scholarly journals Promoting the Effector Function of Natural Killer (NK) Cells with a Focus on Bispecific Redirecting Antibodies

2021 ◽  
Vol 1 (3) ◽  
Author(s):  
Pouya Safarzadeh Kozani ◽  
Pooria Safarzadeh Kozani ◽  
Abdolkarim Sheikhi
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Effector Function

scholarly journals M144, a Murine Cytomegalovirus (Mcmv)-Encoded Major Histocompatibility Complex Class I Homologue, Confers Tumor Resistance to Natural Killer Cell–Mediated Rejection

1999 ◽  
Vol 190 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Erika Cretney ◽  
Mariapia A. Degli-Esposti ◽  
Eloise H. Densley ◽  
Helen E. Farrell ◽  
Nick J. Davis-Poynter ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
Effector Function ◽  
Class I ◽  
Murine Cytomegalovirus ◽  
Cell Effector Function ◽  
Cell Effector

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2–deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell–mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell–mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2–activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Defective Natural Killer (NK) Receptor Expression and Effector Function in Acute Myeloid Leukemia Partially Normalize At Remission and Predict Response to Chemotherapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2591-2591
Author(s):  
Kate Stringaris ◽  
Takuye Sekine ◽  
Ahmad Khoder ◽  
Abdullah Alsuliman ◽  
Pavlu Jiri ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Induction Chemotherapy ◽  
Myeloid Leukemia ◽  
Healthy Donor ◽  
Nk Cell ◽  
Effector Function ◽  
And Function ◽  
Acute Myeloid

Abstract Abstract 2591 Despite favorable initial responses to induction chemotherapy, most patients with acute myeloid leukemia (AML) will relapse. We hypothesized that just as cancers evade immunosurveillance by suppressing the immune response (“immunoediting”), remission and relapse in AML may be determined by similar immune interactions. After stem cell transplantation natural killer (NK) cells exert powerful allogeneic graft vs. leukemia effects. To explore immunosurveillance by autologous NK cells and immunoediting by AML blasts, we prospectively analyzed NK surface phenotype and function in AML patients at presentation and following remission induction. Using multi-color flow cytometry, we analyzed the surface expression of natural cytotoxicity receptors (NCRs), killer immunoglobulin receptors (KIRs) and C-type lectins directly ex-vivo in 32 consecutive patients at presentation and following complete remission (CR) in 12 patients for whom remission samples were available. Results were compared with 15 healthy controls. NK effector function in AML was measured against K562 leukemia targets and autologous AML blasts by CD107a degranulation and interferon-gamma (IFNγ) and TNF-alpha (TNFα) production. NK cells from 32 patients with AML at diagnosis had an abnormal phenotype compared to controls, with downregulation of the activatory receptor NKp46 (MFI 187± 15 vs. 266± 24, p=0.007) and upregulation of the inhibitory receptor NKG2A (mean 45%± 4.2 vs. 32%± 2.7, p=0.046). Moreover, AML-NK cells were defective in their effector function with significantly reduced CD107a degranulation (5% vs. 11%, p=0.0002), TNFα (1% vs. 3%, p=0.008) and IFNγ production (1% vs. 5%, p=<0.0001). In the 12 patients who achieved remission following induction chemotherapy NKp46 expression normalized from an MFI 122 at presentation to 242 (p=0.01); however, NKG2A expression continued to increase (45% vs. 61%, p=0.008). At remission AML-NK cells displayed CD107a degranulation levels comparable to that of healthy donor NK cells (9% vs. 11%, p=0.4). In contrast, TNFα and IFNγ production only partially normalized (2% vs. 3%, p=0.3) and (3% vs. 5%, p=0.04), respectively. AML patients with NKG2A overexpression (> median of 32.6%) at diagnosis were significantly less likely to achieve CR post chemotherapy compared to those with lower NKG2A expression (78% vs. 32% p= 0.041). Furthermore, patients who failed to respond to induction chemotherapy had significantly reduced NK effector function at diagnosis compared to normal controls (mean TNFα production 1% cf. 5% p=0.019). We then sought for evidence of immunoediting by AML on NK cell phenotype and function. Co-incubation of healthy donor NK cells with primary AML blasts for 24 hours resulted in significant reduction in TNFα production (p=0.02), IFNγ production (p=0.01) and a trend to reduced CD107a degranulation (p=0.07) against K562 leukemia targets. Our results indicate that AML blasts can produce long-lasting changes in NK cell subsets and impair their effector function and favouring immune escape from NK cell control. This work justifies larger prospective analyses to relate NK-based prognostic factors to classical predictive factors for remission and relapse, and should guide the development of NK cell based immunotherapy to improve outcome in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deletion of CISH and TGFβR2 in iPSC-Derived NK Cells Promotes High Cytotoxicity and Enhances In Vivo Tumor Killing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2780-2780
Author(s):  
Alexandra Gerew ◽  
Steven Sexton ◽  
Kevin M Wasko ◽  
Mark S Shearman ◽  
Kate Zhang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Gene Editing ◽  
Nk Cell ◽  
Effector Function ◽  
Negative Regulator ◽  
Publicly Traded ◽  
The Past ◽  
Tumor Killing

Abstract Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells. Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity. We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing. In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. Disclosures Gerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Generation of Natural Killer Cells with Enhanced Function from a CRISPR/Cas12a-Edited Induced Pluripotent Stem Cell Line

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-8
Author(s):  
Jung-Il Moon ◽  
Melissa S Chin ◽  
Andrew T Burden ◽  
Steven Sexton ◽  
Kevin Wasko ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Effector Function ◽  
Publicly Traded ◽  
Killer Cells ◽  
Killing Assay ◽  
Induced Pluripotent

Adoptive cell therapy using T cells to treat cancer is efficacious in a number of hematologic malignancies. Recently, natural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. NK cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For instance, KIR mismatching enables allogenic NK cells to kill MHC-positive tumor cells similar to MHC-negative tumor cells. Effector function of allogeneic NK cells are typically diminished by limited functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing, however, can overcome these biological limitations. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene 2, TGFBR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR/Cas12a to generate edited iPSC lines that were differentiated into CD56+ iNK cells. Specifically, we generated TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iPSC clones with bi-allelic gene disruption confirmed by next generation sequencing. Importantly, we also confirmed that the edited clones were pluripotent. In particular, a minimum of 3 clones from each genotype were differentiated to CD56+ iNK cells. After differentiation, &gt;90% of the cells expressed CD56 for all genotypes. Additionally, we observed the expression of canonical natural killer cell markers such as CD16, NKG2A, KIRs, NKp46, NKp44, and NKp30 within this CD56+ population. We tested the effector function of TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iNKs in a variety of molecular and functional assays, including a spheroid killing assay and an in vitro serial killing assay. For example, we utilized a SK-OV-3 spheroid killing assay to determine the intrinsic ability for the iNK cells to kill tumor targets following the differentiation process. TGFβR2-/-, CISH-/-, and TGFβR2-/-/CISH-/- iNKs reduce the size of SK-OV-3 ovarian tumor spheroids more effectively than control iNK cells in the presence of exogenous TGF-β. In conclusion, we have established an iPSC editing platform that can generate a near infinite supply of natural killer cells with enhanced tumor killing function, paving the way for future off-the-shelf cell therapies for application to broad oncology indications. Disclosures Moon: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chin:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Burden:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Borges:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Welstead:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Two-signal requirement for activation and effector function of natural killer cell response to allogeneic tumor cells

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4456-4463 ◽  
Author(s):  
Jian-Xin Gao ◽  
Xingluo Liu ◽  
Jing Wen ◽  
Michael A. Caligiuri ◽  
Iwona Stroynowski ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Cell Response ◽  
Killer Cell ◽  
T Cell Response ◽  
Effector Function ◽  
Class I

AbstractOptimal activation of T cells requires delivery of both antigenic and costimulatory signals. It is unclear, however, if the function of the natural killer (NK) cells is also modulated by these 2 signals. Here we report that efficient control of solid allogeneic tumors by NK cells depends on codelivery of both B7-1 and major histocompatibility complex (MHC) class I on the tumor cells. The codelivery is required for optimal expansion and effector function of NK cells in response to both melanoma and plasmocytoma that expressed allogeneic MHC class I. Our results demonstrate that the 2 signals required for T-cell function also can regulate NK immunity and reveal an important similarity between the innate NK response and the adaptive T-cell response. (Blood. 2003;102:4456-4463)

scholarly journals Natural Killer Cells Are Present in Rag1−/− Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke

2021 ◽  
Author(s):  
Leoni Rolfes ◽  
Tobias Ruck ◽  
Christina David ◽  
Stine Mencl ◽  
Stefanie Bock ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Subset ◽  
Neurological Deficits ◽  
In Vitro Assays ◽  
Experimental Stroke ◽  
Transfer Model

AbstractRag1−/− mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1−/− mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1−/− and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1−/− NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1−/− were comparable in number and function to those in WT mice. Rag1−/− mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1−/− controls. Our results indicate that NK cells from Rag1−/− mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.

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