Generation of a double insulin and somatostatin reporter line, SCSe001-A-3, for the advancement of stem cell-derived pancreatic islets.

Remarkable strides have been made over the past decade on the development of pancreatic ß-cells from human stem cells through directed differentiation, allowing for modeling of ß-cell development, function and disease. However, in vitro models and future therapeutic applications will require the use of stem cell-derived islets with multiple monohormonal endocrine cells types, including α, ß, and δ cells. Using the previously reported Mel1 InsGFP/w human embryonic stem cell (hESC) line, we have knocked-in Red Fluorescence Protein (RFP) under the control of the endogenous somatostatin promoter using CRISPR/Cas9, generating a dual insulin and somatostatin reporter hESC line.

GATA6 suppression enhances lung specification from human pluripotent stem cells.

The transcription factor GATA6 has been shown to be important for lung development and branching morphogenesis in mouse models, but its role in human lung development is largely unknown. Here, we studied the role of GATA6 during lung differentiation using human pluripotent stem cells. We found that the human stem cell lines most efficient at generating NKX2.1+ lung progenitors express lower endogenous levels of GATA6 during endoderm patterning and that knockdown of GATA6 during endoderm patterning increased the generation of these cells. Complete ablation of GATA6 resulted in the generation of lung progenitors displaying increased cell proliferation with up to a 15-fold expansion compared with control cells, whereas the null cell line displayed a defect in further development into mature lung cell types. Furthermore, transgenic expression of GATA6 at the endoderm anteriorization stage skewed development toward a liver fate at the expense of lung progenitors. Our results suggest a critical dosage effect of GATA6 during human endoderm patterning and a later requirement during terminal lung differentiation. These studies offer an approach of modulating GATA6 expression to enhance the production of lung progenitors from human stem cell sources.

Crosstalk between type II NKT cells and T cells leads to spontaneous chronic inflammatory liver disease.

BACKGROUND & AIM: Natural killer T (NKT) cells are CD1d-restricted innate-like T cells that modulate innate and adaptive immune responses. Unlike the well-characterized invariant/type I NKT cells, type II NKT cells with a diverse T cell receptor repertoire are poorly understood. This study defines the pathogenic role of type II NKT cells in the etiology of chronic liver inflammation. METHODS: Transgenic mice with the Lck promoter directing CD1d overexpression on T cells in Jα18 wild-type (Lck-CD1dTgJα18+; type I NKT cell sufficient) and Jα18-deficient (Lck-CD1dTgJα18o, type I NKT cell deficient) mice were analyzed for liver pathology and crosstalk between type II NKT cells and conventional T cells. CD1d expression on T cells in peripheral blood samples and liver sections from autoimmune hepatitis patients and healthy individuals were also examined. RESULTS: Lck-CD1dTgJα18o and Lck-CD1dTgJα18+ mice developed similar degrees of liver pathology resembling chronic autoimmune hepatitis in humans. Increased CD1d expression on T cells promoted the activation of type II NKT cells and other T cells. This resulted in Th1-skewing and impaired Th2 cytokine production in type II NKT cells. Dysfunction of type II NKT cells was accompanied by conventional T cell activation and pro-inflammatory cytokine production, leading to a hepatic T/B lymphocyte infiltration, elevated autoantibodies and hepatic injury in Lck-CD1dTg mice. A similar mechanism could be extended to humans as CD1d expression is upregulated on activated human T cells and increased presence of CD1d-expressing T cells was observed in autoimmune hepatitis patients. CONCLUSIONS: Our data reveals enhanced crosstalk between type II NKT cells and conventional T cells, leading to a Th1-skewed inflammatory milieu, and consequently, to the development of chronic autoimmune liver disease. Lay summary: CD1d overexpression on T cells enhances crosstalk between type II NKT cells and T cells, resulting in their aberrant activation and leading to the development of chronic autoimmune liver disease.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αß transgenic T cells (24αß T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αß T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

The functions of type I and type II natural killer T cells in inflammatory bowel diseases.

CD1d-restricted natural killer T (NKT) cells are a distinct subset of T cells that rapidly produce an array of cytokines on activation and play a critical role in regulating various immune responses. NKT cells are classified into 2 groups based on differences in T-cell receptor usage. Type I NKT cells have an invariant T-cell receptor α-chain and are readily detectable by α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. Type II NKT cells have a more diverse T-cell receptor repertoire and cannot be directly identified. Both types of NKT cells and multiple CD1d-expressing cell types are present in the intestine, and their interactions are likely to be modulated by pathogenic and commensal microbes, which in turn contribute to the intestinal immune responses in health and disease. Indeed, in several animal models of inflammatory bowel disease, type I NKT cells have been shown to make both protective and pathogenic contributions to disease. In contrast, in patients with ulcerative colitis, and a mouse model in which both CD1d expression and the frequency of type II NKT cells are increased, type II NKT cells seem to promote intestinal inflammation. In this review, we summarize the present knowledge on the antigen recognition, activation, and function of NKT cells with a particular focus on their role in inflammatory bowel disease and discuss factors that may influence the functional outcome of NKT cell responses in intestinal inflammation.

dysregulation of CD1d-restricted type ii natural killer T cells leads to spontaneous development of colitis in mice.

BACKGROUND & AIMS: CD1d-restricted natural killer (NK) T cells are a subset of immunoregulatory T cells that comprise type I (express the semi-invariant T-cell receptor [TCR] and can be detected using the α-galactosylceramide/CD1d tetramer) and type II (express diverse TCRs and cannot be directly identified). Studies in mouse models of inflammatory bowel disease revealed a complex role for type I NKT cells in the development of colitis. Type II NKT cells have been associated with intestinal inflammation in patients with ulcerative colitis. METHODS: To investigate whether dysregulation of type II NKT cells, caused by increased expression of CD1d, can contribute to colitis, we generated transgenic mice that express high levels of CD1d and a TCR from an autoreactive, type II NKT cell (CD1dTg/24αßTg mice). RESULTS: CD1dTg/24αßTg mice had reduced numbers of 24αß T cells compared with 24αßTg mice, indicating that negative selection increases among type II NKT cells engaged by abundant self-antigen. The residual 24αß T cells in CD1dTg/24αßTg mice had an altered surface phenotype and acquired a cytokine profile distinct from that of equivalent cells in 24αßTg mice. Interestingly, CD1dTg/24αßTg mice spontaneously developed colitis; adoptive transfer experiments confirmed that type II NKT cells that develop in the context of increased CD1d expression are pathogenic. CONCLUSIONS: Aberrant type II NKT cell responses directly contribute to intestinal inflammation in mice, indicating the importance of CD1d expression levels in the development and regulation of type II NKT cells.