Basic And Clinical Study For NK Cells Receptor KIR

Natural Killer(NK)Cells are lymphocyte in the same family as T and B cells,but play an important role in the innate immune system as providing response to infection,tumor formation,and immune regulation.Although without exquisite antigenic specificity like T and B cells,NK cells discriminate self from non-self through the interaction of killer-cell immunoglobulin-like receptors(KIRs)expressed on cell surface and major histocompatibility complex class ?(MHC-?)molecules on target cells.NK cells recognize and exert cytotoxity to target cell with absence or reduced expression of MHC-I molecules.During development,the interaction between the self MHC-I molecule and its specific inhibitory KIR that allows NK cells to become functionally mature,however,the mechanism for KIR acquisition and regulation remains unclear.Moreover,more and more evidences based on clinical research indicated that in addition to inhibitory KIRs,activating KIRs may come in to play in NK cell function regulation.1.The regulation of KIRs expression and NK cell functionally mature.In peripheral blood,KIR are predominantly expressed on the CD56dimCD16+subset of NK cells,whereas not on CD56briCD16-cells(except KIR2DL4).During NK cells development,their KIR was acquire repertoire in a stochastic manner.And DNA methylation has been shown to play an important role in maintenance of expression patterns of KIR genes.As far,the mechanism of how KIR transcription is regulated is still a puzzle.Given the importance of KIR expression in regulating NK cell function,study for element involved in KIR acquisition would improve our understanding to KIR/MHC related disease and NK cell clinical approach.Our research is focus on the elements influencing KIR expression and regulation in NK cells.Human peripheral NK cells are generated from CD34+ bone marrow progenitors.We separated human cord blood CD34+ hematopoietic stem cell(HSC)and induce its differentiation into mature NK cells in a feeder cell-free in-vitro culture system and stimulated with cytokines cocktail.Interestingly,we found that the NK cells differentiated from this in-vitro system were with lower KIR expression than peripheral NK cells.To study the mechanism of limited KIR expression in some NK cells,we separated KIR+CD56dimCD16+,KIR-CD56dimCD16+ and CD56briCD16-peripheral NK cells,as well as KIR+ and KIR-NK cell differentiated from cord blood CD34+HSC in vitro and screened the differential expression genes(DEGs)of KIR+ and KIR" NK cell through comparative transcriptome analyses with total RNA sequencing.Through comprehensive comparative analysis among the separated five groups NK cells,we found that the in-vitro differentiated NK cells have similar key hallmarks of mature peripheral NK cell such as CD56,CD 16,CD 107a and perforin,however,actually displayed distinct whole transcriptome phenotype from the peripheral.The principal component analysis(PCA)analysis showed that in-vitro differentiated NK cells are with more similar transcriptome phenotype to CD56briCD16-peripheral NK subset when compared with the CD56dimCD16+subset.Beyond our expectation,there was tiny differentiation of transcriptome phenotype between the KIR+ and KIR-NK subsets both for peripheral CD56dimCD16+NK cell and the in-vitro differentiated NK cells when being resting,therefore limited amount of DEGs we can find out.Nevertheless,we found several DEGs such as RASSF4,RUNX2,MYC and ID3 were correlated with KIR genes expression.The RNA-Seq analysis showed that RASSF4 was with positive correlation with KIR gene expression,while the transcription factor RUNX2,MYC and ID3 was shown with negative correlation.In the model of human umbilical cord blood(UCB)-derived CD34+ cells development in humanized mouse,RASSF4 was up-regulated along NK cell differentiation and maintained after NK cells acquiring mature phenotype(e.g.KIR acquisition)while the RUNX2 was up-regulated in the mid-term stage but down-regulated later.However,we found that RASSF4 was preferentially expressed in KIR+mature human NK cells but was down-regulated at later stage during in vitro differentiation of NK cells from human umbilical cord blood(UCB)-derived CD34+cells.In contrast,RUNX2 which was preferentially expressed in KIR-mature human NK cells especially CD56briCD16-NK cells was up-regulated in the mid-term stage.The abnormal expression model of RASSF4 and RUNX2 during NK cell differentiation might impair KIR expression.More evidence for how RASSF4 and RUNX2 influence the expression of KIR family should be found.II.Activating KIRs play a role in response to CHB therapy.NK cells as an important component of innate immune system,play a vital role in defensing early infection.NK cell recognizes viral-infected cells via KIR receptors which are encode by a gene family with highly allelic polymorphism and variable gene content.Multiple HLA and KIR genes have been implicated in association with viral infection(e.g.HIV and HCV)and disease progression.However,no candidate gene has been identified to predict the outcome of anti-HBV treatment.Through observation on therapy for CHB,we wanted to know whether KIR or HLA plays a role in IFN-a therapy for chronic HBV infection and find pretreatment predictor for CHB therapy.In this study,we recruited a total of 119 hepatitis B e antigen(HBeAg)-positive CHB patients who had received anti-HBV treatment for 48 weeks and 96 healthy subjects as normal control,all of whom were Han Chinese.19 KIR genes and three major KIR ligands(HLA-C1,HLA-C2,and HL-Bw4)were genotyped by PCR-SSP.Among the total 119 patients,43 patients achieved sustained response(SR)induced by IFN-a treatment for 48 weeks,while other 76 patients achieved no response(NR).SR was defined as HBeAg loss,with HBV DNA<2,000 IU/ml and ALT normalization at 24 weeks posttreatment(week 72).We analyzed the KIR and ligand HLA genotype of healthy subjects,CHB patients(grouped by SR and NR)as well as the dynamic variations of clinical viro logical and serological indices of the CHB patients during the course of treatment.In the present study,several conclusion have been made.1.HBV infection was highly endemic in Asia,but less prevalence in Europe and North America.Whether KIR genes variance among population categorized by different ethnicity and region contribute to distinct outcome of HBV infection.Activating KIRs in the B haplotype were less prevalent in the Han Chinese(especially in CHB patients who were Han Chinese)than in Caucasians,including both European and US Caucasians.It is reasonable to speculate that there is a link between one or more activating KIRs in the B haplotype and hepatitis B infection clearance.2.Because of the immunomodulatory effects of IFN-a,treatment with IFN-a enhancement of antigen presentation to the immune system,activation of NK cells and other immune cells.To investigate whether KIR contribute to treatment response in this process,we compared a particular KIR or HLA genes between the SR and NR groups and found that KIR3DS1 showed significant differences between the SR and NR groups,being more prevalent in the SR group(51.2%)than the NR group(18.4%)[Odd ratio(OR)= 4.64,P = 0.0002].This finding support a role for KIR3DS1 in enhancing NK cells to defense against HBV.3.Because the interaction between KIR and its HLA ligand determines the transmission of the KIR downstream signal,we further assessed whether the KIR/HLA pairing was correlated with the response to therapy in CHB infection.We found that comparison of the frequencies between CHB patients presented with the KIR3DS1 and HLA-B Bw4-80Ile combination genotype in the SR group(20.9%)and NR group(1.3%)(OR= 19.85,P=0.0008)showed that this genotype contributed to the response to IFN-a therapy in HBeAg-positive chronic HBV infection.Besides,patients possessing the KIR3DS1/HLA-B Bw4-80Ile combination genotype showed more dramatically decreased of HBV DNA and HBeAg levels than those not possessing.4.Multiple logistic regression analysis used to identify predictors of SR in the present study verified a protective effect of KIR3DS1 in combination with HLA-B Bw4-80Ile to achieve SR for HBeAg-positive CHB patients treated with IFN-a(OR =16.98,P= 0.01).In summary,we focused on the acquisition and function of KIRs,an important receptors family of NK cells in this study.First we studied the distinct signature of KIRs,especially inhibitory KIRs,expressed on peripheral NK cells and in-vitro differentiated NK cells.And further screened the regulatory elements for KIR expression and maintaining through bioinformatics analysis.We found that two key molecules RASSF4 and RUNX2 potentially participate in KIR expression regulation during NK cell development with opposite effect.However,it needs further studies on the mechanism of how they effect.Besides,our finding based on clinical observation supported a role for activating KIR genes in the prevalence of HBV infection,extended the present understanding of the effect of the KIR3DS1/HLA-B Bw4-80Ile genotype on HBV infection and therapy response,furthermore,advanced our understanding the role of activating KIRs in NK cells immune response to HBV infection.