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 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.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3642-3642 ◽  
Author(s):  
Purvi Gada ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
Philip B. McGlave ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
High Dose ◽  
Unexpected Result ◽  
Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

R603: A New Low Dose Efficient Anti-CD20 Immunotherapy for CLL Patients ?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4155-4155
Author(s):  
Magali Le Garff-Tavernier ◽  
Julie Decocq ◽  
Christophe de Romeuf ◽  
Jean-Luc Teillaud ◽  
Charles-Antoine Dutertre ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Low Dose ◽  
Surface Expression ◽  
Raji Cells ◽  
Healthy Donors ◽  
The One ◽  
Anti Cd20 ◽  
Functional Capacities

Abstract Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. We previously conducted a complete NK cells phenotypic expertise and functional assays including cytotoxicity against K562 cell line and antibody-dependent cellular cytotoxicity (ADCC) with rituximab, showing no major differences between NK cells from CLL patients and NK cells from healthy donors. We are now interested in functional capacities of NK cells in presence or not of a new anti-CD20 mAb: R603, a chimeric anti-CD20 mAb exhibiting a low fucose content as described for EMAB-6 mAb (C. de Romeuf et al, BJH 2008) in comparison to rituximab. To assess the degranulation of NK cells from CLL patients in response to anti-CD20 mAbs, we examined the surface expression of CD107a (percentage of CD107a+ NK cells) after co-incubation of PBMC from untreated CLL patients (n=8) with Raji cells (E/T ratio: 1/1) at 2 concentrations of each anti-CD20 mAb (10 and 1,000 ng/ml). At the higher mAb dose (1,000 ng/ml), R603 related degranulation of CLL NK cells (median (m): 43.6%; range (r): 27.0–79.8) was similar to the one obtained with rituximab (m: 38.9%; r: 22.4–75.2). At the lower dose (10 ng/ml), R603 related degranulation of CLL NK cells (m: 45.7%; r: 28.7–79.2) was similar to the one obtained with the high mAb concentration, contrary to rituximab related degranulation which was significantly decreased (m: 14.1%; r: 1.4–45.4) (p&lt;0.0001). These results are emphasized by ADCC chromium assay performed with purified CLL NK cells (E/T ratio: 5/1 and 10/1) against Raji cells and with or without anti-CD20 mAbs (at 3 doses: 1, 10 and 1,000 ng/ml). R603 related ADCC levels were high whatever the mAb concentration, contrary to rituximab related ADCC levels which were very low at 1 ng/ml and only reached R603 ADCC levels at 1,000 ng/ml. Similar results were obtained with healthy donors. Without addition of Raji cells (CLL PBMC + mAb), at the lower dose (10 ng/ml), none of the NK cells from CLL patients exhibited degranulation with rituximab, contrary to R603 where 5/8 CLL patients exhibited degranulation (cut-off: more than 10% of CD107a+ NK cells). At the higher dose (1,000 ng/ml), NK cells from 6/8 CLL patients with rituximab and from 7/8 CLL patients with R603 showed degranulation and R603 related degranulation levels (m: 32.3%, r: 0.8–51.0) were significantly superior to rituximab related degranulation levels (m: 12.1%, r: 0.1–30.6) (p=0.0005). These results showed that R603 in the presence of CLL B cells might induce CLL NK degranulation. In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy by the ADCC pathway. Moreover, R603 a new anti-CD20 mAb, induced at low dose a significantly higher in vitro ADCC against Raji cells and autologous CLL B cells, compared to rituximab. This R603 mAb feature may be useful in therapeutic strategy for CLL patients.

Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3928-3928
Author(s):  
Michele Levin ◽  
Janet Ayello ◽  
Frances Zhao ◽  
Andrew Stier ◽  
Lauren Tiffen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. 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.

scholarly journals Absence of NKG2D Ligands Defines Human Acute Myeloid Leukaemia Stem Cells and Mediates Their Immune Evasion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 769-769
Author(s):  
Anna M Paczulla ◽  
Kathrin Rothfelder ◽  
Simon Raffel ◽  
Martina Konantz ◽  
Julia Steinbacher ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Surface Expression ◽  
Leukemic Cells ◽  
Pdx Models ◽  
Acute Myeloid

Abstract Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice. Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p<0.001, n=32 AML cases) and selective abilities to initiate leukemia in NSG mice devoid of functional NK cells (NKG2DLneg, 33/35, 94%; NKG2DLpos, 0/35, 0%; p<0.001, n=13 AML cases, Fig. 1b) and to survive chemotherapy in vivo. In mice, NKG2DLneg AML cells generated both NKG2DLpos and NKG2DLneg progeny of which again only latter induced leukemia in re-transplant assays. Even though similar leukemia-specific mutations were retrieved in NKG2DLneg and NKG2DLpos AML cells derived from the same patient (n=12 analysed patients), published LSC, HSC and 17-genes stemness score signatures were specifically enriched in NKG2DLneg fractions. Mechanistically, expression of poly-ADP-ribose polymerase 1 (PARP1) was identified as enriched in NKG2DLneg compared to NKG2DLpos leukemic subpopulations, and PARP1 inhibition (PARPi) using either siRNAs or pharmacological inhibitors such as AG-14361, veliparib, talazoparib or olaparib, increased NKG2DL mRNA transcripts between 6 and >50 fold. PARP1 binding sites were identified by in silico analysis in NKG2DL promoters and binding was confirmed by chromatin immunoprecipitation in the promoters of MICA and MICB. Importantly, treatment with PARPi induced NKG2DL surface expression on LSCs in vitro and in vivo and co-treatment with PARPi and NK cells efficiently suppressed leukemogenesis in patient derived xenograft (PDX) models (Fig. 1c). These data suggest that PARP1 inhibition sensitizes LSCs to NK cell mediated elimination. Finally, NKG2DL surface expression was found to inversely correlate with favorable molecular AML characteristics (favorable ELN risk group vs. other: p=0.034; inv(16) versus other: p=0.023), complete remission rates after induction chemotherapy (all patients: p=0.002, patients <65 years: p=0.004) and patient overall survival (patients <65 years: p=0.028). Enhanced PARP1 expression in leukemic cells furthermore associated with poor clinical outcome (TCGA data set, p=0.0038). In summary, our data link the concept of LSCs to immune escape in human AML and propose the absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes (including CD34 negative AMLs). This LSC specific mechanism of immune evasion could be overcome by treatment with PARP1 inhibitors, which in conjunction with functional NK cells holds promise to eradicate LSCs and promote immune-mediated cure of AML. Fig. 1: Human AML contain NKG2DLpos as well as NKG2DLneg subpopulations but only latter display leukemia initiation capacity (a: left, analysis of n=175 AML cases using NKG2D-Fc staining, right: exemplary flow cytometry plots; b: leukemic infiltration and survival in mice transplanted with NKG2DLneg or NKG2DLpos AML cells sorted from the same AML cases). PARP1 inhibition with AG-14361 up-regulates NKG2DL on CD34+ LSCs, and in vivo co-treatment with AG-14361 and polyclonal allogeneic NK cells suppresses leukemogenesis in PDX models (c). Figure. Figure. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.

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 &lt; 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 (&lt;30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p&lt;0.01, Figure C), blood (6-fold decrease, p&lt;0.01), and liver (4-fold decrease, p&lt;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&lt;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&lt;0.05), and IFN-γ production was reduced (p&lt;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.

Abstract 49: Over-activation Of Natural Killer Cells Due To Dysfunction Of Cd16-mediated Signaling In Adamts13-deficient Ttp Patients With A History Of Relapse

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
FANG ZHOU ◽  
Darise Farris
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
K562 Cells ◽  
Antibody Treatment ◽  
History Of ◽  
Relapse Group

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threaten vascular autoimmune disease. There is no effective method to treat it in clinical trials since pathogenesis of TTP has not been fully elucidated. Here we investigate the role of NK cells in relapse development of TTP. Our results showed that the frequencies of CD3 − CD56 dim CD16 − and CD3 − CD56 bri CD16 − NK cells are increased in TTP patients with a history of relapse. Expression of CD107a, granzyme A and IFN-γ by CD3 − CD56 dim NK cells following in vitro stimulation with PMA/ionomycin / monensin is improved in the relapse group, compared with those on NK cells derived from TTP patients without relapse development. NK cells isolated from TTP patients with a history of relapse indicated stronger cytotoxicity to target K562 cells than those of NK cells derived from TTP patients without relapse development, suggesting prior activation of NK cells in vivo . Treatment with anti-human CD16 antibody up-regulates cytotoxicity of NK cells derived from TTP patients without relapse development. However, Anti-human CD16 antibody treatment does not affect cytotoxicity of NK cells isolated from TTP patients with a history of relapse, suggesting inability of CD16-mediated signaling in NK cells derived from TTP patients with relapse development. These data provide evidence of altered NK cell activation and/or licensing in TTP patients with a history of relapse modulated by CD16-mediated signaling and a new avenue of investigation into mechanisms of TTP immunopathogenesis.

Optimizing Lentiviral Transduction of Human Natural Killer Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4714-4714 ◽  
Author(s):  
Su Su ◽  
Dawn M Betters ◽  
Muthalagu Ramanathan ◽  
Keyvan Keyvanfar ◽  
Aleah Smith ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Phenotype ◽  
Human Renal Cell Carcinoma ◽  
Hiv 1

Abstract Abstract 4714 The development of an efficient method to genetically modify natural killer (NK) cells could be used to characterize NK cell differentiation, acquisition of self-tolerance, tumor trafficking in vivo, as well as to manipulate NK cells to enhance their activity against infectious diseases and tumors. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on LV transduction of either fresh or in vitro expanded human NK cells or its effects on NK cell phenotype and cytolytic function. In this study, we used an HIV-based LV expressing enhanced green fluorescence protein (EGFP) driven by a murine stem cell virus long terminal repeat (MSCV-LTR) promoter to transduce CD3− and CD56+ and/or CD16+ human NK cells that were either resting, IL-2 activated, or expanded in vitro using an irradiated EBV-LCL feeder cell line. We observed that resting NK cells were difficult to transduce with LVs, even at high multiplicities of infection (MOI), with transduction efficiencies (TE) in the range of only 3–14%. The efficiency of LV transduction improved when the NK cells were pre-stimulated in vitro with IL-2: TE improved to 21±0.2% in NK cells cultured for 24 hours in media containing IL-2 (200 U/mL) and 28.7±12.9% in NK cells that underwent in vitro expansion over 9 days prior to transduction using irradiated EBV-LCL feeder cells and media containing IL-2 (200U/mL). Subsequently, we evaluated incremental MOIs (3-200) to optimize LV transduction of expanded NK cells; optimal transduction was achieved using a spinoculation protocol at a MOI of 25 which resulted in the highest transduction efficiencies with the least amount of cell death. Increasing the MOI above this level resulted in a small increase in transduction, but was offset by an increase in NK cell apoptosis/death. Using a one-round, non-spinoculation protocol and an MOI of 30, we obtained a median transduction efficiency of 29% (range 16–41) with excellent retention of NK cell viability. This optimized protocol was used to transduce expanded NK cells with a LV vector encoding an shRNA targeting a region of the NK cell inhibitory receptor transcript NKG2A. Following transduction, surface expression of NKG2A decreased significantly on expanded NK cells compared to non-transduced expanded NK cells and “scramble transduced” LV controls; at a MOI of 10, the MFI of NKG2A on expanded human NK cells decreased 35% compared to non-transduced and LV transduced scramble controls (median MFI 428, 673, 659 in shRNA, non-transduced and scramble LV control transduced NK cells respectively). A comparison of transduction efficiencies using LVs expressing EGFP driven by MSCV-LTR, EF1a, and Ubi promoters showed MSCV-LTR mediated the highest level of gene expression in expanded NK cells. Transduced NK cells maintained stable EGFP transgene expression in vitro, which peaked 5 days following LV transduction and remained stable for an additional 9 days. The phenotype of lentiviral transduced NK cells was similar to non-transduced NK cells. Specifically, expression of CD56, CD16, granzyme A and B, perforin, the inhibitory receptors NKG2A, KIR3DL1, KIR3DL2, and KIR2DL1/DL2, and the activating receptors NKG2D, NCRs NKp46, and NKp30 were not altered in either fresh or expanded NK cells following LV transduction, although we did observe a significant reduction in NKp44 expression in LV transduced cells (22% compared to 50% on untransduced NK cells; 0.02). Furthermore, NK cell function, as assessed by cytokine production and cytotoxicity vs tumor targets was not altered in LV transduced NK cells. A 51Cr release cytotoxicity assay showed GFP+ NK cells, flow sorted following LV transduction of expanded NK cells, had similar cytotoxicity against K562 cells and human renal cell carcinoma cells (RCC) compared to non-transduced expanded NK cell controls (figures). In conclusion, we show that an HIV-1 based lentiviral vector driven by a MSCV-LTR, mediated efficient and stable gene transfer in IL-2 activated and in vitro expanded human NK cells. This study provides valuable insights for methods to optimize the long-term expression of LV transduced genes in human NK cells which could be used to improve their anti-tumor function in vivo. Target: K562 cells Target: RCC cell line Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro education of human natural killer cells by KIR3DL1

2019 ◽  
Vol 2 (6) ◽  
pp. e201900434
Author(s):  
Jason Pugh ◽  
Neda Nemat-Gorgani ◽  
Zakia Djaoud ◽  
Lisbeth A Guethlein ◽  
Paul J Norman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Surface Expression ◽  
Class I ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Basic Protocol

During development, NK cells are “educated” to respond aggressively to cells with low surface expression of HLA class I, a hallmark of malignant and infected cells. The mechanism of education involves interactions between inhibitory killer immunoglobulin–like receptors (KIRs) and specific HLA epitopes, but the details of this process are unknown. Because of the genetic diversity of HLA class I genes, most people have NK cells that are incompletely educated, representing an untapped source of human immunity. We demonstrate how mature peripheral KIR3DL1+ human NK cells can be educated in vitro. To accomplish this, we trained NK cells expressing the inhibitory KIR3DL1 receptor by co-culturing them with target cells that expressed its ligand, Bw4+HLA-B. After this training, KIR3DL1+ NK cells increased their inflammatory and lytic responses toward target cells lacking Bw4+HLA-B, as though they had been educated in vivo. By varying the conditions of this basic protocol, we provide mechanistic and translational insights into the process NK cell education.

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