RESUMO
Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Células Matadoras Naturais , Leucemia , Linfoma , Membrana Celular/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Interferon gama/metabolismo , Receptores de Células Matadoras Naturais , Humanos , Leucemia/metabolismo , Linfoma/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Serpinas , Humanos , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Proteômica , Estudos Retrospectivos , Serpinas/genéticaRESUMO
Hydroxyurea (HU) is frequently used in the early phase of chronic myeloid leukemia (CML) to achieve cytoreduction prior to tyrosine kinase inhibitor (TKI) therapy. However, its impact on CML stem and progenitor cells (SPC) remains largely unknown. This study utilized targeted proteo-transcriptomic expression data on 596 genes and 51 surface proteins in 60,000 CD14-CD34+ cells from chronic phase CML patients to determine effects of shortterm HU treatment (4-19 days) on CML SPC. Peripheral blood and bone marrow samples were obtained from 17 CML patients eligible for short-term HU treatment (three patients before and after HU; seven patients before HU; and seven patients after HU) and subjected to single-cell CITE-seq and/or flow cytometry analysis. The analysis revealed enhanced frequencies of hemoglobin-expressing (HBA1, HBA2, HBB) erythroid progenitor cells in blood and bone marrow following HU treatment. In addition, there was an accumulation of cell subsets with S/G2/M phase-related gene and protein expression, likely representing cells arrested in, or progressing slowly through, the cell cycle. The increased frequency of cells in S/G2/M phase after HU was observed already among the most immature leukemic stem cells (LSC), and patients with a high fraction of LSC in the S/G2/M phase showed poor responsiveness to TKI treatment. We conclude that short-term HU treatment entails differentiation of erythroid progenitor cells and alters the characteristics of LSC in CML. The results imply that studies of LSC and progenitor populations in CML should take effects of initial HU therapy into account.
RESUMO
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.
Assuntos
Interleucina-2 , Leucemia Mieloide Aguda , Humanos , Antígenos HLA-B/genética , Antígeno HLA-B44 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Recidiva , ImunoterapiaRESUMO
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.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígenos HLA-B/genética , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/terapia , Adolescente , Adulto , Idoso , Antígenos HLA-B/imunologia , Humanos , Imunoterapia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Pessoa de Meia-Idade , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Variantes Farmacogenômicos , Resultado do Tratamento , Células Tumorais Cultivadas , Adulto JovemRESUMO
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.
Assuntos
Imunoterapia/métodos , Leucemia Mieloide/imunologia , Leucemia Mieloide/terapia , Linfócitos T Reguladores/imunologia , Doença Aguda , Adolescente , Adulto , Idoso , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Histamina/imunologia , Histamina/uso terapêutico , Humanos , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucemia Mieloide/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Prognóstico , Modelos de Riscos Proporcionais , Indução de Remissão , Linfócitos T Reguladores/metabolismo , Telômero/genética , Adulto JovemRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Histamina/imunologia , NADPH Oxidases/imunologia , Neoplasias Experimentais/imunologia , Animais , Diferenciação Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Monócitos/citologia , Monócitos/imunologia , Neoplasias Experimentais/enzimologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Imunidade Adaptativa/imunologia , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia , Proteínas de Transporte/imunologia , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Inflamassomos/imunologia , Interferon gama/biossíntese , Subunidade p35 da Interleucina-12/imunologia , Interleucina-18/antagonistas & inibidores , Interleucina-1beta/antagonistas & inibidores , Ativação Linfocitária/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLRAssuntos
Imunoterapia , Quimioterapia de Indução , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/prevenção & controle , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Taxa de SobrevidaRESUMO
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.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Apoptose , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linfócitos T/patologia , Medula Óssea/patologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/enzimologia , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , NADPH Oxidase 2 , Poli(ADP-Ribose) Polimerase-1 , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell-cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.
Assuntos
Apoptose , Citotoxicidade Imunológica , Proteína Ligante Fas/metabolismo , Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neutrófilos/imunologia , Caspase 8/metabolismo , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Humanos , Células Matadoras Naturais/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Light-responsive molecular tools targeting kinases affords one the opportunity to study the underlying cellular function of selected kinases. In efforts to externally control lymphocyte-specific protein tyrosine kinase (LCK) activity, the development of release-and-report LCK inhibitors is described, in which (i) the release of the active kinase inhibitor can be controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the active kinase inhibitor. This introduces an unprecedented all-photonic method for users to both control and monitor real-time inhibitory activity. A functional cellular assay demonstrated light-mediated LCK inhibition in natural killer cells. The use of coumarin-derived caging groups resulted in rapid cellular uptake and non-specific intracellular localisation, while a BODIPY-derived caging group predominately localised in the cellular membrane. This concept of release-and-report inhibitors has the potential to be extended to other biorelevant targets where both spatiotemporal control in a cellular setting and a reporting mechanism would be beneficial.
RESUMO
Cancer/testis antigens (CTAs) are widely expressed in melanoma and lung cancer, emerging as promising targets for vaccination strategies and T-cell-based therapies in these malignancies. Despite recognizing the essential impact of intratumoral heterogeneity on clinical responses to immunotherapy, our understanding of intratumoral heterogeneity in CTA expression has remained limited. We employed single-cell mRNA sequencing to delineate the CTA expression profiles of cancer cells in clinically derived melanoma and lung cancer samples. Our findings reveal a high degree of intratumoral transcriptional heterogeneity in CTA expression. In melanoma, every cell expressed at least one CTA. However, most individual CTAs, including the widely used therapeutic targets NY-ESO-1 and MAGE, were confined to subpopulations of cells and were uncoordinated in their expression, resulting in mosaics of cancer cells with diverse CTA profiles. Coordinated expression was observed, however, mainly among highly structurally and evolutionarily related CTA genes. Importantly, a minor subset of CTAs, including PRAME and several members of the GAGE and MAGE-A families, were homogenously expressed in melanomas, highlighting their potential as therapeutic targets. Extensive heterogeneity in CTA expression was also observed in lung cancer. However, the frequency of CTA-positive cancer cells was notably lower and homogenously expressed CTAs were only identified in one of five tumors in this cancer type. Our findings underscore the need for careful CTA target selection in immunotherapy development and clinical testing and offer a rational framework for identifying the most promising candidates.
Assuntos
Antígenos de Neoplasias , Neoplasias Pulmonares , Melanoma , Análise de Célula Única , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma/imunologia , Melanoma/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/imunologia , Análise de Célula Única/métodos , Masculino , Regulação Neoplásica da Expressão GênicaRESUMO
Late divergence of survival curves of treated patients and controls is commonly seen in successful cancer immunotherapy trials. Although late survival curve divergence may be caused by a delayed action of therapy, it may also be related to early effects of the treatment. We suggest that late survival divergence most often reflects a specific benefit of therapy for patients who suffer from a comparatively slow progression of disease. The occurrence of delayed survival curve divergence has important implications for the statistical analysis of immunotherapy trials. Thus, it leads to non-proportional hazard ratios that make commonly used statistical tests, e.g., the logrank test, suboptimal. It is therefore suggested that the statistical analysis of immunotherapy trials primarily should be based on a test that compares the survival curves at or after a prespecified, fixed, late time point.
Assuntos
Imunoterapia , Estimativa de Kaplan-Meier , Neoplasias/mortalidade , Neoplasias/terapia , Estudos de Casos e Controles , Humanos , Modelos Estatísticos , Neoplasias/imunologia , Resultado do TratamentoRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Estresse Oxidativo/imunologia , Subpopulações de Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/virologia , Comunicação Celular/imunologia , Membrana Celular/imunologia , Membrana Celular/microbiologia , Membrana Celular/virologia , Células Cultivadas , Técnicas de Cocultura , Citomegalovirus/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Humanos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Oxirredução , Compostos de Sulfidrila/metabolismo , Subpopulações de Linfócitos T/microbiologia , Subpopulações de Linfócitos T/virologiaRESUMO
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.
Assuntos
Interleucina-2 , Leucemia Mieloide Aguda , Humanos , Interleucina-2/genética , Interleucina-2/farmacologia , Interleucina-2/uso terapêutico , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Alelos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , CitocinasRESUMO
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.
Assuntos
Leucemia Mieloide Aguda/terapia , Receptores Histamínicos H2/metabolismo , Apoptose/efeitos dos fármacos , Citometria de Fluxo , Seguimentos , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Hibridização in Situ Fluorescente , Interleucina-2/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Indução de Remissão , Taxa de SobrevidaAssuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Medições Luminescentes/métodos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oniocompostos/imunologia , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rituximab , Células Tumorais CultivadasRESUMO
Interleukin-1 beta (IL-1ß), a pro-inflammatory cytokine, has been ascribed a role in the expansion of myeloid progenitors in acute myeloid leukemia (AML) and in promoting myeloid cell-induced suppression of lymphocyte-mediated immunity against malignant cells. This study aimed at defining the potential impact of IL-1ß in the post-remission phase of AML patients receiving immunotherapy for relapse prevention in an international phase IV trial of 84 patients (ClinicalTrials.gov; NCT01347996). Consecutive serum samples were collected from AML patients in first complete remission (CR) who received cycles of relapse-preventive immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2). Low IL-1ß serum levels before and after the first HDC/IL-2 treatment cycle favorably prognosticated leukemia-free survival and overall survival. Serum levels of IL-1ß were significantly reduced in patients receiving HDC/IL-2. HDC also reduced the formation of IL-1ß from activated human PBMCs in vitro. Additionally, high serum levels of the IL-1 receptor antagonist IL-1RA were associated with favorable outcome, and AML patients with low IL-1ß along with high IL-1RA levels were strikingly protected against leukemic relapse. Our results suggest that strategies to target IL-1ß might impact on relapse risk and survival in AML.