Influence of serum and soluble CD25 (sCD25) on regulatory and effector T-cell function in hepatocellular carcinoma

Abstract

Our previous studies showed that high levels of soluble CD25 (sCD25) in the serum of patients with hepatocellular carcinoma (HCC) correlated with blunted effector T-cells (Teff) responses, tumour burden and poor survival. Understanding the interactions between Teff, CD4+CD25+ regulatory T cells (Treg) and soluble factors can identify novel therapeutic targets. In this study, we characterize the mechanisms by which HCC serum and sCD25 mediate suppression of Teff and evaluate the effect of sCD25 on the suppression assays with normal healthy control cells (NHC) at a 1:1 Treg to Teff cell ratio to determine whether sCD25 has any impact on Treg suppression. HCC serum and sCD25 suppressed Teff proliferation and downregulated CD25 expression on HCC Teff in a dose-dependent fashion with sCD25 doses above 3000 pg/ml. Treg from HCC and cirrhosis patients suppressed proliferation of target CD4+CD25- Teff in serum-free medium (SFM). HCC Treg showed a higher degree of suppression than cirrhosis-derived Treg. In contrast, Treg from NHC did not suppress target Teff in SFM. However, isolated Treg from all three study subjects (HCC, cirrhosis and NHC) suppressed CD4+CD25- Teff in serum conditions or in the presence of sCD25 in the range 6000-12,000 pg/ml. In conclusion, downregulation of CD25 cell surface expression on Teff is part of the overall suppressive mechanism of sCD25 and HCC serum on Teff responses. The observed sCD25 and HCC serum-mediated suppression is further influenced via novel immune-inhibitory interaction between CD4+CD25+ Treg and sCD25.

Figure 1. In SFM conditions NHC CD4+CD25+ can not suppress NHC CD4+CD25− cells proliferation

Results are shown as mean cell proliferation ± SE (cpm). NHC CD4+CD25− cells alone (37329.5 ± 4881.5; n = 3). *p<0.05, **p< 0.01, next, CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in SFM conditions, 37235.6 ± 2465.5 no suppression, and finally, CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in autologous serum containing conditions 486 ±254,suppression is observed.

Figure 2. HCC CD4+CD25+ cells can suppress proliferation of HCC CD4+CD25− cells stimulated with PHA in SFM conditions

HCC CD4+CD25− cells from an early stage HCC donor cultured alone and in SFM conditions (134372 ± 911 cpm) and with HCC CD4+CD25− cells 1:1 in SFM (2026 ± 478) and autologous HCC serum conditions (2940.5 ± 216.5). HCC CD4+CD25− cells from an Intermediate stage HCC donor, cultured in SFM conditions (62927.5 ± 405.5 cpm) and with HCC CD4+CD25+ cells 1:1 in SFM (1366 ± 27) and autologous HCC serum (2023.5 ± 405.5). HCC CD4+CD25− cells from an advance stage HCC donor, SFM conditions (5713.5 ± 365.5 cpm) and with HCC CD4+CD25+ cells 1:1 in SFM (126 ± 148 and autologous serum (140 ± 111). *p<0.05, **p< 0.01.

Figure 3. HCC CD4+CD25+ cells are more suppressive that NHC CD4+CD25+ cells in serum free media conditions at various Treg/Teff ratios (1:1, 1:10, 1:20; 1:30)

NHC derived CD4+CD25+ Tregs fail to suppress the baseline proliferative response of target CD4+CD25− Teffs (18330.3 ± 732.2 cpm) at any of the Treg/Teff ratios in the co-cultures: 1:1 ratio 17929.48 ± 1663 (P= 0.63), 1:10 ratio 17651.4 ± 7 1972.9 cpm (P= 0.08), 1:20 ratio 18373.3 ± 1481.3 (P= 0.98) and 1:30 ratio 19590.9± 1597.4 cpm (P= 0.51). In contrast, HCC derived CD4+CD25+ Tregs suppress CD4+CD25− Teff at all the four Treg/Teff ratios examined in a graded fashion: baseline 3020.5 ± 115.4 cpm, 1:1 ratio 191.5 ± 25.5 (P= 0.00016); 1:10 ratio 501.5 ± 22.5 cpm (P= 0.0002); 1:20 ratio 1147.5 ± 67.5 (P= 0.0002); and 1:30 ratio 1990.5 ± 194.5 cpm (P= 0.045)

Figure 4. HCC and DC CD4+CD25+ cells can suppress proliferation of CD4+CD25− cells stimulated with PHA in SFM

Results are shown as mean cell proliferation ± SE (cpm). DC (black bar) and HCC (grey bar) CD4+CD25− cells alone (63841.5 ± 11309.96 and 67457.83 ± 23651.3; n = 3). p<=0.225, next, CD4+CD25− cells at a cell ratio I:I with CD4+CD25+ cells in SFM conditions, DC coculture (black bar) 11514.14 ± 4665.6, HCC coculture 1176.333 ± 371.5; n = 3 both cocultures show suppression and are not statistically different, p= 0.075 and finally, CD4+CD25− cells at a cell ratio I:I with CD4+CD25+ cells in autologous serum containing conditions DC coculture 14603.3 ± 7110.1; n = 3 and HCC coculture 1696.5 ± 537.5, both cocultures show suppression and are not statistically different, p= 0.083 although a statistical trend is observed.

Figure 5. CD4+CD25− cells from NHC are suppressed in SFM conditions when recombinant sCD25 is added to the medium

(A)Suppression is dose dependent and starts to be significant at 6000 pg/ml. NHC CD4+CD25− cells alone (37329.5 ± 4881.5 cpm; n = 3 **p< 0.01), CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in SFM conditions (35383.5 ± 199.5), CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in SFM conditions plus 3000 pg/ml of sCD25 (32407.5 ± 1689.5), CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in SFM conditions plus 6000 pg/ml of sCD25 (15555 ± 1900) and CD4+CD25− cells at a cell ratio I:I with NHC CD4+CD25+ cells in SFM conditions plus 12000 pg/ml of sCD25 (967.5 ± 255.5). (B) Enhanced Suppression of CD4+CD25+ Tregs from NHC in SFM is Specific for sCD25. To evaluate if the effect of sCD25 (alpha chain) in the NHC Tregs/Teffs co-cultures at a 1:1 ratio in serum free conditions is specific for sCD25, 6000 pg/ml of sCD25, beta receptor of CD25 and alpha receptor of IL-6 were added to 1:1 suppression assays. In the absence of soluble receptors, Tregs fail to suppress Teffs under SFM conditions: 18330.3 + 732.2 cpm for CD4+CD25− Teff cells alone; and 17929.5 + 1663 cpm when Treg/Teff cocultured at 1:1 ration, n=4, P= 0.61. While the Tregs in the 1:1 co-cultures with sCD25 showed suppression (393.8 + 169.1 cpm, P<0.001) of the baseline Teff proliferative response (18330.3 + 732.2 cpm), the Tregs in the 1:1 co-cultures containing the beta CD25 receptor (17527.5 + 962.3 cpm, P= 0.53) and the soluble IL-6 alpha receptor (20622.9 + 710.8, P= 0.03) at a similar dose of 6000 pg/ml did not show suppression.

Figure 6. sCD25 and HCC serum decreases induced CD25 expression on HCC CD4+CD25− cells

HCC CD4+CD25− cells were cultured for 3 days after stimulation with PHA and supplementation with recombinant sCD25 and HCC serum, later, cells were stained for CD25 and CD4 and analyzed by flow cytometry. The percentage of CD25 positive cells from the total analyzed is shown. Figures are representative of three separate experiments (three separate donors). First row left, media (non PHA), next PHA only, and NHC serum. Second row left, HCC serum 5%, HCC serum 10% and, HCC serum plus IL2 (500 units per ml). Third row left: sCD25 dose 1 (3000 pg/ml), dose 2 (6000 pg/ml) and dose 3 (12000 pg/ml).

Figure 7. Tregs from HCC Patients Express more TGFβ1 than Tregs from NHC and DC controls

The baseline cell surface expression of TGFβ1 on CD4+CD25+ cells were evaluated by flow cytometry using PBMCs from controls and patients with advanced HCC. The gating strategy and percentage of TGF β1 positive CD4+CD25+ cells for controls and HCC patients are shown. Cell surface staining of Tregs for TGFβ1 showed a 2 to 4 fold higher level of staining in patients with advanced HCC (22.3%, 21.2%, and 24.4%) than controls (6.4% in NHC and 10.3% in DC).