The HLA-B -21 dimorphism impacts on NK cell education and clinical outcome of immunotherapy in acute myeloid leukemia.

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 -21M or 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 NKG2A+ NK cells and displayed superior capacity to degranulate lytic granules against KIR ligand-matched 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-2-based 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-2-based 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.

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy.

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov ) 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency.

Histamine promotes the development of monocyte-derived dendritic cells and reduces tumor growth by targeting the myeloid NADPH oxidase.

The efficiency of immune-mediated clearance of cancer cells is hampered by immunosuppressive mediators in the malignant microenvironment, including NADPH oxidase-derived reactive oxygen species. We aimed at defining the effects of histamine, an inhibitor of the myeloid NADPH oxidase/NOX2, on the development of Ag-presenting dendritic cells (DCs) from myeloid precursors and the impact of these mechanisms for tumor growth. Histamine was found to promote the maturation of human DCs from monocytes by increasing the expression of HLA-DR and costimulatory molecules, which resulted in improved induction of Th cells with Th0 polarity. Experiments using wild-type and NOX2-deficient myelomonoblastic cells showed that histamine facilitated myeloid cell maturation only in cells capable of generating reactive oxygen species. Treatment of mice with histamine reduced the growth of murine EL-4 lymphomas in parallel with an increment of tumor-infiltrating DCs in NOX2-sufficient mice but not in NOX2-deficient (gp91(phox) (-/-)) mice. We propose that strategies to target the myeloid NADPH oxidase may facilitate the development of endogenous DCs in cancer.

TLR-Stimulated Neutrophils Instruct NK Cells To Trigger Dendritic Cell Maturation and Promote Adaptive T Cell Responses.

Polymorphonuclear neutrophils (PMNs) are innate effector cells with pivotal roles in pathogen recognition, phagocytosis, and eradication. However, their role in the development of subsequent immune responses is incompletely understood. This study aimed to identify mechanisms of relevance to the cross talk between human neutrophils and NK cells and its potential role in promoting adaptive immunity. TLR-stimulated PMNs were found to release soluble mediators to attract and activate NK cells in vitro. PMN-conditioned NK cells displayed enhanced cytotoxicity and cytokine production, and responded vigorously to ensuing stimulation with exogenous and endogenous IL-12. The neutrophil-induced activation of NK cells was prevented by caspase-1 inhibitors and by natural antagonists to IL-1 and IL-18, suggesting a role for the NOD-like receptor family pyrin domain containing-3 inflammasome. In addition, PMN-conditioned NK cells triggered the maturation of monocyte-derived dendritic cells, which promoted T cell proliferation and IFN-γ production. These data imply that neutrophils attract NK cells to sites of infection to convert these cells into an active state, which drives adaptive immune responses via maturation of dendritic cells. Our results add to a growing body of evidence that suggests a sophisticated role for neutrophils in orchestrating the immune response to pathogens.

Monocytic AML cells inactivate antileukemic lymphocytes: role of NADPH oxidase/gp91(phox) expression and the PARP-1/PAR pathway of apoptosis.

Dysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91(phox). Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1-dependent apoptosis in adjacent NK cells, CD4(+) T cells, and CD8(+) T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91(phox), did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91(phox) mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10(-11)) or FAB-M2 cells (P < 10(-9)). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity.

Virus-induced appearance of the selectin ligand sLeX in herpes simplex virus type 1-infected T-cells: involvement of host and viral factors.

Circulating leukocytes that express selectin ligands such as the carbohydrate epitope sialyl Lewis X (sLeX) may interact with endothelial selectins, resulting in transmigration of the leukocyte across the endothelial wall to adjacent tissue. Due to the potential of selectin-ligand interactions as targets in viral pathogenesis, we aimed at determining whether herpes simplex virus type 1 (HSV1) is able to induce the appearance of sLeX at the surface of infected leukocytes. We found that HSV1 infection of a T-cell line resulted in transcriptional activation of human fucosyltransferase genes FUT3, FUT6 and FUT7, the two latter genes encoding the fucosyltransferases rate limiting for sLeX synthesis. Flow cytometry and confocal microscopy demonstrated that HSV1 infection resulted in a 2-fold rise in the proportion of sLeX-positive cells. Increased levels of FUT3, FUT6 and FUT7 RNA were detected already at 3 h post infection, and treatment with cycloheximide, a translation inhibitor, blocked a HSV1-induced increase in the expression of FUT3, FUT6 and FUT7 RNA, suggesting involvement of viral or cellular proteins. Studies with infectious viral mutants indicated that the viral immediate early (α) protein ICP0 is essential for the initiation of FUT7 though not for FUT3 or FUT6 transcription. In CD3+ cells, derived from peripheral blood mononuclear cells, HSV1 infection induced expression of FUT3, FUT5 and FUT6, whereas FUT7 was not altered. The mean sLeX fluorescence intensity of CD3+ cells was significantly higher in HSV1-infected CD3+ cells. This suggests that infected leukocytes during HSV1 viremia may express selectin ligands with possible but as yet unproven roles in viral pathogenesis.

Redox remodeling by dendritic cells protects antigen-specific T cells against oxidative stress.

Microorganisms and microbial products induce the release of reactive oxygen species (ROS) from monocytes and other myeloid cells, which may trigger dysfunction and apoptosis of adjacent lymphocytes. Therefore, T cell-mediated immunity is likely to comprise mechanisms of T cell protection against ROS-inflicted toxicity. The present study aimed to clarify the dynamics of reduced sulfhydryl groups (thiols) in human T cells after presentation of viral and bacterial Ags by dendritic cells (DCs) or B cells. DCs, but not B cells, efficiently triggered intra- and extracellular thiol expression in T cells with corresponding Ag specificity. After interaction with DCs, the Ag-specific T cells acquired the capacity to neutralize exogenous oxygen radicals and resisted ROS-induced apoptosis. Our results imply that DCs provide Ag-specific T cells with antioxidative thiols during Ag presentation, which suggests a novel aspect of DC/T cell cross-talk of relevance to the maintenance of specific immunity in inflamed or infected tissue.

Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.

Single-cell gene expression levels show substantial variations among cells in seemingly homogenous populations. Astrocytes perform many control and regulatory functions in the central nervous system. In contrast to neurons, we have limited knowledge about functional diversity of astrocytes and its molecular basis. To study astrocyte heterogeneity and stem/progenitor cell properties of astrocytes, we used single-cell gene expression profiling in primary mouse astrocytes and dissociated mouse neurosphere cells. The transcript number variability for astrocytes showed lognormal features and revealed that cells in primary cultures to a large extent co-express markers of astrocytes and neural stem/progenitor cells. We show how subpopulations of cells can be identified at single-cell level using unsupervised algorithms and that gene correlations can be used to identify differences in activity of important transcriptional pathways. We identified two subpopulations of astrocytes with distinct gene expression profiles. One had an expression profile very similar to that of neurosphere cells, whereas the other showed characteristics of activated astrocytes in vivo.

Remission maintenance in acute myeloid leukemia: impact of functional histamine H2 receptors expressed by leukemic cells.

Post-consolidation immunotherapy with histamine dihydrochloride and interleukin-2 has been shown to improve leukemia-free survival in acute myeloid leukemia in a phase III trial. For this study, treatment efficacy was determined among 145 trial patients with morphological forms of acute myeloid leukemia as defined by the French-American-British classification. Leukemia-free survival was strongly improved in M4/M5 (myelomonocytic/monocytic) leukemia but not in M2 (myeloblastic) leukemia. We also analyzed histamine H(2) receptor expression by leukemic cells recovered from 26 newly diagnosed patients. H(2) receptors were typically absent from M2 cells but frequently expressed by M4/M5 cells. M4/M5 cells, but not M2 cells, produced reactive oxygen species that triggered apoptosis in adjacent natural killer cells. These events were significantly inhibited by histamine dihydrochloride. Our data demonstrate the presence of functional histamine H(2) receptors on human AML cells and suggest that expression of these receptors by leukemic cells may impact on the effectiveness of histamine-based immunotherapy.

Experience and Perception of Patients and Healthcare Professionals on Acute Leukemia in Rwanda: A Qualitative Study.

Purpose: To explore challenges associated with the timely diagnosis, therapy, and prognosis of acute leukemia in Rwanda. Methods: This is a qualitative study using a phenomenological approach that involved patients, patients' guardians, and healthcare professionals such as physicians from district hospitals and specialists from referral hospitals, as well as healthcare administrators. The primary data were collected from district and referral hospitals and central healthcare administration in Rwanda. The data were collected between July and October 2019. In-depth interviews were conducted, and thematic analysis was employed to interpret the results. Results: We identified barriers to seeking healthcare such as (i) insufficient knowledge within the population may lead patients and their guardians to consult traditional healers before seeking qualified medical care, and (ii) financial constraints that preclude payment of healthcare fees or other out-of-pocket cost related to diagnosis and treatment. We also observed that the referral system is tedious and primary healthcare facilities lack the competence and resources for the necessary diagnostic practices. Both may further delay diagnosis and therapy. Accordingly, healthcare professionals at the referral hospitals stated that most patients were seen at an advanced stage of the disease. For the treatment of acute lymphoblastic leukemia (ALL), only chemotherapy is utilized in Rwanda, while bone marrow (BM) transplantation is not available. Palliation is the only available treatment for the vast majority of Rwandan acute myeloid leukemia (AML) patients. Conclusion: ALL and AML are likely under-reported in Rwanda and diagnosis may be delayed, which may be explained by patient-related factors (lack of knowledge, financial constraints), a tedious referral system, and suboptimal diagnostic resources.

Idelalisib Rescues Natural Killer Cells from Monocyte-Induced Immunosuppression by Inhibiting NOX2-Derived Reactive Oxygen Species.

The 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.

Anthracycline-based consolidation may determine outcome of post-consolidation immunotherapy in AML.

Consolidation chemotherapy in acute myeloid leukemia (AML) aims at eradicating residual leukemic cells and mostly comprises high-dose cytarabine with or without the addition of anthracyclines, including daunorubicin. Immunogenic cell death (ICD) may contribute to the efficacy of anthracyclines in solid cancer, but the impact of ICD in AML is only partly explored. We assessed aspects of ICD, as reflected by calreticulin expression, in primary human AML blasts and observed induction of surface calreticulin upon exposure to daunorubicin but not to cytarabine. We next assessed immune phenotypes in AML patients in complete remission (CR), following consolidation chemotherapy with or without anthracyclines. These patients subsequently received immunotherapy with histamine dihydrochloride (HDC) and IL-2. Patients who had received anthracyclines for consolidation showed enhanced frequencies of CD8+ TEM cells in blood along with improved survival. We propose that the choice of consolidation therapy prior to AML immunotherapy may determine clinical outcome.

IFNL4 Genotypes Predict Clearance of RNA Viruses in Rwandan Children With Upper Respiratory Tract Infections.

Polymorphisms in the interferon lambda gene locus (IFNL) such as the IFNL4 genetic variants rs12979860 and rs368234815 are predictive of resolution of hepatitis C virus infection, but information about the impact of these variants in other infections is scarce. This study aimed at determining the potential impact of IFNL4 variation for the clearance of respiratory tract pathogens in Rwandan children (≤5 years old, n = 480) seeking medical care for acute respiratory infections. Nasopharyngeal swabs were retrieved from all children at the first hospital referral and from 161 children at follow-up visits 2 weeks later. The swabs were analyzed for pathogens by real-time PCR and for host cell IFNL4 genotype at rs12979860 and rs368234815. Approximately 1/3 of the children were homozygous for the rs12979860 T allele and the rs368234815 ΔG allele, which are overrepresented in subjects of African descent. These IFNL4 variants were significantly associated with reduced clearance of RNA viruses. Our results suggest that IFNL4 genotypes that are common among subjects of African descent may determine inefficacious clearance of RNA viruses from the respiratory tract.

Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2.

In patients with acute myeloid leukemia (AML), treatment with histamine dihydrochloride (HDC) and low-dose IL-2 (HDC/IL-2) in the post-chemotherapy phase has been shown to reduce the incidence of leukemic relapse. The clinical benefit of HDC/IL-2 is pronounced in monocytic forms of AML, where the leukemic cells express histamine type 2 receptors (H2R) and the NAPDH oxidase-2 (NOX2). HDC ligates to H2Rs to inhibit NOX2-derived formation of reactive oxygen species, but details regarding the anti-leukemic actions of HDC remain to be elucidated. Here, we report that human NOX2+ myelomonocytic/monocytic AML cell lines showed increased expression of maturation markers along with reduced leukemic cell proliferation after exposure to HDC in vitro. These effects of HDC were absent in corresponding leukemic cells genetically depleted of NOX2 (NOX2-/-). We also observed that exposure to HDC altered the expression of genes involved in differentiation and cell cycle progression in AML cells and that these effects required the presence of NOX2. HDC promoted the differentiation also of primary monocytic, but not non-monocytic, AML cells in vitro. In a xenograft model, immunodeficient NOG mice were inoculated with wild-type or NOX2-/- human monocytic AML cells and treated with HDC in vivo. The administration of HDC reduced the in vivo expansion of NOX2+/+, but not of NOX2-/- human monocytic AML cells. We propose that NOX2 may be a conceivable target in the treatment of monocytic AML.

Cytomegalovirus Serostatus Affects Autoreactive NK Cells and Outcomes of IL2-Based Immunotherapy in Acute Myeloid Leukemia.

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.

Dynamics of myeloid cell populations during relapse-preventive immunotherapy in acute myeloid leukemia.

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.

NOX2-dependent immunosuppression in chronic myelomonocytic leukemia.

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.

Dynamics of cytotoxic T cell subsets during immunotherapy predicts outcome in acute myeloid leukemia.

Preventing relapse after chemotherapy remains a challenge in acute myeloid leukemia (AML). Eighty-four non-transplanted AML patients in first complete remission received relapse-preventive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 in an international phase IV trial (ClinicalTrials.gov; NCT01347996). Blood samples were drawn during cycles of immunotherapy and analyzed for CD8+ (cytotoxic) T cell phenotypes in blood. During the first cycle of therapy, a re-distribution of cytotoxic T cells was observed comprising a reduction of T effector memory cells and a concomitant increase of T effector cells. The dynamics of T cell subtypes during immunotherapy prognosticated relapse and survival, in particular among older patients and remained significantly predictive of clinical outcome after correction for potential confounders. Presence of CD8+ T cells with specificity for leukemia-associated antigens identified patients with low relapse risk. Our results point to novel aspects of T cell-mediated immunosurveillance in AML and provide conceivable biomarkers in relapse-preventive immunotherapy.