NKG2D Mediated Cytotoxicity Of Allogeneic NK Cells Against Human Nasopharyngeal Carcinoma Cell CNE2 And Multi-drug Resistant Carcinoma Cell CNE2/DDP

Background and objectivesRadical external radiotherapy and systemic chemotherapy are the major meansfor the treatment of human nasopharyngeal carcinoma (NPC). Although cure rate forpatients with early stage treated by radiotherapy is high, prognosis for patients withlocoregionally advanced stage and distant metastases is still disappointing despitedthe combined radiochemotherapy due to the emergence of cancer cells withmulti-drug resistance. Thus it is necessary to seek effective methods for the treatmentof NPC patients, especially for the multi-drug resistant NPC patients. In allogeneichematopoietic transplantation, donor-derived alloreactive natural killer (NK) cellshave shown improved cure rate in haematopoietic malignancies. Allo-NK cells haverecently been shown to kill melanoma, kidney carcinoma, intestinal carcinoma, andovarian cancer cells in vitro. Nevertheless, it is unknown whether allo-NK cells havean effect on NPC cells.Cytotoxicity against target cells by NK cells is closely associated with receptorsand the corespanding ligands on the target cells. The engagement of theKIRs(inhibitory killer cell immunoglobulin like receptors) expressed by NK cellswith their specific major histocompatibility complex (MHC) classâ… molecules on target cells results in inactivation of NK cells. In the absence of specific ligands ofKIRs, target cells are susceptible to NK-mediated cytotoxicity. NKG2D is a criticalactivating receptor, expressed by all the human NK cells. Human MHC classâ… chain-related molecule A (MICA) or B (MICB)and ULBPs(human cytomegalovirusglycoprotein UL16 binding proteins, including ULBP1, ULBP2, ULBP3) are themain ligands of NKG2D receptor. Ligation of the NKG2D with NKG2D ligands ontumor cells will activate NK cells' cytotoxicity. High expression of NKG2D ligandswas detected on many human epithelial tumors, Wheras it is currently unknown ifthey are expressed on human nasopharyngeal carcinoma cells.In allogeneic hematopoietic transplantation, donor-derived alloreactive NK cellswere shown to mediate anti-leukemic effects and prevent leukemia relapse aftertransplantation when KIR-ligand mismatch existed in the graft-versus-host (GvH)direction. NK cells were shown to inhibit granulocyte macrophage-colony-formingunit (CFU-GM) formation by leukemic CD34⁺ cells but not normal CD34⁺ cells,suggesting that NK cells are capable of killing the leukemic colony forming cells.Based on known NK cells biology, we will investigate wether allogeneicNK-cells could mediate cytotoxicity against the nasopharyngeal carcinoma cell CNE2and the multi-drug resistant carcinoma cell CNE2/DDP in vitro and in vivo. If thecytotoxicity is confirmed, we will further investigate whether there is difference ofthe cytotoxicity mediated by NK cells against CNE2 and CNE2/DDP cells and themolecular mechanism underlining the difference. On elucidating these problems, wewill provide theoretical and experimental data for establishment of a new potentialtherapeutic approach for NPC patients.MethodsChapterâ… The different characteristics of CNE2 and CNE2/DDP cellsThe morphological feature, growth characters and discrepancy of cloneformation between CNE2/DDP and CNE2 were examined. Flow cytometry was usedto evaluate the cell cycle distribution, expression of P-170 and ABCG2 proteins. Drug sensitivity was measured by MTT assay. Tumorigenicity was evaluated throughsubcutaneous injection of 1×10⁶ CNE2/DDP or CNE2 cells into BALB/c nude mice,the changes of tumor volume were observed.Chapterâ…¡Detection of HLA-classâ… molecules and NKG2D ligands in CNE2and CNE2/DDP cellsCNE2 and CNE2/DDP cells were gathered and DNA was extracted withQIAamp DNA extracting kit. The HLA-classâ… genotypes of CNE2 and CNE2/DDPcells were determined by sequence specific primer polymerase chain reproduction(PCR-SSP, Biotest). MRNA expressions of NKG2D ligands (MICA, MICB, ULBP1,ULBP2, ULBP3) were analyzed by RT-PCR, expressions of HLA classâ… moleculesand NKG2D ligands on the surface of CNE2 and CNE2/DDP cells were analyzed byflow cytometry or immunohistochemistery.Chapterâ…¢Cytotoxicity assays of NK Cells against CNE2 andCNE2/DDP cells in VitroNormal peripheral blood(PB) were obtained with informed consent. DNA wasextracted from PB drawn in acid citrate dextrose buffer (ACD-B) by QIAamp DNAextracting kit. The KIR genotypes of the 5 healthy persons were determined byPCR-SSP. Expression of HLA classâ… molecules and NKG2D ligands on the surfaceof K562 cells were analyzed by flow cytometry. NK cells were obtained from PBlymphocytes by CD56 antibody magnetic isolation. NK cells were cultured in thepresence of recombinant human IL-2 (rhIL-2) for 24h. Cytotoxicity of NK cellsagainst CNE2 and CNE2/DDP cells and K562 cells were detected by LDH releasingassay at different effect-to-target cell ratios (E:T). To test whether NK cell killingactivity was associated with the target cell surface NKG2D ligands and HLA classâ… molecules, we blocked NKG2D ligands (MICA, MICB, ULBP1, LBP2, and ULBP3)and HLA classâ… molecules by different monoclonal antibodies and assessed theirkilling activity at 20:1 E:T ratio.The effects of NK cells on the clone formation ofCNE2 and CNE2/DDP cells were analysed at 20:1 E:T ratio. Chapterâ…£Antitumor effect of NK cells on CNE2 and CNE2/DDP cellsxenografts in nude miceTwenty-four BALB/c nude mice were divided into 4 groups: the CNE2 controlgroup, the CNE2/DDP control group, CNE2 plus NK cells group and CNE2/DDPplus NK cells group. NK cells group were injected subcutaneously 1×10⁶ CNE2 orCNE2/DDP cells cells together with 3×10⁷ NK cells (isolated from 3 healthy personsby magnetic bead selection and cultured, amplified with recombinant human IL-2 andPHA in vitro) injected intravenously through tail veins, while the control group wereinjected 1×10⁶ CNE2 or CNE2/DDP cells soly. The time and the rate of tumorformation were observed, the perpencicular tumor diameter was measured withsliding caliper at 3-day intervals and the tumor volume was calculated, then thegrowth curves of tumors were drawn. Three weeks after tumor formation, all the micewere euthanized, human CD45⁺CD56⁺cells in peripheral blood of mice were detectedby FACS, tumor xenografts were dissected and weighed and the histopathologicalchanges of the tumor were observed by H-E staining.ResultsChapterâ… Study of the different characteristics of CNE2 and CNE2/DDP cellsCNE2/DDP cells were smaller, more regular round and longer doubling timethan CNE2 cells (29.46h vs 21.03h). Expression of P-170 and ABCG2 proteins onCNE2/DDP cells were higher than that on the CNE2 cells. Cell cycle distribution ofCNE2/DDP cells is different with parental cells. The ratios of CNE2/DDP cells andCNE2 cells in G₀/G₁ phase were (65.77±0.81)% and (44.9±2.21)%, respectively(t=15.337, P=0.000), the ratios of CNE2/DDP cells and CNE2 cells in S phasewere (23.63±0.42)% and (39.67±1.27)%, respectively (t=20.834, P=0.000), whilethe ratios of CNE2/DDP cells and CNE2 cells in G₂/Mphase were (10.93±0.25)%,(13.23±0.31)%, respectively(t=10.065, P=0.001).The resistance index of CNE2/DDPcells to DDP was 18.15, it also showed various cross-resistance to 5-fluorouracil (5-FU), vincristine (VCR), the resistance indexes were 28.38, 13.55, respectively.The result of clone formation assays demonstrated that the clone formation rates ofCNE2/DDP and CNE2 cells were (38.44±2.68)% and (26.07±2.70)%, respectively(t=9.760, P=0.000). There was no difference of the formation rate of transplantabletumor between CNE2 and CNE2/DDP cells, time of tumorigenicity betweenCNE2/DDP and CNE2 group were (17.17±1.17)d, (10.00±2.68)d, respectively(t=5.998, P=0.000).Chapterâ…¡Expression of HLA-classâ… molecules and NKG2D ligands by CNE2and CNE2/DDP cellsHLA genotypes of CNE2 and CNE2/DDP cells were A2, 24, B18, 35, Cw4, 7.Expression of MICA, MICB, ULBP1, ULBP2, ULBP3 were confirmed by RT-PCR,but ULBP1 and ULBP3 were not detected on the surface of CNE2 and CNE2/DDPcells by FACS. Expression of MICA, MICB on the CNE2 cells were higher than thaton the CNE2/DDP cells, but there were no difference of the expression of ULBP2between these two cells. Expression of HLA classâ… molecules on the CNE2 cellswere (99.77±0.12)%, higher than that on the CNE2/DDP cells, which were(22.40±2.61) (t=73.113, P=0.000). Expression of MICA/MICB and ULBP2 on thesurface of CNE2 and CNE2/DDP cells was confirmed by immunohistochemistery.Chapterâ…¢Cytotoxicity of NK Cells against CNE2 and CNE2/DDP cells inVitroThe 5 healthy donors experssed KIR2DL1, KIR2DL3, KIR3DL1, KIR3DL2.There were mismatches between KIR3DL1, KIR3DL2 and HLA-A, B molecules inthe CNE2 and CNE2/DDP cells.K562 cells expressed all the NKG2D ligands: MICA, MICB, ULBP1, ULBP2,ULBP3 but not expressed HLA classâ… molecules.In vitro, NK cells displayed highly cytotoxicity against K562, CNE2 andCNE2/DDP cells with lysis of (29.02±0.45)%, (10.50±2.17)%, (4.98±0.95)%respectively at E:T ratios of 5:1; (44.43±1.36)%, (27.68±1.47)%, (15.48±2.10)%respectively at E:T ratios of 10:1; (57.82±1.35)%, (36.99±3.13)%, (28.46±4.30)% respectively at E:T ratios of 20: 1; (71.24±2.36)%, (55.00±2.20)%, (40.95±2.21)%,respectively at E:T ratios of 30:1. Cytotoxic effect of NK cells on K562, CNE2 andCNE2/DDP cells was enhanced in relation to the increased E: T ratio. There weredifference of NK-cells mediated cytotoxicity among these three groups. Blockingexperiments confirmed that killing of K562 by NK cells was efficiently inhibited byanti-MICA mAb, anti-MICB mAb, anti-ULBP1 mAb, anti-ULBP2 mAb andanti-ULBP3 mAb with lysis of (46.82±2.62)%, (49.26±0.98)%, (50.42±1.79)%,(50.75±1.23)%, (51.68±1.34)%, respectively at E:T ratios of 20:1, the cytotoxicitywas lower than that prior to the blocking experiment ((71.24±2.36)%). Anti-MICAmAb, anti-MICB mAb, anti-ULBP2 mAb could partially inhibit the cytotoxicity ofNK ceils against CNE2 and CNE2/DDP cells, Wheras anti-ULBP1 mAb andanti-ULBP3 mAb could not inhibit the cytotoxicity of NK cells against CNE2 andCNE2/DDP cells. The cytotoxicity of NK cells against CNE2 and CNE2/DDP cellscould be increased by W6/32(anti-HLA-I molecules mAb). The result of cloneformation assays demonstrated that the clone formation rates of CNE2/DDP andCNE2/DDP cells cocultured with NK cells on the 14th were(38.4±2.68)% and(34.52±1.84)%, respectively(t=3.621, P=0.002), while that of CNE2 cells and CNE2cells cocultured with NK cells were (26.07±2.70) % and (22.48±1.39)%,respectively(t=3.556, P=0.004). The data indicated that NK cells could inhibite theclone formation of CNE2/DDP and CNE2 cells.The data indicated that both KIR-HLA signal system and the NKG2D-ligandsignal system play a role in NK cells-mediated cytotoxicity against CNE2 andCNE2/DDP cells. NK cells displayed higher cytotoxicity against CNE2 cellscompared with CNE2/DDP cells.Chapterâ…£Antitumor effect of NK cells on CNE2 and CNE2/DDP cellsxenografts in nude mice The time of tumor formation in CNE2 and CNE2 plus NKcells group were(10.00±2.68)d, (18.80±1.64)d, respectively(t=6.375, P=0.000), the rate of tumorformation were100% (6/6), 83.33%(5/6), respectively, while the time of tumorformation in the CNE2/DDP and CNE2/DDP plus NK cells group were(17.17±1.17)d,(24.83±1.47)d respectively(t=9.991, P=0.000), the rate of tumor formation were100% for the two groups.Growth curves showed that tumor masses of control group grew more rapidlythan the treatment group.Three weeks after tumor formation, all the mice were killed, human CD56⁺ NKcells could be detected in peripheral blood of mice treated with NK cells but couldnot be detected in the control groups.The average weight of tumors in the CNE2 and CNE2 plus NK cells groupwere(2.22±0.09)g, (1.42±0.09)g, respectively(t=14.475,P=0.000), the tumor growthinhibition rate was 36.04%. The average weight of tumors in the CNE2/DDP andCNE2/DDP plus NK cells group were (1.60±0.22)g, (1.28±0.52)g, respectively(t=3.517, P=0.006), the tumor growth inhibition rate was 20%.HE stain show that tumor tissues were poorly differentiated squamous cellcarcinoma, which were polygon, oval or irregular shaped nucleus, the nuclearseparation were obvious. There were more necrosis regions and lymphocytesinfiltrated in the tumor tissue in the treatment group than in the control group.Keratinization of tumor cells was found in the CNE2 plus NK cells group.Summary1. There were different biologic characteristics between CNE2 and CNE2/DDPcells, CNE2/DDP cells were longer doubling time, higher clone formation rate, higherratios in G₀/G₁ phase compared with CNE2 cells. CNE2/DDP cell was a typicalmulti-drug resistant cell line.2. Expression of MICA, MICB, ULBP1, ULBP2, ULBP3 were confirmed by RT-PCR, but only MICA, MICB and ULBP2 were expressed on the surface ofCNE2,CNE2/DDP cells by FACS. Expression of HLA classâ… molecules, MICA,MICB by CNE2 cells were higher than that by the CNE2/DDP cells.3. NK cells displayed higher cytotoxicity against CNE2 cells compared withCNE2/DDP cells, NK cells could inhibite the clone formation of CNE2 andCNE2/DDP cells.4. Cytotoxicity assay indicated that killing of CNE2 and CNE2/DDP cells byNK cells was through NKG2D-MICA, MICB, ULBP2 interaction, while HLA-classâ… molecules on the CNE2 and CNE2/DDP cells inhibited NK cells mediatedcytotoxicity. NK cells were shown to inhibit the clone formation by CNE2/DDP andCNE2 cells.5. Allogeneic NK cells could inhibite the growth of CNE2 and CNE2/DDPcells xenograft in BALB/c nude mice.Innovation of our research1. We first discovered that KIR-HLA signal system and NKG2D-ligand signalsystem play a role in NK cells-mediated cytotoxicity against the CNE2 andCNE2/DDP cells.2. We first discovered the disparities of HLA classâ… molecules and NKG2Dligands between multi-drug resistant and parental nasopharyngeal carcinoma cells.Our findings elucidated the molecular mechanism of the different killing activitymediated by NK cells.Value of our researchThis will enable us to perspect the relationship between the immune system andthe multi-drug resistant tumor cells. Our findings provide the theoretical andexperimental basis for enhancing the cytotoxicity against the multi-drug resistanttumor cells by the immune system.