RESUMEN
Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.
Asunto(s)
Canales de Calcio , Canales de Potencial de Receptor Transitorio , Humanos , Canales de Calcio/metabolismo , Calcio/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Mitocondrias/metabolismo , Lisosomas/metabolismo , Células Asesinas Naturales/metabolismoRESUMEN
Adoptive transfer of allogeneic NK cells holds great promise for cancer immunotherapy. There is a variety of protocols to expand NK cells in vitro, most of which are based on stimulation with cytokines alone or in combination with feeder cells. Although IL-15 is essential for NK cell homeostasis in vivo, it is commonly used at supraphysiological levels to induce NK cell proliferation in vitro. As a result, adoptive transfer of such IL-15-addicted NK cells is associated with cellular stress because of sudden cytokine withdrawal. In this article, we describe a dose-dependent addiction to IL-15 during in vitro expansion of human NK cells, leading to caspase-3 activation and profound cell death upon IL-15 withdrawal. NK cell addiction to IL-15 was tightly linked to the BCL-2/BIM ratio, which rapidly dropped during IL-15 withdrawal. Furthermore, we observed a proliferation-dependent induction of BIM short, a highly proapoptotic splice variant of BIM in IL-15-activated NK cells. These findings shed new light on the molecular mechanisms involved in NK cell apoptosis following cytokine withdrawal and may guide future NK cell priming strategies in a cell therapy setting.
Asunto(s)
Proteína 11 Similar a Bcl2/metabolismo , Proliferación Celular/efectos de los fármacos , Interleucina-15/farmacología , Células Asesinas Naturales/efectos de los fármacos , Apoptosis , Proteína 11 Similar a Bcl2/genética , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Humanos , Interleucina-15/inmunología , Células K562 , Células Asesinas Naturales/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
BACKGROUND: Natural killer (NK) cells have a unique capability of spontaneous cytotoxicity against malignant cells and hold promise for off-the-shelf cell therapy against cancer. One of the key challenges in the field is to improve NK cell homing to solid tumors. METHODS: To gain a deeper understanding of the cellular mechanisms regulating trafficking of NK cells into the tumor, we used high-dimensional flow cytometry, mass cytometry, and single-cell RNA-sequencing combined with functional assays, creating a comprehensive map of human NK cell migration phenotypes. FINDINGS: We found that the chemokine receptor repertoire of peripheral blood NK cells changes in a coordinated manner becoming progressively more diversified during NK cell differentiation and correlating tightly with the migratory response of the distinct NK cell subsets. Simultaneous ligation of CXCR1/2 and CX3CR1, synergistically potentiated the migratory response of NK cells. Analysis of 9471 solid cancers from publicly available TCGA/TARGET repositories revealed dominant chemokine patterns that varied across tumor types but with no tumor group expressing ligands for more than one chemokine receptor present on mature NK cells. INTERPRETATION: The finding that chemokine stimulation can elicit a synergistic migratory response in NK cells combined with the identified lack of naturally occurring pairs of chemokines-chemokine receptors in human cancers may explain the systematic exclusion of NK cells from the tumor microenvironment and provides a basis for engineering next-generation NK cell therapies against malignancies. FUNDING: The Polish Ministry of Science and Higher Education, the National Science Centre, Poland, The Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, The Swedish Cancer Society, the Swedish Children's Cancer Foundation, The Swedish Research Council, The Center of Excellence: Precision Immunotherapy Alliance, Knut and Alice Wallenberg Foundation and National Cancer Institute.
Asunto(s)
Neoplasias , Receptores de Quimiocina , Niño , Humanos , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Células Asesinas Naturales/metabolismo , Neoplasias/patología , Quimiocinas/metabolismo , Inmunoterapia Adoptiva , Microambiente TumoralRESUMEN
BACKGROUND: Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. RESULTS: ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. CONCLUSIONS: These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.
Asunto(s)
Citotoxicidad Inmunológica , Leucemia Mieloide Aguda , Animales , Ratones , Citotoxicidad Celular Dependiente de Anticuerpos , Células Asesinas Naturales/metabolismo , Leucemia Mieloide Aguda/patología , Receptores KIR/metabolismoRESUMEN
BACKGROUND: Current prognostic variables can only partly explain the large outcome heterogeneity in diffuse large B cell lymphoma (DLBCL). We aimed to investigate the utility of systems-level protein and immune repertoire profiling for outcome prognostication in DLBCL. METHODS: In this retrospective study, we used proximity extension assay technology to quantify 81 immune-related proteins in serum or plasma in 2 independent cohorts in a total 111 DLBCL patients. Protein levels were assessed before and after treatment with rituximab and chemotherapy, and the patients were compared with 19 age- and sex-matched healthy blood donors. In a subset of the patients, we performed a broad mass cytometric characterization of immune cell repertoires in peripheral blood. FINDINGS: Patients displayed large deviations in protein profiles compared with healthy controls. Development of a systemic protein deviation (SPD) score provided a 4-protein-based metric that reflected the overall degree of protein deviations compared with age- and sex-matched healthy blood donors. The SPD score identified patients with very poor overall survival in both cohorts and correlated with increased frequencies of peripheral blood PD-1+ CD8+ T cells, and expansion of myeloid-derived suppressor cells. CONCLUSIONS: Our results show that a simple metric based on measurement of a small set of serum or plasma proteins can be used to probe systemic immune changes associated with poor survival in DLBCL. This finding warrants further investigation in larger, prospective studies to establish a clinical prognostic biomarker.
Asunto(s)
Linfoma de Células B Grandes Difuso , Receptor de Muerte Celular Programada 1 , Biomarcadores , Linfocitos T CD8-positivos/patología , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/inmunología , Neoplasias Colorrectales/prevención & control , Fragmentos de Inmunoglobulinas/uso terapéutico , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/uso terapéutico , Animales , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Tasa de SupervivenciaRESUMEN
Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.
Asunto(s)
Células Asesinas Naturales/metabolismo , Lisosomas/metabolismo , Aminopiridinas/farmacología , Animales , Granzimas/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Ratones , Receptores KIR/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Binding of the coagulation protease factor VIIa to its receptor Tissue Factor (TF) induces intracellular signals in several cell types including HaCaT keratinocytes. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR2 is involved in factor VIIa and factor Xa signal transduction. Our results indicate that the epidermal growth factor receptor (EGFR) and the proline rich tyrosine kinase 2 (PYK2) participate in TF/FVIIa signalling as formation of the TF/FVIIa complex increased the phosphorylation of these proteins. Both FVIIa protease activity and available TF were necessary for generation of the signal. Increased tyrosine phosphorylation of the EGFR was observed following TF/FVIIa complex formation on the cell surface. The EGFR kinase inhibitor tyrphostin AG1478 abrogated the TF/FVIIa-complex induced MAP kinase activation and mRNA increase of egr-1, heparin-binding EGF, and interleukin-8 following FVIIa addition. Using specific antibodies, increased phosphorylation of PYK2 tyrosine residues 402 and 580 was observed. The first site is the major autophosphorylation site and the docking site for Src family kinases. The second site is important for the kinase activity. The Src family kinase Yes and the tyrosine phosphatase SHP-2 were detected in immunoprecipitates using either anti-PYK2 or anti-EGFR antibodies. Their coprecipitation with EGFR increased in the presence of FVIIa. Moreover, the coprecipitation of EGFR and PYK2 increased with FVIIa stimulation. Together, these data suggest that EGFR, PYK2, Yes, and SHP-2 are involved in transduction of the TF/FVIIa signal possibly via transactivation of the EGF receptor.
Asunto(s)
Receptores ErbB/metabolismo , Factor VIIa/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tromboplastina/metabolismo , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Factor de Crecimiento Epidérmico/genética , Factor VIIa/farmacología , Quinasa 2 de Adhesión Focal , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/genética , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Quinazolinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Tirfostinos/farmacologíaRESUMEN
A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.
Asunto(s)
Acetilcisteína/análogos & derivados , Factor VII/química , Factor VII/genética , Mutación , Acetilcisteína/química , Animales , Anticuerpos Monoclonales/química , Encéfalo/metabolismo , Brefeldino A/química , Células CHO , Dicroismo Circular , Cricetinae , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/química , Exones , Factor X/química , Factor Xa/química , Heterocigoto , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Péptidos/química , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/química , Mapeo Restrictivo , Análisis de Secuencia de ADN , Serina Endopeptidasas/química , Resonancia por Plasmón de Superficie , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: The standard treatment of ovarian cancer with chemotherapy often leads to drug resistance and relapse of the disease, and the need for development of novel therapy alternatives is obvious. The MOC31PE immunotoxin binds to the cell surface antigen EpCAM, which is expressed by the majority of epithelial cancers including ovarian carcinomas, and we studied the cytotoxic effects of MOC31PE in ovarian cancer cells. METHODS: Investigation of the effects of MOC31PE treatment on protein synthesis, cell viability, proliferation and gene expression of the ovarian cancer cell lines B76 and HOC7. RESULTS: MOC31PE treatment for 24 h caused a dose-dependent reduction of protein synthesis with ID50 values of less than 10 ng/ml, followed by reduced cell viability. In a gene expression array monitoring the expression of 84 key genes in cancer pathways, 13 of the genes were differentially expressed by MOC31PE treatment in comparison to untreated cells. By combining MOC31PE and the immune suppressor cyclosporin A (CsA) the MOC31PE effect on protein synthesis inhibition and cell viability increased tenfold. Cell migration was also reduced, both in the individual MOC31PE and CsA treatment, but even more when combining MOC31PE and CsA. In tumor metastasis PCR arrays, 23 of 84 genes were differentially expressed comparing CsA versus MOC31PE + CsA treatment. Increased expression of the tumor suppressor KISS1 and the nuclear receptor NR4A3 was observed, and the differential candidate gene expression was confirmed in complementary qPCR analyses. For NR4A3 this was not accompanied by increased protein expression. However, a subcellular fractionation assay revealed increased mitochondrial NR4A3 in MOC31PE treated cells, suggesting a role for this protein in MOC31PE-induced apoptotic cell death. CONCLUSION: The present study demonstrates that MOC31PE may become a new targeted therapy for ovarian cancer and that the MOC31PE anti-cancer effect is potentiated by CsA.