Programmed Expansion And Functional Identification Of NK Cells

Natural killer(NK)cells are important innate immune cells which play vital roles in combating aginst infections and killing tumor cells.During hematopoietic stem cells transplantation(HSCT),NK cells can kill leukemia cells without causing graft versus host diseases(GVHD).Thus,NK cells are promising candidate for adoptive transfer immunotherapy.To acquire quantities of functional NK cells and get better therapeutic effect are the important points about NK cell therapy.In the present research,we got some achievements to solve the issues.1.We apply cord blood CD34+ stem cells as inicial cells to differentiate into NK cells with different cytokines.After five weeks,NK cells can reach at least ninety percent.And the cells can expand about 7000 folds during the culture period.The hierarchical clustering algorithm of differentially expressed transcripts suggested that the cells were closer in first three weeks than in the following two weeks.Depending on the results from the microarray analysis,we define that NK cells undergo a programmed differentiation pattern which can be divided into two stages as program 1 and program 2 during their in vitro differentiation.2.By detecting the known transcription factors(TFs)that regulate NK cell functions,we found that the cells in program 2 have similar transcript profile with primary NK cells.The results implied that the cultured cells can be applied as a model to study NK development and differentiation.To analyze the differential expressed transcription factors in the two stages,we found that the cells of program 1 enriched the TFs of zinc coordinating group,whereas the cells in program 2 enriched the helix-turn-helix group and basic domain group TFs.And the enrichment of different TF groups suggested that program 1 prepares NK cell progenitors,while program 2 regulates NK cell transformation and maturation.3.Analyzing the expression pattern of membrane molecules,chemokine receptors and cytokine receptors during dinstinct programs,we found that the differentiated NK cells expressed higher levels of function-related receptors.The results show that NK cells have the potency to be activated and kill the tumor cells.4.Then we detect the cytotoxic molecules about the cells in different stages,we found that differentiated NK cells expressed much more inflammatory cytokines or cytotoxicity-related molecules.And the differentiated NK cells can efficiently kill the leukemia cell lines.5.With the results of microarray chips,we found that the differentiated NK cells,as the peripheral and cord blood NK cells,expressed kinds of SLAM family receptors(SFRs),including CD244,CD48,CRACC and NTBA.And they all expressed the adaptors of SAP-related molecules which ensure that the SFRs can transmit activating signals.6.To detect different tumor cells with RT-PCR and flow cytometry,we found that leukemia cell lines expressed SLAM related ligands(SFRLs),including CD48,CD244,CRACC and NTBA,whereas solid tumor cells did not.Furthermore,clinical leukemia patients also had different expression levels of SFRLs.The results suggest that NK cells may be more effective to kill leukemia cells by employing SFR-SFRLs.7.By over-expression and knock-down the expression level of SFRLs,we found that the interaction between SFR and SFRLs can promote the conjugate formation and cytotoxicity towards target cells.The differential susceptibilities of NK cells towards leukemia cells suggest that the detection of SFRLs can be used as clinical predictors about the efficiency of NK cell therapy.In conclusion,we build a procedure to acquire quantities of functional NK cells in the in vitro culture system.Microchip analysis of differentially expressed transcripts reveals a previously undescribed NK cell programmed differentiation pattern.This differentiation program can be divided into two stages categorized by distinct expression profiles of transcription factors;in program 1,progenitors prepare to commit to the NK cell lineage,and in program 2,NK cells transform and reach maturity.In addition,we identified that the interaction of SFR-SFRL between NK cells and leukemia cells,supporting adoptive NK cell treatment for hematological malignancies.Furthermore,to detect the expression of SFRL on leukemia cells could serve as a predictor for NK cell clinical therapy.