scholarly journals A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 578-586 ◽  
Author(s):  
Sarah Cooley ◽  
Feng Xiao ◽  
Michelle Pitt ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Cell Lineage ◽  
Transplant Recipients ◽  
Functional Maturation ◽  
Healthy Donors ◽  
Stromal Cell Line ◽  
Immature Cells

Abstract How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56bright cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56dim cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% ± 2.8% of the CD56dim NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56dim NKG2A− KIR− NK cells lack “at least one” inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-γ production. Upon culture with IL-15 and a stromal cell line, CD56dim and CD56bright KIR− NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56bright cells precede CD56dim cells.

scholarly journals Redefining interferon-producing killer dendritic cells as a novel intermediate in NK-cell differentiation

Blood ◽  
2012 ◽  
Vol 119 (19) ◽  
pp. 4349-4357 ◽  
Author(s):  
Fanny Guimont-Desrochers ◽  
Geneviève Boucher ◽  
Zhongjun Dong ◽  
Martine Dupuis ◽  
André Veillette ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Cell Lineage ◽  
Current View ◽  
Antitumoral Activity ◽  
Nk Cell Differentiation ◽  
A Cell

Abstract The cell lineage origin of IFN-producing killer dendritic cells (IKDCs), which exhibit prominent antitumoral activity, has been subject to debate. Although IKDCs were first described as a cell type exhibiting both plasmacytoid DC and natural killer (NK) cell properties, the current view reflects that IKDCs merely represent activated NK cells expressing B220, which were thus renamed B220+ NK cells. Herein, we further investigate the lineage relation of B220+ NK cells with regard to other NK-cell subsets. We surprisingly find that, after adoptive transfer, B220− NK cells did not acquire B220 expression, even in the presence of potent activating stimuli. These findings strongly argue against the concept that B220+ NK cells are activated NK cells. Moreover, we unequivocally show that B220+ NK cells are highly proliferative and differentiate into mature NK cells after in vivo adoptive transfer. Additional phenotypic, functional, and transcriptional characterizations further define B220+ NK cells as immediate precursors to mature NK cells. The characterization of these novel attributes to B220+ NK cells will guide the identification of their ortholog in humans, contributing to the design of potent cancer immunotherapies.

Micrornas Promote Activation and Maturation of Natural Killer Cells Through Toll-Like Receptor Signaling.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2160-2160 ◽  
Author(s):  
Jianhua Yu ◽  
Shun He ◽  
Lai-Chu Wu ◽  
Hsiaoyin Mao ◽  
Sumithira Vasu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Real Time ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Maturation ◽  
Toll Like Receptor ◽  
Rt Pcr ◽  
Tlr Signaling ◽  
Healthy Donors

Abstract Abstract 2160 Introduction: MicroRNAs (miRNAs) are short ribonucleic acids, which consist of an average of 22 nucleotides, and bind to complementary sequences of target mRNAs to result in translational repression or target degradation, thus silencing gene expression. MiRNAs can be abundantly found in circulating blood, yet whether, as a class of regulatory molecules, they may interact with innate immune natural killer (NK) cells has not been explored. Methods: Human NK cells were first enriched from the peripheral blood of healthy donors by negative selection using RosetteSep NK cell enrichment cocktail, followed by positive selection using anti-CD56 microbeads. After purity of ≥ 99% was confirmed by flow cytometry, NK cells were used for experiments. After being isolated from healthy donor serum by ExoQuick Exosome precipitation and verified by immunoblotting for CD9 expression, exosomes were assessed for miRNA content via real-time reverse-transcriptase (RT)-PCR using TaqMan miRNA assays. Purified NK cells were stimulated with either whole exosomes or miRNAs complexed with DOTAP, a liposomal transfection reagent. Downstream activation of Toll-like receptor (TLR) signaling by miRNAs was measured via immunoblotting for NF-kB, and its inhibition was similarly assayed in the presence of TLR blocking antibodies. Flow cytometry was used to assess NK cell activation (via CD69 surface expression) and NK cytotoxicity against tumor cells (in a CD107a degranulation assay). IFN-g production was measured via Real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). For in vivo stimulation, a complex consisting of miRNAs and Lipofectamine 2000 was administered by tail-vein injection. NK cell activation was then measured using the aforementioned in vitro assays. After in vivo stimulation with miRNAs, which was performed in the presence of NK cells or following NK depletion by TM-β1 (IL-2/15Rβ) mAb, development of implanted lymphoma tumor cells was monitored by bioluminescent imaging. NK cells purified from lymphoma patients and from healthy donors were assessed for expression of the NF-kB signaling component, p65, and TLRs via real-time RT-PCR. NK cell maturation was analyzed by flow cytometric staining for surface receptors, such as CD56 and CD94, indicative of NK cell maturation. Results: We found that, in the presence of a low dose IL-12, treatment of human NK cells with several mature miRNAs induced CD69 expression, IFN-g production, and expression of the degranulation marker, CD107a. MiRNA-containing exosomes freshly isolated from normal human donors were also able to activate NK cells, even in the absence of IL-12. In vivo, infusion of several miRNAs into the peripheral blood similarly activated murine NK cells, while T cells were not activated. Furthermore, miRNA administration significantly protected mice from developing tumors, and this occurred in an NK cell-dependent manner. Interestingly, miRNAs also augmented expression of surface markers associated with NK cell maturation, such as CD56 and CD94, suggesting that miRNAs may play a role in promoting NK cell maturation. Mechanistically, we found that stimulation with miRNAs led to downstream activation of NF-kB. This effect was blunted upon blockade of TLR (e.g. TLR1) signaling, and was attenuated in lymphoma patients. Conclusion: Collectively, we provide the first evidence that extrinsic miRNAs, as a class of regulatory molecules, directly activate and may also promote the maturation of NK cells. These effects on NK cell activation and maturation are mediated, at least in part, by the TLR signaling pathway. This phenomenon may be important for normal host defense against infection and/or malignant transformation. Our studies indentify a new function of miRNAs with physiological relevance, and their potential for applications in preventing or treating cancer and infections either alone or as an adjuvant. Disclosures: Jaglowski: Pharmacyclics: Research Funding.

scholarly journals Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Pamela Wong ◽  
Carly C. Neal ◽  
Lily Chang ◽  
Julia A Wagner ◽  
Melissa M. Berrien-Elliott ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Box ◽  
Nsg Mice ◽  
Healthy Donors ◽  
And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

scholarly journals Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF.

1992 ◽  
Vol 175 (3) ◽  
pp. 779-788 ◽  
Author(s):  
M J Robertson ◽  
R J Soiffer ◽  
S F Wolf ◽  
T J Manley ◽  
C Donahue ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Cytolytic Activity ◽  
Stimulatory Factor

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex-unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3876-3876
Author(s):  
Caroline Veuillen ◽  
Jerome Rey ◽  
Rémy Castellano ◽  
Florence Orlanducci ◽  
Françoise Mallet ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Surface Expression ◽  
Resting Cells ◽  
Nk Receptors ◽  
Healthy Donors

Abstract Abstract 3876 Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo. Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study. We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL. We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn't observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing. To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells. Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001). Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2

2016 ◽  
Vol 8 (6) ◽  
pp. 617-629 ◽  
Author(s):  
Wagdi Almishri ◽  
Tania Santodomingo-Garzon ◽  
Tyson Le ◽  
Danuta Stack ◽  
Christopher H. Mody ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Agonistic Antibody ◽  
Adaptive Immune ◽  
Nk Cell Cytotoxicity ◽  
Essential Function ◽  
Tnfα Receptors

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.

scholarly journals Infusion of Umbilical Cord Blood-Derived Natural Killer Cells in Immune-Deficient Mice for the Treatment of Resistant Acute Myeloid Leukaemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5727-5727
Author(s):  
Nadine Khalifé-Saleh ◽  
Meriem Hasmim ◽  
Yanyan Zhang ◽  
Khalil Saleh ◽  
Jean-Henri Bourhis ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Specific Lysis ◽  
Healthy Donors ◽  
Deficient Mice ◽  
Acute Myeloid

Abstract Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukaemia (AML). Success of this NK cell immunotherapy is dependent on obtaining high numbers of functional NK cells that have the potential to survive in vivo. The use of umbilical cord blood (UCB) CD34+ cells as a source of allogenic NK cells is an interesting method that can generate a readily available, non-invasive, off the shelf cellular product. We developed a cytokine-based culture method for the generation of NK cell products derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh UCB units. Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors. UCB-derived NK cells displayed good cytolytic activity against NK-sensitive K562 cells with a percentage of specific lysis varying from 30 to 50%. Cytolysis was directly correlated to CD94 expression since CD94-sorted NK cells were responsible for all the in vitro cytolytic function of differentiated NKs against K562 cells. There was an inconstant susceptibility of patient-derived primary AML cells to UCB-derived NK lysis in vitro with a specific lysis ranging from 0 to 25%. We further characterized UCB-derived NK cells by investigating their toxicity, biodistribution, and anti-leukemic potential in vivo. As adoptive transfer of NK cells is an attractive approach for treating refractory leukemia, immune deficient mice were engrafted with a patient derived AML strain resistant to NK-mediated lysis and doxorubicin. After successful engraftment and randomization, leukemic mice were injected with either UCB- derived NK cells or NK cells from healthy donors (NKhds) or doxorubicin, with one control group that didn't receive any treatment. Mice were sacrificed after 2 weeks of treatment and leukemia load along with NK distribution were evaluated by flow cytometry in the blood, bone marrow (BM) and spleen. There was no evidence of toxicity of UCB-derived or healthy donors NK cells in mice. Both types of cells were preferentially found in the blood and in the spleen, even though NKhds reached much higher levels than UCB-derived NKs. As for treatment efficacy, none of our treatment showed anti-leukemic potential based on the absence of decrease of leukemic cells in BM, blood, and spleen. In vivo microenvironment didn't overcome resistance of the patient derived AML cell to NK lysis or to doxorubicin. Remarkably, all of the UCB derived NK cells founded in vivo expressed the CD94 whereas not more than 20% of the injected cells were positive for this marker. Whether it was by in vivo selection or by in vivo differentiation must be investigated. Interestingly, a small cell population with CD56 and CD34 double staining was distinguished in UCB-derived NK and NK healthy donor treated leukemic mice suggesting in vivo interaction between leukemic and NK cells. Further characterization of this population may help to understand the molecular mechanism of leukemic recognition by NK cells and resistance of leukemic cells to cytolysis. In conclusion, UCB-derived NK generation is feasible. Investigation of the role of CD94 in these cells is needed, as cell sorting by CD94 selection in addition to the CD56 could be an interesting approach in the future to select highly functional expanded NK cells before therapeutic use. Furthermore, infusion of UCB-derived NK cells into immune-deficient mice is achievable and non-toxic. However, in vivo environment didn't overcome primary in vitro resistance of AML cells despite an established interaction. Additional elucidation of AML resistance mechanisms to NK lysis is mandatory before therapeutic application. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3336-3336
Author(s):  
Anudishi Tyagi ◽  
Stanley Ly ◽  
Fouad El-Dana ◽  
Bin Yuan ◽  
Sabrina Grimm ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Treated Group ◽  
Control Treatment ◽  
Poor Overall Survival ◽  
Healthy Donors ◽  
Immunomodulatory Function ◽  
Pdx Models

Abstract Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells. Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs). Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p &lt; 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p &lt; 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p &lt; 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p &lt; 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3. Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. Disclosures Battula: Tolero Pharmaceuticals: Research Funding.

531 AFM13-targeted blood and cord-blood-derived memory-like NK cells as therapy for CD30+ malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A567-A567
Author(s):  
Lucila Kerbauy ◽  
Nancy Marin ◽  
Mecit Kaplan ◽  
Pinaki Banerjee ◽  
Melissa Berrien-Elliott ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Nk Cell ◽  
Specific Antigen ◽  
Hematologic Malignancies ◽  
Cellular Immunotherapy ◽  
Cell Responses ◽  
Healthy Donors

BackgroundNatural killer (NK) cells are a nascent cellular immunotherapy for hematologic malignancies. Target recognition of NK cell-resistant cancers remains a substantial barrier to broad application of NK cell therapy. One solution are bispecific engagers that trigger NK cells via an NK activating receptor when simultaneously engaging a tumor-specific antigen.MethodsHere, we investigated single NK cell responses stimulated by the tetravalent bispecific innate cell engager (ICE®) AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on several types of NK cells.ResultsMultidimensional mass cytometry revealed heterogeneity within AFM13-directed conventional (c)NK cell responses, as well as consistent polyfunctional activation of mature terminally differentiated NK cells across donors. The source of NK cells also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12, IL-15, and IL-18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood expanded NK cells that were pre-activated with IL-12, IL-15 and IL-18 also exhibited enhanced killing with AFM13 stimulation, via upregulation of signaling pathways related to NK cell effector function. These cells were stably pre-loaded with AFM13 enhancing responses to CD30+ lymphomas in vitro and in vivo in immunodeficient NSG mouse models.ConclusionsCollectively, these data identify promising combinations of AFM13 with cytokine-activated adult blood or cord blood NK cells against CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

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