| In Chinese, leukemia ranked the sixth in men, ranked eighth in the women, and in the first place in children as the most common cause of cancer-related death.Leukemia theraphies include radiotherapy and chemotherapy which can reduce tumor load to get remission, transplantation and immunotherapy. The CTL (Cytotoxic T-cell, CTL) cells were mainly used in the peripheral blood stem cell transplantion (PBSCT) and other adoptive immunotherapy in clinical practice currently. Transplantion can be divided into auto-graft and allograft according to the source of graft. Autologous peripheral blood stem cell transplantation has an anti-leukemic effect and without GVHD, but it induces a systemic auto-immune syndrome resembling graft-versus host disease (GVHD), and this syndrome termed autologous GVHD, which has significant anti-tumor activity. It can reduces the incidence of tumor relapse after autologous peripheral blood stem cell transplant (APBSCT). APBSCT has a higher quality of life after transplantation than Allo-HSCT, because of lacking real GVLE (graft-versus leukemia effect, GVLE), it is easy to relapse. If infusing an amount of leukemia antigen-specific CTLs from theirself after APBSCT, to some extent, reducing relapse and enhancing anti-leukemia effects without that autologous GVHD syndrome. On the basis of APBCST, giving patients some donor's specific CTLs, can also increase GVLE. In a way, these methods maybe change the effect of APBCST.Allogeneic stem cell transplant (Allo-SCT), due to GVLE, some patients can survive for long periods. However, accompanied by complications such as:graft-versus-host disease (Graft versus the host disease, GVHD), serious infections, implanted failure, hematopoietic reconstitution delay-related, and hindered the success of transplant. CTL clone cells are GVHD and GVL effective cell that prevent or limit infection during transplant. CTLs can promote engraftment and they are also essential for reconstitution of hematopoiesis.Donor lymphocyte infusion (Donor lymphocyte transfusions, DLT) can induce the GVL effect to eliminate leukemia relapse. CTL cells are the key effector cells. DLI also have a variety of transplant-related complications, and it is a problems faced by clinical workers how to get the best effect in DLI.ALL lacks cell surface lymphocyte function antigen (LFA-1), poorly response to the treatment of NK cells. CTL cell seems to be the only effector for ALL tumor cells. CTL cells have demonstrated their importance in immunotherapy and technologies routes.Clinical found that implantation of the success of GVHD severity and transplantation for receptor T cells increased significantly relevant. The severity of GVHD is most closely correlates with the proportion of donor and recipient T cells. Complete donor chimerism or mixed donor/recipient chimerism did not completely remove the effector cells and memory cells of the recipients. Killed their own tumor cells by the CTL clone what it called RVLE (the Recepient-the versus leukemia effect, RVTE). It is important to find out the best balance between exclusion and RVLE. Recipient's CTL clones are not only can identify tumor cell and executive immune clearance, but also can help the donor immune cells to carry out the function of tumor-killing, while helping us to understand instead of immunization and adoptive immunity concept, to analyse some clinical graft failure cases which patients get long-term survival without disease recurrence, and some patients relapsed in complete donor chimerim.Wilms' tumor Antigen-1(WT1) is over-expressed in70%-900of ALL patients, and such patients have a high relapse rates. Confirmed in vitro and mouse studies, WT1is a significant and widespread expression of leukemia antigen target. Myeloid leukemia and healthy donors have WT1-specific T cells, and indicating that WT1expression is easy to induce T-cell response. WT1-specific CD8+T cells closely related to and leukemia tumor burden. The study found that highly purified CD8+T cells in mixed bone marrow transplant will have a high GVL response and low lethal GVHD. If we could cultivated CTL clone cells with a strong cytotoxicity and leukemia cell clearance capacity that only selectively killed leukemia, but didn't kill normal cells, and they have no GVHD or rejection, and the host is completely tolerated them, and could be efficiently amplificated in vitro. Thus, the specific CTLs could provide a possibly clinically applicable new strategy for the separation of GVHD and GVLE. The main objective of this experiment:to be isolated and culture CD8+/WT1-specific CTL cell clone and identified features and their sources.Method1. In vitro culture and identification of DCsDendritic cells (dendritic cell. DC), their main function is to process antigen material and present it on the surface to CTLs. The traditional culture method of DC generally require about4-10days, Induced proliferative response will usually last from3-9days, improved the maturity of DC for last day. DC can't follow the CTL culture in vitro for the time required DC cultural time too long. We changed the time of DC culture for2days. Compared with the traditional culture method and7days of culture method:(1) DC phenotypic analysis by four-color flow cytometry.(2) Morphological changes of DC were observed under inverted microscope and Wright-guitar dyeing line.(3) Antigen-presenting function of DC were evaluated by cytotoxic of HLA-A*0201Tc cells.2. A single clone culture of CTL derived from the healthy donors(1) Magnetic cell sorting of CD56-CD8+cells:HLA-A*0201healthy donor peripheral blood mononuclear cells (PBMNC). CD56-cells were negatively selected from PBMCs (After stimulation culture in vitro) by magnetic sorting, and CD8+cells were positively selected from CD56-cells.(2) Flow cytometric analysis and sorting:the CD19-cell populations of WT1/HLA-A*0201/Pentamer and CD8double positive cells were special CTL cells and collected them.(3) Preparation of feeder cells:three copies of non-HLA-A*0201-positive healthy donor peripheral blood mononuclear cells irradiated with gamma ray at50Gy, then be prepared feeder cells.(4) Preparation of CTL cell clones: separation of cloning by the limited dilution method. Antigen loading of DC with WT1(irradiated with gamma ray at30Gy) and mixed feeder cells.(5) Phenotypes of CTL clones identified by flow detection. The expression rate of CD45RA/CD45RO, CCR7,-CD3and CD8, CD28, CD27, CD107were detected.(6) Toxic particles (Granzyme B and Perforin) of CTL clone cells were detected by flow cytometry.(7) Cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17of the culture supernatant from CTL clone observed by BDTM Cytometric Bead Array (CBA) Assay.(8) Cytotoxicity detection:To identify CTL cytotoxic function by killing the standard cell strains and the original leukemia cells.3. CTL clone culture of the patients after NACST(1) In vitro, specific stimulate amplification of Tc cells from PBMNC of leukemia patients after NASCT.(2) Flow cytometric sorting:Gating CD3+CD19-cell population WT1/HLA-A*0201/Pentamer and CD8double-positive cell (CTL cells) were collected.(3) Three copies of non-HLA-A*0201-positive healthy donor peripheral blood mononuclear cells irradiated by gamma-ray (50Gy), and be prepared feeder cells.(4) CTL clones were prepared by the limited dilution method and mixed with antigen loaded DC and feeder cells, and then cultured in vitro.(5) Expression rates of phenotype molecular CD45RA/CD45RO, CCR7and CD3and CD8, CD28, CD27, CD107a were detected by flow cytometry technique.(6) The CTL cloning cytoplasmic toxicity particles Granzyme B, Perforin-positive were detected by FCM.(7) Cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, IL-17) were detected by CBA.(8) Cytotoxic function of CTL clone cells were evaluated by killing standard tumor cells and leukemia original tumor.4. Comparative study of two different types clones in healthy donors and leukemia petients.The phenotype, toxic particles, functions of secretion and cytotoxiciity in two different types CTL clones were detected by FCM.5. Studied the origin and function of CTL clone cells by microscopics(1) Sex chromosome of CTL clone were analysed by fluorescence in situ hybridization (FISH):selected5cases after NASCT (donor-recipient gender difference), and cultured CTL clone cells in vitro got in situ hybridization with X and Y chromosome probes to distinguish between donor and recipient on CTL clone cells after opposite sex transplantation, according to the different signals of the X, Y chromosome.(2) Confocal laser scanning microscope to observe the morphology and function of CTL clones:select HLA-A*0201-positive recipients and HLA-A*0201-negative donors patients. WT1-specific CTL clone cells were cultured in vitro. WT1/HLA-A*0201/Pentamer-PE marked CTL cells, PKH26marked dendritic cells, CFSE labeled target cells of NB4(WT1and HLA-A*0201double-positive human acute promyelocytic leukemia tumor cells) were mixed and incubated2h together, and then observed under LSCM.(C) The fine structure and function of CTL clones can be cearly observed by scanning electron microscope (SEM):WT1-positive CTL clone cells were cultured in vitro for4-6weeks and mixed with DC and NB4cells and mix-cells cultured for2h, then collected and treated clone cells. observed clone cells by SEM.6. Protein expressions of CTL clones between healthy donors and patients.Selected three healthy donors and leukemia patients after NASCT, and CTL clone cells cultured for six weeks in vitro. The cells were lysed. Their proteins were exrrated, and analyzed protein concentration.2D gels separating total protein, stained, sweeped gel and image analysis. Establishment of protein difference spectrum detected the differentially expressed proteins. A combination of enzymatic digestion and peptide analysis by mass spectrometry is used to identify differentially expressed proteins. Peripheral blood lymphocytes derived from healthy donors and leukemia patietents after NASCT be used as the basis for protein difference spectrum of control, and biological information retrieval be used to identify reactive proteins.Result1. Dendritic cell culture in vitro(1) The morphologies of immature and mature DC (cultured in three ways) have been not found out especially different properties by optical microscopy.(2) Phenotypic differences of immature DC:Expressions of of CDla and CD86(P<0.01), CD83and HLA-DR (P<0.05) in traditional culture were significantly higher than the expression of2days culture. Expression of CD1a (P<0.01) was significantly higher than the expression of7days culture; Expressions of CD83and of HLA-DR (P<0.05) in7days culture were significantly higher than the expression of2days culture; Expression of CD83(P<0.05) was significantly higher than the expression of traditional method; Phenotype of mature DCs were found any differences between the cultures of2days and7days; Expression of CDla, CD11c, CD80, and HLA-DR in the method of2days was significantly higher than the traditional culture method (P<0.05); Eexpressions of CD80and CD83in7days method were significantly higher than the traditional culture method (P<0.05).(3)3Antigen presentation activity of mature DC:Death rate of target cells due to CD8+Tc cell-mediated killing in2days (75.8±7.1)%and7days culture groups (74.6±3.2)%were significantly higher than the killing rate of traditional method (65.2±1.3)%(P<0.01).2. Culture and Identification of CTL clone cells from healthy donors(1) Phenotype:CD3+CD8+group accounted for (98.7±0.2); CD27+cells accounted for (62.3±10.0)%; CD28+cells accounted for (71.9±7.9)%; CD107a+cells accounted for (49.0±11.0)%; CD45RA+cells accounted for (65.1±8.4), CD45RO+cells accounted for (34.3±4.2)%; CCR7+cells accounted for (18.1±5.2)%.(2) Expression rate of toxic particles:granzyme B:(43.9±7.4)%, Perforin:(32.7±6.8)%.(3) Proportion of subsets: CD45RA+CD27+CCR7+cells accounted for (9.0±2.0)%; of CD45RA-of CD45RO+of CD27+of CCR7+accounted for (13.0±2.4)%; CD45RA-CD45RO+CD27+CCR7-cells accounted for (26.1±5.9)%; CD45RA+CD27+/-CD28+/-cells accounted for (61.3±7.9)%.(4) Pentamer detection:WT1/HLA-A*0201pentamer+and CD8+cell accounted for (76±3.9)%. Secretory function of CTL clone (pg/ml):IL-2:(2129.5±414.7)%, IL-4:(17.5±9.1)%, IL-6:(21.6±9.7), IL-10:(24.8±8.6) and TNF-a:(40.8±21.5), IFN-γ:(102.3±61.0), IL-17:(18.5±8.6).(5) Cytotoxic function:Death rate of NB4(89.7±7.6)%was significantly higher than the death rate of U937and K562(19.0±4.3)%(19.7±5.2)%(P<0.05). The death rates of original leukemia cells AML and ALL:(54.0±16.6)%,(56.5±12.4)%, and there were no difference between them, P>0.05. 3. Culture and identification of the CTL clone cells in patients with leukemia after NASCT(1) Phenotype:CD3+CD8+cells accounted for (98.6±0.5)%; CD27+cells accounted for (66.3±8.1)%; CD28+cells accounted for (70.1±7.8)%; CD107a+cells accounted for (51.4±7.7)%; CD45RA+cells accounted for (65.7±6.3)%; CD45+cells accounted for (33.9.1±4.5)%; CCR7+cells accounted for (17.1±2.7)%.(2) Expression rates of toxicity of particles in the intracellular:granzyme B:(40.6±9)%, perforin:(30.9±6.2)%.(3) The proportion of subsets group:CD45RA+CD27+CCR7+cells accounted for (12.6±3.2)%; CD45RA-CD45RO+CD27+CCR7+cells accounted for (17.5±4.6)%; CD45RA-CD45RO+CD27+CCR7-cells accounted for (19.5±3.6)%; CD45RA+CD27+/-CD28+/-cells accounted for (62.1±7.4)%.(4) Detection of pentamer:WT1/HLA-A*0201pentamer+CD8+cells:(70.2±8.5)%. Secretory function of CTL clone cells (pg/ml):IL-2:(2365.0±246.1), IL-4:(40.5±8.3), IL-6:(1705.3.6±416.2),1L-10:(63.2±22.2), TNF-a:(121.3±35.3), IFN-y:(219.5.3±65.1),1L-17:(14.5±5.0).(5) Cytotoxic function: Death rate of NB4(84.5±5.1)%was significantly higher than the death rates of U937(21.5±5.5)%and K562(22.3±6.5)%,(P<0.05). The death rates of original leukemia cells of AML (66.4±7.6)%was significantly higher than that of ALL (58.6±5.6)%(P <0.05).4. Comparative study of CTL clone between patients and healthy donorsThere were not any differences in expressions of phenotypic and toxic particles between healthy donors and patients. Concentrations of cytokines CTL clones from patients such as IL-4, IL-6, IL-10, TNF-alpha and INF-y were significantly higher than healthy donors's (P<0.01). Function of cytotoxicity did not find any significant differences between them.5. Source and function of CTL clones from patientsWe found CTL clone cells of recipients after NASCT by FISH; We found the CTL cells of recipients after NASCT and directly observed their cytotoxicity by LMST. The CTL clone cells cultured in vitro showed a typical T-cell morphology, filaments contact with the DC and activity of killed tumors observed in cloned cells by SEM.6. Expression differences of protein between two types of CTL clone cellsPeripheral blood lymphocytes between leukemia patients after NASCT and healthy donors were found30different proteins and identified13specific functional proteins. There were44different proteins in CTL clone cells between the leukemia patients (after NASCT) and healthy donor.25functional proteins were identified. Significantly increased proteins included in18. Decreased proteins included in12. Four proteins have not been named. There are four co-reactive proteins between leukemia and transplantion, which are coactosin-like protein, Septin-11, GATA3and purine nucleoside phosphorylase.Conclusion (1) Method of2days culture DC is successful, and morphology and function of mature DC were standard and effective.(2) Culture methods of CTL clone from healthy donors and leukemia patients after NASCT was successful. Effective amplification can increased1-5×107cells. The amplificated rate is about78%. A series of identification in cultured clone cells were identified as funtional CTLs.(3) Subsets of clone cells included in memory cells which be proliferated by one cell and formed different stages of cell subsets.(4) Secretory function of CTL clone cells from leukemia patients after NASCT was stronger than healthy donors's.(5) Immune beads and the pentamer combined with flow sorting can obtained high purity CD8+CTL cells.(6) HLA restriction of peptide-specific pentamer and FISH complemented each other, and can identified source of donor/recipient's CTL clone cells.(7) There were significant differences in protein expression of PBMNC and CTL clone cells between the healthy donors and patients.
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