PVRIG is a novel NK cell immune checkpoint receptor in acute myeloid leukemia

This study explored the novel immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced NK cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK cell PVRIG expression levels were not increased. To understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumour cell recognition, cytokines (IL-2 and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum (ER) and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti- PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIGPVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.

Download Full-text

Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers12082171 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2171

Author(s):

Isabel Valhondo ◽

Fakhri Hassouneh ◽

Nelson Lopez-Sejas ◽

Alejandra Pera ◽

Beatriz Sanchez-Correa ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Healthy Volunteers ◽

Nk Cells ◽

Myeloid Leukemia ◽

Cell Activation ◽

Nk Cell ◽

T Cell Subsets ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

Download Full-text

iPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Blood ◽

10.1182/blood-2021-148618 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1692-1692

Author(s):

Benjamin Goldenson ◽

Manuel Fierro ◽

Somayeh Pouyanfard ◽

Dan S Kaufman

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Immune Checkpoint ◽

Stock Options ◽

Myeloid Leukemia ◽

Nk Cell ◽

Anti Tumor Activity ◽

Acute Myeloid ◽

Privately Held

Abstract Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML. AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01). To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC. To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, "off-the-shelf" cell therapy approach that can be translated into novel clinical therapies. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

Download Full-text

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.852 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A893-A893

Author(s):

Laurent Gauthier ◽

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.

Download Full-text

Role of Glucocorticoid-Induced TNF-Related Protein (GITR) Ligand in the Interaction of Acute Myeloid Leukemia with NK Cells.

Blood ◽

10.1182/blood.v110.11.18.18 ◽

2007 ◽

Vol 110 (11) ◽

pp. 18-18

Author(s):

Matthias Krusch ◽

Katrin M. Baltz ◽

Tina Baessler ◽

Lothar Kanz ◽

Helmut R. Salih

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Myeloid Leukemia ◽

Map Kinases ◽

Nk Cell ◽

Immune Surveillance ◽

Related Protein ◽

Surface Receptors ◽

Enhanced Production ◽

Acute Myeloid

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies. The activity of NK cells is governed by a balance of activating and inhibitory surface receptors. Glucocorticoid-induced TNF-related protein (GITR) and its ligand (GITRL) are members of the TNF/TNF receptor (TNFR) superfamily, which mediates multiple cellular functions including proliferation, differentiation, and cell death. Recently we reported that NK cells express GITR while cancer cells express GITRL and GITR-GITRL interaction down regulates NK cell-mediated anti-tumor immunity (Baltz et al., FASEB J 2007). Here we demonstrate that GITRL is expressed on 6 of 7 investigated acute myeloid leukemia (AML) cell lines and on primary AML cells in 30 of 52 (59%) patients, while no GITRL expression was detected on CD34+ cells of healthy donors (n=5). GITRL expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via GITRL led to phosphorylation of ERK and JNK resulting in significantly (p<0.05, Mann-Whitney U-test) enhanced production of IL-10 and TNF by patient AML cells (n=10). In line, specific inhibitors for JNK and ERK1/2 blocked the cytokine release by AML cells demonstrating that activation of MAP kinases is responsible for the production of the immunoregulatory cytokines following GITRL stimulation. Importantly, blocking GITR-GITRL interaction in cocultures of AML and NK cells significantly (both <0.05 Mann-Whitney U-test) increased cellular cytotoxicity about 70% and IFN-γ production about 60%, and this was due to restored NK cell NF-κB activity. Thus, GITRL substantially influences immunoediting by AML cells and enables the escape of AML cells from NK cell-mediated immune surveillance. The correlation found between GITRL expression and NK cell susceptibility may provide useful information for NK cell-based immunotherapy.

Download Full-text

Role of CD137/4-1BB and Its Ligand in the NK Cell Mediated Immune Surveillance of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.880.880 ◽

2007 ◽

Vol 110 (11) ◽

pp. 880-880

Author(s):

Tina Baessler ◽

Matthias Krusch ◽

Katrin M. Baltz ◽

Benjamin J. Schmiedel ◽

Helga M. Schmetzer ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Nk Cell ◽

Immune Surveillance ◽

Cell Reactivity ◽

Ifn Γ ◽

Tnfr Superfamily ◽

Acute Myeloid

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies including acute myeloid leukemia (AML). NK cell reactivity is governed by a balance of activating and inhibitory receptors including various members of the TNF receptor (TNFR) superfamily. The TNFR superfamily member CD137/4-1BB has been shown to stimulate proliferation and IFN-γ production, but not cytotoxicity of NK cells in mice. Surprisingly, yet nothing is known regarding the consequences of CD137-CD137 ligand (CD137L) interaction for NK cell reactivity in humans. In this study we demonstrate that CD56dimCD16+ but not CD56brightCD16− NK cells express CD137 upon stimulation with the activating cytokines IL-2 and IL-15 with peak expression between 48 and 60h. Furthermore, we found that 5 of 7 investigated AML cell lines and 16 of 51 (33%) primary AML cells of patients expressed substantial CD137L levels, while no CD137L expression was detected on CD34+ cells of healthy donors (n=5). CD137L expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via CD137L into AML cells (n=10) significantly induced the release of the immunoregulatory cytokines IL-10 and TNF (both p<0.05, Mann-Whitney U-test). Surprisingly and in contrast to available data regarding the function of murine CD137, we found that in humans blocking CD137-CD137L interaction caused a significant increase in NK cell cytotoxicity and IFN-γ production about 50% (both p<0.05, Mann-Whitney U-test) in coculture assays with CD137L-expressing patient AML cells and AML cell lines. The inhibitory effect of CD137 on NK cell reactivity was further confirmed in cocultures of NK cells with CD137L-transfectants and by triggering CD137 with an agonistic monoclonal antibody. This indicates that CD137 mediates opposite effects in murine compared to human NK cells. Furthermore we conclude that CD137L expression substantially influences tumor immunoediting by AML cells and diminishes NK cell reactivity against AML.

Download Full-text

RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

Blood ◽

10.1182/blood.v116.21.2164.2164 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2164-2164

Author(s):

Benjamin J Schmiedel ◽

Constantin M Wende ◽

Tina Baessler ◽

Carolin Scheible ◽

Stefan Wirths ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Conflicts Of Interest ◽

Immune Surveillance ◽

Surface Protein ◽

Leukemia Cells ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Over-Expression of CD200 In Acute Myeloid Leukemia Mediates the Expansion of Regulatory T-Lymphocytes and Directly Inhibits Natural Killer Cell Tumor Immunity

Blood ◽

10.1182/blood.v116.21.491.491 ◽

2010 ◽

Vol 116 (21) ◽

pp. 491-491 ◽

Cited By ~ 2

Author(s):

Steven J Coles ◽

Stephen Man ◽

Robert Hills ◽

Eddie CY Wang ◽

Alan Burnett ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Tumor Immunity ◽

Myeloid Leukemia ◽

Nk Cell ◽

Cytolytic Activity ◽

Target Cells ◽

Over Expression ◽

Regulatory T Lymphocytes ◽

Acute Myeloid

Abstract Abstract 491 CD200 is a type-1 transmembrane glycoprotein which suppresses inflammatory and autoimmune responses by signalling through its cognate transmembrane receptor homologue (CD200R). Normally, CD200 expression is restricted to immune privileged sites where it enhances immune tolerance through mechanisms that include modulating the expansion of FOXP3+ regulatory T-lymphocytes (T-regs) and suppressing macrophage cytolytic activity. Furthermore, leukocyte associated CD200 has been reported to suppress Natural Killer (NK) cell activity in vivo. Pathologically, we have previously shown that CD200 over-expression on leukemic blasts in around 50% of acute myeloid leukemia (AML) patients is significantly associated with a poor overall survival (Tonks et al, Leukemia, 2007). Given the existing evidence that T-reg frequency and NK cell function influence blast clearance and long-term survival in AML, we investigated the possibility that CD200 expression in AML may be directly suppressing anti-tumor immunity in this disease. Here we present evidence that CD200+ AML can suppress host anti-tumor responses by augmenting the frequency of AML patient T-regs and by direct inhibition of NK cell anti-tumor activity. We also show that targeting the interaction between CD200 and its receptor might provide a new strategy for the treatment of AML. Bone marrow aspirates from 91 diagnostic AML patients were analysed by multiparameter flow cytometry for blast CD200 protein expression. We found that the level of blast CD200 expression directly correlated with an increased frequency of T-regs (CD4+CD25++FoxP3+; R=0.78, p=0.0008). Measuring 3H-thymidine incorporation, we show that T-regs isolated from AML patients by MACS® separation inhibited T-cell proliferation (induced by CD3 and CD28 stimulation) at ratios <0.1%, thus confirming that patients T-regs were functional. In contrast to T-regs, NK cell frequency (CD45+CD19−CD3−CD56+) did not correlate with the level of AML blast CD200 expression (R=0.15, p=0.851), however, NK cell subpopulation bivariate analysis using CD56 and CD16 demonstrated that the CD56dimCD16+ (the principle active NK population) was significantly reduced by over 50% in CD200+ AML patients (36±5% compared to 15±5%, p=0.009). Furthermore, CD200 expression on target cells appeared to have a direct effect on the cytotoxic activity of NK cells; co-culture of NK cells with CD200+ targets resulted in decreased CD107a expression (a marker for cytolytic granules) in NK cells (23±4% vs 12±5%, p=0.038) and decreased apoptosis of the target cells (19±1% vs 10±1%, p=0.041). Since CD200R was detected on NK cells in AML patients, it was likely that CD200 was having a direct effect on suppression of NK cytotoxicity. This was supported by the significant recovery of NK cytolytic activity against CD200+ blasts in the presence of a CD200 blocking antibody (5±1% vs 11±2% CD107a+ NK cells, p=0.046) whereas there was no change seen with CD200− blasts (19±4% vs 19±3%). In conclusion, these findings suggest that CD200 expression on leukemic blasts plays an influential role in suppressing anti-tumor immunity in AML patients through modulating the expansion of functionally suppressive T-regs and directly suppressing NK cell cytolytic activity. In this study blocking CD200 interaction with its receptor was able to recover a significant proportion of patient NK activity, making CD200 a potential therapeutic target for CD200+ AML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.5294.5294 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5294-5294

Author(s):

Patrick Schlegel ◽

Chihab Klose ◽

Christina Kyzirakos ◽

Ursula J.E. Seidel ◽

Kai Witte ◽

...

Keyword(s):

Cell Culture ◽

Acute Myeloid Leukemia ◽

Nk Cells ◽

Cell Expansion ◽

Myeloid Leukemia ◽

Nk Cell ◽

Residual Disease ◽

Cell Transfer ◽

Minimal Residual ◽

Acute Myeloid

Abstract GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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

Blood ◽

10.1182/blood-2011-01-329508 ◽

2011 ◽

Vol 118 (12) ◽

pp. 3273-3279 ◽

Cited By ~ 237

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.

Download Full-text

Acute myeloid leukemia immune escape by epigenetic CD48 silencing

Clinical Science ◽

10.1042/cs20191170 ◽

2020 ◽

Vol 134 (2) ◽

pp. 261-271 ◽

Cited By ~ 2

Author(s):

Zhiding Wang ◽

Yang Xiao ◽

Wei Guan ◽

Mengzhen Wang ◽

Jinghong Chen ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Nk Cells ◽

Immune Evasion ◽

Myeloid Leukemia ◽

Immune Escape ◽

Nk Cell ◽

Epigenetic Modification ◽

High Expression ◽

Hypomethylating Agent ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a malignant disorder of hemopoietic stem cells. AML can escape immunosurveillance of natural killer (NK) by gene mutation, fusions and epigenetic modification. The mechanism of AML immune evasion is not clearly understood. Here we show that CD48 high expression is a favorable prognosis factor that is down-regulated in AML patients, which can help AML evade from NK cell recognition and killing. Furthermore, we demonstrate that CD48 expression is regulated by methylation and that a hypomethylating agent can increase the CD48 expression, which increases the NK cells killing in vitro. Finally, we show that CD48 high expression can reverse the AML immune evasion and activate NK cells function in vivo. The present study suggests that a combination the hypomethylating agent and NK cell infusion could be a new strategy to cure AML.

Download Full-text