RESUMO
ABSTRACT: Epstein-Barr virus (EBV) is a potent carcinogen linked to hematologic and solid malignancies and causes significant global morbidity and mortality. Therapy using allogeneic EBV-specific lymphocytes shows promise in certain populations, but the impact of EBV genome variation on these strategies remains unexplored. To address this, we sequenced 217 EBV genomes, including hematologic malignancies from Guatemala, Peru, Malawi, and Taiwan, and analyzed them alongside 1307 publicly available EBV genomes from cancer, nonmalignant diseases, and healthy individuals across Africa, Asia, Europe, North America, and South America. These included, to our knowledge, the first natural killer (NK)/T-cell lymphoma (NKTCL) EBV genomes reported outside of East Asia. Our findings indicate that previously proposed EBV genome variants specific to certain cancer types are more closely tied to geographic origin than to cancer histology. This included variants previously reported to be specific to NKTCL but were prevalent in EBV genomes from other cancer types and healthy individuals in East Asia. After controlling for geographic region, we did identify multiple NKTCL-specific variants associated with a 7.8-fold to 21.9-fold increased risk. We also observed frequent variations in EBV genomes that affected peptide sequences previously reported to bind common major histocompatibility complex alleles. Finally, we found several nonsynonymous variants spanning the coding sequences of current vaccine targets BALF4, BKRF2, BLLF1, BXLF2, BZLF1, and BZLF2. These results highlight the need to consider geographic variation in EBV genomes when devising strategies for exploiting adaptive immune responses against EBV-related cancers, ensuring greater global effectiveness and equity in prevention and treatment.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/imunologia , Variação Genética , Genoma Viral , ImunoterapiaRESUMO
Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL. SIGNIFICANCE: Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85.
Assuntos
Linfoma Extranodal de Células T-NK , Células T Matadoras Naturais , Epigenômica , Perfilação da Expressão Gênica , Humanos , Células Matadoras Naturais/patologia , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Células T Matadoras Naturais/patologiaRESUMO
Inadequate diagnostics compromise cancer care across lower- and middle-income countries (LMICs). We hypothesized that an inexpensive gene expression assay using paraffin-embedded biopsy specimens from LMICs could distinguish lymphoma subtypes without pathologist input. We reviewed all biopsy specimens obtained at the Instituto de Cancerología y Hospital Dr. Bernardo Del Valle in Guatemala City between 2006 and 2018 for suspicion of lymphoma. Diagnoses were established based on the World Health Organization classification and then binned into 9 categories: nonmalignant, aggressive B-cell, diffuse large B-cell, follicular, Hodgkin, mantle cell, marginal zone, natural killer/T-cell, or mature T-cell lymphoma. We established a chemical ligation probe-based assay (CLPA) that quantifies expression of 37 genes by capillary electrophoresis with reagent/consumable cost of approximately $10/sample. To assign bins based on gene expression, 13 models were evaluated as candidate base learners, and class probabilities from each model were then used as predictors in an extreme gradient boosting super learner. Cases with call probabilities < 60% were classified as indeterminate. Four (2%) of 194 biopsy specimens in storage <3 years experienced assay failure. Diagnostic samples were divided into 70% (n = 397) training and 30% (n = 163) validation cohorts. Overall accuracy for the validation cohort was 86% (95% confidence interval [CI]: 80%-91%). After excluding 28 (17%) indeterminate calls, accuracy increased to 94% (95% CI: 89%-97%). Concordance was 97% for a set of high-probability calls (n = 37) assayed by CLPA in both the United States and Guatemala. Accuracy for a cohort of relapsed/refractory biopsy specimens (n = 39) was 79% and 88%, respectively, after excluding indeterminate cases. Machine-learning analysis of gene expression accurately classifies paraffin-embedded lymphoma biopsy specimens and could transform diagnosis in LMICs.
Assuntos
Países em Desenvolvimento , Linfoma de Células T Periférico , Biópsia , HumanosRESUMO
Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.
Assuntos
Proliferação de Células , Interleucina-18/metabolismo , Interleucinas/biossíntese , Linfócitos/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Células Dendríticas/fisiologia , Humanos , Imunidade Inata , Interleucina-15/imunologia , Interleucinas/genética , Interleucinas/imunologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo , Interleucina 22RESUMO
Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56(dim) NK cell efficiently kills malignant targets at rest, whereas the less mature CD56(bright) NK cells cannot. In this study, we show that resting CD56(bright) NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56(dim) NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56(bright) NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell-activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell's ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell's PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56(bright) NK cell to the cytolytic CD56(dim) NK cells.
Assuntos
Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/imunologia , PTEN Fosfo-Hidrolase/imunologia , Animais , Células Cultivadas , Citometria de Fluxo , Humanos , Immunoblotting , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called "stage 3" developmental intermediate minimally characterized by a CD34(-)CD117(+)CD94(-) immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that include interleukin-1 receptor (IL-1R1)-positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here, we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes the differentiation of tonsillar IL-22-producing IL-1R1(hi) human ILC3s to CD56(bright)CD94(+) interferon (IFN)-γ-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, we demonstrate the lineage plasticity of human ILCs by identifying AHR as a transcription factor that prevents IL-1R1(hi) ILC3s from differentiating into NK cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Antígeno CD56/genética , Antígeno CD56/metabolismo , Linhagem da Célula , Células Cultivadas , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Células Matadoras Naturais/citologia , Linfócitos/citologia , Tonsila Palatina/citologia , Receptores de Hidrocarboneto Arílico/genética , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Interleucina 22RESUMO
Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/imunologia , Receptores Imunológicos/metabolismo , Western Blotting , Proliferação de Células , Citometria de Fluxo , Humanos , Imunoprecipitação , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Imunológicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
It is known that microRNAs (miRs) are involved in lymphocyte development, homeostasis, activation, and occasionally malignant transformation. In this study, a miR-155 transgene (tg) was driven to be overexpressed off of the lck promoter in order to assess its effects on natural killer (NK) cell biology in vivo. miR-155 tg mice have an increase in NK-cell number with an excess of the CD11b(low)CD27(high) NK subset, indicative of a halt in terminal NK-cell differentiation that proved to be intrinsic to the cell itself. The increase in NK cells results, in part, from improved survival in medium alone and enhanced expansion with endogenous or exogenous interleukin 15. Phenotypic and functional data from miR-155 tg NK cells showed constitutive activation and enhanced target cell conjugation, resulting in more potent antitumor activity in vitro and improved survival of lymphoma-bearing mice in vivo when compared with wild type NK cells. The enhanced NK-cell survival, expansion, activation, and tumor control that result from overexpression of miR-155 in NK cells could be explained, in part, via diminished expression of the inositol phosphatase SHIP1 and increased activation of ERK and AKT kinases. Thus, the regulation of miR-155 is important for NK-cell development, homeostasis, and activation.
Assuntos
Células Matadoras Naturais/imunologia , Linfoma/imunologia , MicroRNAs/genética , Regulação para Cima , Animais , Contagem de Células , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Inositol Polifosfato 5-Fosfatases , Interferon gama/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Linfoma/genética , Linfoma/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , TransgenesRESUMO
The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34âºCD38dimLinâ» cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34âºCD38brightLinâ» cells; (c) CD34âºCD1aâºCD11câ» cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34â»CD1aâºCD3â»CD11câ» cells, which resemble CD4âºCD8⺠double-positive T cells in the thymus; and (e) CD34â»CD1aâºCD3âºCD11câ» cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⺠cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre-T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.