T regulatory cells control T-cell proliferation partly by the release of soluble CD25 in patients with B-cell malignancies

Abstract

Interleukin-2 (IL-2) is one of the most studied cytokines driving T-cell proliferation, activation and survival. It binds to the IL-2 receptor consisting of three chains, the α (CD25), β and common γ (γc). The binding of the CD25 chain to IL-2 is necessary to expose high-affinity binding sites for the β and γc chains, which, in turn, are responsible for downstream signalling. A high level of soluble CD25 (sCD25) has been associated with a poor prognosis in patients with non-Hodgkin's lymphoma. The function and source of origin of this soluble receptor is not well investigated. In the present study we hypothesized that T regulatory (Treg) cells may release CD25 to act as a decoy receptor for IL-2, thereby depriving T-effector cells of IL-2. Peripheral blood from patients with B-cell malignancies (n = 26) and healthy controls (n = 27) was investigated for the presence and function of FoxP3(+) Treg cells and sCD25 by multi-colour flow cytometry and enzyme-linked immunosorbent assay. Further, the proliferative capacity of T cells was evaluated with or without the presence of recombinant sCD25. The results demonstrate that Treg cells from patients had lower CD25 expression intensity and that they released CD25 in vitro. Further, high levels of Treg cells correlated with sCD25 plasma concentration. Recombinant sCD25 could suppress T-cell proliferation in vitro. In conclusion, the release of sCD25 by Treg cells may be a mechanism to deprive IL-2 and thereby inhibit anti-tumour T-cell responses.

Figure 1

Regulatory T (Treg) cells from patients have a decreased cell surface expression of CD25. Peripheral blood mononuclear cells from patients with chronic lymphocytic leukaemia (n = 12) and B-cell lymphoma (n = 14) were stained for CD3, CD4, CD127, FoxP3 and CD25 to investigate the surface expression of CD25 on Treg cells. Patients were compared with healthy age- and gender matched controls (n = 27). Patients had a significant decrease of CD25⁺ Treg cells (a) and lymphoma patients had a decreased surface expression of CD25 compared with healthy controls (b). Differences among groups were analysed using the Mann–Whitney test.

Figure 2

Soluble (s) CD25 is increased in chronic lymphocytic leukaemia (CLL) and lymphoma plasma. Plasma from CLL patients (n = 12), B-cell lymphoma patients (n = 14) and healthy controls (n = 27) was analysed for the presence of sCD25 using enzyme-linked immunosorbent assay. Patients displayed a significantly higher level of soluble CD25 than healthy individuals. Mann–Whitney U-test was used to calculate significant differences.

Figure 3

Soluble (s) CD25 levels correlates with regulatory T (Treg) cell levels in patients with B-cell lymphoma. Levels of sCD25 were correlated with Treg-cell levels using linear regression. No correlation was found between these two factors in healthy controls (a) or patients with chronic lymphocytic leukaemia (CLL) (b). In patients with B-cell lymphoma, a strong correlation was found between sCD25 and Treg-cell levels (c; P < 0·0001). The sCD25 did not correlate with the general CD4⁺ T cells in lymphoma (d) or CLL (e). To investigate if the high levels of sCD25 originated from malignant B cells we performed correlation analysis on sCD25 and white blood cell count. No correlation between these two factors was found (f).

Figure 4

Purified regulatory T (Treg) cells release soluble CD25 (sCD25). Immune cell types were purified from peripheral blood mononuclear cells (PBMCs; n = 4) using magnetic antibody cell sorting beads. Unfractionated PBMCs, CD4⁺ CD127^low CD25^high Treg cells, CD4⁺ T cells, CD8⁺ T cells, CD14⁺ monocytes, CD20⁺ B cells, and PBMCs as well as CD20⁺ B cells from CLL blood were cultured for 2 days in a 96-well plate. Cells were cultured without stimulation (a) or with anti-CD3 stimulation (b). Mann–Whitney U-test was used to analyse differences among groups.

Figure 5

Recombinant soluble (s) CD25 inhibit T-cell proliferation. (a) Recombinant sCD25 was added to OKT-3/interleukin-2 (IL-2) stimulated peripheral blood mononumear cells in triplicates from 11 donors in an Alamar Blue assay. Cells were cultured in final concentrations of 1000 (n = 8), 500 (n = 8) 250 (n = 8) or 100 pg/ml (n = 3) sCD25. The experiment was repeated four times with similar results. (b) PBMCs from six healthy donors were unstimulated, or stimulated with anti-CD3 (OKT3), OKT3 + IL-2, OKT3 + IL-2 + sCD25 (1000 pg/ml) or OKT3 + pre-incubated IL-2/sCD25 to allow complex formation before culture. The OKT3/IL-2 T cells were significantly different from unstimulated (P = 0·002), OKT3-stimulated (P = 0·02), OKT3/IL-2/sCD25-stimulated (P = 0·0152) and OKT3 ⁺ IL-2/sCD25 complex (P = 0·02). The experiment was repeated with six additional donors with similar results. Mann–Whitney U-test was used to analyse differences among groups.

Figure 6

Patient T cells exhibit decreased proliferative capacity. (a) Peripheral blood mononuclear cells were stimulated with anti-CD3/interleukin-2 and cultured in triplicates in 96-well plates in Alamar Blue assays. The mean valuesof 18 patients and 23 controls are shown in the figure. (b) Healthy controls (< 5000 pg/ml) and patients divided into high (> 5000 pg/ml) and low (< 5000 pg/ml) levels of plasma sCD25 and evaluated for proliferative capacity at day 5 of stimulation. Bars represent standard error of the mean and statistic analysis was performed using Mann–Whitney U-test.