The CARD9 Gene in Koalas (Phascolarctos cinereus): Does It Play a Role in the Cryptococcus-Koala Interaction?

Cryptococcus is a genus of fungal pathogens that can infect and cause disease in a range of host species and is particularly prominent in koalas (Phascolarctos cinerus). Like other host species, koalas display a range of outcomes upon exposure to environmental Cryptococcus, from external nasal colonization to asymptomatic invasive infection and, in rare cases, severe clinical disease resulting in death. Host factors contributing to these varied outcomes are poorly understood. Due to their close relationship with eucalypt trees (a key environmental niche for Cryptococcus gattii) and suspected continual exposure to the pathogen, koalas provide a unique opportunity to examine host susceptibility in natural infections. Caspase recruitment domain-containing protein 9 (CARD9) is a key intracellular signaling protein in the fungal innate immune response. Humans with mutations in CARD9 succumb to several different severe and chronic fungal infections. This study is the first to sequence and explore CARD9 variation in multiple koalas using Sanger sequencing. Four CARD9 exons were successfully sequenced in 22 koalas from a New South Wales, Australia population. We found minimal variation between koalas across all four exons, an observation that was also made when CARD9 sequences were compared between koalas and six other species, including humans and mice. Ten single-nucleotide polymorphisms (SNP) were identified in this study and explored in the context of cryptococcal exposure outcomes. While we did not find any significant association with variation in cryptococcal outcomes, we found a high degree of conservation between species at several SNP loci that requires further investigation. The findings from this study lay the groundwork for further investigations of CARD9 and Cryptococcus both in koalas and other species, and highlight several considerations for future studies.

Immunophenotyping, Part I: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures.

Nanoparticles are increasingly used in biomedical applications to influence the way the immune system reacts to tumors and infectious disease-causing agents. Nanoparticles not-intended for immunomodulation can also influence immune responses by affecting immune cell subsets' viability and/or activity. While immunophenotyping is commonly used to assess the effects of drugs and nanoparticles on immune cell subsets, no standardized approach exists due to the breadth of available cell models and instrumentation. In this chapter, we describe a protocol for flow cytometer calibration and reagent qualification prior to its use in the immunophenotyping experiment. The strategies described herein can be adapted to other instruments. The subsequent chapter-immunophenotyping part II (Chap. 25 )-provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood.

Immunophenotyping, Part II: Analysis of Nanoparticle Effects on the Composition and Activation Status of Human Peripheral Blood Mononuclear Cells.

The use of nanoparticles as drug delivery carriers requires analysis of their safety, which among other tests, includes immunotoxicity. Nanoparticles are also increasingly used for applications intended to specifically activate, inhibit, or modify the immune system's responses to improve the treatment of inflammatory and autoimmune disorders, cancer immunotherapy, and vaccines targeting cancer cells and viral and bacterial pathogens. In addition to the safety, the analysis of nanoparticles intended for immune system targeting includes mechanistic immunology investigations. Immunophenotyping provides researchers with a tool to assess the immune cell viability and activation status. These results provide mechanistic insights into nanoparticle efficacy and toxicity and therefore are of interest to the biomedical nanotechnology field. However, no standardized approaches exist due to the breadth of methods and instruments available for this analysis. This chapter provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood. While this experimental strategy is specific to the NovoCyte 3005 flow cytometer, it can be adapted to other instruments. Instructions for instrument setup, calibration, and antibody qualification are described in this book's Chapter 24 , Immunophenotyping, part I.

Multicolor flow cytometry-based immunophenotyping for preclinical characterization of nanotechnology-based formulations: an insight into structure activity relationship and nanoparticle biocompatibility profiles.

Introduction: Immunophenotyping, which is the identification of immune cell subsets based on antigen expression, is an integral technique used to determine changes of cell composition and activation in various disease states or as a response to different stimuli. As nanoparticles are increasingly utilized for diagnostic and therapeutic applications, it is important to develop methodology that allows for the evaluation of their immunological impact. Therefore, the development of techniques such as immunophenotyping are desirable. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry. Methods: We developed two distinct multicolor flow cytometry immunophenotyping panels which allow for the evaluation of the effects of nanoparticles on the composition and activation status of treated human peripheral blood mononuclear cells. These two panels assess the presence of various lymphoid and myeloid-derived cell populations as well as aspects of their activation statuses-including proliferation, adhesion, co-stimulation/presentation, and early activation-after treatment with controls or nanoparticles. To conduct assay performance qualification and determine the applicability of this method to preclinical characterization of nanoparticles, we used clinical-grade nanoformulations (AmBisome, Doxil and Feraheme) and research-grade PAMAM dendrimers of different sizes (G3, G4 and G5) and surface functionalities (amine-, carboxy- and hydroxy-). Results and Discussion: We found that formulations possessing intrinsic fluorescent properties (e.g., Doxil and AmBisome) interfere with accurate immunophenotyping; such interference may be partially overcome by dilution. In the absence of interference (e.g., in the case of dendrimers), nanoparticle size and surface functionalities determine their effects on the cells with large amine-terminated dendrimers being the most reactive.

Change in Lipofectamine Carrier as a Tool to Fine-Tune Immunostimulation of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs' immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics.

How the Potassium Channel Response of T Lymphocytes to the Tumor Microenvironment Shapes Antitumor Immunity.

Competent antitumor immune cells are fundamental for tumor surveillance and combating active cancers. Once established, tumors generate a tumor microenvironment (TME) consisting of complex cellular and metabolic elements that serve to suppress the function of antitumor immune cells. T lymphocytes are key cellular elements of the TME. In this review, we explore the role of ion channels, particularly K+ channels, in mediating the suppressive effects of the TME on T cells. First, we will review the complex network of ion channels that mediate Ca2+ influx and control effector functions in T cells. Then, we will discuss how multiple features of the TME influence the antitumor capabilities of T cells via ion channels. We will focus on hypoxia, adenosine, and ionic imbalances in the TME, as well as overexpression of programmed cell death ligand 1 by cancer cells that either suppress K+ channels in T cells and/or benefit from regulating these channels' activity, ultimately shaping the immune response. Finally, we will review some of the cancer treatment implications related to ion channels. A better understanding of the effects of the TME on ion channels in T lymphocytes could promote the development of more effective immunotherapies, especially for resistant solid malignancies.

Immunophenotyping: Analytical approaches and role in preclinical development of nanomedicines.

Pharmaceutical products can activate immune cells, suppress their function, or change the immune responses to traditional immunologically active agonists such as those present in microbes. Therefore, the assessment of immunostimulation, immunosuppression, and immunomodulation comprises the backbone of immunotoxicity studies of new drug entities. Depending on physicochemical properties (e.g., size, charge, surface functionalities, hydrophobicity), nanoparticles can be immunostimulatory, immunosuppressive, and immunomodulatory. Various methods and experimental frameworks have been established to support preclinical translational studies of nanotechnology-based drug products. Immunophenotyping after the exposure of cells or preclinical animal models to nanoparticles can provide critical information about the changes in both the numbers of immune cells and their activation status. However, this methodology is underutilized in preclinical studies of engineered nanomaterials. Herein, we review current literature about varieties of instrumentation and methods utilized for immunophenotyping, discuss their advantages and limitations, and propose a roadmap for applying immunophenotyping to support preclinical immunological characterization of nanotechnology-based formulations.

Inconsistencies in fertility preservation for young people with cancer in the UK.

OBJECTIVE: To assess the utilisation of and funding structure for fertility preservation for children diagnosed with cancer in the UK. DESIGN: Survey of paediatric oncologists/haematologists. Questionnaires were sent electronically with reminder notifications to non-responders. SETTING: UK Paediatric Oncology Principal Treatment Centres (PTCs). PARTICIPANTS: Paediatric oncologists/haematologists with an interest in the effects of treatment on fertility representing the 20 PTCs across the UK. MAIN OUTCOME MEASURES: Referral practices, sources and length of funding for storage of gametes or gonadal tissue for children diagnosed with cancer in the preceding 12 months. RESULTS: Responses were received from 18 PTCs (90%) with responses to 98.3% of questions. All centres had referred patients for fertility preservation: ovarian tissue collection/storage 100% (n=18 centres), sperm banking 100% (n=17; one centre was excluded due to the age range of their patients), testicular tissue storage 83% (n=15), mature oocyte collection 35% (n=6; one centre was excluded due to the age range of their patients). All centres with knowledge of their funding source reported sperm cryopreservation was NHS funded. Only 60% (n=9) centres reported the same for mature oocyte storage. Of the centres aware of their funding source, half reported that ovarian and testicular tissue storage was funded by charitable sources; this increased in England compared with the rest of the UK. CONCLUSIONS: Inequality exists in provision of fertility preservation for children with cancer across the UK. There is lack of formalised government funding to support international guidelines, with resultant geographical variation in care. Centralised funding of fertility preservation for children and young adults is needed alongside establishment of a national advisory panel to support all PTCs.

Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients.

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca2+ fluxes, and thereby T cell effector functions. In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca2+ signaling and mobility in CD8+ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca2+ signaling in CD8+ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8+ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca2+ fluxes in a subset of HSNCC patients. In CD8+ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca2+ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8+ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca2+ release activated Ca2+ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.

Targeted knockdown of the adenosine A2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells.

In solid malignancies, including head and neck squamous cell carcinoma (HNSCC), the immunosuppressive molecule adenosine, which accumulates in the tumor, suppresses cytotoxic CD8+ T cell functions including chemotaxis and tumor infiltration. Adenosine functions through binding to the adenosine A2A receptor (A2AR) present on T cells. In order to increase T cell migration into the tumor, the negative effect of adenosine must be abrogated. Systemic drug treatments targeting A2AR are available; however, they could lead to negative toxicities due to the broad expression of this receptor. Herein, we developed a lipid nanoparticle (NP)-based targeted delivery approach to knock down A2AR in T cells in order to increase their chemotaxis in the presence of adenosine. By using flow cytometry, immunofluorescence, qRT-PCR, and 3D-chemotaxis, we demonstrated that CD45RO-labeled nanoparticles delivering ADORA2A gene-silencing-RNAs decreased ADORA2A mRNA expression and rescued the chemotaxis of HNSCC CD8+ memory T cells. Overall, the data indicate that targeting the adenosine signaling pathway with lipid NPs is successful at suppressing the inhibitory effect of adenosine on the chemotaxis of HNSCC memory T cells, which could ultimately help increase T cell infiltration into the tumor.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier.

Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.

PD1 blockade enhances K+ channel activity, Ca2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer.

BACKGROUND: Immunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response. METHODS: Functional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor). RESULTS: Pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients' PBTs. Unlike non-responders' PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection. CONCLUSIONS: Pembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

A Compartmentalized Reduction in Membrane-Proximal Calmodulin Reduces the Immune Surveillance Capabilities of CD8+ T Cells in Head and Neck Cancer.

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.

Survival from cancer in young people: An overview of late effects focusing on reproductive health.

This paper provides a summary of the areas of survival from childhood, teenage and young adult cancers and the significant late effects that can arise from treatment; with particular focus on the area of reproductive health and the impact on both fertility and pregnancy. To complete this review, Web of Science and MEDLINE were used. Search terms included: ""survival AND childhood OR teenage OR young adult cancer", "late effects", "childhood cancer", "teenage AND/OR young adult cancer", AND "fertility after cancer" OR "pregnancy AND cancer" OR "fertility preservation". Additionally, the clinical expertise of the authors was drawn upon. Childhood cancer is a thankfully rare occurrence; however, the incidence is increasing. Survival rates remain high and this means that a growing population of childhood and young adult cancer survivors are reaching adulthood. For some of these adults, although cured of their cancer, they are now facing a future with lasting effects on their health from their treatments. These effects, commonly referred to as late effects, are defined as health problems related either directly to the underlying cancer or to its treatment and which occur months or years after treatment has finished. Reproductive health is an important consideration for these patients, and although many will be able to conceive naturally, some will exhibit impaired fertility after their treatments. This can include difficulties at all points along the path from conception to delivery of a live, healthy offspring. High-quality, large-population evidence is sparse in many areas relating to fertility risk from treatment and the maternal and fetal health of childhood cancer survivors. Yet given the potential for complications, the authors advocate consideration of fertility at the time of diagnosis and before potentially gonadotoxic treatment.

A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patients to infiltrate an adenosine-rich microenvironment.

The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of immunotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibits T cell motility through the A2A receptor (A2AR) and suppression of KCa3.1 channels. We conducted three-dimensional chemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8+ T cells from head and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8+ T cells was reduced in the presence of adenosine, and the effect was greater on HNSCC CD8+ T cells than on healthy donor (HD) CD8+ T cells. This response correlated with the inability of CD8+ T cells to infiltrate tumors. The effect of adenosine was mimicked by an A2AR agonist and prevented by an A2AR antagonist. We found no differences in A2AR expression, 3',5'-cyclic adenosine monophosphate abundance, or protein kinase A type 1 activity between HNSCC and HD CD8+ T cells. We instead detected a decrease in KCa3.1 channel activity, but not expression, in HNSCC CD8+ T cells. Activation of KCa3.1 channels by 1-EBIO restored the ability of HNSCC CD8+ T cells to chemotax in the presence of adenosine. Our data highlight the mechanism underlying the increased sensitivity of HNSCC CD8+ T cells to adenosine and the potential therapeutic benefit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors.

Fertility preservation options in prepubertal females: feasibility, safety and outcomes.

Fertility preservation is a developing area of reproductive biology and successes in adult populations has led to an emerging interest in prepubertal populations. Advances in treatment strategies for many disease processes including childhood cancer means that many patients now survive their initial diagnosis. Ovarian insufficiency is a recognized side-effect of chemotherapy and as such advances in reproductive technologies are now possible to attempt to overcome the so called late effects. Questions regarding the outcomes and safety of these treatments still exist. A review of the available literature with regard to fertility preservation options in prepubertal females was undertaken with consideration given to the need for options, availability of options, safety of available techniques and current outcomes. The new discipline of onco-fertility is rapidly advancing. Work to facilitate accurate and robust identification of those in need of fertility preservation and the optimization of strategies for fertility restoration are ongoing. With time it is hoped that the techniques used to freeze-store ovarian tissue and to conduct auto-transplantation will undergo refinement and so advance from the research arena into mainstream clinical practice. Further research is likely to be fruitful and will offer significant hope to those at risk of premature ovarian insufficiency whether from underlying genetic causes or as a result of ablative therapies. It is important that research advancements are disseminated widely as this will help to inform clinical practitioners, nurses and counsellors of strategies and to ensure that appropriate treatments are offered to patients who are at risk of fertility loss.