Safety, tolerability, and pharmacology of AB928, a novel dual adenosine receptor antagonist, in a randomized, phase 1 study in healthy volunteers.

Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5'-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 µM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

Cross-presenting dendritic cells are required for control of Leishmania major infection.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line.

Dendritic cells (DCs) are the most potent antigen presenting cells and possess an incomparable ability to activate and instruct T cells, which makes them one of the cornerstones in the regulation of the cross-talk between innate and adaptive immunity. Therefore, a deep understanding of DC biology lays the foundations to describe and to harness the mechanisms that regulate the development of the adaptive response, with clear implications in a vast array of fields such as the study of autoimmune diseases and the development of new vaccines. However, the great difficulty to obtain large quantities of viable non-activated DCs for experimentation have considerably hindered the progress of DC research. Several strategies have been proposed to overcome these limitations by promoting an increase of DC abundance in vivo, by inducing DC development from DC progenitors in vitro and by generating stable DC lines. In the past years, we have described a method to derive immortalized stable DC lines, named MutuDCs, from the spleens of Mushi1 mice, a transgenic mouse strain that express the simian virus 40 Large T-oncogene in the DCs. The comparison of these DC lines with the vast variety of DC subsets described in vivo has shown that all the MutuDC lines that we have generated so far have phenotypic and functional features of type 1 conventional DCs (cDC1s). With the purpose of deriving DC lines with characteristics of type 2 conventional DCs (cDC2s), we bred a new Batf3-/- Mushi1 murine line in which the development of the cDC1 subset is severely defective. The new MutuDC line that we generated from Batf3-/- Mushi1 mice was phenotypically and functionally characterized in this work. Our results demonstrated that all the tested characteristics of this new cell line, including the expression of subset-determining transcription factors, the profile of cytokine production and the ability to present antigens, are comparable with the features of splenic CD4- cDC2s. Therefore, we concluded that our new cell line, that we named CD4- MutuDC2 line, represents a valuable model for the CD4- cDC2 subset.

Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

Timing is everything: dendritic cell subsets in murine Leishmania infection.

Mouse models of Leishmania major infection have shown that a predominant CD4(+) T helper type 1 cell (Th1) response leads to protection, while T helper type 2 cell (Th2) predominance confers susceptibility. Dendritic cells (DCs) are antigen-presenting cells that orchestrate the T cell response. The immune response to L. major involves direct antigen presentation by migrating DCs or transfer of antigens to resident DCs to prime T cells. In this review, we discuss the timing and consequences of antigen presentation by DC subsets and how this affects Leishmania susceptibility. We propose a model where dermal DCs and Langerhans cells play a role early in infection, followed by inflammatory monocyte-derived DC and lymph node (LN)-resident DCs at later time points of infection to establish the resistant Th1 response.

Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research.

Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.