Deferiprone modulates in vitro responses by peripheral blood T cells from control and relapsing-remitting multiple sclerosis subjects

Abstract

T cells are important mediators of autoimmune inflammation in relapsing-remitting multiple sclerosis (RRMS). Previous studies found that deferiprone, an iron chelator, suppressed disease activity in a mouse model of multiple sclerosis, and inhibition of T cell proliferation was implicated as a putative mechanism. The objective of the present study was to examine the effects of deferiprone on suppressing in vitro responses of T cells from control and RRMS subjects. Peripheral blood T cells were co-stimulated with anti-CD3+anti-CD28 and cultured with or without interleukin 2 (IL-2). Proliferating CD4+ T cells from control and RRMS subjects, cultured with or without IL-2, decreased in response to 75 μM deferiprone, although the extent of decreased proliferation of CD4+ T cells from RRMS subjects was less than for control subjects. Proliferating CD8+ T cells from control subjects, cultured with or without IL-2, also decreased in response to 75 μM deferiprone, and this decrease was seen in proliferating CD8+ T cells from RRMS cultured with IL-2. CD4+CD25+ and CD8+CD25+ cells from control subjects, cultured with or without IL-2, declined in 75 μM deferiprone, but the decrease was smaller than for the CD4+ and CD8+ proliferative responses. CD4+CD25+ and CD8+CD25+ cells from RRMS subjects showed more variability than for control subjects, but CD4+CD25+ cultured with IL-2 and CD8+CD25+ cells cultured without IL-2 significantly declined in 75 μM deferiprone. CD4+FoxP3+ and CD4+CD25+FoxP3+ cells tended to remain constant or increase. In summary, deferiprone induced declines in proliferative responses at a dosage that is within peak serum pharmacological concentrations.

Fig. 1

Proliferation of CD4+ and CD8+ T cells as measured by analyzing CFSE from control and RRMS subjects in the presence of deferiprone. The deferiprone/vehicle (complete culture media without drug) ratio was measured for control or RRMS cells cultured with or without IL-2. A) CD4+ proliferating and B) CD8+ proliferating cells (error bars = SEM). For each subject, the area under the lines over 25–100 μM deferiprone concentrations with or without IL-2 was determined and plotted according to the subject's age; C) CD4+ proliferating and D) CD8+ proliferating cells. There was not a statistically significant difference between the area under the curve for RRMS and control subjects (one tailed Student t-test, significance at p ≤ 0.05). Plots of mean ± s.e. for each group (triangle – control subject; circle – RRMS subject; unfilled symbol – without IL-2; filled symbol – with IL-2). N=5 control subjects (cells from 1 subject had 5 μM as the lowest deferiprone concentration); n=7 RRMS subjects (1 subject was treated with a limited deferiprone profile: 0, 0.5, 1, 5, 10, 50, 100, 200 μM, and its area under the curve was generated from an extrapolated line. Also, CD4+ T cells treated with IL-2 from one subject had 10 μM as the lowest deferiprone concentration).

Fig. 2

Effect of deferiprone on net changes in the percentage of viable CD4+ and CD8+ T cells. T cells were cultured with TCR co-stimulation (e.g., anti-CD3 + anti-CD28) in the presence of varying concentrations of deferiprone. The ratio of data obtained at different concentrations of deferiprone vs. vehicle (complete culture media without drug) was calculated for control or RRMS cells cultured with or without IL-2. A) CD4+ and B) CD8+ cells (viability determined by exclusion of live/dead fixable violet stain, and then gated for live cells). Plots of mean ± s.e. for each group (triangle – control subject; circle – RRMS subject; unfilled symbol – without IL-2; filled symbol – with IL-2). N=5 control subjects (cells from 1 subject had 5 μM as the lowest deferiprone concentration); n=7 RRMS subjects (1 subject was treated with a limited deferiprone profile: 0, 0.5, 1, 5, 10, 50, 100, 200 μM).

Fig. 3

Effect of deferiprone on CD4+CD25+ and CD8+CD25+ T cells. The modulation of double positive CD4+CD25+ or CD8+CD25+ T cells from control and RRMS subjects was analyzed using a ratio of proliferating (CFSE) A) CD4+CD25+ or B) CD8+CD25+ T cells in the presence of deferiprone divided by the equivalent cell populations cultured in the vehicle (complete culture media without drug). At 75 μM deferiprone, less proliferation was observed for RRMS subjects (CD4+CD25+ with IL-2; CD8+CD25+ without IL-2) and for control subjects (CD4+CD25+ or CD8+CD25+ with or without IL-2). Thus, deferiprone mitigates the normal proliferation of T cells upon TCR co-stimulation regardless of mitogenic co-stimulation (presence or absence of IL-2). Plots of mean ± s.e. for each group (triangle – control subject; circle – RRMS subject; unfilled symbol – without IL-2; filled symbol – with IL-2). N=5 control subjects (cells from 1 subject had 5 μM as the lowest deferiprone concentration); n=7 RRMS subjects (1 subject was treated with a limited deferiprone profile: 0, 0.5, 1, 5, 10, 50, 100, 200 μM).

Fig. 4

Raw data illustrating the effects of deferiprone on proliferating CD4+ cells. A) CD4+ cells were cultured from a control or RRMS subject in the absence (0 μM) or presence (75 μM) of deferiprone without IL-2. Gating was performed on live cells that were CD4+. CFSE and CD25 intensities were plotted on the x and y axes, respectively. Proliferating cells appear to the left of 10³ CFSE staining. As revealed in the upper left quadrants, 75 μM deferiprone decreased the proliferation of CD4+CD25+ T cells from control and RRMS subjects. B) Histograms of CFSE fluorescence with gating of live CD4+CD25+ cells in A. The X axis is CFSE level of fluorescence and the Y axis is number of cells. As revealed in the lower left boxes, 75 μM deferiprone greatly decreased the proliferation of CD4+CD25+ T cells.

Fig. 5

Responses of Treg from control and RRMS subjects in the presence of deferiprone with or without IL-2. A) CD4+FoxP3+ and B) CD4+CD25+FoxP3+ cells. Plots of mean ± s.e. for each group (triangle – control subject; circle – RRMS subject; unfilled symbol – without IL-2; filled symbol – with IL-2). N=5 control subjects (cells from 1 subject had 5 μM as the lowest deferiprone concentration); n=7 RRMS subjects (1 subject was treated with a limited deferiprone profile: 0, 0.5, 1, 5, 10, 50, 100, 200 μM, and the 10 μM concentration for CD4+CD25+FoxP3+ was missing for 1 RRMS subject).