Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) enables hematopoietic reconstitution through donor stem cells matched at essential HLA loci. While it offers potential for a graft-versus-leukemia (GvL) effect through elimination of residual malignant cells, it also carries the risk of graft-versus-host disease (GvHD) due to the recognition of healthy tissues. We identified two groups of HLA-II restricted antigens with distinct behavior towards HLA-DM. DM-resistant antigens are presented when HLA-DM is expressed. In contrast, DM-sensitive antigens require inhibition of HLA-DM by HLA-DO. Because HLA-DO expression is confined to hematopoietic antigen-presenting cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues, even under inflammatory conditions. Since HLA-DP is frequently mismatched in unrelated donor transplants, we hypothesized that CD4+ T cells targeting DM-sensitive antigens in a mismatched HLA-DP allele could be an ideal way to achieve GvL effect without inducing GvHD. Methods: To identify T cells recognizing DM-sensitive antigens, isolated CD4⁺ T cells from an HLA-DP–mismatched donor were co-cultured with HeLa cells expressing invariant chain (li) and one of the five most common HLA-DP molecules in the Caucasian population (DPB1*01:01, *02:01, *03:01, 04:01, 04:02). Activated T cells were isolated based on expression of CD137 and clonally expanded. Reactivity of T-cell clones against HeLa + Ii ± HLA-DP ± HLA-DM, malignant hematopoietic and non-hematopoietic cell lines, EBV-infected B cells, and primary AML blasts was characterized, along with cytotoxicity and cytokine profiles. T-cell receptors (TCR) of T-cell clones with the most favorable characteristics were sequenced in order to generate TCR-engineered T cells. TCR reexpression was achieved by orthotopic T-cell receptor replacement (OTR) using CRISPR. Results: Of 105 T-cell clones from nine donors, 79 targeted DM-sensitive and 26 DM-resistant antigens, distinguished by their reactivity in presence of HLA-DM. T-cell clones directed against DM-sensitive antigens did not recognize non-hematopoietic cells, even under inflammatory conditions. In contrast, HLA-DO positive malignant hematopoietic cell lines and primary AML blasts were recognized by T-cell clones targeting DM-sensitive antigens, indicating potential leukemia-specificity. Testing against cell lines from various donors suggests that antigen recognition by T cells was independent of donor-specificity, but rather directed against the mismatched HLA-DP possibly complexed with a monomorphic peptide. Across all experimental conditions, the recognition profile (sensitive or resistant) remained consistent regardless of target cell concentration. Donor-dependent cytotoxicity for isolated T-cell clones was observed and mainly mediated by Granzyme A and B. Extended cytokine analysis revealed secretion of INF-γ, IL-5, IL-13, and IL-6, accompanied by lower levels of IL-4. After reexpression, TCR-engineered T cells demonstrated high functionality as shown by IFN-γ ELISA and activation marker analysis via flow cytometry. Conclusion: We show feasibility to identify T cells directed against DM-sensitive antigens presented in mismatched HLA-DP alleles recognizing malignant hematopoietic cells, which in future may contribute to the development of GvL directed T-cell therapies with reduced risk of GvHD.

BACKGROUND & AIMS:Enteric glial cells regulate gastrointestinal homeostasis and inflammation. Although activated enteric glial cells have been shown to support epithelial and immune balance in preclinical models, their functional status and turnover in inflammatory bowel diseases remain poorly defined. This study aimed to identify enteric glial cell activation markers and assess their susceptibility to cytokine-driven death in inflammatory bowel diseases. METHODS:We analyzed 390 intestinal samples from patients with inflammatory bowel disease using bulk and single-nucleus RNA sequencing and validated findings across public datasets comprising over 1160 patients and 19,000 enteric glial cell transcriptomes. We used multiple mouse models of gut inflammation, reporter-based glial sorting, transcriptomics, and glia-specific Casp8 deletion to dissect mechanisms of enteric glial cell activation and death. Ex vivo stimulation of sorted enteric glial cells was used to assess cytokine-specific effects. RESULTS:We identified novel inflammatory bowel disease subtype- and location-specific enteric glial cell activation markers, including osteopontin (SPP1), enriched in ulcerative colitis. Single-nucleus and single-cell data revealed that activated enteric glial cell clusters selectively upregulate cell death signatures with inflammatory bowel disease enteric glial cells displaying necroptosis via phosphorylation of mixed lineage kinase domain-like pseudokinase. In mice, acute T helper 1 cell/T helper 17-driven inflammation rapidly induced enteric glial cell activation and necroptosis, impairing intestinal motility. Ex vivo, interferon-γ and tumor necrosis factor costimulation, but not individual cytokines, induced mixed lineage kinase domain-like pseudokinase-dependent necroptosis in enteric glial cells. Casp8-deficient enteric glial cells ere hypersensitive to tumor necrosis factor-induced death, confirming a Caspase-8-dependent survival checkpoint. CONCLUSIONS:A proportion of activated enteric glial cells are selectively eliminated in inflammatory bowel disease via cytokine-mediated necroptosis, driven by a coordinated interferon-γ/tumor necrosis factor axis. This process compromises enteric glial support functions and may contribute to inflammatory bowel disease-associated dysmotility. Targeting glial survival may represent a novel therapeutic avenue.

BACKGROUND:The peptide GPR15L is produced by colonic epithelial cells and has been implicated in T cell recruitment to the large intestine. However, its role in chronic colitis has been unclear so far. OBJECTIVE:To explore the role of GPR15L in the pathogenesis of experimental colitis and IBD. DESIGN:We studied how genetic deletion or overexpression of Gpr15l as well as rectal application of recombinant GPR15L alters the course of acute dextran sodium sulfate colitis and T cell transfer colitis. The impact of GPR15L on microbiota was explored with co-housing, littermate and faecal microbiota transfer studies, by 16S rRNA sequencing as well as anti-microbial assays and shotgun metagenomics. The expression of GPR15L was evaluated across three independent cohorts of patients with IBD and correlated to microbial diversity and flare-free survival. RESULTS:GPR15L clearly mitigated experimental colitis, but this was independent of T cell recruitment and GPR15. Instead, we observed that the effects of GPR15L were mediated by altered microbiomes in the large intestine and, consistently, showed that GPR15L acts as an antimicrobial peptide under anaerobic conditions and shapes microbial communities towards a homeostatic phenotype. Rectal supplementation of GPR15L counteracted experimental colitis. In patients with IBD, GPR15L expression was decreased in active inflammation, correlated with microbial diversity and was associated with flare-free survival. CONCLUSIONS:GPR15L is a host-defence peptide that plays a beneficial role in the pathogenesis of intestinal inflammation. It seems promising to further evaluate its potential as a future therapeutic approach in IBD.

ABSTRACT:Calprotectin, a calcium- and zinc-binding protein that comprises the subunits S100A8 and S100A9, has been extensively studied as a biomarker of gastrointestinal (GI) inflammation through fecal and serum analyses. However, its role in intestinal tissue remains poorly understood because of the limited availability of biopsy specimens. In this study, we analyzed S100A8 and S100A9 messenger RNA (mRNA) expression in 579 intestinal biopsy specimens from allogeneic stem cell transplantation recipients and observed a strong association with acute GI graft-versus-host disease (aGI-GVHD; P< .001). Neutrophil infiltration correlated with the severity of aGI-GVHD (P< .001), and calprotectin expression was strongly linked to Toll-like receptor 4 (TLR4; P< .001) and TLR2 (P< .001) expression. Both TLR4 and aGI-GVHD were associated with elevated calprotectin mRNA levels (P< .001). When patients received broad-spectrum antibiotics at disease onset, calprotectin expression was suppressed (S100A8, P = .001; S100A9, P = .01). GI site-specific differences in calprotectin expression were identified: during severe aGI-GVHD, levels increased up to 30-fold in the small intestine and up to fivefold in the large intestine with respect to mild or no aGI-GVHD, whereas under homeostasis, the large intestine exhibited higher baseline calprotectin (P = .001). The high clinical relevance of this finding is evident from the observation that calprotectin expression was prognostic for transplant-related mortality. Our study suggests that (1) calprotectin is a potential biopsy biomarker in aGI-GvHD and (2) calprotectin expression and neutrophil infiltration possibly indicate translocation of microbiota, which (3) may be modulated by antibiotics.

BACKGROUND:IBD is a chronic inflammatory condition driven by complex genetic and immune interactions, yet preclinical models often fail to fully recapitulate all aspects of the human disease. A systematic comparison of commonly used IBD models is essential to identify conserved molecular mechanisms and improve translational relevance. OBJECTIVE:We performed a multimodel transcriptomic analysis of 13 widely used IBD mouse models to uncover coregulatory gene networks conserved between preclinical colitis/ileitis and human IBD and to define model-specific and conserved cellular, subcellular and molecular signatures. DESIGN:We employed comparative transcriptomic analyses with curated and a priori statistical correlative methods between mouse models versus IBD patient datasets at both bulk and single-cell levels. RESULTS:We identify IBD-related pathways, ontologies and cellular compositions that are translatable between mouse models and patient cohorts. We further describe a conserved core inflammatory signature of IBD-associated genes governing T-cell homing, innate immunity and epithelial barrier that translates into the new mouse gut Molecular Inflammation Score (mMIS). Moreover, specific mouse IBD models have distinct signatures for B-cell, T-cell and enteric neurons. We discover that transcriptomic relatedness of models is a function of the mode of induction, not the canonical immunotype (Th1/Th2/Th17). Moreover, the model compendium database is made available as a web explorer (http://trr241.hosting.rrze.uni-erlangen.de/SEPIA/). CONCLUSION:This integrated multimodel approach provides a framework for systematically assessing the molecular landscape of intestinal inflammation. Our findings reveal conserved inflammatory circuits, refine model selection, offering a valuable resource for the IBD research community.

Dysregulation at the intestinal epithelial barrier is a driver of inflammatory bowel disease (IBD). However, the molecular mechanisms of barrier failure are not well understood. Here, we demonstrate dysregulated mitochondrial fusion in intestinal epithelial cells (IECs) of patients with IBD and show that impaired fusion is sufficient to drive chronic intestinal inflammation. We found reduced expression of mitochondrial fusion–related genes, such as the dynamin-related guanosine triphosphatase (GTPase) optic atrophy 1 ( OPA1 ), and fragmented mitochondrial networks in crypt IECs of patients with IBD. Mice with Opa1 deficiency in the gut epithelium ( Opa1 i∆IEC ) spontaneously developed chronic intestinal inflammation with mucosal ulcerations and immune cell infiltration. Intestinal inflammation in Opa1 i∆IEC mice was driven by microbial translocation and associated with epithelial progenitor cell death and gut barrier dysfunction. Opa1 -deficient epithelial cells and human organoids exposed to a pharmacological OPA1 inhibitor showed disruption of the mitochondrial network with mitochondrial fragmentation and changes in mitochondrial size, ultrastructure, and function, resembling changes observed in patient samples. Pharmacological inhibition of the GTPase dynamin-1–like protein in organoids derived from Opa1 i∆IEC mice partially reverted this phenotype. Together, our data demonstrate a role for epithelial OPA1 in regulating intestinal immune homeostasis and epithelial barrier function. Our data provide a mechanistic explanation for the observed mitochondrial dysfunction in IBD and identify mitochondrial fusion as a potential therapeutic target in this disease.

The heterogeneity of conventional dendritic cells type 1 (cDC1s) and type 2 (cDC2s) is well established, yet the identity and origin of CD301b+ cDC2s remain debated. Here, we show that CD301b+ cDC2s and CD103+ cDC1s develop from pre-committed progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF). While CD103+ cDC1s acquire their phenotype and functional properties through GM-CSF-driven differentiation from pre-cDC1s, CD301b+ cDC2s emerge as cytokine-induced states from DC2- and DC3-committed progenitors. CD103+ cDC1s and CD301b+ cDC2s exhibit enhanced T cell priming capacities and distinct cytokine expression profiles upon GM-CSF exposure. In vivo, DC-intrinsic GM-CSF sensing is dispensable for acquiring CD103 and CD301b expression with the notable exception of lung DCs, while specific type 2 cytokines induce CD103 and CD301b ex vivo. These findings identify GM-CSF and specific type 2 cytokines as central regulators of cDC1 and cDC2 effector differentiation and establish CD301b as a marker of a cytokine-driven cDC2 state.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality, and most patients fail to respond to immune checkpoint inhibitors (ICIs). Nutritional factors, particularly dietary lipids, influence cancer progression and immunity. Cholesterol can promote tumor growth and immune evasion, whereas ω−3 polyunsaturated fatty acids (PUFAs) may exert anti-inflammatory and immunostimulatory effects. We aimed to determine how dietary lipids influence NSCLC progression and whether ω−3 PUFA supplementation can enhance the efficacy of PD-1 blockade. Serum lipid profiles from 152 NSCLC patients were analyzed for associations with prognosis. Female murine NSCLC models were fed diets enriched in cholesterol, saturated fats, or ω−3 PUFAs, with or without anti-PD-1 therapy. Tumor growth, immune infiltration, cytokine production, and epithelial–mesenchymal transition (EMT) markers were assessed by flow cytometry, ELISA, and gene expression analysis. In patients, elevated serum triglycerides correlate with poor outcomes, and tumor cholesterol metabolism links to EMT marker expression. In female mice, cholesterol-rich diets accelerate tumor growth, increase EMT-associated genes (SNAIL, Vimentin), and elevate pro-tumoral cytokines (IL-6, IL-10, IL-17A). ω−3 PUFA diets reduce tumor burden, lower immunosuppressive cytokines, decrease regulatory T cells, and enhance cytotoxic T cell activity. Combining ω−3 PUFAs with anti-PD-1 therapy synergistically suppresses tumor growth and improves antitumor immune responses. Dietary lipids modulate NSCLC progression via metabolic, inflammatory, and immune pathways. ω−3 PUFA supplementation counteracts cholesterol-driven tumor promotion and augments PD-1 blockade efficacy, supporting dietary modulation as a complementary strategy to improve immunotherapy outcomes in NSCLC. We study how dietary fats affect lung cancer and its treatment. Lung cancer is the leading cause of cancer deaths, and many patients do not respond well to modern immunotherapies, a type of cancer treatment. Cholesterol can make tumors grow faster and weaken the immune system, while ω−3 fatty acids (found in fish and some plants) are thought to have protective effects. We analyzed blood and tumor samples from lung cancer patients and tested different diets in mice, with or without immunotherapy. We find that cholesterol fuels cancer growth, while ω−3s slow it down, strengthen immune defenses, and make immunotherapy work better. These results suggest that eating more ω−3 fatty acids could help support cancer treatment, though clinical trials are needed to confirm this finding. Harre et al. analyze serum lipids from patients with NSCLC and test how cholesterol- or ω−3 PUFA–enriched diets affect tumor growth and immunity in mouse models, with or without PD-1 therapy. They find that cholesterol drives tumor progression and immune suppression, while ω−3 PUFAs reduce tumor burden and improve PD-1 efficacy

Splenic metallophilic marginal zone macrophages (MMMs) are positioned to control the dissemination of blood-borne threats. We developed a purification protocol to enable characterization of MMMs phenotypically and transcriptionally. MMM gene expression profile was enriched for pathways associated with CD8+ T cell activation and major histocompatibility complex class I (MHC class I) cross-presentation. In vitro, purified MMMs equaled conventional dendritic cells type 1 (cDC1s) in cross-priming CD8+ T cells to soluble and particulate antigens, yet MMMs employed a distinct vacuolar processing pathway. In vivo biphoton and ex vivo light-sheet imaging showed long-standing contacts with cognate T cells differentiating to effectors. MMMs cross-primed protective CD8+ T cell antitumor responses both by capturing blood-borne tumor antigens and by internalizing tumor cells seeding the spleen. This cross-priming required expression of the transcription factor Batf3 by MMMs but was independent of cDC1-mediated capture of tumor material for cross-presentation or MHC class I-dressing. Thus, MMMs combine control of the dissemination of blood-borne pathogens and tumor materials with the initiation of innate and adaptive responses.

Changes in the intestinal microbiome and microbiota-derived metabolites predict clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report that desaminotyrosine (DAT), a product of bacterial flavonoid metabolism, correlates with improved overall survival and reduced relapse rates in patients receiving allo-HSCT. In preclinical mouse models, treatment with synthetic DAT prevents graft-versus-host disease by protecting the intestinal barrier and promoting intestinal regeneration and contributes to graft-vs.-leukemia responses. DAT´s beneficial effects on intestinal regeneration remain effective despite broad-spectrum antibiotics-induced dysbiosis, also when administered by fecal microbiota transfer with flavonoid-degrading F. plautii. Mechanistically, DAT promotes mTORC1-dependent activation and proliferation of intestinal stem cells, with concomitant engagement of the innate immune receptor STING required to mitigate metabolic stress and maintain an undifferentiated stem cell state independently of type-I interferon responses. Additionally, DAT can skew T cells towards an effector phenotype to modulate graft-versus-leukemia responses. Our data uncover DAT's dual, tissue- and immune-modulating properties and underscore its potential in precision microbiome-based therapies to improve tissue regeneration and minimize immune-mediated side effects.

Background Enteric glial cells (EGC) play a crucial role in maintaining gut homeostasis, but their dysregulation in inflammatory bowel diseases (IBD) remains poorly understood. Emerging preclinical data suggests activated EGC have beneficial roles in controlling gut pathophysiology. Objective Understanding EGC activation and adaptation during experimental and clinical IBD. Design We provide the first highly integrated approach to identify EGC activation signature in IBD. Profiling 390 samples from IBD patients via bulk and single-nucleus (sn) transcriptomics and replicate the findings on publicly available bulk and single-cell (sc) datasets from 1160 patients and 19,000 single EGC. Preclinical modelling of Th1/Th17 inflammation, reporter-assisted EGC sorting, analysis of regulated cell death, and Casp8 ablation in EGC was performed Results We identified novel IBD type and sampling associated EGC activation signature. Specific EGC activation markers were shared in biopsies and resection specimens, and were divergent between Crohn’s disease and Ulcerative colitis. Preclinical modelling of intestinal inflammation identified combinatorial TNF and IFN-γ-driven activation of EGC, associated with elevated necroptosis, and negatively impacting gut motility. Genetic-reporter-enabled sorting and downstream analyses confirmed TNF and IFN-γ-driven EGC necroptosis, potentiated by Casp8 deficiency. Furthermore, snRNA-Seq from IBD patient samples confirmed elevated cell death signature in activated but not in rare neuroglia progenitor-like cluster. Conclusion Our findings identify IBD type-associated activated EGC markers involved in immune and epithelial homeoastasis. We uncover necroptosis of activated EGCs as a constituent of intestinal inflammation. Advancing our understanding of activated EGC survival is pivotal in elucidating their complex roles in maintaining gut immune-epithelial homeostasis. What is already known on this topic Activated EGC have emerged as important contributors in maintaining epithelial, immune and neuronal homeostasis. Increasing evidence from mouse studies points to the role of activated EGC in epithelial regeneration, tolerogenic T-cell activation, relaying psychological stress to the enteric nervous system, post-injury neurogenesis, and helminth clearance. Nevertheless, no consensus has emerged on what might define activated EGC in the context of IBD and how EGC turnover is affected in gut inflammation, limiting translation of their disease associated roles. What this study adds By combining bulk with single cell and single nucleus transcriptomes from IBD patients we identified new IBD type– and location-associated EGC activation signatures. Some of these are conserved with mouse EGC in gut inflammation models. We identified osteopontin an immunomodulator and Wnt6 an epithelial morphogen elevated in IBD EGC. We also identified IBD-associated EGC cell clusters, which display higher expression of cell death pathway transcripts. To investigate EGC turnover, we utilized preclinical models and found rapid EGC activation upon Th1/Th17 inflammation. This was associated with elevated EGC activation and caspase-independent necroptotic cell death. Ex vivo experiments showed a combinatorial requirement of IFN-γ and TNF in mediating EGC necroptosis. Our findings were replicated on multiple publicly available sc-RNA sequencing datasets from IBD patients. How this study might affect research, practice or policy Expanding on the available repertoire of EGC activation markers in IBD, both shared and unique to sampling procedure, disease type, and location will provide researchers with tools to identify EGC homeostasis during IBD. Moreover, the nature of the identified markers will stimulate research into specific EGC pathways triggered in inflammation. Adding to this, the rapid induction in pathological death of activated but not naive EGC upon IFN-γ and TNF stimulation will shed light on EGC adaptation and turnover. Our identification of markers of activated EGC with immuno-modulatory and epithelial-regenerative properties, including osteopontin and wingless family of morphogenes will stimulate further research in EGC-immune and EGC-epithelial communication in the context of IBD. ### Competing Interest Statement The authors have declared no competing interest.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine with a complex and multifaceted pathogenesis. While various animal models exist to study specific disease mechanisms relevant to human IBD, a comprehensive comparative framework linking these to IBD pathophysiology is lacking. In this study, we provide a framework that delineates common and unique features encountered at the transcriptomic level in 13 widely used mouse models, employing both curation-based and statistically correlative analyses. Our comparative transcriptomic analyses between mouse models versus established as well as new patient datasets reveal specific disease mechanisms in IBD. Furthermore, we identify IBD-related pathways, ontologies, and cellular processes that are comparable between mouse models and patient cohorts. Our findings provide a valuable resource for selecting the most appropriate experimental paradigm to model unique features of IBD pathogenesis, allowing analysis at the tissue, cellular, and subcellular levels. ### Competing Interest Statement The authors have declared no competing interest.

Einleitung Intraepithelial lymphocytes (IELs) are T cells that patrol within the epithelial layer of the intestine [1]. IELs represent a first line of defense against pathogens and are mainly antigen-experienced CD8+T cells. Previous work suggested that overreaction or dysfunction of IELs might contribute to the pathogenesis of inflammatory bowel diseases (IBD). CXCL10 is a chemokine considered to promote the recruitment of immune cells to sites of inflammation, thereby amplifying local inflammation. However, the interaction of CXCL10 with IELs from IBD patients has not been clarified so far.

Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.

Histomorpholgy is one of the mainstays of acute Graft-versus-host disease (GvHD) diagnosis. However, concerns about reproducibility and the most appropriate grading system question its usefulness. Our aim was to assess histomorphological parameters and previously reported grading systems for GvHD regarding reproducibility and validity. Moreover, we propose that sum scores, derived by combining separately scored morphological parameters into a total score, might provide a simplified but equally effective means to grade GvHD. A total of 123 colon biopsies were assessed across four pathologists for intestinal GvHD using a Round-Robin test and results were correlated with clinical findings. Interobserver reproducibility was high for histological parameters that were evaluated as indicators of acute GvHD. Published grading systems were moderately reproducible (ICC 0.679-0.769) while simplified sum scores, in comparison, showed better interrater reliability (ICC 0.818-0.896). All grading systems and sum scores were associated with clinical signs of GvHD and in part with therapy response and survival. However, they were not able to stratify patients according to the clinical severity of GvHD. In a hot-spot analysis 1 crypt apoptotic body (CAB) in 10 crypts was a reasonable cut-off value for minimal diagnostic criteria of GvHD. In conclusion, histology can contribute to the diagnosis of GvHD and is reproducible. Published grading systems are able to reflect clinical findings as are simplified sum scores, which showed improved reproducibility and might be easier to handle as they are based on adding up histological parameters rather than transferring histological findings into a separate grading system. Sum scores will have to be further tested in a prospective setting.

T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αβ+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.

IntroductionMacrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood.MethodsTaking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence.ResultsWe show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype.DiscussionOur data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions.

Background Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS). Methods Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded. Results Our approach discriminated between healthy (mean MCS = 2.5) and inflamed tissue (mean MCS > 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS. Conclusions Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.