Role Of Leukemia Derived Microvesicles In The Process Of T Cell Exhaustion

PartⅠRole of T lymphocyte in the malignant transformation of hematopoietic stem/progenitor cells induced by leukemic MVsObjective:Microvesicle(MV)is a bilayer lipid vesicle produced by budding,carrying all kinds of proteins,lipids and RNA of maternal cells.It is considered as a"microfilm"maternal cell.Our previous studies found that K562-MV contained BCR-ABL mRNA and a large number of miRNA,which could induce hematopoietic stem/progenitor cells to transform into leukemic cells by continuously inducing donor peripheral blood mononuclear cells(PBMC).As we all know,mobilized donor peripheral blood contains not only a large number of hematopoietic stem cells,but also a wealth of T lymphocytes,but many T cells are unable to prevent malignant transformation of normal cells.Here,based on our previous studies,we clarified the role and changes of T lymphocyte cells in the process of PBMC malignant transformation induced by MVs.Methods:The human CML blast crisis cell line K562 were cultured in RPMI 1640 with10%FBS and 1%FBS at 37°C in 5%CO2.Supernatant of K562 were collected to isolate MV by gradient centrifugation.CD3 microbeads was used to isolated the CD3~+T cells from PBMC,the purity of isolated CD3 T cells were analyzed by flow cytomery.Isolated CD3~+T cells were added into another PBMC.Three groups PBMC,deleted-CD3 PBMC,added-CD3 PBMC were induced continuously by resuspended K562-MV for more than 20days.Asmallamountofcellswereharvestedonday5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 and stained with Giemsa to observe the cell morphology,determining the time points of malignant transformation.The PBMCs were adjusted to 4×10~6cells per well in six-well plate and the total number of cells per well were counted on 0,3,7 days of induction.The percentage of T lymphocyte subsets CD3~+CD4~+and CD3~+CD8~+T cells were examined by flow cytometry on day 0,3,7,14after induction.In addition,the expression of PD-1 on the surface of CD3~+CD4~+and CD3~+CD8~+T cells also were analyzed by flow cytometry and RT-PCR were used to examine the expression of PD-1 and Tim-3 mRNA on T cells isolated from induced PBMCs on day 0,3,7.Results:Isolated CD3~+T cells had more than 98%puriy.However,magnetic bead sorting couldn,t remove all CD3~+T cells in PBMC,but only reduce about CD3~+T cells.After removing part of T cells from PBMCs,the time point of malignant transformation was significantly shorted comparing to MV inducing normal PBMCs(13.67±0.667 vs9.167±0.872,P=0.0021);While Increasing the number of T cells in PBMC didn,t significantly prolong the time of the malignant transformation(13.67±0.667 vs14.5±0.764,P=0.43).The absolute numbers of total cells and the proportion of lymphocyte decreased continuously during induction with MV,while the proportion of CD3~+CD8~+and CD3~+CD4~+T cells achieved were highest on day 3 and then decreased gradually.The expression of PD-1 in CD3~+CD8~+and CD3~+CD4~+T cells were markedly increased after seven days of induction.Similarly,Tim-3 and LAG-3 mRNA significantly increased on days 3 and 7 of incubation with K562-MVs.Conclusion:In summary,reducing T cells could accelerate malignant transformation,but increasing the number of T cells does not delay malignant transformation,which suggested that preventing malignant change is not only the problem of the number of T cells,but also the changes in the function of T cells and significant changes have taken place in T cells during induction.In addition,the expression of inhibitory receptors PD-1,Tim-3 and LAG-3 increased during induction,which indicating the T cell dysfunction.Part Ⅱ Leukemia-derived MVs induce T cell exhaustion in vitro and vivoObjective: Adoptive T cellular therapy,especially chimeric antigen receptor T cell(CAR-T),is promising in the therapy of leukemia and other cancers.However,in the context of chronic antigen exposure in chronic viral infections and cancer,T cells could become exhausted/dysfunction.Here,we intend to prove that leukemia-derived MVs can induce T cell exhaustion in vitro and vivo,and reveal a novel mechanism of T cell exhaustion.Methods: MVs induce T cell exhaustion in Vitro: Leukemia cells K562,KG1 a,Jurkat derived MVs were isolated by gradient centrifugation.Following the principle of informed consent,primary CD3+T and CD8+T cells were isolated from peripheral blood mononuclear cells(PBMCs)by positive selection using CD3 and CD8 microbeads.T cells were examined on day 0,3,7 after induction: CFSE was used to analyzed the proliferation;the cells were labeled with Annexin V and propidium iodide to analyzed the apoptosis;The secretion of IL-2 in intracellular and the expression of CD69,PD-1,Tim-3 on surface of T cell were analyzed by flow cytometry;The BD? Cytometric Bead Array(CBA)Human Th1/Th2/Th17 Cytokine Kit was used to detect the levels of cytokines IL-2,IL-4,IL-6,IL-10,TNF,IFN-γ,and IL-17 A according to the manufacturer’s instructions;RT-PCR was used t detect the expression of PD-1,CTLA-4,LAG3,Tim-3,IL-2、TGF-β、IFN-γm RNA in T cells on day 0,3,7 after induction.Mouse model of T cell exhaustion: Balb/c mice were divided into 6 groups: PBS was injected into the tail vein,K562 cells were injected into the tail vein,K562-MV was injected intraperitoneally,and K562-MV,KG1a-MV and Jurkat-MV were injected into the tail vein,once a day for 20 days.Approximately 100μl peripheral blood was obtained from the internal canthus vein on days 3,5,7,10,and 20 post-injection to analyzed the expression of PD-1,Tim-3 on the surface of CD3+CD4+and CD3+CD8+T cells.Mice were sacrificed on day 7 or day 20 post-injection or 15 days after stopping injection.Spleens were analyzed using real-time PCR and immunohistchemistry.Results: The MVs exerted no significant effect on the proliferation of T cells on day 3 or on day 7 by CFSE assay;MVs induced apoptosis in T cells on day 7 but not on day 3;the marker of active T cells,CD69 and cytokine IL-2 increased significantly on day 3;The indicators of exhaustion PD-1,Tim-3,continuously increased from day 0 to 7.In addition,the expression of PD-1 m RNA was significantly higher on day 7 than day 3 after incubation with K562-MVs,Jurkat-MVs,while there was no significant effect after incubation without and with KG1a-MVs.Expression of CTLA-4,Tim-3,LAG-3 m RNA showed similar trend after induction.The level of cytokine IL-2,IL-4,IL-6,IL-10,TNF,IFN-γ,and IL-17 A decreased on day 7 compared to day 3.Furthermore,RT-PCR detected that the IFN-γ,IL-2,CD107 a were significantly higher on day 3 and inhibitory cytokine TGF-β was expressed at a lower level on day 3 than that on day 7.In mouse model,the proportion of PD-1-expressing on CD3+CD8+and CD3+CD4+T cells in the peripheral blood of mice increased by 3-to 8-fold after incubation with MVs on day7 and 20;After 20 days of injection in vivo,the proportion of PD-1-and Tim3-expressing on CD8+T cells was significantly higher in groups K562-MVs(both intravenous and intraperitoneal),Jurkat-MVs,and KG1a-MVs than that in the control group;the group injected with K562 cells was not significantly different from the control group due to intermittent injection;Spleens in experimental groups were positive for human CD45,which mainly marks interstitial cells;The expression of PD-1 in the spleen tissues was also higher than in the control group on day 7,as detected by RT-PCR;The rate of positivity for anti-human CD45 was significantly reduced,and the expression of PD-1 in the spleens was not significantly different between the experimental group and the control group on day 15 after stopping injection.Conclusion: We have demonstrated that MVs derived from leukemia cells can act as important inhibitory signals to induce T cell exhaustion and repress T cell functions.In mice model,MVs induced persistent immune response could cause spleen enlargement and elevated level of checkpoint inhibitors.Although cancer-derived MVs have been reported to be immunosuppressive,few studies have highlighted the connection between T cell exhaustion and MVs.These results provide novel insights into the mechanism underlying T cell exhaustion.Part Ⅲ Mechanism of leukemia-derived MVs inducing T cells exhaustionObjective: T cell function in cancer patients is usually impaired due to the constitutive activation of immune checkpoint inhibitors.This state is known as ‘exhaustion’ and is often associated with the inefficient control of tumors or persistent infections.These exhausted T cells upregulate multiple inhibitory receptors/immune checkpoints,such as CTLA-4 and PD-1,anti-CTLA-4 and/or anti-PD-1 monoclonal antibodies can successfully reinvigorate tumor-infiltrating T cells and provide clinical benefits to many patients with advanced cancer.However,T cell exhaustion cannot be completely resolved with PD-1inhibitors.Which suggest that there may be important mechanisms in T cells exhaustion that have not been found.Here,we further explore the specific mechanism of MV inducing T cells exhaustion.Methods: RT-PCR was used to detect the expression of BCR-ABL1 after K562-MVs co-incultured with T cells;Confocal microscopy was used to observe the fusion of MV and T cells after co-culture;To determine the gene expression change and the mechanism of T cells exhaustion,we performed RNA-seq for T cells of three key time points,0,3 and 7day,repeat three times at each time point;Based on bioinformatics analysis,we obtained the genes with significant difference during induction.According to the transcription factor spectrum and the prediction of mi RNA target,we constructed a pre feedback loop(FFL)that plays a key role in the induction;SERPINB2,CXCL5,IL-1B in T ells and mi R-182,mi R-16,mi R-126,mi R-21,mi R-92 a in K562-MV were selected according to the bioinformatics analysis.The five mi RNA mimics or inhibitors were co-transfected into T cells to detect the changes of mi RNAs,inhibitory receptors and target genes by RT-PCR;WB was used to examine the level P65 in cytoplasm and nucleus,which was the key protein of NF-κB pathway.Results: By co-culturing T cells with MVs,we microscopically observed the fusion of MVs(green)with T cells;Furthermore,the copies of BCR-ABL1 in T cells were significantly increased after co-culture with MVs,indicating that MVs likely transport BCR-ABL1 m RNA into T cells;According to our previous study,K562-MVs carry many mi RNAs.mi R-92a-3p,mi R-21-5p,mi R-16-5p,mi R-126 and mi R-182-5p,which are abundant in K562-MVs,are transported into T cells and can affect the function of these cells.The target genes of the five identified mi RNAs,such as SERPINB2,IL-1B and CXCL5,in CD8+T cells were down-regulated after co-transfection with five mi RNA mimics;Moreover,compared with that in the negative control(NC),the expression of PD-1,CTLA-4,Tim3 and LAG3 in CD8+T cells was significantly increased after the co-transfection of the five mi RNA mimics into these CD8+T cells;NF-κB was downregulation after co-transfection with five mi RNA mimics.We speculate MV might participate in T cell exhaustion and checkpoints via NF-κB pathway regulation.Conclusion: MVs could merge with and deliver their cargo into T cells in vitro.Our results indicated that functional mi RNAs in MVs,such as mi R-92a-3p,mi R-21-5p,mi R-16-5p,mi R-126 and mi R-182-5p,can be exogenously delivered into T cells to induce the exhaustion phenotype.These mi RNAs can thus be used as novel markers of T cell dysfunction and provide potential targets for new therapeutic approaches to reverse these dysfunctions.According to our bioinformatics analyses,the NF-κB pathway,which is a key regulator involved in T cell activation and exhaustion,was the most significantly regulated pathway during the process of exhaustion.Our results further indicated that Serpin B2,IL-1β and CXCL5 are downstream targets of the MV-associated mi RNAs and they are pivotal in NF-κB pathway.