Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads. We collected peripheral blood from each human donor. We isolated peripheral blood mononuclear cells by Ficoll, and negatively selected for CD4+ T cells using RosettaSep. We then either left cells unstimulated or stimulated them with beads conjugated with anti-CD3 and anti-CD28. Cells from 15 individuals were harvested at up to 3 time points (0hr, 4hr or 48hr), lysed and RNA isolated to be profiled on microarray.
Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of 348 healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads. We collected peripheral blood from each human donor. We isolated peripheral blood mononuclear cells by Ficoll, and negatively selected for CD4+ T cells using RosettaSep. We isolated peripheral blood mononuclear cells by Ficoll, and negatively selected for CD4+ T cells using RosettaSep. We then either left cells unstimulated or stimulated them with beads conjugated with anti-CD3 and anti-CD28 either without additional cytokines, or with IFNb, or with Th17 cocktail. Cells were harvest at 0hr, 4hr (anti-CD3/CD28 +/- IFNb) or 48hr (anti-CD3/CD28 +/- Th17), lysed and RNA isolated to be profiled on Nanostring.
Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads alone or with IFNb or Th17 polarizing cytokines. We collected peripheral blood from each human donor. We isolated peripheral blood mononuclear cells by Ficoll, and negatively selected for CD4+ T cells using RosettaSep. We then either left cells unstimulated or stimulated them with beads conjugated with anti-CD3 and anti-CD28 either without additional cytokines, or with IFNb, or with Th17 cocktail. Cells were harvest at up to 8 time points (0hr, 45min, 2hr, 4hr, 10hr, 24hr, 48hr and 72hr), lysed and RNA isolated to be profiled on microarray.
Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of 348 healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads.
Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads alone or with IFNb or Th17 polarizing cytokines.
Project description:This SubSeries in the ImmVar project investigates the response of selected genes in T cells from healthy human individuals to ascertain the impact of genetic or non-genetic variation on T cell activation parameters. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads alone or with IFNb or Th17 polarizing cytokines.
Project description:To indentify the effect of JPH203 on the gene expression in activated T cells, microarray analysis was performed using activated human T cells derived from peripheral blood. Human CD4+ T cells from peripheral blood in healthy volunteer were activated with anti-CD3 and anti-CD28 in the presence of 1uM JPH203 for 3days. RNA was extracted and gene expression was compared between JPH203-treated cells and control cells. Gene epression of human peripheral blood CD4+ T cells activated with anti-CD3 and anti-CD28 in the presence of 1uM JPH203 for 3days was measured.
Project description:Disorders in regulatory T cell (Treg) function can result in the break-down of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of Treg is of great relevance. We used Treg from individuals carrying the C77G polymorphism as human models to study the role of CD45 molecules. The C77G mutation occurs with low frequency in healthy individuals and prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4+CD25highFoxp3+ Treg from carriers of C77G strongly expressed CD45RA isoforms whereas they were almost absent in cells from individuals with wild-type CD45. The expression patterns of CD45RB and CD45RC did not differ between C77G and control Treg but CD45R0 molecules were reduced in C77G cells. Resting C77G Treg showed diminished upregulation of activation markers and a reduced proliferative potential when stimulated with anti-CD3/TcR or anti-CD3/CD28 mAb suggesting decreased responsiveness to activating stimuli. This is in line with the observation that CD3/CD28-mediated activation of in vitro expanded Treg resulted in lower levels of TGF-? and IL-10 transcripts in C77G cells compared to controls. Furthermore, microarray studies revealed distinct gene expression patterns in Treg from C77G carriers and individuals with wild-type CD45. In addition, the capacity to suppress proliferation of conventional CD4+ T cells was reduced in C77G Treg. These data suggest that the changes in CD45 isoform combination resulting from C77G alter the responsiveness of Treg to CD3/TcR- and/or CD28-mediated signalling. Targeting of CD45 isoforms might be an approach to modulate Treg function. Total RNA was isolated from expanded and stimulated (2h with 1:5 CD3/CD28 beads) Treg of wild-type and C77G carriers using RNeasy Plus Micro Kit (Qiagen). Two pools of three wild-type individuals and three C77G individuals were prepared using equal amounts of RNA from each single donors. The two samples were each hybridised twice on an Agilent human Gene Expression Microarray 4x44K. cell type comparison
Project description:To further develop our gene expression approach to treatment of autoimmunity, we have employed whole genome microarray expression profiling as a discovery platform to identify genes differentially expressed upon immunosuppressor treatment responsible for inhibition of T cell effector functions. Human CD4+CD25- cells from healthy donors were pretreated or not with rapamycin and stimulated with anti-CD3 and anti-CD28 for 12h.
Project description:Comparison of total RNA-seq data from ex vivo unstimulated and stimulated (with anti-CD3/CD28) cells from two primary cell models of HIV latency (resting-cell and wild-type virus models) and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals (ex vivo cells). Two donors were analyzed per model.
