TIM3 is a context-dependent coregulator of cytotoxic T cell function

Cytotoxic T lymphocytes (CTLs) are essential effectors in the antiviral and antitumour immune response and attractive targets in cancer immunotherapy. Although CTLs can directly recognise and kill tumour cells, CTLs become suppressed in the tumour microenvironment. This project investigated the inhibitory receptor T cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is expressed on T cells after chronic antigen exposure and marks the most exhausted tumour infiltrating CTLs in multiple solid tumours. However, it is unclear whether TIM3 directly regulates CTL function. In addition, despite its predominantly inhibitory role in vivo, TIM3 can promote cellular activation in T and non-T cells, and the roles of putative ligands in TIM3 function are disputed. Therefore, we aimed to determine the effect of TIM3 on direct CTL antitumour function and how the TIM3 ligands Galectin9 (GAL9) and CEACAM1 regulate its function. We employed three-dimensional (3D) tumour spheroids that effectively induce CTL suppression similar to the in vivo tumour microenvironment in comparison to conventional two-dimensional (2D) tumour cell culture. In the 3D spheroid model, TIM3 significantly inhibited CTL cytotoxicity and cytoskeletal polarisation as a key mechanism of effective cytolysis in murine and human CTLs. In contrast, in the 2D tumour model, TIM3 stimulated CTL cytotoxicity, cytoskeletal polarisation, and secretion of the immune-stimulatory cytokine interferon γ (IFNγ). Expression of GAL9 and CEACAM1 in trans on tumour cells further suppressed the CTL killing ability in the 3D spheroid model and enhanced costimulatory function in 2D. CEACAM1 in cis neutralised TIM3 functions in both 3D and 2D. We suggest that TIM3 functions as a context-dependent coregulatory receptor, as supported by the engagement of its ligands GAL9 and CEACAM1. In a largely stimulatory signalling context of a CTL, TIM3 functions as a costimulator, and in a more inhibitory context, TIM3 functions as a coinhibitor

Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8(+) cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A(2A)R and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4

Cellular Structures Controlling T Cell Signaling in Time and Space

T cell signaling is characterized by the diverse enrichment of receptors and signaling intermediates at particular subcellular regions of the T cell at specific times, resulting in complex spatiotemporal signaling distributions. These signaling distributions control the flow of information through the T cell signaling network and thus govern the efficiency of cellular activation. Here we discuss principal cellular structures driving the organization of T cell signaling including membrane topology, vesicular trafficking, cytoskeletal structures and protein complexes

In situ characterization of stem cells-like biomarkers in meningiomas

Abstract Background Meningioma cancer stem cells (MCSCs) contribute to tumor aggressiveness and drug resistance. Successful therapies developed for inoperable, recurrent, or metastatic tumors must target these cells and restrict their contribution to tumor progression. Unfortunately, the identity of MCSCs remains elusive, and MSCSs’ in situ spatial distribution, heterogeneity, and relationship with tumor grade, remain unclear. Methods Seven tumors classified as grade II or grade III, including one case of metastatic grade III, and eight grade I meningioma tumors, were analyzed for combinations of ten stem cell (SC)-related markers using immunofluorescence of consecutive sections. The correlation of expression for all markers were investigated. Three dimensional spatial distribution of markers were qualitatively analyzed using a grid, designed as a repository of information for positive staining. All statistical analyses were completed using Statistical Analysis Software Package. Results The patterns of expression for SC-related markers were determined in the context of two dimensional distribution and cellular features. All markers could be detected in all tumors, however, Frizzled 9 and GFAP had differential expression in grade II/III compared with grade I meningioma tissues. Correlation analysis showed significant relationships between the expression of GFAP and CD133 as well as SSEA4 and Vimentin. Data from three dimensional analysis showed a complex distribution of SC markers, with increased gene hetero-expression being associated with grade II/III tumors. Sub regions that showed multiple co-staining of markers including CD133, Frizzled 9, GFAP, Vimentin, and SSEA4, but not necessarily the proliferation marker Ki67, were highly associated with grade II/III meningiomas. Conclusion The distribution and level of expression of CSCs markers in meningiomas are variable and show hetero-expression patterns that have a complex spatial nature, particularly in grade II/III meningiomas. Thus, results strongly support the notion of heterogeneous populations of CSCs, even in grade I meningiomas, and call for the use of multiple markers for the accurate identification of individual CSC subgroups. Such identification will lead to practical clinical diagnostic protocols that can quantitate CSCs, predict tumor recurrence, assist in guiding treatment selection for inoperable tumors, and improve follow up of therapy

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Additional file 3: Table S2. Differentially expressed cancer driver genes [66] in individual tumors compared with three normal brain sample data sets, referenced in Gene Expression Omnibus (GEO) submission GSE77259. Values were generated from previously published data sets [64, 65] using Transcriptome Analysis Console v. 4.0

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Additional file 5: Figure S3. A. Representative immunofluorescence images for consecutive sections for the grade I Jed64_MN meningioma. Sections were double stained for Ki67 (red) with Nestin (green), SOX2 (red) with CD133 (green), Vimentin (green) with FZD9 (red), SSEA4 (green) with SOX2 (red), and SSEA4 (green) with Olig2 (red), and each section was stained with DAPI (blue). Single staining of GFAP (red) or BIIITubulin (red) is also shown. All images were taken at 20x. B. A grid used as a repository of information for categorical staining is shown with a color-coded legend and size dimensions for sub-areas

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Additional file 1: Table S1. The clinical profiles for the included patients and their tumors’ histopathological features

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Additional file 7: Table S3. All combinations of markers observed in consecutive sections and their frequencies in all 15 meningioma samples

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Additional file 2: Figure S1. H&E images for different morphological variants and atypical features for meningiomas used in this study. A. Images showing WHO classified morphological features for different meningioma variants. Meningothelial (Jed39_MN) with neoplastic growth of syncytial epithelial cells with indistinct cell borders arranged in whorls; fibroblastic (Jed40_MN) showing spindle cells with indistinct cell boundaries running in fascicle; transitional (Jed38_MN) with ratios of meningothelial to fibroblastic patterns 40:60; psammomatous (Jed43_MN) composed of whorled clusters of spindle cells with numerous psammoma bodies; chordoid (Jed79_MN), Cords of epithelioid cells with focal clear to foamy cytoplasm on myxoid stroma.; rhabdoid (Jed29_MN) showing hypercellular sheets with rhabdoid morphology. B. Tumors with atypical features. Images show patternless growth (sheeting) in Jed72_MN, necrosis and small cells with high nuclear to cytoplasm ratio in Jed58_MN, and brain invasion in Jed13_MN. Magnifications are indicated above images