Project description:We identified DCIR2+DCs but not DEC205+DCs as able to induce peripheral T cell tolerance in pre-diabetic autoimmune NOD mice. To determine what distinct genetic programs are elicited in the auto-reactive CD4 T cells early after stimulation by these two DC subsets, we utilized adoptive transfer of BDC2.5 CD4 T cells into NOD mice, which were then given chimeric antibody to deliver the beta-cell specific antigen to either DCIR2+DCs or DEC205+DCs, leading to BDC2.5 CD4 T cell specific stimulation in vivo. The analysis shows that the negative transcriptional factor Zbtb32 (ROG) is up-regulated more in BDC2.5 CD4 T cells after stimulated with a antigen via DCIR2+DCs presentation, compared with DEC205+DCs, suggesting the involvement of Zbtb32 in DCIR2+DCs-mediated auto-reactive T cell tolerance in disease ongoing NOD mice. The BDC2.5 CD4 T cells after 14 hour of injection with a 100 ng of anti-DCIR2-BDC, anti-DEC205-BDC antibody, or PBS as a control to NOD mice, were sorted by BD FACSAria and their expression profile was analyzed using Affymetrix chips.
Project description:We identified DCIR2+DCs but not DEC205+DCs as able to induce peripheral T cell tolerance in pre-diabetic autoimmune NOD mice. To determine what distinct genetic programs are elicited in the auto-reactive CD4 T cells early after stimulation by these two DC subsets, we utilized adoptive transfer of BDC2.5 CD4 T cells into NOD mice, which were then given chimeric antibody to deliver the beta-cell specific antigen to either DCIR2+DCs or DEC205+DCs, leading to BDC2.5 CD4 T cell specific stimulation in vivo. The analysis shows that the negative transcriptional factor Zbtb32 (ROG) is up-regulated more in BDC2.5 CD4 T cells after stimulated with a antigen via DCIR2+DCs presentation, compared with DEC205+DCs, suggesting the involvement of Zbtb32 in DCIR2+DCs-mediated auto-reactive T cell tolerance in disease ongoing NOD mice. The BDC2.5 CD4 T cells after 14 hour of injection with a 100 ng of anti-DCIR2-BDC, anti-DEC205-BDC antibody, or PBS as a control to NOD mice, were sorted by BD FACSAria and their expression profile was analyzed using Affymetrix chips. NOD mice with transferred BDC2.5 T cell were injected with 100 ng of anti-DCIR2-BDC (NOD_DCIR2-BDC), anti-DEC205-BDC antibody (NOD_DEC-BDC), or PBS (NOD_PBS) as a control. After 14 hours, BDC2.5 CD4 T cells were sorted by BD FACSAria and their expression profile was analyzed using Affymetrix microarray chips. Each sample has a biological duplicate. Raw data were preprocessed with the RMA algorithm in Partek, and averaged expression values were used for analysis.
Project description:Total pancreatic RNA was isolated from 3 week old NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid animals by GITC method. Targets were produced using standard Affymetrix procedures from about 10ug total RNA. The data from NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid Affymetrix MGU74Av2 cel files was converted into Robust Multi Array (RMA) text file for analysis using GeneSpring 6.1 Keywords: other
Project description:Comparison of gene expression between T regulatory and T effector cells isolated from the pancreatic lesion of 3-4 wk old BDC2.5 tg NOD mice Keywords: cell type comparison
Project description:Total pancreatic RNA was isolated from 3 week old NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid animals by GITC method. Targets were produced using standard Affymetrix procedures from about 10ug total RNA. The data from NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid Affymetrix MGU74Av2 cel files was converted into Robust Multi Array (RMA) text file for analysis using GeneSpring 6.1
Project description:Abstract Two major dendritic cell (DC) subsets have been described in the islets of mice: The immunogenic CD8α-CD11b+ DCs and the tolerogenic CD8α+CD103+ DCs. We have recently reported on reduced numbers of the minor population of tolerogenic CD8α+CD103+ DCs in the pancreas of 5 week old pre-diabetic non-obese diabetic (NOD) mice. Aim: To analyze also the larger subset of CD11c+CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice 1) for maturation and tolerance inducing molecules found abnormally expressed on CD8α+CD103+ DCs, and 2) for genome-wide gene expression to further elucidate abnormalities in underlying gene expression networks. Methods: CD11c+CD8α- DCs were isolated from 5 week old C57BL/6 and NOD pancreas. Expression of cell surface markers including CD86, CCR5, CD11b, CD103, Clec9a, CD24 and CD200R3 were measured by FACS. Genome-wide gene expression by microarray was assessed during the steady state and after in vitro LPS stimulation. Results: The steady state pancreatic CD11c+ CD8α- DCs during the pre-diabetic stage showed: 1) A reduced expression of several gene networks important for the prime functions of the cell, such as for cell renewal, immune stimulation and immune tolerance induction, for migration and for the provision of growth factors for beta cell regeneration. This general deficiency state was corroborated by a reduced in vivo proliferation (BrdU incorporation) of the cells and the reduced expression in FACS analysis of CD86, CCR5, CD103, Clec9a, CD24 and CD200R3 on the cells. 2) A hyper reactivity of these cells to LPS correlated with an enhanced pro-inflammatory state characterized by altered expression of a number of classical pro-inflammatory factors and cytokines. Conclusion: The NOD CD11c+CD8α- DCs seem to be Janus-faced depending on the conditions: Deficient in steady state with reduced immune stimulation capabilities also for tolerance induction; over-inflammatory with a molecular profile suggesting a preferential stimulatory capacity for Th1 cells when encountering a Pathogen-Associated Molecular Pattern (PAMP) in the form of LPS. We used microarray gene expression analysis to explain the abnormal expression of several cell surface markers involved in tolerace, migration and maturation in the steady-state and to measure the effect of a PAMP such as LPS We isolated RNA from FACS sorted CD11c+CD8α- DCs in 10 pooled pancreases from pre-diabetic NOD and non-diabetic C57BL/6 mice at 5 weeks. In addition, we treated in another experiment the isolated pancreas DCs with LPS (and PBS), incubated for 18h and measured gene expression. We compared gene expression between strains NOD vs C57BL/6 under steady-state and after in-vitro LPS/PBS stimulation.
Project description:BDC2.5/NOD mice were treated with cyclophosphamide to induce type 1 diabetes. Their pancreatic islets were analyzed before treatment (Day 0) and as treatment progressed (Days 1 through 3) Keywords = type I diabetes Keywords = cyclophosphamide Keywords = BDC2.5 Keywords = NOD Keywords = pancreas Keywords = islets Keywords: time-course
Project description:How do Treg cells control an autoimmune lesion? We depleted Foxp3+ Treg cells in autoimmune prone TCR transgenic BDC2.5 mice on the NOD background Kinetic of gene-expression changes in CD4 T cells isolated for pancreas and pancreas draining lymph node (PLN) of Foxp3-DTR.BDC2.5/NOD mice after Treg cell ablation Keywords: T cell activation/ differentiation after Treg depletion timecourse
Project description:This SuperSeries is composed of the following subset Series: GSE34934: Expression data from BDC2.5 TCR Tg, preselected Rag-/-.B6 and Rag-/-.NOD.H2b thymocytes upon antigenic stimulation GSE34935: Expression data from BDC2.5 TCR Tg thymocytes on B6g7 and NOD background Refer to individual Series