Succinate acts as an extracellular mediator signaling through the G protein-coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid-specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA+ T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (Nt-PEX5) based on the protein's interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

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The ubiquitin ligase Cbl-b is an established regulator of T cell immune response thresholds. We recently showed that adoptive cell transfer (ACT) of cblb −/− CD8+ T cells enhances dendritic cell (DC) immunization-mediated anti-tumor effects in immune-competent recipients. However, translation of cblb targeting to clinically applicable concepts requires that inhibition of cblb activity be transient and reversible. Here we provide experimental evidence that inhibition of cblb using chemically synthesized siRNA has such potential. Silencing cblb expression by ex vivo siRNA transfection of polyclonal CD8+ T cells prior to ACT increased T cell tumor infiltration, significantly delayed tumor outgrowth, and increased survival rates of tumor-bearing mice. As shown by ex vivo recall assays, cblb silencing resulted in significant augmentation of intratumoral T cell cytokine response. ACT of cblb-silenced polyclonal CD8+ T cells combined with DC-based tumor vaccines predominantly mediated anti-tumor immune responses, whereas no signs of autoimmunity could be detected. Importantly, CBLB silencing in human CD8+ T cells mirrored the effects observed for cblb-silenced and cblb-deficient murine T cells. Our data validate the concept of enhanced anti-tumor immunity by repetitive ACT of ex vivo cblb siRNA-silenced hyper-reactive CD8+ T cells as add-on adjuvant therapy to augment the efficacy of existing cancer immunotherapy regimens in clinical practice.

Whereas some individuals develop immunity to bee sting and mount protective IgG4- mediated antibody responses to bee venom phospholipase A2 (PLA), others produce large amounts of PLA-specific IgE antibodies and become allergic to this, otherwise, innocuous antigen. PLA-specific IgE responses are the result of imbalanced T helper (Th)2-cell differentiation. There are multiple mechanisms driving the differentiation of naive CD4+ T cells into Th1- or Th2-cell phenotypes. Most of them are linked to the conditions occurring during initial or repeated encounters with the allergen, in the context of an antigen-presenting cell (APC). The different types of APC and their availability to display particular cytokine production profiles, pattern recognition receptors, costimulatory molecules and specific HLA haplotypes are key determinants for human Th1- and Th2-cell polarization.

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Blüml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Günther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 527.