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

The adenosine 2a receptor and TIM3 directly inhibit killing of tumor cells by cytotoxic T lymphocytes through interference with cytoskeletal polarization

AbstractTumors 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 focus on CTL killing, blocking A2aR and TIM3 complements therapies that enhance T cell priming, e.g. anti-PD1 and anti-CTLA-4.</jats:p

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

AbstractTumors 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 A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.</jats:p

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

Tumor cell spheroid-induced suppression of primary human cytotoxic T cells as a scalable in vitro model of exhaustion

Background:Cytotoxic T lymphocytes (CTL) are key effectors in the antitumor immune response. However, their function is commonly suppressed in tumorsin the form of exhausted CTL. Understanding mechanisms of suppression and of therapeutics to overcome them is of substantial basic andtranslational importance yet hindered by limited access to large numbers of exhausted CTL in vitro.Methods: Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using functional assays, a21-antibody flow cytometry panel and determination of calcium signaling and CTL tumor cell couple maintenance, we have characterized theirphenotype.Results: We show that these cells closely resemble exhausted CTL from tumors. For a better understanding of in vitro human primary CTLas key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function onmethodology of generation, antigen dose, and affinity across two T–cell receptors and multiple tumor cell lines. As a further determination oftheir phenotype, we have investigated the morphology and subcellular F-actin distributions of CTL as key regulators of effector function. Primaryhuman CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, the gradual stabilization of such cell couples wasassociated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples.Conclusion: This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitatetheir use in basic research, the development of CTL-targeting therapeutics and the determination of their mechanism of action

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

TIM3 is a coregulatory receptor that is highly abundant on multiple immune cell types, including T cells in response to prolonged exposure to antigen, and it marks functionally suppressed cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. TIM3 exhibits inhibitory function in vivo but paradoxically has costimulatory T cell signaling capability in vitro. Here, we found that TIM3 directly inhibited the function of murine and human CTLs in direct interaction with target tumor cell spheroids. TIM3 regulated the ability of suppressed CTLs to polarize their actin cytoskeleton as a required step in cytolysis. Whereas the expression of the proposed TIM3 ligands CEACAM1 and galectin 9 in trans on target tumor cells enhanced TIM3 function, expression of CEACAM1 in cis on CTLs had the opposite effect. TIM3 functioned as an inhibitory receptor on spheroid-suppressed CTLs but not on active CTLs in a two-dimensional tissue culture model. Together, these data suggest that TIM3 enhances T cell function, serving as either a coinhibitory or costimulatory receptor depending on the functional context of the T cell on which it is expressed

MOESM1 of In situ characterization of stem cells-like biomarkers in meningiomas

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