| Objective: To uncover the potential functions and mechanisms of tumor-derived small extracellular vesicles(s EVs)to the immune escape of NPM1-mutated AML.Methods:(1)The bone marrow aspirates of NPM1-mutated AML patients,NPM1-wildtype AML patients,and benign hematologic disease individuals were collected.IHC was used to quantify T cells in the bone marrow aspirates.The CD8+ T cells were extracted from the bone marrow aspirates of NPM1-mutated AML patients,NPM1-wildtype AML patients,and benign hematologic disease individuals.The proliferation of CD8+ T cells was determined by CFSE assay.The IL-2 and IFN-γ secretion of CD8+ T cells were measured by ELISA.The expression of CD25 and Granzyme B of CD8+ T cells were detected by FCM.The serum derived from NPM1-mutated AML patients,NPM1-wildtype AML patients,or benign hematologic disease individuals were co-cultured with CD8+ T cells which extracted from volunteers.The proliferation of CD8+ T cells was determined by CFSE assay.The expression of IL-2,IFN-γ,CD25,and Granzyme B of CD8+ T cells were detected by FCM.The conditioned medium from the NPM1-mutated cell line(OCI/AML3-CM)was co-cultured with CD8+ T cells.The proliferation of CD8+ T cells was determined by CFSE assay.The IL-2 and IFN-γ secretion of CD8+ T cells were measured by ELISA.The expression of CD25 and Granzyme B of CD8+ T cells were detected by FCM.(2)s EVs were removed from OCI/AML3-CM via pharmacologically blocking the s EV secretion of OCI/AML3 using GW4869 or physically removing s EVs by ultracentrifugation.The proliferation of CD8+ T cells was determined by CFSE assay.The IL-2 and IFN-γ secretion of CD8+ T cells were measured by ELISA.The expression of CD25 and Granzyme B of CD8+ T cells were detected by FCM.s EVs were purified from OCI/AML3-CM through ultracentrifugation and confirmed their identity by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and western blot.OCI/AML3-s EVs were co-cultured with CD8+ T cells.The proliferation of CD8+ T cells was determined by CFSE assay.The IL-2 and IFN-γsecretion of CD8+ T cells were measured by ELISA.The expression of CD25 and Granzyme B of CD8+ T cells were detected by FCM.(3)OCI/AML3-s EVs were co-cultured with CD8+ T cells,and the production of total creatine and ATP in CD8+ T cells were detected via indicated kits.The m RNA and protein levels of the creatine metabolize-related enzymes were also assessed via q RT-PCR and western blot.Knockdown of SLC6A8 expression of CD8+ T cells by si RNA and verified via western blot.The creatine metabolism was detected by indicated kits.The proliferation of CD8+ T cells was determined by CFSE assay.The IL-2 and IFN-γsecretion of CD8+ T cells were measured by ELISA.The expression of CD25 and Granzyme B of CD8+ T cells were detected by FCM.Transfection of SLC6A8 into the CD8+ T cells treated with OCI/AML3-s EVs and detected the creatine metabolism and immune function of CD8+ T cells.(4)The s EV-related mi RNAs which participate in the regulation of leukemic cells on CD8+ T cells were explored by bioinformatics analysis,q RT-PCR and IF.The directed regulation of mi RNA on SLC6A8 was determined by luciferase reporter assay.CD8+ T cells were treated with mi RNA mimic and sh RNA,and the expression of SLC6A8,creatine metabolism,and immune function were detected via western blot,indicated kits,and FCM,respectively.q RT-PCR was used to detect the expression of serum s EV-related mi RNA in patients,and assess the correlation of s EV-related mi RNA with the immune function of CD8+T cells.(5)q RT-PCR was used to detect OCI/AML3-derived s EV-related mi RNA after treated with sh NPM1.The University of California Santa Cruz(UCSC)database was used to explore the host gene of mi RNA.The luciferase reporter assay and Ch IP were used to check the regulation of NPM1 c to s EVs-related mi RNA.(6)FCM,Wright’s staining,IF,and western blot were used to analyze the immune escape of NPM1-mutated leukemia in hu HSC-NOG mice treated with OCI/AML3,OCI/AML3 NC,OCI/AML3 mi RNA KD,OCI/AML3 mi RNA KD + s EVs.Results:(1)There was fewer CD8+ T cell and abnormal function as decreased IL-2 and IFN-(?) secretion in NPM1-mutated AML compared with NPM1-wildtype AML and benign hematologic disease individuals.The serum derived from NPM1-mutated AML patients significantly reduced CD8+ T cell function,including proliferation,IL-2 and IFN-γ production,CD25 expression,and Granzyme B production,in comparison with the serum from NPM1-wildtype AML and benign hematologic disease individuals in the co-culture system.Moreover,a similar phenomenon was observed that the conditioned medium from the NPM1-mutated cell line(OCI/AML3-CM)inhibited CD8+ T cell proliferation,IL-2 and IFN-γsecretion,CD25 expression and granzyme B production.(2)Blocking the s EV secretion pharmacologically of OCI/AML3 using GW4869 markedly rescued CD8+ T cell proliferation,IL-2 and IFN-γ secretion,CD25 expression,and granzyme B production.Removing s EVs physically from OCI/AML3-CM by ultracentrifugation also partly restored CD8+ T cell function.s EVs derived from OCI/AML3 cells were approximately 70 nm in diameter with a double layer membrane structure,and the s EVs expressed s EV-specific protein markers TSG101,HSP70,CD9 and CD63,but not calnexin,an endoplasmic reticulum protein.Furthermore,we incubated CD8+ T cells with OCI/AML3-derived s EVs(OCI/AML3-s EVs),and OCI/AML3-s EVs obviously inhibited CD8+ T cell proliferation,IL-2 and IFN-γ secretion,CD25 expression and granzyme B production.(3)OCI/AML3-s EVs decreased total creatine and ATP in CD8+ T cells,and enhanced extracellular creatine levels.Meanwhile,the m RNA and protein levels of the creatine metabolize-related enzymes were also assessed.The expression of SLC6A8,the specific transporter for creatine,was sharply decreased in CD8+ T cells treated with OCI/AML3-s EVs.Knockdown of SLC6A8 expression by si RNA significantly decreased creatine and ATP production,and inhibited CD8+ T cell function.Transfection of SLC6A8 into the CD8+ T cells treated with OCI/AML3-s EVs increased cellular creatine level,ATP production and CD8+ T cell function.(4)mi R-19a-3p,mi R-19b-3p and mi R-29b-3p might regulate SLC6A8 in NPM1-mutated AML by comparing the changed mi RNAs in NPM1-mutated AML patients and the potential upstream mi RNAs targeting SLC6A8 via three mi RNA prediction algorithms(mi Randa,mi RDB,and Target Scan).Further analysis confirmed that mi R-19a-3p was most abundant in OCI/AML3-s EVs and increased most prominently in CD8+ T cells cultured with OCI/AML3-s EVs over a 24 h.A luciferase reporter assay revealed that mi R-19a-3p overexpression significantly inhibited the transcript activity of SLC6A8.The mi R-19a-3p mimic reduced endogenous SLC6A8 expression,creatine production and ATP levels in CD8+ T cells.Furthermore,sh RNA-mediated knockdown of mi R-19a-3p in OCI/AML3 cells markedly decreased mi R-19a-3p levels in s EVs.Treatment of CD8+ T cells with s EVs derived from mi R-19a-3p knockdown OCI/AML3 cells(OCI/AML3-s EVs mi R-19a-3p KD)significantly increased SLC6A8 expression,and enhanced CD8+ T cell function compared with OCI/AML3-s EVs NC treated group.Additionally,in patients,serum s EV-related mi R-19a-3p was significantly increased in NPM1-mutated AML patients compared with NPM1-wildtype AML patients and benign hematologic disease individuals,and was negatively correlated with SLC6A8 expression and the immune function of CD8+ T cells.(5)mi R-19a-3p was higher in both OCI/AML3(harboring NPM1 mutation)and OCI/AML3-releted s EVs than in other AML cell lines(harboring no NPM1 mutation),such as NB4,THP1,KG-1a,and OCI/AML2 cells.Efficiently inhibiting NPM1 c by a lentivirus-mediated sh NPM1 strategy significantly attenuated mi R-19a-3p levels in OCI/AML3 cells and OCI/AML3-s EVs.CTCF could bind to the MIR17HG(the host gene of mi R-19a-3p)promoter at CCCGA(E1)detected by Ch IP assay,and CTCF inhibited MIR17 HG transcription determined by dual-luciferase assay.Suppression of NPM1 c increased the binding ability of CTCF to the MIR17 HG promoter.The binding between PABPC1 and mi R-19a-3p in the OCI/AML3 cells was verified by mi RNA pull down assay and RIP assay.As suppression of NPM1 c decreased not only PABPC1 expression but also the binding of mi R-19a-3p and PABPC1 in OCI/AML3 cells.Suppression of NPM1 c decreased the enrichment of H3K27 ac in the promoter of the PABPC1 gene in OCI/AML3 cells.(6)In xenograft model in hu HSC-NOG mice,s EV-related mi R-19a-3p led to more leukemic cells in peripheral blood and bone marrow,more liver and splenic infiltration,shorter survival times,a lower level of CD8+ T cell contents,also a decreased SLC6A8 expression and creatin production.Conclusions:(1)NPM1-mutated AML cells impair CD8+ T cell function.(2)Leukemic cells impair the immune function of CD8+ T cells via s EV release.(3)Leukemic cell-derived s EVs downregulate SLC6A8-mediated creatine metabolism to impair CD8+ T cell immune function.(4)Leukemic cell-derived s EVs transfer of mi R-19a-3p to inhibit SLC6A8 in CD8+ T cells.(5)NPM1c-mediated mi R-19a-3p expression and packaging into s EVs.(6)s EV-related mi R-19a-3p impairs CD8+ T cell antitumor function in vivo. |
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