Characterization of the molecular composition and in vitro regenerative capacity of platelet-based bioproducts and related subfractions.

The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, as the lack of characterization and detailed production methods of platelet-based bioproducts persists in the literature and precludes cross-study comparisons. We characterized the molecular composition and in vitro regenerative capacity of platelet-rich plasma (PRP) produced in a closed-system. Furthermore, we performed a parallel characterization on different PRP subfractions (plasma and plasma-free platelet lysate), identifying that the fractions containing platelet-derived cargo exert the most potent regenerative capacity. This observation led us to develop a method to obtain a platelet secretome highly enriched in growth factors, free of plasma and cellular components (PCT/IB2022/057936), with the aim of establishing a superior bioproduct. The molecular characterization of secretomes revealed agonist-dependent differences, which correlates with beneficial grades of regenerative capacity. Importantly, secretomes showed general superiority to PRP in vitro. We discuss the variables influencing the bioproduct quality (inter-donor variation, platelet source and processing methods). Finally, we propose that the characteristics of secretomes circumvents certain limitations of PRP (autologous vs allogeneic), and envision that optimizing post-processing protocols (nanoencapsulation, lyophilization), would allow their clinical application even beyond regenerative medicine. STATEMENT OF SIGNIFICANCE: The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, or to improve/personalize them, as the lack of characterization of the bioproducts and their production methods is a constant in the literature, reason that precludes cross-study comparisons. In the present manuscript, we provide a comprehensive molecular and functional characterization of platelet-based bioproducts and subfractions, including platelet rich plasma, plasma fractions and platelet secretomes produced with a methodology developed by our group. Our results show that the molecular composition of each fraction correlates with its regenerative capacity in vitro. Thus, a rigorous characterization of platelet-derived bioproducts will potentially allow universal use, customizing and new applications.

Proteomics-wise, how similar are mouse and human platelets?

The field of proteomics and its application to platelet biology, is rapidly and promisingly developing. Platelets (and megakaryocytes) are postulated as biosensors of health and disease, and their proteome poses as a tool to identify the specific health-disease hallmarks. Furthermore, the clinical management of certain pathologies where platelets are active players demands the development of alternative treatments, such is the case in patients where the balance thrombosis-bleeding is compromised, and a proteomics approach might aid at the identification of novel targets. Hereby, the mouse and human platelet proteomes and secretomes from public databases are compared, which shows that human and mouse platelets share a highly conserved proteome, considering identified proteins, and most importantly, their relative abundance. These supports, also interspecies wise, the use of the proteomics tool in the field, substantiated by a growing number of clinically relevant studies in humans or preclinical models. While the study of platelets through proteomics seems accessible and direct (i.e. noninvasive blood sampling, enucleated), there are some points of concern regarding the quality control of samples for such proteomics studies. Importantly, the quality of the generated data is improving over the years, which will allow cross-study comparisons. In parallel, the application of proteomics to the megakaryocyte compartment has a promising but long journey ahead. We foresee and encourage the application of platelet proteomics for diagnostic/prognostic purposes even beyond hematopoiesis and transfusion medicine, and as a tool that will procure the improvement of current therapies and the development of alternative treatment options.

The unbiased identification and quantitation of the protein profile (the so-called proteome) of cells, tissues, or organs, has gained attention from different fields because it gives additional valuable information to research questions. Understanding the protein building blocks of a biological system in normal physiological processes and how this may be altered in disease, may allow the discovery of biomarkers that could be used in diagnosis (early diagnosis), prognosis of disease or response to treatment. Furthermore, it may allow the identification of novel targets to develop personalized treatment options. Platelets, the anucleate cell components of the blood in charge of maintaining the body hemostasis, are postulated as biosensors of health and disease, and their proteome poses as a tool to identify health-disease hallmarks. Since platelets are in the circulation, a noninvasive blood sample is sufficient to obtain platelets from donors or patients in order to acquire information of the platelet proteome. Still, some research questions might require the use of animal preclinical models, where researchers may phenocopy human disorders, pathologies or diseases, to better understand the mechanisms behind these traits and to test potential novel treatments. How meaningful the studies in preclinical models are depends on how similar the biological systems of study are, interspecies wise. Hereby, the mouse and human platelet proteomes from available databases obtained by different research groups are compared, which shows that human and mouse platelets share a highly conserved proteome, considering identified proteins, and most importantly, their relative abundance. These supports, also interspecies wise, the use of the proteomics tool in the field, an approach with growing clinical relevance, as discussed.

Clinical Response After Treatment of Knee Osteoarthritis With a Standardized, Closed-System, Low-Cost Platelet-Rich Plasma Product: 1-Year Outcomes.

Background: Intra-articular infiltration of platelet-rich plasma (PRP) is an alternative therapeutic option to classic hyaluronic acid for the treatment of symptomatic knee osteoarthritis (KOA). However, variation in preparation methods and quality assessment of PRP makes the study of its real clinical efficacy difficult. Purpose: To (1) evaluate the clinical efficacy of a characterized PRP product prepared in a standardized manner and in a closed-system for the treatment of KOA and to (2) evaluate the association of the clinical response to PRP-related variables. Study Design: Case series; Level of evidence, 4. Methods: We recruited 130 patients with nonoperative KOA and evaluated them for 1 year. PRP was prepared from a donation of autologous blood, obtaining 3 aliquots of approximately 10mL of product, which were frozen, allowing platelet disruption, platelet factor release, and long-term storage, until administration. Patients were treated 3 consecutive times every 4 weeks with an intra-articular PRP knee injection under sterile conditions. Complete blood count was performed on the whole-blood sample and the processed PRP before freezing it, for product quality assessment. Patients were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and basic satisfaction scale at 3 months, 6 months, and 1 year after intervention. Results: Quality assessment confirmed a leukocyte-poor PRP product (white blood cell count, 0.09 ± 0.09 × 109/L) with a high platelet purity (platelet count, 630.86 ± 191.75 × 109/L). WOMAC scores improved, and basic satisfaction was achieved in 70% of patients. No adverse events were reported. No correlations were observed between PRP quality parameters and clinical results. PRP complete treatment production costs were €108/US$125 (€36/US$41.6 per injection). Conclusion: This standardized PRP production method resulted in improved WOMAC scores at 1 year postoperatively in 70% of patients with KOA. This technique was safe and affordable and ensured consecutive infiltrations with the same product to each patient.

Platelet number and function alterations in preclinical models of sterile inflammation and sepsis patients: implications in the pathophysiology and treatment of inflammation.

Platelets are the blood cells in charge of maintaining the body hemostasis, recognising the damaged vessel wall, and providing the appropriate cellular surface for the coagulation cascade to act locally. Additionally, platelets are active immunomodulators. At the crossroads of hemostasis and inflammation, platelets may exert either beneficial actions or participate in pathological manifestations, and have been associated with the prothrombotic nature of multi-organ failure in systemic inflammation. Platelet number alterations have been reported in septis, and platelet transfusions are given to thrombocytopenic patients. However, the risk to develop transfusion related acute lung injury (TRALI) is higher in sepsis patients. In this manuscript we show that platelets produced during inflammation in preclinical mouse models of sterile inflammation display lower aggregation capacity when stimulating certain receptors, while responses through other receptors remain intact, and we name them "inflammation-conditioned" platelets. In a cohort of sepsis patients, we observed, as previously reported, alterations in the number of platelets and platelet hyperreactivity. Furthermore, we identified a receptor-wise platelet aggregation response disbalance in these patients, although not similar to platelets from preclinical models of sterile inflammation. Interestingly, we generated evidence supporting the notion that platelet aggregation capacity disbalance was partially triggered by plasma components from sepsis patients. Our findings have implications in the indication of platelet transfusions in sepsis patients: Are fully functional platelets suitable for transfusion in sepsis patients? Current Clinical Trials (RESCUE) will answer whether platelet production stimulation with thrombopoietin receptor agonists (TPO-RAs) could be a substitute of platelet transfusions.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even >64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Clinical Management of Hypertension, Inflammation and Thrombosis in Hospitalized COVID-19 Patients: Impact on Survival and Concerns.

The most severe clinical manifestations of the Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are due to an unbalanced immune response and a pro-thrombotic hemostatic disturbance, with arterial hypertension or diabetes as acknowledged risk factors. While waiting for a specific treatment, the clinical management of hospitalized patients is still a matter of debate, and the effectiveness of treatments to manage clinical manifestations and comorbidities has been questioned. In this study, we aim to assess the impact of the clinical management of arterial hypertension, inflammation and thrombosis on the survival of COVID-19 patients. The Spanish cohorts included in this observational retrospective study are from HM Hospitales (2035 patients) and from Hospital Universitario Central de Asturias (72 patients). Kaplan Meier survival curves, Cox regression and propensity score matching analyses were employed, considering demographic variables, comorbidities and treatment arms (when opportune) as covariates. The management of arterial hypertension with angiotensin-converting enzyme 2 (ACE2) inhibitors or angiotensin receptor blockers is not detrimental, as was initially reported, and neither was the use of non-steroidal anti-inflammatory drugs (NSAIDs). On the contrary, our analysis shows that the use on itself of corticosteroids is not beneficial. Importantly, the management of COVID-19 patients with low molecular weight heparin (LMWH) as an anticoagulant significantly improves the survival of hospitalized patients. These results delineate the current treatment options under debate, supporting the effectiveness of thrombosis prophylaxis on COVID-19 patients as a first-line treatment without the need for compromising the treatment of comorbidities, while suggesting cautiousness when administering corticosteroids.

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells.

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs.

On the Quest for In Vitro Platelet Production by Re-Tailoring the Concepts of Megakaryocyte Differentiation.

The demand of platelet transfusions is steadily growing worldwide, inter-donor variation, donor dependency, or storability/viability being the main contributing factors to the current global, donor-dependent platelet concentrate shortage concern. In vitro platelet production has been proposed as a plausible alternative to cover, at least partially, the increasing demand. However, in practice, such a logical production strategy does not lack complexity, and hence, efforts are focused internationally on developing large scale industrial methods and technologies to provide efficient, viable, and functional platelet production. This would allow obtaining not only sufficient numbers of platelets but also functional ones fit for all clinical purposes and civil scenarios. In this review, we cover the evolution around the in vitro culture and differentiation of megakaryocytes into platelets, the progress made thus far to bring the culture concept from basic research towards good manufacturing practices certified production, and subsequent clinical trial studies. However, little is known about how these in vitro products should be stored or whether any safety measure should be implemented (e.g., pathogen reduction technology), as well as their quality assessment (how to isolate platelets from the rest of the culture cells, debris, microvesicles, or what their molecular and functional profile is). Importantly, we highlight how the scientific community has overcome the old dogmas and how the new perspectives influence the future of platelet-based therapy for transfusion purposes.

In vitro platelet production for transfusion purposes: Where are we now?

Over the last decade there has been a worldwide increase in the demand of platelet concentrates (PCs) for transfusion. This is, to a great extent, due to a growing and aging population with the concomitant increase in the incidence of onco-hematological diseases, which require frequent platelet (PLT) transfusions. Currently, PLTs are sourced uniquely from donations, and their storage time is limited only to a few days. The necessity to store PCs at room temperature (to minimize loss of PLT functional integrity), poses a major risk for bacterial contamination. While the implementation of pathogen reduction treatments (PRTs) and new-generation PLT additive solutions have allowed the extension of the shelf life and a safer PLT transfusion product, the concern of PCs shortage still pressures the scientific community to find alternative solutions with the aim of meeting the PLT transfusion increasing demand. In this concise report, we will focus on the efforts made to produce, in in vitro culture, high yields of viable and functional PLTs for transfusion purposes in a cost-effective manner, meeting not only current Good Manufacturing Practices (cGMPs), but also transfusion safety standards.

Platelet-derived bio-products: Classification update, applications, concerns and new perspectives.

Platelet derived bio-products in the form of platelet rich plasma, plasma rich in growth factors, or plasma-free platelet releasates, are being studied worldwide with the aim of proving their efficacy in tissue regeneration within many different clinical areas, such as traumatology, maxillofacial surgery, ophthalmology, dermatology and otorhinolaryngology, amongst others. The current lack of consensus in the preparation method and application form, or in the quality assessment of each bio-product, precludes adequate interpretation of the relevance of reported clinical outcomes, and, while many in clinicians are very positive about them, many are sceptic. Relevant aspects of these products are considered to propose a classification nomenclature which would aid a comprehensive comparison of clinical outcomes of bio-products of the same characteristics. Finally, the uses of platelet-derived bio-products in in vitro culture (for cell therapy purposes) as a substitute of animal-origin sera, and other future perspectives of applications of platelet-derived bio-products are discussed.

Optimising platelet secretomes to deliver robust tissue-specific regeneration.

Promoting cell proliferation is the cornerstone of most tissue regeneration therapies. As platelet-based applications promote cell division and can be customised for tissue-specific efficacy, this makes them strong candidates for developing novel regenerative therapies. Therefore, the aim of this study was to determine if platelet releasate could be optimised to promote cellular proliferation and differentiation of specific tissues. Growth factors in platelet releasate were profiled for physiological and supraphysiological platelet concentrations. We analysed the effect of physiological and supraphysiological releasate on C2C12 skeletal myoblasts, H9C2 rat cardiomyocytes, human dermal fibroblasts (HDF), HaCaT keratinocytes, and chondrocytes. Cellular proliferation and differentiation were assessed through proliferation assays, mRNA, and protein expression. We show that supraphysiological releasate is not simply a concentrated version of physiological releasate. Physiological releasate promoted C2C12, HDF, and chondrocyte proliferation with no effect on H9C2 or HaCaT cells. Supraphysiological releasate induced stronger proliferation in C2C12 and HDF cells compared with physiological releasate. Importantly, supraphysiological releasate induced proliferation of H9C2 cells. The proliferative effects of skeletal and cardiac muscle cells were in part driven by vascular endothelial growth factor alpha. Furthermore, supraphysiological releasate induced differentiation of H9C2 and C2C12, HDF, and keratinocytes. This study provides insights into the ability of releasate to promote muscle, heart, skin, and cartilage cell proliferation and differentiation and highlights the importance of optimising releasate composition for tissue-specific regeneration.

Implementation of a closed platelet-rich-plasma preparation method using the local blood bank infrastructure at the Principality of Asturias (Spain): Back to basic methodology and a demographics perspective after 1 year.

Platelet-rich plasma (PRP) is used with increasing demand as autologous adjuvant therapy in many areas of regenerative medicine, thanks to the platelet rich content of growth factors and bio-active molecules. However, to date there is a lack of consensus on PRP preparation methods, on processing and application forms, on clinical application guidelines and on knowledge-based composition at the cellular and molecular level, making difficult the assessment of clinical results from different groups in different clinical areas. Here we describe the implementation of PRP production on a closed-system using the infrastructure of a certified blood bank, detailing methodology, and validation and production results 1 year after its implementation. Our methodology provides a reproducible, safe, practical and yet affordable PRP bio-product that will allow further studies to better define PRP applications in regenerative medicine and personalized therapeutic regimes.

Drug-induced hyperploidy stimulates an antitumor NK cell response mediated by NKG2D and DNAM-1 receptors.

Formation of polyploid or aneuploid cells is a pathological hallmark of malignant tumors. Cell cycle checkpoint mechanisms play a crucial role in ensuring genomic integrity during mitosis, avoiding the generation of aneuploid cells. Additionally, cancer cell DNA ploidy is subjected to extrinsic controls operated by activation of adaptive immune responses mediated by T cells. NK cells exert a central role in the innate anticancer immunity; however, the mechanisms involved in the recognition of tumor cells by NK cells have not been fully elucidated. Herein, we report that drug-induced polyploidy in cancer cells activates antitumor responses mediated by NK cells. Thus, hyperploidy-inducing chemotherapeutic agents strongly upregulate the tumor expression of ligands for the NK cell activating receptors NKG2D and DNAM-1. Drug-induced hyperploidy modulated the repertoire of activating receptors and the cytokine profile of NK cells, rendering tumor cells more susceptible to NK cell-mediated lysis through the activation of NKG2D and DNAM-1 receptors. In addition, hyperploidization stimulated the production of IL-2 by CD4 T cells, which induced NK cell proliferation and activity. The stimulation of MICA, a key NKG2D ligand, in hyperploid cells was mainly mediated by ATM protein kinase. Likewise, pharmacological inhibition of key regulators of endoplasmic reticulum stress in certain cell models supports a role for this pathway in NKG2D ligand upregulation. Overall, our findings indicate that, besides the cytotoxic effect on tumor cells, the therapeutic activity of anti-mitotic drugs may be mediated by the induction of a coordinated antitumor immune response involving NK and T cells.

Analysis of clinical prognostic variables for Chronic Lymphocytic Leukemia decision-making problems.

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is a disease with highly heterogeneous clinical course. A key goal is the prediction of patients with high risk of disease progression, which could benefit from an earlier or more intense treatment. In this work we introduce a simple methodology based on machine learning methods to help physicians in their decision making in different problems related to CLL. MATERIAL AND METHODS: Clinical data belongs to a retrospective study of a cohort of 265 Caucasians who were diagnosed with CLL between 1997 and 2007 in Hospital Cabueñes (Asturias, Spain). Different machine learning methods were applied to find the shortest list of most discriminatory prognostic variables to predict the need of Chemotherapy Treatment and the development of an Autoimmune Disease. RESULTS: Autoimmune disease occurrence was predicted with very high accuracy (>90%). Autoimmune disease development is currently an unpredictable severe complication of CLL. Chemotherapy Treatment has been predicted with a lower accuracy (80%). Risk analysis showed that the number of false positives and false negatives are well balanced. CONCLUSIONS: Our study highlights the importance of prognostic variables associated with the characteristics of platelets, reticulocytes and natural killers, which are the main targets of the autoimmune haemolytic anemia and immune thrombocytopenia for autoimmune disease development, and also, the relevance of some clinical variables related with the immune characteristics of CLL patients that are not taking into account by current prognostic markers for predicting the need of chemotherapy. Because of its simplicity, this methodology could be implemented in spreadsheets.

Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042).

Demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (DIG-MSK) is a recently isolated analogue of mithramycin A (MTA) that showed differences with MTA in the DNA binding strength and selectivity. These differences correlated with a better therapeutic index and less toxicity in animal studies. Herein, we show that DIG-MSK displays a potent anti-tumor activity against different types of cancer cell lines, ovarian tumor cells being particularly sensitive to this drug. Of relevance, DIG-MSK exerts low toxicity on fibroblasts and peripheral blood mononuclear cells, this toxicity being significantly lower than that of MTA. In correlation with its antitumor activity, DIG-MSK strongly inhibited Sp1-mediated transcription and endogenous Sp1 mRNA expression, which correlated with the inhibition of the expression of key Sp1-regulated genes involved in tumorigenesis, including VEGFA, BCL2L1 (Bcl-XL), hTERT, BRCA2, MYC and SRC in several ovarian cells. Significantly, DIG-MSK was a stronger inhibitor of VEGFA expression than MTA. Accordingly, DIG-MSK also exhibited potent anti-angiogenic activity on microvascular endothelial cells. Likewise, it significantly inhibited the gene expression of VEGFR1, VEGFR2, FGFR, PDGFB and PDGFRA and, additionally, it induced the expression of the anti-angiogenic factors angiostatin and tunstatin. These effects correlated with a pro-apoptotic effect on proliferating microvascular endothelial cells and the inhibition of the formation of endothelial capillary structures. Overall, the pleiotropic activity of DIG-MSK in inhibiting key oncogenic and angiogenic pathways, together with its low toxicity profile, highlight the therapeutic potential of this new drug.

NKG2D signaling in cancer immunosurveillance.

The immune system is able to detect and eliminate transformed cells. The activating receptor NKG2D is particularly relevant for cancer immunosurveillance. NKG2D ligand expression renders tumor cells more susceptible to be killed by NK and T cells, and correlates with the clinical outcome of the disease. However, tumors develop mechanisms to overcome the NKG2D-mediated immune response, which has been associated with poor prognosis and impairment of the clinical benefits of immunotherapy in many human cancers. The highly specific pattern of expression displayed by the NKG2D ligands, mainly confined to tumor cells, together with the strong immune response triggered by this receptor clearly supports the idea that the NKG2D-mediated pathway may be a powerful target for the treatment of cancer. This review draws together the most recent discoveries concerning the biology of the NKG2D signaling and their therapeutic relevance in the context of cancer.

Lenalidomide induces immunomodulation in chronic lymphocytic leukemia and enhances antitumor immune responses mediated by NK and CD4 T cells.

Lenalidomide is an immunomodulatory drug with therapeutic activity in chronic lymphocytic leukemia (CLL). However, it has pleiotropic effects, and the mechanism of action responsible for its therapeutic activity has not been well defined yet. Herein, we show that lenalidomide treatment does not have an effect on the proliferation of leukemia cells, but it increases the proliferation of B cells from healthy donors. Lenalidomide did not exert a direct effect on the apoptosis of leukemia cells obtained from CLL patients, although it indirectly induced their apoptosis through the activation of nonmalignant immune cells. Thus, lenalidomide markedly increased the proliferation of NK and CD4 T cells. The effect of lenalidomide on NK cells was secondary to the induction of IL-2 production by CD4 T cells. Accordingly, depletion of T cells or blockade of IL-2 activity completely abrogated the proliferation of NK cells. Additionally, lenalidomide enhanced NK and NKT-like cell-mediated natural cytotoxicity against leukemia cells from CLL patients. Lenalidomide also upregulated CD20 expression on leukemia cells and, accordingly, it had a synergistic effect with rituximab on promoting antibody-dependent cell-mediated cytotoxicity against primary leukemia cells. Overall, these observations provide a support for combining lenalidomide with rituximab as a treatment in CLL.

Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease.

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.

Molecular Bases for the Regulation of NKG2D Ligands in Cancer.

NKG2D is an activating receptor expressed by NK and T cells primarily involved in the elimination of transformed and infected cells. NKG2D ligands are self-proteins restrictedly expressed in healthy tissues, but induced in response to signaling pathways commonly associated with transformation. Proliferative, tumor suppressor, and stress signaling pathways linked to the tumorigenic process induce the expression of NKG2D ligands, initiating an immune response against the incipient tumor. Nevertheless, the activity of NKG2D ligands is counter-regulated in vivo by the immunoediting of cancer cells, resulting in the expression of multiple mechanisms of immune evasion in advanced tumors. The redundancy of NKG2D ligands, besides increasing the complexity of their regulation, may impair the generation of these immune evasion mechanisms. In this review, we attempt to integrate the mechanisms and pathways involved in the regulation of NKG2D ligand expression in cancer.

Regulation of NKG2D signaling during the epithelial-to-mesenchymal transition.

The plasma membrane receptor natural killer group 2 member D (NKG2D) underpins a major mechanism whereby natural killer (NK) and T cells recognize malignant cells. We have recently demonstrated that the epithelial-to-mesenchymal transition, one of the first steps of metastatic dissemination, is under the control of an immunological checkpoint that relies on NKG2D-mediated immune responses.