RESUMEN
Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.
Asunto(s)
Citidina Desaminasa/genética , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Regulación hacia Arriba , Animales , Modelos Animales de Enfermedad , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MutaciónRESUMEN
Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.
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Antineoplásicos Inmunológicos/efectos adversos , Citidina Desaminasa/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/efectos adversos , Pirimidinas/efectos adversos , Adenina/análogos & derivados , Anciano , Proliferación Celular/efectos de los fármacos , Citidina Desaminasa/biosíntesis , Regulación hacia Abajo , Femenino , Humanos , Masculino , Persona de Mediana Edad , PiperidinasRESUMEN
Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.
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Leucemia Linfocítica Crónica de Células B/inmunología , Lisofosfolípidos/inmunología , Oxazinas/farmacología , Piridinas/farmacología , Receptores de Lisoesfingolípidos/inmunología , Esfingosina/análogos & derivados , Estilbenos/farmacología , ADP-Ribosil Ciclasa 1/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Linfocitos B , Ligando de CD40/biosíntesis , Movimiento Celular , Quimiocina CXCL12/biosíntesis , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Lisofosfolípidos/biosíntesis , Masculino , Glicoproteínas de Membrana/biosíntesis , Ratones , Persona de Mediana Edad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcr/biosíntesis , Receptores CXCR4 , Receptores de Lisoesfingolípidos/biosíntesis , Esfingosina/biosíntesis , Esfingosina/inmunología , Receptores de Esfingosina-1-Fosfato , Quinasa Syk , Células Tumorales Cultivadas , Microambiente TumoralAsunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Aspergillus fumigatus/inmunología , Candida albicans/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Macrófagos/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Neutrófilos/inmunología , Inhibidores de Proteínas Quinasas/efectos adversos , Agammaglobulinemia Tirosina Quinasa/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Macrófagos/patología , Proteínas de Neoplasias/inmunología , Neutrófilos/patología , Inhibidores de Proteínas Quinasas/administración & dosificaciónRESUMEN
The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.
Asunto(s)
ADN/análisis , Colorantes Fluorescentes/química , Verde de Metilo/química , Coloración y Etiquetado/economía , Coloración y Etiquetado/métodos , Animales , ADN/química , Microscopía Fluorescente , Factores de Tiempo , Pez Cebra/embriologíaRESUMEN
Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al.1.
RESUMEN
Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4(+) T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4(+) T cells from CLL patients, similarly in T cells from ZAP-70(+) to ZAP-70(-) patients. Autologous nurse-like cells establish a close contact with CD4(+) T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Quimiocina CXCL12/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos/inmunología , Antígenos CD/biosíntesis , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Separación Celular , Quimiocina CXCL12/metabolismo , Citometría de Flujo , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/metabolismo , Microscopía ConfocalRESUMEN
Hairy cell leukemia (HCL) is an incurable, rare lymphoproliferative hematological malignancy of mature B cAlthough first line therapy with purine analogues leads to positive results, almost half of HCL patients relapse after 5-10 years, and standard treatment may not be an option due to intolerance or refractoriness. Proliferation and survival of HCL cells is regulated by surrounding accessory cells and soluble signals present in the tumor microenvironment, which actively contributes to disease progression. In vitro studies show that different therapeutic approaches tested in HCL impact the tumor microenvironment, and that this milieu offers a protection affecting treatment efficacy. Herein we explore the effects of the tumor microenvironment to different approved and experimental therapeutic options for HCL. Dissecting the complex interactions between leukemia cells and their milieu will be essential to develop new targeted therapies for HCL patients.
RESUMEN
Small extracellular vesicle (sEV, or exosome) communication among cells in the tumor microenvironment has been modeled mainly in cell culture, whereas their relevance in cancer pathogenesis and progression in vivo is less characterized. Here we investigated cancer-microenvironment interactions in vivo using mouse models of chronic lymphocytic leukemia (CLL). sEVs isolated directly from CLL tissue were enriched in specific miRNA and immune-checkpoint ligands. Distinct molecular components of tumor-derived sEVs altered CD8+ T-cell transcriptome, proteome, and metabolome, leading to decreased functions and cell exhaustion ex vivo and in vivo. Using antagomiRs and blocking antibodies, we defined specific cargo-mediated alterations on CD8+ T cells. Abrogating sEV biogenesis by Rab27a/b knockout dramatically delayed CLL pathogenesis. This phenotype was rescued by exogenous leukemic sEV or CD8+ T-cell depletion. Finally, high expression of sEV-related genes correlated with poor outcomes in CLL patients, suggesting sEV profiling as a prognostic tool. In conclusion, sEVs shape the immune microenvironment during CLL progression. SIGNIFICANCE: sEVs produced in the leukemia microenvironment impair CD8+ T-cell mediated antitumor immune response and are indispensable for leukemia progression in vivo in murine preclinical models. In addition, high expression of sEV-related genes correlated with poor survival and unfavorable clinical parameters in CLL patients. See related commentary by Zhong and Guo, p. 5. This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/genética , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Transcriptoma , Inmunidad , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Microambiente Tumoral/genéticaRESUMEN
Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.
Asunto(s)
Citidina Desaminasa/sangre , Citidina Desaminasa/genética , Cambio de Clase de Inmunoglobulina , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Secuencia de Bases , Biomarcadores de Tumor/genética , Proliferación Celular , Cartilla de ADN/genética , Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Mutación , Pronóstico , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Neoplásico/sangre , ARN Neoplásico/genéticaRESUMEN
Aplidin is a novel cyclic depsipeptide, currently in Phase II/III clinical trials for solid and hematologic malignancies. The aim of this study was to evaluate the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common leukemia in the adult. Although there have been considerable advances in the treatment of CLL over the last decade, drug resistance and immunosuppression limit the use of current therapy and warrant the development of novel agents. Here we report that Aplidin induced a dose- and time-dependent cytotoxicity on peripheral blood mononuclear cells (PBMC) from CLL patients. Interestingly, Aplidin effect was markedly higher on monocytes compared to T lymphocytes, NK cells or the malignant B-cell clone. Hence, we next evaluated Aplidin activity on nurse-like cells (NLC) which represent a cell subset differentiated from monocytes that favors leukemic cell progression through pro-survival signals. NLC were highly sensitive to Aplidin and, more importantly, their death indirectly decreased neoplasic clone viability. The mechanisms of Aplidin-induced cell death in monocytic cells involved activation of caspase-3 and subsequent PARP fragmentation, indicative of death via apoptosis. Aplidin also showed synergistic activity when combined with fludarabine or cyclophosphamide. Taken together, our results show that Aplidin affects the viability of leukemic cells in two different ways: inducing a direct effect on the malignant B-CLL clone; and indirectly, by modifying the microenvironment that allows tumor growth.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Depsipéptidos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucocitos Mononucleares/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/patología , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclofosfamida/administración & dosificación , Depsipéptidos/administración & dosificación , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Péptidos Cíclicos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
OBJECTIVE: Richter's transformation of B-cell chronic lymphocytic leukemia (CLL) to cutaneous diffuse large B-cell lymphoma (DLBCL) is very rare. We took the advantage of one of these cases to test the hypothesis that the chemokine receptor CCR4 is involved in the homing of CLL cells to skin. PATIENTS AND METHODS: We evaluated CCR4 expression by flow cytometry in both circulating and skin CD19(+) leukemic cells from a patient with cutaneous DLBCL. As controls, we used peripheral blood samples from CLL patients without skin manifestations and from elderly healthy donors. RESULTS: We found that both DLBCL cells derived from the original CLL clone and circulating CLL cells from this patient expressed CCR4. Although it was previously reported that CCR4 is not expressed in CLL cells, we found that a low but significant proportion of leukemic cells from CLL patients with no skin manifestations do express CCR4. There was a positive correlation between the expression of CCR4 and the percentage of ZAP-70 of each sample. Moreover, we consistently observed a higher expression of CCR4 within CD19(+)CD38(+) and CD19(+)Ki67(+) subsets compared to CD19(+)CD38(-) and CD19(+)Ki67(-) lymphocytes from the same sample, respectively. CONCLUSION: We conclude that the chemokine receptor CCR4 is not a special feature of CLL cells with skin manifestation, but rather it is expressed in a low but significant proportion of peripheral blood CLL cells.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma de Células B Grandes Difuso/inmunología , Receptores CCR4/metabolismo , Neoplasias Cutáneas/inmunología , Anciano , Anciano de 80 o más Años , Antígenos CD19/sangre , Antígenos CD19/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/sangre , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Receptores CCR4/sangre , Piel/inmunología , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/patología , Proteína Tirosina Quinasa ZAP-70/sangre , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.
Asunto(s)
Inmunoglobulinas Intravenosas/inmunología , Inmunomodulación , Leucemia Linfocítica Crónica de Células B/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Inmunoglobulinas Intravenosas/farmacología , Inmunomodulación/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/sangre , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Current standard treatment of patients with hairy cell leukemia (HCL), a chronic B-cell neoplasia of low incidence that affects the elderly, is based on the administration of purine analogs such as cladribine. This chemotherapy approach shows satisfactory responses, but the disease relapses, often repeatedly. Venetoclax (ABT-199) is a Bcl-2 inhibitor currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in adult patients ineligible for intensive chemotherapy. Given that HCL cells express Bcl-2, our aim was to evaluate venetoclax as a potential therapy for HCL. We found that clinically relevant concentrations of venetoclax (0.1 and 1 µM) induced primary HCL cell apoptosis in vitro as measured by flow cytometry using Annexin V staining. As microenvironment induces resistance to venetoclax in CLL, we also evaluated its effect in HCL by testing the following stimuli: activated T lymphocytes, stromal cells, TLR-9 agonist CpG, and TLR-2 agonist PAM3. We found decreased levels of venetoclax-induced cytotoxicity in HCL cells exposed for 48 h to any of these stimuli, suggesting that leukemic B cells from HCL patients are sensitive to venetoclax, but this sensitivity can be overcome by signals from the microenvironment. We propose that the combination of venetoclax with drugs that target the microenvironment might improve its efficacy in HCL.
RESUMEN
BACKGROUND: T cells from patients with chronic lymphocytic leukemia may play an important role in contributing to the onset, sustenance, and exacerbation of the disease by providing survival and proliferative signals to the leukemic clone within lymph nodes and bone marrow. DESIGN AND METHODS: By performing chemotaxis assays towards CXCL12, CCL21 and CCL19, we sought to evaluate the migratory potential of T cells from chronic lymphocytic leukemia patients. We next analyzed the chemokine-induced migration of T cells, dividing the chronic lymphocytic leukemia samples according to their expression of the poor prognostic factors CD38 and ZAP-70 in leukemic cells determined by flow cytometry. RESULTS: We found that T cells from patients with chronic lymphocytic leukemia are less responsive to CXCL12, CCL21 and CCL19 than T cells from healthy adults despite similar CXCR4 and CCR7 expression. Following separation of the patients into two groups according to ZAP-70 expression, we found that T cells from ZAP-70-negative samples showed significantly less migration towards CXCL12 compared to T cells from ZAP-70-positive samples and that this was not due to defective CXCR4 down-regulation, F-actin polymerization or to a lesser expression of ZAP-70, CD3, CD45, CD38 or CXCR7 on these cells. Interestingly, we found that leukemic cells from ZAP-70-negative samples seem to be responsible for the defective CXCR4 migratory response observed in their T cells. CONCLUSIONS: Impaired migration towards CXCL12 may reduce the access of T cells from ZAP-70-negative patients to lymphoid organs, creating a less favorable microenvironment for leukemic cell survival and proliferation.
Asunto(s)
Inhibición de Migración Celular/fisiología , Quimiocina CXCL12/fisiología , Quimiotaxis de Leucocito/fisiología , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos T/patología , Proteína Tirosina Quinasa ZAP-70/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Técnicas de Cocultivo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4(+) T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.
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Anemia Hemolítica Autoinmune , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Anciano , Células Presentadoras de Antígenos , Proliferación Celular , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos , Persona de Mediana Edad , Unión Proteica , Linfocitos T/inmunologíaRESUMEN
Extracellular vesicles (EV), comprising microvesicles and exosomes, are particles released by every cell of an organism, found in all biological fluids, and commonly involved in cell-to-cell communication through the transfer of cargo materials such as miRNA, proteins, and immune-related ligands (e.g., FasL and PD-L1). An important characteristic of EV is that their composition, abundance, and roles are tightly related to the parental cells. This translates into a higher release of characteristic pro-tumor EV by cancer cells that leads to harming signals toward healthy microenvironment cells. In line with this, the key role of tumor-derived EV in cancer progression was demonstrated in multiple studies and is considered a hot topic in the field of oncology. Given their characteristics, tumor-derived EV carry important information concerning the state of tumor cells. This can be used to follow the outset, development, and progression of the neoplasia and to evaluate the design of appropriate therapeutic strategies. In keeping with this, the present brief review will focus on B-cell malignancies and how EV can be used as potential biomarkers to follow disease progression and stage. Furthermore, we will explore several proposed strategies aimed at using biologically engineered EV for treatment (e.g., drug delivery mechanisms) as well as for impairing the biogenesis, release, and internalization of cancer-derived EV, with the final objective to disrupt tumor-microenvironment communication.
RESUMEN
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells arrested in G0/G1 stages that coexist with proliferative B cells. We identified one of these proliferative subsets in the peripheral blood from patients with unmutated disease (UM). Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis of the mRNA and microRNAs in this leukemic subpopulation and compared results with those for the quiescent counterpart. Our results suggest that proliferation of this subset mainly depends on microRNA-22 overexpression, which induces phosphatase and tensin homolog (PTEN) down-regulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. These results underline the role of the PI3K/AKT pathway at the origin of this proliferative pool in patients with UM CLL and provide additional rationale for the use of PI3K inhibitors.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Mutación , Transducción de Señal , Microambiente Tumoral/genética , Antígenos CD40/metabolismo , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Masculino , MicroARNs/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Survivin , TranscriptomaRESUMEN
Chronic lymphocytic leukemia (CLL) is the main cause of autoimmune hemolytic anemia (AHA). However, the cellular basis underlying this strong association remains unclear. We previously demonstrated that leukemic B cells from patients with CLL recognize the erythrocyte protein Band 3, a prevalent autoantigen in AHA. Here we show that the major binding site of Band 3 on leukemic cells is an extrinsic protein identified as high-mobility group nucleosome binding protein 2 (HMGN2), a nucleosome-interacting factor which has not been previously reported at the cell surface. T lymphocytes do not express HMGN2 or bind Band 3. Removal of HMGN2 from the cell membrane abrogated the capacity of Band 3-pulsed CLL cells to induce CD4 + T cell proliferation. We conclude that surface HMGN2 in leukemic B cells is involved in Band 3 binding, uptake and presentation to CD4 + T lymphocytes, and as such may favor the initiation of AHA secondary to CLL.