CD137L Reverse the Immunological Synapse Defects of Natural Killer Cells in Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 246-246
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Dean A. Lee ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Leukemic Cells ◽  
Cytolytic Function ◽  
Immunological Synapses ◽  
Acute Myeloid

Abstract Abstract 246 Natural killer (NK) cells are an innate component of immune system that can produce a graft vs. leukemia (GVL) effect after stem cell transplantation. NK cells derived from acute myeloid leukemia (AML) patients are defective in their cytolytic function against leukemic cells. In order to better understand the mechanism of this defect, we performed functional assays examining immunological synapse formation of AML patient NK cells with autologous and allogeneic primary AML cells acting as antigen-presenting cells (APCs). Confocal microscopy was used to image and score F-actin polymerization at the immunological synapse between patient NK cells and leukemic cells. Accumulation of F-actin beneath the area of the NK: APC contact site is a hallmark of NK lytic synapses and allows signaling molecules to regulate appropriate activation and effector function. AML patient derived NK cells (AML-NK cells) formed significantly fewer synapses with autologous leukemia cells than healthy donor NK cells (12% versus 30%, n = 16. p > 0.001). Moreover, AML-NK cells were defective in their ability to recruit the key receptor NKG2D and the signaling molecule phosphotyrosine to immunological synapse contact sites. Signaling through the costimulatory ligand4-1BB-L (CD137L) has been shown to activate T cells, enhance antitumor responses and has multiple immunomodulatory effects on dendritic cells and NK cells. We postulated that AML-NK cells could be activated for enhanced cytolytic activity using artificial APCs generated to express CD137L. To test this, we setup co-culture assays using AML-NK cells and artificial CD137L-APCs before subsequent examination of immunological synapse function with AML blasts. Stimulated AML-NK cells that formed cell conjugate interactions with AML blasts, showed a significant increase in formation of immunological synapses compared to unstimulated AML-NK cells. The number of AML-NK/AML blast immunological synapses increased 16 hours after stimulation and peaked at approximately 72 hours. CD137L stimulation of AML-NK cells was also associated with increased cytotoxic function against primary AML cells (n = 6, p <0.01). Furthermore, CD137L stimulation increased recruitment of tyrosine-phosphorylated proteins at AML-NK immunological synapses compared with unstimulated control experiments (RRI 4.1 versus 2.3, n = 3, p < 0.01). Taken together, our data suggests that immune functional suppression of AML-NK cells in leukemia patients can be reversed by CD137L activation signaling, resulting in enhanced F-actin synapse formation, phosphotyrosine signaling, and cytolytic function. Thus, enhanced recruitment of signaling molecules to the NKIS may represent a novel immunomodulatory function of CD137L in the NK cell–mediated killing of AML cells. These findings should aid development of new immune based therapies for leukemia. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2663-2663
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Immune Suppression ◽  
Direct Contact ◽  
Myeloid Leukemia ◽  
Synapse Formation ◽  
Nk Cell ◽  
Immune Synapse ◽  
Acute Myeloid

Abstract Abstract 2663 Poster Board II-639 Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures: No relevant conflicts of interest to declare.

Defective expression and function of natural killer cell–triggering receptors in patients with acute myeloid leukemia

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3661-3667 ◽  
Author(s):  
Régis T. Costello ◽  
Simona Sivori ◽  
Emanuela Marcenaro ◽  
Marina Lafage-Pochitaloff ◽  
Marie-Joelle Mozziconacci ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Killer Cell ◽  
Cytolytic Activity ◽  
Receptor Interaction ◽  
Tumor Escape ◽  
Cytolytic Function ◽  
Acute Myeloid

The cytolytic function of natural killer (NK) cells is induced by the engagement of a series of activating receptors and coreceptors some of which have recently been identified and collectively termed natural cytotoxicity receptors (NCRs). Here, we analyzed the cytolytic function of NK cells obtained from patients with acute myeloid leukemia (AML). In sharp contrast with healthy donors, in most (16 of 18) patients with AML the majority of NK cells displayed low NCR surface density (NCRdull). This phenotype correlated with a weak cytolytic activity against autologous leukemic cells that could not be reversed by the monoclonal antibody-mediated disruption of HLA class I/killer immunoglobulinlike receptor interaction. The remaining 2 patients were characterized by NK cells having an NCRbright phenotype. Surprisingly, although displaying NCR-mediated cytolytic activity, these NCRbright NK cells were unable to kill autologous leukemic blasts. Importantly, the leukemic blasts from these 2 patients were also resistant to lysis mediated by normal NCRbrightallogeneic NK cells. Our study suggests that in most instances the inability of NK cells to kill autologous leukemic blasts is consequent to low NCR surface expression. In few cases, however, this failure appears to involve a mechanism of tumor escape based on down-regulation of ligands relevant for NCR-mediated target cell recognition.

Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients

Blood ◽  
2011 ◽  
Vol 118 (12) ◽  
pp. 3273-3279 ◽  
Author(s):  
Antonio Curti ◽  
Loredana Ruggeri ◽  
Alessandra D'Addio ◽  
Andrea Bontadini ◽  
Elisa Dan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Median Number ◽  
Active Disease ◽  
Acute Myeloid

Abstract Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56+CD3− natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor–ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 106/Kg. T cells were < 105/Kg. No NK cell–related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipient's targets. Adoptively transferred NK cells were alloreactive against recipient's cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

scholarly journals Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia

2020 ◽  
Author(s):  
Adeline Crinier ◽  
Pierre-Yves Dumas ◽  
Bertrand Escalière ◽  
Christelle Piperoglou ◽  
Laurine Gil ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Single Cell ◽  
Natural Killer ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Single Cell Rna Sequencing ◽  
Acute Myeloid

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

Adoptive Immunotherapy with Haploidentical Kir Ligand-Mismatched Natural Killer Cells In Elderly High Risk Acute Myeloid Leukemia Patients: Biological and Clinical Results of A Pilot Study

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4287-4287
Author(s):  
Antonio Curti ◽  
Loredana Ruggeri ◽  
Alessandra D'Addio ◽  
Andrea Bontadini ◽  
Valeria Giudice ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Active Disease ◽  
Acute Myeloid

Abstract Abstract 4287 Purpose: To evaluate safety, feasibility and anti-leukemia potential of haploidentical KIR-L mismatched natural killer (NK) cell infusion in elderly high risk acute myeloid leukemia (AML) patients. Patients and Methods: Thirteen patients (5 active disease, 2 molecular relapse and 6 complete remissions) with median age 62 years (range 53–73) received NK cell infusion after immunosuppressive chemotherapy (fludarabine/cyclophosphamide), followed by interleukin-2. Highly purified CD56+CD3- NK cells from haploidentical KIR-L mismatched donors were used. The median number of infused NK cells was 2.74 × 106/Kg. T cells were less than 105/Kg. NK cell chimerism, phenotyping, and functional assays were performed. Results: No significant toxicity, including graft versus host disease, related to NK cell infusion was observed. Among patients with active disease, 1/5 obtained transient complete remission (CR), whereas 4/5 patients had no clinical benefit. Both patients in molecular relapse obtained CR, which lasted 9 and 4 months. Three/6 patients in morphologic CR are disease-free after 34, 32 and 18 months. Donor NK cells were demonstrated in the peripheral blood (PB) of all evaluable patients with a peak at day 10 after infusion and, in some cases, also in the bone marrow (BM). NK alloreactivity was demonstrated in vivo by the detection of donor-derived postinfusion NK clones capable of killing recipient targets. Conclusion: Infusion of purified CD56+CD3- NK cells is feasible and safe in elderly high risk AML patients. Adoptively transferred NK cells, which can be detected in PB and BM after infusion, are alloreactive against recipient cells and may induce an anti-leukemic activity. Disclosures: No relevant conflicts of interest to declare.

Defective killing of dendritic cells by autologous natural killer cells from acute myeloid leukemia patients

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2186-2188 ◽  
Author(s):  
Cyril Fauriat ◽  
Alessandro Moretta ◽  
Daniel Olive ◽  
Régis T. Costello
Keyword(s):  
Acute Myeloid Leukemia ◽  
Dendritic Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
T Lymphocyte ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Lymphocyte Responses ◽  
Acute Myeloid

Abstract At the frontier between innate and adaptive immunity, dendritic cells (DCs) secrete numerous cytokines and express costimulatory molecules that initiate or enhance natural killer (NK) and T-lymphocyte responses. NK cells also regulate DC physiology by killing immature DCs (iDCs), thus limiting inflammation and inappropriate T-lymphocyte tolerization. In a previous study, we have reported that NK cells from acute myeloid leukemia patients (AML-NK cells) have deficient natural cytotoxicity receptor (NCR) expression. Herein, we analyzed the consequences of such a defect regarding the regulatory role of AML-NK cells in DC physiology. We show that NK cells display poor cytolytic capacities against DCs derived from healthy donor monocytes or derived from autologous leukemic blasts. These data point to a novel defect in the regulation of adaptive immune responses initiated by DCs in AML patients. This may lead to specific T-lymphocyte tolerization by spontaneous or ex vivo expanded iDCs expressing leukemia-derived antigens. (Blood. 2005;106: 2186-2188)

CD137 ligand mediates opposite effects in human and mouse NK cells and impairs NK-cell reactivity against human acute myeloid leukemia cells

Blood ◽  
2010 ◽  
Vol 115 (15) ◽  
pp. 3058-3069 ◽  
Author(s):  
Tina Baessler ◽  
Jean Enno Charton ◽  
Benjamin Joachim Schmiedel ◽  
Frank Grünebach ◽  
Matthias Krusch ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Tumor Necrosis Factor Receptor ◽  
Interleukin 10 ◽  
Leukemic Cells ◽  
Monocytic Differentiation ◽  
Cell Reactivity ◽  
Acute Myeloid

Abstract Natural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34+ cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-γ production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.

scholarly journals CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

Haematologica ◽  
2020 ◽  
Author(s):  
Mark Gurney ◽  
Arwen Stikvoort ◽  
Emma Nolan ◽  
Lucy Kirkham-McCarthy ◽  
Stanislav Khoruzhenko ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Chimeric Antigen Receptor ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cd38 Expression ◽  
Nk Cell Therapy ◽  
Acute Myeloid

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

scholarly journals Natural Killer Cells Enhance Transplantation Outcomes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-27-SCI-27
Author(s):  
Sarah A. Cooley
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Gene Content ◽  
Graft Vs Host Disease ◽  
Cytokine Stimulation ◽  
Cmv Reactivation ◽  
Acute Myeloid

Natural killer (NK) cells, the first lymphocyte subset to reconstitute after hematopoietic cell transplantation (HCT), may enhance transplant outcomes by killing virally-infected or malignant cells to reduce relapse and treatment-related mortality by promoting engraftment and by reducing graft vs. host disease. The function of NK cells is regulated by the net balance of signaling via several families of activating or inhibitory receptors. The killer-cell immunoglobulin-like receptor (KIR) family is of particular importance in HCT because of its interactions with class I human leukocyte antigen (HLA) molecules. HLA-C, HLA-Bw4 and some HLA-A allotypes function as ligands for the inhibitory KIR receptors, thus mediating tolerance to self. HLA-mismatched HCT may generate alloreactive NK cells in the recipient when there is a KIR-ligand mismatch at HLA-C, B or A. The proof of concept for this principle was established by the Perugia group in haploidentical transplants, where KIR-ligand mismatch was associated with reduced relapse in patients with acute myeloid leukemia. Subsequent groups have studied this and other algorithms, including KIR ligand absence or KIR-KIR ligand genotyping to evaluate the role of NK cells in mediating protection after allogeneic HCT. Human KIR haplotypes are defined by gene content, where the A haplotype contains mainly inhibitory KIR, and B haplotypes contain more activating KIR. Unrelated donors with more KIR B haplotype genes have been associated with protection from relapse and improved survival in AML, an effect which is enhanced in recipients expressing HLA-C1. Although NK cells mediate beneficial effects after HCT, compared to adult donor NK cells, engrafting NK cells are immature and hyporesponsive when exposed to tumor targets. Recently human cytomegalovirus (CMV) has been shown to drive the expansion of a population of long-lived CD57+NKG2C+ NK cells with attributes of immunologic memory. These cells have heightened capacity for cytokine production or cytolytic response to tumor targets. Several groups have shown that CMV reactivation correlates with reduced relapse after allogeneic HCT, suggesting that these CMV “adaptive” NK cells may contribute to the graft vs. leukemia effect. Strategies to isolate and expand “adaptive” NK cells without clinical CMV reactivation are being explored. Lastly, adult donor NK cells are an appealing population to exploit for adoptive cellular therapy. Donors can be chosen based on predicted NK cell alloreactivity or based on KIR gene content. Adoptively transferred NK cells which expand and proliferate in vivo in response to cytokine stimulation (IL-2 or IL-15) are potent anti-tumor effectors which do not mediate graft vs. host disease. Several groups are using haploidentical adult NK cell products to treat acute myeloid leukemia, multiple myeloma, lymphoma, myelodysplasic syndrome and a variety of solid tumors. Various approaches using adoptive transfer of NK cells, together with HCT, targeting antibodies which mediate antibody dependent cellular cytotoxicity, or cytokine stimulation are being tested clinically. In summary, we are just beginning to define the complexity of NK cell interactions with HLA and other ligands and to describe different properties of various NK cell subsets to develop more sophisticated strategies to exploit NK cells to treat cancer. Disclosures No relevant conflicts of interest to declare.

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