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Radiotherapy can elicit abscopal effects in non-irradiated metastases, particularly under immune checkpoint blockade (ICB). We report on two elderly patients with oligometastatic melanoma treated with anti-PD-1 and stereotactic body radiation therapy (SBRT). Before treatment, patient 1 showed strong tumor infiltration with exhausted CD8+ T cells and high expression of T cell-attracting chemokines. This patient rapidly mounted a complete response, now ongoing for more than 4.5 years. Patient 2 exhibited low CD8+ T cell infiltration and high expression of immunosuppressive arginase. After the first SBRT, his non-irradiated metastases did not regress and new metastases occurred although neoepitope-specific and differentiation antigen-specific CD8+ T cells were detected in the blood. A second SBRT after 10 months on anti-PD-1 induced a radiologic complete response correlating with an increase in activated PD-1-expressing CD8 T cells. Apart from a new lung lesion, which was also irradiated, this deep abscopal response lasted for more than 2.5 years. However, thereafter, his disease progressed and the activated PD-1-expressing CD8 T cells dropped. Our data suggest that oligometastatic patients, where a large proportion of the tumor mass can be irradiated, are good candidates to improve ICB responses by RT, even in the case of an unfavorable pretreatment immune signature, after progression on anti-PD-1, and despite advanced age. Besides repeated irradiation, T cell epitope-based immunotherapies (e.g., vaccination) may prolong antitumor responses even in patients with unfavorable pretreatment immune signature. Electronic supplementary material The online version of this article (10.1007/s00262-020-02587-8) contains supplementary material, which is available to authorized users.

Background: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X 7 and promotes inflammasome assembly and interleukin-1β expression. We hypothesized a functional role of the signal axis ATP–P2X 7 in inflammasome activation and the chronic inflammation driving atherosclerosis. Methods: P2X 7 -competent and P2X 7 -deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X 7 may have a role in atherosclerosis, P2X 7 expression was analyzed in aortic arches from low density lipoprotein receptor -/- mice consuming a high-cholesterol or chow diet. P2X 7 +/+ and P2X 7 −/− low density lipoprotein receptor −/− mice were fed a high-cholesterol diet to investigate the functional role of P2X 7 knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X 7 . Results: Lipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1β in P2X 7 -competent macrophages. In contrast, P2X 7 -deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1β. P2X 7 receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X 7 -deficient mice showed smaller atherosclerotic lesions than P2X 7 -competent mice (0.162 cm 2 ±0.023 [n=9], P2X 7 −/− low density lipoprotein receptor −/− : 0.084 cm 2 ±0.01 [n=11], P =0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X 7 −/− mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X 7 -deficient mice. Last, we observe increased P2X 7 expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease. Conclusions: P2X 7 deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X 7 represents an interesting potential new target to combat atherosclerosis.

// Andreas Zech 1,* , Benjamin Wiesler 1,* , Cemil Korcan Ayata 1 , Tilmann Schlaich 1 , Thorsten Durk 1 , Madelon Hosfeld 1 , Nicolas Ehrat 1 , Sanja Cicko 1 and Marco Idzko 1 1 Department of Pneumology, University Medical Centre Freiburg, Germany * These authors have contributed equally to this work Correspondence to: Marco Idzko, email: // Keywords : asthma, P2RX4, ATP, dendritic cells, IL-1s, Immunology and Microbiology Section, Immune response, Immunity Received : September 30, 2016 Accepted : November 09, 2016 Published : November 15, 2016 Abstract Compelling evidences point out a crucial role for extracellular nucleotides such as adenosine triphosphate (ATP) during inflammatory conditions. Once released into the extracellular space, ATP modulates migration, maturation and function of various inflammatory cells via activating of purinergic receptors of the P2Y- and P2X- family. P2RX4 is an ATP-guided ion channel expressed on structural cells such as alveolar epithelial and smooth muscle cells as well as inflammatory cells including macrophages, dendritic cells (DCs) and T cells. P2RX4 has been shown to interact with P2RX7 and promote NLRP3 inflammasome activation. Although P2RX7 has already been implicated in allergic asthma, the role of P2RX4 in airway inflammation has not been elucidated yet. Therefore, we used a selective pharmacological antagonist and genetic ablation to investigate the role of P2RX4 in an ovalbumin (OVA) driven model of allergen-induced airway inflammation (AAI). Both, P2RX4 antagonist 5-BDBD treatment and P2rx4 deficiency resulted in an alleviated broncho alveolar lavage fluid eosinophilia, peribronchial inflammation, Th2 cytokine production and bronchial hyperresponsiveness. Furthermore, P2rx4 -deficient bone marrow derived DCs (BMDCs) showed a reduced IL-1s production in response to ATP accompanied by a decreased P2rx7 expression and attenuated Th2 priming capacity compared to wild type (WT) BMDCs in vitro . Moreover, mice adoptively transferred with P2rx4 -deficient BMDCs exhibit a diminished AAI in vivo . In conclusion our data suggests that P2RX4-signaling contributes to AAI pathogenesis by regulating DC mediated Th2 cell priming via modulating IL-1s secretion and selective P2RX4-antagonists might be a new therapeutic option for allergic asthma.

Recently, ceramide-1-phosphate (C1P) has been shown to modulate acute inflammatory events. Acute lung injury (Arnalich et al. 2000. Infect. Immun. 68: 1942–1945) is characterized by rapid alveolar injury, lung inflammation, induced cytokine production, neutrophil accumulation, and vascular leakage leading to lung edema. The aim of this study was to investigate the role of C1P during LPS-induced acute lung injury in mice. To evaluate the effect of C1P, we used a prophylactic and therapeutic LPS-induced ALI model in C57BL/6 male mice. Our studies revealed that intrapulmonary application of C1P before (prophylactic) or 24 h after (therapeutic) LPS instillation decreased neutrophil trafficking to the lung, proinflammatory cytokine levels in bronchoalveolar lavage, and alveolar capillary leakage. Mechanistically, C1P inhibited the LPS-triggered NF-κB levels in lung tissue in vivo. In addition, ex vivo experiments revealed that C1P also attenuates LPS-induced NF-κB phosphorylation and IL-8 production in human neutrophils. These results indicate C1P playing a role in dampening LPS-induced acute lung inflammation and suggest that C1P could be a valuable candidate for treatment of ALI.

Acute respiratory distress syndrome (ARDS) is a life-threating lung condition resulting from a direct and indirect injury to the lungs [1, 2]. Pathophysiologically it is characterized by an acute alveolar damage, an increased permeability of the microvascular-barrier, leading to protein-rich pulmonary edema and subsequent impairment of arterial oxygenation and respiratory failure [1]. This study examined the role of extracellular ATP in recruiting inflammatory cells to the lung after induction of acute lung injury with lipopolysaccharide (LPS). However, the precise mechanism is poorly understood. Our objective was to investigate the functional role of the P2X7 receptor in the pathogenesis of acute respiratory distress syndrome (ARDS/ acute lung injury (ALI)) in vitro and in vivo. We show that intratracheally applied LPS causes an acute accumulation of ATP in the BALF (bronchoalveolar lavage) and lungs of mice. Prophylactic and therapeutic inhibition of P2X7R signalling by a specific antagonist and knock-out experiments was able to ameliorate the inflammatory response demonstrated by reduced ATP-levels, number of neutrophils and concentration of pro-inflammatory cytokine levels in the BALF. Experiments with chimeric mice showed that P2X7R expression on immune cells was responsible for the observed effect. Consistently, the inflammatory response is diminished only by a cell-type specific knockdown of P2X7 receptor on non-stationary immune cells. Since the results of BALF from patients with acute ARDS or pneumonia simulated the in vivo data after LPS exposure, the P2X7 receptor may be a new therapeutic target for treatment in acute respiratory distress syndrome (ARDS/ALI).

Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2XP2XLipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1β in P2XP2X

This study examined the role of extracellular ATP in recruiting inflammatory cells to the lung after induction of acute lung injury (ALI) with lipopolysaccharide (LPS). Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. However, the precise mechanism is poorly understood. Our objective was to investigate the functional role of P2X7 receptor in the pathogenesis of acute lung injury (ALI) in vivo. Here we show that inhalative LPS cause an acute accumulation of ATP in the airways of mice. Purinergic receptor antagonists have been used in both therapeutic and prophylactic models and leaded reduction in the ATP-Level and the total cell count in bronchoalveolar lavage (BALF). A succession of experiment with P2X7R-/- and WT chimera animals revealed that the hematopoetic system plays a pivatol role in the observed effect. Finally, we elucidated the contribution of different cell-type specific knockdown of P2X7 receptor, such as LysMCre (macrophages/neutrophils) P2X7fl, CCTCre P2X7fl (conducting airway epithelia), CD4Cre P2X7fl (lymphocytes), SPCTetoCre P2X7fl (airway-epithelial cells), in the pathogenesis of ALI. This decision of P2X7 Receptor may be a new therapeutic target for a treatment in acute lung injury (ALI). Key words: acute lung inflammation, P2X7, KN62, cell-type-specific knockdown of P2X7, BAL, ATP.