IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo - PubMed (original) (raw)

. 2006 Sep 1;108(5):1571-9.

doi: 10.1182/blood-2006-02-004747. Epub 2006 Apr 27.

Erik A Nelson, Mehrdad Mohseni, Fabrice Porcheray, Haesook Kim, Despina Litsa, Roberto Bellucci, Elke Raderschall, Christine Canning, Robert J Soiffer, David A Frank, Jerome Ritz

Affiliations

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

Emmanuel Zorn et al. Blood. 2006.

Abstract

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

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Figures

Figure 1.

Figure 1.

Recombinant IL-2 regulates FOXP3 expression in CD4+CD25+ Tregs. (A) CD4+CD25- (▪) and CD4+CD25+ (formula image) T cells purified from healthy individuals were cultured for 6 hours in the presence of recombinant IL-2 with or without anti-CD3 stimulation. FOXP3 expression was then assessed by quantitative PCR. (B) Purified CD4+CD25- and CD4+CD25+ cells were cultured for 18 hours with or without IL-2. Levels of FOXP3 protein were subsequently assessed by Western blotting. Control blotting of α-tubulin is also shown in this figure. (C) Purified CD4+CD25- (□) or CD4+CD25+ (○) cells were cultured for 6 hours with varying concentrations of IL-2 and then assessed for FOXP3 expression by quantitative PCR.

Figure 2.

Figure 2.

IL-2 signaling targets FOXP3 through STAT3 and STAT5 proteins. (A) The location of the human FOXP3 gene on chromosome X is illustrated at top of figure. The middle box shows the proximal region of FOXP3 including exons 1 to 4 as obtained from the University of California Santa Cruz genome assembly Web site and corresponds to a segment located between bases 48 870 000 and 48 878 500 (

http://genome.ucsc.edu

). The degree of interspecies conservation of the DNA within this segment is represented by peak histograms. The bottom box illustrates a segment of the first intron of FOXP3 located between bases 48 873 406 and 48 873 819, which includes 2 consensus STAT-binding motifs (in blue). The red sequence identifies mutations of the 2 STAT-binding sites introduced in the luciferase constructs. (B) Plasmid constructs were generated that contained wild-type (WT) FOXP3 intronic segment encompassing the 2 STAT motifs upstream of the luciferase cDNA or a mutated version of this segment with disrupted STAT elements. WT (▪) as well as mutated constructs (formula image) were cotransfected in 293 cells together with a plasmid containing a constitutively activated form of STAT3, STAT3C, or a constitutively activated form of STAT5, STAT5a1*6. Transcriptional activity was assessed after 24 hours. Error bars indicate SD.

Figure 3.

Figure 3.

Low-dose administration of IL-2 induces the expansion of Tregs in vivo. (A) Percent CD4+CD25+ in blood CD3+ cells was measured by flow cytometry for each patient in samples collected before and 4 to 11 weeks after beginning daily IL-2 therapy. Results are presented as percent change in the frequency of CD4+CD25+ in total CD3+ T cells between the 2 samples. formula image indicates patients with solid tumors. ▪ indicates patients who received IL-2 after allogeneic T-cell-depleted HSCT. Flow cytometry values for one patient (number 17) were not available. (B) FOXP3 expression was assessed by quantitative PCR assays using the same patient samples as mentioned in panel A. Percent change following IL-2 therapy was calculated using values obtained from samples before and after treatment. (C) Intracellular staining of patient 9′s PBMCs collected before and during IL-2 therapy with anti-FOXP3 mab. Cells were also stained with anti-CD4 mab. Events were acquired after gating on lymphocytes.

Figure 4.

Figure 4.

Effect of IL-2 on Tregs in vivo is transient. (A) Percent changes in frequencies of CD4+CD25+/CD3+ were calculated using phenotypic values obtained from samples collected during IL-2 treatment and samples collected at later time points, 4 to 10 months after IL-2 therapy (Table 1). Patients with solid tumors are represented with formula image; patients after allogeneic HSCT are represented with ▪. (B) FOXP3 expression was assessed in the same samples collected during IL-2 treatment and after completion of therapy (Table 1). Percent changes were calculated using values obtained with these 2 sets of samples.

Figure 5.

Figure 5.

Low-dose IL-2 induces the expansion of FOXP3-CD56+ cells in vivo. (A) Percent change in frequencies of CD56+ cells in total blood lymphocytes was determined using values obtained by flow cytometry from samples collected before and during IL-2 treatment. Values for one patient (number 11) were not available. (B) CD4+ T cells and CD3-CD56+ NK cells were purified by cell sorting from 5 patient samples collected during IL-2 treatment. FOXP3 expression was assessed by quantitative PCR in both cell subsets for the 5 patients.

Figure 6.

Figure 6.

Treatment with 5-aza-2′-deoxycytidine allows IL-2 induction of FOXP3 expression in NK cells. (A) Human NKL cells were treated with 2 different doses of 5-aza-2′-deoxycytidine for 3 days and then incubated with or without IL-2 for 18 hours. FOXP3 expression was then assessed by quantitative PCR. (B) Primary CD3-CD56+ NK cells were isolated from the blood of a healthy donor and treated with 5-aza-2′-deoxycytidine for 3 days at 10 μM. Cells were then incubated with or without IL-2 for an additional 18 hours before FOXP3 gene expression was tested by quantitative PCR.

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