Tumor-specific hepatic stellate cells (tHSCs) induces DIgR2 expression in dendritic cells to inhibit T cells

Tumor-specific hepatic stellate cells (tHSCs) contributes to tumorigenesis and progression of hepatocellular carcinoma (HCC). The potential function of tHSCs on dendritic cells (DCs) was studied here. We discovered that tHSCs co-culture induced upregulation of DIgR2 (dendritic cell-derived immunoglobulin receptor 2) in bone marrow-derived DCs (mDCs). Activation of MEK-ERK is required for DIgR2 expression in mDCs. MEK-ERK inhibitors or shRNA-mediated silence of MEK1/2 in mDCs inhibited tHSCs-induced DIgR2 expression. Meanwhile, tHSCs stimulation decreased production of multiple cytokines (CD80, CD86 and IL-12) in mDCs. Such an effect was almost reversed by DIgR2 shRNA in mDCs. Further, tHSCs-stimulated mDCs induced T-cell hypo-responsiveness, leading to decreased cytotoxic T lymphocyte (CTL) activity and reduced IFN-γ production in splenic T cells. T cell proliferation inhibition and apoptosis were also noticed. These actions on T cells were again largely inhibited by DIgR2 shRNA in mDCs. Together, our results indicate that tHSCs directly induces DIgR2 expression in DCs to inhibit T cells.

Recently studies, including ours [10,11], have also proposed a key function of tHSCs in tumor immunology [7][8][9]. We have previously shown that tHSCs could induce T cell apoptosis [10]. Further, tHSCs also inhibits T cell response, causing HCC cell migration and invasion [11]. Dendritic cells (DCs) are specialized antigen-presenting cells (APCs), which are responsible for the initiation and regulation of immune response [12,13]. Activation of DCs is important for anti-tumor activity [12,13]. Dendritic cell-derived immunoglobulin receptor 2, or DIgR2, is a key inhibitory receptor that inhibits DCs-induced antigen-specific T-cell responses [14]. DC-derived DIgR2 is shown to bind to the receptor in T cells, suppressing T-cell proliferation, cytokine production and cytotoxic T lymphocyte (CTL) activity [14]. In the current study,

Research Paper
we show that tHSCs induces DIgR2 expression in DCs, causing T-cell hypo-responsiveness.

RESULTS tHSCs co-culture induces DIgR2 expression in bone marrow-derived dendritic cells (mDCs)
As described in our previous studies [10], the primary HSCs were derived from livers of control normal Buffalo rats or the xenograft HCC tissues, which were named as quiescent HSCs (qHSCs) and tumor HSCs (tHSCs), respectively [10]. Primary cultured bone marrow-derived dendritic cells (mDCs) were also derived from Buffalo rats [14]. mDCs were then co-cultured with tHSCs or qHSCs (mDCs to HSCs ratio, 20: 1). Expression of DIgR2, the key immune inhibitory receptor of immunoglobulin superfamily [14], was then tested. Quantitative real-time PCR ("qRT-PCR") assay results in Figure 1A demonstrated that DIgR2 mRNA expression in mDCs was significantly elevated after co-culture of tHSCs. DIgR2 mRNA level increased over 9-10 fold higher in mDCs with tHSCs challenge ( Figure 1A). On the other hand, qHSCs co-culture had no significant effect on DIgR2 mRNA expression ( Figure 1A). Further, DIgR2 protein expression in mDCs was also dramatically induced when co-cultured with tHSCs (but not the qHSCs, three sets of repeated data were quantified in Figure 1B). These results suggest that DIgR2 expression is induced in mDCs after tHSCs co-culture.

MEK-ERK activation is required for DIgR2 expression in tHSCs-stimulated mDCs
Next, we studied the potential mechanism of DIgR2 expression in mDCs with tHSCs co-culture. Western blotting assay results showed that co-culture with tHSCs induced significant activation of MAPK/ ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mDCs ( Figure 2A). Phosphorylated protein expression (Three sets of repeated blot data were quantified in (B) of DIgR2 in bone marrow-derived dendritic cells (mDCs), co-cultured with/out quiescent HSCs (qHSCs) or tumor HSCs (tHSCs) for applied time, were shown. "Ctrl" stands for mDCs only. "Tubulin" stands for loading control β-Tubulin. *P < 0.05 vs. "Ctrl" group.
(p-) MEK1/2 and p-ERK1/2 in mDCs were significantly increased following co-culture of tHSCs ( Figure 2A). To study the link between MEK-ERK activation and DIgR2 expression, pharmacological MEK-ERK inhibitors were first applied, including PD98059, U0126 and MEK-162 [15,16]. As shown in Figure 2B, treatment with these inhibitors almost completely blocked MEK-ERK cascade activation in mDCs with tHSCs co-culture. Consequently, DIgR2 mRNA ( Figure 2C) and protein ( Figure 2D) expressions were also largely inhibited. The pharmacological evidences suggest that activation of MEK-ERK signaling is required for DIgR2 expression in tHSCs-stimulated mDCs.

shRNA knockdown of DIgR2 in mDCs
The aim of this study is to test the potential effect of tHSCs on the activity of DCs. More specifically, we wanted to know if tHSCs-induced DIgR2 expression could influence the functions of mDCs. Thus, lentiviral shRNA method was applied to selectively knockdown DIgR2 in mDCs. A set of three lentiviral DIgR2 shRNAs, targeting the non-overlapping sequence of DIgR2, were established. The three shRNAs were named as "DlgR2-shRNA Sq1/2/3". qRT-PCR assay results in Figure 3A confirmed that adding each of the three shRNAs significantly inhibited DIgR2 mRNA expression in mDCs with co-culture of tHSCs. Consequently, DIgR2 protein expression was also silenced (Three sets of repeated data were quantified in Figure 3B). Among three tested DIgR2 shRNAs, the DIgR2 shRNA "Sq3" showed highest efficiency in knocking down DIgR2 ( Figure 3A and 3B). This DlgR2-shRNA ("Sq3") was then selected for further functional studies. Notably, the non-sense scramble control shRNA ("c-sh") failed to decrease DIgR2 expression in mDCs ( Figure 3A and 3B). Notably, DIgR2 shRNA failed to inhibit survival of mDCs (tested by the trypan blue assay).
These results indicate that DlgR2 induction by tHSCs is important for subsequent inhibition of above cytokine expression. Further ELISA assay results showed that productions of CD80 ( Figure 4D), CD86 ( Figure 4E) and IL-12 ( Figure 4F) were also decreased in tHSCs-stimulated mDCs. Such effects were again almost abolished with shRNA knockdown of DlgR2 ( Figure 4D-4E). Notably, the non-sense scramble control shRNA ("c-sh") was in-effective ( Figure 4A-4F).

tHSCs-stimulated DlgR2 expression in mDCs inhibits splenic T cells
It has been previously shown that DC-derived DIgR2 binds to the receptor in T cells, which shall inhibit normal T cell functions. Thus, we then co-cultured splenic T cells with mDCs (T cells/mDCs ration, 20:1). OVA-II peptide CTL assay results in Figure 5A confirmed that co-culture with tHSCs-stimulated mDCs significantly inhibited the CTL activity of splenic T cells. Further, lipopolysaccharide (LPS)-induced interferon γ (IFN-γ) production in splenic T cells was also largely inhibited with the existence of tHSCs-stimulated mDCs ( Figure  5B). Remarkably, such effects in splenic T cells were almost completely reversed with silence of DlgR2 in tHSCs-stimulated mDCs ( Figure 5A and 5B). These results indicate that tHSCs-stimulated DlgR2 expression/ production in mDCs should mediate inhibition of splenic T cells. Further studies confirmed that co-culture of tHSCs-stimulated mDCs induced proliferation inhibition ( Figure 5C and 5D) and apoptosis ( Figure 5E) in splenic T cells, which were again largely attenuated with DlgR2 shRNA in mDCs ( Figure 5C-5E). Notably, qHSCsstimulated mDCs had no significant inhibition on splenic T cells ( Figure 5A-5E). Also, the non-sense scramble control shRNA ("c-sh") failed to rescue splenic T cells from tHSCs-treated mDCs ( Figure 5A-5E).
Immune evasion of HCC cells [24][25][26] and other tumor cells is critical for cancer progression [26,27]. One underling mechanism is to decrease the number and activity of anti-tumor immune cells, at both the tumor site and in the lymphoid organs [26,27]. tHSCs could induce T-cell hypo-responsiveness, an effect that is not observed with the qHSCs. Previous studies have shown that B7-H1 (a key member of the B7 family of co-stimulatory molecules) upregulation in tHSCs could result in increased ligation of PD-1 receptor on activated T-cells, causing T cell apoptosis and inhibition of T-cell-mediated tumor cell apoptosis [28][29][30]. Meanwhile, activated tHSCs could also increase the number of in T-reg cells to exert immunosuppressive functions [31,32].
We here discovered another key mechanism of tHSCs in immuno-regulation: via direct communicating with DCs. tHSCs stimulation induced upregulation of DIgR2 in mDCs, which is a key negative regulator of DC-initiated T-cell responses [14]. Further, tHSCsstimulated mDCs induced T-cell hypo-responsiveness, causing decreased CTL activity and reduced IFN-γ production as well as T cell proliferation inhibition and apoptosis. Remarkably, such effects were largely attenuated with DIgR2 shRNA in mDCs. Based on these results, we propose that tHSCs stimulation directly induces DIgR2 expression and production in DCs, which binds to T-cells, causing T-cell hypo-responsiveness and apoptosis.
Intriguingly, we showed that activation of MEK-ERK cascade is required for tHSCs-stimulated DIgR2 expression in mDCs. MEK-ERK inhibitors (PD98059, U0126 and MEK-162) or shRNA-mediated knockdown of MEK1/2 largely attenuated tHSCs-induced DIgR2 expression in mDCs. Further studies will be needed to further characterize the underlying signaling mechanisms of DIgR2 expression in mDCs.

Chemicals, reagents and antibodies
All the antibodies utilized in this study were obtained from Abcam (Suzhou, China). The cell culture reagents were obtained from Invitrogen (Shanghai, China). MEK-ERK inhibitors, PD98059, U0126 and MEK-162 [33,34], were provided by Selleck (Beijing, China). The mRNA primers were designed and synthesized by the Genepharm (Shanghai, China).

Rat HCC tumor model
The detailed protocol of rat tumor model was described in detail in our previous studies [10,11]. Briefly, buffalo rats (4-5 week-old) were obtained from the Animal Center of Soochow University (Suzhou, China). The MRH HCC cells [10,11] were injected into the right flanks of the Buffalo rats. Four weeks after the initial injection, xenograft tumors were established. Surgeryisolated fresh HCC tumors were then cut into small piece (2×1×1 mm 3 ), and were transplanted to the rat livers [10], using the described procedures [10]. Tumor extension was then determined by ultrasound on days 7, 14, 21 and 28 after tumor establishment. The animal protocols were approved by Institutional Animal Care and Use Committee (IACUC) and Ethics Board of Anhui Medical University. All surgical procedures were performed with anesthesia. All efforts were made to minimize suffering.

Primary culture of spleen T cells
As described in our previous studies [10,11], the minced rat spleens were filtered. The splenocytes were then isolated from erythrocytes via centrifugation of the cell suspension on a Ficoll gradient (Histopaque 1083, Sigma) [37]. The cells were layered onto the top of the gradient in a 10-ml Falcon tube, followed by centrifugation at 800×g for 20 min at room temperature without braking. Lymphocytes (mainly T-cells) were collected, and washed twice in PBS-1% FBS (washing buffer). Cells were then cultured.

Western blotting assay
Cell lysates (30-40 μg each condition) were separated by the 10-12% Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) gels, which were transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Suzhou, China) [45]. The blots were blocked (with 5% non-fat milk) and incubated with designated primary plus secondary antibodies. Enhanced chemiluminescence (ECL, Roche, Shanghai, China) detection kit was utilized to visualize the targeted band/s under x-ray films. The intensity (total gray) of each band was quantified via ImageJ software, and was normalized to the loading control (β-Tubulin) [40].

Enzyme-linked immunosorbent assay (ELISA) assay
Cytokine production was examined by the corresponding ELISA kit (R&D Systems), and detailed protocol was described previously [46].

MEK1/2 shRNA
Two lentiviral MEK1/2 shRNAs were also provided by Genepharm (Shanghai, China), which were also added to cultured mDCs for 24 hours. MEK1/2 expression in the resulting cells was tested by Western blotting assay.

Statistical analysis
All statistical analyses were conducted using SPSS 15.0 software. Values were expressed as the mean ± standard deviation (SD). P < 0.05 was considered to indicate a statistically significant result.

CONCLUSIONS
These results indicate that tHSCs directly induces DIgR2 expression in DCs to inhibit T cells.

Authorsʼ contributions
All authors listed carried out the experiments, participated in the design of the study and performed the statistical analysis, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.