IFN-Gamma Producing Regulatory T Cells Counterbalance T Cell-Mediated Injury to the Intestinal Stem Cell Compartment in Mice and Humans

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Such inflammatory diseases are fostered by damage to the intestinal barrier after transplantation. Consequently, the integrity and regeneration of the intestinal barrier is a key factor in the prevention of GVHD. On one side the main driver for regeneration of damaged gut epithelium are intestinal stem cells (ISC), but on the other side these cells are themselves primary targets of donor-derived T cells. One known mechanism of T cell mediated damage to the stem cell compartment is through IFN-γ dependent ISC toxicity. Yet, little is known about how T cells are contributing to the regeneration of damaged tissue after allo-HSCT and GVHD. Methods: To address this, we used preclinical models for allo-HSCT and GVHD including transplantation of recipient mice with escalating doses of Wildtype or IFN-γ-deficient allogeneic T cells and in the presence or absence of the JAK-1/2 inhibitor ruxolitinib. Intestinal regeneration was assessed by RNA-seq, flow cytometry and a newly established ex vivo organoid recovery assay. GVHD outcome was assessed by clinical scoring, histology and survival. Additionally, we established an allogeneic co-culture system of murine or human intestinal organoids with CD4+ conventional T cells or T regs -/+ Ruxolitinib. Effects on organoid growth and cell death were assessed by size measurements and manual counting after passaging. Results: We here demonstrate that recipient mice with increasingly dense intestinal infiltration by allogeneic T cells not only developed more severe GVHD (Fig. 1A), but also showed augmented recovery potential early after allo-HSCT (Fig. 1B). This was associated with intestinal gene signatures related to epithelial regeneration and protection from GVHD. Utilizing ex vivo cultures of intestinal organoids generated from murine allo-HSCT recipients, we found that development of GVHD but also regenerative capacity of ISCs were dependent on interferon (IFN)-γ-producing T cells in the intestine (Fig. 2A-B). Mice with fulminant GVHD and enhanced organoid recovery showed accumulation of intestinal regulatory T cells (Tregs) (Fig. 2C). Ex vivo, T regs nurtured growth of intestinal organoids in an IFN-γ dependent manner (Fig. 2D-E). This effect was diminished in intestinal organoids lacking IFNγR signaling, but was independent of T reg intrinsic IFNγR signaling (Fig. 2E-F). Intriguingly, treatment of murine allo-HSCT recipients with the JAK-1/2 inhibitor ruxolitinib enhanced epithelial organoid regeneration and numbers of intestinal Tregs (Fig. 3A-B). Similarily, growth of human intestinal organoids co-cultured with allogeneic T cells could be augmented by ruxolitinib treatment (Fig. 3C). We thus propose that the level and differentiation of infiltrating intestinal T cells determines both ISC damage and epithelial regeneration during immune-mediated tissue injury, leading to a sensitive equilibrium that can be modulated by therapeutic intervention. We also provide evidence that ruxolitinib improves ISC regeneration via IFNγ-producing Treg cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Naked mole-rat hematopoietic stem and progenitors are highly quiescent with an inherent myeloid bias

Naked mole-rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which is a neotenic trait. A myeloid bias towards granulopoiesis in concert with decreased B-lymphopoiesis defines the marrow composition, resembling fetal leukopoiesis. Very similar to primates, the primitive stem cell compartment is marked by CD34 and THY1. Remarkably, stem and progenitor respiration rates are as low as in human cells, while NMR cells show a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. Our work provides a platform to study immunology and stem cell biology in an animal model of exceptional longevity.

Novel Pituitary Organoid Model as Powerful Tool to Unravel Pituitary Stem Cell Biology Across Ages and Disease

The pituitary gland harbors a population of stem cells. However, role and regulation of these cells remain poorly understood. We recently established organoids from mouse pituitary as a novel research tool to explore pituitary stem cell biology (Cox et al., J. Endocrinol. 2019; 240:287-308). In general, organoids represent 3D in vitro cell configurations that develop and self-organize from (single) tissue stem cells under well-defined culture conditions that typically mirror the stem cell niche and/or embryogenic processes. Organoids reliably recapitulate key aspects of the original organ, including of its stem cell compartment. Moreover, organoids are long-term expandable while retaining these properties. We demonstrated that pituitary organoids originate from the resident (SOX2+) stem cells, largely phenocopy these cells and retain the stemness phenotype during expansive culture. Interestingly, the organoids show confident in vivo translatability and, when developed from transgenically damaged gland, recapitulate the activation status of the stem cells as observed in situ following injury. Now, we found that the organoids also mirror the stem cells’ phenotype and biology in physiological conditions in which the stem cell compartment is either activated or compromised. Organoids from the neonatal maturing pituitary reproduce phenotypical and functional aspects of its activated stem cells, whereas organoids from aging gland mimic the declined functional state of the stem cells in old pituitary. Interestingly, this functional decay was found to be reverted during organoid culture, indicating that the old pituitary stem cells retain intrinsic functionality but are in vivo restrained by an obstructive microenvironment, not present in the organoid culture. Indeed, using single-cell transcriptomics and in vivo analysis, we found that the aging pituitary suffers from a prevailing inflammatory state (inflammaging) which appears to raise the threshold for stem cell activation. Interestingly, comparison of young and old pituitary led us to the discovery of pituitary stem cell activators. Finally, we found that activated parameters of organoid formation are also observed when tumorigenesis takes place in the gland, again mimicking the in situ stem cell activation that is occurring in this perturbed, pathological condition. Taken together, we identified, and applied, our new pituitary organoid model as advanced and powerful tool to gain profound insight into pituitary stem cell behavior across life and disease, which is expected to eventually translate into restorative and rejuvenative tactics when pituitary function is compromised by damage or age. In this context, our single-cell transcriptome database has strong potential to unveil appealing targets.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression

The overactivation of Wnt/b-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of b-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-kB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.