Recalling the Biological Significance of Immune Checkpoints on NK Cells: A Chance to Overcome LAG3, PD1, and CTLA4 Inhibitory Pathways by Adoptive NK Cell Transfer?

Immune checkpoint receptors (IC) positively or negatively regulate the activation of the host immune response, preventing unwanted reactions against self-healthy tissues. In recent years the term IC has been mainly used for the inhibitory ICs, which are critical to control Natural Killer (NK) and Cytotoxic CD8(+) T cells due to its high cytotoxic potential. Due to the different nature of the signals that regulate T and NK cell activation, specific ICs have been described that mainly regulate either NK cell or T cell activity. Thus, strategies to modulate NK cell activity are raising as promising tools to treat tumors that do not respond to T cell-based immunotherapies. NK cell activation is mainly regulated by ICs and receptors from the KIR, NKG2 and NCRs families and the contribution of T cell-related ICs is less clear. Recently, NK cells have emerged as contributors to the effect of inhibitors of T cell-related ICs like CTLA4, LAG3 or the PD1/PD-L1 axes in cancer patients, suggesting that these ICs also regulate the activity of NK cells under pathological conditions. Strikingly, in contrast to NK cells from cancer patients, the level of expression of these ICs is low on most subsets of freshly isolated and in vitro activated NK cells from healthy patients, suggesting that they do not control NK cell tolerance and thus, do not act as conventional ICs under non-pathological conditions. The low level of expression of T cell-related ICs in "healthy" NK cells suggest that they should not be restricted to the detrimental effects of these inhibitory mechanisms in the cancer microenvironment. After a brief introduction of the regulatory mechanisms that control NK cell anti-tumoral activity and the conventional ICs controlling NK cell tolerance, we will critically discuss the potential role of T cell-related ICs in the control of NK cell activity under both physiological and pathological (cancer) conditions. This discussion will allow to comprehensively describe the chances and potential limitations of using allogeneic NK cells isolated from a healthy environment to overcome immune subversion by T cell-related ICs and to improve the efficacy of IC inhibitors (ICIs) in a safer way

Influencia de la necrosis tumoral en neuroblastoma sobre la migración de células NK.

El neuroblastoma es el tumor sólido extracraneal más común en niños, suponiendo el 8% de los casos de cáncer infantil. Mientras los casos de neuroblastoma de bajo riesgo tienen una tasa de supervivencia superior al 90%, el neuroblastoma de alto riesgo, con una alta probabilidad de no responder a los tratamientos convencionales, tiene una tasa de supervivencia a 5 años del 50%. Por lo tanto, es muy necesario el desarrollo de terapias específicas contra el neuroblastoma de alto riesgo. La terapia adoptiva con células NK es una de las líneas de investigación contra el cáncer infantil, ya que por lo general se tratan de tumores poco inmunogénicos, con una baja tasa de mutaciones, y contra los que las células NK se presentan como una herramienta terapéutica potente y segura. El neuroblastoma, como tumor sólido, presenta unas características en su microentorno tumoral que modulan la actividad de las células inmunes, como la presencia de necrosis tumoral y la oposición a la infiltración debido a la estructura del tumor, que pueden alterar los resultados de la terapia adoptiva con células NK. Además, mientras la inmunoterapia con anticuerpos monoclonales contra la vía de inmunosupresión PD1/PD-L1 ha sido ampliamente estudiada en linfocitos T, mostrando una gran relevancia, no ha sido tan estudiada en las células NK. Debido a esto, en este trabajo se ha estudiado la actividad citotóxica de las células NK expandidas in vitro con dos protocolos frente a cultivos tanto en 2D como en 3D de líneas celulares de neuroblastoma. Los cultivos en 3D presentan ciertas características del microentorno tumoral, mostrando mayor grado de semejanza con la complejidad de un tumor sólido. También se ha estudiado la expresión de PD1 en las células NK. La comparación de los protocolos de expansión mostró diferencias entre los niveles de citotoxicidad de las células NK observados con ambos procedimientos. Los cultivos en 3D mostraron mayor resistencia a la citotoxicidad de las células NK que los cultivos en 2D. Respecto al estudio de la expresión de PD1 en las células NK, se ha observado el aumento de la expresión en la membrana tras la incubación de las células NK con células tumorales, observándose niveles análogos a los presentes en pacientes con cáncer. Además, se observó una recuperación parcial de la actividad citotóxica de las células NK al añadir el anticuerpo anti-PD1. Estos resultados indican la relevancia de la vía PD1/PD-L1 en las células NK y su influencia por lo tanto en los tratamientos con inmunoterapia con células NK. <br /

Caracterización de un modelo in vitro de cultivo celular 3D de neuroblastoma para el estudio de inmunoterapia con células NK

La inmunoterapia se presenta como un nuevo y prometedor tratamiento contra cánceres infantiles como el neuroblastoma (NB). Una de estas nuevas terapias en desarrollo es el uso de células asesinas naturales (NK) modificadas genéticamente para la expresión de receptores de antígenos quiméricos (NK-CAR). En concreto, en nuestro grupo de investigación se está desarrollando un NK-CAR frente a las proteínas CD47 y calreticulina, las cuales son sobreexpresadas en NB. Para el desarrollo de esta terapia, se estudió como paso inicial un modelo de la línea celular SH-SY5Y cultivado en tres dimensiones (3D). Mediante cultivo en 3D se generaron agregados celulares compactos, llamados esferoides, que mimetizan de forma más exacta la arquitectura de un tumor real. Los esferoides de la línea celular SH-SY5Y presentaron diferencias respecto al cultivo tradicional en dos dimensiones en cuanto a sus características fenotípicas y la resistencia frente a los tratamientos estudiados. Se aplicaron las quimioterapias usadas normalmente contra el NB y el tratamiento con células NK activadas para usarlos como referencia para en un futuro evaluar el nivel de actividad de las células NK-CAR desarrolladas. La importancia del modelo celular seleccionado quedó patente en los resultados, y se aceptó el modelo de cultivo de la línea celular SH-SY5Y en 3D como modelo adecuado para el desarrollo de la terapia con células NK-CAR.<br /

Oxidative phosphorylation dysfunction modifies the cell secretome

Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell’s secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials

All about (nk cell-mediated) death in two acts and an unexpected encore: initiation, execution and activation of adaptive immunity

NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences

PD-1 is expressed in cytotoxic granules of NK cells and rapidly mobilized to the cell membrane following recognition of tumor cells

5 figures.-- Supplementary information available.The contribution of the T cell-related inhibitory checkpoint PD-1 to the regulation of NK cell activity is still not clear with contradictory results concerning its expression and role in the modulation of NK cell cytotoxicity. We provide novel key findings on the mechanism involved in the regulation of PD-1 expression on NK cell membrane and its functional consequences for the elimination of cancer cells. In contrast to freshly isolated NK cells from cancer patients, those from healthy donors did not express PD-1 on the cell membrane. However, when healthy NK cells were incubated with tumor target cells, membrane PD-1 expression increased, concurrent with the CD107a surface mobilization. This finding suggested that PD-1 was translocated to the cell membrane during NK cell degranulation after contact with target cells. Indeed, cytosolic PD-1 was expressed in freshly-isolated-NK cells and partly co-localized with CD107a and GzmB, confirming that membrane PD-1 corresponded to a pool of preformed PD-1. Moreover, NK cells that had mobilized PD-1 to the cell membrane presented a significantly reduced antitumor activity on PD-L1-expressing-tumor cells in vitro and in vivo, which was partly reversed by using anti-PD-1 blocking antibodies. Our results indicate that NK cells from healthy individuals express cytotoxic granule-associated PD-1, which is rapidly mobilized to the cell membrane after interaction with tumor target cells. This novel finding helps to understand how PD-1 expression is regulated on NK cell membrane and the functional consequences of this expression during the elimination of tumor cells, which will help to design more efficient NK cell-based cancer immunotherapies.Work in the JP laboratory is funded by FEDER (Fondo Europeo de Desarrollo Regional), Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación y Universidades (MCUN), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente Inocente, ASPANOA, and Carrera de la Mujer de Monzón. Postdoctoral Juan de la Cierva Contract (MA and LS) and Predoctoral Grant from AECC (CP). JP is supported by ARAID Foundation; Fundación Agencia Aragonesa para la investigación y el Desarrollo; Fundación Científica Asociación Española Contra el Cáncer.Peer reviewe

Pathological Features in Paediatric Patients with TK2 Deficiency

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype

All about (NK cell-mediated) death in two acts and an unexpected encore: initiation, execution and activation of adaptive immunity

3 figures.NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences.This research was supported by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU, FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RB-I00), ASPANOA, Donación Javier Saura Carceller, and Carrera de la Mujer de Monzón. JP was supported by Fundacion Aragon I+D (ARAID). MA was granted by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (IJC2019-039192-I). LS was granted by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJC2020-046181-I). IU-M and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC), MG-T by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. CO was granted by AEI -Agencia Estatal de Investigación- (PTA2020-018510-I / AEI / 10.13039/501100011033).Peer reviewe

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required

GDF-15 is elevated in children with mitochondrial diseases and is induced by mitochondrial dysfunction

Background We previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction. Methods We analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA. Results Our results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated lev- els of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated. Conclusions Our data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochon- drial dysfunction and that its levels correlate in vitro with FGF-21 level