miR-21 expression in AML patients (n = 16) compared to HDs (n = 9). (B) MiR-21 is upregulated in AML patient T lymphocytes (n = 19) compared to HD lymphocytes (n = 9). Data obtained by quantitative RT-PCR amplification of miR-21 are plotted. All data were collected and statistically analyzed using Student’s t-test. Values are presented as the means of at least three independent experiments. The level of statistical significance was set at p < 0.01 (**) for BM-circulating AMLD vs HD and p ≤ 0.05 (*) for BM CD3 AML vs HD.
Figure 4
Figure 4
miR-21 induces cell death in T lymphocytes. (A) T cell lentivirus transduction efficiency: analysis using cytometry of the percentage of transduced human T lymphocytes (=3) by lentiviral vectors containing GFP and hsa-miR-21 or scramble as a control (=3). (B) Relative expression of miR-21 in lenti-miR-21-transduced cells (=3) compared to the control (=3). (C) MiR-21 induces HD T lymphocyte cell death. (D) Inhibition of miR-21 expression increases resistance to cell death in T lymphocytes (=3). All data were collected and statistically analyzed using Student’s t-test. Values are presented as the means of at least three independent experiments. The levels of statistical significance were set at p ≤ 0.03 and p < 0.01.
Figure 5
Figure 5
Gene expression analysis of cell death pathways induced by miR-21 (A–E). Analysis of the gene expression of cell death pathways in lenti-miR-21-transduced T lymphocytes (=3) compared to lenti-scramble-transduced T lymphocytes (=3). Relative expression of upregulated proapoptotic (A), antiapoptotic (B), autophagy (C), necrosis (D), and downregulated genes (E). All data were collected and statistically analyzed using Student’s t-test. The level of statistical significance was set at p < 0.05.
Figure 6
Figure 6
miR-21 induces a protumoral response in T lymphocytes. (A–C) Analysis of the expression of immune response genes in lenti-miR-21-transduced T lymphocytes (=3) compared to lenti-scramble-transduced T lymphocytes (=3). Relative expression of common genes related to T regulatory and immunosuppressive markers (A), Th2 immune genes (B) and genes related to the protumoral immune response (C). (D) Relative expression of genes related to the protumoral response in AML T lymphocytes (=27) compared to HD lymphocytes (=11). All data were collected and statistically analyzed using Student’s t-test. The level of statistical significance was set at p < 0.05.
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