Targeting pediatric cancers via T-cell recognition of the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers.

Corticosteroids impair epithelial regeneration in immune-mediated intestinal damage.

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.

Determinants of epidemic size and the impacts of lulls in seasonal influenza virus circulation.

During the COVID-19 pandemic, levels of seasonal influenza virus circulation were unprecedentedly low, leading to concerns that a lack of exposure to influenza viruses, combined with waning antibody titres, could result in larger and/or more severe post-pandemic seasonal influenza epidemics. However, in most countries the first post-pandemic influenza season was not unusually large and/or severe. Here, based on an analysis of historical influenza virus epidemic patterns from 2002 to 2019, we show that historic lulls in influenza virus circulation had relatively minor impacts on subsequent epidemic size and that epidemic size was more substantially impacted by season-specific effects unrelated to the magnitude of circulation in prior seasons. From measurements of antibody levels from serum samples collected each year from 2017 to 2021, we show that the rate of waning of antibody titres against influenza virus during the pandemic was smaller than assumed in predictive models. Taken together, these results partially explain why the re-emergence of seasonal influenza virus epidemics was less dramatic than anticipated and suggest that influenza virus epidemic dynamics are not currently amenable to multi-season prediction.

Chemotherapy-induced intestinal injury promotes Galectin-9-driven modulation of T cell function.

The intestine is vulnerable to chemotherapy-induced toxicity due to its high epithelial proliferative rate, making gut toxicity an off-target effect in several cancer treatments, including conditioning regimens for allogeneic hematopoietic cell transplantation (allo-HCT). In allo-HCT, intestinal damage is an important factor in the development of Graft-versus-Host Disease (GVHD), an immune complication in which donor immune cells attack the recipient's tissues. Here, we developed a novel human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced intestinal epithelial damage on T cell behavior. Chemotherapy treatment using busulfan, fludarabine, and clofarabine led to damage responses in organoids resulting in increased T cell migration, activation, and proliferation in ex- vivo co-culture assays. We identified galectin-9 (Gal-9), a beta-galactoside-binding lectin released by damaged organoids, as a key molecule mediating T cell responses to damage. Increased levels of Gal-9 were also found in the plasma of allo-HCT patients who later developed acute GVHD, supporting the predictive value of the model system in the clinical setting. This study highlights the potential contribution of chemotherapy-induced epithelial damage to the pathogenesis of intestinal GVHD through direct effects on T cell activation and trafficking promoted by galectin-9.

Potential impacts of prolonged absence of influenza virus circulation on subsequent epidemics.

Background: During the first two years of the COVID-19 pandemic, the circulation of seasonal influenza viruses was unprecedentedly low. This led to concerns that the lack of immune stimulation to influenza viruses combined with waning antibody titres could lead to increased susceptibility to influenza in subsequent seasons, resulting in larger and more severe epidemics. Methods: We analyzed historical influenza virus epidemiological data from 2003-2019 to assess the historical frequency of near-absence of seasonal influenza virus circulation and its impact on the size and severity of subsequent epidemics. Additionally, we measured haemagglutination inhibition-based antibody titres against seasonal influenza viruses using longitudinal serum samples from 165 healthy adults, collected before and during the COVID-19 pandemic, and estimated how antibody titres against seasonal influenza waned during the first two years of the pandemic. Findings: Low country-level prevalence of influenza virus (sub)types over one or more years occurred frequently before the COVID-19 pandemic and had relatively small impacts on subsequent epidemic size and severity. Additionally, antibody titres against seasonal influenza viruses waned negligibly during the first two years of the pandemic. Interpretation: The commonly held notion that lulls in influenza virus circulation, as observed during the COVID-19 pandemic, will lead to larger and/or more severe subsequent epidemics might not be fully warranted, and it is likely that post-lull seasons will be similar in size and severity to pre-lull seasons. Funding: European Research Council, Netherlands Organization for Scientific Research, Royal Dutch Academy of Sciences, Public Health Service of Amsterdam. Research in context: Evidence before this study: During the first years of the COVID-19 pandemic, the incidence of seasonal influenza was unusually low, leading to widespread concerns of exceptionally large and/or severe influenza epidemics in the coming years. We searched PubMed and Google Scholar using a combination of search terms (i.e., "seasonal influenza", "SARS-CoV-2", "COVID-19", "low incidence", "waning rates", "immune protection") and critically considered published articles and preprints that studied or reviewed the low incidence of seasonal influenza viruses since the start of the COVID-19 pandemic and its potential impact on future seasonal influenza epidemics. We found a substantial body of work describing how influenza virus circulation was reduced during the COVID-19 pandemic, and a number of studies projecting the size of future epidemics, each positing that post-pandemic epidemics are likely to be larger than those observed pre-pandemic. However, it remains unclear to what extent the assumed relationship between accumulated susceptibility and subsequent epidemic size holds, and it remains unknown to what extent antibody levels have waned during the COVID-19 pandemic. Both are potentially crucial for accurate prediction of post-pandemic epidemic sizes.Added value of this study: We find that the relationship between epidemic size and severity and the magnitude of circulation in the preceding season(s) is decidedly more complex than assumed, with the magnitude of influenza circulation in preceding seasons having only limited effects on subsequent epidemic size and severity. Rather, epidemic size and severity are dominated by season-specific effects unrelated to the magnitude of circulation in the preceding season(s). Similarly, we find that antibody levels waned only modestly during the COVID-19 pandemic.Implications of all the available evidence: The lack of changes observed in the patterns of measured antibody titres against seasonal influenza viruses in adults and nearly two decades of epidemiological data suggest that post-pandemic epidemic sizes will likely be similar to those observed pre-pandemic, and challenge the commonly held notion that the widespread concern that the near-absence of seasonal influenza virus circulation during the COVID-19 pandemic, or potential future lulls, are likely to result in larger influenza epidemics in subsequent years.

Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy.

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

Organoids can be established reliably from cryopreserved biopsy catheter-derived endometrial tissue of infertile women.

RESEARCH QUESTION: Can organoids be established from endometrial tissue of infertile women and does tissue cryopreservation allow for establishment of organoids comparable to organoids derived from freshly biopsied endometrial tissue? DESIGN: Endometrial tissue was obtained from six infertile women through minimally invasive biopsy using a Pipelle catheter and subjected to organoid development, immediately after biopsy as well as after tissue cryopreservation. Organoid formation efficiency, morphology, expandability potential, endometrial marker expression (immunostaining and reverse transcription quantitative real-time polymerase chain reaction) and hormonal responsiveness (after oestradiol and progesterone treatment) were assessed. RESULTS: Organoids established from both fresh and frozen tissue at comparable efficiency could be passaged long-term and showed similar morphology, i.e. cystic with a central lumen lined by a single epithelial cell layer. They also exhibited comparable expression of endometrial markers and proliferative activity (Ki67 expression). Finally, organoids from freshly biopsied and cryopreserved endometrial tissue showed similar responses to oestradiol and progesterone treatment. CONCLUSIONS: Organoids can be established from cryopreserved endometrial tissue of infertile women and cryopreservation of the biopsy does not affect organoid formation and overall organoid characteristics. Cryopreservation of biopsies for later organoid development facilitates sample collection from any fertility clinic, not just the ones near an organoid laboratory.

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells.

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

BACKGROUND & AIMS: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. METHODS: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. RESULTS: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. CONCLUSIONS: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.