NK Cells of Acute Myeloid Leukemia Patients Exhibit Exhausted Phenotype with Impaired Functional Activity

Abstract Acute myeloid leukemia (AML) accounts for about one third of all leukemias, with half of all leukemia deaths. The low 5-year survival rate and manifestation of this deadly disease in old age reinforce the need for new safe and effective AML therapies. Considering the exceptional role of immune cells in the recognition and elimination of tumor cells, one of these methods is immunotherapy. However, for the development of immunotherapeutic approaches, it is necessary to clearly understand the role of certain effector cells in the pathogenesis of the disease, as well as to have the knowledge about the phenotypic characteristics of these cells. Natural killer (NK) cells play important roles in innate cancer immunosurveillance, and some published data indicate that the antitumor function of NK cells is reduced in AML patients. To expand upon previous work, we performed a comprehensive analysis of the phenotypic and functional characteristics of NK cells in previously untreated AML patients that took into account a wide variety of biomarkers. The goals of this study were to define the phenotypic and functional differences in NK cells from AML patients and healthy donors and determine how these parameters affect outcome of the disease. We used 14-color flow cytometry to assess more than 30 measurable markers on NK cells from the peripheral blood of 33 untreated AML patients and age-matched healthy controls. In addition, NK cells were tested for interferon-γ responses under antibody-dependent cellular cytotoxicity conditions. Results in AML patients were then compared to healthy donors. We found that the NK cells of patients with AML have a significantly lower capacity to secrete IFN-γ and showed numerous signs of an exhausted phenotype, as compared to healthy controls. These included increased surface expression of CD39, PD-1, and LILRB1 on NK cells from AML patients. Interestingly, surface expression of the TIGIT checkpoint receptor did not differ between AML patients and healthy donors, but surface expression of the activating receptor DNAM-1, which shares the same ligands on tumor cells, was decreased. All of these data confirm that the NK cells of AML patients are functionally impaired. We also noted that the frequency of CD57 + NKG2C + KIR + memory-like "adaptive" NK cells was greater in the blood of AML patients. The proportion of adaptive NK cells did not correlate with the age of donors. Phenotypic features of this cell subpopulation from the AML patients included significantly increased expression levels of CD56 and significantly lower expression of the activating receptor DNAM-1. Disclosures Zhigarev: Janssen R&D: Research Funding; Immunitas Therapeutics: Consultancy; Tavotek Biotherapeutics: Consultancy. Drenberg: Janssen R&D: Current Employment. Campbell: Janssen R&D: Research Funding.

Download Full-text

Changes in the Main Bone Marrow Cell Subpopulations May Potentially Contribute to the Initiation of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-128097 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2494-2494

Author(s):

Shlomit Yehudai-Reshef ◽

Rawan Sabah ◽

Tal Gabay ◽

Tsila Zuckerman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Cell Viability ◽

Myeloid Leukemia ◽

Healthy Controls ◽

Healthy Donors ◽

Cluster 2 ◽

Acute Myeloid

Introduction: In acute myeloid leukemia (AML), differentiation and proliferation of early progenitors is inhibited, leading to blast accumulation in the bone marrow (BM) and in the peripheral blood (PB). Hematopoietic stem cells (HSCs) reside in specialized BM microenvironment (ME), mainly containing osteocytes and adipocytes, derived from BM mesenchymal stem cells (MSCs). Recent studies suggest a possible role of the ME in various hematological malignancies, that either serves as a potential source of an altered milieu or mediates abnormal interaction with HSCs. BM ME contributes to disease pathogenesis, chemotherapy resistance and recurrence in certain types of hematological cancers, but there is limited evidence that BM cells themselves may initiate AML. BM derived MSCs are characterized by a potential to mainly differentiate to adipocytes, osteocytes or chondrocytes. Our experiments have revealed an imbalance between osteocytes and adipocytes in the BM of AML patients that is not observed in the BM of healthy donors. We hypothesize that this imbalance could result in the development of leukemic stem cells/pre-leukemic cells, leading to leukemia initiation. The current study aimed to characterize the composition of AML BM derived adipocytes and osteocytes and evaluate the ability of these subpopulations to support AML cell viability. Methods: Secreted levels of adipogenic and osteogenic biomarkers in the BM plasma were measured using ELISA. The potential of AML MSCs to differentiate to adipocytes and osteocytes was assessed using FACS analysis. Exome sequencing was applied for genetic alteration screening in AML MSCs. Obtained adipocytes and osteocytes were separately co-cultured with AML cells and colony forming unit (CFU) assays were used to evaluate AML cell viability. ProcartaPlex immunoassay was used to evaluate levels of cytokines and other potential mediators secreted to the conditioned media. Results: The study included BM samples of 10 AML patients and 13 healthy donors. Levels of secreted FABP4 and osteocalcin biomarkers were reduced in AML plasma (279.7±164 pg/ml and 0.00057±0.017 pg/ml, respectively) compared to that of healthy controls (1230±1502 pg/ml and 0.0016±0.00052 pg/ml, respectively), with P=0.02 for FABP4 and P=0.017 for osteocalcin. According to adipocyte cell differentiation, patients were divided into two clusters. Cluster-1 demonstrated a higher expression level of FABP4 (mean 65.2±11.4%) compared to healthy controls (mean: 47.5±19.2 %; P=0.05), while cluster-2 exhibited a lower level of FABP4 expression (mean 30.78±6.1%) than healthy controls (mean 47.6±19.2%; P=0.01). A nonsynonymous somatic mutation in the AHNAK2 gene (V3209L) was found in patients' mesenchymal stromal cells. A statistically significant reduction in AML cell proliferation (P<0.01) was revealed following co-culture with adipocyte cells of cluster-1 patients. Of the 10 cytokines tested, only ANG1 levels were significantly elevated in the co-culture of adipocytes from cluster-1 (189.5±17.3pg/ml), cluster-2 (258.08±34.33pg/ml) or osteocytes (392.04±58.3pg/ml) with AML cells of the same patients compared to the levels observed in controls (0.002±0.052pg/ml for adipocytes and 168.663pg/ml for osteocytes; P<0.01). Conclusions: The current study has demonstrated that changes in the secretion of stromal specific biomarkers and in the MSC ability to differentiate to adipocytes and osteocytes indicate impaired functional properties of BM MSCs in AML. Within the adipocyte subpopulation in the AML BM, clusters with either increased or inhibited adipogenesis have been revealed. These variations in adipocyte and osteocyte cells reflect interpatient heterogeneity in AML BM subpopulations. The AHNAK2 gene mutation found in AML MSCs in our study and the previously suggested role of this gene in adipocyte differentiation regulation, may explain in part the phenotype diversity observed in AML patients. Adipocyte cells, derived from an AML patient, were found to inhibit normal hematopoiesis in HSCs of the same patient. This specific crosstalk may be related to the elevated levels of ANG1 mediating abnormal signals from adipocytes to HSCs. Overall, the findings of this study may support the contribution of abnormal composition of the main BM subpopulations to leukemogenesis and could ultimately pave the way to novel therapeutic strategies targeting the BM niche. Disclosures No relevant conflicts of interest to declare.

Download Full-text

TCRab Deficient CAR T-Cells Targeting CD123: An Allogeneic Approach of Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v126.23.2555.2555 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2555-2555 ◽

Cited By ~ 1

Author(s):

Roman Galetto ◽

Céline Lebuhotel ◽

Agnès Gouble ◽

Nuria Mencia-Trinchant ◽

Cruz M Nicole ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Surface Expression ◽

Car T Cells ◽

Healthy Donors ◽

Car T ◽

Acute Myeloid

Abstract The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products. In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency. Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR. We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested. Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients. Disclosures Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

Download Full-text

High Blood BAALC Copy Numbers Determined By Digital Droplet PCR at Timepoint of Allogeneic Transplantation in Complete Remission Predicts Relapse in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.517.517 ◽

2016 ◽

Vol 128 (22) ◽

pp. 517-517

Author(s):

Madlen Jentzsch ◽

Marius Bill ◽

Julia Schulz ◽

Juliane Grimm ◽

Stefanie Beinicke ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Research Funding ◽

Residual Disease ◽

Absolute Quantification ◽

Prognostic Impact ◽

Healthy Controls ◽

Copy Numbers ◽

Acute Myeloid

Abstract Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC (Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALC copy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR. We identified 82 AML pts with PB in first (60%) or second CR (23%) or CRi (17%) up to 28 days prior to HSCT available. Median age at HSCT was 63.9 (range 50.8-76.2) years (y). All pts received non-myeloablative (NMA) conditioning (fludarabine 3x30 mg & 2 Gy total body irradiation). At diagnosis, mutation status (mut) of the NPM1, CEBPA, IDH1, IDH2,& DNMT3A gene & presence of FLT3-ITD or FLT3-TKD were assessed. In pre-HSCT PB, absolute quantification of BAALC copy numbers was performed by ddPCR & results were normalized to ABL1 copy numbers.Additionally, absolute BAALC copy numbers wereassessedin PB of healthy controls (n=7) with a median age of 62.7 (range 39.6-82.0) y. Pts were grouped according to the European LeukemiaNet (ELN) classification in 21% favorable, 23% intermediate-I, 24% intermediate-II, 23% adverse & 9% unknown. Pts & healthy control were evenly matched in age (P=1) & sex (P=1). BAALC/ABL1 copy numbers did not differ between AML pts at HSCT (median 0.03 [range 0.01-2.48]) & the healthy controls (median 0.04 [range 0.03-0.10], P=.34, Figure 1). A cut-off point of 0.14absolute BAALC/ABL1 copies was determined using the R package 'OptimalCutpoints' & used to define pts with high (26%) & low (74%) pre-HSCT BAALC/ABL1 copy numbers. The copy number at this cut-off point was higher than the two-fold standard deviation over the median of the healthy controls (0.10 BAALC/ABL1). Pts with high & low pre-HSCT BAALC/ABL1 copy numbers did not differ significantly in pre-treatment characteristics (i.e. hemoglobin, white blood count, platelets, blasts in bone marrow or PB, ELN genetic group, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 mut) or remission status at HSCT (CR1 vs. CR2 vs. CRi). However, pts with high pre-HSCT BAALC/ABL1 copy numbers had a significantly higher cumulative incidence of relapse (CIR, P=.02, Figure 2a) & shorter overall survival (OS, P=.02, Figure 2b). High pre-HSCT BAALC/ABL1 copy numbers especially impacted on CIR when we restricted our analysis to pts with normal cytogenetics (P=.003). In multivariate analysis for the entire cohort, high pre-HSCT BAALC/ABL1 copy numbers retained the prognostic impact on CIR (Hazard Ratio [HR] 3.6, Confidence Interval [CI] 1.6-8.2, P=.002) after adjustment for disease status at HSCT (P=.006) & the prognostic impact on OS (HR 2.2, CI 1.1-4.3, P=.02). In conclusion, ddPCR is a feasible method for absolute quantification of BAALC copy numbers in PB, which may indicate residual disease burden in AML pts. High PB BAALC/ABL1 copy numbers (>0.14) in AML pts in hematologic CR at HSCT associated with higher CIR & shorter OS in univariate & multivariate models. AML pts with high PB BAALC/ABL1 copy numbers at HSCT should be closely monitored for relapse in the post-transplant period. In the future prospective studies will be required to validate the absolute PB BAALC/ABL1 copy number cut-off point & to evaluate whether AML pts with high BAALC/ABL1 copy numbersmight benefit from additional treatment before HSCT. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Poenisch: Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Download Full-text

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

Blood ◽

10.1182/blood-2005-03-1270 ◽

2005 ◽

Vol 106 (6) ◽

pp. 2186-2188 ◽

Cited By ~ 35

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)

Download Full-text

Anti-Tumor Effects of BDH1 in Acute Myeloid Leukemia

Frontiers in Oncology ◽

10.3389/fonc.2021.694594 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fei Han ◽

Huanhuan Zhao ◽

Jun Lu ◽

Weina Yun ◽

Lingling Yang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Pcr Analysis ◽

Rt Pcr ◽

Poor Survival ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Worse Prognosis ◽

Acute Myeloid

Dysregulation of ketone metabolism has been reported in various types of cancer. In order to find out its role in acute myeloid leukemia (AML) pathogenesis, we first analyzed the expression levels of 10 key genes involved in ketone metabolism in AML blasts and CD34+ hematopoietic stem cells (HSCs) from healthy donors. We found that the expression level of BDH1 was significantly lower in AML than in normal HSCs. The downregulation of BDH1 gene expression in AML cell lines as compared with normal HSCs was further confirmed with real-time RT-PCR. Analysis of TCGA and other database revealed that the downregulation of BDH1 was associated with worse prognosis in AML patients. In addition, we showed that overexpression of BDH1 inhibited the viability and proliferation of AML cells. In contrast, BDH1 knock-down promoted AML cell growth. Collectively, our results suggest the previously unappreciated anti-tumor role of BDH1 in AML, and low BDH1 expression predicts poor survival.

Download Full-text

Role of Polymorphisms of NKG2D Receptor and Its Ligands in Acute Myeloid Leukemia and Human Stem Cell Transplantation

Frontiers in Immunology ◽

10.3389/fimmu.2021.651751 ◽

2021 ◽

Vol 12 ◽

Author(s):

Alena Machuldova ◽

Monika Holubova ◽

Valentina S. Caputo ◽

Miroslava Cedikova ◽

Pavel Jindra ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Myeloid Leukemia ◽

Nk Cell ◽

Nkg2d Receptor ◽

Acute Myeloid

Natural killer cells possess key regulatory function in various malignant diseases, including acute myeloid leukemia. NK cell activity is driven by signals received through ligands binding activating or inhibitory receptors. Their activity towards elimination of transformed or virally infected cells can be mediated through MICA, MICB and ULBP ligands binding the activating receptor NKG2D. Given the efficiency of NK cells, potential target cells developed multiple protecting mechanisms to overcome NK cells killing on various levels of biogenesis of NKG2D ligands. Targeted cells can degrade ligand transcripts via microRNAs or modify them at protein level to prevent their presence at cell surface via shedding, with added benefit of shed ligands to desensitize NKG2D receptor and avert the threat of destruction via NK cells. NK cells and their activity are also indispensable during hematopoietic stem cell transplantation, crucial treatment option for patients with malignant disease, including acute myeloid leukemia. Function of both NKG2D and its ligands is strongly affected by polymorphisms and particular allelic variants, as different alleles can play variable roles in ligand-receptor interaction, influencing NK cell function and HSCT outcome differently. For example, role of amino acid exchange at position 129 in MICA or at position 98 in MICB, as well as the role of other polymorphisms leading to different shedding of ligands, was described. Finally, match or mismatch between patient and donor in NKG2D ligands affect HSCT outcome. Having the information beyond standard HLA typing prior HSCT could be instrumental to find the best donor for the patient and to optimize effects of treatment by more precise patient-donor match. Here, we review recent research on the NKG2D/NKG2D ligand biology, their regulation, description of their polymorphisms across the populations of patients with AML and the influence of particular polymorphisms on HSCT outcome.

Download Full-text

Targeting and Depletion of Acute Myeloid Leukemia Blasts By MEN1112, a Novel Humanized Defucosylated Monoclonal Antibodies with Specificity for Bst1/CD157 Antigen

Blood ◽

10.1182/blood.v124.21.2235.2235 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2235-2235

Author(s):

Adriano Venditti ◽

Francesco Buccisano ◽

Luca Maurillo ◽

Maria Ilaria Del Principe ◽

Andrea Coppola ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Ex Vivo ◽

Antigen Expression ◽

Distinct Pattern ◽

Healthy Donors ◽

Acute Myeloid

Abstract MEN1112 is a new humanized, defucosylated, monoclonal antibody (mAb) with high specific affinity for Bst1/CD157 antigen. Bst1/CD157 antigen expression on blood cells of acute myeloid leukemia (AML) patients and healthy donors was investigated by flow cytometry using a PE-labeled MEN1112 mAb. Twenty three patients affected with AML have been tested, 18 at diagnosis, 4 at relapse, 1 resistant. In 16 out of 23 patients both bone marrow (BM) and peripheral blood (PB) specimens were evaluated. PB and BM samples from healthy donors (N=2) were also assessed. In healthy donors and AML patients, PB and BM lymphocytes were Bst1/CD157 antigen negative whereas monocytes and neutrophils showed a distinct pattern of MEN1112 mean fluorescence intensity (MIF), with monocytes having the brightest expression. In the stem cell compartment, an intermediate level of MFI was observed (p<0.001). Prevalence of expression of the antigen on patients’ samples was over 90%. On AML blast cells from each single patient, MEN1112 expression was heterogeneous; indeed the antigen was expressed on 50%±29% and 47% ±39% of blasts in BM and PB, respectively. The anti-leukemia activity of MEN1112 on AML cell lines was tested, in vitro, by a flow cytometry-based cell depletion assay in the presence of lymphokine activated immune effector cells: a strong depletion of leukemia cells was demonstrated suggesting that MEN1112 might exert anti-leukemia activity through antibody dependent cell-mediated cytotoxicty (ADCC). The activity of MEN1112 was also tested ex vivo on whole PB showing that the antibody was able to deplete AML blasts in 9 out of 23 patients (47.4 %) with a percentage of AML blast depletion ranging between 4.3 - 66 %. In whole BM from 2 out of 11 evaluable patients MEN1112 induced 68% and 23% of AML blast depletion. Bst1/CD157 shedding assessment showed that, in the sera from AML samples, the concentration of Bst1/CD157 antigen was comparable to that measured in healthy donors. Moreover, since Fcγ receptor (CD16) genotype might be a factor contributing to the antitumor activity of the antibody, the polymorphism CD16-158Phe/Val was analyzed. Five out of 19 samples were homozygous for CD16-158 Phe; 5 were homozygous for CD16- 158 Val and 9 were heterozygous for CD16-158. MEN1112-induced blast depletion was observed for each genotype. Moreover, in an attempt to identify the determinant of MEN1112 activity, % in PB of blast (antigen positive), NK cells or residual normal cells were evaluated. Altogether, these results are promising suggesting the potential for an ADCC-mediated MEN1112 antileukemic effect and they support the clinical development of MEN1112. Disclosures Venditti: Menarini Ricerche SpA: Research Funding. Buccisano:Menarini Ricerche SpA: Research Funding. Del Principe:Menarini Ricerche SpA: Research Funding. Coppola:Menarini Ricerche SpA: Research Funding. Palomba:Menarini Ricerche SpA: Research Funding. Aureli:Menarini Ricerche SpA: Research Funding. Arriga:Menarini Ricerche SpA: Research Funding. Bellarosa:Menarini Spa: Employment. Bressan:Menarini Ricerche SpA: Employment. Manzini:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment. Amadori:Menarini Ricerche SpA: Research Funding. Sconocchia:Menarini Ricerche SpA: Research Funding.

Download Full-text

Unraveling the Role of Innate Lymphoid Cells in Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers13020320 ◽

2021 ◽

Vol 13 (2) ◽

pp. 320

Author(s):

Matthew R. Lordo ◽

Steven D. Scoville ◽

Akul Goel ◽

Jianhua Yu ◽

Aharon G. Freud ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Cancer Progression ◽

Myeloid Leukemia ◽

Nk Cell ◽

Lymphoid Cells ◽

Effector Cells ◽

And Function ◽

Acute Myeloid

Over the past 50 years, few therapeutic advances have been made in treating acute myeloid leukemia (AML), an aggressive form of blood cancer, despite vast improvements in our ability to classify the disease. Emerging evidence suggests the immune system is important in controlling AML progression and in determining prognosis. Natural killer (NK) cells are important cytotoxic effector cells of the innate lymphoid cell (ILC) family that have been shown to have potent anti-leukemic functions. Recent studies are now revealing impairment or dysregulation of other ILCs in various types of cancers, including AML, which limits the effectiveness of NK cells in controlling cancer progression. NK cell development and function are inhibited in AML patients, which results in worse clinical outcomes; however, the specific roles of other ILC populations in AML are just now beginning to be unraveled. In this review, we summarize what is known about the role of ILC populations in AML.

Download Full-text

Expanded Natural Killer Cells for Cellular Therapy of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.2743.2743 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2743-2743 ◽

Cited By ~ 1

Author(s):

Hiroyuki Fujisaki ◽

Harumi Kakuda ◽

Timothy Lockey ◽

Paul W. Eldridge ◽

Wing Leung ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Cell Expansion ◽

Myeloid Leukemia ◽

Nk Cell ◽

Target Cells ◽

Healthy Donors ◽

Acute Myeloid

Abstract Approximately half of the patients with acute myeloid leukemia (AML) harbor occult disease during therapy, leading to overt relapse. Novel treatments are needed to advance cure rates. AML cells are sensitive to natural killer (NK) cell cytotoxicity if they express HLA Class I molecules that do not bind killer-inhibitory receptors (KIR) on NK cells. The demonstration that haploidentical NK cells can expand in vivo and exert anti-AML activity when infused after non-myeloablative conditioning (Miller et al., Blood105: 3051, 2005), provided impetus to further explore their clinical potential and develop ways to increase their efficacy. The success of NK cell therapy depends on: i) mismatch in recipient HLA and donor KIR phenotype, allowing NK cell alloreactivity; ii) infusion of sufficient numbers of NK cells to achieve an effector: target (E:T) ratio that produces a significant leukemia cytoreduction. We found that K562 cells genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) induced expansion of human NK cells (Imai et al., Blood106: 376, 2005). In the present study, we first tested the stimulatory capacity of irradiated K562-mb15-41BBL in 34 additional healthy donors: CD56+ CD3− NK cell expansion after 7–10 days of culture was 5–87 fold (median, 22); after 21 days, NK cells could expand >1000 fold. CD3+ T cells expanded minimally or not at all. NK cells derived from 12 healthy donors were tested against the AML cell lines K562, KG-1, U937 and HL-60. Expanded NK cells were consistently cytotoxic at low E:T ratios. Thus, mean (± SD) cytotoxicity after 4 hrs at 4: 1 was 85.1% ± 8.7% for K562, 83.7% ± 9.4% for KG-1, 78.8% ± 15.2% for U937 and 94.8% ± 5.1% for HL-60. Expanded NK cells were effective even when outnumbered by target cells: at a 0.5: 1 ratio, cytotoxicities were 34.1% ± 14.7% with K562, 51.5% ± 16.5% with KG-1, 24.5% ± 14.8% with U937 and 52.1% ± 9.8% with HL-60. We next tested cytotoxicity of expanded NK cells from 10 donors against primary cells obtained from the bone marrow of 9 newly diagnosed patients with AML. Median cytotoxicity after 4 hrs of culture at a 4: 1 ratio was high, although interdonor variability was observed, with cytoxicities ranging from 22% to 90%. When expanded NK cells were cultured for 7 days with primary AML cells in the presence of bone marrow mesenchymal cells (to prevent spontaneous apoptosis of the AML cells) we could detect cytotoxicity at a 0.01:1 E:T ratio. Expanded NK cells were consistently more cytotoxic than primary NK cells from the same donor. Gene expression studies revealed marked changes in expression of adhesion molecules and cytokine transcripts after expansion. Expanded NK cells exerted considerable antileukemic effect in NOD-SCID-IL2Rgammanull mice engrafted with human AML cells, providing a strong rationale for their clinical testing. To this end, the K562-mb15-41BBL stimulatory cell line is currently being made under cGMP conditions and conditions for large-scale NK cell expansion have been established in support of a pilot protocol in which expanded haploidentical NK cells with be administered to patients with refractory AML.

Download Full-text

CD47-Sirpα Interaction Modulates Homing and Engraftment of Human Acute Myeloid Leukemia Stem Cells in Mice.

Blood ◽

10.1182/blood.v114.22.476.476 ◽

2009 ◽

Vol 114 (22) ◽

pp. 476-476

Author(s):

Alexandre PA Theocharides ◽

Liqing Jin ◽

Armando G Poeppl ◽

Tatiana K Prasolava ◽

Olga I Gan ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Immune System ◽

Nk Cells ◽

Innate Immune System ◽

Myeloid Leukemia ◽

Research Funding ◽

Innate Immune ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 476 Introduction: A large body of work has shown that acute myeloid leukemia (AML) clones are hierarchically organized and maintained by leukemia initiating cells (AML-LSC). However, little is known about molecular regulators that govern AML-LSC fate. Using the non-obese diabetic (NOD)–severe combined immunodeficiency (SCID) xenotransplantation model, our group recently found that CD47-SIRPα protein interaction is essential for repopulation of normal hematopoietic stem cells (HSC) in mice (Takenaka et al, Nat Immunol 2007). The NOD background conferred the best support for human engraftment, whereas mice with other polymorphisms of Sirpa could not be engrafted (i.e. NOD.NOR-Idd13.SCID). CD47 on human AML contributes to pathogenesis by inhibiting phagocytosis of leukemia cells through CD47-SIRPα interaction (Jaiswal et al, Cell 2009). CD47 is increased on human AML LSCs compared to normal HSCs, and high levels of CD47 are associated with poor patient outcome (Majeti et al, Cell 2009). This indicates that CD47 may function as an important molecular regulator of AML-LSC fate through communication with SIRPα protein expressed on cells of the innate immune system. Results: Consistent with published results, we observed increased CD47 expression in primary AML cells compared to normal cord blood cells. We next investigated the functional relevance of CD47 for AML-LSCs by xenografting primary human AMLs into NOD.SCID and NOD.NOR-Idd13.SCID (Idd) mice. Following intravenous (i.v.) transplantation, none of three primary human AML samples could engraft Idd mice while robust engraftment in NOD.SCID mice was observed, consistent with our previous data using normal human HSCs. When the same samples were transplanted intrafemorally (i.f.), 2 of 6 Idd mice showed engraftment in the injected femur but no engraftment in other bones or the spleen; evidence of the latter is linked to stem cell function. To assess the role of innate immunity, we pre-treated mice with antibody against murine CD122 which depletes host natural killer (NK) cells and macrophages. Engraftment in the injected femur was observed in NOD.SCID mice (43/43) for all 10 AML samples tested, and interestingly in 31 of 42 Idd mice (8/10 AML samples tested), with similar engraftment levels (Idd 37.4±6% vs NOD.SCID 53.8±5%, p=0.33). As expected, NOD.SCID mice supported migration to non-injected bones (38/43 mice, 10/10 AML samples tested). In contrast to results obtained in the absence of anti-CD122 pre-treatment, engraftment in non-injected bones was now detectable in 8 of 42 Idd mice (2/10 AML samples tested), pointing to improved migration of AML-LSCs following depletion of NK cells and macrophages. However, the engraftment level in non-injected bones was significantly lower in Idd compared to NOD.SCID mice (2.4±4% vs 63.2±6%, p=0.001). Moreover, AML-LSCs were unable to repopulate the spleens of Idd mice. Homing assays revealed decreased homing to BM and spleen 16 hours following i.v. injection in Idd compared to NOD.SCID mice. Conclusion: Our results support the hypothesis that CD47-SIRPα interaction is critical for engraftment of AML-LSCs. Attenuation of CD47-SIRPα interaction (as in Idd mice) leads to decreased engraftment ability of AML-LSCs. Enhancement of engraftment in the injected femur and in some cases migration to other bones in Idd mice following anti-CD122 treatment is likely mediated through interference with cells of the innate immune system. Interruption of CD47-SIRPα signaling through targeting of either CD47 or SIRPα provides a potential therapeutic approach for eradication of AML-LSCs. Disclosures: Dick: Roche: Research Funding; CSL Ltd: Research Funding.

Download Full-text