Pouring petrol on the flames: Using oncolytic virotherapies to enhance tumour immunogenicity.

Oncolytic viruses possess the ability to infect, replicate and lyse malignantly transformed tumour cells. This oncolytic activity amplifies the therapeutic advantage and induces a form of immunogenic cell death, characterized by increased CD8 + T-cell infiltration into the tumour microenvironment. This important feature of oncolytic viruses can result in the warming up of immunologically 'cold' tumour types, presenting the enticing possibility that oncolytic virus treatment combined with immunotherapies may enhance efficacy. In this review, we assess some of the most promising candidates that might be used for oncolytic virotherapy: immunotherapy combinations. We assess their potential as separate agents or as agents combined into a single therapy, where the immunotherapy is encoded within the genome of the oncolytic virus. The development of such advanced agents will require increasingly sophisticated model systems for their preclinical assessment and evaluation. In vivo rodent model systems are fraught with limitations in this regard. Oncolytic viruses replicate selectively within human cells and therefore require human xenografts in immune-deficient mice for their evaluation. However, the use of immune-deficient rodent models hinders the ability to study immune responses against any immunomodulatory transgenes engineered within the viral genome and expressed within the tumour microenvironment. There has therefore been a shift towards the use of more sophisticated ex vivo patient-derived model systems based on organoids and explant co-cultures with immune cells, which may be more predictive of efficacy than contrived and artificial animal models. We review the best of those model systems here.

The Ussing chamber system for measuring intestinal permeability in health and disease.

BACKGROUND: The relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn's disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues. METHODS: An Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3DTR mice) were used to validate the system along with human colonic biopsy samples. RESULTS: Distinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system. CONCLUSIONS: The Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn't available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.

Mbd2 enables tumourigenesis within the intestine while preventing tumour-promoting inflammation.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2-/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2-/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells.

The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.

CD200 receptor restriction of myeloid cell responses antagonizes antiviral immunity and facilitates cytomegalovirus persistence within mucosal tissue.

CD200 receptor (CD200R) negatively regulates peripheral and mucosal innate immune responses. Viruses, including herpesviruses, have acquired functional CD200 orthologs, implying that viral exploitation of this pathway is evolutionary advantageous. However, the role that CD200R signaling plays during herpesvirus infection in vivo requires clarification. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that CD200R facilitates virus persistence within mucosal tissue. Specifically, MCMV infection of CD200R-deficient mice (CD200R(-/-)) elicited heightened mucosal virus-specific CD4 T cell responses that restricted virus persistence in the salivary glands. CD200R did not directly inhibit lymphocyte effector function. Instead, CD200R(-/-) mice exhibited enhanced APC accumulation that in the mucosa was a consequence of elevated cellular proliferation. Although MCMV does not encode an obvious CD200 homolog, productive replication in macrophages induced expression of cellular CD200. CD200 from hematopoietic and non-hematopoietic cells contributed independently to suppression of antiviral control in vivo. These results highlight the CD200-CD200R pathway as an important regulator of antiviral immunity during cytomegalovirus infection that is exploited by MCMV to establish chronicity within mucosal tissue.

The paradox of NKp46+ natural killer cells: drivers of severe hepatitis C virus-induced pathology but in-vivo resistance to interferon α treatment.

OBJECTIVE: There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated. DESIGN: We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα. RESULTS: Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure. CONCLUSIONS: NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance.

Interleukin-6 limits influenza-induced inflammation and protects against fatal lung pathology.

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.

Rapid innate control of antigen abrogates adaptive immunity.

Natural killer (NK) cells provide an immediate first line of defence against viral infections. Memory responses, maintained by CD4(+) T cells, require exposure to viral antigen and provide long-term protection against future infections. It is known that NK cells can promote the development of the adaptive response through cytokine production and cross-talk with antigen-presenting cells. In this paper however, we summarize a series of recent publications, in mouse models and for the first time in man, with the unifying message that rapid viral antigen control by the innate immune system limits antigen exposure to CD4(+) cells thereby abrogating the development of a memory response. We discuss the significant implication of these studies on viral treatment strategies and immunization models.

Avidity of influenza-specific memory CD8+ T-cell populations decays over time compromising antiviral immunity.

Decline of cell-mediated immunity is often attributed to decaying T-cell numbers and their distribution in peripheral organs. This study examined the hypothesis that qualitative as well as quantitative changes contribute to the declining efficacy of CD8(+) T-cell memory. Using a model of influenza virus infection, where loss of protective CD8(+) T-cell immunity was observed 6 months postinfection, we found no decline in antigen-specific T-cell numbers or migration to the site of secondary infection. There was, however, a large reduction in antigen-specific CD8(+) T-cell degranulation, cytokine secretion, and polyfunctionality. A profound loss of high-avidity T cells over time indicated that failure to confer protective immunity resulted from the inferior functional capacity of remaining low avidity cells. These data imply that high-avidity central memory T cells wane with declining antigen levels, leaving lower avidity T cells with reduced functional capabilities.

Rapid early innate control of hepatitis C virus during IFN-α treatment compromises adaptive CD4+ T-cell immunity.

The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4(+) T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4(+) T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection.

The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4⁺ and CD8⁺ T-cell immunity.

Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4(+) T-cell-driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3-deficient (DR3(KO)) mice and their DR3(WT) littermates with the ß-herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T-cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8(+) T cells, with TCR activation increasing its levels 4-fold and altering the ratio of DR3 splice variants. T-cell responses were reduced up to 90% in DR3(KO) mice during acute infection. Adoptive transfer experiments indicated this was dependent on T-cell-restricted expression of DR3. DR3-dependent CD8(+) T-cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3(KO) hosts on a C57BL/6 background was associated with 4- to 7-fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus-specific T-cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.

Anti-CD8 antibodies can trigger CD8+ T cell effector function in the absence of TCR engagement and improve peptide-MHCI tetramer staining.

CD8(+) T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8(+) T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8(+) T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8(+) T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8(+) T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8(+) T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8(+) T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8(+) T cell signaling.

Structural definition of HLA class II-presented SARS-CoV-2 epitopes reveals a mechanism to escape pre-existing CD4+ T cell immunity.

CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.

Seven mysteries of LAG-3: a multi-faceted immune receptor of increasing complexity.

Despite three decades of research to its name and increasing interest in immunotherapies that target it, LAG-3 remains an elusive co-inhibitory receptor in comparison to the well-established PD-1 and CTLA-4. As such, LAG-3 targeting therapies have yet to achieve the clinical success of therapies targeting other checkpoints. This could, in part, be attributed to the many unanswered questions that remain regarding LAG-3 biology. Of these, we address: (i) the function of the many LAG-3-ligand interactions, (ii) the hurdles that remain to acquire a high-resolution structure of LAG-3, (iii) the under-studied LAG-3 signal transduction mechanism, (iv) the elusive soluble form of LAG-3, (v) the implications of the lack of (significant) phenotype of LAG-3 knockout mice, (vi) the reports of LAG-3 expression on the epithelium, and (vii) the conflicting reports of LAG-3 expression (and potential contributions to pathology) in the brain. These mysteries which surround LAG-3 highlight how the ever-evolving study of its biology continues to reveal ever-increasing complexity in its role as an immune receptor. Importantly, answering the questions which shroud LAG-3 in mystery will allow the maximum therapeutic benefit of LAG-3 targeting immunotherapies in cancer, autoimmunity and beyond.

Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity.

BACKGROUND: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. METHODS: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. RESULTS: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. CONCLUSIONS: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection.

Type I interferon (IFN alpha) acts directly on human memory CD4+ T cells altering their response to antigen.

Despite its use widely as a therapeutic agent, and proposed use as vaccine adjuvant, the effect of IFNalpha on T cell function is poorly understood. As a pleiotropic innate cytokine produced rapidly in response to pathogens, it is well placed to impinge on specific immune responses. The aim of this study was to examine the impact of IFNalpha on the function of human memory CD4(+) T cells using the recall Ags purified protein derivative, tetanus toxoid, and hemagglutinin. IFNalpha administered either in vivo or added exogenously in vitro tended to enhance proliferative responses of purified protein derivative-specific T cells in marked contrast to the other cognate populations whose responses were often diminished. Purifying the memory CD4(+)CD45RO(+) T cells confirmed IFNalpha acted directly on these cells and not via an intermediate. The T cells could be divided into two broad categories depending on how IFNalpha effected their responses to cognate Ag: 1) enhanced proliferation and a striking increase in IFNgamma-production compared with smaller increases in IL-10 (increased ratio of IFNgamma:IL-10), and 2) neutral or diminished proliferation coupled with a smaller increase in IFNgamma relative to the increase in IL-10 (reduced IFNgamma:IL-10 ratio). IFNalpha has a role in modifying memory T cell responses when they are exposed to cognate Ag and may be important in vaccination strategies designed to augment particular Th memory responses.

CD59 blockade enhances antigen-specific CD4+ T cell responses in humans: a new target for cancer immunotherapy?

CD59, a broadly expressed GPI-anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4(+) T cell activity in vivo. In this study, we explored the role of CD59 on human CD4(+) T cells. Our data demonstrate that CD59 is up-regulated on activated CD4(+) T cells and serves to down-modulate their activity in response to polyclonal and Ag-specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4(+) T cell response to two different tumor Ags. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.

Neutrophilia, lymphopenia and myeloid dysfunction: a living review of the quantitative changes to innate and adaptive immune cells which define COVID-19 pathology.

Destabilization of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils and eosinophils represents an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response that bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of intensive care unit admission and death; therefore, this parameter is particularly important for clinical decision-making. Mild and severe diseases differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decisions or predict disease outcomes.

T cell phenotypes in COVID-19 - a living review.

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients' long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.