RESUMO
Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Evolução Clonal/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Linfoma Folicular/etiologia , Linfoma Folicular/metabolismo , Rearranjo Gênico , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma Folicular/patologia , RecidivaRESUMO
Antigenic stimulation via the B-cell receptor (BCR) is a major driver of the proliferation and survival of chronic lymphocytic leukemia (CLL) cells. However, the precise mechanisms by which BCR stimulation leads to accumulation of malignant cells remain incompletely understood. Here, we investigated the ability of BCR stimulation to increase messenger RNA (mRNA) translation, which can promote carcinogenesis by effects on both global mRNA translation and upregulated expression of specific oncoproteins. Re-analysis of gene expression profiles revealed striking upregulation of pathways linked to mRNA translation both in CLL cells derived from lymph nodes, the major site of antigen stimulation in vivo, and after BCR stimulation in vitro. Anti-IgM significantly increased mRNA translation in primary CLL cells, measured using bulk metabolic labeling and a novel flow cytometry assay to quantify responses at a single-cell level. These translational responses were suppressed by inhibitors of BTK (ibrutinib) and SYK (tamatinib). Anti-IgM-induced mRNA translation was associated with increased expression of translation initiation factors eIF4A and eIF4GI, and reduced expression of the eIF4A inhibitor, PDCD4. Anti-IgM also increased mRNA translation in normal blood B cells, but without clear modulatory effects on these factors. In addition, anti-IgM increased translation of mRNA-encoding MYC, a major driver of disease progression. mRNA translation is likely to be an important mediator of the growth-promoting effects of antigen stimulation acting, at least in part, via translational induction of MYC. Differences in mechanisms of translational regulation in CLL and normal B cells may provide opportunities for selective therapeutic attack.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos B/imunologia , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Anticorpos Anti-Idiotípicos/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Piperidinas , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/genética , Quinase Syk , Células Tumorais CultivadasRESUMO
The vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca(2+) flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM(+) and IgG(+) cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site.
Assuntos
Linfócitos B/metabolismo , Sinalização do Cálcio , Moléculas de Adesão Celular/metabolismo , Região Variável de Imunoglobulina/metabolismo , Lectinas Tipo C/metabolismo , Linfoma Folicular/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores de Superfície Celular/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Moléculas de Adesão Celular/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Região Variável de Imunoglobulina/imunologia , Lectinas Tipo C/imunologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Receptores de Superfície Celular/imunologia , Células Tumorais CultivadasRESUMO
B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.
Assuntos
Infecções Bacterianas/complicações , Região Variável de Imunoglobulina/imunologia , Lectinas/imunologia , Linfoma Folicular/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Infecções Bacterianas/imunologia , Citometria de Fluxo , Glicosilação , Humanos , Região Variável de Imunoglobulina/química , Linfoma Folicular/complicações , Infecções Oportunistas/complicações , Infecções Oportunistas/imunologia , Polissacarídeos/metabolismo , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismoAssuntos
Antígenos CD/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfonodos/imunologia , Microambiente Tumoral/imunologia , Antígenos CD/sangue , Linfócitos B/patologia , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica/métodos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Estadiamento de Neoplasias/métodos , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Resposta a Proteínas não Dobradas , Tirosina Quinase da Agamaglobulinemia , Linfócitos B/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Transdução de Sinais , Quinase SykRESUMO
Chronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to bead-bound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCγ2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive "resting" population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success.
Assuntos
Linfócitos B/patologia , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores CXCR4/metabolismo , Anticorpos Imobilizados , Linfócitos B/metabolismo , Células Clonais/metabolismo , Células Clonais/patologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores de Superfície Celular/metabolismoRESUMO
Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.
Assuntos
Membrana Celular/metabolismo , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Western Blotting , Ciclo Celular , Proliferação de Células , Humanos , Técnicas Imunoenzimáticas , Leucemia Linfocítica Crônica de Células B/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Anticorpos Anti-Idiotípicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
CD180 is a toll-like receptor that is highly expressed in complex with the MD-1 satellite molecule on the surface of B cells. In chronic lymphocytic leukaemia (CLL) however, the expression of CD180 is highly variable and overall, significantly reduced when compared to normal B cells. We have recently shown that reduced CD180 expression in CLL lymph nodes is associated with inferior overall survival. It was therefore important to better understand the causes of this downregulation through investigation of CD180 at the transcriptional and protein expression levels. Unexpectedly, we found CD180 RNA levels in CLL cells (n = 26) were comparable to those of normal B cells (n = 13), despite heterogeneously low expression of CD180 on the cell surface. We confirmed that CD180 RNA is translated into CD180 protein since cell surface CD180-negative cases presented with high levels of intracellular CD180 expression. Levels of MD-1 RNA were, however, significantly downregulated in CLL compared to normal controls. Together, these data suggest that changes in CD180 cell surface expression in CLL are not due to transcriptional downregulation, but defective post-translational stabilisation of the receptor due to MD-1 downregulation.
Assuntos
Antígenos CD , Regulação para Baixo , Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Antígenos CD/metabolismo , Antígenos CD/genética , Membrana Celular/metabolismo , Regulação Leucêmica da Expressão GênicaRESUMO
The B-cell receptor (BCR) is a key survival molecule for normal B cells and for most B-cell malignancies. Recombinatorial and mutational patterns in the clonal immunoglobulin (Ig) of chronic lymphocytic leukemia (CLL) have revealed 2 major IgMD-expressing subsets and an isotype-switched variant, each developing from distinct B-cell populations. Tracking of conserved stereotypic features of Ig variable regions characteristic of U-CLL indicate circulating naive B cells as the likely cells of origin. In CLL, engagement of the BCR by antigen occurs in vivo, leading to down-regulated expression and to an unanticipated modulation of glycosylation of surface IgM, visible in blood cells, especially in U-CLL. Modulated glycoforms of sIgM are signal competent and could bind to environmental lectins. U-CLL cases express more sIgM and have increased signal competence, linking differential signaling responses to clinical behavior. Mapping of BCR signaling pathways identifies targets for blockade, aimed to deprive CLL cells of survival and proliferative signals. New inhibitors of BCR signaling appear to have clinical activity. In this Perspective, we discuss the functional significance of the BCR in CLL, and we describe strategies to target BCR signaling as an emerging therapeutic approach.
Assuntos
Antineoplásicos/uso terapêutico , Fatores Imunológicos/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Ensaios Clínicos como Assunto , Regulação Leucêmica da Expressão Gênica , Humanos , Fatores Imunológicos/antagonistas & inibidores , Fatores Imunológicos/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca(2+). Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.
Assuntos
Lectinas/imunologia , Linfoma Folicular/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/imunologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Imunidade Inata , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Técnicas In Vitro , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Linfoma Folicular/etiologia , Linfoma Folicular/genética , Manose/química , Receptor de Manose , Lectina de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/imunologia , Modelos Imunológicos , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Hipermutação Somática de ImunoglobulinaRESUMO
Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the mu-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of mu chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface mu chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.
Assuntos
Imunoglobulina M/química , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Apresentação de Antígeno , Linfócitos B/imunologia , Estudos de Casos e Controles , Glicosilação , Humanos , Imunoglobulina M/genética , Cadeias mu de Imunoglobulina/química , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Modelos Imunológicos , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Prognóstico , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
CD30, a non-death domain-containing member of the tumor necrosis factor receptor superfamily, triggers apoptosis in anaplastic large cell lymphoma cells. The CD30 signaling pathways that lead to the induction of apoptosis are poorly defined. Here, we show that the induction of apoptosis by CD30 requires concurrent inhibition of p38 mitogen-activated protein kinase, which itself is activated by engagement of CD30 with CD30 ligand. Treatment of anaplastic large cell lymphoma cells with CD30 ligand and pharmacologic inhibitors of p38 mitogen-activated protein kinase, but not with CD30 ligand or inhibitors alone, triggered the activation of caspase-8 and the induction of apoptosis. Caspase-8 activation occurred within a few hours (2.5-4 h) after receptor triggering, was unaffected by the neutralization of ligands for the death domain-containing receptors TNFR1, Fas, DR3, DR4, or DR5, but was abolished by the expression of a dominant-negative form of the adaptor protein FADD. Importantly, we show that expression of the caspase-8 inhibitor c-FLIP(S) is strongly induced by the CD30 ligand, and that this is dependent on the activation of p38 mitogen-activated protein kinase. Thus, we provide evidence that the induction of apoptosis by CD30 in anaplastic large cell lymphoma cells is normally circumvented by the activation of p38 mitogen-activated protein kinase. These findings have implications for CD30-targeted immunotherapy of anaplastic large cell lymphoma.
Assuntos
Apoptose/efeitos dos fármacos , Antígeno Ki-1/farmacologia , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Antígeno Ki-1/imunologia , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Isotiocianatos/farmacologia , Leucemia/genética , Leucemia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Anticorpos Anti-Idiotípicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genes myc , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Células MCF-7 , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Estresse Fisiológico , Transcrição Gênica/efeitos dos fármacosRESUMO
Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8(+) cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hospedeiro Imunocomprometido , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos do Interstício Tumoral/imunologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária , Depleção Linfocítica , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fenótipo , Baço/imunologia , Subpopulações de Linfócitos T/patologia , Fatores de TempoRESUMO
BACKGROUND: Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM. RESULTS: We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A+ or AG-EB+) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-6, interferon (IFN)-gamma, transforming growth factor (TGF)-beta1, tumor necrosis factor (TNF)-alpha and IL-10. They can be sub-divided into glycophorin A positive (Gl A+) and erythroblast antigen positive (AG-EB+) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-beta1 and TNF-alpha production were found between Gl A+ and AG-EB+ BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-gamma and IL-2 production specifically by the AG-EB+ ENC. Thus, Gl A+ and AG-EB+ ENC produce IL-1beta, IL-2, IL-4, IL-6, IFN-gamma, TGF-beta1 and TNF-alpha. Gl A+ ENC also produce IL-10. CONCLUSION: Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM.
Assuntos
Antígenos/fisiologia , Células da Medula Óssea/metabolismo , Citocinas/biossíntese , Eritroblastos/imunologia , Glicoforinas/fisiologia , Linfócitos B/fisiologia , Células Cultivadas , Citocinas/fisiologia , Humanos , Interferon gama/biossíntese , Interferon gama/fisiologia , Interleucina-1/biossíntese , Interleucina-1/fisiologia , Interleucina-10/biossíntese , Interleucina-10/fisiologia , Interleucina-2/biossíntese , Interleucina-2/fisiologia , Interleucina-4/biossíntese , Interleucina-4/fisiologia , Interleucina-6/biossíntese , Interleucina-6/fisiologia , Linfócitos T/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection.
Assuntos
Citocinas/análise , Imunoensaio/métodos , Anticorpos , Anticorpos Monoclonais , Meios de Cultivo Condicionados , Citocinas/sangue , Citocinas/normas , Humanos , Imunoensaio/normas , Imunoensaio/estatística & dados numéricos , Interferon gama/análise , Interferon gama/sangue , Interleucina-10/análise , Interleucina-10/sangue , Interleucina-2/análise , Interleucina-2/sangue , Interleucina-4/análise , Interleucina-4/sangue , Leucócitos Mononucleares/imunologia , Medições Luminescentes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The presence of mRNA of cytokines IL-1alpha, IL-1beta, IL-4, IL-6, IFN-gamma, TGF-beta, GM-CSF and the absence of mRNA of IL-2, IL-3 and IL-5 were identified in erythroid cells isolated from spleen of mice subjected to erythropoiesis-stimulating actions (treated with phenylhydrazine, acute hypoxia) and from spleen of newborn mice. Gene expression of cytokines in erythroid cells therewith differed both qualitatively and quantitatively depending on erythropoiesis-stimulating action. Erythroid cells of newborn mice contained mRNA of GM-CSF, but did not contain mRNA of IFN-gamma, whereas erythroid cells of mice under acute hypoxia or treated with phenylhydrazine had mRNA of IFN-gamma, but no mRNA of GM-CSF. After cell cultivation with erythropoietin, qualitative and quantitative changes in gene expression of cytokines were observed including the appearance of mRNA of IL-2, IL-3 but not of IL-5. The fact of gene expression of cytokines was confirmed on cells from single erythroid colonies. The level of mRNA expression of cytokines in erythroid cells was comparable with, and for a number of cytokines even higher than in splenocytes of mice immunized with T-dependent antigen. Moreover, it was demonstrated that erythroid cells are capable of translating proteins themselves. The production of GM-CSF and IFN-gamma by erythroid cells extracted from spleen of newborn mice, as well as from mice after erythropoiesis-stimulating actions (treatment with phenylhydrazine, acute hypoxia, acute blood loss) was detected. The level of IFN-gamma production was comparable with the level of production of this cytokine by mitogen-stimulated splenocytes. Thus, erythroid cells, as well as the cells of practically all hemopoietic lineages are capable to express and produce a number of hemo- and immunoregulatory cytokines by means of which they can participate in the regulation of hemo- and immunopoiesis, as well as exert a self-regulatory lineage restricted effect on cell proliferation and differentiation.