Project description:To analyse the influence of pentanoate on the differentiation of naive CD4 T cells under Th17 inducing conditions, murine CD4 T cells were isolated from spleen and lymphnodes of FIR/TIGER mice (IL-10 (GFP) - Foxp3 (RFP) - Reporter mice). The T cells were kept for 3 days under Th17 inducing conditions either with 4 mM pentanoate or without pentanoate (control).
Project description:miRNA expression profiling in highly purified murine CD4+ Tconv and Treg cells. FoxP3-GFP-hCre1a(high) reporter mice were used to separate both populations based on surface markers and presence or absence of GFP.
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.
Project description:Naturally occurring CD25+CD4+ regulatory T cells (T reg cells) are currently intensively characterized because of their major importance in modulating host responses to tumors and infections, in preventing transplant rejection, and in inhibiting the development of autoimmunity and allergy. Originally, CD4+ T reg cells were identified exclusively by the constitutive expression of CD25, and many in vivo experiments have been performed using depleting antibodies directed against CD25. However, both the existence of CD25– T reg cells, especially within peripheral tissues, as well as the expression of CD25 on activated conventional T cells, which precludes discrimination between T reg cells and activated conventional T cells, limits the interpretation of data obtained by the use of anti-CD25 depleting antibodies. The most specific T reg cell marker currently known is the forkhead box transcription factor Foxp3, which has been shown to be expressed specifically in mouse CD4+ T reg cells and acts as a master switch in the regulation of their development and function. To address the question of the in vivo role of T reg cells in immunopathology, we have generated bacterial artificial chromosome (BAC)–transgenic mice termed depletion of regulatory T cell (DEREG) mice, which express a diphtheria toxin receptor (DTR) enhanced GFP (eGFP) fusion protein under the control of the foxp3 locus, allowing both detection and inducible depletion of Foxp3+ T reg cells. The gene expression profile of both CD4+eGFP+FoxP3+ and CD4+eGFPnegFoxP3neg cells isolated from DEREG mice was here analyzed by micro array. Keywords: DEREG, FoxP3, FoxP3-EGFP, mouse, regulatory T cell, CD4 CD4+GFP+FoxP3+ and CD4+GFPnegFoxP3neg cells were isolated from DEREG mice by negative selection of CD4+ T cells (Invitrogen Kit) and subsequent FACS sorting for GFP+ and GFPneg cells. Purity was greater than 99 %. cRNA was prepared according to the Affymetrix Labeling Protocol, fragmented and hybridized to Affymetrix GeneChip Mouse Genome 430.
Project description:There is much controversy about the role of T-regulatory cells (Treg) in human colon cancer. High densities of tumor-infiltrating Treg can correlate with better or worse clinical outcomes depending on the sutdy. Treg have potent anti-inflammatory functions that have been shown to control cancer progression. However, Treg isolated from patient with colon cancer or in mouse models of polyposis do not have the ability to suppress inflammation and instead promote cancer. Gene expression was preformed to determine differences between Treg isolated from healthy mice and Treg isolated from polyp-ridden mice. Treg (CD4+Foxp3-GFP+) and CD4+ non-Treg (CD4+Foxp3-GFP-) were isolated from various organs of healthy Foxp3-GFP reporter mice or polyp-ridden Foxp3-GFPxAPCΔ468 reporter mice
Project description:To analyze gene expression in in regulatory T cell precursors that develop in the absence of a functional Foxp3 protein as compared to that of normal regulatory T cells Experiment Overall Design: Murine wild-type and mutant (delta) Foxp3 alleles were tagged with EGFP reporters. CD4 positive T cells expressing the respective tagged Foxp3 allele (Foxp3/EGFP and delta Foxp3/EGFP) were isolated from hemizygous male mice by cell sorting using FACS. CD4 positive cells that did not express the EGFP tag were a isolated. Total RNA was prepared and used in the array studies.
Project description:miRNA expression profiling in highly purified murine CD4+ Tconv and Treg cells. FoxP3-GFP-hCre1a(high) reporter mice were used to separate both populations based on surface markers and presence or absence of GFP. Two-condition experiment, Tconv vs. Treg. Biological replicates: 1 Tconv, 1 Treg, purified from the same pooled mice. One replicate on 1 array.
Project description:iTreg cells from Tbmc mLN mice treated with one week of 1% Oral Ova were compared to Total Treg from WT mice. Tbmc mice were treated with 1% Oral Ova and Foxp3 GFP+ CD4+ cells were sorted from mLN. CD4+ Foxp3+ cells were also sorted from WT Foxp3 GFP Balb/c mice.
Project description:Single-cell RNAseq (10x Genomics) analysis of mouse splenic CD4+ T cells in WT and ∆Foxp3 mice and in WT/∆Foxp3 bone marrow chimeras. Mouse CD4+T cells in 21-day-old male WT and ∆Foxp3 mice were isolated from spleen by flow cytometry as DAPI–TCRβ+CD4+ cells for 10x Genomics Single Cell 3′ Reagent Kit (V2 chemistry, one sample per channel). For bone marrow chimera experiment: 7 week-old CD45.2-recipient mice were irradiated with 1000 Rad, reconstituted with 4 million CD3-depleted bone marrow cells: 50% CD45.1 x Foxp3-IRES-GFP (WT, 21d-old male) and 50% Foxp3DeltaEGFPiCre/RFP x ROSA-YFP x CD45.1/2 (scurfy, 21d-old male). 10 weeks later, spleen were harvested and tagged using a different Hashtags fo each mouse. ∆Foxp3 CD4+ T cells were sorted as DAPI–TCRb+CD4+CD45.1+CD45.2+. WT CD4+ T cells were sorted as DAPI–TCRb+CD4+CD45.1+CD45.2–. Control WT DAPI–TCRb+CD4+GFP+ Treg cells and GFP- Tconvs cells were also tagged and sorted. Samples with different hastags were pooled before single cell encapsulation using 10x Genomics Single Cell 3′ Reagent Kit (V3 chemistry).
Project description:Subcutaneous tumors from mock transfected LLC or b8 transfected LLC were established on flanks of C57B/6 FoxP3-Ires GFP mice. Mice were treated with isotype control antibodies or anti-b8 and Tregs (CD4+ GFP+) were sorted, pooled and total RNA isolated for RNAseq.