Project description:Methods: Foxp3Cre-ERT2 x Tnfrsf1bFlox/Flox (icKO) and Foxp3Cre-ERT2 control (Ctrl) mice were immunized with MOG35-55 peptide to induce active EAE. From day 7 to 14, they were treated with Tamoxifen to delete TNFR2 only in Tregs. At day 14, Tregs and Tconvs were purified by flow cytometry from the inflamed central nervous system (CNS) or draining lymph nodes (dLN) for RNA extraction Results: In the CNS, TNFR2-deficient Tregs have lower expression of several Treg cell-signature genes and increased expression of genes normally expressed by Tconvs. The expression of 185 and 229 genes was respectively down- and up-regulated in Tconvs isolated from the CNS of mutant animals, compared to controls. Tconvs from icKO mice displayed a highly activated phenotype.
Project description:Foxp3 expressing regulatory T cells (Tregs) are the central regulator of immune homeostasis and tolerance. As it is believed that proper Treg function is compromised under inflammatory conditions, exploring a pathway that enhances Treg function is of great importance. In this study, we report that IL-27, an IL-12 family cytokine known to play both pro- and anti-inflammatory role in T cells, plays a pivotal role in Treg function to control T cell-induced colitis. Unlike WT Tregs capable of inhibiting colitogenic T cell expansion and inflammatory cytokine expression, IL-27R-deficient Tregs were unable to downregulate inflammatory T cell responses. Tregs stimulated with IL-27 expressed substantially enhanced suppressive function both in vitro and in vivo. IL-27 stimulation of Tregs induced expression of LAG3, a surface molecule implicated in negatively regulating immune responses. LAG3 expression in IL-27-stimulated Tregs was critical to mediate suppressive Treg function. Finally, human Tregs also displayed enhanced suppressive function and LAG3 expression in response to IL-27 stimulation. Taken together, our results highlight a novel function of the IL-27/LAG3 axis in Treg regulation of inflammatory responses in the intestine. FACS purified Foxp3+ Tregs were stimulated in the presence of media or IL-27 to compare IL-27 induced gene profiles. Four samples (media stimulated or IL-27-stimulated) were collected from four independent experiments. Genes altered by IL-27 treatment were compared to those of media stimulated Tregs.
Project description:Foxp3 expressing regulatory T cells (Tregs) are the central regulator of immune homeostasis and tolerance. As it is believed that proper Treg function is compromised under inflammatory conditions, exploring a pathway that enhances Treg function is of great importance. In this study, we report that IL-27, an IL-12 family cytokine known to play both pro- and anti-inflammatory role in T cells, plays a pivotal role in Treg function to control T cell-induced colitis. Unlike WT Tregs capable of inhibiting colitogenic T cell expansion and inflammatory cytokine expression, IL-27R-deficient Tregs were unable to downregulate inflammatory T cell responses. Tregs stimulated with IL-27 expressed substantially enhanced suppressive function both in vitro and in vivo. IL-27 stimulation of Tregs induced expression of LAG3, a surface molecule implicated in negatively regulating immune responses. LAG3 expression in IL-27-stimulated Tregs was critical to mediate suppressive Treg function. Finally, human Tregs also displayed enhanced suppressive function and LAG3 expression in response to IL-27 stimulation. Taken together, our results highlight a novel function of the IL-27/LAG3 axis in Treg regulation of inflammatory responses in the intestine.
Project description:Tregs and Tcons were isolated by flow cytometry from the spleen of naïve mice and from the spleen and central nervous system (brain + spinal cord) (CNS) of mice at the peak of experimental autoimmune encephalomyelitis (EAE) induced by immunization with MOG(35-55). Samples at EAE peak were collected at day 16-18 after immunization. Purity was >99.5%. Foxp3.eGFP mice (provided by A. Rudensky) were used to identify Tregs. A bin channel excluded CD11b+ cells during the sort. Mice were 6-10 weeks of age at the time of immunization. The goal was to identify genes differentially expressed between Tregs and Tcons from naive mice, and genes differentially expressed by these cells in the inflamed tissue (central nervous system) at the peak of the disease.
Project description:Comparison of gene expression profile of CD4+ CD25+, CD4+ CD25- CD45RBlow LAG3+ and CD4+ CD25- CD45RBlow LAG3- T cells. Naive CD4+CD25-CD45RBhigh T cells were used as a reference for pair comparison with values from the three other subsets.
Project description:Combined nivolumab (anti-PD1) and relatlimab (anti-LAG3) have shown enhanced effectiveness in melanoma patients. However, how these two receptors work together to hinder anti-tumor immunity remains unclear. Our study demonstrates that PD1/LAG3-deficient CD8+ T cells with more effectively clearing tumors and survive longer in melanoma mouse models. These PD1/LAG3-deficient CD8+ T cells have unique transcriptional profiles, broad TCR clonality, and enriched effector-like and interferon-responsive genes, leading to increased IFN gamma release indicating functionality. PD1 and LAG3 together drive T cell exhaustion, with a significant impact on TOX modulation. Mechanistically, autocrine IFN gamma signaling is crucial for enhancing anti-tumor immunity in PD1/LAG3-deficient CD8+ T cells, providing insights into the enhanced efficacy of combined PD1 and LAG3 targeting.
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: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.