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 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 852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A893-A893
Author(s):  
Laurent Gauthier ◽  
Angela Virone-Oddos ◽  
Angela Virone-Oddos ◽  
Jochen Beninga ◽  
Benjamin Rossi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Antitumor Activity ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Activating Receptors ◽  
Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4082-4082
Author(s):  
Michel Van Gelder ◽  
Peter Frings ◽  
Catarina Matos ◽  
Gerard Bos ◽  
Harry Schouten ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Bone Marrow ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Nk Cell ◽  
Tumor Stem Cells ◽  
4T1 Breast Cancer

Abstract Abstract 4082 Poster Board III-1017 Background Patients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. Methods The 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. Results Ninety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence). Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. Conclusions This report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. 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.

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 Maintenance with Histamine and IL-2 Induces a Marked Expansion of Activated CD56bright NK Cells in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1422-1422
Author(s):  
Angelica Cuapio ◽  
Mirte Post ◽  
Sabine Cerny-Reiterer ◽  
Markus Osl ◽  
Volker Huppert ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Leukemic Cells ◽  
Cell Numbers ◽  
Before And After ◽  
Cell Subpopulations ◽  
Maintenance Of Remission

Abstract In acute myeloid leukemia (AML), residual leukemic (stem) cells that escape initial chemotherapy are considered a major source of relapse. Clinical trials have used IL-2 for AML patients with the aim to reduce relapse rates by eliminating residual leukemic cells through activation of NK and T cells. However, monotherapy with IL-2 has led to disappointing results. Nevertheless, recent clinical trials have shown that the co-administration of IL-2 and histamine dihydrochloride (HDC) provides maintenance of remission in AML. Histamine suppresses the formation of reactive oxygen species (ROS) thereby protecting NK and T cells from ROS-induced dysfunction and apoptosis. In addition, IL-2 is considered to maintain the anti-leukemic activity of NK cells. However, the direct effect of this treatment on NK cell numbers and anti-AML activity has not been studied in detail so far. In this study, we analyzed the immunophenotype and function of NK cells in a cohort of 7 AML patients (FAB M2, n=1; M4, n=2; M4-eo with inv 16, n=2; M5, n=2) treated with HDC plus IL-2. All patients had received induction chemotherapy with daunorubicin (45 mg/m² i.v. days 1-3), cytosine arabinoside, ARA-C (2 x 100 mg/m² i.v., days 1-7) and etoposide (100 mg/m² i.v., days 1-5) as well as at least 3 cycles of consolidation chemotherapy with high dose or intermediate dose ARA-C (Sperr et al, Clin Cancer Res 2004;10:3965-3971 and Krauth et al, J Immunol 2006;176:1759-1768). After having achieved a complete hematologic remission, patients were treated with HDC (0.5 mg) plus recombinant IL-2 (0.9 x 106 IU) twice daily s.c. for 21 days per cycle. Blood was drawn before and during treatment with HDC plus IL-2. We found that after one week of treatment with HDC plus IL-2, NK cell numbers increased in peripheral blood (from 101.8 ± 28.25 cells/µl before therapy to 208.2 ± 38.27 cells/µl after therapy, p<0.05). In the NK cell fraction, we observed an astonishing increment of CD56bright NK cells in all treated patients (from 7.2±0.97% or 17.6±5.8 cells/µl before therapy to 38.8±4.4% or 104±19.4 cells/µl after therapy, p<0.05; see Fig.1A/B). The cytotoxic activity of the CD56bright cells, as determined by NK cell degranulation and target cell lysis using the cell line K562, showed a significant increase in comparison to cells obtained before treatment (p<0.05). This was associated with an increased expression of KIR as well as the activation markers NKp44 (see Fig.1C), NKp46, and CD25 on NK cells. Furthermore, we observed a significant increase in expression of CD56 on NK cells after treatment with HDC plus IL-2 in our AML patients (2.5 ± 0.55 fold increase in the mean fluorescence intensity of CD56, p=0.02), whereas CD16 expression did not change significantly. In addition, treatment with HDC+IL-2 also induced an increased proportion of circulating CD4+CD25highCD127low/neg regulatory T cells (Treg). Finally, in vitro stimulation of NK cells with histamine plus IL-2 mimicked the effects observed in vivo. Interestingly, the in vitro treatment was also associated with an increased expression of CD56 without altered expression of CD16, suggesting that this effect could be a specific and reliable indicator of in vivo responses of NK cells to HDC plus IL-2 therapy. In conclusion, treatment with HDC plus IL-2 causes a striking increase in CD56bright NK cells. These specifically expanded NK cells exhibit an activated phenotype with enhanced potential to kill leukemic cells. We propose that the maintenance of remission in AML patients treated with HDC plus IL-2 might, at least in part, be the result of an improved anti-leukemic NK cell function. Fig 1. Effect of HDC plus IL2 on NK cells of AML patients. A) Representative dot plots of the CD56bright and CD56dim NK cell subpopulations from an AML patient treated with histamine+IL2 before and after treatment. B) Absolute cell numbers of CD56bright NK cells of 7 AML patients before and after treatment, *** p<0.01. C) Follow-up of NKp44 and KIR expression after HDC plus IL-2 therapy. Shown are histograms for NKp44 and KIR expression on total CD56+ CD3- NK cells of one patient representative for the majority of patients tested. Fig 1. Effect of HDC plus IL2 on NK cells of AML patients. A) Representative dot plots of the CD56bright and CD56dim NK cell subpopulations from an AML patient treated with histamine+IL2 before and after treatment. B) Absolute cell numbers of CD56bright NK cells of 7 AML patients before and after treatment, *** p<0.01. C) Follow-up of NKp44 and KIR expression after HDC plus IL-2 therapy. Shown are histograms for NKp44 and KIR expression on total CD56+ CD3- NK cells of one patient representative for the majority of patients tested. Disclosures Sperr: MEDA Pharma GmbH & Co. KG: Speakers Bureau. Valent:MEDA Pharma GmbH & Co. KG: Speakers Bureau.

scholarly journals Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities

2021 ◽  
Vol 9 (12) ◽  
pp. e003894
Author(s):  
Ilias Christodoulou ◽  
Won Jin Ho ◽  
Andrew Marple ◽  
Jonas W Ravich ◽  
Ada Tam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Flow Analysis ◽  
Surface Expression ◽  
Myelogenous Leukemia ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Transgenic Expression

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

scholarly journals Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities

2021 ◽  
Author(s):  
Ilias Christodoulou ◽  
Win Jin Ho ◽  
Andrew Marple ◽  
Jonas W Ravich ◽  
Ada Tam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Flow Analysis ◽  
Surface Expression ◽  
Myelogenous Leukemia ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Transgenic Expression

Background: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence. Methods: We characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models. Results: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.ζ/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.ζ/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model. Conclusion: Transgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing.

scholarly journals Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

2021 ◽  
Vol 8 (6) ◽  
pp. 110
Author(s):  
Nathalie Meijerink ◽  
Jean E. de Oliveira ◽  
Daphne A. van Haarlem ◽  
Guilherme Hosotani ◽  
David M. Lamot ◽  
...  
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Relative Abundance ◽  
Nk Cell ◽  
Feed Additives ◽  
Microbiota Composition ◽  
In Ovo ◽  
Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

scholarly journals Therapeutic effects of sesamolin on leukemia induced by WEHI-3B in model mice

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Senthil Nagarajan ◽  
Jae Kwon Lee
Keyword(s):  
Nk Cell ◽  
Neoplastic Cell ◽  
Positive Control ◽  
Cytolytic Activity ◽  
Leukemic Cells ◽  
Therapeutic Effects ◽  
Control Drug ◽  
Lysis Activity

AbstractSesamolin is one of the lignans derived from sesame oil. It has demonstrated significant antioxidant, anti-aging, and anti-mutagenic properties. It also reportedly augments natural killer (NK) cell lysis activity. We previously reported that sesamolin also exerts anticancer effects in vitro and induces enhanced NK cell cytolytic activity against tumor cells. Herein, we aimed to determine the mechanism by which sesamolin prevents and retards tumorigenesis in BALB/c mouse models of leukemia induced by murine (BALB/c) myelomonocytic leukemia WEHI-3B cells. Banded neutrophils, myeloblasts, and monocytic leukemic cells were more abundant in the leukemia model than in normal mice. Sesamolin decreased the number of leukemic cells by almost 60% in the leukemia model mice in vivo; additionally, sesamolin and the positive control drug, vinblastine, similarly hindered neoplastic cell proliferation. Spleen samples were ~ 4.5-fold heavier in leukemic mice than those obtained from normal mice, whereas spleen samples obtained from leukemic mice treated with sesamolin had a similar weight to those of normal mice. Moreover, sesamolin induced a twofold increase in the cytotoxic activity of leukemic mouse NK cells against WEHI-3B cells. These results indicated that sesamolin exerts anti-leukemic effects in vivo.

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