RESUMEN
Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).
Asunto(s)
Epidermólisis Ampollosa Distrófica , Humanos , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Empalme del ARN , Piel , Intrones , Precursores del ARN , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Colágeno Tipo VII/genéticaRESUMEN
Machine learning has been proven to be a powerful tool in the identification of diagnostic tumor biomarkers but is often impeded in rare cancers due to small patient numbers. In patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), early-in-life development of particularly aggressive cutaneous squamous-cell carcinomas (cSCCs) represents a major threat and timely detection is crucial to facilitate prompt tumor excision. As miRNAs have been shown to hold great potential as liquid biopsy markers, we characterized miRNA signatures derived from cultured primary cells specific for the potential detection of tumors in RDEB patients. To address the limitation in RDEB-sample accessibility, we analyzed the similarity of RDEB miRNA profiles with other tumor entities derived from the Cancer Genome Atlas (TCGA) repository. Due to the similarity in miRNA expression with RDEB-SCC, we used HN-SCC data to train a tumor prediction model. Three models with varying complexity using 33, 10 and 3 miRNAs were derived from the elastic net logistic regression model. The predictive performance of all three models was determined on an independent HN-SCC test dataset (AUC-ROC: 100%, 83% and 96%), as well as on cell-based RDEB miRNA-Seq data (AUC-ROC: 100%, 100% and 91%). In addition, the ability of the models to predict tumor samples based on RDEB exosomes (AUC-ROC: 100%, 93% and 100%) demonstrated the potential feasibility in a clinical setting. Our results support the feasibility of this approach to identify a diagnostic miRNA signature, by exploiting publicly available data and will lay the base for an improvement of early RDEB-SCC detection.
RESUMEN
Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.
Asunto(s)
Epidermólisis Ampollosa Distrófica , Epidermólisis Ampollosa , Humanos , Trans-Empalme , Piel/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa/genética , Queratinocitos/metabolismo , Colágeno Tipo VII/genética , MutaciónRESUMEN
Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.
Asunto(s)
Epidermólisis Ampollosa de la Unión , Epidermólisis Ampollosa , Animales , Humanos , Epidermólisis Ampollosa de la Unión/genética , Edición Génica , Piel , Mutación , Queratinocitos , Epidermólisis Ampollosa/genéticaRESUMEN
Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.
Asunto(s)
Autoantígenos , Epidermólisis Ampollosa de la Unión , Epidermólisis Ampollosa , Colágenos no Fibrilares , Autoantígenos/genética , Desoxirribonucleasa I/genética , Epidermólisis Ampollosa/metabolismo , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/terapia , Homocigoto , Humanos , Laminina/genética , Mutación , Colágenos no Fibrilares/genética , Eliminación de Secuencia , Colágeno Tipo XVIIRESUMEN
: Introduction: Antibody treatment with anti-thymocyte globulin (ATG) has been shown to be cardioprotective. We aimed to evaluate which single anti-T-cell epitope antibody alters chemokine expression at a level similar to ATG and identified CD3, which is a T-cell co-receptor mediating T-cell activation. Based on these results, the effects of anti-CD3 antibody treatment on angiogenesis and cardioprotection were tested in vitro and in vivo. METHODS: Concentrations of IL-8 and MCP-1 in supernatants of human peripheral blood mononuclear cell (PBMC) cultures following distinct antibody treatments were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). In vivo, anti-CD3 antibodies or vehicle were injected intravenously in rats subjected to acute myocardial infarction (AMI). Chemotaxis and angiogenesis were evaluated using tube and migration assays. Intracellular pathways were assessed using Western blot. Extracellular vesicles (EVs) were quantitatively evaluated using fluorescence-activated cell scanning, exoELISA, and nanoparticle tracking analysis. Also, microRNA profiles were determined by next-generation sequencing. RESULTS: Only PBMC stimulation with anti-CD3 antibody led to IL-8 and MCP-1 changes in secretion, similar to ATG. In a rat model of AMI, systemic treatment with an anti-CD3 antibody markedly reduced infarct scar size (27.8% (Inter-quartile range; IQR 16.2-34.9) vs. 12.6% (IQR 8.3-27.2); p < 0.01). The secretomes of anti-CD3 treated PBMC neither induced cardioprotective pathways in cardiomyocytes nor pro-angiogenic mechanisms in human umbilical vein endothelial cell (HUVECs) in vitro. While EVs quantities remained unchanged, PBMC incubation with an anti-CD3 antibody led to alterations in EVs miRNA expression. CONCLUSION: Treatment with an anti-CD3 antibody led to decreased scar size in a rat model of AMI. Whereas cardioprotective and pro-angiogenetic pathways were unaltered by anti-CD3 treatment, qualitative changes in the EVs miRNA expression could be observed, which might be causal for the observed cardioprotective phenotype. We provide evidence that EVs are a potential cardioprotective treatment target. Our findings will also provide the basis for a more detailed analysis of putatively relevant miRNA candidates.
Asunto(s)
Complejo CD3/inmunología , Cicatriz/tratamiento farmacológico , Leucocitos Mononucleares/efectos de los fármacos , MicroARNs/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/inmunología , Neovascularización Fisiológica/efectos de los fármacos , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Suero Antilinfocítico/inmunología , Suero Antilinfocítico/uso terapéutico , Cardiotónicos/inmunología , Quimiocina CCL2/metabolismo , Cicatriz/inmunología , Cicatriz/prevención & control , Modelos Animales de Enfermedad , Exosomas/efectos de los fármacos , Exosomas/inmunología , Exosomas/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interleucina-8/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , MicroARNs/genética , Neovascularización Fisiológica/inmunología , Proteoma/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Epidermolytic ichthyosis is a skin fragility disorder caused by dominant-negative mutations in KRT1 or KRT10. No definitive restorative therapies exist that target these genetic faults. Gene editing can be used to efficiently introduce frameshift mutations to inactivate mutant genes. This can be applied to counter the effect of dominantly inherited diseases such as epidermolytic ichthyosis. In this study, we used transcription activator-like effector nuclease technology, to disrupt disease-causing mutant KRT10 alleles in an ex vivo cellular approach, with the intent of developing a therapy for patients with epidermolytic ichthyosis. A transcription activator-like effector nuclease was designed to specifically target a region of KRT10, upstream of a premature termination codon known to induce a genetic knockout. This proved highly efficient at gene disruption in a patient-derived keratinocyte cell line. In addition, analysis for off-target effects indicated no promiscuous gene editing-mediated disruption. Reversion of the keratin intermediate filament fragility phenotype associated with epidermolytic ichthyosis was observed by the immunofluorescence analysis of correctly gene-edited single-cell clones. This was in concurrence with immunofluorescence and ultrastructure analysis of murine xenograft models. The efficiency of this approach was subsequently confirmed in primary patient keratinocytes. Our data demonstrate the feasibility of an ex vivo gene-editing therapy for more than 95.6% of dominant KRT10 mutations.
Asunto(s)
Edición Génica/métodos , Hiperqueratosis Epidermolítica/terapia , Filamentos Intermedios/metabolismo , Queratina-10/genética , Piel/patología , Alelos , Animales , Biopsia , Línea Celular , Modelos Animales de Enfermedad , Exones/genética , Estudios de Factibilidad , Femenino , Terapia Genética/métodos , Humanos , Hiperqueratosis Epidermolítica/genética , Hiperqueratosis Epidermolítica/patología , Queratina-10/metabolismo , Queratinocitos/patología , Queratinocitos/trasplante , Masculino , Ratones , Mutación , Cultivo Primario de Células , Estabilidad Proteica , Piel/citología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
PURPOSE: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need.Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. RESULTS: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. CONCLUSIONS: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/etiología , Epidermólisis Ampollosa Distrófica/complicaciones , Epidermólisis Ampollosa Distrófica/genética , Glicina/análogos & derivados , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/etiología , Sulfonas/uso terapéutico , Antineoplásicos/farmacología , Apoptosis , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Silenciamiento del Gen , Genes Recesivos , Glicina/farmacología , Glicina/uso terapéutico , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Terapia Molecular Dirigida , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero , ARN Interferente Pequeño , Neoplasias Cutáneas/diagnóstico , Sulfonas/farmacología , Quinasa Tipo Polo 1RESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.
Asunto(s)
Desaminasas APOBEC/genética , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Citosina Desaminasa/genética , Epidermólisis Ampollosa Distrófica/enzimología , Epidermólisis Ampollosa Distrófica/genética , Mutación/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Variaciones en el Número de Copia de ADN/genética , Reparación del ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis/genética , Tasa de Mutación , Transcriptoma/genéticaRESUMEN
Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/sangre , Vesículas Extracelulares/metabolismo , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Neoplásico/sangre , ARN Neoplásico/genética , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/genética , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/biosíntesisRESUMEN
With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.
Asunto(s)
Sistemas CRISPR-Cas , Epidermólisis Ampollosa Simple/terapia , Edición Génica/métodos , Queratina-14/genética , Queratinocitos/metabolismo , Reparación del ADN por Recombinación , Secuencia de Bases , Células Cultivadas , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/metabolismo , Epidermólisis Ampollosa Simple/patología , Exones , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones , Queratina-14/metabolismo , Queratinocitos/patología , Queratinocitos/trasplante , Terapia Molecular Dirigida , Mutación , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/genéticaRESUMEN
Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vß7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.
Asunto(s)
Antígenos CD1d/metabolismo , Linfocitos T CD8-positivos/citología , Leucemia Linfocítica Crónica de Células B/metabolismo , Células T Asesinas Naturales/citología , Anciano , Anciano de 80 o más Años , Animales , Complejo CD3/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions. METHODS: VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined. RESULTS: About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro. CONCLUSIONS: Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration, but only a limited role in their protection by stromal cells.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Médula Ósea/patología , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Infiltración Leucémica/patología , ADP-Ribosil Ciclasa 1/sangre , Animales , Apoptosis , Linfocitos B/inmunología , Médula Ósea/metabolismo , Adhesión Celular , Recuento de Células , Femenino , Humanos , Inmunohistoquímica , Integrina alfa4beta1/sangre , Antígeno Ki-67/metabolismo , Leucemia Linfocítica Crónica de Células B/sangre , Tejido Linfoide/patología , Masculino , Ratones , Factores de Riesgo , Células del Estroma/patología , Análisis de SupervivenciaRESUMEN
The combination of cytotoxic treatment with strategies for immune activation represents an attractive strategy for tumour therapy. Following reduction of high tumour burden by effective cytotoxic agents, two major immune-stimulating approaches are being pursued. First, innate immunity can be activated by monoclonal antibodies triggering antibody-dependent cellular cytotoxicity. Second, tumour-specific T cell responses can be generated by immunization of patients with peptides derived from tumour antigens and infused in soluble form or loaded onto dendritic cells. The choice of cytotoxic agents for such combinatory regimens is crucial since most substances such as fludarabine are considered immunosuppressive while others such as cyclophosphamide can have immunostimulatory activity. We tested in this study whether fludarabine and/or cyclophosphamide, which represent a very effective treatment regimen for chronic lymphocytic leukaemia, would interfere with a therapeutic strategy of T cell activation. Analysis of peripheral blood samples from patients prior and during fludarabine/cyclophosphamide therapy revealed rapid and sustained reduction of tumour cells but also of CD4(+) and CD8(+) T cells. This correlated with a significant cytotoxic activity of fludarabine/cyclophosphamide on T cells in vitro. Unexpectedly, T cells surviving fludarabine/cyclophosphamide treatment in vitro had a more mature phenotype, while fludarabine-treated T cells were significantly more responsive to mitogenic stimulation than their untreated counterparts and showed a shift towards T(H)1 cytokine secretion. In conclusion, fludarabine/cyclophosphamide therapy though inducing significant and relevant T cell depletion seems to generate a micromilieu suitable for subsequent T cell activation.