Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy.

T memory stem cells (TSCM) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated ß-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.

Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade.

OBJECTIVE: Investigating the effect of ferroptosis in the tumour microenvironment to identify combinatory therapy for liver cancer treatment. DESIGN: Glutathione peroxidase 4 (GPx4), which is considered the master regulator of ferroptosis, was genetically altered in murine models for hepatocellular carcinoma (HCC) and colorectal cancer (CRC) to analyse the effect of ferroptosis on tumour cells and the immune tumour microenvironment. The findings served as foundation for the identification of additional targets for combine therapy with ferroptotic inducer in the treatment of HCC and liver metastasis. RESULTS: Surprisingly, hepatocyte-restricted GPx4 loss does not suppress hepatocellular tumourigenesis. Instead, GPx4-associated ferroptotic hepatocyte death causes a tumour suppressive immune response characterised by a CXCL10-dependent infiltration of cytotoxic CD8+ T cells that is counterbalanced by PD-L1 upregulation on tumour cells as well as by a marked HMGB1-mediated myeloid derived suppressor cell (MDSC) infiltration. Blocking PD-1 or HMGB1 unleashes T cell activation and prolongs survival of mice with Gpx4-deficient liver tumours. A triple combination of the ferroptosis inducing natural compound withaferin A, the CXCR2 inhibitor SB225002 and α-PD-1 greatly improves survival of wild-type mice with liver tumours. In contrast, the same combination does not affect tumour growth of subcutaneously grown CRC organoids, while it decreases their metastatic growth in liver. CONCLUSION: Our data highlight a context-specific ferroptosis-induced immune response that could be therapeutically exploited for the treatment of primary liver tumours and liver metastases.

Ubiquitin carboxyl-terminal esterase L1 promotes proliferation of human choroidal and retinal endothelial cells.

PURPOSE: We aimed: (1) to establish endothelial expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in human choroid and retina and; (2) to investigate a role for UCHL1 in basic processes involved in intraocular neovascularization. DESIGN: Controlled translational experimental study. METHODS: Ethanol-fixed human choroid and retina (n = 3 eyes) were indirectly immunostained with rabbit anti-human UCHL1 antibody. Endothelial proliferation and migration assays were performed using cultured human choroidal and retinal endothelial cells (n = 6 isolates/assay). Cells were transfected with UCHL1-targeted or non-targeted small interfering (si)RNA and a commercially available transfection system, and used 48 hours later in experiments. Cell proliferation was evaluated using an assay in which cellular DNA was fluorescently tagged for quantification by microplate reader. Cell migration was examined in an assay that involved counting the number of endothelial cells moving across a perforated membrane. Transcript silencing was verified by Western blot for all assays. RESULTS: Immunohistochemistry confirmed expression of UCHL1 by endothelium in human choroid and retina in vivo. UCHL1-specific knockdown resulted in significantly less proliferation (p < 0.0001) for 3 human choroidal endothelial isolates and 3 human retinal endothelial isolates, and significantly less migration (p ≤ 0.016) for 2 of 3 human choroidal endothelial isolates and 1 of 3 human retinal endothelial isolates. CONCLUSIONS: Our results suggest that UCHL1 may be involved in choroidal and retinal endothelial proliferation in most persons, and endothelial migration in some persons. UCHL1 may be a suitable target for a new treatment of intraocular neovascularisation.

Toxoplasma gondii migration within and infection of human retina.

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5 × 10(7) parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110-250 live tachyzoites; 64.5-95.2% (mean  =79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p ≤ 0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.

Imaging Retinal Vascular Changes in the Mouse Model of Oxygen-Induced Retinopathy.

PURPOSE: Oxygen-induced retinopathy in the mouse is the standard experimental model of retinopathy of prematurity. Assessment of the pathology involves in vitro analysis of retinal vaso-obliteration and retinal neovascularization. The authors studied the clinical features of oxygen-induced retinopathy in vivo using topical endoscopy fundus imaging (TEFI), in comparison to standard investigations, and evaluated a system for grading these features. METHODS: Postnatal day (P)7 mice were exposed to 75% oxygen for five days to induce retinopathy or maintained in room air as controls. Retinal vascular competence was graded against standard photographs by three masked graders. Retinal photographs were obtained at predetermined ages using TEFI. Postmortem, retinal vaso-obliteration was measured in whole mounts with labeled vasculature, and retinal neovascularization was quantified in hematoxylin- and eosin-stained ocular cross sections. RESULTS: Fundus photography by TEFI was possible from P15, when retinal vascular incompetence, including dilatation and tortuosity, was significant in mice with oxygen-induced retinopathy in comparison to controls. Vascular incompetence peaked in severity at P17 and persisted through P25. Comparison with in vitro analyses indicated that vascular changes were most severe after retinal avascularity had begun to decrease in area, and coincident with the maximum of retinal neovascularization. A weighted Fleiss-Cohen kappa indicated good intra- and interobserver agreement for a 5-point grading system. CONCLUSIONS: Topical endoscopy fundus imaging demonstrates retinal vascular incompetence in mice with oxygen-induced retinopathy. The technique complements standard postmortem analysis for following the course of the model. TRANSLATIONAL RELEVANCE: Topical endoscopy fundus imaging has application in the evaluation of novel biologic drugs for retinopathy of prematurity.