Transient regulatory T-cell (Treg) depletion with IL-2 diphtheria toxin fusion protein enhances clearance of acute myeloid leukemia by haploidentical natural killer (NK) cells

Green tea (GT) treatment was evaluated for its effect on the immune and antineoplastic response of elderly acute myeloid leukemia patients with myelodysplasia-related changes (AML-MRC) who are ineligible for aggressive chemotherapy and bone marrow transplants. The eligible patients enrolled in the study (n = 10) received oral doses of GT extract (1000 mg/day) alone or combined with low-dose cytarabine chemotherapy for at least 6 months and/or until progression. Bone marrow (BM) and peripheral blood (PB) were evaluated monthly. Median survival was increased as compared to the control cohort, though not statistically different. Interestingly, improvements in the immunological profile of patients were found. After 30 days, an activated and cytotoxic phenotype was detected: GT increased total and naïve/effector CD8+ T cells, perforin+/granzyme B+ natural killer cells, monocytes, and classical monocytes with increased reactive oxygen species (ROS) production. A reduction in the immunosuppressive profile was also observed: GT reduced TGF-β and IL-4 expression, and decreased regulatory T cell and CXCR4+ regulatory T cell frequencies. ROS levels and CXCR4 expression were reduced in bone marrow CD34+ cells, as well as nuclear factor erythroid 2–related factor 2 (NRF2) and hypoxia-inducible factor 1α (HIF-1α) expression in biopsies. Immune modulation induced by GT appears to occur, regardless of tumor burden, as soon as 30 days after intake and is maintained for up to 180 days, even in the presence of low-dose chemotherapy. This pilot study highlights that GT extracts are safe and could improve the immune system of elderly AML-MRC patients.

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Expression of interleukin-3 receptor subunits on defined subpopulations of acute myeloid leukemia blasts predicts the cytotoxicity of diphtheria toxin interleukin-3 fusion protein against malignant progenitors that engraft in immunodeficient mice

Blood ◽

10.1182/blood-2006-04-013813 ◽

2006 ◽

Vol 108 (10) ◽

pp. 3530-3537 ◽

Cited By ~ 41

Author(s):

Leman Yalcintepe ◽

Arthur E. Frankel ◽

Donna E. Hogge

Keyword(s):

Acute Myeloid Leukemia ◽

Fusion Protein ◽

Diphtheria Toxin ◽

Myeloid Leukemia ◽

Relative Level ◽

Immunodeficient Mice ◽

Interleukin 3 ◽

Qrt Pcr ◽

Cell Fractions ◽

Acute Myeloid

AbstractThe interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Rα and βc subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT388IL3). Among 19 patient samples, the relative level of the IL-3Rα was higher than the IL-3Rβc and highest in CD34+CD38-CD71- cells, enriched for candidate leukemia stem cells, compared with cell fractions depleted of such progenitors. Overall, the amount of IL-3Rβc subunit did not vary among sorted subpopulations. However, expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rβc expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34+CD38-CD71- cells from different samples; this value was correlated (r = .76, P = .05) with the ability of DT388IL3 to kill AML progenitors that engraft in β2-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus, quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors.

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Targeting of a B7-1 (CD80) immunoglobulin G fusion protein to acute myeloid leukemia blasts increases their costimulatory activity for autologous remission T cells

Blood ◽

10.1182/blood.v97.10.3138 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3138-3145 ◽

Cited By ~ 35

Author(s):

Michael Notter ◽

Tim Willinger ◽

Ulrike Erben ◽

Eckhard Thiel

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Fusion Protein ◽

Immunoglobulin G ◽

Myeloid Leukemia ◽

Costimulatory Molecule ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Acute Myeloid

Abstract Transfection of tumor cells with the gene encoding the costimulatory molecule B7-1 (CD80), the ligand for CD28 and cytotoxic T lymphocye antigen-4 on T cells, has been shown to result in potent T-cell–mediated antitumor immunity. As an alternative approach, this study analyzed the costimulatory capacity of a human B7-1 immunoglobulin G (IgG) fusion protein targeted to the cell membrane of human acute myeloid leukemia (AML) blasts. Flow cytometric analysis revealed a low constitutive expression of B7-1 on human AML blasts (on average, 3.0 ± 4.3%; n = 50). In contrast, the expression of B7-2 (CD86) was highly heterogeneous and higher in AML blasts of French-American-British classification types M4 and M5 (P < .0001). The B7-1 IgG fusion protein used in this study efficiently costimulated the proliferation of resting and preactivated T cells when immobilized on plastic. After preincubation with B7-1 IgG, specific binding of the fusion protein to the high-affinity Fcγreceptor I (CD64) on leukemic cells was demonstrated and was found to increase the proliferation of both allogeneic and autologous T cells in costimulation experiments. Furthermore, targeting of B7-1 IgG to the tumor membrane resulted in increased proliferation of autologous remission T cells and had the potential to generate an enhanced redirected cytotoxic T-cell response against autologous AML blasts. In summary, the targeting of B7-1 IgG fusion protein described in this study represents a strategy alternative to gene therapy to restore the expression of the costimulatory molecule B7-1 on human AML blasts, thereby enhancing their immunogenicity for autologous T cells. This new approach may have implications for T-cell–mediated immunotherapy in AML.

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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.

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

Blood ◽

10.1182/blood.v118.21.246.246 ◽

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.

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Upregulation of CD200 is associated with Foxp3+ regulatory T cell expansion and disease progression in acute myeloid leukemia

Tumor Biology ◽

10.1007/s13277-012-0578-x ◽

2012 ◽

Vol 34 (1) ◽

pp. 531-542 ◽

Cited By ~ 31

Author(s):

Ali Memarian ◽

Maryam Nourizadeh ◽

Farimah Masoumi ◽

Mina Tabrizi ◽

Amir Hossein Emami ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

T Cell ◽

Disease Progression ◽

Regulatory T Cell ◽

Cell Expansion ◽

Myeloid Leukemia ◽

T Cell Expansion ◽

Acute Myeloid

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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.

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

Cellular and Molecular Immunology ◽

10.1038/s41423-020-00574-8 ◽

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.

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Induction of remission in patients with acute myeloid leukemia without prolonged myelosuppression using diphtheria toxin-interleukin 3 fusion protein

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.7068 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 7068-7068 ◽

Cited By ~ 2

Author(s):

A. E. Frankel ◽

M. A. Weir ◽

P. D. Hall ◽

M. Holguin ◽

C. Cable ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Fusion Protein ◽

Diphtheria Toxin ◽

Myeloid Leukemia ◽

De Novo ◽

Cell Transplant ◽

Interleukin 3 ◽

History Of ◽

Acute Myeloid

7068 The recombinant diphtheria toxin fusion protein, DT388IL3, composed of the catalytic and translocation domains of diphtheria toxin (DT388) fused to human interleukin-3 (IL3) showed selective cytotoxicity to acute myeloid leukemia (AML) stem cells both in vitro and in vivo and was prepared for a phase I clinical study (Urieto, Protein Exp Purif 33, 123, 2004). FDA approval (BB IND#11314) and IRB approvals were obtained. Seventy-five AML patients were screened and thirty-one patients treated. The median age of treated patients was 62 years (range, 25- 81 years). There were sixteen males and fifteen females. Disease was de novo in three, first relapse in ten, second relapse in eight, and refractory in ten patients. Four patients had a history of MDS, and one had a history of secondary AML. One patient each had previously received an autologous or allogeneic stem cell transplant. Cytogenetics were unfavorable in ten, intermediate in nineteen, and not done in two. Seven patients were treated with 4 μg/kg, eight patients were treated with 5.3 μg/kg, thirteen patients treated with 7.1 μg/kg, and three patients treated with 9.4 μg/kg DT388IL3. Drug-related toxicities were mild to moderate and transient including fever, chills, hypotension, hypoxemia, and hypoalbuminemia. Consistent with an absence of toxicity to normal hematopoietic progenitors, responses occurred in the absence of prolonged myelosuppression. Among thirty evaluable patients, we have observed one CR of 8 months duration, two partial remissions (PRs) lasting one and three months and three minimal responses with clearance of peripheral blasts and marrow blast cytoreductions of 89%, 90% and 93% lasting one to two months. Dose escalation is proceeding. No significant financial relationships to disclose.

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

Blood ◽

10.1182/blood.v114.22.2663.2663 ◽

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.

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