Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25- effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.

Fig. 1

Sorafenib increases T cell responses in HCC patients. a Low doses of sorafenib significantly improved T cell proliferation in the PBMC isolated from the patients with HCC (n = 20) from 25,823 ± 3,820 cpm (no sorafenib) to 31,278 ± 3,820 cpm (p = 0.025). Results are shown as mean cell proliferation ± SE (cpm). b A subset of patients (n = 6) exhibited a high degree of improvement in their T cell responses. In this group, low doses of sorafenib led to a significant increase in T cell proliferation from a baseline 28,780 ± 2,108 cpm to 41,374 ± 6,278 cpm (p = 0.028). c CD4+CD25− Teff from patients with advanced HCC (n = 6) were cultured without (38,520.5 ± 4,838 cpm) and with three concentrations of sorafenib—0.1, 1 and 3 μM. A significant increase in Teff proliferation was observed with all three low doses of sorafenib. The low doses of sorafenib resulted in a general improvement in Teff proliferation: 51,764 ± 1,847 cpm with 0.1 μM (p = 0.04); 57,953 ± 5,225 cpm with 1 μM (p = 0.02) and 59,701 ± 5,138 cpm with 3 μM (p = 0.013). *p < 0.05; **p < 0.01. d Extended dose response of sorafenib on CD4+CD25− Teff from patients with HCC (n = 2). Low doses of sorafenib result in improved T cell responses: 18,565 ± 49 cpm with 0.1 μM (p = 0.008); 19,768 ± 145 cpm with 1 μM (p = 0.005); (18,937 ± 1,402 cpm with 3 μM (p = 0.13); 15,848 ± 652 cpm with 6 μM (p = 0.65); while higher doses result in a stepwise decline in T cell responses: (9,381 ± 909 cpm with 12 μM (p = 0.02); 3,821.5 ± 492 cpm with 15 μM (p = 0.002); 256 ± 154 cpm with 18 μM (p = 0.004); 41 ± 492 cpm with 21 μM (p = 0.0003); and 34 ± 6 cpm with 25 μM (p = 0.0003)

Fig. 2

Sorafenib stimulates HCC Teff to secrete IL-2. a IL-2 levels were analyzed in the supernatants from CD4+CD25− Teff cultures treated with sorafenib (n = 2). IL-2 content was significantly higher in the supernatants from the Teff cultures containing 0.1 and 3 μM sorafenib when compared to the cultures without sorafenib. The IL-2 levels in the Teff cultures without sorafenib (62 ± 12 pg/ml) significantly increased with the addition of low doses of sorafenib: 112 ± 12 pg/ml with 0.1 μM (p = 0.05); 109 ± 23 pg/ml with 1 μM (p = 0.15); 110 ± 10 pg/ml with 3 μM (p = 0.02). b This figure represents the IL2 levels recovered from the sample of HCC Teff cells plotted in Fig. 1d. Baseline IL2 secretion from—HCC Teff without sorafenib 111 ± 3 pg/ml; 0.1 μM sorafenib 104 ± 5 pg/ml (p = 0.4); 2 μM 10 ± 2 pg/ml (p = 0.5); 3 μM 114 ± 5 pg/ml (p = 0.4), 6 μM 87 ± 9 pg/ml (p = 0.12); 12 μM 49 ± 4 pg/ml (p = 0.007);15 μM 42 ± 0.6 pg/ml (p = 0.002); 18 μM 17 ± 1 pg/ml (p = 0.001); 21 μM 7 ± 0.7 pg/ml (p = 0.0008); and 25 μM (11 ± 0.9 pg/ml, p = 0.00091)

Fig. 3

Sorafenib increases the cell surface expression of CD25 on HCC Teff. HCC PBMC cells were stimulated with PHA and cultured without sorafenib or with increasing concentrations of sorafenib ranging from 0.1 to 12 μM. Flow cytometry was used to determine the surface expression of CD25 on the CD4+CD127+ T effector cell population in the cultures. A representative patient is shown. The addition of sorafenib significantly increased the percentage of CD25 expression on the CD4+CD127+ Teff compartment from 9.5 (no sorafenib) to 16.0 % with 1 μM. Further supplementation of sorafenib above 1uM failed to substantially increase CD25 expression

Fig. 4

Sorafenib decreases HCC Treg-mediated suppression. a Suppression assays were performed with CD4+CD25− Teff and CD4+CD25+ Tregs from patients with HCC. Teff cultured alone and stimulated with PHA proliferate up to 47,458 ± 2,462 cpm. The addition of Treg decreased cell proliferation. 1Treg:1Teff; 14,668 ± 161 cpm (p = 0.047). The addition of sorafenib to the cocultures (0.1 μM) did not decreases HCC Treg-mediated suppression 16,253 ± 161 cpm (p = 0.13). In contrast, sorafenib 1 μM and recombinant IL2 significantly decreases HCC Treg-mediated suppression, 25,750 ± 1,253 cpm (p = 0.013) and 27,220 ± 1,760 cpm (p = 0.02) respectively. b CD4+CD25− Teff cells from a patient with HCC were stimulated with PHA (27,432 ± 1,415 cpm) and also cocultured with CD4+CD25+ T cells at ratios of 1:10 and 1:1. The suppression cocultures showed a progressive decrease in target T cell proliferation from 19,620 ± 2,493 cpm with the 1:10–1,230 ± 121 cpm with the 1:1 cultures. The CD4+CD25− target T cell proliferation was restored by the addition of low doses of sorafenib as follows: 8,314 ± 1,309 cpm with 0.1 μM (p = 0.016); 45,011 ± 6,896 cpm with 1 μM (p = 0.023); 34,662 ± 6,141 cpm with 2 μM (p = 0.032); and 28,648 ± 2,155 cpm with 3 μM (p = 0.006). c HCC PBMC cells were stimulated with PHA and cultured alone or supplemented with increasing concentrations of sorafenib ranging from 0.1 to 25 μM. Flow cytometry was used to determine the Treg (CD4+CD25+CD127−) frequency in the cultures. A representative patient is shown. The percentage of CD4+CD25+CD127− T cells at baseline (PHA stimulated in the absence of sorafenib) was 5.3 %. Addition of increasing doses of sorafenib did not significantly change the frequency of Tregs which was 6.5 % with 0.1 μM, 4.5 % with 1 μM, 6.2 % with 3 μM, 4.7 % with 6 μM, 5.7 % with 12 μM, and 6.5 % with 25 μM

Fig. 5

Sorafenib significantly increases IL-2 content in HCC Teff/Treg cocultures. a IL2 content in the supernatants from the suppression assays was determined by ELISA. Teff cells alone produced at the end of the culture 32 ± 6 pg/ml of IL2. IL-2 levels in the 1:1 cocultures alone, with 0.1 μM of sorafenib and 1 μM of sorafenib were 36 ± 1 (p = 0.6), 51.3 ± 1 (p = 0.07) and 73.5 ± 3.4 (p = 0.024) pg/ml, respectively. The IL-2 control showed the largest content of IL2 at 582 ± 52 (p = 0.008), although the degree of cell proliferation was similar to the cocultures treated with 1 μM sorafenib. b This figure represents the IL2 levels from the supernatants of Fig. 4b. The introduction of Tregs to cocultures with target CD4+CD25− Teff cells lead to a twofold decrease in IL2 secretion from 74 + 13 pg/ml by CD4+CD25− Teff to 33 ± 6 pg/ml (p = 0.11) in the 1:10 and to even lower levels in the 1:1 (3.2 ± 1 pg/ml) cocultures. The addition of sorafenib to the 1:1 cocultures significantly elevates IL2 levels, particularly with 1 μM (73 ± 0.5, p = 0.00005), 2 μM (103 ± 21, p = 0.04), and 3 μM (73 ± 2, p = 0.0008)

Fig. 6

Sorafenib significantly increases IL-6 content in HCC Teff/Treg cocultures. ELISA was used to determine IL6 concentration in the supernatants from the suppression assays. Teff cells from patients with HCC alone generated at the end of the culture 1,241 ± 108 pg/ml of IL 6. Next, the IL-6 levels in the first coculture 1–1 without sorafenib and with 0.1 and 1 μM sorafenib were 1,612 ± 95 (p = 0.12), 1,734 ± 54 (p = 0.15) and 1,896 ± 78 (p = 0.039) pg/ml, respectively. Finally, the IL2 control showed not significant increase in IL6 content when compared to HCC Teff cells alone, 1,633 ± 29 (p = 0.86)