HLA-B alleles are associated with outcomes in various pathologies, including autoimmune diseases and malignancies. The encoded HLA-B proteins are pivotal in antigen presentation to cytotoxic T cells, and some variants containing a Bw4 motif also serve as ligands to the killer immunoglobulin-like receptors (KIR) 3DL1/S1 of NK cells. We investigated the potential impact of HLA-B genotypes on the efficacy of immunotherapy for relapse prevention in acute myeloid leukemia (AML). Seventy-eight non-transplanted AML patients receiving HDC/IL-2 in the post-consolidation phase were genotyped for HLA-B and KIR genes. HLA-B*44 heralded impaired LFS (leukemia-free survival) and overall survival (OS), but the negative association with outcome was not shared across alleles of the HLA-B44 supertype. Notably, HLA-B*44 is one of few HLA-B44 supertype alleles containing a Bw4 motif with a threonine at position 80, which typically results in weak binding to the inhibitory NK receptor, KIR3DL1. Accordingly, a strong interaction between KIR3DL1 and Bw4 was associated with superior LFS and OS ( p = 0.014 and p = 0.027, respectively). KIR3DL1 + NK cells from 80 T-Bw4 donors showed significantly lower degranulation responses and cytokine responses than NK cells from 80I-Bw4 donors, suggesting impaired KIR3DL1-mediated education in 80 T-Bw4 subjects. We propose that presence of a strong KIR3DL1 + –Bw4 interaction improves NK cell education and thus is advantageous in AML patients receiving HDC/IL-2 immunotherapy for relapse prevention.

Supplementary Figures and Table

Abstract HLA-B proteins interact with key immune receptors on both T and NK cells, and variants of the encoding gene are associated with outcomes in various pathologies, including autoimmune diseases and malignancies. HLA-B is pivotal in antigen presentation to cytotoxic T cells, and some variants containing a Bw4 motif also serve as ligands to the killer immunoglobulin-like receptors (KIR) 3DL1/S1 of NK cells. We investigated the potential impact of HLA-B genotypes on the efficiency of immunotherapy for relapse prevention in acute myeloid leukemia (AML). Seventy-nine non-transplanted AML patients receiving HDC/IL-2 in the post-consolidation phase were genotyped for HLA-B and KIRs genes. HLA-B*07 associated with improved leukemia-free survival (LFS), while HLA-B*44 heralded impaired LFS and overall survival (OS). The negative association with outcome was not shared across alleles of the HLA-B44 supertype. Notably, HLA-B*44 is one of few HLA-B44 supertype alleles containing a Bw4 motif with a threonine at position 80, which typically results in weak binding to the inhibitory NK receptor, KIR3DL1. Accordingly, a strong interaction between KIR3DL1 and Bw4 was associated with superior LFS and OS (p = 0.014 and p = 0.027, respectively). KIR3DL1 + NK cells from 80T-Bw4 donors showed significantly lower degranulation responses and cytokine responses than NK cells from 80I-Bw4 donors, suggesting impaired KIR3DL1-mediated education in 80T-Bw4 subjects. We propose that presence of a strong KIR3DL1 + - Bw4 interaction is advantageous in AML patients receiving immunotherapy for relapse prevention. This is likely achieved by improved NK cell education and a reduced expression of HLA-Bw4 on malignant leukemic cells.

BACKGROUND:The natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at determining to what extent NKC haplotypes impact on NK cell repertoire and function, and whether such gene variants impact on outcome of IL-2-based immunotherapy in acute myeloid leukemia (AML).METHODS:Genotype status of NKG2D rs1049174 and NKG2A rs1983526 was determined using the TaqMan-Allelic discrimination approach. To dissect the impact of single nucloetide polymorphim (SNP) on NK cell function, we engineered the K562 cell line with CRISPR to be killed in a highly NKG2D-dependent fashion. NK cells were assayed for degranulation, intracellular cytokine production and cytotoxicity using flow cytometry.RESULTS:In AML patients receiving immunotherapy, the NKG2A gene variant, rs1983526, was associated with superior leukemia-free survival and overall survival. We observed that superior NK degranulation from individuals with the high-cytotoxicity NKG2D variant was explained by presence of a larger, highly responsive NKG2A+ subset. Notably, NK cells from donors homozygous for a favorable allele encoding NKG2A mounted stronger cytokine responses when challenged with leukemic cells, and NK cells from AML patients with this genotype displayed higher accumulation of granzyme B during histamine dihydrochloride/IL-2 immunotherapy. Additionally, among AML patients, the NKG2A SNP defined a subset of patients with HLA-B-21 TT with a strikingly favorable outcome.CONCLUSIONS:The study results imply that a dimorphism in the NKG2A gene is associated with enhanced NK cell effector function and improved outcome of IL-2-based immunotherapy in AML.

Natural killer cells are important effector cells in the immune response against myeloid malignancies. Previous studies show that the expression of activating NK cell receptors is pivotal for efficient recognition of blasts from patients with acute myeloid leukemia (AML) and that high expression levels impact favorably on patient survival. This study investigated the potential impact of activating receptor gene variants on NK cell receptor expression and survival in a cohort of AML patients receiving relapse-preventive immunotherapy with histamine dihydrochloride and low-dose IL-2 (HDC/IL-2). Patients harboring the G allele of rs1049174 in the KLRK1 gene encoding NKG2D showed high expression of NKG2D by CD56 bright NK cells and a favorable clinical outcome in terms of overall survival. For DNAM-1, high therapy-induced receptor expression entailed improved survival, while patients with high DNAM-1 expression before immunotherapy associated with unfavorable clinical outcome. The previously reported SNPs in NCR3 encoding NKp30, which purportedly influence mRNA splicing into isoforms with discrete functions, did not affect outcome in this study. Our results imply that variations in genes encoding activating NK cell receptors determine receptor expression and clinical outcome in AML immunotherapy.

AbstractThe phosphatidylinositol-4,5-bisphosphate-3 kinase-δ (PI3Kδ) inhibitor idelalisib, used alone or in combination with anti-CD20, is clinically efficacious in B-cell lymphoma and chronic lymphocytic leukemia (CLL) by promoting apoptosis of malignant B cells. PI3K regulates the formation of reactive oxygen species (ROS) by the myeloid NADPH oxidase NOX2, but the role of PI3Kδ in myeloid cell–induced immunosuppression is unexplored. We assessed the effects of idelalisib on the spontaneous and IgG antibody–induced ROS production by human monocytes, on ROS-induced cell death of human natural killer (NK) cells, and on tumor cell clearance in an NK cell–dependent mouse model of metastasis. Idelalisib potently and efficiently inhibited the formation of NOX2-derived ROS from monocytes and rescued NK cells from ROS-induced cell death. Idelalisib also promoted NK cell cytotoxicity against anti–CD20-coated primary human CLL cells and cultured malignant B cells. Experiments using multiple PI3K inhibitors implicated the PI3Kδ isoform in regulating NOX2-induced ROS formation and immunosuppression. In B6 mice, systemic treatment with idelalisib significantly reduced the formation of lung metastases from intravenously injected melanoma cells but did not affect metastasis in B6.129S6-Cybbtm1Din (Nox2−/−) mice or in NK cell–deficient mice. Our results imply that idelalisib rescues NK cells from NOX2/ROS-dependent immunosuppression and thus exerts antineoplastic efficacy beyond B-cell inhibition.

Immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) reduces the risk of relapse in the post-chemotherapy phase of acute myeloid leukemia (AML). Here we report the results of exploratory analyses of the clinical efficacy of HDC/IL-2 in AML with focus on the impact of karyotype aberrations in leukemic cells. Post-hoc analyses of phase III trial data suggested that HDC/IL-2 is primarily beneficial for patients with AML of normal karyotype. These results may be helpful in the selection of patients who are suitable for therapy and in the design of future immunotherapy protocols aiming at further defining the mechanism of relapse prevention by HDC/IL-2.

Background: Contemporary studies have identified >1 cycle of induction chemotherapy to induce complete remission (CR) as an independent risk factor for relapse and mortality in younger adults with acute myeloid leukemia (AML; Othus et al., Leukemia 2019). This study aimed at defining the potential impact of previous induction chemotherapy on the clinical benefit of relapse-preventive immunotherapy in AML. Methods: In a previously reported phase 3 trial (Brune et al., Blood 2006; Martner et al., Blood Rev 2013), two hundred and sixty-one patients with AML in first CR (18-84 years, median 55) who were not eligible for allogeneic transplantation were randomly assigned to receive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) or standard of care (no treatment, control). The HDC component of this regimen aims at countering immunosuppression induced by reactive oxygen species, which are formed by the NOX2 enzyme expressed by normal and malignant myeloid cells. HDC, a NOX2 inhibitor, improves the function and viability of anti-leukemic lymphocytes such as natural killer cells and cytotoxic T cells (Martner et al., J Pathol 2019). The immunotherapy was initiated in CR1 after the completion of chemotherapy with the aim to prevent relapse in the post-consolidation phase. Cycles 1 to 3 comprised 3 weeks on treatment and 3 weeks off treatment. In cycles 4 to 10 the off-treatment periods were extended to 6 weeks. In each cycle, patients in the treatment arm received HDC (Noventia Pharma, Milan, Italy) at 0.5 mg and human recombinant low-dose IL-2 (aldesleukin; 16 400 IU/kg; Chiron Corporation, Emeryville, CA) subcutaneously twice a day. All patients were followed for at least 36 months after random assignment. The median follow-up time was 48 months. Results: The number of previous induction cycles was known in 260 patients, and 203 of these attained CR after 1 induction cycle (78%). Among the 57 remaining patients, forty-seven attained CR after 2 cycles, and 3 (n=7) or 4 (n=3) cycles were required to induce CR in 10 patients. We compared outcomes of patients who had achieved CR1 after 1 vs >1 cycle of induction chemotherapy and observed a non-significant trend towards inferior outcome in control arm patients who had required >1 induction with a similar trend in younger control patients (<60 years old). In the HDC/IL-2 arm, outcome was significantly superior in younger patients who had attained CR1 after 1 vs >1 induction (leukemia-free survival, LFS, P=0.0007, hazard ratio, HR, 1.67-6.77; overall survival, OS, P=0.009, HR 1.30-6.33 in multivariable analyses correcting for potential confounding factors for relapse and death). When comparing outcomes between treatment and control arms within groups of patients who had attained CR1 after 1 cycle of induction chemotherapy, we observed that the LFS of patients in the HDC/IL-2 arm was significantly superior over controls (P=0.01 in multivariable analysis, N=203). In patients who were <60 years old at random assignment (N=130), treatment with HDC/IL-2 entailed significantly improved LFS (P=0.0008 vs control, HR 0.29-0.72) and OS (P=0.01, HR 0.28-0.75). HDC/IL-2 did not improve LFS or OS in patients who had required >1 cycle of induction to attain CR1 (P>0.5 vs control) and was not significantly beneficial in older patients. A test of interaction supported that the effect size of HDC/IL-2 vs control for LFS was more pronounced in younger patients who had attained CR after 1 (HR=0.48) vs >1 (HR=1.33) cycle of induction (P=0.04). Figure 1 shows Kaplan-Meier analyses of outcome, with unadjusted P-values from logrank tests, in the phase 3 trial arms among all patients attaining CR1 after 1 induction (A and B), in corresponding patients <60 years old (C and D) and in all patients who had attained CR1 after >1 cycle of induction (E and F). Conclusions: These findings imply that the efficiency of previous induction chemotherapy independently determines the clinical benefit of relapse-preventive immunotherapy in adult AML patients who are not candidates for allogeneic transplantation. The results may have implications for the selection of patients who are likely to benefit from treatment with HDC/IL-2 and for the design of clinical trials that evaluate relapse-preventive immunotherapy. Figure 1 Disclosures Thorén: Cytovia, New York: Patents & Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. Martner:Cytovia, New York: Patents & Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. Hellstrand:Cytovia, New York: Patents & Royalties: Holds patents protecting the use of NOX2 inhibitors in cancer. OffLabel Disclosure: HDC/IL-2 is approved for the prevention of AML relapse within the EU but is not yet approved in the US.

Natural killer (NK) cell function is regulated by inhibitory receptors, such as the family of killer immunoglobulin-like receptors (KIRs) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules on potential target cells, and recent studies imply that an HLA-B dimorphism at position -21 in the gene segment encoding the leader peptide dictates whether NK cell regulation primarily relies on the KIRs or the NKG2A/CD94 receptor. The impact of this HLA-B dimorphism on NK cell-mediated destruction of leukemic cells or on the course of leukemia is largely unknown. In a first part of this study, we compared functions of NK cells in subjects carrying HLA-B -21Mor 21T using interleukin-2 (IL-2)-activated NK cells and leukemic cells from patients with acute myeloid leukemia (AML). Subjects carrying HLA-B -21M harbored better-educated NKG2A1 NK cells and displayed superior capacity to degranulate lytic granules against KIR ligandmatched primary leukemic blasts. Second, we aimed to define the potential impact of HLA-B -21 variation on the course of AML in a phase 4 trial in which patients received IL-2based immunotherapy. In keeping with the hypothesis that 21M may be associated with improved NK cell functionality, we observed superior leukemia-free survival and overall survival in -21M patients than in -21T patients during IL-2based immunotherapy. We propose that genetic variation at HLA-B -21 may determine the antileukemic efficacy of activated NK cells and the clinical benefit of NK cell-activating immunotherapy.

Mutations leading to constitutive RAS activation contribute in myeloid leukemogenesis. RAS mutations in myeloid cells are accompanied by excessive formation of reactive oxygen species (ROS), but the source of ROS and their role for the initiation and progression of leukemia have not been clearly defined. To determine the role of NOX2-derived ROS in RAS-driven leukemia, double transgenic LSL-KrasG12D × Mx1-Cre mice expressing oncogenic KRAS in hematopoietic cells (M-KrasG12D) were treated with Nα-methyl-histamine (NMH) that targeted the production of NOX2-derived ROS in leukemic cells by agonist activity at histamine H2 receptors. M-KrasG12D mice developed myeloid leukemia comprising mature CD11b+Gr1+ myeloid cells that produced NOX2-derived ROS. Treatment of M-KrasG12D mice with NMH delayed the development of myeloproliferative disease and prolonged survival. In addition, NMH-treated M-KrasG12D mice showed reduction of intracellular ROS along with reduced DNA oxidation and reduced occurence of double-stranded DNA breaks in myeloid cells. The in vivo expansion of leukemia was markedly reduced in triple transgenic mice where KRAS was expressed in hematopoietic cells of animals with genetic NOX2 deficiency (Nox2−/− × LSL-KrasG12D × Mx1-Cre). Treatment with NMH did not alter in vivo expansion of leukemia in these NOX2-deficient transgenic mice. We propose that NOX2-derived ROS may contribute to the progression of KRAS-induced leukemia and that strategies to target NOX2 merit further evaluation in RAS-mutated hematopoietic cancer.

0 0 1 300 1716 University of Gothenburg 14 4 2012 14.0 Normal 0 false false false SV JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:SV;} Mutations leading to Ras gene activation are important contributors to myeloid leukemogenesis. Kras is one of three homologues of the Ras family, and oncogenic Kras is frequently found in monocytic forms of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myeloproliferative diseases (MPD). Earlier studies show that excessive amounts of reactive oxygen species (ROS) are produced in Kras-mutated myeloid malignancies. Myeloid cells, including Kras-mutated leukemic cells, express the ROS-generating enzyme NOX2. Relatively little is known about the impact of ROS formation, in particular NOX2-derived ROS formation, on leukemogenesis. Mice where oncogenic Kras was induced in hematopoietic cells of LSL-KrasG12D and Mx1-Cre double transgenic mice with ensuing myeloproliferation were treated with a histamine H2-receptor selective NOX2 inhibitor, Nα -methyl histamine (NMH) to assess whether NOX2 is a feasible therapeutic target in Kras-driven leukemia. Spleens of diseased Kras mice were infiltrated with mature CD11b+Gr1+ myeloid cells that expressed NOX2 and produced ROS. The NOX2-dependent formation of ROS in CD11b+Gr1+ cells was inhibited by NMH. In vivo administration of NMH delayed the development of myeloproliferative disease and significantly prolonged survival of Kras mice (p=0.003). In contrast, treatment with NMH did not alter the survival of mice when Kras expression was induced in hematopoietic cells of NOX2-deficient mice that did not produce ROS (Nox2-/-, LSL-KrasG12D and Mx1-Cre triple transgenic mice). In Kras-mutated Nox+/+ mice, but not in Kras-mutated Nox-/- mice, the in vivo administration of NMH entailed diminished ROS levels and reduced oxidative stress as reflected by reduced DCFDA and anti-8 Hydroxyguanosine staining. We propose that NOX2-derived ROS in Kras-mutated hematopoetic cells mediate genomic instability that may contribute to the progression of leukemia. Strategies to target NOX2 should be further evaluated in Ras-mutated hematopoietic cancer. Citation Format: Ebru Aydin, Alexander Hallner, Hanna Grauers Wiktorin, Anna Staffas, Kristoffer Hellstrand, Anna Martner. NOX2 inhibition reduces oxidative stress and prolongs survival of mice with Kras induced myeloproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 441.

Abstract Human cytomegalovirus (CMV) infection is reported to promote NK cell differentiation and education. The CMV-induced generation of highly differentiated adaptive-like NK cells has been proposed to affect favorably on the maintenance of remission in patients with acute myeloid leukemia (AML) after allogeneic stem cell transplantation (allo-SCT). The impact of CMV infection and adaptive-like NK cells on relapse and survival of patients with AML not receiving allo-SCT remains unknown. We assayed CMV IgG serostatus to determine past CMV infection in 81 nontransplanted AML patients who were receiving relapse-prevention immunotherapy comprising histamine dihydrochloride and low-dose interleukin-2 (HDC/IL2; NCT01347996). CMV seropositivity correlated negatively with leukemia-free and overall survival of patients receiving HDC/IL2, but did not correlate with outcomes in a contemporary control cohort. Analysis of outcome after stratification of patients based on concordant or discordant killer immunoglobulin-like receptor (KIR) and HLA genotypes implied that the negative impact of CMV seropositivity was restricted to patients lacking a ligand to inhibitory KIRs (iKIR). Previous CMV infection was also associated with fewer NK cells expressing only nonself iKIRs (NS-iKIR). We propose that CMV-driven NK cell education depletes the population of NS-iKIR NK cells, which in turn reduces the clinical benefit of relapse-preventive immunotherapy in AML. Cancer Immunol Res; 6(9); 1110–9. ©2018 AACR.

NK cells are negatively regulated by inhibitory receptors, such as the family of Killer Immunoglobulin-like Receptors (KIR) and the NKG2A/CD94 heterodimer. These receptors recognize cognate HLA class I molecules, and recent studies suggest that an HLA-B dimorphism in the leader peptide at position -21 dictates whether NK cell regulation mainly relies on the KIRs or the NKG2A/CD94 receptor. Thus, haplotypes with -21M delivers functional peptides to HLA-E, but will rarely harbor genes encoding the KIR ligand HLA-C2. However, little is known regarding the clinical implications of this dimorphism. For this study, we performed HLA typing and an extensive flow cytometry analysis of the NK cell repertoire in 80 AML patients who received immunotherapy with histamine dihydrochloride (HDC) and IL-2 for the prevention of relapse in the post-consolidation phase. Below median levels of HLA class I expression, reflecting low-grade NK cell inhibition, were found to correlate with significantly better outcome in HDC/IL-2-treated patients, indicating that the efficacy of the immunotherapy is NK cell-dependent. In contrast to healthy controls, the majority of NK cells in AML patients in complete remission displayed expression of NKG2A, which was even more prominent in patients after 3 weeks of HDC/IL-2 treatment, or after in vitro culture of NK cells with IL-2. Thirty-eight patients (47.5 %) carried the M/x genotype in HLA B -21 (predicting NKG2A dependence) and 42 patients (52.5 %) carried the T/T genotype (KIR dependence). Despite the high percentage of NKG2A+ NK cells, M/x patients showed significantly improved leukemia-free survival (p=0.04) and overall survival (p=0.007) compared with T/T patients. Only patients with M/x genotype benefited from low expression of HLA class I. The IL-2-induced NKG2A expression was associated with enhanced NKG2A-mediated inhibition as pure populations of NKG2A-expressing NK cells were inhibited by a leukemic cell line transfected to express the NKG2A ligand HLA E, which was reverted by antibody blockade of NKG2A. Our results suggest that the outcome of AML immunotherapy is affected by (i) expression levels of HLA class I, and (ii) the HLA B-21 dimorphism and, hence, whether the KIR or NKG2A-CD94 are the dominant inhibitory pathway for NK cells. Furthermore, the pronounced expression of NKG2A in HDC/IL-2-treated patients suggests that combination strategies, including checkpoint inhibitors targeting NKG2A together with HDC/IL-2, may be feasible for relapse prevention in AML.

Relapse of leukemia in the postchemotherapy phase contributes to the poor prognosis and survival in patients with acute myeloid leukemia (AML). In an international phase IV trial (ClinicalTrials.gov; NCT01347996), 84 patients with AML in first complete remission who had not undergone transplantation received immunotherapy with histamine dihydrochloride (HDC) and low-dose IL-2 with the aim of preventing relapse. The dynamics of myeloid cell counts and expression of activation markers was assessed before and after cycles of immunotherapy and correlated with clinical outcome in terms of relapse risk and survival. During cycles, a pronounced increase in blood eosinophil counts was observed along with a reduction in monocyte and neutrophil counts. A strong reduction of blood monocyte counts during the first HDC/IL-2 treatment cycle predicted leukemia-free survival. The HDC component of the immunotherapy exerts agonist activity at histamine type 2 receptors (H2Rs) that are expressed by myeloid cells. It was observed that the density of H2 R expression in blood monocytes increased during cycles of immunotherapy and that high monocyte H2R expression implied reduced relapse risk and improved overall survival. Several other activation markers, including HLA-DR, CD86, and CD40, were induced in monocytes and dendritic cells during immunotherapy but did not predict clinical outcome. In addition, expression of HLA-ABC increased in all myeloid populations during therapy. A low expression of HLA-ABC was associated with reduced relapse risk. These results suggest that aspects of myeloid cell biology may impact clinical benefit of relapse-preventive immunotherapy in AML.

Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival of less than 15 months, emphasizing the need for better treatments. Immunotherapy as a treatment for improving or aiding the patient's own immune defense to target the tumor has been suggested for GBM. A randomized clinical trial of adoptive cell transfer using ALECSAT (Autologous Lymphoid Effector Cells Specific Against Tumor Cells) is currently ongoing in Sweden. Here we performed a paired pre-clinical study to investigate the composition and in vitro effect of ALECSAT and identify determinants for the effect using autologous GBM-derived cancer stem cells (CSC), immunocytochemistry and flow cytometry. We show a clear dose-response relationship of ALECSAT on CSC, suggesting that the number of infused cells is of importance. In addition, the in vitro effect of ALECSAT on CSC correlated significantly to the blood count of T helper (Th) cells in the patient indicating a potential benefit of collecting cells for ALECSAT preparation at an even earlier stage when patients generally have a better blood count. The factors identified in this study will be important to consider in the design of future immunotherapy trials to achieve prolonged survival.

Chronic myelomonocytic leukemia (CMML) is a myeloproliferative and myelodysplastic neoplasm with few treatment options and dismal prognosis. The role of natural killer (NK) cells and other antileukemic lymphocytes in CMML is largely unknown. We aimed to provide insight into the mechanisms of immune evasion in CMML with a focus on immunosuppressive reactive oxygen species (ROS) formed by the myeloid cell NADPH oxidase-2 (NOX2). The dominant population of primary human CMML cells was found to express membrane-bound NOX2 and to release ROS, which, in turn, triggered extensive PARP-1-dependent cell death in cocultured NK cells, CD8(+) T effector memory cells, and CD8(+) T effector cells. Inhibitors of ROS formation and scavengers of extracellular ROS prevented CMML cell-induced lymphocyte death and facilitated NK cell degranulation toward Ab-coated, primary CMML cells. In patients with CMML, elevation of immature cell counts (CD34(+)) in blood was associated with reduced expression of several NK cell-activating receptors. We propose that CMML cells may use extracellular ROS as a targetable mechanism of immune escape.

Interactions between killer-immunoglobulin-like receptors (KIRs) and their HLA class I ligands are instrumental in natural killer (NK) cell regulation and protect normal tissue from NK cell attack. Human KIR haplotypes comprise genes encoding mainly inhibitory receptors (KIR A) or activating and inhibitory receptors (KIR B). A substantial fraction of humans lack ligands for inhibitory KIRs (iKIRs), that is, a 'missing ligand' genotype. KIR B/x and missing ligand genotypes may thus give rise to potentially autoreactive, unlicensed NK cells. Little is known regarding the impact of such genotypes in untransplanted acute myeloid leukemia (AML). For this study, NK cell phenotypes and KIR/HLA genotypes were determined in 81 AML patients who received immunotherapy with histamine dihydrochloride and low-dose IL-2 for relapse prevention (NCT01347996). We observed that presence of unlicensed NK cells impacted favorably on clinical outcome, in particular among patients harboring functional NK cells reflected by high expression of the natural cytotoxicity receptor (NCR) NKp46. Genotype analyses suggested that the clinical benefit of high NCR expression was restricted to patients with a missing ligand genotype and/or a KIR B/x genotype. These data imply that functional NK cells are significant anti-leukemic effector cells in patients with KIR/HLA genotypes that favor NK cell autoreactivity.

The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL.