The signaling pathway involved in wild type and 2D2-gut CD4+ T cell development
ABSTRACT: To find the signalling pathway involved in WT/2D2 CD4+ gut T cell (CD4+ intraepithelial lymphocytes, CD4+IEL) differentiation, particulary to determine whether aryl hydrocarbon receptor (AHR) pathway is involved. Gene expression was analyzed ex vivo in CD4+ T cells that were sorted from Wild type-Gut / 2D2 mouse-Gut / Wild type-Spleen / 2D2 mouse-Spleen (N=3 per group). The genes highly expressed in WT/2D2 Gut compared to WT/2D2 Spleen were selected by k-means clustering. AHR pathway related genes (IPA software) were further selected.
Project description:To find the signalling pathway involved in WT/2D2 CD4+ gut T cell (CD4+ intraepithelial lymphocytes, CD4+IEL) differentiation, particulary to determine whether aryl hydrocarbon receptor (AHR) pathway is involved. Gene expression was analyzed ex vivo in CD4+ T cells that were sorted from Wild type-Gut / 2D2 mouse-Gut / Wild type-Spleen / 2D2 mouse-Spleen (N=3 per group). The genes highly expressed in WT/2D2 Gut compared to WT/2D2 Spleen were selected by k-means clustering. AHR pathway related genes (IPA software) were further selected.
Project description:Specific pathogen-free IL-10 KO mice failed to develop inflammatory bowel disease (IBD), whereas IL-10/vitamin D receptor (VDR) double KO mice developed fulminating IBD. WT CD4 T cells inhibited experimental IBD, while VDR KO CD4 T cells failed to suppress IBD. VDR KO mice had normal numbers and functions of regulatory T cells. The percentages of IL-17- and IFN-gamma-secreting T cells in the gut of mice reconstituted with WT and VDR KO CD4 T cells were also not different. Instead, there were twice as many CD8alphaalpha intraepithelial lymphocytes (IEL) in mice that were reconstituted with WT CD4 T cells than in mice reconstituted with VDR KO CD4 T cells. Furthermore, VDR KO mice had reduced numbers of CD8alphaalpha IEL, absent CD4/CD8alphaalpha populations, and as a result low IL-10 production in the IEL. The lack of CD8alphaalpha IEL was due in part to decreased CCR9 expression on T cells that resulted in the failure of the VDR KO T cells to home to the small intestine. We conclude that the VDR mediates T cell homing to the gut and as a result the VDR KO mouse has reduced numbers of CD8alphaalpha IEL with low levels of IL-10 leading to increased inflammatory response to the normally harmless commensal flora.
Project description:The intestinal epithelium harbors large populations of activated and memory lymphocytes, yet these cells do not cause tissue damage in the steady state. We investigated how intestinal T cell effector differentiation is regulated upon migration to the intestinal epithelium. Using gene loss- and gain-of-function strategies, as well as reporter approaches, we showed that cooperation between the transcription factors T-bet and Runx3 resulted in suppression of conventional CD4(+) T helper functions and induction of an intraepithelial lymphocyte (IEL) program that included expression of IEL markers such as CD8?? homodimers. Interferon-? sensing and T-bet expression by CD4(+) T cells were both required for this program, which was distinct from conventional T helper differentiation but shared by other IEL populations, including TCR??(+)CD8??(+) IELs. We conclude that the gut environment provides cues for IEL maturation through the interplay between T-bet and Runx3, allowing tissue-specific adaptation of mature T lymphocytes.
Project description:Bach2 regulates homeostasis of foxp3+ regulatory T cells and protects against fatal lung disease in mice. Cells from WT and Bach2 KO spleen were isolated. CD4+ CD25+ GITR+ (Treg) cells were sorted by FACS sorting. Total RNAs were extracted from sorted Treg cells using by Rneasy Kit (Qiagen).
Project description:Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.
Project description:Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30<sup>+</sup>γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated T<sub>H</sub>17 but decreased CD8<sup>+</sup>T, γδT, T<sub>FH</sub> and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8<sup>+</sup>, as well as reduced CD4<sup>+</sup>T cells with an elevated T<sub>H</sub>17 over Treg/T<sub>FH</sub> ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.
Project description:Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated ?? T cell compartments. Thus, the development of signature, murine TCR??+ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin ?? IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal ??+ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.V?7+ IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective ?? IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.
Project description:Acute graft versus host disease is a serious condition caused by allo-reactive donor CD4+ T cells from allogenic hematopoietic stem cell transplantation. To understand the developmental relationships between T-helper states in mesenteric lymph nodes (mLN), TCR transgenic CD4+ T cells specific for a single allo-peptide (TEa cells) from mice were recovered at Days 0, 1, 2, 3, and 4 from mLN, and Day 5 from the gut and underwent processing to generate scRNA-seq dataset. TEa cells were also recovered at Day 5 from mLN and were either treated with and without IEL-isolation pre-digestion buffer as controls.
Project description:Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural T-IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to compare the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8<sup>+</sup> T cells from lymph nodes. This data exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; upregulated cholesterol and lipid metabolic pathways, leading to high cholesterol levels in T-IEL; suppression of T cell antigen receptor signalling and expression of the transcription factor TOX, reminiscent of chronically activated T cells. These novel findings illustrate how T-IEL integrate multiple tissue-specific signals to maintain their homeostasis and potentially function.
Project description:Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells residing in the epithelium at the interface between the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes, ranging from pathogen control to tissue stability. However, despite their critical importance in mucosal immune responses, very little is known about the homeostasis of different IEL subpopulations. The phosphoprotein osteopontin is important for critical physiological processes, including cellular immune responses, such as survival of Th17 cells and homeostasis of NK cells among others. Because of its impact in the immune system, we investigated the role of osteopontin in the homeostasis of IEL. In this study, we report that mice deficient in the expression of osteopontin exhibit reduced numbers of the IEL subpopulations TCR??+, TCR?+CD4+, TCR?+CD4+CD8?+, and TCR?+CD8??+ cells in comparison with wild-type mice. For some IEL subpopulations, the decrease in cell numbers could be attributed to apoptosis and reduced cell division. Moreover, we show in vitro that exogenous osteopontin stimulates the survival of murine IEL subpopulations and unfractionated IEL derived from human intestines, an effect mediated by CD44, a known osteopontin receptor. We also show that iCD8? IEL but not TCR??+ IEL, TCR?+ IEL, or intestinal epithelial cells, can promote survival of different IEL populations via osteopontin, indicating an important role for iCD8? cells in the homeostasis of IEL.