Traumatic brain injury (TBI) is a major cause of death throughout the world and currently there are no approved drugs to treat this debilitating condition emphasising the clear unmet substantial clinical need. It is well recognised that microglial activation and the hostile neuroinflammatory response arising after the initial insult provides a therapeutic window for pharmacological intervention. The purinergic P2X7 receptor (P2X7R) is a key driver of neuroinflammation in a range of animal models of TBI. To generate translational evidence for the role of the P2X7R, we optimised two human inflammatory models; human cells (monocyte-derived microglia (hMDM)) in vitro and human brain tissue ex vivo, to test the impact of clinical-stage brain-penetrant P2X7R antagonists. Using lipopolysaccharide (LPS)-primed hMDM, the P2X7R agonist, BzATP, evoked a concentration-dependent increase in pro-inflammatory IL-1β and IL-18 release, which was antagonised in a concentration-dependent manner by selective P2X7R antagonists, and also inhibitors of either the NLRP3 complex or caspase-1, implicating a role for the inflammasome in P2X7R-mediated cytokine release. Using slices of human brain tissue, BzATP similarly evoked cytokine release in a concentration-dependent manner that was also antagonised by selective P2X7R antagonists at pharmacologically relevant concentrations. The present study demonstrates the ability of P2X7R antagonists to suppress the neuroinflammatory response from primed hMDM and human brain slices to offer direct translational data that central P2X7R antagonism may limit pathology-driven pro-inflammatory responses in the brain. This is predicted to improve the clinical outcomes for patients with TBI and other neuroinflammatory pathologies.

Psychedelic drugs that activate 5-HT2A receptors have been long used for cultural, medicinal and recreational purposes. Interest in psychedelics for treating psychiatric disorders has resurged recently and is well documented; less well recognised are their anti-inflammatory properties. Growing evidence now demonstrates that psychedelics modulate immune responses, including inhibiting pro-inflammatory cytokine release. Furthermore, in vivo studies demonstrate that psychedelics, like (R)-DOI, reduce inflammation in animal models of acute and chronic inflammatory disease such as asthma. Likewise, some clinical studies with psychedelic drugs (e.g. psilocybin) demonstrate an impact upon circulating cytokine levels, supporting a translation from the animal models to the clinical arena. Such data emphasise the promise of therapeutic approaches targeting inflammation. Interestingly, recent research has also uncovered compounds that maintain therapeutic potential without likely causing psychedelic effects. These discoveries suggest that drugs informed by psychedelic drugs, but which do not evoke psychedelic experiences, which we term PIPI drugs (Psychedelic drug Informed but Psychedelic experience Inactive), could offer effective treatments for mental health and inflammation, presenting new avenues for therapeutic development. LINKED ARTICLES: This article is part of a themed issue Emerging Therapeutic Opportunities for Psychedelic and Related Drugs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v183.14/issuetoc.

To understand the impact of purinergic signaling on the regulation of pro-inflammatory cytokine release in human microglia.

Introduction: In toxicology, steps are being taken towards more mechanism-focused and human relevant approaches to risk assessment, requiring new approaches and methods. Additionally, there is increasing emphasis by regulators on risk assessment of immunotoxicity.Methods: Here we present data from a peripheral blood mononuclear cell (PBMC) system whereby a varied set of stimuli, including those against the TCR and Toll-like receptors, enable readouts of cytokine and prostaglandin E2 (PGE2) production with monocyte, T cell and B cell viability, proliferation, and associated activation markers. In addition to results on the impact of the stimuli used, initial profiling data for a case study chemical, curcumin, is presented, illustrating how the system can be used to generate information on the impact of exogenous materials on three major constituent immune cell subsets for use in risk assessment and to direct follow-on studies.Results: The different stimuli drove distinct responses, not only in relation to the “quantity” of the response but also the “quality”. Curcumin had a limited impact on the B cell parameters measured, with the stimuli used, and it was noted that in contrast to T cells where there was either no impact or a reduction in viability and proliferation with increasing concentration, for B cells there was a small but significant increase in both measurements at curcumin concentrations below 20 µM. Similarly, whilst expression of activation markers by T cells was reduced by the highest concentration of curcumin, they were increased in B cells. Curcumin only impacted the viability of stimulated monocytes at the highest concentration and had differential impact on different activation markers. Levels of all cytokines and PGE2 were reduced at higher concentrations.Discussion: Although the platform has certain limitations, it nevertheless enables assessment of healthy baseline monocyte, T-, and B-cell responses, and scrutiny of the impact of different stimuli to detect potential immune suppression or enhancement from exogenous materials. In the case of curcumin, a pattern of responses indicative of immune suppressive / anti-inflammatory effects was detected. It is an accessible, highly modifiable system that can be used to screen materials and guide further studies, providing a holistic, integrated picture of effects.

Abstract Within the tumour microenvironment, regulatory T cells diminish anti-tumour responses by inhibiting effector T cell activation. Immunotherapies that enhance the effector T cell response may also enhance Treg activity, limiting the overall action of the therapy. It is therefore important to consider the ‘net-effect’ of immune activation in systems containing both cell types. To better understand responder T cell activation in a relevant cellular environment, we have investigated human effector T cell activation in the presence of regulatory T cells in vitro exploiting multiple readouts from multi-cell type suppression assays (MR-MCTM suppression assay). These assays have been performed in the absence or presence of IL-2 (hypothesised to boost both effector and regulatory T cell responses), and the absence or presence of IL-7 (hypothesised to skew towards effector T cell responses), to assess potential for biased activation of effector T cells over regulatory T cells. For certain activation markers, e.g. CD25 and CD71, the ability to restore effector T cell expression in the presence of regulatory T cells was greater for IL-7 compared to IL-2. We have also investigated the impact of these two cytokines on a range of early and later activation readouts of the responder T cell populations to add granularity to overall impact. Interestingly and in contrast to IL-7, IL-2 evoked greater increases in activation marker expression by regulatory T cells, indicating enhanced regulatory T cell function may contribute to the differential impact of these cytokines upon the responder T cell response. These results demonstrate how potential immunotherapies may differentially impact effector and regulatory T cell populations and highlight the considerable utility of investigating potential immunotherapies (e.g. checkpoint inhibitors) in MR-MCTM assays to better understand impact upon the overall balance of immune activation. Citation Format: Emma Welsh, Zhi Li, Leila Sallie, Ashley Pegg, Hujo Chan, Jamie Cowley, Arshpreet Kaur, Neale Harrison, David J Smith, Mark Bryant, Catherine A Brady, John Gordon, Nicholas Barnes, Omar Qureshi. Preferential ability of IL-7 versus IL-2 to restore effector T cell responses in the presence of regulatory T cells assessed using MR-MCTM assays in vitro [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A029.

Neuroinflammation is generally accepted as a major feature of Alzheimer’s disease (AD) with genetic evidence linking ‘myeloid’ associated genes (e.g. TREM2, CD33, ABCA7 and CR1) with AD risk and supporting a role for microglia (and infiltrating monocytes) in mediating pathology. The excitatory P2×7 receptor, expressed by microglia in the brain, upon activation by pathological concentrations of ATP drives a pro-neuroinflammatory response primarily through the release of IL-1β and IL-18. In contrast P2Y receptor signaling has been associated with anti-inflammatory effects such as reduction in TNFa release. The present study investigated the role of purinergic signalling in the modulation of cytokine release from human microglia derived from monocytes (iMDM). Human microglia (iMDM) were differentiated from human peripheral blood monocytes by culture in a cocktail of CNS-associated cytokines over 5-10 days. The immuno-phenotype of the iMDM versus the originating monocytes were assessed by immuno-cytochemistry. iMDM were primed with LPS before stimulation of purinergic receptors with selected agonists and antagonists to investigate cytokine release. Findings were extended using human brain slices. iMDM displayed an upregulation of microglial phenotypic markers (e.g. TMEM119, IBA1, CX3CR1, P2YR12 and TREM-2). Following priming of iMDM with LPS, the P2×7 receptor agonist, BzATP, evoked a concentration-dependent release of pro-inflammatory cytokines (including IL-1α, IL-1β and IL-18) that was prevented in a concentration-dependent manner by P2×7 receptor antagonists and NLRP3 inhibitors. Although the P2×7 receptor was the major driver of IL-1β release, TNFa release (evoked by LPS) and sensitivity to NLRP3 inhibitors, was modulated by purinergic agonists suggestive of a role for P2Y receptors in this system. The importance of P2×7-receptor in mediating IL-1β release was further confirmed by the use of human brain slices. Modulation of purinergic signalling has the potential to regulate neuroinflammation and the ability of P2×7 receptor antagonists to suppress a pro-inflammatory response by microglia suggests this pharmacological action may offer benefit to patients with AD.

IntroductionThe engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells, and, therefore, blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine elicited serum antibodies. The emergence of SARS-CoV-2 variants has necessitated the development of adaptable assays that can be applied to assess the effectiveness of antibody-based therapeutics.MethodsThrough the testing of a range of recombinant spike proteins, we have developed a cell-based, ACE2/spike protein interaction assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell-bound spike protein fluorescence.ResultsUsing spike proteins from the original "Wuhan" SARS-CoV-2 strain and the Delta and Omicron variants, we identified differential blocking activity of three monoclonal antibodies directed against the spike receptor-binding domain. Importantly, biological activity in the spike interaction assay translated to efficacy in a SARS-CoV-2 infection assay.DiscussionThe spike protein interaction assay can be used to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable for quantification of the impact of antibodies against new and emerging SARS-CoV-2 variants.

Supplementary Data from Treatment of Chronic Lymphocytic Leukemia with a Hypomethylating Agent Induces Expression of NXF2, an Immunogenic Cancer Testis Antigen

The engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells and accordingly blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine-elicited serum antibodies. The emergence of SARS-CoV-2 variants necessitates the development of adaptable assays that can be applied to assess the effectiveness of therapeutics. Through testing of a range of recombinant spike proteins, we have developed a cell based, ACE2/spike protein binding assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell bound spike protein fluorescence. Using spike proteins from the original ‘Wuhan’ SARS-CoV-2 virus, as well as the delta and omicron variants, we identify differential blocking activity of three monoclonal antibodies directed against the spike receptor binding domain. Importantly, biological activity in the spike binding assay translated to efficacy in a SARS-CoV-2 infection assay. Hence, the spike binding assay has utility to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable to quantify impact of antibodies against new and emerging SARS-CoV-2 variants.

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

The metabolism of L-tryptophan to N-formyl-L-kynurenine by indoleamine-2,3-dioxygenase 1 (IDO1) is thought to play a critical role in tumour-mediated immune suppression. Whilst there has been significant progress in elucidating the overall enzymatic mechanism of IDO1 and related enzymes, key aspects of the catalytic cycle remain poorly understood. Here we report the design, synthesis and biological evaluation of a series of tryptophan analogues which have the potential to intercept putative intermediates in the metabolism of 1 by IDO1. Functionally-relevant binding to IDO1 was demonstrated through enzymatic inhibition, however no IDO1-mediated metabolism of these compounds was observed. Subsequent T-m-shift analysis shows the most active compound, 17, exhibits a distinct profile from known competitive IDO1 inhibitors, with docking studies supporting the hypothesis that 17 may bind at the recently-discovered S-i site. These findings provide a start-point for development of further mechanistic probes and more potent tryptophan-based IDO1 inhibitors.

The cellular uptake, intracellular processing, and presentation of foreign antigen are crucial processes for eliciting an effective adaptive host response to the majority of pathogens. The effective recognition of antigen by T cells requires that it is first processed and then presented on MHC molecules that are expressed on other cells. A critical step leading to the presentation of antigen is delivering the foreign cargo to an intracellular compartment where the antigen can be processed and loaded onto MHC molecules. Fc-gamma receptors (FcγRs) recognize IgG-coated targets, such as opsonized pathogens or immune complexes (ICs). Cross-linking leads to internalization of the cargo with associated activation of down-stream signaling cascades. FcγRs vary in their affinity for IgG and intracellular trafficking, and therefore have an opportunity to regulate antigen presentation by controlling the shuttling and processing of their cargos. In this way, they critically influence physiological and pathophysiological adaptive immune cell functions. In this review, we will cover the contribution of FcγRs to antigen-presentation with a focus on the intracellular trafficking of IgG-ICs and the pathways that support this function. We will also discuss genetic evidence linking FcγR biology to immune cell activation and autoimmune processes as exemplified by systemic lupus erythematosus (SLE).

Abstract Prolonged exposure to antigens can lead to a state of reduced T cell responsiveness termed exhaustion. Restoring T cell functions from this state of reduced functionality by, for example, blockade of inhibitory immune checkpoints is now an established therapeutic modality with lasting impact on survival for some patients. Modelling human T cell exhaustion and its reversal in vitro can be challenging in medium or high-throughput assays. We have therefore sought to develop and characterize an in vitro model of T cell exhaustion using human T cells. Long-term (14-day) prolonged stimulation of human T cells in vitro led to both an up-regulation of established exhaustion markers PD-1, LAG-3 and TIM-3, and a lack of responsiveness to subsequent stimulation with human allogeneic monocyte-derived dendritic cells (DCs) as assessed by, for instance, IFNγ and TNFα production. Subsequent addition of blocking antibodies against PD-1 (pembrolizumab and nivolumab) led to a robust reversal of exhausted state by, for instance, restoration of cytokine secretion. The magnitude and nature of the response also correlated with the response of dissociated cells from lung and colon carcinomas. This response also showed greater sensitivity to PD-1 blockade compared to freshly isolated T cells or when using PBMCs stimulated with a pool of EBV peptides. By performing intracellular cytokine staining of human exhausted T cells stimulated with allogeneic dendritic cells, we further noted that PD-1 blockade using either pembrolizumab or nivolumab increased the percentage of cells producing IFNγ suggesting a greater responding pool of T cells. However, whilst the levels of the IFNγ in the supernatants of the exhausted T cell/allogeneic dendritic cell cultures was fully restored when compared to ‘fresh' T cell/allogeneic dendritic cell cultures, the percentage of IFNγ+ cells was not, nor was it fully restored by the inclusion of PMA/ionomycin. Taken together, this would suggest that PD-1 blockade induces responding cells to produce more IFNγ. We suggest that this chronic activation model of human T cell exhaustion with a robust assay window, and responsive to the clinically validated anti-PD-1 antibodies, nivolumab and pembrolizumab, may provide a platform for the discovery of new immuno-oncology therapeutics as well as the assessment of differences in mechanism of action between them. Citation Format: Omar S. Qureshi, Alexander Roberts, Lindsay Bentley, Tina Tang, Fay Stewart, Graham Wallace, Alison Cooper, Aaron Scott, David Thickett, Babu Naidu, Thomas Pinkney, Graham Taylor, Kristian Brock, Louise Healy, Zania Stamataki, Catherine Brady, S J. Curnow, John Gordon, Nicholas M. Barnes. A robust enhancement of cytokine production in a human chronic activation model of T cell exhaustion in vitro through blockade of PD-1/PDL-1 interactions using pembrolizumab or nivolumab; correlation with dissociated tumor immune cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3274.

Inosine Acedoben Dimepranol (IAD), licensed for the treatment of cell-mediated immune deficiencies associated with viral infections, has been reported to impact a variety of immune parameters both in vitro and in vivo. Here we report the results from a clinical trial where multiple lymphocyte subsets - CD19+ B cells, CD3+ T cells, CD4+ T-helper cells, FoxP3hi/CD25hi/CD127lo regulatory T cells (Tregs), CD3-/CD56+ NK cells, and CD3+/CD56+ NKT cells - were, together with serum immunoglobulins and IgG subclasses, followed during 14days of IAD administration to ten healthy volunteers; these selected from 27 individuals pre-screened in vitro for their capacity to respond to IAD as gauged by increases in the percentage of Treg and/or NKT cells arising in PHA-stimulated cultures. While a transient spike and dip in Treg and T-helper fractions, respectively, was noted, the outstanding consequence of IAD administration (1g po, qds) was an early and durable rise in NK cells. For half the cohort, NK cells increased as a percentage of total peripheral blood lymphocytes within 1.5h of receiving drug. By Day 5, all but one of the volunteers displayed higher NK cell percentages, such elevation - effectively a doubling or greater - being maintained at termination of study. The IAD-induced populations were as replete in Granzyme A and Perforin as basal NK cells. The novel finding of IAD boosting phenotypically competent NK numbers in healthy individuals supports the drug's indicated benefit in conditions associated with viral infection and reinforces the potential for uplift where immune performance may be compromised.

Background and PurposeThe 5‐HT3 receptor is a ligand‐gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non‐selective effects at pharmacologically active concentrations. The present study identifies 5‐chloroindole (Cl‐indole) as a potent PAM of the 5‐HT3 receptor.Experimental Approach5‐HT3 receptor function was assessed by the increase in intracellular calcium and single‐cell electrophysiological recordings in HEK293 cells stably expressing the h5‐HT3A receptor and also the mouse native 5‐HT3 receptor that increases neuronal contraction of bladder smooth muscle.Key ResultsCl‐indole (1–100 μM) potentiated agonist (5‐HT) and particularly partial agonist [(S)‐zacopride, DDP733, RR210, quipazine, dopamine, 2‐methyl‐5‐HT, SR57227A, meta chlorophenyl biguanide] induced h5‐HT3A receptor‐mediated responses. This effect of Cl‐indole was also apparent at the mouse native 5‐HT3 receptor. Radioligand‐binding studies identified that Cl‐indole induced a small (∼twofold) increase in the apparent affinity of 5‐HT for the h5‐HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl‐indole was able to reactivate desensitized 5‐HT3 receptors. In contrast to its effect on the 5‐HT3 receptor, Cl‐indole did not alter human nicotinic α7 receptor responses.Conclusions and ImplicationsThe present study identifies Cl‐indole as a relatively potent and selective PAM of the 5‐HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5‐HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.Linked ArticlesRecent reviews on allosteric modulation can be found at:Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476‐5381.2012.02223.xRoche D, Gil D and Giraldo J (2013). Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two‐state and operational models. British Journal of Pharmacology 169: 1189–1202. doi: 10.1111/bph.12231

Objectives To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS). Methods Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry. Results Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1–3 months of treatment with rituximab. Conclusions ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.