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Purpose of review Premature intracellular activation of pancreatic zymogens leads to the initiation of pancreatitis, which in up to 25% leads to chronic tissue destruction, exocrine and endocrine organ failure, and a moderate increased risk of pancreatic cancer development. Whereas in many cases, the trigger of organ damage is identified, diagnostic workup in a significant number of patients does not reveal the underlying etiology of pancreatic inflammation. In these cases, alterations in different pancreatic susceptibility genes have been described to be directly or indirectly involved in disease development. In this review, we want to give an update on the most important pancreatitis risk genes and their impact on clinical diagnostics and risk stratification as well as possible treatment options. Recent findings Genetic testing is not routinely implemented in the diagnostic workup of acute or chronic pancreatitis, as most genetic variations are not considered causative for pancreatitis development but confer increased susceptibility and genetic testing rarely changes disease management. However, in patients with recurrent pancreatitis episodes of unknown etiology after intensive diagnostic work-up, in patients with a family history of pancreatitis, relatives of patients with hereditary pancreatitis, and patients with disease onset at young age, genetic testing and counseling is recommended. Besides well-established susceptibility genes such as PRSS1, SPINK1, CPA1, and CFTR, additional genes such as TRPV6 and rare genetic alterations in established risk genes have been recently identified which significantly contribute to the risk of pancreatitis, involving different molecular mechanisms. Summary When genetic testing is considered, we propose screening at least for PRSS1, SPINK1, CPA1, and CFTR gene variants. The emergence of next-generation sequencing methods could also render larger gene panels possible and clinically meaningful to detect rare variants with high-risk phenotypes. Here we summarize, evaluate, and convey in the form of practical recommendations the current level of knowledge with respect to definition, etiology, and genetic diagnostics of all forms of inherited pancreatitis.

Der Einzug der Checkpoint-Inhibitoren (CPI) in die onkologische Therapielandschaft stellte einen wesentlichen Fortschritt der Immuntherapie in der letzten Dekade dar. In diesem Beitrag wird der Wirkmechanismus der CPI und deren aktuelle klinische Anwendung in der Systemtherapie unterschiedlicher Tumorentitaten erlautert. Relevante Artikel wurden in der PubMed-Datenbank identifiziert. Informationen zu klinischen Studien wurden aus dem Studienregister Clinicaltrials.gov extrahiert, Arzneimittelzulassungen aus den offentlichen Schreiben der European Medicines Agency (EMA) und der US Food and Drug Administration (FDA). Obwohl durch CPI wesentliche Fortschritte in der Behandlung vieler Malignome erzielt wurden, konnen robuste Ansprechraten und ein Langzeituberleben nur bei wenigen Patienten beobachtet werden. Die Entwicklung pradiktiver Biomarker zur Maximierung der Effektivitat und Minimierung potenzieller immunvermittelter Nebenwirkungen kann neben neuen Kombinationstherapien dazu beitragen, das volle Potenzial der CPI zu entfalten.

Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists.

Background Unmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy. Materials and Methods T, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed. Results Analyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5 Conclusions We could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands. References O’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60. Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106. Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249. Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181. Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065. Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93. Disclosure Information J. Grun: None. I. Piseddu: None. C. Perleberg: None. N. Rohrle: None. S. Endres: None. D. Anz: None.

CCL22 is a key mediator of leukocyte trafficking in inflammatory immune responses, allergy, and cancer. It acts by attracting regulatory T cells and Th2 cells via their receptor CCR type 4 (CCR4). Beyond its role in inflammation, CCL22 is constitutively expressed at high levels in lymphoid organs during homeostasis, where it controls immunity by recruiting regulatory T cells to dendritic cells (DCs). In this study, we aimed to identify the mechanisms responsible for constitutive CCL22 expression. We confirmed that CD11c+ DCs are the exclusive producers of CCL22 in secondary lymphatic organs during homeostasis. We show that in vitro both murine splenocytes and human PBMCs secrete CCL22 spontaneously without any further stimulation. Interestingly, isolated DCs alone, however, are unable to produce CCL22, but instead require T cell help. In vitro, only the coculture of DCs with T cells or their supernatants resulted in CCL22 secretion, and we identified T cell–derived GM-CSF as the major inducer of DC-derived CCL22 expression. In vivo, Rag1−/− mice, which lack functional T cells, have low CCL22 levels in lymphoid organs, and this can be restored by adoptive transfer of wild-type T cells or administration of GM-CSF. Taken together, we uncover T cell–derived GM-CSF as a key inducer of the chemokine CCL22 and thus, to our knowledge, identify a novel role for this cytokine as a central regulator of immunity in lymphatic organs. This knowledge could contribute to the development of new therapeutic interventions in cancer and autoimmunity.

Rapp et al. demonstrate that dendritic cells in the lymph node secrete CCL22 to build cell–cell contacts with CCR4-expressing regulatory T cells, leading to immune suppression. Conversely, CCL22 deficiency results in enhanced T cell immunity, shown here in the setting of vaccination, cancer, and inflammatory disease., Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell–cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22–CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity., Graphical Abstract

The chemokine CCL22 is a well known modulator of immune responses. By binding to its receptor CCR4, which is highly expressed on several T cell subtypes as well as dendritic cells (DC), CCL22 mediates chemotaxis of responsive cells, thus influencing the distribution of these cells within tissues. For instance, CCL22 is known to be expressed in human and murine tumors contributing to the recruitment of regulatory T cells. Given that many chemokines show effects exceeding pure chemotaxis we hypothesized that CCL22 mediates modulation of immune responses by further activities. In order to identify additional immune modulating properties of CCL22 on DCs and T cells we stimulated murine splenocytes isolated from healthy donor mice with recombinant CCL22 in vitro. We did not find any significant alteration in expression of cell surface activation markers like CTLA-4, PD-1 or CD62L on CD4+, CD8+ and FoxP3+ T cells. However, splenic CD11c+ DCs showed a significant reduction in MHC class I molecules on cell surface upon stimulation with CCL22. Notably, this reduction was most prominent in CD11c+ CD8α+ DCs which also showed decreased levels of MHC class II molecules as well as the costimulatory receptor CD80 after encountering CCL22. In summary, our data indicate an alternative mode of action for CCL22 on Dendritic cells, especially on CD11c+ CD8α+ DCs. The CCL22-mediated modification of DC’s capabilities to present antigens and to activate T cells might represent a fine-tuning mechanism of adaptive immune responses.

Regulatory T cells (Tregs) are highly potent modulators of immune responses by various mechanisms including direct interaction with dendritic cells. Thereby, Tregs play a critical role not only in prevention of autoimmunity but also in tumor immune escape. By binding to its only known receptor CCR4 the chemokine CCL22 is a potent chemoattractant for Tregs, influencing their localization and activity. However, the regulation of CCL22 expression is still poorly understood. Here we demonstrate that under homeostatic conditions cells of the dendritic cell lineage are the main producers of CCL22. In vitro, coincubation of dendritic cells with CD4+ CD25− T cells strongly induced CCL22 expression in a paracrine manner. Using neutralizing antibodies we identified the cytokine family IL-3, IL-5 and GM-CSF, as well as IL-4 to be the main inducers of CCL22 in dendritic cells. In vivo, transfer of CD4+ T cells into Rag−/−-mice lacking mature T and B cells was able to increase CCL22 levels in several lymphoid organs. In summary, we identified the aforementioned T cell-derived factors as the main inducers of the Treg-associated chemokine CCL22, in vitro and in vivo. Therefore, our data suggests that paracrine signalling of T cells might indirectly enhance Treg activity by induction of CCL22 expression in dendritic cells. In conclusion, the data presented here potentially shows a self-limiting mechanism of adaptive immune responses.