IL-18Rα-deficient CD4+T cells induce intestinal inflammation in the CD45RBhitransfer model of colitis despite impaired innate responsiveness

IL-18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4+ T-cell function remains unclear. Here we show that murine intestinal CD4+ T cells express high levels of IL-18Rα and provide evidence that IL-18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RBhi T-cell transfer colitis model, we show that IL-18Rα is expressed on IFN-γ+, IL-17+, and IL-17+IFN-γ+ effector CD4+ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN-γ-producing cells in the cLP. In the steady state and during colitis, TCR-independent cytokine-induced IFN-γ and IL-17 production by intestinal CD4+ T cells was largely IL-18Rα−dependent. Despite these findings however, IL-18Rα−deficient CD4+ T cells induced comparable intestinal pathology to WT CD4+ T cells. These findings suggest that IL-18-dependent cytokine induced activation of CD4+ T cells is not critical for the development of T-cell-mediated colitis

Intestinal dendritic cell migration and induction of T cell responses

The intestine represents the body’s largest surface exposed to the outer world and is thus a major entry site for pathogens such as bacteria and viruses. The intestinal immune system has the important task of protecting us against infection while maintaining tolerance against the vast amount of commensal microbes populating the intestinal tract and the multitude of foreign antigen present in the diet. Intestinal dendritic cells (DCs) have a central role in maintaining homeostasis and their continual migration from the intestinal mucosa to the draining mesenteric lymph nodes (MLN) is required for the induction of adaptive immune responses to orally derived antigen as well as the development of oral tolerance to food proteins. Migratory DCs are also thought to have a central role in the induction of gut-homing receptors on T cells, allowing the T cells to home to the gut and perform effector functions. In this thesis I investigate the signals that drive DC migration to the MLN under homeostatic and inflammatory conditions. The results presented show that in the steady state, DC migration is driven by the adaptor protein MyD88, which mediates signaling through pattern recognition receptors of the Toll-like receptor family as well as receptors of the cytokines IL-1β, IL-18 and IL-33. While optimal DC migration requires MyD88 signaling in CD11c+ cells, including DCs, it is independent of the microbiota, caspase-1-induced maturation of IL-1β and IL-18 and signaling through the IL-18 receptor. Additionally, we find that DC migration in response to an inflammatory stimulus requires TNF-α signaling, in contrast to the steady state migration that is TNF-α independent. We further find that induction of the gut-homing receptor CCR9 on T cells in an inflammatory setting requires TNF-α signaling in stromal cells, but is independent of TNF-α-induced DC migration from the intestine. As TNF-α is involved in the pathology of inflammatory bowel disease (IBD), and TNF-α-neutralizing therapies are used in IBD with great success, these findings could aid the understanding of the mechanisms involved in intestinal inflammation and anti-TNF therapy efficacy. Although IL-18 signaling did not drive steady state DC migration, we found that it had a role in the generation of IFN-γ producing T cells and in cytokine-induced TCR-independent innate-like cytokine production by T cells in a T cell-dependent mouse model of intestinal inflammation. Impaired IL-18 signaling in the T cells did however not have an impact on disease severity. This data could also aid the understanding of disease mechanisms in IBD

Rationale and clinical development of CD40 agonistic antibodies for cancer immunotherapy

Introduction: CD40 signaling activates dendritic cells leading to improved T cell priming against tumor antigens. CD40 agonism expands the tumor-specific T cell repertoire and has the potential to increase the fraction of patients that respond to established immunotherapies. Areas covered: This article reviews current as well as emerging CD40 agonist therapies with a focus on antibody-based therapies, including next generation bispecific CD40 agonists. The scientific rationale for different design criteria, binding epitopes, and formats are discussed. Expert opinion: The ability of CD40 agonists to activate dendritic cells and enhance antigen cross-presentation to CD8+ T cells provides an opportunity to elevate response rates of cancer immunotherapies. While there are many challenges left to address, including optimal dose regimen, CD40 agonist profile, combination partners and indications, we are confident that CD40 agonists will play an important role in the challenging task of reprogramming the immune system to fight cancer

MyD88 Signaling Regulates Steady-State Migration of Intestinal CD103⁺Dendritic Cells Independently of TNF-α and the Gut Microbiota

Intestinal homeostasis and induction of systemic tolerance to fed Ags (i.e., oral tolerance) rely on the steady-state migration of small intestinal lamina propria dendritic cells (DCs) into draining mesenteric lymph nodes (MLN). The majority of these migratory DCs express the α integrin chain CD103, and in this study we demonstrate that the steady-state mobilization of CD103(+) DCs into the MLN is in part governed by the IL-1R family/TLR signaling adaptor molecule MyD88. Similar to mice with complete MyD88 deficiency, specific deletion of MyD88 in DCs resulted in a 50-60% reduction in short-term accumulation of both CD103(+)CD11b(+) and CD103(+)CD11b(-) DCs in the MLN. DC migration was independent of caspase-1, which is responsible for the inflammasome-dependent proteolytic activation of IL-1 cytokine family members, and was not affected by treatment with broad-spectrum antibiotics. Consistent with the latter finding, the proportion and phenotypic composition of DCs were similar in mesenteric lymph from germ-free and conventionally housed mice. Although TNF-α was required for CD103(+) DC migration to the MLN after oral administration of the TLR7 agonist R848, it was not required for the steady-state migration of these cells. Similarly, TLR signaling through the adaptor molecule Toll/IL-1R domain-containing adapter inducing IFN-β and downstream production of type I IFN were not required for steady-state CD103(+) DC migration. Taken together, our results demonstrate that MyD88 signaling in DCs, independently of the microbiota and TNF-α, is required for optimal steady-state migration of small intestinal lamina propria CD103(+) DCs into the MLN

Transcriptomic profiling of T-cell populations in non-muscle invasive and muscle invasive bladder cancer.

Background: Bladder cancer is categorized as non-muscle invasive (NMIBC) or muscle invasive (MIBC). NIMBC makes up around 70% of the cases and although it is less aggressive, the recurrence rate is 50-70%, thus requiring extensive monitoring. Additionally, there is a risk of progression into MIBC with a 5-year survival of only 50% even when treated with radical cystectomy. Immune checkpoint inhibitors have shown promising results for treatment of bladder cancer; however, only around 30% of patients have a therapeutic effect and novel therapies are thus required. With the aim of pinpointing novel targets for T-cell based therapy, we have performed transcriptomic profiling of specific T cell populations in MIBC and NMIBC, as well as in control bladder tissue.Methods: Muscle-invasive (n=7) as well as non-muscle invasive (n=13) bladder tumor biopsies were obtained from untreated patients and control bladder tissue (n=7). Upon digestion, cells were stained with an antibody panel to enable sorting of CD8+ cytotoxic T-cells (CD8T), CD4+ T-helper cells (Th) and regulatory T-cells (Treg) using fluorescence activated cell sorting. RNA was extracted and subject to sequencing. Differential gene expression analysis was performed, using DESeq2 (genes with padj<0.05 were considered significant). Variance stabilizing transformation was used for PCA-plots and heatmaps. Gene set enrichment analysis (GSEA) was performed using the fgsea package in R with genes pre-ranked according to the log2foldchange from the differential gene expression analysis.Results: Principal component analysis demonstrated that CD8T, unlike Th and Tregs, cluster according to the invasiveness of the disease. Accordingly, many genes were significantly differentially expressed between CD8T in MIBC and NMIBC compared to control, and also between CD8T in MIBC compared to NMIBC. Several genes associated with CD8 T-cell exhaustion were significantly upregulated in MIBC compared to both NMIBC and control. Further, GSEA results indicated biological differences of the CD8T compartment between different tumor stages. Conclusion: The gene expression profiles of CD8 T-cells were significantly different in NMIBC, MIBC and control. The transcriptional profiles give clues on biological differences and disease progression and can be relevant for development of novel treatment strategies

Transcriptional profiling demonstrates altered characteristics of CD8 + cytotoxic T-cells and regulatory T-cells in TP53-mutated acute myeloid leukemia

Background: Acute myeloid leukemia (AML) patients have limited effect from T-cell-based therapies, such as PD-1 and CTLA-4 blockade. However, recent data indicate that AML patients with TP53 mutation have higher immune infiltration and other immunomodulatory therapies could thus potentially be effective. Here, we performed the transcriptional analysis of distinct T-cell subpopulations from TP53-mutated AML to identify gene expression signatures suggestive of altered functional properties.Methods: CD8+ cytotoxic T lymphocytes (CTLs), conventional helper T cells (Th), and regulatory T cells (Tregs) were sorted from peripheral blood of AML patients with TP53 mutation (n = 5) and healthy donors (n = 3), using FACS, and the different subpopulations were subsequently subjected to RNA-sequencing. Differentially expressed genes were identified and gene set enrichment analysis (GSEA) was performed to outline altered pathways and exhaustion status. Also, expression levels for a set of genes encoding established and emerging immuno-oncological targets were defined.Results: The results showed altered transcriptional profiles for each of the T-cell subpopulations from TP53-mutated AML as compared to control subjects. IFN-α and IFN-γ signaling were stronger in TP53-mutated AML for both CTLs and Tregs. Furthermore, in TP53-mutated AML as compared to healthy controls, Tregs showed gene expression signatures suggestive of metabolic adaptation to their environment, whereas CTLs exhibited features of exhaustion/dysfunction with a stronger expression of TIM3 as well as enrichment of a gene set related to exhaustion.Conclusions: The results provide insights on mechanisms underlying the inadequate immune response to leukemic cells in TP53-mutated AML and open up for further exploration toward novel treatment regimens for these patients

IRF4 Transcription-Factor-Dependent CD103(+)CD11b(+) Dendritic Cells Drive Mucosal T Helper 17 Cell Differentiation.

CD103(+)CD11b(+) dendritic cells (DCs) represent the major migratory DC population within the small intestinal lamina propria (SI-LP), but their in vivo function remains unclear. Here we demonstrate that intestinal CD103(+)CD11b(+) DC survival was dependent on interferon regulatory factor 4 (IRF4). Mice with a DC deletion in Irf4 displayed reduced numbers of intestinal interleukin 17 (IL-17)-secreting helper T 17 (Th17) cells and failed to support Th17 cell differentiation in draining mesenteric lymph nodes (MLN) following immunization. The latter was associated with a selective reduction in CD103(+)CD11b(+) MLN DCs and DC derived IL-6. Immunized Il6(-/-) mice failed to support Th17 cell differentiation in MLN in vivo and CD103(+)CD11b(+) MLN DCs supported IL-6-dependent Th17 cell differentiation in vitro. Together, our results suggest a central role for IRF4-dependent, IL-6 producing CD103(+)CD11b(+) DCs in intestinal Th17 cell differentiation

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation

Abstract Background The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. Methods ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. Results ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. Conclusions By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467)