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Whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice

ABSTRACT: To understand the whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice, the total RNA was extracted from indicated Treg cells using the Direct-zol miniprep kit (Zymo Research, R2060). The RNA samples were firstly enriched by Oligo(dT) magnetic beads and used to construct BGISEQ-500 libraries. RNA-seq libraries sequenced using the 50bp single-end protocol (in vivo isolated Treg cells) or 100bp paired-end protocol (TGF-β induced Treg cells) via the BGISEQ-500 sequencer per the manufacturer’s protocol. After filtering of adaptors and low quality reads, clean reads (>26 Million reads per sample for in vivo isolated Treg cells and >40 Million reads per sample for TGF-β induced Treg cells) are mapped to mouse reference genome using HISAT /Bowtie2 tool. Mapping results are stored in BAM files using SAMtools. Total read counts in gene level were summarized using featureCounts function in the Rsubread in R environment, with the R package biomaRt for gene and transcripts mapping. The differential expression (DE) genes were analyzed by DESeq2 package with default setting using total read counts as input, and the adjusted p value (padj) less than 0.05.

ORGANISM(S): Mus musculus

PROVIDER: GSE133019 | GEO | 2020/12/04

REPOSITORIES: GEO

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Project description:Regulatory T (Treg) cells play an important role in the induction and maintenance of peripheral tolerance. Treg cells also suppress a variety of other immune responses, including anti-tumor and alloimmune responses. We have previously reported that tumor-activated Treg cells express granzyme B and that granzyme B is important for Treg cell-mediated suppression of anti-tumor immune responses (GSE13409). Here, we report that allogeneic mismatch also induces the expression of granzyme B. Granzyme B-deficient mice challenged with fully mismatched allogeneic P815 mastocytoma cells have markedly improved survival compared to WT and other granzyme- or perforin-deficient mice, suggesting an immunoregulatory role for granzyme B in this setting. Treg cells harvested from the tumor environment of P815-challenged mice express granzyme B. Treg cells also express granzyme B in vitro during mixed lymphocyte reactions and in vivo in a mouse model of graft-versus-host disease (GVHD). However, in contrast to findings from our previously published tumor model, granzyme B is not required for the suppression of effector T cell (Teff) proliferation in in vitro Treg suppression assays stimulated by either Concanavalin A or allogeneic antigen presenting cells. Additionally, in an ex vivo assay, sort-purified in vivo-activated CD4+Foxp3+ Treg cells from mice with active GVHD -- under conditions known to induce granzyme B expression in Treg cells -- suppressed Teff cell proliferation in a granzyme B-independent manner. Adoptive transfer of naive granzyme B-deficient CD4+CD25+ Treg cells into a mouse model of GVHD rescued hosts from lethatlity equivalently to naive wild-type Treg cells. Serum analysis of GVHD-associated cytokine production in these recipients also demonstrated that Treg cells suppressed production of IL-2, IL-4, IL-5, GM-CSF, and IFN-gamma in a granzyme B-independent manner. In order to determine whether the context in which Treg cells are activated alters the intrinsic properties of Treg cells, we used Foxp3 reporter mice to obtain gene expression profiles of CD4+Foxp3+ Treg cells purifed from naive resting spleens, spleens from mice with acute GVHD, and from ascites fluid of mice challenged intraperitoneally with allogeneic P815 tumor cells. Unsupervised analyses revealed distinct activation signatures of Treg cells among the 3 experimental groups. Taken together, these findings demonstrate that granzyme B is not required for Treg cell-mediated suppression of GVHD, which is in contrast to what we have previously reported for Treg cell function in the setting of tumor challenge. Cell intrinsic differences could partially account for these differential phenotypes. These data also suggest the therapeutic potential of targeting specific Treg cell suppressive functions in order to segregate GVHD and graft-versus-tumor effector functions. Experiment Overall Design: Six replicates of Naive CD4+Foxp3+ Treg cells were purified from resting spleens, five replicates of allogeneic tumor-activated Treg cells and three samples of GVHD-activated Treg cells. Experiment Overall Design: Naive reps 1-3 are controls for the GVHD-activated samples. Experiment Overall Design: Naive reps 4-6 are controls for the Allogeneic tumor-activated samples.

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Whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice

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