Tumor lineage-specific immune response in brain metastatic disease: opportunities for targeted immunotherapy regimen?

Metastases in the brain are the most severe and devastating complication of cancer. The incidence of brain metastasis is increasing. Therefore, the need of finding specific druggable targets for brain metastasis is demanding. The aim of this study was to compare the brain (immune) response to brain metastases of the most common tumor lineages, viz., lung adenocarcinoma and breast cancer. Targeted gene expression profiles of 11 brain metastasis of lung adenocarcinoma (BM-LUAD) were compared to 11 brain metastasis of breast cancer (BCBM) using NanoString nCounter PanCancer IO 360™ Panel. The most promising results were validated spatially using the novel GeoMx™ Digital Spatial Profiler (DSP) Technology. Additionally, Immune cell profiles and expression of drug targets were validated by multiplex immunohistochemistry. We found a more active immune response in BM-LUAD as compared to BCBM. In the BM-LUAD, 138 genes were upregulated as compared to BCBM (adj. p ≤ 0.05). Conversely, in BCBM 28 genes were upregulated (adj. p ≤ 0.05). Additionally, genes related to CD45 + cells, T cells, and cytotoxic T cells showed to be expressed higher in BM-LUAD compared to BCBM (adj. p = 0.01, adj. p = 0.023, adj. p = 0.023, respectively). The spatial quantification of the immune cells using the GeoMx DSP technique revealed the significantly higher quantification of CD14 and CD163 in tumor regions of BM-LUAD as compared to BCBM. Importantly, the immune checkpoint VISTA and IDO1 were identified as highly expressed in the BM-LUAD. Multiplex immunohistochemistry confirmed the finding and showed that VISTA is expressed mainly in BM-LUAD tumor cells, CD3 + cells, and to fewer levels in some microglial cells in BM-LUAD. This is the first report on differences in the brain immune response between metastatic tumors of different lineages. We found a far more extensive infiltration of immune cells in BM-LUAD as compared to BCBM. In addition, we found higher expression of VISTA and IDO1 in BM-LUAD. Taken together, targeted immune therapy should be considered to treat patients with BM-LUAD

Liver ischemia and reperfusion induce periportal expression of necroptosis executor pMLKL wich is associated with early allograft dysfunction after transplantation

Background: Early allograft dysfunction (EAD) following liver transplantation (LT) remains a major threat to the survival of liver grafts and recipients. In animal models, it is shown that hepatic ischemia-reperfusion injury (IRI) triggers phosphorylation of Mixed Lineage Kinase domain-like protein (pMLKL) inducing necroptotic cell death. However, the clinical implication of pMLKL-mediated cell death in human hepatic IRI remains largely unexplored. In this study, we aimed to investigate the expression of pMLKL in human liver grafts and its association with EAD after LT. Methods: The expression of pMLKL was determined by immunohistochemistry in liver biopsies obtained from both human and rat LT. Human liver biopsies were obtained at the end of preservation (T0) and ~1 hour after reperfusion (T1). The positivity of pMLKL was quantified electronically and compared in rat and human livers and post-LT outcomes. Multiplex immunofluorescence staining was performed to characterize the pMLKL-expressing cells. Results: In the rat LT model, significant pMLKL expression was observed in livers after IRI as compared to livers of sham-operation animals. Similarly, the pMLKL score was highest after IRI in human liver grafts (in T1 biopsies). Both in rats and humans, the pMLKL expression is mostly observed in the portal triads. In grafts who developed EAD after LT (n=24), the pMLKL score at T1 was significantly higher as compared to non-EAD grafts (n=40). ROC curve revealed a high predictive value of pMLKL score at T1 (AUC 0.70) and the ratio of pMLKL score at T1 and T0 (pMLKL-index, AUC 0.82) for EAD. Liver grafts with a high pMLKL index (>1.64) had significantly higher levels of serum ALT, AST, and LDH 24 hours after LT compared to grafts with a low pMLKL index. Multivariate logistical regression analysis identified the pMLKL-index (Odds ratio=1.3, 95% CI 1.1-1.7) as a predictor of EAD development. Immunohistochemistry on serial sections and multiplex staining identified the periportal pMLKL-positive cells as portal fibroblasts, fibrocytes, and a minority of cholangiocytes. Conclusion: Periportal pMLKL expression increased significantly after IRI in both rat and human LT. The histological score of pMLKL is predictive of post-transplant EAD and is associated with early liver injury after LT. Periportal non-parenchymal cells (i.e. fibroblasts) appear most susceptible to pMLKL-mediated cell death during hepatic IRI

Evidence for a Role of CCR6+ T Cells in Chronic Thromboembolic Pulmonary Hypertension

Introduction: Previous studies have shown an increase of T cells and chemokines in vascular lesions of patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, detailed characterization of these T cells is still lacking, nor have treatment effects been evaluated. Methods: We included 41 treatment-naive CTEPH patients at diagnosis, 22 patients at 1-year follow-up, and 17 healthy controls (HCs). Peripheral blood T cells were characterized by flow cytometry for subset distribution, cytokine expression and activation marker profile. We used multiplex immunofluorescence to identify CCR6+ T cells in endarterectomy tissue from 25 patients. Results: At diagnosis, proportions of CCR6+ CD4+ T cells were increased in CTEPH patients compared with HCs. Patients displayed a significantly reduced production capacity of several cytokines including TNFα, IFNγ, GM-CSF and IL-4 in CD4+ T cells, and TNFα and IFNγ in CD8+ T cells. CD4+ and CD8+ T cells showed increased expression of the immune checkpoint protein CTLA4. Multivariate analysis separated CTEPH patients from HCs, based on CCR6 and CTLA4 expression. At 1-year follow-up, proportions of CCR6+CD4+ T cells were further increased, IFNγ and IL-17 production capacity of CD4+ T cells was restored. In nearly all vascular lesions we found substantial numbers of CCR6+ T cells. Conclusion: The observed increase of CCR6+ T cells and modulation of the IFNγ and IL-17 production capacity of circulating CD4+ T cells at diagnosis and 1-year follow-up – together with the presence of CCR6+ T cells in vascular lesions - support the involvement of the Th17-associated CCR6+ T cell subset in CTEPH