Arginase 1 (Arg1), which converts l-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly ...characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4/IL-13- and intestinal microbiota-dependent manner. Tie2-Cre Arg1fl/fl mice lacking Arg1 in hematopoietic and endothelial cells recovered faster from colitis than Arg1-expressing (Arg1fl/fl) littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells, and an accumulation of intraluminal polyamines that promote epithelial healing. The proresolving effect of Arg1 deletion was reduced by an l-arginine-free diet, but rescued by simultaneous deletion of other l-arginine-metabolizing enzymes, such as Arg2 or Nos2, demonstrating that protection from colitis requires l-arginine. Fecal microbiota transfers from Tie2-Cre Arg1fl/fl mice into WT recipients ameliorated intestinal inflammation, while transfers from WT littermates into Arg1-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of l-arginine as well as altered intestinal microbiota and metabolic products accounts for the accelerated resolution from colitis in the absence of Arg1. Consequently, l-arginine metabolism may serve as a target for clinical intervention in IBD patients.
Type 1 diabetes (T1D) is a chronic multi-factorial disorder characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. Variations at a large number of genes ...influence susceptibility to spontaneous autoimmune T1D in non-obese diabetic (NOD) mice, one of the most frequently studied animal models for human disease. The genetic analysis of these mice allowed the identification of many insulin-dependent diabetes (Idd) loci and candidate genes, one of them being Cd101. CD101 is a heavily glycosylated transmembrane molecule which exhibits negative-costimulatory functions and promotes regulatory T (Treg) function. It is abundantly expressed on subsets of lymphoid and myeloid cells, particularly within the gastrointestinal tract. We have recently reported that the genotype-dependent expression of CD101 correlates with a decreased susceptibility to T1D in NOD.B6 Idd10 congenic mice compared to parental NOD controls. Here we show that the knockout of CD101 within the introgressed B6-derived Idd10 region increased T1D frequency to that of the NOD strain. This loss of protection from T1D was paralleled by decreased Gr1-expressing myeloid cells and FoxP3+ Tregs and an enhanced accumulation of CD4-positive over CD8-positive T lymphocytes in pancreatic tissues. As compared to CD101+/+ NOD.B6 Idd10 donors, adoptive T cell transfers from CD101-/- NOD.B6 Idd10 mice increased T1D frequency in lymphopenic NOD scid and NOD.B6 Idd10 scid recipients. Increased T1D frequency correlated with a more rapid expansion of the transferred CD101-/- T cells and a lower proportion of recipient Gr1-expressing myeloid cells in the pancreatic lymph nodes. Fewer of the Gr1+ cells in the recipients receiving CD101-/- T cells expressed CD101 and the cells had lower levels of IL-10 and TGF-β mRNA. Thus, our results connect the Cd101 haplotype-dependent protection from T1D to an anti-diabetogenic function of CD101-expressing Tregs and Gr1-positive myeloid cells and confirm the identity of Cd101 as Idd10.
T lymphocytes and myeloid cells express the immunoglobulin-like glycoprotein cluster of differentiation (CD)101, notably in the gut. Here, we investigated the cell-specific functions of CD101 during ...dextran sulfate sodium (DSS)-induced colitis and Salmonella enterica Typhimurium infection. Similar to conventional CD101−/− mice, animals with a regulatory T cell-specific Cd101 deletion developed more severe intestinal pathology than littermate controls in both models. While the accumulation of T helper 1 cytokines in a CD101-deficient environment entertained DSS-induced colitis, it impeded the replication of Salmonella as revealed by studying CD101−/− x interferon-g−/− mice. Moreover, CD101-expressing neutrophils were capable to restrain Salmonella infection in vitro and in vivo. Both cell-intrinsic and -extrinsic mechanisms of CD101 contributed to the control of bacterial growth and spreading. The CD101-dependent containment of Salmonella infection required the expression of Irg-1 and Nox2 and the production of itaconate and reactive oxygen species. The level of intestinal microbial antigens in the sera of inflammatory bowel disease patients correlated inversely with the expression of CD101 on myeloid cells, which is in line with the suppression of CD101 seen in mice following DSS application or Salmonella infection. Thus, depending on the experimental or clinical setting, CD101 helps to limit inflammatory insults or bacterial infections due to cell type-specific modulation of metabolic, immune-regulatory, and anti-microbial pathways.
Type 1 diabetes (T1D) is a chronic multi-factorial disorder characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. Variations at a large number of genes ...influence susceptibility to spontaneous autoimmune T1D in non-obese diabetic (NOD) mice, one of the most frequently studied animal models for human disease. The genetic analysis of these mice allowed the identification of many insulin-dependent diabetes (Idd) loci and candidate genes, one of them being Cd101. CD101 is a heavily glycosylated transmembrane molecule which exhibits negative-costimulatory functions and promotes regulatory T (Treg) function. It is abundantly expressed on subsets of lymphoid and myeloid cells, particularly within the gastrointestinal tract. We have recently reported that the genotype-dependent expression of CD101 correlates with a decreased susceptibility to T1D in NOD.B6 Idd10 congenic mice compared to parental NOD controls. Here we show that the knockout of CD101 within the introgressed B6-derived Idd10 region increased T1D frequency to that of the NOD strain. This loss of protection from T1D was paralleled by decreased Gr1-expressing myeloid cells and FoxP3.sup.+ Tregs and an enhanced accumulation of CD4-positive over CD8-positive T lymphocytes in pancreatic tissues. As compared to CD101.sup.+/+ NOD.B6 Idd10 donors, adoptive T cell transfers from CD101.sup.-/- NOD.B6 Idd10 mice increased T1D frequency in lymphopenic NOD scid and NOD.B6 Idd10 scid recipients. Increased T1D frequency correlated with a more rapid expansion of the transferred CD101.sup.-/- T cells and a lower proportion of recipient Gr1-expressing myeloid cells in the pancreatic lymph nodes. Fewer of the Gr1.sup.+ cells in the recipients receiving CD101.sup.-/- T cells expressed CD101 and the cells had lower levels of IL-10 and TGF-beta mRNA. Thus, our results connect the Cd101 haplotype-dependent protection from T1D to an anti-diabetogenic function of CD101-expressing Tregs and Gr1-positive myeloid cells and confirm the identity of Cd101 as Idd10.
TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp‐76 on iNKT‐cell biology. Compared to B6 ...controls, slp‐76ace/ace mice carrying a missense mutation (Thr428Ile) within the SH2‐domain of slp‐76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP‐deficient mice. Like ADAP−/− iNKT cells, slp‐76ace/ace iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate‐early gene Nur77 and a decreased adhesion to ICAM‐1. Furthermore, CD11b‐intrinsic effects inhibited cytokine release, concanavalin A‐mediated inflammation, and iNKT‐cell accumulation in the liver. Unlike B6 and ADAP−/− mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP‐1‐expression was enhanced in slp‐76ace/ace mice. Blockade of NP‐1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP‐1 as an iNKT‐cell‐specific adhesion factor. Thus, slp‐76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP‐dependent and ‐independent mechanisms.
The adaptor protein slp‐76 regulates the tissue distribution of iNKT cells via ADAP‐dependent and ‐independent mechanisms. The reduced ADAP signal in slp‐76ace/ace iNKT cells triggers the enhanced expression of CD11b and blocks the accumulation of iNKT cells in the spleen and liver. The concomitant, ADAP‐independent upregulation of neuropilin‐1 promotes the redistribution of iNKT cells to the peripheral lymph nodes
While colorectal cancer (CRC) patients with localized disease have a favorable prognosis, the five-year-survival rate in patients with distant spread is still below 15%. Hence, a detailed ...understanding of the mechanisms regulating metastasis formation is essential to develop therapeutic strategies targeting metastasized CRC. The notch pathway has been shown to be involved in the metastatic spread of various tumor entities; however, the impact of its target gene HEYL remains unclear so far.
In this study, we functionally assessed the association between high HEYL expression and metastasis formation in human CRC. Therefore, we lentivirally overexpressed HEYL in two human patient-derived CRC cultures differing in their spontaneous metastasizing capacity and analyzed metastasis formation as well as tumor cell dissemination into the bone marrow after xenotransplantation into NOD.Cg-Prkdc
Il2rg
/SzJ (NSG) mice.
HEYL overexpression decreased tumor cell dissemination and the absolute numbers of formed metastases in a sub-renal capsular spontaneous metastasis formation model, addressing all steps of the metastatic cascade. In contrast, metastatic capacity was not decreased following intrasplenic xenotransplantation where the cells are placed directly into the blood circulation.
These results suggest that HEYL negatively regulates metastasis formation in vivo presumably by inhibiting intravasation of metastasis-initiating cells.
Highlights • Phenotypic differentiation in vitro does not affect tumorigenicity of spheroids. • CRC TIC propagation is feasible in conditions favoring phenotypic differentiation. • Phenotypic ...plasticity of primary CRC TIC enriched in spheroids.
A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In ...this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.
Patient‐derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their ...widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV‐associated B‐lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV‐infected B‐lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B‐lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify “bona fide” carcinoma xenografts.
What's new?
Despite the routine and extensive use of patient‐derived cancer xenografts (PDX) in preclinical cancer research, no universal guidelines for quality testing are available. This study however demonstrates that Epstein‐Barr virus‐associated B‐lymphoproliferations frequently develop following xenotransplantation of colorectal and pancreatic cancer tissue in highly immunodeficient mice. Even minor numbers of residual EBV‐infected B‐lymphocytes present in the initial PDX can fully overgrow epithelial cancer cells during serial xenotransplantation. In addition, patient‐derived B‐lymphocytes can proliferate in culture conditions optimized for primary epithelial cancer cells. B‐lymphoproliferations represent a serious confounding factor, making repeated phenotypic assessments mandatory to verify “bona fide” carcinoma xenografts.
In highly aggressive malignancies like pancreatic cancer (PC), patient-derived tumor models can serve as disease-relevant models to understand disease-related biology as well as to guide clinical ...decision-making. In this study, we describe a two-step protocol allowing systematic establishment of patient-derived primary cultures from PC patient tumors. Initial xenotransplantation of surgically resected patient tumors (n = 134) into immunodeficient mice allows for efficient in vivo expansion of vital tumor cells and successful tumor expansion in 38% of patient tumors (51/134). Expansion xenografts closely recapitulate the histoarchitecture of their matching patients' primary tumors. Digestion of xenograft tumors and subsequent in vitro cultivation resulted in the successful generation of semi-adherent PC cultures of pure epithelial cell origin in 43.1% of the cases. The established primary cultures include diverse pathological types of PC: Pancreatic ductal adenocarcinoma (86.3%, 19/22), adenosquamous carcinoma (9.1%, 2/22) and ductal adenocarcinoma with oncocytic IPMN (4.5%, 1/22). We here provide a protocol to establish quality-controlled PC patient-derived primary cell cultures from heterogeneous PC patient tumors. In vitro preclinical models provide the basis for the identification and preclinical assessment of novel therapeutic opportunities targeting pancreatic cancer.
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