TGFb dependent Treg conversion and the inhibition of conversion by IL6
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE21027 | GEO | 2010/03/25
SECONDARY ACCESSION(S): PRJNA125781
REPOSITORIES: GEO
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Project description:Analysis of CD4+ cells activated with anti-CD3 and cultured in the presence of IL2. The effects of TGFb and IL6 on Treg conversion were analyzed to discover methods of inhibiting Treg conversion and/or Treg functional inhibition. CD4+ T-cells were enriched from lymph nodes of Foxp3-GFP mice and cultured under the under the following conditions: 1) anti-CD3+IL2; 2) anti-CD3+IL2+TGFβ; 3) anti-CD3+IL2+TGFβ+IL6; and 4) anti-CD3+IL2+IL6. On day 4 cells were collected, stained with anti-CD4-PE, and sorted into CD4+FOXP3- and CD4+FOXP3+ populations (>95% purity). For each treatment condition T-effector cells and Tregs were labeled independently (either cy3 or cy5) and hybridized together for a two-color array experiment. Each treatment condition had dye swapped replicates, and a minimum of 3 replicate in total. Two color experiment. Four conditions : 1) anti-CD3+IL2, 3 samples 2) anti-CD3+IL2+TGFβ, 3 samples 3) anti-CD3+IL2+TGFβ+IL6, 4 samples 4) anti-CD3+IL2+IL6, 3 samples
2010-04-07 | E-GEOD-21027 | biostudies-arrayexpress
Project description:The tolerogenic anti-CD3 monoclonal antibodies (anti-CD3) are promising compounds for the treatment of type 1 diabetes (T1D). Anti-CD3 administration induces transient T-cell depletion both in preclinical and in clinical studies. Notably, said depletion mainly affects CD4+ but not CD8+ T cells. Moreover, T1D reversal in preclinical models is accompanied by the selective expansion of CD4+FOXP3+ T regulatory (Treg) cells, which are fundamental for the long-term maintenance of anti-CD3-mediated tolerance. The mechanisms that lead to this immune-shaping by affecting mainly CD4+ T effector cells while sparing CD4+FOXP3+ Treg cells have still to be fully elucidated. This study shows that CD3 expression levels differ from one T-cell subset to another. CD4+FOXP3- T cells contain higher amounts of CD3 molecules than do CD4+FOXP3+ and CD8+ T cells both in mice and in humans. Said differences may explain the anti-CD3-mediated immune resetting that occurs in vivo after anti-CD3 administration in mice. In addition, transcriptome analysis demonstrates that CD4+FoxP3+ Treg cells are significantly less responsive than CD4+FoxP3- T cells to anti-CD3 treatment at molecular levels. Thus, heterogeneity in CD3 expression likely confers the various T-cell subsets differing susceptibility to in vivo tolerogenic anti-CD3-mediated modulation. This data sheds new light on the molecular mechanism that underlies anti-CD3-mediated immune resetting, and thus may open new opportunities to improve this promising treatment.
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Project description:Goals and objectives of this study: to identify genes preferentially induced in human CD4+CD25hi Treg cells following T-cell activation with potential role for stabililization & maintenance of the regulatory program. Keywords: T-cell receptor stimulation, gene-regulation, comparative gene expression profiling Homo sapiens CD4+CD25- and CD4+CD25hi T cells were sorted from healthy individual donors and analyzed ex vivo (non-activated) or 1 day following T-cell receptor activation using anti-CD3/IL2 and anti-CD3/-CD28/IL2 (activated), respectively.
2009-04-20 | E-GEOD-13017 | biostudies-arrayexpress
Project description:Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells are likely to be involved, and we have shown a strong effect of anti-CD3 on homeostatic control of CD4+ FoxP3+ regulatory T (Treg) cells. To analyze the early consequences of anti-CD3 treatment, we sorted and profiled Treg and conventional CD4+ T (Tconv) cells in the first hours and days after anti-CD3 treatment of NOD mice. In practice, NOD mice carrying the Foxp3-GFP reporter were treated with anti-CD3 mAb KT3 (50 ug iv) and CD4+ T cells were sorted from pooled spleen and lymph nodes after 2, 8, 24 and 72 hrs, separating Treg and Tconv cells on the basis of GFP expression. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most antigen-induced responses. Most transcripts were similarly induced in Treg and Tconv cells, but several were differential, in particular those encoding the IL7 receptor (IL7R) and transcription factors Id2/3 and Gfi1, upregulated in Treg but repressed in Tconv cells. In parallel experiments, we tested the effect of soluble anti-CD3 added to cultures of fresh splenocytes, sorting Treg and Tconv cells at the same time points. Many of the anti-CD3 elicited changes, and of the differential response observed in vivo, were also observed in vitro. Two independent replicate series; Treg and Tconv samples abbreviated TR and TC, respectively. Keywords: Transcriptional activation, TCR All gene expression profiles were obtained from highly purified T cell populations sorted by flow cytometry. RNA from 5 x 104 cells was amplified, labeled, and hybridized to Affymetrix ST1.0 Gene arrays. Raw data were preprocessed with the RMA algorithm in GenePattern, and averaged expression values were used for analysis.
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Project description:Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells are likely to be involved, and we have shown a strong effect of anti-CD3 on homeostatic control of CD4+ FoxP3+ regulatory T (Treg) cells. To analyze the early consequences of anti-CD3 treatment, we sorted and profiled Treg and conventional CD4+ T (Tconv) cells in the first hours and days after anti-CD3 treatment of NOD mice. In practice, NOD mice carrying the Foxp3-GFP reporter were treated with anti-CD3 mAb KT3 (50 ug iv) and CD4+ T cells were sorted from pooled spleen and lymph nodes after 2, 8, 24 and 72 hrs, separating Treg and Tconv cells on the basis of GFP expression. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most antigen-induced responses. Most transcripts were similarly induced in Treg and Tconv cells, but several were differential, in particular those encoding the IL7 receptor (IL7R) and transcription factors Id2/3 and Gfi1, upregulated in Treg but repressed in Tconv cells. In parallel experiments, we tested the effect of soluble anti-CD3 added to cultures of fresh splenocytes, sorting Treg and Tconv cells at the same time points. Many of the anti-CD3 elicited changes, and of the differential response observed in vivo, were also observed in vitro. Two independent replicate series; Treg and Tconv samples abbreviated TR and TC, respectively. Keywords: Transcriptional activation, TCR
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Project description:CD4+ cells from Foxp3.eGFP mice containing Foxp3- Teff and Foxp3+ Treg cells were treated with anti-CD3/CD28 monoclonal antibodies or soluble OX40L and JAG1 for 3 days to induce TCR-dependent vs TCR-independent Treg proliferation. Untreated fresh CD4+ T-cells used as control. Post treatment T-cell proliferation was confirmed by Cell Trace violet dilution and Foxp3+ (Treg) and Foxp3-(Teff) were sorted. Differential gene expression profiling between Tregs and Teff cells among control, anti-CD3/CD28 and OX40L-JAG1 treated cultured was performed using affymetrix mouse gene 2.0 ST micro array. We used microarrays to detail the global programme of gene expression underlying Treg proliferation and identified distinct classes of up-regulated genes during this process under different methods of expansion.
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Project description:Treatment with anti-CD3 is a promising therapeutic approach for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells may be involved, but the evidence has been conflicting, and there is great uncertainty as to possible mechanistic connections. We investigated this issue in mice derived from the NOD model, which were engineered mice in which Treg populations were perturbed, or could be manipulated by acute ablation or transfer. The data highlighted the involvement of Foxp3+ cells in anti-CD3 action. Rather than a generic influence on all Treg cells, the therapeutic effect seemed to involve an striking expansion of previously constrained Treg cell populations; this expansion occurred not through conversion from Foxp3- Tconv cells but from a dramatic proliferative expansion. We found that Treg cells are normally constrained by TCR-specific niches in secondary lymphoid organs, and that intraclonal competition restrains their possibility for conversion and expansion in the spleen and lymph nodes, much as niche competition limits their selection in the thymus. The strong perturbations induced by anti-CD3 overcame these niche limitations, in a process dependent on receptors for trophic cytokines, interleukin-2 receptor (IL-2R) and IL-7R. Keywords: Treatment response
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Project description:Treatment with anti-CD3 is a promising therapeutic approach for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells may be involved, but the evidence has been conflicting, and there is great uncertainty as to possible mechanistic connections. We investigated this issue in mice derived from the NOD model, which were engineered mice in which Treg populations were perturbed, or could be manipulated by acute ablation or transfer. The data highlighted the involvement of Foxp3+ cells in anti-CD3 action. Rather than a generic influence on all Treg cells, the therapeutic effect seemed to involve an striking expansion of previously constrained Treg cell populations; this expansion occurred not through conversion from Foxp3- Tconv cells but from a dramatic proliferative expansion. We found that Treg cells are normally constrained by TCR-specific niches in secondary lymphoid organs, and that intraclonal competition restrains their possibility for conversion and expansion in the spleen and lymph nodes, much as niche competition limits their selection in the thymus. The strong perturbations induced by anti-CD3 overcame these niche limitations, in a process dependent on receptors for trophic cytokines, interleukin-2 receptor (IL-2R) and IL-7R. Keywords: Treatment response All gene expression profiles were obtained from highly purified T cell populations sorted by flow cytometry. RNA from 5 - 50 x 104 cells was amplified, labeled, and hybridized to Affymetrix M430v2. Raw data were preprocessed with the RMA algorithm in GenePattern, and averaged expression values were used for analysis.
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