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.

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). Electronic supplementary material The online version of this article (10.1186/s40425-019-0570-8) contains supplementary material, which is available to authorized users.

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.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103 þ CD11b þ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103 þ CD11b À (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed a 4 b 7 þ , suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

IL-18 has been implicated in inflammatory bowel disease (IBD),Keywords: IFN-γ r IL-18 receptor signaling r Innate responsiveness r T-cell transfer colitis Additional supporting information may be found in the online version of this article at the publisher's web-site

BackgroundAlligator has developed a new concept, Neo-X’, to enable antigen presenting cells to efficiently enhance priming of neoantigen-specific T cells, which may be the missing aspect in tumors that lack T cell infiltration. We hypothesize that binding of the CD40 x EpCAM bsAb (4224) to CD40 on DCs and EpCAM on tumor exosomes or tumor debris leads to i) activation of the DC, ii) uptake of the tumor material, iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and iiii) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool. CD40 crosslinking by engagement with a tumor antigen on a tumor cell is required to achieve a functional agonistic effect, and subsequent DC activation will therefore only be achieved in the presence of tumor antigens.Methods4224 evaluated in vitro using human monocyte-derived DC, co-cultured with cells expressing EpCAM. In addition the functional effects were evaluated using tumor cell lines and B-cell lines expressing CD40. In vivo, the anti-tumor efficacy of the CD40 x EpCAM bsAb was determined in human CD40 transgenic mice bearing MB49 bladder carcinoma tumors transfected with human EpCAM or controls.ResultsIn vitro, we have demonstrated that the CD40 x EpCAM bsAb induces tumor target dependent activation of dendritic cells, as analyzed by flow cytometry measuring HLA-DR and CD86 expression on the DC and by measuring IL-12p40 levels in the supernatant. Further, the ability of bsAbs within the Neo-X’ concept to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells depends on the receptor density of the tumor target. In vivo, 4224 displayed a potent, EpCAM-dependent anti-tumor effect with significantly reduced tumor growth and improved survival compared to an equivalent dose of the combination of the monospecific CD40 Ab and EpCAM targeting antibody. The tumor-localizing property of 4224 also shows potential for improved safety compared to CD40 monospecific antibodies. A biodistribution analysis demonstrated that the bispecific 4224 in the RUBY-format displayed similar half-life as the monospecific CD40 mAb in mice.ConclusionsIn conclusion, the Neo-X’ concept, by targeting CD40 and a tumor specific antigen, has the potential to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.

BackgroundIndications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation, generate new tumor-specific T cells, and activate tumor-infiltrating myeloid cells to remodel the tumor microenvironment, represent a promising opportunity to meet this need. In this study, we present the first in vivo data supporting a role for tumor-associated antigen (TAA)-mediated uptake and cross-presentation of tumor antigens to enhance tumor-specific T-cell priming using CD40×TAA bispecific antibodies, a concept we named Neo-X-Prime.MethodsBispecific antibodies targeting CD40 and either of two cell-surface expressed TAA, carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) or epithelial cell adhesion molecule (EpCAM), were developed in a tetravalent format. TAA-conditional CD40 agonism, activation of tumor-infiltrating immune cells, antitumor efficacy and the role of delivery of tumor-derived material such as extracellular vesicles, tumor debris and exosomes by the CD40×TAA bispecific antibodies were demonstrated in vitro using primary human and murine cells and in vivo using human CD40 transgenic mice with different tumor models.ResultsThe results showed that the CD40×TAA bispecific antibodies induced TAA-conditional CD40 activation both in vitro and in vivo. Further, it was demonstrated in vitro that they induced clustering of tumor debris and CD40-expressing cells in a dose-dependent manner and superior T-cell priming when added to dendritic cells (DC), ovalbumin (OVA)-specific T cells and OVA-containing tumor debris or exosomes. The antitumor activity of the Neo-X-Prime bispecific antibodies was demonstrated to be significantly superior to the monospecific CD40 antibody, and the resulting T-cell dependent antitumor immunity was directed to tumor antigens other than the TAA used for targeting (EpCAM).ConclusionsThe data presented herein support the hypothesis that CD40×TAA bispecific antibodies can engage tumor-derived vesicles containing tumor neoantigens to myeloid cells such as DCs resulting in an improved DC-mediated cross-priming of tumor-specific CD8+T cells. Thus, this principle may offer therapeutics strategies to enhance tumor-specific T-cell immunity and associated clinical benefit in indications characterized by poor T-cell infiltration or deficiencies in T-cell priming.

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.