Probes for CD4

Abstract BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

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.