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.

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 to uninfected controls. (b) Cytokine production was quantified using enzyme-linked immunosorbent assays. Interferon (IFN)-γ secretion after 12 h of CD4⁺ T cell culture was reduced significantly in LC compared to controls in both peripheral blood and spleen. Lower levels of both IFN-γ and IL-10 from spleen were observed when compared to peripheral blood in LC, but not in controls. IL-4 secretion was not detected in each group (data not shown) (*P < 0·05; **P < 0·01; ***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 as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (c) and programmed death-1 (PD-1) (d) were significantly more expressed in liver cirrhosis (LC) than controls in both peripheral blood and spleen (***P < 0·001).

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 treatment with a mixture of monoclonal antibodies against PD-L1 and PD-L2; the blocking effect is expressed as the ratio of the mean value after blockage relative to the basal condition (n-fold-increase) (*P < 0·05).

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 analysed using flow cytometry. The expression of PD-L1 and PD-L2 was significantly higher in liver cirrhosis (LC) compared to controls (*P < 0·05). (b) Representative gated plots of both PBMC and SMC are shown. (c) Spleen immunostaining from LC and controls confirmed the distribution of PD-1 and PD-1 ligands. Arrowheads indicate positive cells. (d) Their expressions were scored 0 as negative, 1 as positive and 2 as strongly positive to differentiate LC from controls. Spleens from hepatitis C virus (HCV)-related LC showed higher expression of PD-1 and PD-L2 than controls (*P < 0·05; **P < 0·01).

Fig. 5

CD4⁺ CD25⁺ forkhead box P3 (FoxP3)⁺ regulatory T cell (T_reg) frequency. FoxP3⁺ T_reg frequency was analysed using flow cytometry in peripheral blood and spleen and expressed as percentage. Higher frequency of T_reg was observed in both peripheral blood and spleen from liver cirrhosis (LC) patients (**P < 0·01), whereas no differences between peripheral blood and spleen were observed in hepatitis C virus (HCV) patients.

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 secretion (b) increased significantly after splenectomy, similar to IFN-γ/interleukin (IL)-10 ratio (d) (*P < 0·05; **P < 0·01).

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 changes in the CTLA-4⁺ (a), but the frequencies of PD-1⁺ CD4⁺ T cells decreased significantly after splenectomy (b) (***P < 0·001).