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Kinetic analysis of the response to anti-CD3 in conventional and regulatory CD4+ T cells, in mice and in vitro.

ABSTRACT: 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

ORGANISM(S): Mus musculus

PROVIDER: GSE48210 | GEO | 2013/07/01

SECONDARY ACCESSION(S): PRJNA209205

REPOSITORIES: GEO

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Kinetic analysis of the response to anti-CD3 in conventional and regulatory CD4+ T cells, in mice and in vitro.

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.

2013-07-01 | E-GEOD-48210 | biostudies-arrayexpress

Expression Data of Treg and Tconv Cells from FoxP3-GFP Chimeric and FoxP3-ires-GFP B6 and NOD Mice

Project description:The aim of this study was to quantify the impact of chimeric Foxp3-GFP protein on the Treg cell transcriptional program. Duplicate samples of Tconv (CD3+CD4+GFP-) and Treg (CD3+CD4+GFP+) splenocytes were double-sorted to achieve > 99.0% purity, from 6 weeks old male Foxp3-Fusion-GFP and Foxp3-ires-GFP mice of both B6 and NOD backgrounds. Following cell sorting into Trizol, RNA was purified, labeled and hybridized to Affymetrix arrays.

2012-04-26 | E-GEOD-37605 | biostudies-arrayexpress

Comparison of expression profiles of Foxp3(+)epigenetics(-) T cells, Foxp3(-)epigenetics(+) T cells, and Foxp3(+)epigenetics(+) T cells

Project description:Analysis of Foxp3(+)epigenetics(-) T cells, Foxp3(-)epigenetics(+) T cells, and Foxp3(+)epigenetics(+) T cells. Results indicate regulatory T cell (Treg) ontogenesis requires two independent processes, expression of the transcription factor Foxp3 and establishment of Treg epigenetic programs induced by T cell receptor (TCR) stimulation. GFP+CD4+ and GFP-CD4+ splenocytes were sorted from DEREG and DEREG/Scurfy mice. These cells were activated with anti-CD3/CD28 antibodies, and then transduced with Foxp3-expressing retrovirus (pGCSamIN, NGFR marker). NGFR+ T cells sorted were subjected to microarray analysis (Affymetrix, mouse genome 430 2.0 array). To normalize the experimental conditions, Tregs (GFP+ T cells from DEREG) and Tconv (GFP- T cells from DEREG) were also activated and transduced with empty vector. Two replicates each.

2012-11-05 | E-GEOD-25252 | biostudies-arrayexpress

TGFb dependent Treg conversion and the inhibition of conversion by IL6

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+TGFÎ²; 3) anti-CD3+IL2+TGFÎ²+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+TGFÎ², 3 samples 3) anti-CD3+IL2+TGFÎ²+IL6, 4 samples 4) anti-CD3+IL2+IL6, 3 samples

2010-04-07 | E-GEOD-21027 | biostudies-arrayexpress

TGFb dependent Treg conversion and the inhibition of conversion by IL6

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.

2010-03-25 | GSE21027 | GEO

Gene expression profiles of CD4+CD25+ Tregs from NOD and B6.H2g7 mice

Project description:The NOD (nonobese diabetic) mouse strain develops a characteristic autoimmune syndrome that closely resembles human type I diabetes. It has been suggested that NOD mice exhibit both numerical deficiency in CD4+CD25+ regulatory T cells (Treg) and reduced suppressive activity. We compared sorted CD4+CD25+ Tregs from the spleens of 6-7 week-old female NOD and nondiabetic B6.H2g7 mice. Tregs were 93±2% and 95±1% Foxp3+ in NOD and B6.H2g7 cells, respectively, on post-sort reanalysis. "Conventional" CD4+CD25- T cells (Tconv) are included as reference populations. Surprisingly, Treg "signature" is similar between the two strains, with only a few probesets that subtly deviate. Keywords: Cell population comparison from two mouse strains. For each strain (NOD and B6.g7), we analyzed two populations: CD4+CD25+ Treg and CD4+CD25- Tconv cells, for a total of four distinct populations. RNA from three mice were pooled for each replicate; there are three independent replicates for each population. After RMA normalization, intensity values were averaged across the three replicates and analyzed. We calculated the ratio of Treg/Tconv intensity values for each strain and compared the results.

2008-12-19 | E-GEOD-6813 | biostudies-arrayexpress

Anti-CD3 therapy permits regulatory T cells to surmount T cell receptor-specified peripheral niche constraints

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.

2010-07-29 | E-GEOD-22527 | biostudies-arrayexpress

Immune Gene Expression Profile of Conventional and Regulatory T cells

Project description:Understanding human regulatory T cells (Tregs) heterogeneity may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Treg (tTreg) and CD4+FOXP3+Helios- peripherally-induced Treg (pTreg), followed by comparison to CD4+FOXP3-Helios- T conventional (Tconv) cells. This analysis revealed that the coinhibitory receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) was highly expressed on tTreg. In this study CD4 T cells were stained for the Treg-associated transcription factors FOXP3 and Helios, and subsequently FACS sorted to yield three populations: tTreg (CD4+FOXP3+Helios+), pTreg (CD4+FOXP3+Helios–) and the reference population Tconv (CD4+FOXP3–Helios–). A direct transcriptional profile was obtained from the recovered RNA from the populations defined as tTreg, pTreg, and Tconv.

2015-04-30 | E-GEOD-61834 | biostudies-arrayexpress

Heterogeneous CD3 expression levels in differing T-cell subsets account for in vivo anti-CD3-mediated immune resetting

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.

2015-01-19 | E-MTAB-2437 | biostudies-arrayexpress

Gene profiling data of CD4+ T cells doubly transduced with EOS+LEF1 or GATA1+SATB1

Project description:The transcription factor FoxP3 partakes dominantly in the specification and function of FoxP3+ CD4+ T regulatory cells (Tregs), but is neither strictly necessary nor sufficient to determine the characteristic Treg transcriptional signature. Computational network inference and experimental testing assessed the contribution of several other transcription factors (TFs). Enforced expression of Helios or Xbp1 elicited specific signatures, but Eos, Irf4, Satb1, Lef1 and Gata1 elicited exactly the same outcome, synergizing with FoxP3 to activate most of the Treg signature, including key TFs, and enhancing FoxP3 occupancy at its genomic targets. Conversely, the Treg signature was robust to inactivation of any single cofactor. A redundant genetic switch thus locks-in the Treg phenotype, a model which accounts for several aspects of Treg physiology, differentiation and stability. To study the impact of the combination of two transcription factors on the expression of the Treg transcriptional signature, CD4+ Tconv cells activated with anti-CD3+CD28 beads were retrovirally transduced with cDNAs encoding EOS and LEF1, or GATA1 and SATB1. The cells were then sorted into Trizol, and RNA was purified, labeled and hybridized to Affymetrix arrays.

2012-08-23 | E-GEOD-40277 | biostudies-arrayexpress

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