Immunosenescence: limitations of natural killer cell-based cancer immunotherapy.

Cancer is primarily considered a disease of old age. Immunosenescence refers to the age-associated changes in the immune system, and its contribution to the increased risk of cancer in old individuals has been discussed for many years. Natural killer (NK) cells are cytotoxic innate immune cells specialized in defence against tumour and virus-infected cells. NK cell cytotoxicity is the result of a fine balance between activating and inhibitory receptors. Several activating receptors have been identified that recognize different ligands frequently found over-expressed on tumour cells or virus-infected cells. The most important NK cell inhibitory receptors interact with major histocompatibility complex class I molecules expressed on almost all nucleated cells preventing NK cell-mediated lysis of healthy cells. NK cell immunosenescence is characterized by a redistribution of NK cell subsets, a diminished expression of several activating receptors and lower per-cell cytotoxicity. Altered expression of activating receptors has also been described in young and elderly cancer patients probably due to chronic exposure to ligands on tumour cells. Thus, the effect of both age and cancer may act synergistically to diminish NK cell-mediated tumour immunosurveillance. Different strategies harnessing the power of NK cells to target tumour cells have been designed including adoptive therapy with autologous or allogeneic expanded NK cells. In addition, checkpoint blockade of inhibitory receptors and the use of agonist antibodies to stimulate activating receptors are emerging areas of research. In this context, the effect of immunosenescence should be considered to improve the efficiency of cancer immunotherapy.

Natural killer cell immunosenescence in acute myeloid leukaemia patients: new targets for immunotherapeutic strategies?

Several age-associated changes in natural killer (NK) cell phenotype have been reported that contribute to the defective NK cell response observed in elderly patients. A remodelling of the NK cell compartment occurs in the elderly with a reduction in the output of immature CD56(bright) cells and an accumulation of highly differentiated CD56(dim) NK cells. Acute myeloid leukaemia (AML) is generally a disease of older adults. NK cells in AML patients show diminished expression of several activating receptors that contribute to impaired NK cell function and, in consequence, to AML blast escape from NK cell immunosurveillance. In AML patients, phenotypic changes in NK cells have been correlated with disease progression and survival. NK cell-based immunotherapy has emerged as a possibility for the treatment of AML patients. The understanding of age-associated alterations in NK cells is therefore necessary to define adequate therapeutic strategies in older AML patients.

Cytokine profiles in acute myeloid leukemia patients at diagnosis: survival is inversely correlated with IL-6 and directly correlated with IL-10 levels.

BACKGROUND: Several evidences support the existence of cytokine deregulation in acute myeloid leukemia (AML) patients that may be associated with pathogenesis, disease progression and patient survival. METHODS: In the present study, we analyzed plasma levels of pro- and anti-inflammatory cytokines in AML patients and age-matched healthy donors. TNF-α, IL-6, IL-1ß, IL-2, IFN-γ, IL-17A, IL-12p70, IL-8, IL-10, IL-4 and IL-5 were analyzed using fluorescent bead-based technology and TGF-ß by ELISA technique. Because age-associated differences in cytokine profiles have been described, patients and healthy individuals were divided into two age groups: up to 65 years and over 65 years. RESULTS: Our results showed that plasma TNF-α, IL-6 and IL-10 levels were higher in AML patients from both groups of age. IL-8 was increased in AML patients less than 65 years while the plasma concentrations of IL-4, IL-5 and IL-12p70 were significantly higher only in elderly AML patients compared with aged-matched healthy controls. Moreover, plasma levels of IL-6 and IL-10 were associated with patient survival and event-free survival. CONCLUSIONS: An aberrant production of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 is observed in AML patients. Low levels of IL-6 and high levels of IL-10 represent favorable prognostic factors for survival in AML patients. These results support the idea that cytokine deregulation may be useful as a marker for predicting clinical evolution in AML patients.

Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients.

This study tested the hypothesis that the expression of CD112 and CD155 (DNAM-1 ligands) on leukemic blasts induces a decreased expression of the activating receptor DNAM-1 on natural killer (NK) cells from acute myeloid leukemia (AML) patients. DNAM-1 is a co-receptor involved in the activation of NK cell cytotoxicity after its interaction with its ligands CD112 and CD155 on target cells. Here we study the expression of DNAM-1 on NK cells and DNAM-1 ligands on blasts from AML patients stratified by age. The results demonstrate that NK cells from AML patients younger than 65 years have a reduced expression of DNAM-1 compared with age-matched controls. The analysis of DNAM-1 ligands showed a high expression of CD112 and CD155 on leukemic blasts. An inverse correlation between CD112 expression on leukemic blasts and DNAM-1 expression on NK cells was found. Furthermore, downregulation of DNAM-1 was induced on healthy donors' NK cells after in vitro culture with leukemic blasts expressing DNAM-1 ligands. In conclusion, these results support the hypothesis that receptor-ligand crosslinking downregulates DNAM-1 expression on NK cells from patients <65 years of age. Considering the relevance of DNAM-1 in NK recognition and killing of leukemic cells, the reduced expression of this receptor on NK cells from AML patients can represent an additional mechanism of tumor escape.

Association between cortical thickness and anxiety measures: A scoping review.

Cortical thickness has been increasingly studied in the context of structural-brain-behavior associations, such as anxiety; however, the literature is scattered across methods and research fields. This scoping review aims to summarize the available data concerning the association between cortical thickness and anxiety-related measures and identify the current research gaps. Searches were conducted in PubMed, PsycINFO/PsycARTICLES, Web of Science, OpenGrey and Networked Digital Library of Theses and Dissertations, and reference lists of key studies. Two researchers independently screened the abstracts and full-text reports according to the eligibility criteria, as well as extracted and charted the data. Quantitative and descriptive syntheses were conducted. The included publications (n = 18) reported cross-sectional studies, and 17 used surface-based approaches to estimate cortical thickness. Differences in regional cortical thickness were found to be associated with different anxiety-related measures/processes. Brain regions of interest include the medial orbitofrontal cortex, the ventromedial prefrontal cortex, the insula, the temporo-parietal areas, and the anterior cingulate cortex. However, caution should be warranted when interpreting the available results, as there is high variability in the field across anxiety-related measures, distinctive anxiety disorders, and data processing conditions and analysis. More research into this association is needed, to replicate and clarify existing findings.

Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activating receptors and their ligands.

Natural killer (NK) cell activation is strictly regulated to ensure that healthy cells are preserved, but tumour-transformed or virus-infected cells are recognized and eliminated. To carry out this selective killing, NK cells have an ample repertoire of receptors on their surface. Signalling by inhibitory and activating receptors by interaction with their ligands will determine whether the NK cell becomes activated and kills the target cell. Here, we show reduced expression of NKp46, NKp30, DNAM-1, CD244 and CD94/NKG2C activating receptors on NK cells from acute myeloid leukaemia patients. This reduction may be induced by chronic exposure to their ligands on leukaemic blasts. The analysis of ligands for NK cell-activating receptors showed that leukaemic blasts from the majority of patients express ligands for NK cell-activating receptors. DNAM-1 ligands are frequently expressed on blasts, whereas the expression of the NKG2D ligand MICA/B is found in half of the patients and CD48, a ligand for CD244, in only one-fourth of the patients. The decreased expression of NK cell-activating receptors and/or the heterogeneous expression of ligands for major receptors on leukaemic blasts can lead to an inadequate tumour immunosurveillance by NK cells. A better knowledge of the activating receptor repertoire on NK cells and their putative ligands on blasts together with the possibility to modulate their expression will open new possibilities for the use of NK cells in immunotherapy against leukaemia.

Oncolytic adenovirus with hyaluronidase activity that evades neutralizing antibodies: VCN-11.

Tumor targeting and intratumoral virus spreading are key features for successful oncolytic virotherapy. VCN-11 is a novel oncolytic adenovirus, genetically modified to express hyaluronidase (PH20) and display an albumin-binding domain (ABD) on the hexon. ABD allows the virus to self-coat with albumin when entering the bloodstream and evade neutralizing antibodies (NAbs). Here, we validate VCN-11 mechanism of action and characterize its toxicity. VCN-11 replication, hyaluronidase activity and binding to human albumin to evade NAbs was evaluated. Toxicity and efficacy of VCN-11 were assessed in mice and hamsters. Tumor targeting, and antitumor activity was analyzed in the presence of NAbs in several tumor models. VCN-11 induced 450 times more cytotoxicity in tumor cells than in normal cells. VCN-11 hyaluronidase production was confirmed by measuring PH20 activity in vitro and in virus-infected tumor areas in vivo. VCN-11 evaded NAbs from different sources and tumor targeting was demonstrated in the presence of high levels of NAbs in vivo, whereas the control virus without ABD was neutralized. VCN-11 showed a low toxicity profile in athymic nude mice and Syrian hamsters, allowing treatments with high doses and fractionated administrations without major toxicities (up to 1.2x1011vp/mouse and 7.5x1011vp/hamster). Fractionated intravenous administrations improved circulation kinetics and tumor targeting. VCN-11 antitumor efficacy was demonstrated in the presence of NAbs against Ad5 and itself. Oncolytic adenovirus VCN-11 disrupts tumor matrix and displays antitumor effects even in the presence of NAbs. These features make VCN-11 a safe promising candidate to test re-administration in clinical trials.

[Active surveillance for patients with localized prostate cáncer. Adherence and protocol drop analysis.] / La vigilancia activa en pacientes con cáncer de próstata localizado. Análisis de adherencia y salida del protocolo.

OBJECTIVE: VA is currently considered the treatment of choice for patients with low and very low risk prostate cancer. We analyzed the evolution of this treatment strategy in our series and adherence to the protocol. MATERIAL AND METHODS: Ambispective study of patients in VA in our center between 2014- 2019. 237 meet inclusion criteria, of which 142 (60%) have a minimum of 12 months of follow- up. Mean age: 68.5 (4678), median PSA 6.37 ng / ml (1-33). 229 (96.6%) are ISUP 1 and 8 (3.4%) ISUP 2. Objectives are proposed to assess our adherence to the protocol. Descriptive statistics are used to communicate the results. RESULTS: According to the classification by risk groups of the NCCN, 145 (61.2%), 49 (20.7%) and 42 (17.7%) were very low risk, low risk and favorable intermediate risk patients, respectively. The median of follow-up is 14 months (0-66). Of the patients with a minimum follow-up of 12 months, 107 (75.4%) were re-biopsied. 80 (33.8%) leave the protocol in these 5 years, 31.3% (25) by their own decision, 55% (44) due to medical criteria, and 11.3% (9) go to WW. After 5 years of follow-up, 99.2% of patients are still alive, 0.8% died of specific non-cancer causes. Of the objectives to assess adherence, 8 are achieved, 1 partially and 1 is not evaluable. CONCLUSIONS: VA in our center is already the treatment of choice for very low-risk patients, with a constant increase from year to year. Adherence to the protocol has been favorable during the period of time studied.

OBJETIVO: La VA se ha convertido en uno de los tratamientos de elección del CP localizado de bajo y muy bajo riesgo. Analizamos la evolución de esta estrategia de tratamiento en nuestra serie, así como la adherencia al protocolo.MATERIAL Y MÉTODOS: Estudio ambispectivo de los pacientes incluidos en VA en nuestro centro entre los años 2014-2019. 237 pacientes cumplen los criterios de inclusión en VA, de los cuales 142 (60%) tienen un seguimiento mínimo de 12 meses. Edad media: 68,5(46-78), mediana PSA 6,37 ng/ml (1-33). 229 pacientes (96,6%) son ISUP 1 y 8 (3,4%) ISUP 2. Se proponen unos objetivos para valorar nuestra adherencia al protocolo. Se utiliza estadística descriptiva y contraste de hipótesis para comunicar los resultados.RESULTADOS Y DISCUSIÓN: Atendiendo a la clasificación por grupos de riesgo de la NCCN, 145 (61,2%), 49 (20,7%) y 42 (17,7%) eran pacientes muy bajo riesgo, bajo riesgo y riesgo intermedio favorable respectivamente. El tiempo (mediana) de permanencia en el programa es de 14 meses (0-66). De los pacientes con un seguimiento mínimo de 12 meses, 107 (75,4%) son re ­ biopsiados. 80 pacientes (33,8%) salen del protocolo en estos 5 años, 31,3% (25) por decisión propia, 55% (44) por criterios médicos, y 11,3% (9) pasan a WW. Tras 5 años de seguimiento, el 99,2% de los pacientes continúan vivos, el 0,8% falleció por causas no cáncer específicas. De los objetivos para evaluar la adherencia, 8 de ellos se alcanzan, 1 parcialmente y 1 no es evaluable. CONCLUSIONES: La VA en nuestro centro constituye actualmente el tratamiento de elección para los pacientes con muy bajo riesgo. La adherencia al protocolo ha sido favorable durante el periodo de tiempo estudiado.

VCN-01 disrupts pancreatic cancer stroma and exerts antitumor effects.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplastic stroma that limits the delivery of anticancer agents. VCN-01 is an oncolytic adenovirus designed to replicate in cancer cells with a dysfunctional RB1 pathway and express hyaluronidase. Here, we evaluated the mechanism of action of VCN-01 in preclinical models and in patients with pancreatic cancer. METHODS: VCN-01 replication and antitumor efficacy were evaluated alone and in combination with standard chemotherapy in immunodeficient and immunocompetent preclinical models using intravenous or intratumoral administration. Hyaluronidase activity was evaluated by histochemical staining and by measuring drug delivery into tumors. In a proof-of-concept clinical trial, VCN-01 was administered intratumorally to patients with PDAC at doses up to 1×1011 viral particles in combination with chemotherapy. Hyaluronidase expression was measured in serum by an ELISA and its activity within tumors by endoscopic ultrasound elastography. RESULTS: VCN-01 replicated in PDAC models and exerted antitumor effects which were improved when combined with chemotherapy. Hyaluronidase expression by VCN-01 degraded tumor stroma and facilitated delivery of a variety of therapeutic agents such as chemotherapy and therapeutic antibodies. Clinically, treatment was generally well-tolerated and resulted in disease stabilization of injected lesions. VCN-01 was detected in blood as secondary peaks and in post-treatment tumor biopsies, indicating virus replication. Patients had increasing levels of hyaluronidase in sera over time and decreased tumor stiffness, suggesting stromal disruption. CONCLUSIONS: VCN-01 is an oncolytic adenovirus with direct antitumor effects and stromal disruption capabilities, representing a new therapeutic agent for cancers with dense stroma. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005556-42 and NCT02045589.

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines.

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the "European Searchable Tumour Cell Line and Data Bank" (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/ ) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.

Functional implications of HNK-1 expression on invasive behaviour of melanoma cells.

HNK-1 epitope, also known as CD57, is expressed on a wide range of cells and has been related to cell to cell and cell to extracellular matrix interactions. Expression of the HNK-1 epitope has been considered a prognostic factor in several tumours, as it has been associated with the risk of metastasis. HNK-1 has been found to be expressed on uveal and cutaneous melanoma and proposed as a useful marker of the risk of metastasis. We have analysed the HNK-1 expression on a large panel of melanoma cell lines, the involvement of this epitope in melanoma cell adhesion, as well as its migrative and invasive behaviour. HNK-1 was highly expressed in 12.9% of melanoma cell lines, and in vitro experiments using invasive melanoma cell lines demonstrated that an HNK-1 blockade reduces cell adhesion to extracellular matrix as well as their migrative and invasive ability. These data support the functional relevance of HNK-1 expression in metastatic melanoma.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes.

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

Effect of age and CMV on NK cell subpopulations.

NK cells represent an important component of the innate immune response against infection and tumors. Age-associated changes in NK cell phenotype have been previously reported that can be responsible of functional NK cell deficiency. The aim of this work was to analyze the effect CMV seropositivity and aging on the distribution of NK cell subsets with a focus on the expression of cytotoxicity-related molecules and on the expression of CD94/NKG2 heterodimers and CD57 on these NK cell subsets. Our results show that CMV seropositivity in young individuals does not significantly affect peripheral blood NK cell percentage and NK cell subsets defined by the use of CD56 and CD16 markers. In contrast a significant increase in the percentage of NK cells is observed in elderly donors, all of them are CMV seropositive, when compared with young CMV seropositive subjects. A decrease in the percentage of CD56bright NK cells, either fully immature CD16 negative or CD16+ and an increase in the CD56-CD16+ subset are also found in the elderly. CMV seropositivity either in healthy young or elderly individuals is associated to the expression of CD94/NKG2C dimers and high expression of CD57on the CD56dimCD16+ NK cell subset. CD56-CD16+ NK cells, which are expanded in the elderly, show a decreased expression of granzymes A and B and an increased expression of CD94/NKG2C and CD57 in CMV seropositive young donors when compared with CMV seronegative young individuals. These results indicate that CMV and age have a different effect on NK cell phenotype and emphasize the relevance of including the determination of CMV serostatus in those studies addressed to analyze the immune response in the elderly.

Human adipose-derived stem cells impair natural killer cell function and exhibit low susceptibility to natural killer-mediated lysis.

Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared.

Immunosenescence of human natural killer cells.

Natural killer (NK) cells are a key component of innate immunity involved not only in the elimination of virus-infected or tumor cells but also in the regulation of the immune response by producing cytokines and chemokines that can activate other cellular components of innate and adaptive immunity. NK cell subsets are differentially affected by aging. Whereas CD56(bright) cells are decreased in healthy elderly individuals, the CD56(dim) subset is expanded. The expression of CD57, a marker of highly differentiated NK cells, is increased in the elderly; this supports the notion that a remodeling process of NK cell subsets occurs in aging with a gradual decrease in more immature CD56(bright) NK cells and an increase in highly differentiated CD56(dim) CD57+ NK cells. This NK cell redistribution can explain many of the phenotypic and functional changes in NK cells associated with healthy aging such as decreased proliferation and the maintenance of CD16-dependent cytotoxicity.

NK cell recognition and killing of melanoma cells is controlled by multiple activating receptor-ligand interactions.

The role of natural killer (NK) cells in tumor immunosurveillance has been recently underlined. A better understanding of the receptor-ligand interactions between NK cells and solid tumor cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practice. We previously analyzed the surface expression of ligands for NK cell-activating receptors and costimulatory molecules in a large panel of melanoma cell lines. Although the expression of ligands for NK cell-activating receptors is variable, the majority of melanoma cell lines express ligands for NKG2D and for DNAX accessory molecule-1 (DNAM-1). While the NKG2D receptor has been described as the principal entity responsible for the lysis of several melanoma cell lines, the role of natural cytotoxicity receptors (NCRs) and DNAM-1 receptors in NK cell recognition and killing of melanoma cells has been recently emphasized. Antibody-mediated masking of NKG2D, NCRs, and DNAM-1 has proven that NKG2D, NCRs, and DNAM-1 frequently cooperate in the lysis of melanoma cells. In this work, we provide an overview of recent advances in the study of melanoma cells' susceptibility to NK cell-mediated lysis and how multiple receptor-ligand interactions participate in melanoma cell elimination.

Melatonin enhances the immune response to vaccination against A1 and C strains of Dichelobacter nodosus.

Melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. Here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of A1 and C strains of Dichelobacter nodosus in sheep. Subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. Administration of melatonin to vaccinated animals enhanced both the titer of antibodies and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Our results suggest that melatonin increased the immune response to vaccination and open new perspectives in the design of prophylactic strategies.