Temporal, Quantitative and Functional Characteristics of Single-KIR Positive Alloreactive NK Cell Recovery Account for Impaired Graft Versus Leukemia Activity after Haploidentical HSCT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3274-3274
Author(s):  
Luca Vago ◽  
Barbara Forno ◽  
Elisabetta Zino ◽  
Simona Di Terlizzi ◽  
Maria T. Lupo Stanghellini ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Option ◽  
Flow Cytometric Analysis ◽  
Cell Activity ◽  
Biological Data ◽  
Cell Recovery ◽  
Hematopoietic Stem

Abstract Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) is a promising therapeutic option for patients lacking a fully compatible donor. Due to extensive T cell depletion, Natural Killer (NK) cell activity represents the only immunological protection against disease relapse for the first months after haplo-HSCT. Clinical studies have associated donor-recipient incompatibility for Human Leukocyte Antigen (HLA) ligands of Killer Immunoglobulin-like Receptors (KIR), with a marked anti-leukemic activity. Alloreactive donor NK cells carrying a single KIR whose ligand is missing in the recipient mediate a potent graft vs. leukemia effect, resulting in reduced incidence of relapse and increased Overall Survival (OS). These exciting results have recently been challenged by conflicting clinical and biological data from different groups. In the present study, we have characterized reconstitution of NK cells, in particular of alloreactive single-KIR+ NK cells, in 58 patients who received CD34+ selected haplo-HSCT for high-risk hematologic malignancies. One month after haplo-HSCT CD56bright/CD56dim NK cell subsets were subverted in their proportions and phenotypic features, accounting for enrichment in maturation intermediates. We show that CD25 and CD117 deregulation by CD56bright, and NKG2A and CD62L by CD56dim, are intrinsic to NK cell physiologic differentiation and support a sequential CD56bright-to-CD56dim NK cell maturation. Consistently, the in vitro functional potential of these maturation intermediates against leukemic blasts was heavily impaired, both in terms of cytotoxicity and of cytokine release. Full mature receptor repertoire reconstitution took at least three months. Alloreactive single-KIR+ NK cells had highly variable frequency ranging from less than 1% to more than 30% of NK cells circulating at 90–120 days after transplantation, independently from predicted NK alloreactivity. Importantly, out of three patients with predicted NK alloreactivity, none had a relative expansion of alloreactive single-KIR+ cells, accounting for less than 1% of circulating NK cells in two of them. As demonstrated by flow cytometric analysis of NK cell CD107a mobilization in response to the HLA class I negative target 721.221, single-KIR+ NK cells at three months after haplo-HSCT showed a not yet fully developed functional reactivity, which was recovered to donor-levels only at later time-points. In line with these observations, clinical outcome of haplo-HSCT was not affected in any way by the presence of donor NK alloreactivity. The incidence of relapse was virtually identical in patients transplanted from alloreactive or non-alloreactive donors. Taken together, our data shed new light onto the kinetics of NK cell differentiation in vivo and suggest that NK alloreactivity could be best exploited by the use of mature donor single-KIR+ selected alloreactive NK cells.

scholarly journals Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul D. Bates ◽  
Alexander L. Rakhmilevich ◽  
Monica M. Cho ◽  
Myriam N. Bouchlaka ◽  
Seema L. Rao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

scholarly journals RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii194-ii195
Author(s):  
Nazanin Majd ◽  
Maha Rizk ◽  
Solveig Ericson ◽  
Kris Grzegorzewski ◽  
Sharmila Koppisetti ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
In Vitro Cytotoxicity ◽  
Orthotopic Mouse Model ◽  
Hematopoietic Stem ◽  
Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

Azacytidine Compromises NK-Cell Activity in AML and MDS Patients Undergoing MRD-Based Pre-Emptive Treatment After Allogeneic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4122-4122
Author(s):  
Katja Sockel ◽  
Claudia Schönefeldt ◽  
Sieghart Sopper ◽  
Martin Wermke ◽  
Marc Schmitz ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Activating Receptors

Abstract Abstract 4122 The hypomethylating agent azacytidine (AZA) represents the standard treatment for many high-risk MDS and AML patients. While the clinical efficacy has been confirmed in several studies, the precise molecular mechanism of action has not been fully understood yet. Human NK-cells play an important role in the regulation of immune responses against malignant cells. Their function is controlled by a complex interplay of activating and inhibitory receptors - some of them being regulated by methylation of the respective genes. We, therefore explored, whether AZA modulates in vitro NK-cell function as well as in vivo during minimal-residual disease (MRD)-guided treatment of imminent relapse in MDS and AML patients treated within the prospective RELAZA trial (NCT00422890). Methods: After purifying NK-cells of healthy donors by MACS (magnetic cell sorting), NK-cells were exposed in vitro to different concentrations of AZA (100nM, 1μM, 3μM) with or without IL-2. In parallel, the NK-cell phenotype of patients (n=12) with AML or MDS, undergoing MRD-guided treatment with AZA after stem cell transplantation was monitored by FACS from peripheral blood samples on day 1, 5 and 7 of the first and second AZA cycle. All patients were still in complete haematological remission at the time of therapy. Results: In vitro, we observed a significant reduction (3,1% to 1,8% p=0.028) of the immature and cytokine-regulating CD56bright NK-cell subpopulation with increasing concentrations of AZA. There was a trend towards a reduced expression of the death-ligand TRAIL, the activating receptors NKG2D and NKp46 and for an increased expression of the inhibitory KIR CD158b1/b2, whereas we could not detect any changes in the expression of FAS-L, Perforin, Granzyme B, NKp30, NKp44, CD69, CD57, DNAM-1, CD16, and NKG2A-CD94. Confirmatory, we observed a significant decrease in the expression of TRAIL (p=0.003), NKG2D (p=0.03) and NKp46 (p=0.006) during AZA treatment in-vivo. Interestingly, these changes appeared to be reversible. The observed reduction of NK-cell activating receptors and TRAIL during AZA treatment correlated with a reduction or stable course of MRD in all analyzed patients. Conclusion: In summary these data suggest that the clinical effects of AZA are not mediated by enhancing NK-cell activity. In fact, the drug may have inhibitory effects on NK-cell function which should be considered when applying AZA in the post-transplant setting. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Modulation of Natural Killer Cell Activity in the Setting of Oncolytic Virotherapy and with a Chimeric Antigen Receptor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 210-210 ◽  
Author(s):  
Chen Xilin ◽  
Jianfeng Han ◽  
Chu Jianhong ◽  
Walter Meisen ◽  
Zhang Jianying ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Nk Cells ◽  
Tumor Cells ◽  
Mouse Models ◽  
Nk Cell ◽  
Cell Activity ◽  
Innate Immune ◽  
The Brain

Abstract Natural killer (NK) cells are innate lymphocytes that can rapidly eradicate tumor cells, especially those lacking MHC Class I molecules. NK cells can also rapidly eradicate herpes virus-infected cells. We designed an oncolytic herpes virus (oHSV) to selectively infect, replicate within, and lyse glioblastoma (GBM), a devastating brain tumor with a median survival of only 15 months following diagnosis. We have shown that the rapid influx of NK cells limits oHSV efficacy in GBM as they impede oHSV replication and spread [Alvarez-Breckenridge et al., Nat Med, 2012, 18(12):1827-34]. In the current study, we developed NK cell-based novel GBM therapies by decreasing the brain influx of NK cells to enhance the efficacy of oHSV, while arming NK cells in the brain with a chimeric antigen receptor (CAR) that targets both the wild-type EGFR and its mutant form EGFRvIII, two GBM tumor-associated antigens. We then investigated the synergistic effects between EGFR-CAR NK cells and oHSV. Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine of NK cells [Yu et al, Immunity, 2006, 24(5):575-90]. We first determined if oHSV efficacy for treatment of GBM would be augmented by inhibiting anti-oHSV activity of NK cells with TGF-β pre-treatment. In vitro, NK cells pre-treated with TGF-β displayed less cytolytic capacity against oHSV-infected GBM cell lines and patient-derived GBM stem-like cells. In viral replication assays, co-culturing oHSV-infected GBM cells with NK cells pre-treated with TGF-β significantly increased virus titers. In an immunocompetent syngeneic GBM mouse model,administration of TGF-β to GBM-bearing mice prior to oHSV injection significantly inhibited intracranial infiltration and activation of NK cells (P < 0.05). In orthotopic human GBM xenograft mouse models and in syngeneic GBM mouse models, TGF-β treatment in vivo prior to oHSV therapy resulted in inhibition of NK cell infiltration, suppression of tumor growth and significantly prolonged survival of GBM-bearing mice (P < 0.05). Furthermore, depletion of NK cells incompletely blocked the positive effects of in vivo treatment of GBM with TGF-β on survival, suggesting that TGF-β may also directly act on other innate immune cells such as macrophages/microglia. These data demonstrate a single dose of TGF-β prior to oHSV administration enhances anti-tumor efficacy for GBM at least in part through the transient inhibition of the innate immune responses to oHSV infection. We next investigated whether NK cell activity could be enhanced to more directly target brain tumors while sparing eradication of oHSV. We therefore infected both human NK-92 cells and primary human NK cells to express the second generation CAR targeting both EGFR and EGFRvIII that we designed. Further, we asked if the treatment with EGFR-CAR NK cells plus oHSV could create a therapeutic synergy for the treatment to brain tumors. In vitro, compared with mock-transduced CAR-NK-cells, EGFR-CAR NK cells exhibited significantly higher cytotoxicity and IFN-γ production when co-cultured with tumor cells, for both NK-92 and primary NK cells (P < 0.01). Further, significantly higher cytolytic activity against tumor cells was obtained when CAR NK cells were combined with oHSV-1 infection of tumor cells, compared to either of the monotherapies alone (P < 0.05). In mice, to avoid oHSV clearance by the EGFR-CAR NK cells following the inoculation of the mouse with tumor cells, we administered these two agents sequentially; administering EGFR-CAR NK cells directly into the tumor first as a single injection of 2 × 106 cells, followed by intracranial infection with 2 × 105 plaque-forming units oHSV five days later, presumably after EGFR-CAR NK survival has diminished. Compared to vehicle controls, intracranial administration of either EGFR-CAR NK cells or oHSV blunted tumor growth. However, the combination of EGFR-CAR NK cells followed by oHSV infection resulted in significantly more efficient killing of tumor cells (P < 0.05) and significantly longer survival for tumor-bearing mice when compared to either monotherapy alone. Collectively, our studies demonstrate that in animal tumor models, we can combine novel NK cell and oHSV therapies to significantly improve survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Heterogeneity of natural killer cells in the mouse.

1981 ◽  
Vol 154 (2) ◽  
pp. 306-317 ◽  
Author(s):  
J A Lust ◽  
V Kumar ◽  
R C Burton ◽  
S P Bartlett ◽  
M Bennett
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Cell Activity ◽  
Spleen Cells ◽  
Murine Spleen ◽  
Total Body

Mice were treated with the bone-seeking isotope, 89Sr, cyclophosphamide, and short-term lethal irradiation in vivo, and murine spleen cells are treated with anti-Nk-1.2 plus complement (C) in vitro. Fresh spleen cell suspensions from the above groups and from beige and neonatal mice were subsequently tested for natural killer (NK) cell activity against a panel of lymphoid and nonlymphoid tumor cell target. NK cell reactivities against YAC-1, MPC-11, and Cl.18 tumors were markedly and consistently reduced in (a) mice treated with 89Sr, (b) spleen cells treated with anti-Nk-1.2 plus C, and (c) C57BL/6 bg/bg mice. In contrast, NK activities against FLD-3 and WEHI-164.1 tumors were usually normal in mice treated with 89Sr, in beige mutant mice, and in spleen cells after treatment with anti-Nk-1.2 antibody and C. It appears, therefore, that two major groups of NK cells exist in fresh mouse spleen cells suspensions. NK-A cells are marrow dependent, Nk antigen positive, and deficient in beige mice; these lyse YAC-1, MPC-11, and Cl.18 tumors. NK-B cells, which are responsible for the lysis of WEHI-164.1 and FLD-3, are Nk antigen negative, marrow independent, and unaffected by the bg/bg mutation. Other features of NK-B cells, suggest that these NK cells, although they share the characteristics mentioned above, differ among themselves especially with respect to age of maturation and susceptibility to cyclophosphamide and total body irradiation. The NK-B group may therefore induce subsets that remain to be defined.

Influence of Neuroleptics on Cytotoxic Activity of Rat Natural Killer Cells

1998 ◽  
Vol 11 (2) ◽  
pp. 57-62
Author(s):  
A.J. Madej ◽  
J. Kowalski ◽  
D. Belowski ◽  
Z. S. Herman
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Vitro Experiment ◽  
In Vitro Experiment ◽  
In Vivo Experiment

The aim of the study was to evaluate the in vivo and in vitro effects of three neuroleptics (chlorpromazine, haloperidol, and sulpiride) on the activity of rat spleen NK cells. In the in vivo experiment, rats were injected with different intraperitoneal doses of neuroleptics given once, for 14 or 28 days. In the in vitro experiment rat spleen NK cells were cultured in medium containing two different concentrations of neuroleptics for three days. The cytotoxic activity of NK cells was evaluated by measuring 51Cr release from YAC-1 target cells after 4-hour incubation. We also measured, using fluorescein-labelled anti-NK monoclonal antibody, the percentage of NK cells in the splenocyte population before and after single intraperitoneal injections of neuroleptics. In the in vitro experiment, both haloperidol (1×10−5 M and 1×10−6 M) and sulpiride (1.5×10−3 M and 1.5×10−4 M) induced a statistically significant decrease in the cytotoxic activity of NK cells. The lower dose of chlorpromazine (6×10−6 M) decreased the cytotoxic activity of NK cells, while the higher dose (6×10−5 M) did not. In the in vivo experiment, both single and repeated doses of chlorpromazine (2 mg /kg /day), haloperidol (0.5 mg/kg/day) and sulpiride (50 mg/kg/day) increased NK cell activity. That effect reflected an increase in NK cell activity but not in the number of NK cells. The study has shown that the immunomodulatory effect of neuroleptics on NK cell activity depends mainly on drug concentrations and experimental conditions.

scholarly journals Augmentation of Cytolytic Activity in Murine Natural Killer Cells and Inhibition of Tumor Growth by the Ethanol Fraction of Oyster Extract

2016 ◽  
Vol 17 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Kaito Sakaguchi ◽  
Ming Zhong ◽  
Saeko Kawai ◽  
Yoshio Shimizu ◽  
Eiichi Gohda
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Cell Activity ◽  
Cytolytic Activity ◽  
Proteinase K ◽  
Nk Cell Activity

A reduced number and/or reduced activity of natural killer (NK) cells, which are important for defense against a variety of cancers and viral infections, occur under various stress conditions and in patients with various diseases. In this article, we report that the 30% to 50% ethanol precipitate of oyster extract (EPOE50) dose-dependently enhanced the activity of mouse spleen NK cells in vitro and in vivo. The activity of EPOE50 was eluted with a molecular weight of about 2000 by gel filtration and was inactivated by periodate but not by proteinase K. The activity of highly purified NK cells was also augmented by EPOE50 but not by oligodeoxyribonucleotide 1585, which mimics bacterial DNA. Administration of EPOE50 to mice stimulated splenic NK cell activity without a change in splenic NK cell populations. Although the proliferation of B16 tumor cells in vitro was slightly stimulated by EPOE50, the growth of B16 melanoma in vivo was dose-dependently suppressed by administration of EPOE50. Taken together, our results indicate that EPOE50 augmented NK cell activity and that its administration to mice inhibited tumor growth presumably through the activation of NK cells and also suggest that the active substance is a sugar-containing oligomer or polymer and is not of bacterial origin.

scholarly journals Increased Concentrations of Circulating Soluble MHC Class I-Related Chain A (sMICA) and sMICB and Modulation of Plasma Membrane MICA Expression: Potential Mechanisms and Correlation With Natural Killer Cell Activity in Systemic Lupus Erythematosus

2021 ◽  
Vol 12 ◽  
Author(s):  
Baptiste Hervier ◽  
Matthieu Ribon ◽  
Nadine Tarantino ◽  
Julie Mussard ◽  
Magali Breckler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nk Cells ◽  
Mhc Class I ◽  
Lupus Erythematosus ◽  
Nk Cell ◽  
Cell Activity ◽  
Healthy Individuals ◽  
Mica Expression

Systemic lupus erythematosus (SLE) is a severe autoimmune disease of unknown etiology. The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are stress-inducible cell surface molecules. MICA and MICB label malfunctioning cells for their recognition by cytotoxic lymphocytes such as natural killer (NK) cells. Alterations in this recognition have been found in SLE. MICA/MICB can be shed from the cell surface, subsequently acting either as a soluble decoy receptor (sMICA/sMICB) or in CD4+ T-cell expansion. Conversely, NK cells are frequently defective in SLE and lower NK cell numbers have been reported in patients with active SLE. However, these cells are also thought to exert regulatory functions and to prevent autoimmunity. We therefore investigated whether, and how, plasma membrane and soluble MICA/B are modulated in SLE and whether they influence NK cell activity, in order to better understand how MICA/B may participate in disease development. We report significantly elevated concentrations of circulating sMICA/B in SLE patients compared with healthy individuals or a control patient group. In SLE patients, sMICA concentrations were significantly higher in patients positive for anti-SSB and anti-RNP autoantibodies. In order to study the mechanism and the potential source of sMICA, we analyzed circulating sMICA concentration in Behcet patients before and after interferon (IFN)-α therapy: no modulation was observed, suggesting that IFN-α is not intrinsically crucial for sMICA release in vivo. We also show that monocytes and neutrophils stimulated in vitro with cytokines or extracellular chromatin up-regulate plasma membrane MICA expression, without releasing sMICA. Importantly, in peripheral blood mononuclear cells from healthy individuals stimulated in vitro by cell-free chromatin, NK cells up-regulate CD69 and CD107 in a monocyte-dependent manner and at least partly via MICA-NKG2D interaction, whereas NK cells were exhausted in SLE patients. In conclusion, sMICA concentrations are elevated in SLE patients, whereas plasma membrane MICA is up-regulated in response to some lupus stimuli and triggers NK cell activation. Those results suggest the requirement for a tight control in vivo and highlight the complex role of the MICA/sMICA system in SLE.

Real-Time In Vivo Biodistribution of Multipotent Adult Progenitor Cells (MAPC): Role of the Immune System in MAPC Resistance in Non-Transplanted and Bone Marrow Transplanted Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 507-507
Author(s):  
Jakub Tolar ◽  
Scott Bell ◽  
Ron McElmurry ◽  
Lily Xia ◽  
R. Scott McIvor ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Sleeping Beauty ◽  
Whole Body ◽  
Term Survival ◽  
Hematopoietic Stem

Abstract MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion&gt;&gt; Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

scholarly journals CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy

2021 ◽  
Vol 9 (3) ◽  
pp. e001975
Author(s):  
Xuan Guo ◽  
Tanel Mahlakõiv ◽  
Qian Ye ◽  
Srinivas Somanchi ◽  
Shuyang He ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Tumor Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Negative Regulator ◽  
Hematopoietic Stem ◽  
Nsg Mice

BackgroundTumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b (CBLB), a negative regulator of lymphocyte activity, on placental CD34+ cell-derived NK (PNK) cell cytotoxicity against tumor cells.MethodsUsing CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34+ hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD-scid IL2R gammanull (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated.Results94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34+ cells differentiated into PNK cells with high cell yield and >90% purity determined by CD56+ CD3− cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells.ConclusionCBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.

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