The Anti-leukemia Activity Of CTL Induced By Exosomes Derived From Leukemia Cells

Leukemia is the most common malignant diseases of hematological system. The incidence of leukemia has an increasing trend these years. In lately 1900s, the gradually consummate inducing chemotherapy and the supportive treatment have made the most of the patients to get complete remission (CR). Now the key problem is that some patients can't get complete remission after the inducing chemotherapy because of their original drug-resistance and the patients who have gotten CR might relapse because of the secondary drug-resistance of the remaining leukemia cells. This is the most difficult problem in the treatment of leukemia and so the other tumors. So the biotherapy and the tumor vaccine to exert the immune function of the patients themselves to eliminate the residual leukemia/tumor cells have became one of the focuses of tumor treatment and studies.Studies have showed that tumor cells, solute antigen of tumor cells, tumor proteins or peptides and DNA of tumor cells can produce anti-tumor responses as tumor vaccine. However the immune responses induced by these vaccines are often very weak, they can't solve the problem really. The effect of dendritic cells (DCs) activated by tumor antigen as tumor vaccine has been validated, but some problems such as the origin of DCs and the maintenance of its function haven't been solved well.Zitvegel and Raposo group found that exosomes derived from DCs have the same effect of inducing anti-tumor as DCs. Moreover, exosomes have some characteristics such as steady to heat, can be refrigerated for a long time and can be quantitative analysis etc. So it has more potential as a tumor vaccine. Exosomes are vesicles of 30-100nm in diameter. It can be secreted by many kinds of cells. Exosomes derived from DCs accumulate MHC-ⅠandⅡmolecules,costimulatory molecules and other molecules for antigen present. Exosomes derived from DCs exposed to tumor-derived antigenic peptides induce potent immune responses. The responses mediated by cytotoxic T lymphocyte and induced potent T cell-dependent anti-tumor effects in tumor bearing hosts. Exosomes derived from tumor cells accumulate MHC-ⅠandⅡmolecules,costimulatory molecules and other molecules for antigen present, while it take along tumor antigen. It's another selection of tumor antigen presentation.At present, the study about exosomes in. our country is at the beginning stage and has no report on leukemia. And it has been reported that acute myeloid leukemia cells and chronic myelocytic leukemia cells can be induced to DCs, which not only have the characteristics of leukemia cells but also have the features and functions of DCs. So it might be more effect and convenient to get exosomes from these cells. In this article we reported the anti-leukemia effects of CTL induced by exosomes derived from leukemia cells in different ways, which might be a basic work to choice the suitable exosomes for clinical uses.Materials and methods:The first part1. Induction and identification of DCs derived from K562 cells (K562-DCs)There are some reports concerning that K562 cells were cultured with multiple cytokines (rhG-CSF, rhIL-4, rhTNF) to generate DCs in vitro. We induced K562 cells to differentiate to DCs using cytokines rhG-CSF, rhIL-4 and rhTNF. After cultured for 14 days, the morphology of these cells was observed under invert microscopy and electric microscopy. The phenotype of the cells was analyzed by flow ceremony with anti-MHC classⅠ, MHC classⅡ, CD40, CD80, CD86 and CD54 mAb. 2. Isolation and identification of exosomes derived from K562 cells and K562-DCsExosomes were isolated from the supernatant of K562 cells and K562-DCs. It were purified by a series of centrifugations at 300g, 1200g, and 10 000g to eliminate cells and debris, followed by centrifugation at 100 000g to get the sediment, that is the exosomes we want. The amount of exosomes proteins was measured by spectropolarimeter. Total proteins of exosomes were analyzed. The presence of differential molecules was analyzed by western blot. The morphology was observed by electron microscope.3. The killing effect of CTL induced by exosomes derived from K562 cells to the K562 cells and HL60 cells.The DCs induced from PBMC of normal volunteers stimulated with Kexo was experimental group and the DCs un-stimulated with Kexosomes was control group. The killing activity of CTL to K562 cell and HL60 cells induced by the DCs were detected by MTT assay.4. The killing effect of CTL induced by different exosomes derived from K562 cellsThe different exosomes, which derived from K562 cells,hot shock K562 cells,K562 cells freeze-thawing antigen and RNA antigen stimulated DCs induced from K562 cells, was used to induce special CTL. The activity of the CTL to kill the K562 cells was detected by MTT assay.5. The killing effect of CTL to K562 cells induced by different exosomes or/and CpG.The normal volunteers' PBMC was cultured for 5 days and divided to 7 groups. Three of them were cultured with 20μg/ml of Kexo,DCexo,Rexo and 15ng/ml of CpG as experiment groups (Kexo+CpG,DCexo+CpG and Rexo+CpG). The other three of them were cultured with 20μg/ml of Kexo,DCexo and Rexo only as experiment groups also. The last one was cultured with nither exo or CpG as control group. These groups of DCs were cultured for another three days, and then were cultured with T lymphocytes of normal volunteers to induce CTL. The killing activity of the CTL to K562 cells was assayed by MTT assay. The second part1 .The expression of chemokine-like factor1 (CKLF 1) and CKLF superfamily fatter 8(CKLFSF8) in K562-DCs and K562 cells.The expression of CKLF1 was assayed by using CKLF1 mAb and immunocytochemistry. The expression of CKLFSF8 was assayed by RT-PCR.2.The proliferation and exosomes production of K562-DCs and K562 cells wasassayed after transfected by CKLFSF8 gene.After the K562-DCs and K562 cells were transfected by CKLFSF8, the cellswere observed under invert microscopy and the proliferation was assayed by MTTassay and the expression of EGFR was detected by immunocytochemical method andthe exosomes was obtained and then was observed and monitored.The third part1. The activity of CTL induced by exosomes derived from CML patients' leukemia cellsThe activity of CTL induced from CML patients' T lymphocytes by exosomes derived from CML patients' leukemia cells (Pexo) and exosomes derived from K562 cells by MTT assay.2. The activity of CTL to kill the patients' leukemia cells induced by different exosomes derived from CML patients' leukemia cells.The activity of CTL to kill the patients' leukemia cells induced by Pexo,PDCexo and PFexo and/or CPG by MTT assay.3. The CTL of patients' and their HLA coincide sibling' induced by isogenesis DCs actived by PFexo. The killing activity of those CTL to patients' leukemia cells were assayed by MTT assay.RESULTSThe first part: The killing effect of CTL induced by exosomes derived from K562 cells in vitro.1. DCs can be induced from K562 cells Afer cultured and induced by rhGM-CSF,rhIL-4 and rhTNF-αfor 12-14 days, the cells displayed typical morphology feature of DCs under invert microscope as well as electronic microscope.K562-DCs had large nucleus and many typical dendrites on cell surfaces.This state of K562-DCs expressed high-level surface phenotypes,including MHC classⅠ,MHC classⅡ,co-stimulating molecules CD86 and CD80.adhesion molecule CD54.2.The identification of exosomes derived from K562 cells and DC induced from K562 cells.The analysis of the proteins with SDS-PAGE,the structure and morphology observed by transmission electron microscope,the phenotypes expression analyzed by western blot of exosomes derived from K562 cells and DC induced from K562 cells (K562-DCs),which were all consistent with the reports in other articles.3.The CTL induced by Kexo has a special killing effect to K562 cells and cross-killing effect to HL60 cells.4.The killing effect to K562 cells of CTL actiVated by different exosomes is stronger than Kexo.The killing effect of CTL activated by exosomes of DCs derived from K562 cells(KDCexo),hot shork K562 cells(Hexo),DCs derived from K562 cells activated by total RNA(Rexo) or freeze thawing antigen of K562 cells(Rexo) is stronger than CTL activated by exosomes derived from K562 cells.Of which,The killing effect of CTL activated by exosomes derived from DCs derived from K562 cells is fhe lowest,and the exosomes derived from DCs derived from K562 cells activated by total RNA of K562 cells is the highest.But there is no statistic difference.The P value is less 0.05. These exosomes have the similar antigenic.5.CpG ODN adjuvant can reinforoe the killing effect induced by exosomesExosomes can induce killing effect to K562 cells.After the CpG ODN was joined,the killing activity was reinforced.Indicated that CpG ODN can act as adjuvant to reinforced the exosomes' killing activity. The second part: Effect of CKLFSF8 gene transfaction on K562 cells and DCs induced from K562 cells and their exosomes secret.To investigate whether exosomes of K562 cells and K562-DCs were affected after transfacted with CKLFSF8 gene, we studied the expression of human novel cytokines CKLF1 and CKLFSF8 on K562 cells and K562-DCs with CKLF1 Mab and immunocytochemistry and RT-PCR assays. We discovered that CKLF1 and CKLFSF8 expressed on both K562 cells and K562-DCs. And the CKLF1 and CKLFSF8 expression on the K562-DCs had no change after antigens pulsed. Then K562 cells and K562-DCs were transfacted with CKLFSF8 gene. But we could't got content result after K562 cells and K562-DCs were transfeeted with CKLFSF8 gene. The transfect rate was very low when we used many kinds of transfect reagent. Even after we used JetPEI-Man which has special binding receptor to DCs, we could only get very low transfect rate yet. The transfect of CKLFSF8 gene can inhibit the proliferation and the growth of K562 cells and K562-DCs and the expression of the EGFR on the surface of these calls get lower. The phenomenon was also found in Ecal09 cells and HL60 cells. But the exosomes haven't been found significant different before and after transfected.The third part the killing activity of CTL to CML patients' leukemia cells induced by exosomes derived from CML patients' leukemia cells Our results showed that the CML patients' leukemia cells can secret exosomes. The killing activity of the CTL induced by the exosomes derived from the patients' leukemia cells(Pexo) is stronger than the Kexo. And the exosomes, which derived from DCs induced from CML patients' leukemia cells and DCs induced from CML patients' leukemia calls stimulated with patients' leukemia cells freeze thawing antigen, induced stronger effect than Pexo. CpG ODN can promote the killing effects. The killing activity of CTL induced from the patients' HLA coincide sibling' T lymphocytes activated by PFexo is stronger than Pexo.Conclusion:1. K562 cells and CML patients' leukemia cells can be induced to DCs and secret exosomes.2. The exosomes derived from leukemia cells and DCs induced from leukemia cells take along MHC and co-stimulating molecules which is necessary for antigen presention and have the capacities of stimulating T cells to antigen specific CTLs and special killing effect and cross killing effect. And CpG ODN can promote the effects.3. CKLFSF8 can express on K562 cells and K562-DCs. Exosomes can be get from K562 cells before and after transfacted with CKLFSF8 and have the same effects. CKLFSF8 inhibit the proliferation and growth of leukemia cells. The EGFR was down-regulated and inhibit the proliferation of K562 cells after transtacted with CKLFSF8.4. Exosomes is hopeful for the remedy of leukemia.