Dataset Information

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

ABSTRACT:

Background

Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype.

Methods

We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell-specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed.

Results

CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC.

Conclusions

Although CD45RA Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4CD127CD45RA Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.

SUBMITTER: Arroyo Hornero R

PROVIDER: S-EPMC5265687 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Publications

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

Arroyo Hornero Rebeca R Betts Gareth J GJ Sawitzki Birgit B Vogt Katrin K Harden Paul N PN Wood Kathryn J KJ

Transplantation 20170201 2

<h4>Background</h4>Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD ...[more]

PMID: 28118317

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Project description:We have previously developed an approach that fractionates genomic DNA fragments depending on their CpG density (methyl-CpG-immunoprecipitation, MCIp), and adapted this approach to identify regions that are differentially methylated in the two closely related regulatory T-cells (Treg cells) and conventional T-cells (Tconv cells). Because Treg cells naturally occur at a relatively low frequency, we used a previously established protocol to expand Treg cells from a stable naÃ¯ve Treg population that is characterized by the co-expression of CD4, CD25 and CD45RA. We separated gDNA of both expanded T cell lineages (Tregexp and Tconvexp) into unmethylated (CpG) and methylated pools (mCpG) using MCIp and compared cell type-specific differences in DNA methylation by co-hybridization of the two umethylated or the two methylated DNA subpopulations of Treg and Tconv, respectively, to these locus-wide custom tiling arrays. As enriched DNA-fragments from a cell type in the methylated fraction should be depleted in the unmethylated fraction, the signal intensities in CpG pool and mCpG pool hybridizations should complement themselves (âMirror-Imageâ approach) and thereby allow the identification of differentially methylated regions (DMR). Because we expected to find lineage-specific methylation differences with greater probability in regions associated with differential transcriptional activity, we limited our analysis to gene loci that showed cell type-specific gene expression in Treg versus Tconv cells plus a handful of control regions that were equally expressed in both cell types. The microarray used in this study covered 12 megabases of the human genome and contained 69 regions (with a median size of 100.000 kb) and 128 proximal promoter regions and 181 genes, which included a number of well known and functionally relevant genes like CD40LG, IFNG, FOXP3, IL2RA and CTLA4. Keywords: MCIp-on-chip; comparative genomic hybridization With MCIp gDNA from Treg or Tconv cells was separated into hypo- and hypermethylated pools. On each array, wheather the two hypomethylated fractions- one from Treg, the other from Tconv cells- or the two hypermethylated fractions were cohybridized. Two biological replicates.

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Project description:OBJECTIVE:Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127(low) FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin-17 (IL-17)-producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function. METHODS:The presence and phenotype of CD4+CD45RO+CD25+CD127(low) T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence-activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti-CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme-linked immunosorbent assay, and proliferation assays. RESULTS:In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127(low) Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro-activated monocytes induced an increase in the percentage of IL-17-positive, interferon-? (IFN?)-positive, and tumor necrosis factor ? (TNF?)-positive Treg cells as well as IL-10-positive Treg cells. The observed increase in IL-17-positive and IFN?-positive Treg cells was driven by monocyte-derived IL-1?, IL-6, and TNF? and was mediated by both CD14+CD16- and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL-17 production. CONCLUSION:Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

Dataset Information

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

Background

Methods

Results

Conclusions

Publications

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

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