Oncolytic Newcastle disease virus activation of the innate immune response and priming of antitumor adaptive responses in vitro.

Oncolytic virus (OV) therapy is an emerging approach with the potential to redefine treatment options across a range of cancer indications and in patients who remain resistant to existing standards of care, including immuno-oncology (IO) drugs. MEDI5395, a recombinant Newcastle disease virus (NDV), engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibits potent oncolytic activity. It was hypothesized that activation of immune cells by MEDI5395, coupled with its oncolytic activity, would enhance the priming of antitumor immunity. Using MEDI5395 and recombinant NDVs encoding fluorescent reporter genes, we demonstrated preferential virus uptake and non-productive infection in myeloid cells, including monocytes, macrophages, and dendritic cells (DCs). Infection resulted in immune-cell activation, with upregulation of cell surface activation markers (e.g., CD80, PD-L1, HLA-DR) and secretion of proinflammatory cytokines (IFN-α2a, IL-6, IL-8, TNF-α). Interestingly, in vitro M2-polarized macrophages were more permissive to virus infection than were M1-polarized macrophages. In a co-culture system, infected myeloid cells were effective virus vectors and mediated the transfer of infectious NDV particles to tumor cells, resulting in cell death. Furthermore, NDV-infected DCs stimulated greater proliferation of allogeneic T cells than uninfected DCs. Antigens released after NDV-induced tumor cell lysis were cross-presented by DCs and drove activation of tumor antigen-specific autologous T cells. MEDI5395 therefore exhibited potent immunostimulatory activity and an ability to enhance antigen-specific T-cell priming. This, coupled with its tumor-selective oncolytic capacity, underscores the promise of MEDI5395 as a multimodal therapeutic, with potential to both enhance current responding patient populations and elicit de novo responses in resistant patients.

Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

TSLP production by dendritic cells is modulated by IL-1ß and components of the endoplasmic reticulum stress response.

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of TH 2-cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and ß-glucans requires dectin-1, Syk, NF-κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL-1ß, but not TNF-α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol-requiring transmembrane kinase/endonuclease 1 and protein kinase R-like ER kinase, which are activated by dectin-1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin-1, the IL-1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC-controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4ß7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

Characterization of a novel potency endpoint for the evaluation of immune checkpoint blockade in humanized mice.

Introduction: Humanized mice are emerging as valuable models to experimentally evaluate the impact of different immunotherapeutics on the human immune system. These immunodeficient mice are engrafted with human cells or tissues, that then mimic the human immune system, offering an alternative and potentially more predictive preclinical model. Immunodeficient NSG mice engrafted with human CD34+ cord blood stem cells develop human T cells educated against murine MHC. However, autoimmune graft versus host disease (GvHD), mediated by T cells, typically develops 1 year post engraftment. Methods: Here, we have used the development of GvHD in NSG mice, using donors with HLA alleles predisposed to autoimmunity (psoriasis) to weight in favor of GvHD, as an endpoint to evaluate the relative potency of monoclonal and BiSpecific antibodies targeting PD-1 and CTLA-4 to break immune tolerance. Results: We found that treatment with either a combination of anti-PD-1 & anti-CTLA-4 mAbs or a quadrivalent anti-PD-1/CTLA-4 BiSpecific (MEDI8500), had enhanced potency compared to treatment with anti-PD-1 or anti-CTLA-4 monotherapies, increasing T cell activity both in vitro and in vivo. This resulted in accelerated development of GvHD and shorter survival of the humanized mice in these treatment groups commensurate with their on target activity. Discussion: Our findings demonstrate the potential of humanized mouse models for preclinical evaluation of different immunotherapies and combinations, using acceleration of GvHD development as a surrogate of aggravated antigenic T-cell response against host.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

Dendritic Cell-Derived TSLP Negatively Regulates HIF-1α and IL-1ß During Dectin-1 Signaling.

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (TH) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1ß production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1ß expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

ß-Glucan Size Controls Dectin-1-Mediated Immune Responses in Human Dendritic Cells by Regulating IL-1ß Production.

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes ß-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. ß-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate ß-glucans are thought to be poor activators of innate immunity. We show that ß-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large ß-glucan-stimulated DC generate significantly more IL-1ß, IL-6, and IL-23 compared to those stimulated with the smaller ß-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to ß-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1ß production determined by ß-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that ß-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

IRE1α mediates PKR activation in response to Chlamydia trachomatis infection.

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-ß production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.