Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease

Abstract

CD4(+)Foxp3(+) regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation. We recently reported that daily administration of low-dose interleukin-2 (IL-2) induces selective expansion of functional Tregs and clinical improvement of chronic graft-versus-host disease (GVHD). To define the mechanisms of action of IL-2 therapy, we examined the immunologic effects of this treatment on homeostasis of CD4(+) T cell subsets after transplant. We first demonstrated that chronic GVHD is characterized by constitutive phosphorylation of signal transducer and activator of transcription 5 (Stat5) in conventional CD4(+) T cells (Tcons) associated with elevated amounts of IL-7 and IL-15 and relative functional deficiency of IL-2. IL-2 therapy resulted in the selective increase of Stat5 phosphorylation in Tregs and a decrease of phosphorylated Stat5 in Tcons. Over an 8-week period, IL-2 therapy induced a series of changes in Treg homeostasis, including increased proliferation, increased thymic export, and enhanced resistance to apoptosis. Low-dose IL-2 had minimal effects on Tcons. These findings define the mechanisms whereby low-dose IL-2 therapy restores the homeostasis of CD4(+) T cell subsets and promotes the reestablishment of immune tolerance.

Figure 1. Phenotypic and functional characterization of human Treg and Tcon after in vitro cytokine activation

(A) Representative lymphocyte gate for identification of CD4 T cell subsets. Within the CD4 T cell gate, Treg are identified as CD25^med-highCD127^low and Tcon are identified as CD25^neg-lowCD127^med-high. (B) Gated CD4 T cell subsets were examined for intracellular Foxp3 expression. Representative data is shown. Closed histogram represents isotype control. Blue and red histograms depict Tcon and Treg, respectively. (C) Tregor Tcon isolated from peripheral blood were cultured with responder Tcon from the same donor and stimulated with irradiated allogeneic PBMCs for 5 days. Method for calculating percentage suppression of proliferation is described in Methods. Data are representative of 5 independent experiments. (D) Purified Treg and Tcon were cultured for 15 minutes in various concentrations of IL-2, IL-7 and IL-15. The level of intracellular Stat5 phosphorylation (pStat5) was determined by flow cytometry. Cytokine dose-dependent phosphorylation of Stat5 in each CD4 T cell subset is shown. Data are representative of 3 independent experiments. (E) Purified Treg and Tcon were cultured in low (10 IU/ml) or high (100 IU/ml) concentrations of IL-2 for 5 days and cell proliferation was measured by thymidine incorporation. Data are representative of 3 independent experiments.

Figure 2. Altered cytokine milieu and activation of intracellular Stat5 in chronic GVHD

(A–B)Lymphocyte counts and CD4 T cell counts in peripheral blood from healthy donors and chronic GVHD patients (Cohort 1). Box plots in each figure depict the 75th percentile, median and 25th percentile values and whiskers represent maximum and minimum values. (C) Plasma concentrations of IL-2, IL-7 and IL-15 from healthy donors and HSCT patients. (D) Ratio ofplasma cytokine concentration to CD4 T cell number for each patient group. (E) Expression of phosphorylated Stat5 (pStat5) in CD4 gated Tcon (blue) and Treg (red) from healthy donors and patients with and without chronic GVHD. Representative panels are shown. (F) Expression of pStat5 in Tcon and Treg subsets from 25 healthy donors and 45 HSCT patients. The expression of pStat5 in Tcon was significantly greater than Treg in patients with severe cGVHD (p < 0.005, Wilcoxon-signed-rank test), while pStat5 expression in other groups did not show significant differences between Treg and Tcon. (G) Ratio of Treg-pStat5 MFI to Tcon-pStat5 MFI in healthy donors and transplant patients. Treg-pStat5/Tcon-pStat5 ratio in severe chronic GVHD was significantly lower than in patients without GVHD (p < 0.001, Wilcoxon-rank-sum test).

Figure 3. Selective activation of Treg in patients with chronic GVHD receiving low-dose IL-2

(A) Expression ofpStat5 in gated Tcon (blue) and Treg (red) from patients before and after the start of IL-2 administration. A representative panel from a single patient is shown. (B) MFI of pStat5 in Treg and Tcon were compared before and 1 week after starting IL-2 therapy. Treg pStat5 MFI is significantly elevated after IL-2. Tcon pStat5 was reduced after IL-2 but this change was not statistically significant. Median values are shown in red. (C) Ratio of Treg-pStat5 MFI / Tcon-pStat5 MFI were compared before and 1 week after starting IL-2 therapy. The ratio is significantly increased in all patients examinedafter IL-2 administration (p = 0.0002, Wilcoxon-signed-rank test). Median values are shown in red. (D) Changes of pStat5 MFI in Treg and Tcon during IL-2 therapy. Median values from 13 patients are shown. (E) Ratio of Treg-pStat5 MFI / Tcon-pStat5 MFI ratio during IL-2 therapy. Median values for MFI ratios were measured in 13 patients. Green range depicts the interquartile range of 14 patients without GVHD (Figure 2E).

Figure 4. Effects of low-dose IL-2 on Treg and Tcon in vivo

(A) Concentrations of plasma IL-2 and absolute numbers of Treg during IL-2 therapy. Median values for 14 patients are shown. (B) Concentrations of plasma IL-7 and IL-15 during IL-2 therapy. Median values for 14 patients are shown. (C) Representative flow cytometry histograms for identification of Ki-67⁺ proliferating cells in Treg and Tcon subsets. Percentage of Ki-67⁺ cells is shown for each histogram. (D) Percentage of Ki-67⁺ proliferating cells in Treg and Tcon subsets during IL-2 therapy (median values). *Treg vs. Tcon (p < 0.001, Wilcoxon-signed-rank test). (E) Recent thymic emigrants (CD45RA⁺CD31⁺) within Treg and Tcon subsets during IL-2 therapy. Median fold changes are shown for each subset. *Treg vs. Tcon (p < 0.005, Wilcoxon-signed-rank test). (F) Levels of intracellular Bcl-2 in Treg and Tcon subsets during IL-2 therapy. Median % increase in mean fluorescence intensity is shown. *Treg vs. Tcon (p < 0.05, Wilcoxon-signed-rank test).

Figure 5. Increased resistance of Treg to apoptosis during low-dose IL-2

(A) Spontaneous and Fas-induced apoptosis in Treg before and after IL-2 therapy. Treg and Tcon were isolated by cell sorting and cultured separately for 6 hours with control medium or anti-Fas antibody. Apoptosis was assessed by annexin V/7-AAD co-staining. Representative histograms for Treg are shown. (B) Spontaneous and Fas-induced apoptosis in Tcon and Treg subsets isolated from 3 healthy control donors (Ctrl) and 8 patients with chronic GVHD before and after IL-2 therapy (Pre and Post, respectively). Median values in each group are shown in red (exact Wilcoxon-rank-sum test, Wilcoxon-signed-rank test).

Figure 6. Efficient suppressive function of IL-2-expanded Treg in vitro

(A) IL-2 expandedTreg suppress in vitro proliferation of stimulated Tcon from the same patient. Cells were harvested after 5 days and the proliferation of responder Tcon was examined by CFSE dilution. Top panel: Unstimulated Tcon maintain high level CFSE-labeling in vitro. Middle panel: Anti-CD3/CD28 stimulated Tcon exhibit extensive CFSE-dye dilution. Lower panel: Addition of Treg at 1:1 ratio inhibits proliferation of CD3/CD28 stimulated Tcon. A representative result from 6 independent experiments is shown. (B) Treg from patients receiving IL-2 were cultured with Tcon from the same patient at 1:1 ratio in the presence of anti-CD3 and anti-CD28 antibody. Suppressive activity wasassessed by the measuring the ability of Treg to reduce secretion of IFN-γ by stimulated Tcon. IFN-γ in the culture supernatants was measured by ELISA. (ND = not detected). Result of 3 independent experiments is shown (p < 0.04, exact Wilcoxon-rank-sum test).(C) Treg-mediated suppression of Tcon IFN-γ secretion was measured at various Treg/Tcon ratios. Result of 3 independent experiments is shown.* p < 0.05 vs. no Treg setting, exact Wilcoxon-rank-sum test, Savage test.