Utilizing murine dendritic cell line DC2.4 to evaluate the immunogenicity of subunit vaccines in vitro.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.

Predicting Tacrolimus Concentrations in the Skin of Adult Kidney Transplant Recipients: A Feasibility Study.

Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0-21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin.

Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin.

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG.

Peritumoral administration of immunomodulatory antibodies as a triple combination suppresses skin tumor growth without systemic toxicity.

BACKGROUND: Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity. METHODS: The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections. RESULTS: The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4+FoxP3+ cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor. CONCLUSIONS: Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies.

Checkpoint kinase 1 inhibitor + low-dose hydroxyurea efficiently kills BRAF inhibitor- and immune checkpoint inhibitor-resistant melanomas.

Treatment of melanomas with targeted and immunotherapies has proven effective, but resistance to both treatments is a common outcome leaving a high proportion of patients without effective alternative treatment options. Replication stress is a common feature of melanomas, and this is effectively targeted using a combination of checkpoint kinase 1 (CHK1) inhibitor and low-dose hydroxyurea (LDHU). This combination also promotes inflammatory and anti-tumour immune responses in vivo. Melanoma cell lines resistant to BRAF inhibitor (BRAFi) or immune checkpoint inhibitors (ICI) retain their sensitivity to CHK1i + LDHU, with sensitivity similar to that of parental tumours. In vivo, BRAFi-resistant and BRAFi-sensitive parental tumours produce an identical immune response with treatment.

Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

BACKGROUND: Immunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus also suppresses T-cell function in the skin, contributing to a high incidence of skin cancer and associated mortality and morbidity in solid organ transplant recipients. Here, we aimed to identify a compound capable of re-establishing antitumor T-cell control in the skin despite the presence of tacrolimus. METHODS: In this study, we performed time-resolved fluorescence resonance energy transfer to identify molecules capable of antagonizing the interaction between tacrolimus and FKBP12. The capacity of these molecules to rescue mouse and human T-cell function in the presence of tacrolimus was determined in vitro, and the antitumor effect of the lead compound, Q-2361, was assessed in "regressor" models of skin cancer in immunosuppressed mice. Systemic CD8 T-cell depletion and analyses of intratumoral T-cell activation markers and effector molecule production were performed to determine the mechanism of tumor rejection. Pharmacokinetic studies of topically applied Q-2361 were performed to assess skin and systemic drug exposure. RESULTS: Q-2361 potently blocked the interaction between tacrolimus and FKBP12 and reversed the inhibition of the nuclear factor of activated T cells activation by tacrolimus following T-cell receptor engagement in human Jurkat cells. Q-2361 rescued T-cell function in the presence of tacrolimus, rapamycin, and everolimus. Intratumoral injection of Q-2361-induced tumor regression in mice systemically immune suppressed with tacrolimus. Mechanistically, Q-2361 treatment permitted T-cell activation, proliferation, and effector function within tumors. When CD8 T cells were depleted, Q-2361 could not induce tumor regression. A simple solution-based Q-2361 topical formulation achieved high and sustained residence in the skin with negligible drug in the blood. CONCLUSIONS: Our findings demonstrate that the local application of Q-2361 permits T-cells to become activated driving tumor rejection in the presence of tacrolimus. The data presented here suggests that topically applied Q-2361 has great potential for the reactivation of T-cells in the skin but not systemically, and therefore represents a promising strategy to prevent or treat skin malignancies in immunosuppressed organ transplant recipients.

Mono-phosphorylation at Ser4 of barrier-to-autointegration factor (Banf1) significantly reduces its DNA binding capability by inducing critical changes in its local conformation and DNA binding surface.

Barrier-to-autointegration factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1's DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1's N-terminal domain.

Liposomal Formulations of a Polyleucine-Antigen Conjugate as Therapeutic Vaccines against Cervical Cancer.

Human papilloma virus (HPV) is responsible for all cases of cervical cancer. While prophylactic vaccines are available, the development of peptide-based vaccines as a therapeutic strategy is still under investigation. In comparison with the traditional and currently used treatment strategies of chemotherapy and surgery, vaccination against HPV is a promising therapeutic option with fewer side effects. A peptide derived from the HPV-16 E7 protein, called 8Qm, in combination with adjuvants showed promise as a therapeutic vaccine. Here, the ability of polymerized natural amino acids to act as a self-adjuvating delivery system as a therapeutic vaccine was investigated for the first time. Thus, 8Qm was conjugated to polyleucine by standard solid-phase peptide synthesis and self-assembled into nanoparticles or incorporated in liposomes. The liposome bearing the 8Qm conjugate significantly increased mice survival and decreased tumor growth after a single immunization. Further, these liposomes eradicated seven-day-old well-established tumors in mice. Dendritic cell (DC)-targeting moieties were introduced to further enhance vaccine efficacy, and the newly designed liposomal vaccine was tested in mice bearing 11-day-old tumors. Interestingly, these DCs-targeting moieties did not significantly improve vaccine efficacy, whereas the simple liposomal formulation of 8Qm-polyleucine conjugate was still effective in tumor eradication. In summary, a peptide-based anticancer vaccine was developed that stimulated strong cellular immune responses without the help of a classical adjuvant.

Prevalence of AAV2.5 neutralizing antibodies in synovial fluid and serum of patients with osteoarthritis.

Osteoarthritis (OA) is a leading cause of disability with no cure and only supportive therapy. Adeno-associated virus (AAV) serotype 2.5 is being used in a Phase I clinical trial to deliver the interleukin-1 receptor antagonist into knee joints with OA. Neutralizing antibodies (Nab) directed against AAV2.5, if present, could inhibit gene transfer. Here, we report the prevalence of AAV2.5 Nab in the sera and synovial fluids of patients with OA. Nab titers were measured by their ability to inhibit in vitro transduction by AAV2.5 encoding GFP. Of 44 synovial fluids from patients with mid-stage and advanced OA, 43% had undetectable Nab; 25% had low titers (<1:100), 16% had medium titers (1:100-1:1000) and 16% had high titers (>1:1000) of Nab. Titers of AAV2.5 Nabs correlated with those of AAV2, but not with those of AAV5. Serum titers of AAV2.5 Nab correlated positively with titers in synovial fluid, and were never less than the matched synovial fluid titers. These findings suggest that high titers of Nab against AAV2.5 are uncommon in the synovial fluids of patients with OA, and individuals with high synovial fluid Nab titers can be identified by measuring titers in the serum.

Assessment of patient-to-patient and intra-individual human abdominal skin immune cell variability.

Introduction: The objective of the study was to characterize the baseline intra-individual and inter-individual variability of immune cell subsets within abdominoplasty skin specimens. Methods: Abdominoplasty biopsies were taken from 5 patients and analysed using the Vectra 3 automated quantitative pathology imaging system with inForm software. Results: Adjacent skin regions demonstrated intra-patient variability in immune subset counts ranging from 1- to 5-fold. Inter-variability between patients was approximately 2- to 7-fold for most subsets, except for HLA-DR+ antigen presenting cells, which varied 19-fold. Conclusions: Our data highlight the importance of including multiple patients and multiple patient samples when designing dermatological studies that utilise abdominoplasty skin.

Cytokine/Chemokine assessment as a complementary diagnostic tool for inflammatory skin diseases.

Inflammatory skin conditions are the 4th leading cause of non-fatal health burden in the general population worldwide. The diagnosis of skin lesions due to systemic drug reactions, viral or bacterial exanthems, or in patients with psoriasis, atopic dermatitis or contact dermatitis is often difficult and relies heavily upon conventional histopathologic examination. Conversely, it is widely accepted that the cutaneous profile of inflammatory markers, or 'inflammatory signature', is differentially expressed in various skin conditions. In this pilot study, we investigated the possibility of inflammatory skin disease diagnosis from an immunological perspective in small punch biopsies. We collected lesional and perilesional punch biopsies from 139 patients suffering from a variety of inflammatory skin conditions and attending the Dermatology Department at the Princess Alexandra Hospital in Brisbane, Australia. Using bead-based immunoassays we were able to measure 13 out of 17 inflammatory markers from a pre-selected multi-analyte panel and to detect significant differences between lesional and perilesional biopsies from each individual patient. Hierarchical and unbiased clustering methods based on inflammatory signatures grouped psoriasis and atopic dermatitis lesions into individual clusters in contrast to other skin conditions, highlighting the potential of inflammatory signatures to be used as diagnostic differentiators and to inform alternative targets in anti-inflammatory treatment strategies.

Distribution of Conformational States Adopted by DNA from the Promoter Regions of the VEGF and Bcl-2 Oncogenes.

We employed a previously described procedure, based on circular dichroism (CD) spectroscopy, to quantify the distribution of conformational states adopted by equimolar mixtures of complementary G-rich and C-rich DNA strands from the promoter regions of the VEGF and Bcl-2 oncogenes. Spectra were recorded at different pHs, concentrations of KCl, and temperatures. The temperature dependences of the fractional populations of the duplex, G-quadruplex, i-motif, and coiled conformations of each promoter were then analyzed within the framework of a thermodynamic model to obtain the enthalpy and melting temperature of each folded-to-unfolded transition involved in the equilibrium. A comparison of the conformational data on the VEGF and Bcl-2 DNA with similar results on the c-MYC DNA, which we reported previously, reveals that the distribution of conformational states depends on the specific DNA sequence and is modulated by environmental factors. Under the physiological conditions of room temperature, neutral pH, and elevated concentrations of potassium ions, the duplex conformation coexists with the G-quadruplex conformation in proportions that depend on the sequence. This observed conformational diversity has biological implications, and it further supports our previously proposed thermodynamic hypothesis of gene regulation. In that hypothesis, a specific distribution of duplex and tetraplex conformations in a promoter region is fine-tuned to maintain the healthy level of gene expression. Any deviation from a healthy distribution of conformational states may result in pathology stemming from up- or downregulation of the gene.

Targeting Replication Stress Using CHK1 Inhibitor Promotes Innate and NKT Cell Immune Responses and Tumour Regression.

Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response.

PD-1 and beyond to Activate T Cells in Cutaneous Squamous Cell Cancers: The Case for 4-1BB and VISTA Antibodies in Combination Therapy.

Non-melanoma skin cancers (NMSC) have a higher incidence than all other cancers combined with cutaneous squamous cell carcinoma (cSCC), capable of metastasis, representing approximately 20% of NMSCs. Given the accessibility of the skin, surgery is frequently employed to treat localized disease, although certain localities, the delineation of clear margins, frequency and recurrence of tumors can make these cancers inoperable in a subset of patients. Other treatment modalities, including cryotherapy, are commonly used for individual lesions, with varying success. Immunotherapy, particularly with checkpoint antibodies, is increasingly a promising therapeutic approach in many cancers, offering the potential advantage of immune memory for protection against lesion recurrence. This review addresses a role for PD-1, 4-1BB and VISTA checkpoint antibodies as monotherapies, or in combination as a therapeutic treatment for both early and late-stage cSCC.

Cellular responses at the application site of a high-density microarray patch delivering an influenza vaccine in a randomized, controlled phase I clinical trial.

Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier to use and more cost-effective method for administration of vaccines when compared to the needle and syringe. Since MAPs deliver vaccine to the dermis and epidermis, a degree of local immune response at the site of application is expected. In a phase 1 clinical trial (ACTRN 12618000112268), the Vaxxas high-density MAP (HD-MAP) was used to deliver a monovalent, split inactivated influenza virus vaccine into the skin. HD-MAP immunisation led to significantly enhanced humoral responses on day 8, 22 and 61 compared with IM injection of a quadrivalent commercial seasonal influenza vaccine (Afluria Quadrivalent®). Here, the aim was to analyse cellular responses to HD-MAPs in the skin of trial subjects, using flow cytometry and immunohistochemistry. HD-MAPs were coated with a split inactivated influenza virus vaccine (A/Singapore/GP1908/2015 [H1N1]), to deliver 5 µg haemagglutinin (HA) per HD-MAP. Three HD-MAPs were applied to the volar forearm (FA) of five healthy volunteers (to achieve the required 15 µg HA dose), whilst five control subjects received three uncoated HD-MAPs (placebo). Local skin response was recorded for over 61 days and haemagglutination inhibition antibody titres (HAI) were assessed on days 1, 4, 8, 22, and 61. Skin biopsies were taken before (day 1), and three days after HD-MAP application (day 4) and analysed by flow-cytometry and immunohistochemistry to compare local immune subset infiltration. HD-MAP vaccination with 15 µg HA resulted in significant HAI antibody titres compared to the placebo group. Application of uncoated placebo HD-MAPs resulted in mild erythema and oedema in most subjects, that resolved by day 4 in 80% of subjects. Active, HA-coated HD-MAP application resulted in stronger erythema responses on day 4, which resolved between days 22-61. Overall, these erythema responses were accompanied by an influx of immune cells in all subjects. Increased cell infiltration of CD3+, CD4+, CD8+ T cells as well as myeloid CD11b+ CD11c+ and non-myeloid CD11b- dendritic cells were observed in all subjects, but more pronounced in active HD-MAP groups. In contrast, CD19+/CD20+ B cell counts remained unchanged. Key limitations include the use of an influenza vaccine, to which the subjects may have had previous exposure. Different results might have been obtained with HD-MAPs inducing a primary immune response. In conclusion, influenza vaccine administered to the forearm (FA) using the HD-MAP was well-tolerated and induced a mild to moderate skin response with lymphocytic infiltrate at the site of application.

Evolution of Cancer Vaccines-Challenges, Achievements, and Future Directions.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient's autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.

Investigating T Cell Immunity in Cancer: Achievements and Prospects.

T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8+ Cytotoxic T lymphocytes (CTL), as well as therapeutic strategies trialled to overcome these tumour-evasion mechanisms. Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models.

Allostery of atypical modulators at oligomeric G protein-coupled receptors.

Many G protein-coupled receptors (GPCRs) are therapeutic targets, with most drugs acting at the orthosteric site. Some GPCRs also possess allosteric sites, which have become a focus of drug discovery. In the M2 muscarinic receptor, allosteric modulators regulate the binding and functional effects of orthosteric ligands through a mix of conformational changes, steric hindrance and electrostatic repulsion transmitted within and between the constituent protomers of an oligomer. Tacrine has been called an atypical modulator because it exhibits positive cooperativity, as revealed by Hill coefficients greater than 1 in its negative allosteric effect on binding and response. Radioligand binding and molecular dynamics simulations were used to probe the mechanism of that modulation in monomers and oligomers of wild-type and mutant M2 receptors. Tacrine is not atypical at monomers, which indicates that its atypical effects are a property of the receptor in its oligomeric state. These results illustrate that oligomerization of the M2 receptor has functional consequences.

IFN-γ Critically Enables the Intratumoural Infiltration of CXCR3+ CD8+ T Cells to Drive Squamous Cell Carcinoma Regression.

Ultraviolet (UV) radiation-induced tumours carry a high mutational load, are highly immunogenic, and often fail to grow when transplanted into normal, syngeneic mice. The aim of this study was to investigate factors critical for the immune-mediated rejection of cutaneous squamous cell carcinoma (SCC). In our rejection model, transplanted SCC establish and grow in mice immunosuppressed with tacrolimus. When tacrolimus is withdrawn, established SCC tumours subsequently undergo immune-mediated tumour rejection. Through the depletion of individual immune subsets at the time of tacrolimus withdrawal, we established a critical role for CD8+ T cells, but not CD4+ T cells, γδ T cells, or NK cells, in driving the regression of SCC. Regression was critically dependent on IFN-γ, although IFN-γ was not directly cytotoxic to SCC cells. IFN-γ-neutralisation abrogated SCC regression, significantly reduced CD8+ T cell-infiltration into SCC, and significantly impaired the secretion of CXCL9, CXCL10 and CCL5 within the tumour microenvironment. A strong positive correlation was revealed between CXCL10 expression and CD8+ T cell abundance in tumours. Indeed, blockade of the CXCL10 receptor CXCR3 at the time of tacrolimus withdrawal prevented CD8+ T cell infiltration and the regression of SCC. Chimeric models revealed an important role for immune cells as producers of IFN-γ, but not as recipients of IFN-γ signals via the IFN-γ receptor. Together, these findings suggest a key role for IFN-γ in driving the expression of chemokines within the tumour environment essential for the destruction of established SCC by CD8+ T cells.

Manganese-Doped Silica-Based Nanoparticles Promote the Efficacy of Antigen-Specific Immunotherapy.

Prophylactic human papillomavirus (HPV) vaccines are commercially available for prevention of infection with cancerogenic HPV genotypes but are not able to combat pre-existing HPV-associated disease. In this study, we designed a nanomaterial-based therapeutic HPV vaccine, comprising manganese (Mn4+)-doped silica nanoparticles (Mn4+-SNPs) and the viral neoantigen peptide GF001 derived from the HPV16 E7 oncoprotein. We show in mice that Mn4+-SNPs act as self-adjuvants by activating the inflammatory signaling pathway via generation of reactive oxygen species, resulting in immune cell recruitment to the immunization site and dendritic cell maturation. Mn4+-SNPs further serve as Ag carriers by facilitating endo/lysosomal escape via depletion of protons in acidic endocytic compartments and subsequent Ag delivery to the cytosol for cross-presentation. The Mn4+-SNPs+GF001 nanovaccine induced strong E7-specific CD8+ T cell responses, leading to remission of established murine HPV16 E7-expressing solid TC-1 tumors and E7-expressing transgenic skin grafts. This vaccine construct offers a simple and general strategy for therapeutic HPV and potentially other cancer vaccines.