Influence of membrane CD25 stability on T lymphocyte activity: implications for immunoregulation

Abstract

Background: CD25, a component of the IL-2 receptor, is important in T cell proliferation, activation induced cell death, as well as the actions of both regulatory (Treg) and effector (Teff) T cells. Recent genome wide association studies have implicated the CD25 locus as an important region for genetic susceptibility to a number of autoimmune disorders, with serum levels of soluble CD25 receptor (sCD25) serving as a potential phenotypic marker for this association. However, the functional impact of CD25 cleavage, as well as the influence of sCD25 on immunoregulatory activities, remain largely unknown and form the basis of this effort.

Methodology/principal findings: The generation of sCD25 by Treg (CD4(+)CD25(+)) and Teff (CD4(+)CD25(-)) cells was examined during in vitro suppression assays, efforts that demonstrated constitutive and stable surface CD25 expression on Treg throughout the period of in vitro assessment. In contrast, Teff cells increased CD25 expression during the process of in vitro suppression, with supernatant sCD25 levels correlating to the amount of cellular proliferation. Interestingly, under serum-free conditions, Tregs partially lost their characteristic anergic and suppressive properties. sCD25 supplementation at physiological concentrations to serum free in vitro suppression assays reduced Teff proliferation without specifically influencing suppression. Indeed, sCD25 production within these cultures correlated with cell death.

Conclusions/significance: These results support the notion that sCD25 functions as both a surrogate marker of T cell activation as well as an indicator of subsequent cellular death. In addition, the role of CD25 in immunomodulation is likely dependent on the local inflammatory milieu, with molecules capable of modulating surface CD25 expression playing a key role in defining immune responsiveness.

Figure 1. CD4⁺CD25⁺ Treg cells require serum for suppression of T cell proliferation.

Suppression assays were conducted utilizing FACS sorted cells run in parallel under either (A) SFM or (B) SFM supplemented with 1.0% autologous serum (n = 7 healthy controls). Treg cells were plated alone (5×10³ cells/well), and in decreasing ratios (1∶1, ½∶1, and 0∶1) to a constant number of Teff cells. Cells were stimulated with soluble anti-CD3 (5.0 µg/ml) and anti-CD28 (2.5 µg/ml) in the presence of a ten-fold excess (5×10⁴) of irradiated accessory cells. (C) Graph indicates data plotted from panels (A) and (B) to highlight the differences in proliferation between serum free media (open bars) and 1% serum (closed bars). Cells proliferated significantly less in SFM at Treg to Teff ratios of ½∶1 and 0∶1 (p<0.001 for both conditions). (D). Graph indicates the percent suppression calculated under serum-free conditions (open squares) and supplemented with 1.0% serum (closed squares) at a ratio of 1∶1-Treg to Teff cells (left data points) and at a ratio of ½∶1 Treg∶Teff cells (right points). Under serum free conditions, Treg fail to suppress proliferation and often lead to increased proliferation in the co-culture (% suppression  =  mean + SEM, −197.8+270.6 for SFM vs. 70.4+17.3 with 1.0% serum; *p = 0.04 at a ratio 1∶1 Treg to Teff. This trend continued at a ratio of ½∶1 Treg to Teff cell (−103.1+91.5 vs. 34.8+18.12, for SFM and 1.0% serum, respectively; **p = 0.01). (*p<0.05, **p<0.01, and ***p<0.001).

Figure 2. Contribution of Treg and Teff cells to increased proliferation in suppression assay co-cultures.

Peripheral blood CD4⁺ T cells ((A), left plot) were FACS sorted based on surface expression of CD25 (y-axis) and CD127 (x-axis) to yield CD4⁺CD127^lo/−CD25⁺ T cells ((A), middle plot) and CD4⁺CD127⁺CD25⁻ Teff cells ((A), right plot). Following isolation, Teff cells were labeled with CFSE ((B), left plot, y-axis) and Treg cells labeled with PKH26 ((B), left plot, x-axis). Proliferation of each gated CD4⁺ T cell population was then assessed following activation conditions utilized in the suppression assay. (B) Left plot indicates fluorescence levels of unstimulated cells (depicted in red for Treg and green for Teff), and following 96 h of in vitro activation ((B), right plots). Proliferation of Treg and Teff cells are shown at a ¼∶1 Treg to Teff cell ratio and plotted under conditions of SFM (dashed lines) or in the presence of 5% serum (solid lines). (C) For all ratios of Treg to Teff cells, Treg maintain increased suppressive potential and anergic properties in the presence of serum as measured by division index (DI). (D) In vitro suppression calculated from DI indicated increased suppression by Treg in the presence of serum. Data plotted represent one example of five independent experiments. (E) A standard suppression assay was set up under SFM conditions with either standard or irradiated (3300 rads) Treg and Teff cells as indicated. This analysis demonstrated a synergistic response at a 1∶1 Treg to Teff ratio, with near maximal proliferation following addition of irradiated Teff cells. Statistical significance indicated as *p<0.05, **p<0.01, and ***p<0.001.

Figure 3. Production of sCD25 during the in vitro suppression assay.

Supernatants from triplicate wells were pooled and analyzed for the production of sCD25 by ELISA (n = 7 control subjects). Under conditions of both serum-free and 1.0% serum, the highest levels of sCD25 were detected in the 1∶1 Treg to Teff co-culture condition at the early 48 h time point, but were only significantly higher in media containing 1% serum ((A) and (B)). At the 96 h time point ((C) and (D)), the pattern of sCD25 production more closely resemble the responses observed in the proliferation assay assessed by uptake of ³H-thymidine. (*p<0.05, **p<0.01, and ***p<0.001).

Figure 4. Levels of cellular proliferation correlate with the production of sCD25.

Levels of sCD25 produced during the in vitro suppression assay were plotted versus the amount of proliferation detected at the 96 h time point. Under both (A) SFM and (B) 1.0% serum conditions, for all ratios of Treg to Teff cells (1∶0, 1∶1, ½∶1, and 0∶1, n = 7), the levels of sCD25 detected (x-axis) correlate with cellular proliferation as assessed by the incorporation of ³H-Thymidine (y-axis). Isolated Treg and Teff cells were then assessed for their capacity to proliferate and produce sCD25 in an autocrine fashion over a 5 day time period. Purified CD4⁺CD25⁺ (open bars) and CD4⁺CD25⁻ T cells (closed bars) were cultured under expansion conditions utilizing beads coated with anti-CD3 and anti-CD28 as well as exogenous human recombinant IL-2 (300 U/ml) under serum-free conditions. Shown are (C) proliferation and (D) sCD25 production at 48, 72, 96, and 120 h time points by each indicated cell population. The data plotted represent the mean CPM and pooled sCD25 levels of replicate cultures (n = 6 individuals) with situations identifying significance between Treg and Teff cells indicated (*p<0.05, **p<0.01, and ***p<0.001).

Figure 5. Alteration of MMP-2 and MMP-9 activity alters Treg and Teff cell proliferation and in vitro suppression.

FACS isolated Treg (A) and Teff (B) were activated with anti-CD3 and anti-CD28 coated microbeads in the presence of vehicle control or the selective MMP-2/9 chemical inhibitor II (IC₅₀ = 250 nM). Addition of active recombinant MMP-2 and MMP-9 (1 µg/ml each) led to reduced proliferation of Treg (A) and significantly reduced proliferation of Teff (B) cells. The inhibitor reduced proliferation of Tregs (p<0.01) while having no effect on Teffs. (C) The addition of MMP-2/9 inhibitor to the in vitro suppression assay resulted in increased Teff proliferation while at the same time, augmenting Treg-mediated suppression. Percent (%) suppression at 1∶1 (Treg/Teff) and ½∶1 ratios are noted above the bars, as a comparison to vehicle (0∶1) or MMP-2/9 inhibitor (0∶1) CPM. Graphs show one representative experiment of three individual experiments. (*p<0.05, **p<0.01, and ***p<0.001).

Figure 6. Addition of soluble CD25 differentially effects proliferation of Teff cells and PBMC.

A suppression assay was set up under serum-free media ((A), open bars), or in the presence of a human recombinant sCD25 protein ((A), closed bars). Addition of sCD25 did not significantly restore Teff cell responses or the anergic properties of Treg in serum-free conditions (p = NS for all Treg to Teff cell ratios, 1∶0, 1∶1, ½∶1, and 0∶1). In the 0∶1 condition, sCD25 did appear to reduce proliferation. sCD25 was added to PBMC cultures (n = 10) in the presence of increasing concentrations of sCD25, with anti-CD3/28 microbeads ((B), open bars) or PHA ((B), hatched bars) stimulation. Proliferation increased in some culture conditions and decreased in others. Statistically significant increases were seen by ANOVA under 3/28 stimulation in RPMI (p = 0.05), AIMV (p = 0.001), and XVIVO (p = 0.001) media, and under PHA stimulation in XVIVO medium (p = 0.01) when comparing 0 and 20 ng of sCD25 added. Comparisons among the same stimulation showed that cells cultured in RPMI and AIMV media had significant differences in proliferation regardless of the amount of sCD25 added, with both PHA and CD3/28 stimulation. Under PHA stimulation, CTL cultures proliferated less than either XVIVO or AIMV cultures. To determine if this differential proliferation was due to altered cell death, we measured the amount of nuclear matrix proteins (NMPs) released into the supernatant by dying cells (C). Addition of sCD25 did not result in a dose-responsive increase in cell death. The outcome varied depending on the culture media and stimulus. Plotting the stimulation index versus the production of NMP showed a negative correlation between proliferation and cell death under CD3/28 stimulation ((D), bottom panel). *p<0.05, **p<0.01, and ***p<0.001.

Figure 7. sCD25 production in the absence of exogenous recombinant protein correlates with cell death, not proliferation, of PBMC.

To determine if the variable results seen previously were present in other areas, we measured the production of sCD25 in the 0 ng/ml condition with anti-CD3/28 microbeads ((A), open bars) or PHA ((A), hatched bars) stimulation. sCD25 production varied dependent on the stimulus provided in RPMI and AIMV media conditions (RPMI: (PHA) 18952±2872, (3/28) 7457±916.8, p<0.001; AIMV: 21482±2114, (3/28) 10957±893.8, p<0.001), but did not significantly differ between media formulations. In (B), sCD25 production did not correlate with proliferation as seen previously. Rather, sCD25 was generated at levels correlating with cell death as measured by NMP production, (C) (PHA: r = 0.7182, p<0.001, CD3/28: r = 0.3882, p = 0.0133). ***p<0.001.