Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis.

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial.

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5-20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment-response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients.

A phase I trial of cyclosporine for hospitalized patients with COVID-19.

BACKGROUNDCOVID-19 remains a global health emergency with limited treatment options, lagging vaccine rates, and inadequate healthcare resources in the face of an ongoing calamity. The disease is characterized by immune dysregulation and cytokine storm. Cyclosporine A (CSA) is a calcineurin inhibitor that modulates cytokine production and may have direct antiviral properties against coronaviruses.METHODSTo test whether a short course of CSA was safe in patients with COVID-19, we treated 10 hospitalized, oxygen-requiring, noncritically ill patients with CSA (starting at a dose of 9 mg/kg/d). We evaluated patients for clinical response and adverse events, measured serum cytokines and chemokines associated with COVID-19 hyperinflammation, and conducted gene-expression analyses.RESULTSFive participants experienced adverse events, none of which were serious; transaminitis was most common. No participant required intensive care unit-level care, and all patients were discharged alive. CSA treatment was associated with significant reductions in serum cytokines and chemokines important in COVID-19 hyperinflammation, including CXCL10. Following CSA administration, we also observed a significant reduction in type I IFN gene expression signatures and other transcriptional profiles associated with exacerbated hyperinflammation in the peripheral blood cells of these patients.CONCLUSIONShort courses of CSA appear safe and feasible in patients with COVID-19 who require oxygen and may be a useful adjunct in resource-limited health care settings.TRIAL REGISTRATIONThis trial was registered on ClinicalTrials.gov (Investigational New Drug Application no. 149997; ClinicalTrials.gov NCT04412785).FUNDINGThis study was internally funded by the Center for Cellular Immunotherapies.

The tumor immune microenvironment of primary and metastatic HER2- positive breast cancers utilizing gene expression and spatial proteomic profiling.

BACKGROUND: The characterization of the immune component of the tumor microenvironment (TME) of human epidermal growth factor receptor 2 positive (HER2+) breast cancer has been limited. Molecular and spatial characterization of HER2+ TME of primary, recurrent, and metastatic breast tumors has the potential to identify immune mediated mechanisms and biomarker targets that could be used to guide selection of therapies. METHODS: We examined 15 specimens from eight patients with HER2+ breast cancer: 10 primary breast tumors (PBT), two soft tissue, one lung, and two brain metastases (BM). Using molecular profiling by bulk gene expression TME signatures, including the Tumor Inflammation Signature (TIS) and PAM50 subtyping, as well as spatial characterization of immune hot, warm, and cold regions in the stroma and tumor epithelium using 64 protein targets on the GeoMx Digital Spatial Profiler. RESULTS: PBT had higher infiltration of immune cells relative to metastatic sites and higher protein and gene expression of immune activation markers when compared to metastatic sites. TIS scores were lower in metastases, particularly in BM. BM also had less immune infiltration overall, but in the stromal compartment with the highest density of immune infiltration had similar levels of T cells that were less activated than PBT stromal regions suggesting immune exclusion in the tumor epithelium. CONCLUSIONS: Our findings show stromal and tumor localized immune cells in the TME are more active in primary versus metastatic disease. This suggests patients with early HER2+ breast cancer could have more benefit from immune-targeting therapies than patients with advanced disease.