Immunotherapy using chimeric antigen receptor (CAR)‐engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models ...are needed that allow to test CAR‐mediated cytotoxicity in a tissue‐like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3‐dimensional (3D) patient‐derived colon organoids. Luciferase‐based measurement served as a quantitative read‐out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR‐engineered NK‐92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen‐specific cytotoxicity was studied with CAR‐NK‐92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor‐specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.
Synopsis
Establishment of a preclinical in vitro organoid model allows testing the efficacy and safety of solid tumor cancer immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a tissue‐like environment. 3D live‐imaging of single colorectal cancer (CRC) organoids is highly sensitive and allows to validate treatment assumptions to minimize therapy side effects.
Co‐culture of human natural killer (NK) cells with normal or CRC organoids on an ECM layer allows stable effector‐target cell interaction.
Luciferase‐based cytotoxicity assessment is benchmarked with EPCAM‐directed effector NK cells.
Low expression of EGFRvIII neoantigen is sufficient to render CRC organoids susceptible to lysis.
CAR targeting of FRIZZLED receptors in intestinal organoids reveals off‐target cytotoxicity against wild‐type epithelia of human origin.
A preclinical organoid co‐culture allows testing of immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a personalised tissue‐like environment.
Mast cells (MCs) are crucial players in the relationship between the tumor microenvironment (TME) and cancer cells and have been shown to influence angiogenesis and progression of human colorectal ...cancer (CRC). However, the role of MCs in the TME is controversially discussed as either pro- or anti-tumorigenic. Genetically engineered mouse models (GEMMs) are the most frequently used in vivo models for human CRC research. In the murine intestine there are at least three different MC subtypes: interepithelial mucosal mast cells (ieMMCs), lamina proprial mucosal mast cells (lpMMCs) and connective tissue mast cells (CTMCs). Interepithelial mucosal mast cells (ieMMCs) in (pre-)neoplastic intestinal formalin-fixed paraffin-embedded (FFPE) specimens of mouse models (total lesions n = 274) and human patients (n = 104) were immunohistochemically identified and semiquantitatively scored. Scores were analyzed along the adenoma-carcinoma sequence in humans and 12 GEMMs of small and large intestinal cancer. The presence of ieMMCs was a common finding in intestinal adenomas and carcinomas in mice and humans. The number of ieMMCs decreased in the course of colonic adenoma-carcinoma sequence in both species (p < 0.001). However, this dynamic cellular state was not observed for small intestinal murine tumors. Furthermore, ieMMC scores were higher in GEMMs with altered Wnt signaling (active β-catenin) than in GEMMs with altered MAPK signaling and wildtypes (WT). In conclusion, we hypothesize that, besides stromal MCs (lpMMCs/CTMCs), particularly the ieMMC subset is important for onset and progression of intestinal neoplasia and may interact with the adjacent neoplastic epithelial cells in dependence on the molecular environment. Moreover, our study indicates the need for adequate GEMMs for the investigation of the intestinal immunologic TME.
ABSTRACT
Regulatory T cells (Tregs) are essential for the inhibition of immunity and the maintenance of tissue homeostasis. Signals from the T‐cell antigen receptor (TCR) are critical for early Treg ...development, their expansion, and inhibitory activity. Although TCR‐engaged activation of the paracaspase MALT1 is important for these Treg activities, the MALT1 effector pathways in Tregs remain ill‐defined. Here, we demonstrate that MALT1 protease activity controls the TCR‐induced upregulation of the transcription factor MYC and the subsequent expression of MYC target genes in Tregs. These mechanisms are important for Treg‐intrinsic mitochondrial function, optimal respiratory capacity, and homeostatic Treg proliferation. Consistently, conditional deletion of Myc in Tregs results similar to MALT1 inactivation in a lethal autoimmune inflammatory syndrome. Together, these results identify a MALT1 protease‐mediated link between TCR signaling in Tregs and MYC control that coordinates metabolism and Treg expansion for the maintenance of immune homeostasis.
In murine CD4+Foxp3+ regulatory T cells (Tregs), T‐cell receptor (TCR)/CD28‐induced MALT1 protease activity promotes MYC and MYC‐controlled gene expression in a c‐Rel‐dependent manner. This MALT1‐MYC axis drives mitochondrial metabolism and the respiratory capacity of Tregs, which is critical for Treg proliferation and the maintenance of immune homeostasis.
Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological ...interventions, we describe an unexpected dual role of the tumor necrosis factor receptor–associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.
LRH-1 (liver receptor homolog-1/NR5a2) is an orphan nuclear receptor, which regulates glucose and lipid metabolism, as well as intestinal inflammation via the transcriptional control of intestinal ...glucocorticoid synthesis. Predominantly expressed in epithelial cells, its expression and role in immune cells are presently enigmatic. LRH-1 was found to be induced in immature and mature T lymphocytes upon stimulation. T cell-specific deletion of LRH-1 causes a drastic loss of mature peripheral T cells. LRH-1-depleted CD4
T cells exert strongly reduced activation-induced proliferation in vitro and in vivo and fail to mount immune responses against model antigens and to induce experimental intestinal inflammation. Similarly, LRH-1-deficient cytotoxic CD8
T cells fail to control viral infections. This study describes a novel and critical role of LRH-1 in T cell maturation, functions, and immopathologies and proposes LRH-1 as an emerging pharmacological target in the treatment of T cell-mediated inflammatory diseases.
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