p21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer.

BACKGROUND & AIMS: T cells are crucial for the antitumor response against colorectal cancer (CRC). T-cell reactivity to CRC is nevertheless limited by T-cell exhaustion. However, molecular mechanisms regulating T-cell exhaustion are only poorly understood. METHODS: We investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in cluster of differentiation (CD) 4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I to IV CRC. In vitro coculture models were used to understand the effector function of p21-deficient CD4+ T cells. RESULTS: We observed that the activation of cell cycle regulator p21 is crucial for CD4+ T-cell cytotoxic function and that p21 deficiency in type 1 helper T cells (Th1) leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1-/- mice using ex vivo-treated p21-deficient T cells with palbociclib, an inhibitor of cyclin-dependent kinase 4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. CONCLUSIONS: Our data reveal the importance of p21 in controlling the cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of cyclin-dependent kinase inhibitors such as palbociclib to reduce T-cell exhaustion for future treatment of patients with colorectal cancer.

STAT3 activation through IL-6/IL-11 in cancer-associated fibroblasts promotes colorectal tumour development and correlates with poor prognosis.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood. DESIGN: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11). RESULTS: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts. CONCLUSION: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.

IFN-γ drives inflammatory bowel disease pathogenesis through VE-cadherin-directed vascular barrier disruption.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with rising incidence. Diseased tissues are heavily vascularized. Surprisingly, the pathogenic impact of the vasculature in IBD and the underlying regulatory mechanisms remain largely unknown. IFN-γ is a major cytokine in IBD pathogenesis, but in the context of the disease, it is almost exclusively its immune-modulatory and epithelial cell-directed functions that have been considered. Recent studies by our group demonstrated that IFN-γ also exerts potent effects on blood vessels. Based on these considerations, we analyzed the vessel-directed pathogenic functions of IFN-γ and found that it drives IBD pathogenesis through vascular barrier disruption. Specifically, we show that inhibition of the IFN-γ response in vessels by endothelial-specific knockout of IFN-γ receptor 2 ameliorates experimentally induced colitis in mice. IFN-γ acts pathogenic by causing a breakdown of the vascular barrier through disruption of the adherens junction protein VE-cadherin. Notably, intestinal vascular barrier dysfunction was also confirmed in human IBD patients, supporting the clinical relevance of our findings. Treatment with imatinib restored VE-cadherin/adherens junctions, inhibited vascular permeability, and significantly reduced colonic inflammation in experimental colitis. Our findings inaugurate the pathogenic impact of IFN-γ-mediated intestinal vessel activation in IBD and open new avenues for vascular-directed treatment of this disease.

Contrast-Enhanced µCT for Visualizing and Evaluating Murine Intestinal Inflammation.

Rationale: To develop a simple and fast protocol for the assessment of acute and chronic experimental intestinal inflammation using contrast-enhanced µCT. Methods: For the imaging studies, an acute 2% and 3% dextran sodium sulfate (n = 15, female, 8-12 weeks) and a chronic adoptive transfer colitis model (n = 10, female, 8-9 weeks) were established over 9 days or 6 weeks, respectively. Throughout the experiments, longitudinal measurement of murine intestinal wall thickness and time dependent perfusion was performed on a small animal µCT system (90 kV, 160 µA, FOV: 60 mm, scan time: 17 s, image size: 512x512, layer thickness: 118 µm) between 0.5 and 30 min after intravenous bolus injection of an iodine contrast agent. Weight development, small animal endoscopy, and histological ex vivo analysis were compared to contrast-enhanced µCT imaging findings. Results: Murine intestinal wall thickness was significantly increased in inflamed colons of acute colitis at day 9 in comparison to pre-inflamed state. Perfusion analysis revealed a late contrast enhancement in acute inflamed colons and the renal medulla at day 9 compared to control mice. An increasing intestinal wall thickness was monitored 3, 5 and 6 weeks after on-set of chronic colitis in comparison to controls. A good correlation with endoscopic (r = 0.75, p < 0.0001) and histologic degree of inflammation (r = 0.83, p = 0.04) was found. Conclusion: Contrast-enhanced µCT is a simple and fast method to assess acute intestinal inflammation and to monitor disease progression in experimental models of chronic colitis. According to our findings, one single contrast-enhanced µCT-scan is a valid non-invasive modality to quantify the degree of inflammation in the entire digestive tract in murine inflammatory models.

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold.