Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer

BackgroundPatient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinical oncology research method. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scid IL2Rgammanull (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. MethodsThe immunophenotype of lymphoproliferations arising in the lung TRACERx PDX pipeline were characterised. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).ResultsLymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma with plasma cell features. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. DiscussionOverall, these data suggest that B cell clones with lymphoproliferative potential are present within primary NSCLC tumours, and that these are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines

The effect of ageing on the acute inflammatory response

The elderly present with a state of low-level chronic inflammation termed inflamm-ageing. The aetiology of this phenotype is unknown. Most efforts have focused on the mechanisms by which inflammation starts, whereas little is known regarding the processes that switch off inflammation – resolution – in this context. I therefore investigated the inflammatory response in healthy young and aged individuals using the cantharidin skin blister model of acute, sterile inflammation. Inflammatory onset was unchanged, but neutrophil clearance – a key step in resolution – was impaired in the aged. I observed no defects in neutrophil apoptosis. However, the clearance defect coincided with increased prevalence of HLA-DR+/CD14hi/CD16lo/CD163+ mononuclear phagocytes (MP) in aged compared to young volunteers. Ex vivo incubation of blister MPs with autologous apoptotic neutrophils revealed significantly impaired efferocytosis in aged compared to young individuals. Comparison of key cell surface proteins that mediate efferocytosis revealed that while MerTK, CD14 and CD36 expression was unchanged with age, TIM-4, a key phosphatidylserine receptor, exhibited significantly lower expression on MPs from aged individuals. I also observed a failure of MPs to undergo pro-resolution signaling resulting in a failure to upregulate CCR7, produce TGF-β and release pro-resolving cytochrome P450-derived lipid mediators by resolution in the aged; all known consequences of successful apoptotic cell clearance by MPs. Contrary to a plethora of published literature, inflammatory onset in the skin of aged humans was unimpaired. However, resolution pathways revolving around efferocytosis are severely inhibited resulting in chronic inflammation. This could underpin the aetiology of inflamm-ageing and provide a pharmacologically tractable target to improve immune responses in the elderly

CYP450-derived oxylipins mediate inflammatory resolution

Resolution of inflammation has emerged as an active process in immunobiology, with cells of the mononuclear phagocyte system being critical in mediating efferocytosis and wound debridement and bridging the gap between innate and adaptive immunity. Here we investigated the roles of cytochrome P450 (CYP)-derived epoxy-oxylipins in a well-characterized model of sterile resolving peritonitis in the mouse. Epoxy-oxylipins were produced in a biphasic manner during the peaks of acute (4 h) and resolution phases (24–48 h) of the response. The epoxygenase inhibitor SKF525A (epoxI) given at 24 h selectively inhibited arachidonic acid- and linoleic acid-derived CYP450-epoxy-oxlipins and resulted in a dramatic influx in monocytes. The epoxI-recruited monocytes were strongly GR1+, Ly6chi, CCR2hi, CCL2hi, and CX3CR1lo. In addition, expression of F4/80 and the recruitment of T cells, B cells, and dendritic cells were suppressed. sEH (Ephx2)−/− mice, which have elevated epoxy-oxylipins, demonstrated opposing effects to epoxI-treated mice: reduced Ly6chi monocytes and elevated F4/80hi macrophages and B, T, and dendritic cells. Ly6chi and Ly6clo monocytes, resident macrophages, and recruited dendritic cells all showed a dramatic change in their resolution signature following in vivo epoxI treatment. Markers of macrophage differentiation CD11b, MerTK, and CD103 were reduced, and monocyte-derived macrophages and resident macrophages ex vivo showed greatly impaired phagocytosis of zymosan and efferocytosis of apoptotic thymocytes following epoxI treatment. These findings demonstrate that epoxy-oxylipins have a critical role in monocyte lineage recruitment and activity to promote inflammatory resolution and represent a previously unidentified internal regulatory system governing the establishment of adaptive immunity

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injur

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE 2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4 +/CD44 +/CD62L + and CD4 +/CD44 +/CD62L -/CD27 + T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE 2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation.</p