Project description:IL-12 and IL-18 synergize to promote TH1 responses and have been implicated as accelerators of autoimmune pathogenesis in type 1 diabetes (T1D). We therefore investigated the influence of these cytokines on phenotype and function of immune cells that are involved in disease progression. To understand how IL-12 and IL-18 may synergize to impair Treg function and phenotype, we conducted transcriptional profiling of Treg expanded under normal conditions or in the presence of IL-12 and IL-18. This analysis revealed increased expression of IFNG, GZMB, GZMA, and IL18RAP and decreased FOXP3 in Tregs expanded with IL-12 and IL-18.
Project description:To determine the differentially expressed genes between these subsets and identify the core human in vivo differentiated Tr1 cell gene signature
Project description:Purpose: To identify gene expression patterns in ex vivo isolated human Tr1 cells. Method: RNA sequencing of total mRNA. Results: Differential gene expression of Tr1 and non-Tr1 CD4+ T memory cells. Conclusions: ex vivo type 1 regulatory T cells have a distinct gene expression profile compared to non-Tr1 CD4+ T cell memory cells.
Project description:Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.
Project description:This experiment was to determine if we could observe changes in gene expression following the attenuation of one of the primary redox regulators of cells - thioredoxin reductase 1. The experiment was set up with two cell lines that expressed different basal levels of TR1. LOX cells represent an aggressive melanoma in xenograft models and are high expressors of TR1 while SK Mel-28 cells represent a non-aggressive melanoma in xenografts adn are low expressors of TR1. The experimental design was to set up direct comparisons for melanoma cells with the induction of a control miRNA or the miRNA directed against TR1 - with an evaluation of the uninduced situation. Melanoma cell lines stabily transfected with tetracyclin-inducible microRNA - directed at either control, or to TR1.
Project description:We identified Pparg as a major orchestrator of the phenotype of adipose-tissue resident regulatory T cells (VAT Tregs). To establish the role of Pparg in shaping the VAT Tregs gene profile and cell dynamics, Tregs from lymph nodes and visceral adipose tissue of mice sufficient and deficient of Pparg expression in Tregs were double sorted for microarray analysis. All gene expression profiles were obtained from highly purified (double-sorted) T cell populations sorted by flow cytometry. To reduce variability, cells from multiple mice were pooled. Triplicates were generated for all groups. Raw data were preprocessed with the RMA algorithm in GenePattern and averaged expression values were used for analysis.
Project description:The objective of this study was to determine if a subset of regulatory T cells (Tregs) expressing the transcription factor, Zbtb20, played a unique role in the function of the immune system. Genetic reporter mice were used to isolate Zbtb20-expressing Tregs as well as activated (CD62Llo) and naive (CD62Lhi) Tregs. The gene expression in these cells was determined with RNA-seq.
Project description:We identified Pparg as a major orchestrator of the phenotype of adipose-tissue resident regulatory T cells (VAT Tregs). To establish the role of Pparg in shaping the VAT Tregs gene profile and cell dynamics, Tregs from lymph nodes and visceral adipose tissue of mice sufficient and deficient of Pparg expression in Tregs were double sorted for microarray analysis.
Project description:Interleukin-10 (IL-10) is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and being therefore currently examined in clinical trials as T-cell therapy in patients with inflammatory bowel disease (IBD). However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified in order to optimize the efficiency and to ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo. Double IL-10eGFP Foxp3mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of TR1 cells in a murine IBD model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human TR1 cells, currently employed for cell therapy, to confirm our results. We found that murine TR1 cells expressed functional IL-10 receptor α. TR1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. TR1 cells required IL-10 receptor signaling in order to activate p38 MAP kinase, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human TR1 cells. In conclusion TR1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that in order to optimize TR1 cell-based therapy, IL-10 receptor expression has to be taken into consideration.