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Immune checkpoint inhibition (ICI) is an important treatment modality in metastatic NSCLC and management of immunotherapy-related adverse effects (irAEs) can be challenging. Retreatment after discontinuation of ICI because of irAEs is a frequent clinical dilemma with limited available data.This single-center retrospective observational study reviewed the clinical course of 30 patients with metastatic NSCLC in whom ICI had to be discontinued owing to a serious irAE after an initial objective response to therapy.After ICI discontinuation, 14 patients (47%) developed a durable response of more than 6 months, seven patients (23%) developed oligoprogression treated with local radiotherapy leading to disease control, six patients (20%) had progression of disease within 6 months, and three patients (10%) died owing to a severe irAE.A watchful waiting approach is justified after discontinuation of ICI owing to irAEs in patients with metastatic NSCLC with an initial response to therapy.

IntroductionAutoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology.MethodsMice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton’s tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry.ResultsFollowing pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5+ follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5+ Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population.ConclusionsOur study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression.

Background Recent studies have provided evidence for an important contribution of the immune system in the pathophysiology of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). In this report, we investigated whether the inflammatory profile of pulmonary hypertension patients changes over time and correlates with patient WHO subgroups or survival. Methods 50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients and 18 healthy controls (HCs) were included in the study. Plasma inflammatory markers at baseline and after 1-year follow-up were measured using ELISAs. Subsequently, correlations with hemodynamic parameters and survival were explored and data sets were subjected to unbiased multivariate analyses. Results At diagnosis, we found that plasma levels of interleukin-6 (IL-6) and the chemokines (C-X3-C) motif legend CXCL9 and CXCL13 in CTD-PAH patients were significantly increased, compared with HCs. In idiopathic PAH patients the levels of tumor growth factor-β (TGFβ), IL-10 and CXCL9 were elevated, compared with HCs. The increased CXCL9 and IL-8 concentrations in CETPH patients correlated significantly with decreased survival, suggesting that CXCL9 and IL-8 may be prognostic markers. After one year of treatment, IL-10, CXCL13 and TGFβ levels changed significantly in the PAH subgroups and CTEPH patients. Unbiased multivariate analysis revealed clustering of PH patients based on inflammatory mediators and clinical parameters, but did not separate the WHO subgroups. Importantly, these multivariate analyses separated patients with 3 years survival, in particular when inflammatory mediators were combined with clinical parameters. Discussion Our study revealed elevated plasma levels of inflammatory mediators in different PAH subgroups and CTEPH at baseline and at 1-year follow-up, whereby CXCL9 and IL-8 may prove to be prognostic markers for CTEPH patients. While this study is exploratory and hypothesis generating, our data indicate an important role for IL-8 and CXCL9 in CHD and CTEPH patients considering the increased plasma levels and the observed correlation with survival. Conclusion In conclusion, our studies identified an inflammatory signature that clustered PH patients into WHO classification-independent subgroups that correlated with patient survival.

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton’s tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-kB ligand (RANKL), is used as a biomarker for assessing severity of liver fibrosis. However, its expression and role in pulmonary fibrosis are unknown. We hypothesized that OPG also has a role in pulmonary fibrosis.Human and mouse control and fibrotic lung tissue were used to examine OPG expression, and mouse precision-cut lung slices to study OPG regulation in pulmonary fibrosis. Serum from idiopathic pulmonary fibrosis (IPF) patients and controls was analysed to investigate whether OPG levels correlate with disease status as measured by lung function.OPG-protein levels were significantly higher in mouse and human fibrotic lung tissue compared to control. OPG-mRNA and protein production were induced in mouse precision-cut-lung slices upon TGFβ stimulation and could be inhibited with galunisertib, a TGFβ receptor kinase inhibitor. OPG-protein levels in fibrotic mouse lung tissue correlated with degree of fibrosis. Isolated lung fibroblasts from IPF patients had higher OPG-protein levels than control fibroblasts. Serum OPG levels in IPF patients, at first presentation, negatively correlated with diffusing capacity to carbon monoxide. Finally, serum OPG levels higher than 1234 pg/ml at first presentation were associated with progression of disease in IPF patients.In conclusion, OPG is produced in lung tissue, associates with fibrosis, and may be a potential prognostic biomarker for IPF disease progression. Validation in a larger cohort is warranted to further explore the role of OPG in pulmonary fibrosis and its potential for assessing the prognosis of fibrotic lung disease in individual patients.Take home messageOsteoprotegerin is present in fibrotic lung tissue and high serum levels correlate with low lung function and IPF disease progression in this small study, indicating osteoprotegerin may have value as a biomarker to predict IPF progression

Idiopathic pulmonary fibrosis (IPF) is a progressive, and ultimately fatal, chronic interstitial lung disease characterized by enhanced extracellular matrix deposition. Repetitive alveolar epithelial injury triggers the early development of fibrosis. These injuries, in combination with dysregulated wound repair and fibroblast dysfunction, lead to ongoing tissue remodelling and fibrosis seen in end-stage pulmonary fibrosis. Although the exact etiology in IPF is unknown and probably diverse, all stages of fibrosis are accompanied by innate and adaptive immune responses. The role of inflammation as an important component in IPF etiology is controversial and sometimes seen as an epiphenomenon of fibrosis. This view is partly the result of negative multicenter trials of anti-inflammatory drugs for IPF treatment. However, new insights on the role of macrophages, the loss of T-cell and B-cell tolerance leading auto-immune responses in IPF, and the interaction of immune cells with (myo)fibroblasts have led to a slow change of this opinion. Clearly, more insight is needed to integrate basic immune mechanisms into translational research and finally new IPF therapies. In this concise review, we will focus on the role of our innate and adaptive immune system in the initiation and perpetuation of IPF pathobiology. Next, we will discuss how immune responses are influenced by current anti-fibrotic treatments, such as pirfenidone and nintedanib and end with an overview of recent and upcoming therapeutic trials that target and modulate our immune system in patients with IPF.

Pulmonary arterial hypertension (PAH) is characterized by a high pulmonary arterial pressure leading to hypertrophy of the right ventricle (RV) and ultimately death due to RV failure. The most common form of PAH is idiopathic PAH (IPAH) for which no identified risk factor has been found until date. In IPAH, changes in CD4+ T helper (Th)-cell subsets, regulatory Th-cell (Treg) function, and the presence of pulmonary tertiary lymphoid aggregates were observed. We hypothesized that in PAH, Th-cells harbor a proinflammatory phenotype and signs of chronic activation. To investigate this hypothesis, we used a combined cytokine and transcription factor staining on peripheral blood mononuclear cells (PBMCs) from IPAH and healthy controls (HC). The proportion of activated and memory Th-cells were unaltered, however an increase in activated Tregs was observed in IPAH compared to HC. Moreover, activated Th-cells showed an increased expression of inhibitory molecule CTLA-4 and activation marker CD25 in IPAH compared to HC. Strikingly, IPAH Th-cells were less capable of producing IL-6, IFNγ and IL-17 compared to HC. Specifically, RORγt-positive Th17-cells were less qualified to produce IL-17 in IPAH compared to HC. In conclusion, elevated CD25 and CTLA-4 expression on activated Th-cells possibly point at chronic activation leading to an exhausted Th-cell phenotype. The reduced cytokine production can be caused by I) Th-cell exhaustion, II) an increase in activated Tregs, III) homing of cytokine-producing Th-cells towards lungs, or IV) a negative feedback response due to an high cytokine milieu. These Th-cell alterations suggest chronic activation and immune imbalance which could be involved in PAH pathology.

Background Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. Methods First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. Results Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. Conclusions These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases. Electronic supplementary material The online version of this article (10.1186/s12931-018-0798-8) contains supplementary material, which is available to authorized users.

The presence of tertiary lymphoid organs (TLOs) in lungs of IPAH patients suggest a key role for (auto)immunity in its pathogenesis. Activated dendritic cells (DCs) play a crucial role in the induction of TLOs. DC activation leads to NF-kB activation, which is negatively regulated by the A20 enzyme. In this study, peripheral blood of PAH patients and healthy controls (HC) was examined for DC number and activation. Furthermore, mice harboring activated A20-deficient cDC1s ( Dngr1 -cre X Tnfaip3 fl/fl mice (DNGR1-A20)) were examined at 31 weeks of age. PAH development was monitored using Right Heart Catheterization (RHC), Echocardiography, RV hypertrophy, and Histology. Peripheral CD141+ DCs (cDC1) of IPAH patients showed an increased activation status by CD86 expression compared to HC. DNGR1-A20KO mice showed an significantly increased RV hypertrophy, measured both by echocardiography and Fulton index. RHC showed a trend towards increased RVSP in the DNGR1-A20 KO mice in comparison to the wildtype mice. Furthermore, lungs of DNGR1-A20KO mice exhibited increased perivascular infiltration, muscle wall thickening and collagen deposition around the pulmonary vessels compared to wildtype mice. Our IPAH patient data show a significantly increased activation of cDC1s in peripheral blood samples and our DNGR1-A20 KO mouse model shows progression towards a PAH phenotype with significantly increased RVH and a trend of increased RVSP. The pulmonary vascular infiltrates show a possible role for (auto)immunity in the development of PH. In conclusion, investigating DC activation in the development or progression of PAH will be valuable to gain more insight in the mechanisms driving the pathophysiology of IPAH.

Alveolar macrophages express Receptor Activator of NF-κB (RANK), which is a known regulator of bone matrix turnover. Upon binding of its ligand RANKL, extracellular matrix (ECM) degradation can be induced. This interaction is inhibited by osteoprotegerin (OPG), the decoy receptor of RANKL and previously found to be elevated in pulmonary fibrosis (PF). We hypothesized that OPG in PF dampens ECM degradation by macrophages leading to fibrosis and proceeded to characterize and influence the system in lung tissue. We compared RANKL and OPG protein levels in lung tissue of PF patients (n=11) and controls (n=16), and control and PF mice (n=6). Exact sources were determined by immunohistochemistry, ELISA, and flow cytometry. Furthermore, mice with established PF were treated with soluble RANKL followed by total lung collagen analysis to assess PF. PF patients and mice had significantly higher OPG levels in lung tissue compared to their respective controls (p ECM modulator OPG is differentially expressed in healthy versus fibrotic conditions and induction of OPG following RANKL treatment suggests that the balance with RANKL is tightly regulated. These results support the importance of a RANKL-OPG balance in PF and may constitute a new target for treatment.

Background: Loss of tolerance to auto-antigens after vascular injury could trigger vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) patients. Endothelial cell-specific auto-antibodies produced by B cells could influence the early development of IPAH. Similarities with IPAH pathology have been observed in mice in which B-cell-receptor (BCR) signaling is continuously increased by Bruton9s tyrosine kinase (Btk) overexpression. Therefore, the aim of our study is to investigate if patients with IPAH have activated B cells and if Btk-Tg mice develop signs of PH after pulmonary injury. Methods: Using flow cytometry we determined Btk expression in peripheral B cells from healthy controls (HC) after stimulation with F(ab)2 anti-IgM and in different B-cell subsets in PB mononuclear cells inpatients with IPAH and HC. Finally, we assessed right ventricular weight (RVW) in Btk-Tg and wild type (WT) mice 56 days after bleomycine-induced lung injury. Results: IgM stimulation induced robust Btk protein upregulation in B-cells. IPAH patients (n=13) showed an increased Btk expression in B-cells compared with HC (P Conclusion: Our data suggest that IPAH pathogenesis in humans is driven by B-cell-dependent auto-immune responses. Interestingly, the combination of Btk overexpression in B cells and lung injury in mice could be a novel model to study IPAH and this may be a new tool to study future experimental therapies.