| BandgroudNatural killer (NK) cells, a group of large granular lymphocytes, play essential roles in immune system against malignant and infected targets. Recognition of target cells is governed by a complex set of activating and inhibitory receptors expressed on NK cell surface. The balance between activation and inhibition signaling decides the outcome of NK cell cytolytic response to target cells. NK cells act as innate immune cells, and conduct rapid "natural" killing of cellular targets (tumor cells and infected cells), which lack major histocompatibility complex (MHC) class ? expression, without prior sensitization steps. Parallel to the improved understanding of NK cell biology, NK cell-based immune therapies in clinical settings have also been developed, improved, and tested in cancer patients, including hematological cancers, and some promising results have been reported. Owning to the "missing-self characteristics of NK cell activation, allogeneic NK cell transfusion resulted in more promising outcome than autologous ones in the treatment of cancer patients. NK cell only accounts for 5-15 of peripheral lymphocytes (PBL). Apparently, developing a protocol for efficient activation and expansion of NK cells to achieve sufficient number of functional NK cells is curial for adoptive allo-and auto-NK cell therapies. CD 16 is one type of Fc? receptors, a low-affinity receptor (Fc?R?a) for the Fc portion of immunoglobulin. and cross-linking of CD 16 to target cells induces NK cell antibody-dependent cellular cytotoxicity (ADCC) and lysis of the target cells. Binding of CD16 agonist anti-CD 16 antibody on NK cells is capable of triggering NK cell activation that results in NK cell cytotoxic activity against cancer cells and releasing of cytokines, such as interferon ? (IFN?). Moreover, blocking CD 16 cleavage by inhibiting ADAM metallopeptidase domain 17 (ADAM 17) to boost NK cell cytotoxicity towards cancers has been suggested by a recent study. Therefore, activation of NK through CD 16 can be a promising strategy for in vitro induction of NK cell activation and expansion.Targeting on immune checkpoint molecules such as programmed cell death protein 1 (PD1) and its ligands PD-L1 and PD-L2 by antibodies to block their inhibitory signaling has achieved great success in treatment of several solid tumors and hematological malignancies. Engagement of PD1 with PD-L1/L2 expressed on antigen presenting cells (APCs) delivers inhibition signaling, and this negative regulation of immune response pathway plays crucial roles in induction and maintenance of peripheral immune tolerance. In symptomatic cancer patients, T cells in tumor microenvironment often express PD1, and interaction between PD1 and PD-L1 on cancer cells creates a network blocking T cell-mediated eradication of cancer cells. Such PD1 positive T cells are considered to be a group of exhausted T cells, characterized by reduced effector function and proliferation index. In addition to the findings observed in T cells, NK cells from cancer patients such as multiple myeloma (MM) were also shown to express PD1. Concerning PD1 expression on T cells is inducible upon T cell priming, it is presumable that in vitro activation and expansion procedures may also induce and up-regulate PD1 expression on NK cells. Therefore, it would be of great interest to evaluate PD1 expression on NK cells and the functional changes of NK cells in relation to PD1 blockage in a NK cell expansion system.MM is a hematologic tumor characterized by an uncontrolled clonal expansion of malignant plasma cells. With the development and clinical application of new anti-MM drugs, such as bortezomib and lenalidomide, outcome of MM therapy has been markedly improved, but MM still remains incurable. Similar to other malignancies, relapse cannot be effectively prevented due to minimal residue disease (MRD), in which those remaining cancer cells are usually resistant to conventional therapies. Immunotherapies including NK cell transfusion in combination with PD1/PD-L1/2 blockage may offer a potential solution for eradication of MRD in MM and other tumors.Objective The aim of this study is to develop a novel protocol to highly expand NK cells from peripheral blood and detect their cytolyic effect towards myeloma cells, to investigate the effects of PDl blockage on NK cell activity against myeloma cells and to explore the underling mechanisms and to test whether transfusion expanded NK (exNK) cells combination with PD1 blockage suppress tumor growth and prolong survival of myeloma bearing mice.Methods1 To develop a protocol to expand NK cells and detect their cytotoxicity1.1 A novel protocol to highly expand NK cells. PBMCs from human peripheral blood were isolated using Ficoll, re-suspended with complete medium and plated onto the six-well dishes at the concentration of 1×106/well. The PBMCs were stimulated with anti-CD16 mAb, and IL-2 was added to the medium throughout the whole culture period.1.2 To investigate NK cell activity and its effect against myeloma cells. At day 14 and 21, the cultured cells were collected and analyzed for NK cell purity, number and function. NK cell phenotype and expression of activating (NKp44, NKp46, NKG2D) and inhibitory receptors (CD158a, CD158b, NKB1) were analyzed by flow cytometry. CD 107a translocation assay was applied to detect perforin/granzymes containing granule degranulation activity of NK cells. Apoptotic cell detection assay. Annexin-V-FlUOS was used to detect apoptotic cells induced by NK cells. Myeloma cell line RPMI8226 and leukemia cell line K562 were as target cells.2. To investigate the effects of PD1 blockage on NK cell function against myeloma cells2.1 To examine whether PD-L1 and PD-L2 mAb blockade could affect NK cell-mediated apoptosis of RPMI8226 cells. Monitor PD-L1 and PD-L2 expression on RPMI8226 cells with by flow cytometry. The target RPMI8226 cells were first incubated with anti-PD-L1 mAb or PD-L2 mAb at the concentrations indicated for two hours and then cocultured with NK cells to examine whether PD-L1 and PD-L2 mAb blockade could increase NK cell-mediated apoptosis using colorimetric MTT assay.2.2 To test PD1 expression on expanded NK cell and the effect of PD1 blockage on NK cell activity. PD1 expression on NK cells were tested on day 0,14 and 21 using flow cytometry, respectively. Anti-PD1 mAb was added to the expansion medium on day 7 of culture at the concentration of 2.5 ?g/ml. At day 14 and 21, the cultured cells were collected and analyzed for NK cell purity, number and function. The expanded NK cells (exNK) and PD1 blocked NK cells (exNK+PD1 blockage) were collected on day 21 of culture for phenotypic analysis, degranulation and cytotoxicity assays.3. To detect the anti myeloma effect of expanded NK cell in vivo.3.1 To establish SCID mouse model bearing MM tumor. Human myeloma cell line RPMI8226 cells at logarithmic growth phase were collected and suspended in normal saline (NS) at 1 ×108 cells/ml. Each SCID mouse (female,6-8 weeks old) was injected subcutaneously near the right foreleg with 100?l of the cell suspension. Treatment was initiated when tumor volume reached to approx.300 mm3.3.2 To investigate the inhibitory effects of expanded NK cell combination with PD1-PDL1/2 blockage on tumor development. The myeloma-implanted mice were randomly divided into four treatment groups:normal saline (NS, control), exNK cells, exNK+PD1 blockage, and exNK+PD-L2 blockage. The mice received NK cells via tail vein injection, PD-L2 mAb via intratumorly (IT) injection and IL-2 via intraperitoneally (i.p.) injection. For a humanity reason, the mouse was euthanized by cervical dislocation when the mouse reached endpoint of our observation, which was defined as when a mouse was unable to creep for taking food and/or water. Tumor size and body weight were monitored every 3-4 days up to the day when the first mouse in control group reached the endpointResult1. To develop a protocol to expand NK cells and detect their cytotoxicity.1.1 NK cell expansion from healthy peripheral blood. NK cell expanded highly after stimulation of CD 16 mAb and IL-2. Expansion rate of PBMCs peaked on day 21 of PBMC culture, with the cell number increased by 1002.2±394.53-fold. Flow cytometric NK cell phenotyping showed that NK cell purity (CD3-CD56+) also reached the peak (79.6%±3.7%) on day 21 of culture. Furthermore results from seven independent experiments showed that NK cells were expanded by 549.9±154.7-fold on day 14 and by 4011.5±1082.4-fold on day 21.1.2 Expanded NK cell was all survival when detected with trypan blue. Both activating receptors and inhibiting receptors expression on exNK cells was significantly increased. Our results showed that the exNK cells presented a strong degranulation capacity after cocultured with K562 (38.12%±6.16%) and RPMI8226 (36.25%±7.69%) as targets, respectively. ExNK cells markedly induced apoptosis of RPMI8226 cell (Annexin-V+RPMI8226 cells) in a NK cell concentration-dependent manner (27.93% ±3.33% and 59.1%±3.6%at E:T ratio of 0.3:1 and 1:1).2. PD1-PD-L1/L2 pathway blocking enhances the expanded NK cell cytotoxicity.2.1 PD-L1 and PD-L2 mAbs blocking enhances the expanded NK cell cytotoxicity. Our results showed that 58.5%±13.0% and 73.1%±7.5% of RPMI8226 expressed PD-L1 or PD-L2, respectively. The lower concentration of the anti-PD-L1 antibody (1.25?g/ml) treatment resulted in more pronounced lysis of RPMI8226 mediated by the exNK cells than the control (without antibody) and higher antibody dose (2.5 ?g/ml) treatment group, respectively (P<0.05 for the two comparisons made). Pre-blocking PD-L2 by anti-PD-L2 antibody at two concentrations (2.5?g/ml and 5.0?g/ml) tested significantly improved the exNK cell mediated lysis of RPMI8226 cells (31.2%±6.6% and 24.1%±3.9%, respectively). However, no dosage effects were observed in this setting of PD-L2 blocking.2.2 PD1 expression on the expanded NK cells and PD1 blocking enhances the expanded NK cell degranulation and cytolytic activity against myeloma cells. Our results showed that PD1 was barely present on resting NK cells, but its expression was gradually induced along with the expansion process. Approximately 26% of NK cells on day 21 of culture were positive for PD1. Adding an anti-PDl antibody into the expansion system markedly improved degranulation activity of NK cells upon incubation with RPMI 8226 as target cells. Compared with the exNK cells without PD1 blocking in expansion system, our results showed that the exNK-PDl blockage presented a significant higher percentage of degranulated (CD3- CD56+CD107a+) cells (64.0%±1.0% vs.53.1%±2.7%, P<0.05). Consistently, exNK+PD1 blockage induced more pronounced apoptosis of RPM18226 than the exNK without PDl blocking (Figure 5E) and the difference between exNK and exNK+PD1 blockage groups was statistically significant (63.2%±6.2% vs.44.9%±7.5%, P<0.05). To elucidate the mechanisms underlying PDl blockade-enhanced NK cell degranulation and cytolytic activity, we next examined the expression of several NK cell activation receptors using flow cytometry technique. Results showed that PD1 blocking markedly enhanced NKp44, NKp46 and NKG2D expression on the exNK cells. Moreover, the percentages of NKp44, NKp46 and NKG2D positive cells in the exNK+PD1 blockage group were significantly higher than those in the exNK group without the PD1 antibody in culture, respectively.3. Adoptive transfer of the exNK, exNK+PD1 blockage and exNK cells plus PD-L2 blocking suppressed myeloma growth and improved survival of myeloma mice.Our results demonstrated that between two and three weeks after subcutaneous inoculation of RPMI8226 cells, all sixteen mice developed tumors with a size approx.300 mm3 and were randomly assigned into control. exNK, exNK-PD1 blockage and exNK+PD-L2 blocking groups, respectively. We found that tumor volumes in the control group grew more rapidly in a time-dependent manner than that in the exNK, exNK+PD-L2 blocking and exNK+PD1 blockage groups, respectively. The exNK and exNK+PD1 blockage transfusion more rapidly sustained the tumor growth than the exNK+PD-L2 blocking. On week 2,3 and 4 after treatment, all the mice from the three treatment groups showed markedly smaller tumor volumes than the control. The exNK+PD1 blockage group yielded much greater efficacy in suppression of tumor growth than exNK cells alone group (1297.0±118.1 vs.2747.2±568.8, P< 0.05)We further compared changes in survival among the control and treatment groups using Kaplan-Meier curve. Our results showed that the mean survival time of the mice treated by the control (saline) group, cxNK. exNK+PD-L2 blocking and exNK+PD1 blockage were 30 days,44 days,49 days and 57 days, respectively. The last myeloma bearing mouse in control group was dead at day 36. The first mouse died in exNK. exNK+PD-L2 blockage and exNK+PD1 blockage groups was on days 35,42 and 47. respectively. On day 53 after treatment, all the mice from the four groups were died. All three treatment regimens significantly prolonged survival of the myeloma mice when compared with the control. Moreover, exNK+PD1 blockage treatment significantly prolonged mice survival than the control (P< 0.01) and exNK alone (P< 0.05), and slightly prolonged the survival rate than the exNK+PD-L2 blockage (P>0.05).Conclusion1. With this protocol using anti-human CD 16 antibody and interleukin (IL)-2, NK (CD3-CD56+) cells could be expanded about 4000-fold with over 70% purity during a 21-day culture. The expanded NK (exNK) cells were shown to be highly cytotoxic to K562 and RPMI8226 cell.2. PD-L1 and PD-L2 were highly expressed on RPMI8226 cells. The expression of PD1 was gradually induced along with the expansion process. PD1 blocking enhanced the expanded NK cell degranulation and cytolytic activity against myeloma cells which maybe related with the enhanced activating receptors.3. Adoptive transfer of the NK cells and combination with PD1/PD-L1/2 pathway blocking suppressed myeloma growth and improved survival of myeloma micc. |
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