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Clin Exp Immunol. Aug 2011; 165(2): 243–250.
PMCID: PMC3142649

Modulation of CD4+ T cell responses following splenectomy in hepatitis C virus-related liver cirrhosis

Abstract

Dysfunction of T cells is a common feature in chronic persistent viral infections, including hepatitis C virus (HCV), and although hepatic and peripheral T cells have been studied extensively in chronic HCV hepatitis, the role of splenic T cell responses in such patients is poorly defined. This is an important issue, as thrombocytopenia is a complication of HCV-related liver cirrhosis (LC), due to splenic platelet sequestration and bone marrow suppression; splenectomy has been proposed to treat such patients. Herein, we studied peripheral blood mononuclear cells (PBMC) and splenic lymphoid subpopulations from a total of 22 patients, including 15 with HCV-related LC with marked thrombocytopenia treated with splenectomy, and seven controls. CD4+ T cells from peripheral blood and spleen were isolated and phenotype and function evaluated. Splenic CD4+ T cells in patients with LC expressed molecules associated with inhibitory signalling, including increased frequency of negative markers such as cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and programmed death 1 (PD-1) and decreased production of cytokines. Patients with LC manifest higher levels of splenic CD4+ regulatory T cells and PD-L1- and PD-L2-expressing cells than controls. Blocking of PD-1/PD-1 ligand interaction reconstituted proliferative and cytokine responses of splenic mononuclear cells (SMC) from patients with LC. Splenectomy was followed by an increase in the ratio of interferon (IFN)-γ to interleukin (IL)-10 and a reduction of PD-1-expressing CD4+ T cells in peripheral blood. Our data suggest that peripheral tolerance is promoted by the spleen in LC via the up-regulated expression of PD-1 ligands.

Keywords: HCV, liver cirrhosis, PD-1, PD-1 ligand, splenectomy

Introduction

Hepatitis C virus (HCV) is a major public health problem, involving approximately 170 million people worldwide [1]. HCV is highly efficient at establishing persistent infection, and 70–80% of subjects are unable to clear the virus, resulting in the development of chronic liver disease and liver cancer [2]. It has long been accepted that the host immune system plays a unique role in HCV infection because of its potential to contribute to liver injury, and that HCV-specific CD4+ T cells are essential in the generation of a successful HCV-specific immune response [3]. Indeed, impaired CD4+ T cell responses have been associated with a higher rate of chronic disease and persistent viraemia [4,5]. The finding that hepatocytes are the primary site of HCV viral replication has led to the study of the role of liver HCV-specific T cell responses [4,6]. However, there are relatively few studies on the immune profile of human splenocytes and, even though the spleen has an important role in the induction and regulation of immune responses, the phenotypic and functional aspects of splenic CD4+ T cells in patients with HCV are, to a large extent, unknown.

Splenomegaly occurs occasionally in patients with liver cirrhosis (LC), including HCV infection, and is associated with the development of thrombocytopenia, due to both splenic platelet sequestration [7] and bone marrow suppression [8]. Interferon (IFN)-α/ribavirin combination therapy is the treatment of choice for HCV infection and is known to reduce the stage of disease and prevent the occurrence of hepatocellular carcinoma [911]. However, patients with low levels of platelets tolerate treatment with IFN-α poorly, which itself can cause thrombocytopenia [12,13]; splenectomy has been proposed as an efficient therapeutic option in patients with HCV-related LC [14] prior to anti-viral therapy. Nevertheless, this practice is not accepted widely and is even considered inappropriate [15]. The purpose of this report is to evaluate splenic immune subpopulations and compare them to peripheral blood mononuclear cells in HCV-related LC and to assess the role of splenectomy in altering T cell responses in such patients, to focus and clarify whether splenectomy would be appropriate supportive therapy.

Materials and methods

Subjects

Splenic tissues from a total of 22 patients were included in this study; 15 patients were diagnosed as HCV-related LC and seven were controls who either underwent post-traumatic splenectomy or gastric cancer-associated splenectomy and lymphadenectomy (Table 1). The splenectomy in patients with HCV-related LC was performed to improve pancytopenia prior to the institution of IFN-α therapy. All control patients had normal spleen size and were considered to be immunologically intact hosts. Splenic mononuclear cells (SMC) and peripheral blood mononuclear cells (PBMC) were isolated from each of the 22 samples. In addition, in a nested substudy, PBMC were harvested from 11 of the 15 patients with HCV-related LC 2 months after splenectomy and before the administration of IFN therapy. Splenic tissue samples were subjected to phenotypic analysis utilizing immunohistochemical techniques. All samples were studied after obtaining appropriate institutional informed consent and all experimental protocols were conducted under the Guidelines of Research Ethics Committee of Kyushu University.

Table 1
Characteristics of the subjects enrolled in the study

Preparation of cells

Freshly obtained splenic tissue was subjected to mechanical digestion and dissociated cells filtered through a 150-µm nylon mesh. The splenic cells and heparinized peripheral blood were then subjected to Ficoll-Hypaque gradient centrifugation and enriched populations of SMC and PBMC harvested from the interface, respectively. All mononuclear cells were washed and cryopreserved in media containing 7·5% dimethylsulphoxide (DMSO) and stored in liquid nitrogen until use. CD4+ T cells were negatively isolated using magnetic beads (CD4 isolation kit II; Miltenyi Biotec, Aubum, CA, USA).

Isolated T cells exhibited > 95% viability confirmed by trypan blue dye exclusion and > 90% purity by flow cytometry. Myeloid-derived dendritic cells (mDC) were separated with an mDC isolation kit (Miltenyi Biotec) performed by two magnetic separation steps. The frequency of the isolated CD1c+CD19- mDC was determined to be > 80% by flow cytometry. These procedures are standardized in our laboratory and have been described previously in detail [16].

Proliferation assay and cytokine production

Isolated CD4+ T cells were cultured (1 × 105/well) in 96-well plates precoated with 10 µg/ml of monoclonal antibody to CD3 (OKT3; R&D Systems, Minneapolis, MN, USA) in the presence of human interleukin (IL)-2 (10 U/ml) for 5 days and then pulsed with 1·0 µCi of [3H]-thymidine per well during the last 12 h of culture and subsequently harvested, and counted in a scintillation counter (Betaplate; Wallac, Inc., Waltham, MA, USA). Supernatant fluids were harvested before a thymidine pulse and analysed for levels of IFN-γ, IL-4 or IL-10 production by sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems), using a combination of unlabelled and biotinor enzyme-coupled monoclonal antibody to IFN-γ, IL-4 or IL-10. In all instances, known positive and negative controls were used throughout and all assays were performed in triplicate.

Additionally, a programmed death 1 (PD-1)/PD-1 ligand blocking assay was performed. Briefly, PBMC and SMC were incubated for 45 min at 37°C with a mixture of anti-PD-L1 and PD-L2 antibody (10 µg/ml each) or isotype control antibody (e-Biosciences, San Diego, CA, USA). Proliferation and IFN-γ production was performed as described above. Appropriate positive and negative controls were used throughout.

Flow cytometric analysis of the cell surface and intracellular antigens

Two- or three-colour multi-parameter flow cytometry was performed using a fluorescence activated cell sorter (FACS)Caliber Flow Cytometer (BD Biosciences, San Diego, CA, USA). Cell surface monoclonal antibodies utilized included CD4, CD25, CD28, CD154, PD-1, PD-L1 and PD-L2, and intracellular monoclonal antibodies (mAb) against cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and forkhead box P3 (FoxP3) (BD Biosciences). mAbs specific for CD25, CD28, CD154, CTLA-4, PD-1 and FoxP3 were utilized for the analysis of CD4+ T cells and the mAbs specific for CD40, CD80, CD83 and CD86, PD-L1 and PD-L2 (BD Biosciences) were utilized for the analysis of mDCs.

Cryopreserved mononuclear cells (2·5 – 5·0 × 105) were stained for cell surface antigen expression at 4°C in the dark for 30 min, washed twice in 2 ml phosphate-buffered saline containing 1% bovine serum albumin and 0·01% sodium azide and fixed in 200 µl of 1% paraformaldehyde. Intracellular staining for CTLA-4 was performed following membrane permeabilization of fixed cells with 0·02% saponin (BD Biosciences). FoxP3 staining was performed according to the manufacturer's instructions. Isotype-matched control antibodies were used to determine the background levels of staining. We chose intracellular staining for CTLA-4 because CTLA-4 contains an extracellular V domain, transmembrane domain and a cytoplasmic tail; the frequencies of intracellular CTLA-4 was similar to the frequency of surface CTLA-4+ T cells.

Immunohistochemical staining of spleen specimens

Spleen specimens were formalin-fixed, paraffin-embedded, and used for immunostaining. Deparaffinized and rehydrated sections were used for immunostaining for the cell surface markers PD-1, PD-L1, and PD-L2. Endogenous peroxidases were first blocked by incubation in normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 20 min; monoclonal antibodies were diluted 1:100 (Dako, Kyoto, Japan), and immunostaining was performed on coded sections and were scored 0 as negative, 1 as positive and 2 as strongly positive by a ‘blinded’ and qualified pathologist.

Statistical evaluation

The data obtained were analysed using Prism software (version 5.0; GraphPad Software Inc., La Jolla, CA, USA) and then compared using the Mann–Whitney U-test, a non-parametric test that does not assume Gaussian variation. ELISA and proliferation data represent the average of triplicate wells. P values less than 0·05 were considered to be statistically significant.

Results

Splenic CD4+ T cells from patients with HCV-related LC demonstrate lower levels of activation

Purified splenic CD4+ T cells from patients with HCV-related LC and controls were stimulated in vitro with anti-CD3 mAb in the presence of IL-2. As illustrated in Fig. 1a, while splenic CD4+ T cell proliferation was significantly lower in patients with HCV-related LC compared to uninfected controls (P < 0·05), there was no statistical difference in peripheral CD4+ T cell proliferation between patients and controls. In addition, significantly lower levels of proliferation in splenic CD4+ T cells, compared with their corresponding peripheral CD4+ T cell counterparts, were detected in patients with HCV-related LC (P < 0·05) but not in controls, which is consistent with previous reports [3,17].

Fig. 1
Proliferation and cytokine production of CD4+ T cells upon CD3 and interleukin (IL)-2 stimulation. (a) CD4 T cell proliferation, determined by [3H]-thymidine incorporation, was significantly lower in spleen from liver cirrhosis (LC) patients compared ...

Moreover, IFN-γ secretion after 12 h of CD4+ T cell culture was reduced significantly in cultures from both peripheral blood and spleen from patients with HCV-related LC compared to controls (P < 0·05) (Fig. 1b). Conversely, peripheral CD4+ cell cultures from patients with HCV-related LC synthesized higher relative levels of IL-10 compared to both splenic CD4+ cells (P < 0·001) and healthy controls (P < 0·05), whereas there was no statistical difference in the levels of IL-10 synthesized by the splenic CD4+ T cells (Fig. 1c). There were undetectable levels of IL-4 synthesized by either peripheral or splenic CD4+ T cells (data not shown).

Expression of inhibitory signalling molecules by CD4+ T cells from LC patients

It was reasoned that reduced levels of CD4+ T cell activation could be secondary to immune exhaustion and/or the expression of molecules associated with negative signalling. Therefore, expression of CD28, CD154, CTLA-4 and PD-1 [1820] was analysed in peripheral and splenic CD4+ T cells from patients with HCV-related LC and controls using standard flow cytometry.

As seen in Fig. 2, there was no difference in the relative levels of CD28 or CD154 expression by CD4+ T cells from patients with HCV-related LC compared to controls either in peripheral blood or spleen (Fig. 2a,b). In contrast, the frequencies of CTLA-4- and PD-1-expressing CD4+ T cells were increased significantly in peripheral blood and spleen in patients with HCV-related LC compared to controls (P < 0·001) (Fig. 2c,d). Moreover the frequency of both CTLA-4- and PD-1-expressing CD4+ T cells was higher in spleen compared to peripheral blood from patients with HCV-related LC (P < 0·001).

Fig. 2
CD4+ T cells phenotype. Cell surface markers were determined as the percentages of CD4+ T cells by flow cytometry. No differences in the frequencies of CD28 (a) and CD154 (b), which positively regulate CD4+ T cells, were observed. Negative markers such ...

These results indicate that impaired proliferative responses and reduced synthesis of IFN-γ in patients with HCV-related LC are associated with increased levels of expression of the inhibitory cell surface markers known to be associated with decreased immune function and exhaustion, such as CTLA-4 and PD-1.

Blocking of PD-1/PD-1 ligand interaction reconstitutes proliferative and cytokine responses of SMC from patients with HCV-related LC

To explore the hypothesis that decreased proliferation and IFN-γ production from splenic CD4+ T cells in patients with HCV-related LC were indeed associated with PD-1 increased expression, blocking experiments were performed using mAbs to both PD-L1 and PD-L2. As seen in Fig. 3, blocking of PD-1 ligands resulted in a two- to fourfold enhancement of the proliferative response ratio of PBMC and SMC, respectively (Fig. 3a) and a fourfold enhancement of IFN-γ production by SMC from LC patients (P < 0·05) (Fig. 3b). Similar blocking experiments failed to show any significant increase in proliferative response in cultures of PBMC and SMC from controls.

Fig. 3
Blockage of programmed death-1(PD-1)/PD-1 ligands antagonizes T cell inhibition of splenic mononuclear cells (SMC) from hepatitis C virus (HCV)-related liver cirrhosis. Comparison of proliferation (a) and interferon (IFN)-γ production (b) after ...

The increased ratio of proliferation seen in cultures of SMC was significantly higher than that seen with PBMC with the use of mAbs against the PD-1 ligands (PD-L1 and PD-L2). These results in concert suggest that the decreased T cell responsiveness of the spleen in patients with HCV-related LC is associated at least partially with the up-regulation of PD-1/PD-1 ligands.

PD-1 ligand expression is higher in patients with HCV-related LC

Binding of the co-stimulatory molecules PD-L1 and PD-L2 on the surface of antigen-presenting cells (APC) by PD-1 expressing T cells induces down-regulation of immune responses [19,21,22]. As outlined above, CD4+ T cells from patients with HCV-related LC demonstrated a marked increase of PD-1 expression compared to controls. We therefore examined PD-L1 and PD-L2 expression on unfractionated PBMC and SMC. As seen in Fig. 4a, a higher frequency of PD-L1- and PD-L2-expressing cells in SMC samples compared to PBMC was observed in both patients with HCV-related LC and control donors. The frequency of PD-L1 and PD-L2 expressing cells was higher in the PBMC and SMC from patients with HCV-related LC compared with the normal donors (P < 0·05), assessed with FACS (Fig. 4b). These results were confirmed by immunohistochemical analyses of splenic tissues from patients with HCV-related LC and normal control donors (Fig. 4c,d).

Fig. 4
Expression of negative co-stimulation molecules programmed ligand death-1 (PD-L1) and PD-L2. (a) The expression of negative co-stimulatory molecules PD-L1 and PD-L2 on peripheral blood mononuclear cells (PBMC) and splenic mononuclear cells (SMC) were ...

CD4+ CD25+ FoxP3+ regulatory T cells (Treg) are increased in patients with HCV-related LC

To assess the role of Treg and molecules involved in co-stimulation expressed by myeloid dendritic cells, we examined the frequencies of CD4+ CD25+ FoxP3+ Treg in peripheral blood and spleen from patients with HCV-related LC and controls. As noted in Fig. 5, higher levels of CD4+ CD25+ FoxP3+ T cells were observed in patients with HCV-related LC compared to controls in both peripheral blood and spleen. However, no differences were determined in patients with HCV-related LC between peripheral blood and spleen. Secondly, we analysed the frequency and mean density of CD40, CD80, CD83 and CD86 expressing mDC in peripheral blood and spleen from patients with HCV-related LC and control donors. There was no statistical difference in the frequency of CD1c+ CD19- in the peripheral blood compared to spleen (data not shown).

Fig. 5
CD4+ CD25+ forkhead box P3 (FoxP3)+ regulatory T cell (Treg) frequency. FoxP3+ Treg frequency was analysed using flow cytometry in peripheral blood and spleen and expressed as percentage. Higher frequency of Treg was observed in both peripheral blood ...

Effects of splenectomy on the response of CD4+ T cells in patients with HCV-related LC

To study the consequences of splenectomy on T cell function, an enriched population of CD4+ T cells from peripheral blood was examined before and after the removal of spleen. As seen in Fig. 6a, while there was some increase in the rate of anti-CD3-induced proliferation by the CD4+ T cells post-splenectomy, the data were found to be statistically non-significant. Conversely, the ratio of the levels of IFN-γ synthesized by CD4+ T cells post-splenectomy were markedly higher (P < 0·05) (Fig. 6b). Of interest to note was the finding that, whereas there was no statistical difference in the levels of IL-10 synthesized pre- versus post-splenectomy, there was clearly an increase in the ratio of IFN-γ to IL-10 post-splenectomy (P < 0·01) (Fig. 6c).

Fig. 6
Proliferation and cytokine production of CD4+ T cells before and after splenectomy. Proliferation of CD4+ T cells in peripheral blood was studied before and after splenectomy in patients with hepatitis C virus (HCV)-related cirrhosis. Interferon (IFN)-γ ...

Interestingly, when the frequency of CTLA-4- and PD-1-expressing CD4+ T cells were examined, while there were no differences in the frequencies of CTLA-4-expressing CD4+ T cells, there was a marked decrease in the levels of PD-1-expressing CD4+ T cells post-splenectomy (P < 0·001) (Fig. 7).

Fig. 7
Expression of programmed death-1 (PD-1) but not cytotoxic T lymphocyte associated antigen-4 (CTLA-4) on CD4+ T cells change after splenectomy. CTLA-4 and PD-1 expression on CD4+ T cells were analysed before and after splenectomy. There were no remarkable ...

Discussion

It has been long been suggested that liver injury and disease progression in HCV infection are due in part to immune-mediated events [3]. Impaired adaptive immune responses have been documented in patients with chronic HCV infection and reasoned to be secondary to persistent antigenic stimulation [17,23]. Exhaustion of HCV-specific T cells [24,25], increased frequency of Tregs[26,27] and cytokine production patterns are critical host factors affecting the outcome of disease [28,29]. DC functions in HCV infection remain controversial, although most studies describe either reduced expression levels of co-stimulatory molecules [30] or a decreased allostimulatory capacity of DC [31]. Very little is known about the role of splenic T cells in HCV-related disease. The present study was therefore aimed to clarify the role of splenic CD4+ T cells in patients with HCV related LC. To this end we took advantage of a cohort of patients who underwent splenectomy to treat severe thrombocytopenia in HCV-related LC prior to IFN-α therapy. This report demonstrates for the first time that markers associated traditionally with peripheral tolerance are also promoted by the spleen in HCV-related disease. Indeed, patients with HCV-related LC demonstrate splenic CD4 inhibitory signalling have higher levels of splenic CD4 Tregs, and of PD-L1- and PD-L2-expressing cells. Moreover, blocking of PD-1/PD-1 ligand interaction reconstitutes proliferative and cytokine responses.

Importantly, splenectomy is followed by an increasing ratio of IFN-γ to IL-10 and a reduction of PD-1-expressing CD4+ T cells in peripheral blood.

One of the major mechanisms involved in the induction of peripheral tolerance is thought to involve the function of Tregs[32,33] by their interaction with APCs, including the generation of co-stimulation signals and a role for the programmed cell death pathway (PD-1/PD-1 ligands) [22,34]. Up-regulation of PD-1 on CD4+ and CD8+ T cells has been reported in the livers of HCV hepatitis patients [28]. A significant up-regulation of PD-1 expression was detected on exhausted virus-specific T cells in a mouse model with chronic lymphocytic choriomeningitis virus infection, and promoted viral persistence [20]. Our data demonstrate that this pathway is also involved in T cell signalling among splenic cells of patients with HCV-related LC. Subjects described herein have reduced T cell proliferation and lower levels of IFN-γ production upon CD3 stimulation of CD4+ T cells, and this is associated with high frequencies of CTLA-4+ or PD-1+ T cells, but no correlation was noted with the frequency of CD28+ or CD154+ T cells. This result correlates with the up-regulation of PD-L1 and PD-L2 in SMC. Blocking of PD-1 signalling on exhausted T cells results in the restoration of T cell function, with an increased proliferation, cytotoxicity and cytokine production [18,34].

Blockage of the programmed cell death pathway has also been suggested as a potential immune therapy to enhance the effector T cell responses during persistent HCV infection in a murine HCV model [35]. The inhibitory molecules PD-L1 and PD-L2 play a prominent role in suppressing activated T cells, via a cell contact-mediated mechanism [36]. In addition, these PD-L1/2-mediated suppressive functions are considered to be due to the suppression of IL-2 production [36]. PD-L1/2 has been identified by their distinct and different expression patterns.

Thus, whereas PD-L1 expression is noted primarily in haematopoietic and parenchymal cells [21], PD-L2 expression is restricted mainly to DC and macrophages [21]. Moreover, recent studies have also identified PD-L2 expression in endothelial cells which mediate immune tolerance [37,38]. The findings of functional studies of the differentiation between PD-L1 and PD-L2 still remain controversial, but indicate that PD-L1 and PD-L2 exert overlapping effects on T cell responses [22,38].

The immunohistochemistry findings in the current study reflect high PD-1 expression on spleen from patients with HCV-related LC. Interestingly, PD-1 ligand expression, especially PD-L2, is up-regulated in the spleen with HCV-related LC. The expression of PD-L1 and PD-L2 in SMC was high in comparison to expression in PBMC. Therefore, PD-L1 and PD-L2 expression by splenic cells may contribute to T cell responses, suggested by the recovery of the proliferation and IFN-γ production of SMC by blocking PD-1/PD-1 ligands. The spleen is a reservoir of PD-L1 and PD-L2 in patients with HCV-related LC; splenectomy may induce the recovery of IFN-γ production and peripheral CD4+ T cell proliferation. Therefore, splenectomy in patients with HCV-related LC may not alter the generation of adaptive immune response of CD4+ T cells which thus allows them to achieve a virological response with IFN therapy. Our data reveal more details of the immunobiology of HCV-specific immune response in the spleen. Clearly, more work is needed before a clinical recommendation.

Importantly, our study demonstrates that peripheral tolerance in patients with HCV-related LC is at least partially promoted by the splenic up-regulation of PD-1 ligands.

Disclosure

The authors have no conflicts of interest to declare.

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