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. 2021 Jun 4;11(7):313. doi: 10.1007/s13205-021-02817-5

Acute lymphoblastic leukemia-derived exosome inhibits cytotoxicity of natural killer cells by TGF-β signaling pathway

Huijun Yu 1,2, Tingting Huang 2, Daming Wang 2, Lei Chen 2, Xi Lan 2, Xintong Liu 2, Keyan Chen 2, Haihong He 2, Shaobo Li 2, Yiwen Zhou 2, Jiansheng Xie 1,✉
PMCID: PMC8178429  PMID: 34109098

Abstract

This study was conducted to explore whether acute lymphoblastic leukemia (ALL)-derived exosomes affect natural killer (NK) cells. Exosomes were isolated and identified from Jurkat cells and co-cultured with NK cells. Then, the cytotoxicity, viability, and release of perforin and granzyme B in NK92-MI cells were measured. PCR arrays were used to detect gene expression alterations in the transforming growth factor (TGF)-β pathway of NK92-MI cells treated or not treated with exosomes. The morphology and size of the exosomes isolated from Jurkat cells showed typical characteristics of exosomes, and the expression of cluster of differentiation 63 was detected. Jurkat-derived exosomes were internalized by NK92-MI cells, further inhibiting the proliferation and cytotoxicity of NK92-MI cells. An enzyme-linked immunosorbent assay revealed that the release of perforin and granzyme B from NK92-MI cells decreased after co-culture with exosomes. Similarly, western blot and immunofluorescence staining verified that Jurkat-derived exosomes inhibited the expression of granzyme B and perforin. Furthermore, Jurkat-derived exosomes enhanced the signaling of the TGF-β pathway in NK92-MI cells via the MDS1 and EVI1 complex loci and homeodomain interacting protein kinase 2. In conclusion, we found that ALL-derived exosomes inhibit the biological function of NK cells and provide support for the immunotherapy of ALL.

Keywords: Acute lymphoblastic leukemia, Natural killer, Exosome, TGF-β signaling pathway, Cytotoxicity

Introduction

Acute lymphoblastic leukemia (ALL) is a hematological malignant tumor characterized by the clonal expansion and aggregation of abnormal immature lymphoid precursor cells in the bone marrow and blood (Malard and Mohty 2020). There were 437,033 new cases of and 309,006 deaths from leukemia worldwide in 2018 (Bray et al. 2018). In China, there were approximately 75,300 new cases of and 53,400 deaths because of ALL in 2015, and its incidence in children aged 0–14 years was estimated at 3–4 per 100,000 people, accounting for approximately one-third of all children with malignant tumors (Chen et al. 2016; Katz et al. 2015). The predisposing factors of ALL, including environmental exposure and inherited genetic susceptibility, have been identified in only a few patients, while most cases appear to arise in otherwise healthy individuals (Malard and Mohty 2020). Currently, the first-line treatment for ALL is induction, consolidation, intensification, and long-term maintenance over two or 3 years. Usually, these four phases are based on a combination of chemotherapy with cytarabine and anthracycline. However, relapse remains a huge challenge for patients with ALL. Although prognosis has improved significantly in the past 40 years, approximately half of all patients experience relapse, resulting in a 5-year overall survival rate of approximately 40% for adults and 85% for children (Barwe et al. 2017; Inaba et al. 2013). Therefore, in-depth exploration of the mechanism of leukemia deterioration and relapse after treatment is of great significance for improving the survival time of patients with ALL.

Exosomes, as important mediators of intercellular communication by delivering membrane proteins and other contents (such as proteins, lipids, mRNAs, and non-coding RNAs), not only participate in tumorigenesis caused by tumor metastasis and angiogenesis, but also play an important role in the process of immune escape (Steinbichler et al. 2017). Exosomes not only accelerate the progression of leukemia by creating a microenvironment for the growth of leukemia cells, supporting leukemia drug resistance, cell metastasis, and immune escape, but can also hinder the progression of leukemia cells by loading molecules that intercept these pathways (Prieto et al. 2017; Raimondo et al. 2015; Yang et al. 2019). Huang et al. found that leukemia-derived exosomes enhance anti-leukemia immunity by downregulating the expression of TGF-β1 (Huang et al. 2017). Similarly, leukemia-derived exosomes are loaded with RNA directed to hematopoietic progenitor cells to inhibit their function and remodel the bone marrow niche into a microenvironment that is leukemia growth-permissive (Boyiadzis and Whiteside 2018; Kumar et al. 2018). In contrast, miR-34c-5p selectively targets rat sarcoma (Ras)-associated binding 27B (RAB27B) and causes the inhibition of exosome shedding to induce senescence and the eradication of acute myeloid leukemia stem cells (Peng et al. 2018). However, the function and mechanism of exosomes derived from ALL cells remain unclear.

Natural killer (NK) cells play an immune monitoring and first-line defense role in the control of tumor growth, metastasis, and spread. In the treatment of ALL with hematopoietic cell transplantation, NK cells are the first immune cells to be remodeled, which may control the recurrence of leukemia in the first few months before T cell reconstitution (Shilling et al. 2003). Insufficient or impaired NK cells can cause serious infections or promote the development of malignant tumors. In addition, NK cells may be a prognostic factor for ALL in children (Mizia-Malarz and Sobol-Milejska 2019). Human immune cells have high allele diversity to recognize different antigens (He et al. 2014). In the tumor microenvironment, NK cells recognize tumor antigens mainly through surface-activated receptors and rely on the secretion of cytotoxic molecules, such as perforin and granzyme, to directly lyse and kill tumor cells. Chimeric antigen receptors (CAR) modified by NK cells have selective cytotoxicity to fms-like tyrosine kinase 3 (FLT3)-positive B-ALL cells and inhibit the development of leukemia in vivo (Oelsner et al. 2019). Importantly, extracellular vesicles extracted from NK cells can target and kill tumor cells through a variety of cytotoxic proteins and killing mechanisms (Wu et al. 2019). However, the toxicity of NK cells in the tumor microenvironment can be regulated by cancer cells, causing tumor immune escape. For example, extracellular vesicles derived from lung cancer cells transport transforming growth factor β1 (TGF-β1) to reduce NK cell surface-activated receptor NK group 2D (NKG2D), thereby inhibiting the cytotoxicity of NK cells (Berchem et al. 2016). However, it remains unclear whether and how leukemia cells regulate NK cytotoxicity to achieve immune escape. Therefore, in-depth exploration of the regulatory mechanism of leukemic exosomes on NK cells in the tumor microenvironment will help to identify molecular targets for leukemia treatment and provide a theoretical basis for the prevention of post-treatment relapse. In this study, the exosomes from ALL cells were used to incubate NK cells, and cytotoxicity experiments, western blotting, and immunofluorescence assays were used to explore the effect of exosomes on NK cytotoxicity. This study provides a new perspective for blocking the immune escape of ALL cells and improving the treatment efficiency of ALL.

Materials and methods

Cell culture

The human T-ALL cell line Jurkat (Procell, CL-0129, China) and NK cell line NK92-MI (Procell, CL-0533, China) were obtained from Procell Life Science Technology (Procell, CL-0129, China). NK92-MI cells were cultured in alpha modification of minimum essential medium (Procell, CM-0533, China) supplemented with 0.2 mM inositol, 0.1 mM β-mercaptoethanol, 0.02 mM folic acid, 12.5% fetal bovine serum (FBS), 12.5% horse serum, and 1% penicillin–streptomycin. Jurkat cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA) and then were placed in an incubator at a constant temperature of 37 °C and 5% CO2.

Exosome isolation and identification

Exosomes were isolated from medium supernatant of Jurkat cells treated with differential centrifugation as previously described (Xie et al. 2019). Purified exosomes were immediately fixed with 4% glutaraldehyde and 1% osmium tetroxide, followed by dehydration with conventional ethanol and acetone. Subsequently, exosomes were impregnated with epoxy resin, embedded, and polymerized. Finally, the polymerized products were cut into 0.5 µm semi-thin sections to localize the exosomes under a light microscope and further prepared into 60 nm ultra-thin sections. All sections were stained with uranium acetate and lead citrate and observed under a transmission electron microscope (TEM). Moreover, nanoparticle tracking analysis (NTA) was used to determine the distribution, size, and number of particles in the exosomal formulations using ZetaView Nanoparticle Tracking (Particle Metrix, Germany).

Labeling of exosomes and tracing the NK92-MI uptake of labeled exosomes

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil; Beyotime, Shanghai, China) enables extended periods of staining of the cytoplasm and intracellular membrane without causing cytotoxicity or destroying cell function. The Jurkat-exosome suspension from Jurkat cells was removed by ultracentrifugation at 100,000×g for 120 min twice, and then incubated with 4 mg/mL Dil at room temperature for 15 min, and the incubation fluid was then passed through the Exosome Spin Columns (MW3000, Life, ThermoFisher, USA) to obtain the Dil-labeled Jurkat-derived exosomes. Next, NK92-MI cells were incubated with Dil-labeled Jurkat-derived exosomes for 24 h. After incubation, the chamber cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeated with 0.2% TritonX-100 at room temperature for 10 min, and then stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Finally, the cells were examined under a fluorescent microscope (Axio Scope.A1, Carl Zeiss, Germany).

Cytotoxicity assay

Plasma membrane integrity was assessed by lactate dehydrogenase (LDH) release (Korzeniewski and Callewaert 1983), which is widely used in cytotoxicity detection. To verify the effect of Jurkat-derived exosomes on NK92-MI cytotoxicity, cells were incubated with or without Jurkat-derived exosomes (50 μg/mL). An LDH kit (Beyotime, Shanghai, China) was used to detect NK92-MI cytotoxicity according to the manufacturer’s instructions. The optical density was measured at 490 nm using an Epoch 2 (BioTek Instruments, USA).

CCK-8 assay

The effect of the Jurkat-derived exosomes on NK92-MI cell viability was determined using the Cell Counting Kit-8 (CCK-8; Beyotime, C0038, China). The exosomes (50 μg/mL) derived from Jurkat cells were incubated with NK92-MI cells in 96-well plates for 48 h. Then, 100 μL CCK-8 solution was added to each well, which were then placed in an incubator for further incubation. Samples were taken at 0, 24, 48, 72, and 96 h to measure the absorbance at 450 nm using a microplate reader. Each group set had six repeated wells.

Western blotting

The exosomes (50 μg/mL) derived from Jurkat cells were incubated with NK92-MI cells in 96-well plates for 48 h. Then, the total protein contents of NK92-MI cells after incubation with Jurkat-derived exosomes were analyzed using the BCA protein assay kit (Thermo scientific, USA). Western blotting was used to analyze the isolated proteins as follows: briefly, an equal amount of 20 μg of protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for nonspecific binding with 5% nonfat milk and incubated with primary antibodies at 4 °C overnight, followed by incubation with goat anti-mouse IgG H&L (1:1000, Beyotime, A0216) at room temperature for 1 h. Finally, bands were visualized using the Bio-rad ChemiDoc XRS system. The primary antibodies used were anti-perforin (1:1000, Santa Cruz Biotechnology, sc-373943), anti-granzyme B (1:10,000, Abcam, ab134933), anti-cluster of differentiation (CD63; 1:500, Abcam, ab216130), and anti-GAPDH (1:2000, Proteint, 60,004–1-Lg). GAPDH was used to normalize the expression. The experiment was repeated three times.

Enzyme-linked immunosorbent assay (ELISA)

To evaluate the effect of ALL-derived exosomes on the expression of NK92-MI cytotoxic molecules, the concentrations of perforin and granzyme B of NK92-MI cells after incubation with Jurkat-derived exosomes (50 μg/mL) for 48 h were determined by ELISA with three replicates. Granzyme B and perforin were determined using Solarbio ELISA kits (SEKH0193, Solarbio® LIFE SCIENCES, Beijing, China) and Arigo ELISA kits (ARG80173, Arigo Biolaboratories, Shanghai, China).

Immunofluorescence imaging

The exosomes (50 μg/mL) derived from Jurkat cells were incubated with NK92-MI cells in 96-well plates for 48 h. NK92-MI cells were fixed in 4% formalin at 4 °C for 8 h and embedded in paraffin blocks for cutting into 3 µm sections. Then, all sections were permeabilized in 0.2–0.5% Triton X-100 and blocked in 5% normal donkey serum at room temperature for 1 h. Next, sections were stained with perforin (1:250, Santa Cruz Biotechnology, sc-373943) and granzyme B (1:250, Abcam, ab134933) overnight and detected using fluorescent-conjugated goat anti-mouse IgG H&L (1:500, Abcam, ab150117). Finally, all sections were fixed with fluorescence mounting medium (Sangon, Shanghai, China) and imaged on a Zeiss LSM880 NLO microscope.

PCR array

Total RNA from NK92-MI cells after incubation with Jurkat-derived exosomes or control was extracted for human TGF-β/BMP signaling pathway PCR array (PAHS-035Z, SABiosciences, China) using TRIZOL reagent (Invitrogen, USA). The PCR array analysis was commissioned by Yingbio Biotechnology Company and applied to detect the differential expression of mRNAs in NK92-MI cells between the two groups. Briefly, 25 ng to 5 μg of RNA was reverse transcribed into cDNA using a RT2 First Strand Kit (330,401, Qiagen, China), and then subjected to RT-PCR using a RT2 SYBR Green MasterMix kit (330,401, Qiagen, China).

Statistical analysis

All data were processed using the Statistical Product and Service Solutions (SPSS) 16.0 software. Data are presented as mean ± standard deviation (SD). The significant difference between the two groups was determined by t test. P < 0.05 was considered statistically significant.

Results

Characterization of ALL-derived exosomes

The morphology of exosomes from the Jurkat cells was detected by TEM, as shown in Fig. 1a. The typical morphology of the population of microvesicles was similar to that of the exosomes. The size of the microvesicles with mean 129.3 ± 53.8 nm in diameter was further determined by NTA (Fig. 1b). In addition, western blotting analysis suggested that CD63, a typical exosomal protein, was expressed in these microvesicles, but not in cells (Fig. 1c). These results indicated that Jurkat-derived exosomes were successfully isolated.

Fig. 1.

Fig. 1

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Verification of isolated Jurkat-derived exosomes. a Morphology of isolated Jurkat-derived exosomes was determined by transmission electron microscopy (scale bar, 100 nm). b The size of Jurkat-derived exosomes was measured by nanoparticle tracking analysis (NTA). c The exosome marker CD63 was only expressed in Jurkat-derived exosomes (Exo), not in Jurkat cells (cell) and, compared to GAPDH, CD63 was significantly upregulated in Exo

Jurkat-derived exosomes inhibit NK92-MI cell proliferation and cytotoxicity

To investigate Jurkat-derived exosome internalization, NK92-MI cells were treated with Dil-labeled Jurkat-derived exosomes. As shown in Fig. 2a, exosomes were significantly observed in the cytoplasm of NK92-MI cells under fluorescent microscopy. Jurkat-derived exosomes were internalized by NK92-MI cells. Furthermore, to explore the effect of altering Jurkat-derived exosomes on the biological function of NK cells, we conducted CCK-8 and LDH assays. The proliferation ability and cytotoxicity of NK92-MI cells decreased significantly after incubation with Jurkat-derived exosomes, compared with the control, indicating that Jurkat-derived exosomes inhibited the biological function of NK cells (Fig. 2a, b).

Fig. 2.

Fig. 2

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Jurkat-derived exosomes inhibit NK92-MI cell viability. a Fluorescence microscopy analysis of Jurkat-derived exosome internalization by natural killer (NK) 92-MI cells. Magnification is × 40. The proliferation (b) and cytotoxicity (c) of NK92-MI cells were, respectively, analyzed by CCK-8 and lactate dehydrogenase (LDH) assay. Compared with the control, the proliferation and cytotoxicity of NK92-MI cells were significantly suppressed in the Exo group. Data are mean ± SD (*p < 0.05; **p < 0.01). Exo and control mean NK92-MI cells pretreated with Jurkat-derived exosomes (50 μg/mL) and blank for 48 h, respectively

Jurkat-derived exosomes inhibit the expression of NK92-MI cell cytotoxic molecules

The proteins of granzyme B and perforin are toxic molecules released by NK cells and can induce target cell death. In this study, to evaluate the effects of ALL-derived exosomes on the killing effects of NK92-MI, the expression of perforin and granzyme B in NK92-MI cells was detected by ELISA, western blot, and immunofluorescence after incubation with Jurkat-derived exosomes. The results of ELISA and western blotting indicated that the release of granzyme B and perforin decreased in NK92-MI cells pretreated with Jurkat-derived exosomes, compared with the control (Fig. 3). Moreover, similar results were observed by immunofluorescence (Fig. 4). These results suggested that Jurkat-derived exosomes inhibited the cytotoxicity of NK92-MI cells.

Fig. 3.

Fig. 3

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Jurkat-derived exosomes inhibit cytotoxic molecule expression in NK92-MI cells. The concentrations of granzyme B (a) and perforin (b) in NK92-MI cells (normal and pretreated with Jurkat-derived exosomes) were inhibited by Exo, as revealed by ELISA assay. Exo and control, respectively, refer to NK92-MI cells pretreated with Jurkat-derived exosomes (50 μg/mL) and blank for 48 h. c The protein levels of granzyme B and perforin-1 in NK92-MI cells were inhibited by Exo, as reveled by western blotting. The protein expressions of granzyme B and perforin-1 were quantified by the optical density value relative to GAPDH and were statistically significant. Experiments were performed in triplicate, and all data are mean ± SD (*p < 0.05)

Fig. 4.

Fig. 4

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Jurkat-derived exosomes inhibit cytotoxic molecule expression in NK92-MI cells. Immunofluorescence detection of granzyme B (a) and perforin (b). The fluorescence intensity of the Exo group was significantly decreased, compared with the control group. Exo and control refer to the NK92-MI cells pretreated with Jurkat-derived exosomes (50 μg/mL) and blank for 48 h, respectively. Magnification is × 40

Jurkat-derived exosomes induce the TGF-β signaling pathway

To investigate the alteration of NK92-MI cell signaling pathways by Jurkat-derived exosome treatment, a PCR array was used to integrate the genes related to the TGF-β signaling pathway, a classical immune signaling pathway. In total, 18 differentially expressed genes (DEGs) were identified (Fig. 5), and details of these DEGs are provided in Table 1. Only growth differentiation factor 3 (GDF3) was downregulated after Jurkat-derived exosomal treatment in NK92-MI cells, compared with the control treatment, while the rest were upregulated. In addition, the most significant upregulation was observed for myelodysplastic syndrome 1 (MDS1) and ecotropic viral integration site-1 (EVI1) complex locus (MECOM; approximately 7.31-fold), followed by homeodomain interacting protein kinase 2 (HIPK2; approximately 7.18-fold). These data revealed that Jurkat-derived exosomes enhanced the expression of TGF-β signaling pathway in NK92-MI cells.

Fig. 5.

Fig. 5

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Heatmap of 18 upregulated genes in the TGF-β signaling pathway after Jurkat-derived exosome treatment of NK92-MI cells, as revealed by PCR array. The vertical column indicates grouping, NC represents the control group of NK92-MI cells incubated with blank with three repetitions, and Exo represents the Exo group of NK92-MI cells incubated with exosomes (50 μg/mL) for 48 h with three repetitions. The horizontal column indicates genes, red indicates upregulation, and green indicates downregulation

Table 1.

Details of differentially expressed genes

Symbol AVG ΔCt (Ct(GOI)—Ave Ct (HKG)) 2^− ΔCt Fold up- or down-regulation
Jurkat-Exo NC Jurkat-Exo NC Jurkat-Exo/NC
GDF3 12.5215 10.85609 0.00017 0.000539 − 3.17203
TGFBI 12.14025 13.25682 0.000222 0.000102 2.168317
CDKN1A 7.927692 9.006256 0.004107 0.001945 2.111933
EMP1 7.77744 9.082122 0.004558 0.001845 2.470292
HIPK2 7.685843 10.52933 0.004857 0.000677 7.177539
LTBP2 7.793824 10.0151 0.004506 0.000966 4.663048
LEFTY1 10.01887 11.62783 0.000964 0.000316 3.05033
ENG 5.135473 6.605132 0.028449 0.010272 2.769563
AMHR2 5.775096 7.004807 0.018261 0.007787 2.3452
ACVR1 6.566564 7.956112 0.01055 0.004027 2.619967
BMP2 8.851978 11.04442 0.002164 0.000473 4.570795
MECOM 7.265263 10.13494 0.0065 0.000889 7.309023
BMPR1B 12.18351 13.48741 0.000215 8.71E−05 2.468955
INHBB 9.624945 11.30653 0.001266 0.000395 3.207809
IGF1 9.592112 10.8576 0.001296 0.000539 2.404082
BMP3 10.8929 12.28507 0.000526 0.0002 2.624736
SOX4 8.659646 10.57823 0.002473 0.000654 3.780517
DCN 9.475064 11.00725 0.001405 0.000486 2.892242
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Fold-change, (2^(− Delta Delta Ct)) is the normalized gene expression (2^(− Delta Ct)) in the test sample (Jurkat-Exo) divided the normalized gene expression (2^(− Delta Ct)) in the control sample (NC). Fold-Regulation represents fold-change results in a biologically meaningful way. Fold-change values greater than one indicate a positive- or an up-regulation, and the fold-regulation is equal to the fold-change. Fold-change and fold-regulation values greater than 2 are indicated in italics (up-regulation); fold-change values less than 0.5 and fold-regulation values less than − 2 are indicated in bold (down-regulation)

Discussion

ALL is a hematological malignancy that seriously threatens human health (Malard and Mohty 2020). At present, ALL lacks an effective cure, because the immune evasion of tumor cells seriously hinders therapeutic effects. Exosomes not only participate in tumorigenesis caused by tumor metastasis and angiogenesis, but also play a vital role in the process of immune escape (Steinbichler et al. 2017). NK cells are the main immune defense cells against tumors, and studies have found that tumor cells, including leukemia cells, achieve immune escape by inducing abnormal NK cell function (Dulphy et al. 2016). Importantly, we revealed for the first time, to our knowledge, that ALL-derived exosomes regulate the biological function of NK cells by inducing the TGF-β pathway to promote immune evasion.

In this study, we found that Jurkat-derived exosomes significantly inhibited the biological function of NK92-MI cells, including the proliferation, cytotoxicity, and release of cytotoxic molecules. Similarly, an increasing number of studies have shown that tumor-derived exosomes trigger the dysfunction of NK cells. For instance, Briand et al. found that tumor-derived exosomes induced by radiotherapy decrease granzyme B secretion by NK cells via loading miR-378a-3p (Briand et al. 2020). In addition, melanoma cell-derived exosomes inhibit the functional/phenotype of CD8 T cells and NK cells with downregulated NKG2D expression (Sharma et al. 2020). Moreover, the leukemia cell supernatant inhibits NK cell function, but it is currently unclear whether ALL regulates the cell function through exosomes. Leukemia-derived exosomes can mediate different pathways in many types of target cells to aid survival. For example, microRNA-4532-containing exosomes are released by acute myeloid leukemia (CML) by activating the signal transducer and activator of transcription 3 (STAT3) pathway to mediate normal hematopoiesis in hematopoietic stem cells (Zhao et al. 2019). CML-derived exosomes can also mediate the vascular endothelial growth factor (VEGRF) pathway to increase angiogenesis in the bone marrow (Roma-Rodrigues et al. 2019). These results again support our inference that Jurkat-derived exosomes can affect the biological function of NK cells through a certain pathway.

Exosomes complete intercellular communication by delivering cargo and activating target pathways. TGF-β is a powerful inhibitor of the NK cell anti-tumor response and is highly expressed in patients with ALL (Rouce et al. 2016). We further investigated via PCR array whether exosomes inhibit NK cell toxicity by activating the TGF-β pathway. The results showed that all 17 classical molecules of the TGF-β pathway in NK92-MI cells were upregulated after incubation with Jurkat-derived exosomes, and only GDF3 was downregulated. TGF-β inhibits the phenotype of NK cells by regulating various cell killing factors and activating receptors. For example, TGF-β1 reduces the release of perforin and granzyme and downregulates the expression of some activated receptors, including CD16, DNAM-1, NKG2D, TRAIL, and NKp30, to decrease NK cell cytotoxicity (Tran et al. 2017). Moreover, the expression levels of MECOM (approximately 7.31-fold) and HIPK2 (approximately 7.18-fold) significantly respond to Jurkat-derived exosome induction in NK92-MI cells. MECOM is composed of MDS1 and the EVI1 complex locus, and the latter is an aggressive oncogene associated with leukemia (Mitani 2004). The expression of EVI1 prolongs the phosphorylation of STAT1 and the activation of interferon (IFN)-dependent reporter genes, as well as completely eliminates the anti-proliferative and apoptotic effects of IFN-α (Buonamici et al. 2005). Therefore, MECOM may contribute to immune inhibition in NK cells induced by ALL-derived exosomes. In addition, HIPK2 is indispensable for the precise regulation of immune homeostasis by IFN (Cao et al. 2019). The expression of HIPK2 can be induced by TGF-β both in vivo and in vitro to activate the expression of downstream signaling pathways, including the Notch, NF-κB, Smad, and Wnt/β-catenin pathways, thereby controlling cell function (Chang et al. 2017). Therefore, HIPK2 may be another contributing gene for immune inhibition in NK cells induced by ALL-derived exosomes.

In conclusion, ALL-derived exosomes inhibit the proliferation, cytotoxicity, and release of cytotoxic granules in NK cells through the TGF pathway to evade innate immune surveillance. This study highlights the importance of developing novel therapies focusing on NK cell immunosuppression and restoring anti-leukemia cytotoxicity.

Author contributions

Conceptualization and funding acquisition: JX. Data curation: HY, TH, DW, LC, and XL. Formal analysis: XL, KC, HH, SL, and YZ. Experimental studies: HY, TH, KC, HH, SL, and YZ. Software: DW, LC, XL, and XL. Writing—original draft and review and editing: all authors. All authors read and approved the final manuscript.

Funding

This work was supported by Science, Technology and Innovation Bureau of Bao’an District, Basic Research Project of Healthcare in Bao'an District (no. 2019JD450).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no conflict on interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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