Probes for INS

Sarcoidosis is an idiopathic inflammatory disorder that is commonly treated with glucocorticoids. An imprecise understanding of the immunologic changes underlying sarcoidosis has limited therapeutic progress. Here in this open-label trial (NCT03910543), 10 patients with cutaneous sarcoidosis are treated with tofacitinib, a Janus kinase inhibitor. The primary outcome is the change in the cutaneous sarcoidosis activity and morphology instrument (CSAMI) activity score after 6 months of treatment. Secondary outcomes included change in internal organ involvement, molecular parameters, and safety. All patients experience improvement in their skin with 6 patients showing a complete response. Improvement in internal organ involvement is also observed. CD4+ T cell-derived IFN-γ is identified as a central cytokine mediator of macrophage activation in sarcoidosis. Additional type 1 cytokines produced by distinct cell types, including IL-6, IL-12, IL-15 and GM-CSF, also associate with pathogenesis. Suppression of the activity of these cytokines, especially IFN-γ, correlates with clinical improvement. Our results thus show that tofacitinib treatment is associated with improved sarcoidosis symptoms, and predominantly acts by inhibiting type 1 immunity.

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells.

Many cancer patients experience toxicity during checkpoint blockade immunotherapy, which often leads to treatment discontinuation. To this end, understanding the mechanisms mediating immune-related adverse events (irAE) should ultimately enable improvement in clinical outcomes. Recent work has revealed that tissue-resident memory T (TRM) cells are locally expanded in irAE-dermatitis and -colitis.

A major target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the epipharyngeal mucosa. Epipharyngeal abrasive therapy (EAT) is a Japanese treatment for chronic epipharyngitis. EAT is a treatment for chronic epipharyngitis in Japan that involves applying zinc chloride as an anti-inflammatory agent to the epipharyngeal mucosa. Here, we present a case of a 21-year-old man with chronic coughing that persisted for four months after a diagnosis of mild coronavirus disease 2019 (COVID-19), who was treated by EAT. We diagnosed chronic epipharyngitis as the cause of the chronic cough after the SARS-CoV-2 infection. SARS-CoV-2 spike RNA had persisted in the epipharyngeal mucosa of this Long COVID patient. EAT was performed once a week for three months, which eliminated residual SARS-CoV-2 RNA and reduced epipharyngeal inflammation. Moreover, a reduction in the expression of proinflammatory cytokines was found by histopathological examination. We speculate that the virus was excreted with the drainage induced by EAT, which stopped the secretion of proinflammatory cytokines. This case study suggests that EAT is a useful treatment for chronic epipharyngitis involving long COVID.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors, and one major challenge is T cell exhaustion. To address this challenge, we performed a candidate gene screen using a hypofunction CAR-T cell model and found that depletion of basic leucine zipper ATF-like transcription factor (BATF) improved the antitumor performance of CAR-T cells. In different types of CAR-T cells and mouse OT-1 cells, loss of BATF endows T cells with improved resistance to exhaustion and superior tumor eradication efficacy. Mechanistically, we found that BATF binds to and up-regulates a subset of exhaustion-related genes in human CAR-T cells. BATF regulates the expression of genes involved in development of effector and memory T cells, and knocking out BATF shifts the population toward a more central memory subset. We demonstrate that BATF is a key factor limiting CAR-T cell function and that its depletion enhances the antitumor activity of CAR-T cells against solid tumors.

Cutaneous leishmaniasis is a parasitic infection that causes significant maternal morbidity, and even fetal mortality, during pregnancy, yet there are limited therapeutic options. Here, we report a case of leishmaniasis in a pregnant immigrant with exuberant mucocutaneous lesions with favorable response to liposomal amphotericin B.

To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally ( Oryctolagus cuniculus ) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 hours post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 hours post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1β within the liver and spleen through 48 hours post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.