SummaryExposure of bone‐marrow‐derived dendritic cells (BMDC) to high‐dose ultrapure lipopolysaccharide for 24 hr (LPS‐primed BMDC) enhances their potency in preventing inter‐photoreceptor retinoid binding protein: complete Freund's adjuvant‐induced experimental autoimmune uveoretinitis (EAU). LPS‐primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK‐binding kinase 1, interferon regulatory factor 3 (IRF3), c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF‐κB) and IRF3, resulting in reduced tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), IL‐12 and interferon‐β secretion. LPS‐primed BMDC also show reduced surface expression of Toll‐like receptor‐4 and up‐regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)‐2 signalling. LPS‐primed BMDC are not only homotolerant to LPS but are heterotolerant to alternative pathogen‐associated molecular pattern ligands, such as mycobacterial protein extract (Mycobacterium tuberculosis). Specifically, while M. tuberculosis protein extract induces secretion of IL‐1β, TNF‐α and IL‐6 in unprimed BMDC, LPS‐primed BMDC fail to secrete these cytokines in response to M. tuberculosis. We propose that LPS priming of BMDC, by exposure to high doses of LPS for 24 hr, stabilizes their tolerogenicity rather than promoting immunogenicity, and does so by multiple mechanisms, namely (i) generation of tolerogenic apoptotic BMDC through CD14:NFATc signalling; (ii) reduction of NF‐κB and IRF3 signalling and downstream pro‐inflammatory cytokine production; and (iii) blockade of inflammasome activation.

The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this “two-step” model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity.

Regulatory T (Treg) cells help to maintain tolerance and prevent the development of autoimmune diseases. Retinoic acid (RA) can promote peripheral conversion of naïve T cells into Foxp3+ Treg cells. Here, we show that RA can act as an adjuvant to induce antigen‐specific type 1 Treg (Tr1) cells, which is augmented by co‐administration of IL‐2. Immunization of mice with the model antigen KLH in the presence of RA and IL‐2 induces T cells that secrete IL‐10, but not IL‐17 or IFN‐γ, and express LAG‐3, CD49b and PD‐1 but not Foxp3, a phenotype typical of Tr1 cells. Furthermore, immunization of mice with the autoantigen MOG in the presence of RA and IL‐2 induces Tr1 cells, which suppress pathogenic Th1 and Th17 cells that mediate the development of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease of the CNS. Furthermore, immunization with a surrogate autoantigen, RA and IL‐2 prevents development of spontaneous autoimmune uveitis. Our findings demonstrate that the induction of autoantigen‐specific Tr1 cells can prevent the development of autoimmunity.

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and β-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases.

Introduction: The human gut microbiota plays a role in modulating both intestinal and systemic immunity. Its importance in regulating anti-tumorigenic responses is now being explored, supported by evidence that the functional profile of the gut microbiome influences response to cancer immunotherapy. We previously demonstrated that the gut microbiome-derived Live Biotherapeutic Enterococcus gallinarum, MRx0518, had strong immunostimulatory properties in vitro, and was a TLR5 and NF-κB activator. We further investigated whether this bacterial strain displayed anti-tumor efficacy in a murine mammary carcinoma model. Experimental procedure: The EMT-6 syngeneic mammary carcinoma tumor model was used to investigate the anti-tumor activity of MRx0518. Female BALB/c mice were engrafted subcutaneously at day 0 with tumor cells, and tumor volumes and body weights were measured twice a week until termination. Animals were orally dosed daily with MRx0518 from D-14 until the end of experiments. Faeces, blood and tissues (ileum, colon, tumor) were collected at study start, midpoint and/or end. NanoString technology was used to study gene expression and the associated pathways modulated, and to evaluate the relative abundance of specific immune cell populations in the tumor, ileum and colon. The plasma and tumour cytokine/chemokine profiles were evaluated using a fluorescent bead-based (Luminex) immunoassay kit. 16S rRNA-based metagenomic analysis was performed on stool samples. Results: MRx0518 monotherapy induces a reduction in EMT-6 murine tumor burden. MRx0518 therapeutic activity was associated with upregulation of genes involved in immune cell adhesion and migration, such as Cd38 and Clec14a, and increased Tlr5 expression in the tumor. At the intestinal level, lymphoid compartment and cell adhesion pathways were upregulated, in particular Spp1, Cd19, Ms4a1 and Sell genes. Immune cell populations including NK, T cells and cytotoxic cells were increased by MRx0518 treatment in both intestinal tissue and the tumor microenvironment. Conclusion: Our preclinical studies demonstrate that the ability of oral MRx0518 to reduce tumor growth in the EMT-6 syngeneic model of breast cancer is mainly related to changes in innate (increase in NK cells, upregulation of tumour TLR5 expression) and adaptive (increase of ratio CD8+ T cells/FoxP3+ regulatory T cells) immune responses locally in the gut but also in the distal tumour microenvironment. In this context, MRx0518 monotherapy and combination with immune checkpoint inhibitors is a promising therapeutic approach for enhancing antitumor immune responses. Citation Format: Jean-Francois Mirjolet, Sylvie Maubant, Sophie Bourdot, Loîc Morgand, Aurélie Couturier-Maillard, Delphine Lauté-Caly, Emma Hennessy, Lorenzo Pavarini, Maria Christofi, Tamas Sukei, Elisabeth Logan, Marsilio Adriani, Imke Mulder. Live biotherapeutic MRx0518 as a modulator of immune responses in intestinal tissue and breast tumor microenvironment [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 6201.