KIR-mismatch Induced By RNAi-Ly49C Enhances GVL Effects Exerted By Allogeneic Natural Killer Cells

Background and ObjectivesAllo-HSCT is one of the most effective treatment strategies for malignant hematologic disease.As for the high risk leukemia,its relapse ratio still came as high as 81%.The key to improve therapeutic efficacy of malignant hematology is to enhance GVL effects and cut down the relapse ratio.Allo-NK cells are important effectors of GVL effects in allo-HSCT because of its efficient killing function against malignant cells.Cytotoxicity of NK cells is regulated both by HLA-Killer cell Immunoglobulin-like Receptors(KIRs)(HLA-KIR) inhibitory signals and NKG2D ligand-NKG2D(NKG2DL-NKG2D) activating signals.Due to the existence of HLA-KIR inhibitory signals,cytotoxic effects of haplo-identical donor NK cells were represse,Focusing on this point,we took the advantage of inbred strains and hybrid strains definite genetic background to investigate the cytotoxicity of NK cells. Cytolytic effects were enhanced by human-induced KIR mismatch. MethodsPart 1 Regulation effects of Ly49—H-2 signal passway on cytotoxicity of Allo-NK cells against tumor cellsNK cells were positive selected in MACS from splenocytes of CB6F1 strain mouse.The purity of isolated cells was detected by Flow cytometry(FCM).NK cells' cytolytic effects were detected by LDH releasing assay against YAC-1 in order to measure their biological function.Cytotoxicity against FBL-3 cells(erythroleukemia cell line from C57BL/6 mouse) from NK cells,antibody-blocked NK cells(NK cells cocultured with 14B11 antibody),edited NK cells(effetcors and targets cocultured for 24h at the E:T ratio of 10:1 ) and Ab-blocked edited NK cells were measured by LDH releasing assay.Part 2 Cytotoxic effects against tumor cells affected by RNAi-Ly49 on NK cells in vitroNK cells were isolated as usual and then were transfected by siRNA numbered as 124,335,589,91.Effective siRNA oligo was sieved by FCM measurement on Ly49C expression.Cytotoxicity against YAC-1 and FBL-3 cells were detected by LDH releasing assay from most-inhabitory NK cells.Part 3 GVL effects affected by RNAi-Ly49 on NK cells in haplo-identical transplantation modelHaploidentical HSCT was exerted from male CB6F1 mice to female C57BL/6 mice with leukemia.50 recipients were randomly divided into 5 groups with 10 mice per group.Transplatation groups were used to observe GVL effects.group A,un-treated leukemia groupgroup B,simply irradiated groupgroup C,simply BMC(bone marrow cells)-transplanted group after irradiation(1×10⁷ per mouse at 4h after irradiation for BMC)group D,Both un-treated NK cells infusion and BMC-transplanted group after irradiation(infusion at 1h point after irradiation for 2×10⁶ NK cells per mouse and 1×10⁷ per mouse for BMC at 4h point after irradiation)group E,Both RNAi-treated NK cells infusion and BMC-transplanted group after irradiation(infusion at 1h point after irradiation for 2×10⁺ RNAi-treated NK cells per mouse and 1×10⁷ per mouse for BMC at 4h point after irradiation)Statistical analysis The calculation was performed using SPSS 13.0 software package.The data were represented as the mean±standard deviation.Comparisons in experiments were performed using one-way analysis of variance(ANOVA), independent-samples t-test.Survival curve was made by Kaplan-Meier assay. Differences with P＜0.05 were considered to be significant.ResultsPart 1 Regulation effects of Ly49—H-2 signal passway on cytotoxicity of Allo-NK cells against tumor cells1.1 Cytotoxic effects of isolated NK cellsCytotoxic effects of isolated NK cells against YAC-1 cells were enhanced in relation to the increased E:T ratios as(24.80±0.85)%in 5:1,(45.95±2.09)%in 10:1, (60.71±1.04)%in 20:1 and(79.54±0.94)%in 40:1.Data above indicated that isolated NK cells were competent.1.2 Cytotoxic effects of groups of NK cells against tumor cellsCytotoxicity exerted by groups of NK cells were significantly different(F=228.390,P=0.000).Took the untreated NK cells' cytotoxicity (20.57±1.22)%as baseline data,Ab-blocked NK cells showed markedly higher cytotoxicity(P=0.000),while edited NK ceils displayed noticeably lower cytotoxicity(P=0.000).Ly49C expression in edited NK cells were(84.98±1.25)%, significantly higher than(52.27±2.57)%in untreated NK cells.Ab-blocked edited NK cells cytotoxicity was significantly higher than edited NK cells(P=0.000). These findings suggested that NK cells have different cytotoxic effects against YAC-1 and FBL-3 cells.The cytotoxicity of NK cells was regulated by both inhibitory and activating signal pathways.Up-regulation of KIR(Ly49) in edited NK cells induced by tumor cells coculturation caused lower cytotoxicity of NK cells. Decrease in the expression of inhibitory signals exerted by antibody 14B11 blocking caused higher cytotoxicity of NK cells.Part 2 Cytotoxic effects against tumor cells affected by RNAi-Ly49 on NK cells in vitro2.1.Ly49C expression in groups of NK cells after transfectionLy49C expressions in group of NK cells individually transfected with siRNA numbered in 124,335,91,589 were(27.48±2.79)%,(52.73±2.14)%,(40.98±4.29) %,(33.26±2.77)%.2.2.Cytotoxicity of RNAi-NK cells against tumor cellsThe expressions of Ly49 in NK cells were most effectively inhibited by siRNA numbered 124,which had the inhibitory ratio of 48%.Cytotoxicity of RNAi-NK cells against YAC-1 was(58.77±1.60)%(E:T=20:1 ),showed no significant difference when compared to the un-treated one in(60.71±1.04)%(t=1.757,P=0.154).But markedly difference displayed between cytotoxicity against FBL-3 cells from RNAi-NK cells(29.08±2.91)%and un-treated NK cells(20.57±1.22)%(t=4.664, P=0.010).These findings suggested that RNAi-NK cells cytotoxic effects against YAC-1 cells had no significant difference compared to un-treated NK cells,which coincided with the totally mismatch genetic background between A/Sn(YAC-1 cells derived from A/Sn mouse strain) and CB6F1.When it came to the haploidentical FBL-3, things were different.RNAi-NK cells expressed lower level of KIR,which made NK cells exert more cytolytic effects. Part 3 GVL effects affected by RNAi-Ly49 on NK cells in hapio-identical transplantation modelSuivival condition for each experimental group of micegroup A,un-treated group;leukemia disease advanced quickly;all the mice died at 8-11d,survival time(9.43±1.06)dgroup B,simply irradiated without BMC-transplantation;weight of the mice decreased quickly;survival time(9.23±0.40)d;all died because hematogenesis exhaustion(WBC＜0.5×10⁹/L).group C,two of them lived longer than 30d;survival time(23.88±8.65)dgroup D,four of them lived longer than 30d;survival time(30.14±8.56)d; inhibitory rate was 319.62%when compared to group A;inhibitory rate was 126.21% when compared to group Cgroup E,five of them lived longer than 30d;survival time(33.74±6.71)d; inhibitory rate was 357.79%when compared to group A;inhibitory rate was 141.29% when compared to group CAnti-tumor effects were observed in mouse model with leukemia.Took the survival time as survey index,RNAi-NK cells group showed 357.79%inhibitory rate, which was 38.17%higher than un-treated group A;RNAi-NK cells group showed 15.08%higher inhibitory rate when compared to un-treated NK cells group.Which meant siRNA interference enhanced GVL effects exerted by NK cells.Conclusions1 Functional experiments in vitro showed that cytotoxicity of NK cells was regulated by both inhibitory and activating signal pathways.Decrease in the expression of inhibitory signals exerted by antibody 14B11 blocking caused higher cytotoxicity of NK cells.RNAi-Ly49 developed the same effects as it depressed the expression of inhibitory signals as well.2 Anti-tumor effects were observed in mouse model with leukemia.Took the survival time as survey index,RNAi-NK cells group showed 357.79%inhibitory rate, which was 38.17%higher than un-treated group A;RNAi-NK cells group showed 15.08%higher inhibitory rate when compared to un-treated NK cells group.Which meant siRNA interference enhanced GVL effects exerted by NK cells via human-induced KIR-mismatch.Innovation of the current studyIn the theoretical aspect,we put forward the assumption of inducing KIR-mismatch by KIR-expression-blocking in order to enhance anti-tumor effects and decrease relapse ratio of leukemia after transplantation.In the experimental method aspect,the transplantation was performed from F1(C57BL/6×BALB/c) (H-2^{b/d,Ly49A+Ly49C+} to C57BL/6(H-2^b,Ly49C⁺).Donor NK cells with the expression of Ly49A⁺Ly49C⁺ can recognize its own H-2^d after Ly49C-blocked because its Ly49A expression could be remained.This benefited the success of transplantation greatfully.Significance of the researchOur study provided a potentially effective point for enhancement in anti-tumor effects by NK cells.This may also expanded to other immune effector cells.In short, our study presented theoretical and experimental basis for establishment of a potentially feasible genetic-based adoptive cellular immunotherapy.