Suppressor CD4+ T cells expressing HLA-G are expanded in the peripheral blood from patients with acute decompensation of cirrhosis

Abstract

Objective: Identifying components of immuneparesis, a hallmark of chronic liver failure, is crucial for our understanding of complications in cirrhosis. Various suppressor CD4⁺ T cells have been established as potent inhibitors of systemic immune activation. Here, we establish the presence, regulation and mechanism of action of a suppressive CD4⁺ T cell subset expressing human leucocyte antigen G (HLA-G) in patients with acute decompensation of cirrhosis (AD).

Design: Flow cytometry was used to determine the proportion and immunophenotype of CD4⁺HLA-G⁺ T cells from peripheral blood of 20 healthy controls (HCs) and 98 patients with cirrhosis (28 with stable cirrhosis (SC), 20 with chronic decompensated cirrhosis (CD) and 50 with AD). Transcriptional and functional signatures of cell-sorted CD4⁺HLA-G⁺ cells were delineated by NanoString technology and suppression assays, respectively. The role of immunosuppressive cytokine interleukin (IL)-35 in inducing this population was investigated through in vitro blockade experiments. Immunohistochemistry (IHC) and cultures of primary human Kupffer cells (KCs) were performed to assess cellular sources of IL-35. HLA-G-mediated T cell suppression was explored using neutralising antibodies targeting co-inhibitory pathways.

Results: Patients with AD were distinguished by an expansion of a CD4⁺HLA-G⁺CTLA-4⁺IL-35⁺ immunosuppressive population associated with disease severity, clinical course of AD, infectious complications and poor outcome. Transcriptomic analyses excluded the possibility that these were thymic-derived regulatory T cells. IHC analyses and in vitro cultures demonstrate that KCs represent a potent source of IL-35 which can induce the observed HLA-G⁺ phenotype. These exert cytotoxic T lymphocyte antigen-4-mediated impaired responses in T cells paralleled by an HLA-G-driven downregulation of T helper 17-related cytokines.

Conclusion: We have identified a cytokine-driven peripherally derived suppressive population that may contribute to immuneparesis in AD.

Keywords: immunology in hepatology; immunoregulation.

Figure 1

Expansion of CD4⁺HLA-G⁺ T cell population in patients with acute decompensation of cirrhosis (AD). Peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) (n=20) and patients (stable cirrhosis (SC), n=28; chronic decompensated cirrhosis (CD), n=20 and AD, n=50) were assessed for surface levels of human leucocyte antigen G (HLA-G) using flow cytometry (gating strategy online supplemental figure S1A). (A) Representative flow cytometry histograms used to determine HLA-G levels, all gated based on fluorescence-minus-one (FMO) controls (left panel). Percentage of HLA-G expressing cells in CD3⁺CD4⁺CD8^-T cells in HCs compared with patients with SC, CD and AD (right panel). (B) Representative histograms of immunoglobulin-like transcript 4 (ILT4) levels on monocytes in HCs and patients (SC, CD and AD) (left panel). Distribution of ILT4⁺ monocytes in HCs and in patients (right panel). (C) Correlation of the frequency of CD4⁺HLA-G⁺ T cells with clinical parameters and disease severity scores in patients with AD (model for end-stage liver disease (MELD) scores, Child-Pugh (CP), white cell count (WCC) and C reactive protein (CRP)). (D) Distribution of CD4⁺HLA-G⁺ T cells with increasing disease severity in patients within the AD cohort (AD-No ACLF, n=25; AD-acute-on-chronic-liver failure (ACLF), n=25) compared with SC (n=28) (top panel). Distribution of CD4⁺HLA-G⁺ T cells across the clinical phenotypes of AD (stable decompensated cirrhosis (SDC), n=8; unstable decompensated cirrhosis (UDC), n=13) and AD-ACLF (n=25) (no analyses of the pre-ACLF were performed due to the limited number of this phenotype in the patient cohort) (bottom panel). (E) Distribution of CD4⁺HLA-G⁺ T cells in non-surviving (n=24) and surviving patients (n=23) with AD within 90 days following admission. (F) HLA-G expression was assessed in patients with AD who developed culture-positive primary infections (n=11) and the ones who developed culture-negative infections (n=9) (left panel). Distribution of HLA-G⁺ T cells was compared in patients withh AD who developed short-term secondary infections (n=9) (<28 days) and the ones who developed it in >28 days (n=6) (right panel). Non-parametric statistical analysis was used (Mann-Whitney U test for two group comparison and Kruskal-Wallis followed by a Dunn’s test for multiple comparisons between more than two groups). Data are presented as median values with IQR. Correlation coefficients (r) and correlation p values were tested using non-parametric Spearman’s correlation test. *P<0.05; ***p<0.0005.

Figure 2

Immunophenotyping to characterise CD4⁺HLA-G⁺ population in patients with acute decompensation of cirrhosis (AD) demonstrates that the population is CTLA-4^highIL35^highIL-10^low. (A) Representative flow dot plots and histograms of surface levels of inhibitory marker CTLA-4 assessed in CD4⁺HLA-G⁺ (left panel). CTLA-4 levels on CD4⁺HLA-G⁺ T cells in healthy controls (HCs) and in patients with AD (right panel). (B) Representative dot plots of intracellular cytokine staining used to define levels of interleukin (IL)-35 in the CD4⁺HLA-G⁺ population (left panel). Co-expression of HLA-G and IL-35 in HCs compared with patients with AD (right panel). (C) CD4⁺HLA-G⁺ T cells assessed for their co-expression of IL-35 and IL-10 in patients with AD (n=14). Mann-Whitney U test for two group comparison. Data are presented as median values with IQR. HLA-G, human leucocyte antigen G; CTLA-4, cytotoxic T lymphocyte antigen-4; FMO, fluorescence minus one.

Figure 3

Transcriptional and functional features of CD4⁺HLA-G⁺ T cells from patients with acute decompensation of cirrhosis (AD). (A) Quantitative analysis of immune-related gene in HLA-G⁺ compared to thymus-derived regulatory T cells (tTregs) and or HLA-G^- counterparts from patients with AD (n=4) using NanoString Technologies. Data show Venn diagrams of significantly differentially expressed (DE) genes. (B) Volcano plots comparing HLA-G⁺ T cells to either tTregs or HLA-G^- T cells. Gene names are listed for DE genes showing that gene expression pattern of immune-related genes in circulating CD4⁺HLA-G⁺ T cells are distinct from Tregs and HLA-G-negative counterparts. (C) HLA-G⁺ cells suppressive capacity on CPD-labelled responder peripheral blood mononuclear cells (PBMCs) proliferation. Representative histograms of live CD3⁺ T cells proliferating in the absence or presence of α-CD3 stimulation (top left panel). Representative flow histograms of proliferating CD3⁺ T cells in the presence of HLA-G⁺ fractions at the tested ratios (bottom left panel). Suppressive capacity of HLA-G⁺ (n=4) isolated from patients with AD after 5 days of co-culture (right panel). HLA-G, human leucocyte antigen G.

Figure 4

Sera conditioning and the role of interleukin (IL)-35 in inducing CD4⁺HLA-G⁺ suppressor cells. (A) Assessment of the effect of sera at inducing HLA-G⁺ phenotype in cultured CD4⁺ T cells from healthy controls (HCs) following 48 hours of culture in the presence of 25% sera from HCs and patients with acute decompensation of cirrhosis (AD) (n=15 per group). (B) Proliferation of HC peripheral blood mononuclear cells (PBMCs) in the presence of HC or AD sera-induced human leucocyte antigen G (HLA-G) expression in CD4⁺ T cells (results are representative of seven independent experiments). (C) Concentrations of IL-35 in sera samples were measured in HCs (n=25) and patients with AD (n=25). (D) Measurement of the role of IL-35 in driving the HLA-G-positive phenotype (left panel) and its effect on proliferation responses (right panel). Anti-IL-35 neutralising antibody (α-IL-35, used at 10 µg/mL) (n=12) was used to block IL-35 prior to sera exposure. This was suppressed when sera from patients with AD were pretreated with neutralising IL-35 antibody. Mann-Whitney U test for two group comparison and Wilcoxon matched pairs signed rank test was used for all paired non-parametric tests. Data are presented as median values with IQR.

Figure 5

Immunohistochemical and in vitro evaluation of sources of interleukin (IL)-35 from diseased liver. (A) Immunohistochemistry (IHC) was used to detect and quantify IL-35 (EBI3) in liver explants tissues of patients with acute decompensation of cirrhosis (AD) compared with pathological stable cirrhosis (SC) control (alcohol-related cirrhosis). Single stain for IL-35, detected using DAB (brown), nuclei detected using haematoxylin (blue) with 200× magnification. (B) Double stain for IL-35 (brown) and intrahepatic CD68⁺ tissue Kupffer cells (KCs) (CD68 detected using Permanent Red (red)). Nuclei were detected using haematoxylin (blue) with 200× magnification (top panels). For pseudofluorescence, IL-35, CD68 and nuclei were visualised by red, green and blue, respectively. Co-localisation of IL-35 and CD68 was visualised by yellow (bottom panels). (C) Human primary KCs were assessed for their capacity to secrete IL-35 in vitro following no stimulation (n=9), stimulation with high mobility group box 1 (HMGB1) (n=9) or Escherichia coli lipopolysaccharide (LPS) (n=10) and simultaneous stimulation with both LPS+HMGB1 (n=9). ELISA was used to detect IL-35 concentrations in collected supernatants following 48 hours incubation. (D) Receptors involved in the signalling pathways were tested for their role in the LPS-induced IL-35 secretion through blockade of CD14 (n=6) and toll-like receptor 4 (TLR-4) receptors (n=6). Kruskal-Wallis followed by a Dunn’s test for multiple comparisons between more than two groups. Data are presented as median values with IQR. *P<0.05; **p<0.005; ***p<0.0005.

Figure 6

CD4⁺HLA-G⁺ T cells suppressive capacity is reversed following blockade of cytotoxic T lymphocyte antigen-4 (CTLA-4), whereas blockade of human leucocyte antigen G (HLA-G) impairs T helper 17 (Th17)-related cytokine secretion. (A) HLA-G expressing cells generated following preconditioning of CD4⁺ T cells in sera from patients with acute decompensation of cirrhosis (AD) were tested for their capacity to suppress proliferating peripheral blood mononuclear cells (PBMCs) in the presence of absence of α-CTLA-4 (10 µg/mL) (n=8). (B) Levels of cytokines playing a role in T cell proliferation/function in supernatants collected following 5-day co-cultures of CD4⁺HLA-G⁺ T cells with PBMCs with or without α-CTLA-4 were measured using multiplex cytokine detection system (n=8). (C) Blockade of HLA-G restored production of Th17-related cytokines/chemokines. Wilcoxon matched pairs signed rank test was used for all paired non-parametric tests. Data are presented as median values with IQR.