The Role And Regulatory Mechanism Of Helios-positive Regulatory T Cells In Acute Lymphoblastic Leukemia

BackgroundAcute lymphoblastic leukemia(ALL)is due to a disruption in the regulation of cell growth as well as the failure of the host to provoke a sufficient antitumor immune response.Although many aspects of immunoregulation abnormalities in leukemia have been elucidated,the specific immunological mechanism involved in the multistep leukemogenesis remains unclear.Recent studies have focused on the role of immune negative regulation in human diseases.Regulatory T cells(Tregs),defined based on the expression of CD4 and CD25 and the transcription factors FoxP3,are essential for maintaining peripheral tolerance,but they also suppress sterilizing immunity and weaken anti-tumor immunity.Strong Treg infiltration in tumors is generally associated with poor clinical outcome.Thus,immunosuppression mediated by Tregs is a key facilitator of tumor immune evasion,but the mechanism underlying the contribution of Tregs to human cancer progressionremains unclear.Recent studies have shown that Tregs contribute to tumor angiogenesis throughboth indirect and direct mechanisms.Tregs can promote angiogenesis indirectly by blocking effector-cell-derived angiostatic cytokines,such as interferon ? and C-X-C motif chemokine 10.Tregs that have been recruited to hypoxic areas can also directly stimulate angiogenesis through the production of vascular endothelial growth factor(VEGF).The association of Tregs infiltration with VEGF overexpression and increased microvessel density in endometrial and breast cancers provided clinical cues for a link between Tregs and angiogenesis.VEGF could promote the proliferation of Tregs,and VEGF/VEGF receptor(VEGFR)antibodies or inhibitors could decrease the number of Tregs both in patients with colon cancer and in mouse models.Sustained angiogenesis is also critical for leukemogenesis.Helios,combined with FoxP3,is considered a good marker for discriminating functional Tregs.Helios was originally cloned from a mouse thymoma line and is mainly expressed in the T-cell lineage.Previous studies have demonstrated that Helios upregulates FoxP3 by binding to the FoxP3 promoter,and partially silenced Helios expression in Tregs resulted in decreased FoxP3 levels.These findings have led to the increased interest in Helios,which may play a critical role in controlling certain aspects of Tregs,including their suppressive function,differentiation,and survival.However,there were very few studies have addressed the regulation of Helios in ALL Tregs.Objective1.To discover the association between Helios expression in Tregs and B-precursor(pre-B)ALL pathogenesis.2.To study the effect of Helios expression in Treg function.3.To study the biological characteristics and functions of umbilical cord blood(UCB)derived Tregs.4.To study the impact of Helios+ Tregs on the pathogenesis and angiogenesis in ALL mice.Procedures1 Proportion of Helios+ Tregs in childhood pre-B ALL24 Peripheral blood(PB)samples were obtained from patients who were diagnosed with pre-B ALL.As controls,10 PB consenting age-matched individuals who underwent orthopedic surgery with no history of tumors,antecedent primaryhematologic abnormalities,or immunodeficiency diseases were sampled.1.1 To discover the proportion of Helios+ Tregs in pre-B ALL,CD4 CD25+FoxP3+ and CD4 FoxP3 Helios+ Tregs were detected by flow cytometry.1.2 CD4 CD25+ Tregs and CD4 CD25-helper T(Th)cells were isolated usingCD4+CD25+ Treg isolation kits.Real-time fluorescence quantitative reverse transcription polymerase chain reactio(RT-qPCR)was performed to examine the expression of Helios,FoxP3,and related transcription inhibitors in ALL-Tregs and ALL-Th.1.3 The lentiviral vectors GV358-enhanced green fluorescent protein(EGFP)-Helios and siRNA-Helios were constructed.The transfection efficiency was detected by flow cytometry.The expression of Helios mRNA and protein was detected by RT-qPCR and Western blot.1.4 RT-qPCR was used to detect the expression of transcription factors and immunosuppressive factors.Flow cytometry was performed to detect the expression levels of FoxP3 and transforming growth factor ?1(TGF-?1).2 Helios expression affects the biological function of ALL-Treg cells5 bone marrow(BM)samples were obtained from patients who were diagnosed with pre-B ALL.As controls,5 BM consenting age-matched individuals who underwent orthopedic surgery with no history of tumors,antecedent primary hematologic abnormalities,or immunodeficiency diseases were sampled.2.1 The effect of Helios on proliferation and apoptosis of Treg cells was detected by flow cytometry.The effect of Helios on the immunosuppressive ability of Tregs was detected by CFSE immunosuppressive assay.2.2 Western blot was performed to verify the protein levels of VEGF-VEGFR pathway in bone marrows of pre-B ALL samples.2.3 Endothelial-tube formation assay was performed to test the regulation of Helios on angigenesis.RT-qPCR was performed on the HUVECs to detect downstream genes of VEGF-VEGFR pathway.2.4 In order to study the direct effect of Tregs on leukemia cells,Tregs were co-cultured with Nalm-6 cells,the apoptosis rate of Nalm-6 cells was detected by flow cytometry,and the expression of Bcl-2 and Bax apoptotic proteins was detected by Western blot.3 Biological characteristics and functions of UCB-derived Tregs3.1 We quantified the amount and proliferation ability of CD4+CD25+FoxP3 Helios+ Tregs derived from UCB during the culture period.3.2 The shRNA-Helios lentiviral vector was constructed to regulate the expression of Helios in UCB-Tregs.Transfection efficiency and Helios expression were detected by flow cytometry;3.3 We quantified the immunosuppression ability of CD4 CD25+FoxP3 Helios+Tregs derived from UCB during the culture period3.4 Endothelial-tube formation assay was performed to test the regulation of Helios on angiogenesis.Western blot was performed to verify the levels of CD31 protein in HUVECs.4 Effect and mechanism of Helios+ Tregs on the pathogenesis of ALL model miceThere are four animal groups.Animals in Helioshigh group were received UCB-derived HelioshighTregs and Nalm-6 cells.Animals in Helioslow group were received UCB-derived Helioslow Tregs and Nalm-6 cells.Only Nalm-6 cells were inj ected in model group.Animals in blank group received only PBS.4.1 Mouse weights were determined once a week.We weighed the liver,spleen,and kidneys to calculate the organ index.The proportion of human CD19+ cells and Tregs in PB of four groups were tested by flow cytometry.The proportions of lymphoblast in BM were tested by bone marrow smear.4.2 Western blot was performed to verify the protein levels of VEGF-VEGFR pathway in bone marrows,livers,spleens,and kidneys of four mice groups.The microvessel density(MVD)of the bone marrow was next determined by immunohistochemical assessment.RT-qPCR was performed to detect the expression of angiogesis genes in bone marrow.C-C motif chemokine 22(CCL22)protein levels were measured by ELISA kits.Results1 Proportion of Helios+ Tregs in childhood pre-B ALL1.1 CD4+FoxP3+Helios+T cells took up 12.29± 0.63%of the total CD4+ T cells in pre-B ALL while they accounted for 5.94± 0.61%of the total CD4+ T cells in HCs.Further analysis indicated that CD4+FoxP3+Helios+ T cells accounted for a higher percentage in very high risk ALL(16.14±0.81%)than in standerd risk(10.77±0.65%)and high risk(12.72±1.07%)ALL.1.2 Results of RT-qPCR showed that the expression levels of Helios,FoxP3 and TGF-?1 mRNA in Tregs of ALL were significantly higher than those of Tregs in the control group.1.3 The lentiviral-EGFP-Helios overexpression vector infection rate was about 15%,and the expression of Helios protein was significantly increased.The siRNA-Helios infection rate was about 30%,and the expression of Helios RNA and protein was significantly decreased.1.4 The siRNA-mediated depletion of Helios in ALL-Tregs not only influenced FoxP3 production,but also inhibited TGF-?1 mRNA and protein expression.The expression of FoxP3 and TGF-?1 statistically increased when Helios was upregulated in normal Tregs.2 Effect and mechanism of Helios expression on the biological function of ALL-Tregs2.1 Compared with that in ALL Tregs or a blank control,the inhibition of Helios down regulated the immunosuppressive function of ALL Tregs by improving the proliferation abilities of Th cells.The upregulation of Helios induced a significant increase in the immunosuppressive capacity of HC Tregs.2.2 Levels of VEGFA,VEGFR2,and phosphorylated VEGFR2(p-VEGFR2)proteins were all significantly increased in pre-B ALL.High expression of Helios in HC Tregs led to higher VEGFA secretion in HUVECs,while low expression of Helios resulted in decreased VEGFA production.Overexpression of Helios promoted the phosphorylation of VEGFR2,whereas Helios knockdown in Tregs showed an opposite effect.2.3 Matrigel plugs enriched with supernatants from Helioshigh Tregs significantly accumulated more endothelial cells over 6 h than Matrigel enriched with Helioslow Tregs supernatants.Upregulation of Helios in Tregs significantly increased chemokine(C-X-C motif)ligand 5(CXCL5)and CCL22 expression in HUVECs.Moreover,downregulation of Helios resulted in the opposite effect.2.4 It is verified that slight suppression of normal Tregs to leukemia cells led to apoptosis after 72 h co-culture.Helios up-regulation in Tregs resulted in a reduction of nalm-6 cells population in early apoptosis.An efficient increase in apoptosis level following RNAi-mediated silencing of Helios in Tregs was also evident in nalm-6 cells.Anti-apoptotic protein Bcl-2 was obviously increased in nalm-6 cells in Lenti-Helios group and downregulation of Helios led to the opposite result.3 Biological characteristics and functions of UCB derived Tregs3.1 The number of induced CD4 CD25 Treg cells has risen steadily from day 0 to day 28.At day 14,the proliferation capability of UCB Tregs was significantly enhanced both in UCB and PB.The ratio of Helios+FoxP3+ T cells to isolated CD4+CD25+ T cells increased gradually from day 7 to day 21,and reached a peak of almost 50%at day 21.3.2 The shRNA-Helios lentiviral vector infection rate was about 40%,and the expression of Helios protein in the shRNA-Helios lentiviral vector group was significantly decreased.3.3 The suppression ability of UCB Tregs was significantly enhanced at day 14.In addition,the cell yield and immunosuppressive ability of Tregs in UCB were not lower than PB Tregs after 14 days of culture.3.4 The results showed that compared with the normal Tregs,the supernatant from Helioshigh Tregs promoted angiogenesis.In contrast,blocking the expression of Helios in UCB-Tregs with shRNA-Helios reduced the angiogenic ability.Further,compared with control or blank groups,HUVECs incubated in supernatants from Helioshigh Tregs exhibited higher levels of CD31 protein.In contrast,when shRNA-Helios functioned,CD3 1 expression decreased.4 Role and mechanism of Helios+ Tregs on the pathogenesis of ALL model mice4.1 Mouse weights with ALL were lower after five weeks compared to that in the blank group;however,weights in the three ALL groups were not significantly different.Next,ALL mice exhibited a significantly increase in the liver,spleen,and kidney index compared with normal nude mice.However,among the three ALL groups,no differences were observed in the liver,spleen,and kidney indexes.One week after Treg infusion,neither human nor murine Tregs could be detected in the PB of all groups.90%(9/10)of the recipients developed extensive bone marrow lymphoblastic infiltration in the Helioshigh group.This compared with 83%(10/12)in the Helioslow group,64%(7/11)in the model group,and 0%in the blank group.Furthermore,we detected human CD19+ cells in BM by flow cytometry.Compared with model group and Helioslow group,the proportion of human CD19+ cells in the bone marrow was increased in the Helioshigh group in ALL mice.We also detected human CD19+ cells in PB,but no differences were found between the Helioshigh and Helioslow groups.4.2 In the bone marrow,levels of VEGFA,VEGFR2,and phosphorylated(p)-VEGFR2 were increased both in the mouse models infused with UCB-derived Tregs,and the expression of these three proteins increased more in the Helioshighgroup.Mice in the Helioshigh group had a higher MVD of the bone marrow.We then identified downstream genes regulated by Helios+ Tregs.Higher expression of Helios in Tregs significantly increased CCL22 mRNA expression in the bone marrow.Further,we tested the secretion levels of CCL22 protein in the plasma of mice.Higher expression of Helios in Tregs significantly increased CCL22 protein expression in the plasma.When shRNA-Helios blocked Helios expression in Tregs,the expression of CCL22 in the plasma decreased.Conclusion1.Helios in combination with FoxP3 is currently a better method to identify functional Treg numbers and higher risk in patients with pre-B ALL.2.Helios expression in Tregs participates in a variety of biological processes including immunosuppression,angiogenesis,and apoptosis of tumor cells in pediatric leukemia.Helios may serve as a target for Treg-dependent cancer immunotherapy and an indepth study on the therapeutic application of Helios+ Tregs still need to be elucidated3.In this study,we successfully constructed a culture system of UCB-induced Tregs in vitro.The UCB-Tregs had similar proliferative capacity,immunosuppressive ability and angiogenic ability with PB-Tregs.Helios also participated in the function of UCB-Tregs.