Cytotoxicity Effects And Mechanisms Studies Of Vγ9Vδ2T Cells On Human Acute Myeloid Leukemia Cells

Acute myeloid leukemia(AML)is the most common type of adult acute leukemia(AL). With the improvement of chemotherapy, the complete remission rate CR) has been up to about70%. However, still about20%of refractory AML patients can not achieve CR, with a very low5-year disease-free survival rate. And most of the initial treatment CR patients will relapse resulting in poor prognosis. The presence of drug-resistance as well as leukemia stem cells (LSC) is the root cause of refractoriness and relapse. The development of novel therapeutic agents override the resistance or directly targeting LSC is an urgent need to solves thisl problem. Cellular immunotherapy provides a promising novel therapy.Vγ9Vδ2T cell is one of the T-cell subsets attracting much attention in recent years. It has been confirmed that Vγ9Vδ2T cells can efficiently kill a wide range of tumor cells, even tumor stem cells in a MHC non-restrictive way. It is now easy to obtain a large number of Vγ9Vδ2T cells via induction in vivo or in vitro. Preclinical studies have shown that Vγ9Vδ2T cells based immunotherapy may be safe and effective for several advanced solid tumors. Based on the above basis, we speculate that Vγ9Vδ2T cells based immunotherapy may be a new way for refractory or resistant AML.So in this study we intend to solve the following three scientific problems:The first is to explore the cytotoxicity effects of Vγ9Vδ2T cells against refractory or resistant AML cells; The second is to clarify the recognition and cytotoxicity mechanisms of Vy9V82T cells against AML cells as well as refractory or resistant cells; The third is to explore the potential feasibility of Vy9V82T cells amplificated in vitro in combination.with tyrosine kinase inhibitor (TKI). In order to solve these issues, we conducted the following researches:Part one Cytotoxicity of Vy9V52T cells against human acute myeloid leukemia (AML) cellsFirst, we tested the cytotoxicity effects of Vy9V82T cells against AML cell lines Kasumi-1, K562, NB4and HL-60. We found that Vy9V82T cells exerted cytotoxicity effects against these cell lines in a efficacy to target (E/T) ratio-and time-dependent way. Vy9V82T cells showed different cytotoxicity sensitivity against different types of AML cell lines:they exerted the strongest cytotoxicity against Kasumi-1cell line, moderate cytotoxicity against K.562and NB4cell lines, and weak cytotoxicity against HL-60cell line. There was no significant difference between the cytotoxicity of Vy9V82T cells and cytokines induced killer (CIK) cells against Kasumi-1, K562and NB4cell lines.Next, we tested the cytotoxicity effects of Vy9V82T cells against refractory AML cell lines K562/AO2, HL-60/ADR and NB4/R1. K562/AO2and HL-60/ADR highly expressed P-glycoprotein (P-gp) and are both resistant to anthracyclines, whereas NB4/R1cells were mutated in retinoic acid receptor (RAR) and resistant to retinoic acid. We found that the cytotoxicity of Vy9V82T cells to these cell lines was neither related to the cell types nor drug resistance. Moreover, we had collected11bone marrow samples from refractory AML patients, and found that Vy9V82T cells also exerted ex vivo cytotoxicity against leukemic blasts and CD34+leukemia stem cells (LSC), or when E/T ratio was no less than200:1. However, Vy9V82T cells showed no cytotoxicity effect against normal bone marrow primary monocytes or fibroblasts. To study the cytotoxicity of Vγ9Va2T cells againt refractory AML cells in vivo, we established humanized NOD/SCID mice refractory AML model using K562/AO2. We found that the mice treated with zoledronic acid and IL-2combined with Vy9V82T cells showed unchanged body weight, less tumor load, and longer survival compare to untreated control or the treated with zoledronic acid and IL-2alone.The above results demonstrated that Vy9V82T cells had both ex vivo and in vivo cytotoxicity effect to AML cells, including refractory AML cells. The cytotoxicity effect of Vy9V82T cells against refractory AML cells was neither related to the types nor drug resistance of the cell lines.Part two The recognition and cytotoxicity mechanisms of Vγ9Vδ2T cells against AML cellsWe monitored the interaction of V γ9V δ2T cells with Kasumi-1cells which Vy9V82T cells exerted the strongest cytotoxicity effect using BD Pathway85. After1h coculture, an obvious deformation appeared on V γ9V δ2T cells. After2h coculture, fluorescence derived from Kasumi-1cell membranes could be detected on V γ9V δ2T cells, indicating the trogocytosis phenomenon of V γ9V δ2T cells, hich was also confirmed by flow cytometric analysis. After4h coculture, we visualized the apoptotic morphology of Kasumi-1cells by confocal microscopy.To identify the cytotoxicity mechanism of V γ9V δ2T cells against AML cells, Kasumi-1, K562, HL-60, K562/AO2, as well as leukemic blasts from the bone marrow samples of two refractory AML patients were cocultured with V γ9V δ2T cells for4h. The expression of CD107a on V γ9V δ2T cells increased significantly after cocultured with the above AML cells. Concanamycin A (CMA) which could blocke the expression of CD107a, significantly impared the cytotoxicity effects of V γ9V δ2T cells. Blockade of FasL-Fas pathway by anti-FasL antibody did not affect their cytotoxic effects against Kasumi-1or NB4cells. The level of secreted TNF-a (sTNF-a) in the supernatant raised notably after incubating V γ9V δ2T cells with Kasumi-1and NB4cells for8h. However, blockade of TNF-a-TNF-a receptor pathway by anti-TNF-a antibody failed to affect the cytotoxic effects of V γ9V δ2T cells. Immunoblotting assay revealed that phosphorylation of ERK was induced5to20mins after V γ9V δ2T cells cocultured with K562cells and K562/AO2cells, which was not found in HL-60cell line which Vy9V82T cells exerted very weak cytotoxicity effect. Blocking ERK pathway by PD98059or blocking Akt pathway which functioned in the upstream of ERK by LY294002attenuated the expression of CD107a on V γ9V δ2T cells cocultured with K562and K562/AO2cell lines for4h, which was also not found in HL-60cell line.In order to understand the recognition mechanisms of V Y9V82T cells sagainst AML cells, we blocked the composition of IPP (isopentenyl diphosphate), the ligand of TCR in cell lines Kasumi-1, K562, NB4and K562/AO2by atorastatin, and revealed that it significantly impared the cytotoxicity effects of V γ9V δ2T cells. Flow cytometric analysis presented high expression of NKG2D、CD11a、CD226on the surface of V γ9V δ2T cells, which are all cytotoxicity associated receptors. We also observed that the receptors of NKG2D (MICA/B, ULBP-1, ULBP-2, ULBP-4), CD11a (ICAM-1、 ICAM-2) and CD226(Nectin-2、PVR) were expressed at differerrnt levels in different AML cell lines, among which all the receptors were expressed on Kasumi-1. In addition, the expression levels between sensitive cell lines and their corresponding drug-resistant cell lines were quite different. Correlation analysis using linear regression model revealed that the expression of ULBP-4and Nectin-2had relevance to the cytotoxicity of V γ9V δ2T cells. Blocking NKG2D-ULBP4pathway by anti-NKG2D antibody, we found that the cytotoxicity of V γ9V δ2T cells at4h against Kasumi-1cells which expressed ULBP-4was significantly reduced, however, no reduction was found in K562, NB4and K562/AO2which all had low level or even no expression of ULBP-4. Blocking CD226-Nectin-2pathway by anti-CD226antibody, the cytotoxicity against Kasumi-1, K562, NB4and K562/AO2, which all expressed Nectin-2, were all remarkably reduced. Blocking CD11a-ICAM-1/2pathway by anti-CD11a antibody, the cytotoxicity against Kasumi-1, K562, NB4and K562/AO2, which all expressed ICAM-1/2, revealed no reduction.The above results suggested that the cytotoxicity of V γ9V δ2T cells against AML cells was depended on direct contact between cells and trogocytosis of Vγ9V δ2T cells, which may activated the phosphorylation of ERK pathway, followed by perforin and granzyme release, and finally induced the apoptosis of target cells in4hours. The TCR-IPP、NKG2D-ULBP-4and CD226-Nectin-2recognition pathways may take part in the cytotoxicity of V γ9V δ2T cells against AML cells. The cytotoxicity effect was related to the expression of ULBP-4and Nectin-2on the surface of AML cells instead of cell types or drug resistance.Part three The potential feasibility of Vγ9Vδ2T cells amplificated in vitro in combination.with TKIWe first tested the influence of imatinib, nilotinib and dasatinib on the amplification efficiency of Vy9V82T cells induced in vitro at50%inhibiting concentrations (IC50, related to the inhibitory capacity toward the ABL kinase) and plasma peak levels reached in patients of either compound. We found that only dasatinib at IC50concentration significantly increases the amplification efficiency of Vy9V82T cells, while maintaining a ideal induced purity. We next detected the expression of activation associated molecules CD69, HLA-DR and CD25on Vy9V82T cells, and found only dasatinib at IC50concentration enhanced the expression of activation associated molecules CD69and HLA-DR, before the proliferation assay results, which was consistent with the results of proliferation assays before. The level of CD25in each group were not significantly different. We also tested the expression of activation-induced apoptosis-related molecular Fas on Vy9V82T cells, and found that the dasatinib at50%IC50and plasma peak levels both have significant inhibitory effects.As we found in the above that among3TKIs only dasatinib at IC50(10nmol/L) concentration significantly increased the amplification efficiency of Vy9V82T cells, while at plasma peak level (200nmol/L) the amplification efficiency was inhibited, which dedicated the amplification may be significantly different by different concentrations of dasatinib. To explore the best induced program of Vy9V82T cells by dasatinib, we further detected the effect of dasatinib in gradient concentrations on Vy9V82T cells proliferation and anti-AML function. We found dasatinib promoted the absolute number of Vy9V82T cells induced in vitro within the scope of10nmol/L, which reached a peak at2nmol/L, while dasatinib above10nmol/L had concentration-dependent inhibition effects. The induced purity of Vy9V82T cell were maintained by dasatinib within the scope of10nmol/L, while dasatinib above10nmol/L had concentration-dependent inhibition effects. Dasatinib had more degranulation effects of Vy9V82T cells at all the concentrations than those of control group. This enhanced role of dasatinib showed a first decreased and then rising trend, with an increasing concentrations of dasatinib, which was closely associated with the proportion of CD27-subsets of Vy9V82T cells. We further found that dasatinib at lower doses, primarily increased the proportion of CD27-CD45RA-subsets, while at higher doses, mainly increased CD27-CD45RA+subsets.The above results showed that dasatinib within a scope of appropriate dose range increased the amplification efficiency of Vy9V82T cells, which may be related to upregulation of activation associated molecules, and inhibited the expression of Fas to keep away from activation mediated apoptosis. Dasatinib also enhanced the degranulation effects against AML cells, which was closely associated with increasing the proportion of CD27-subsets of Vy9V82T cells. So optimizing the induced program of Vγ9Vδ2T cells in combination with dasatinib may be reasonable.In summary, this study confirms the cytotoxicity effect of Vγ9Vδ2T cells against refractory or resistant AML cells, reveals the recognition and cytotoxicity mechanisms of Vγ9Vδ2T cells against AML cells, and also clarifies the combination effect and mechanisms of Vγ9Vδ2T cells combined with dasatinib during an appropriate concentration range. This study may bring a new idea of treating refractory or resistant AML by Vγ9Vδ2T cell-based therapy, and provide experimental basis for enriching the understanding of Vγ9Vδ2T cell anti-tumor biological behavior, also provide valuable experiment basis of optimizing the program of Vγ9Vδ2T cells induced in vitro.