Characterization of HCV-specific CD4+Th17 immunity in recurrent hepatitis C-induced liver allograft fibrosis

Abstract

Hepatitis C virus (HCV) recurrence with accelerated fibrosis following orthotopic liver transplantation (OLT) is a universal phenomenon. To evaluate mechanisms contributing to HCV induced allograft fibrosis/cirrhosis, we investigated HCV-specific CD4+Th17 cells and their induction in OLT recipients with recurrence utilizing 51 HCV+ OLT recipients, 15 healthy controls and 9 HCV- OLT recipients. Frequency of HCV specific CD4+ Tcells secreting IFN-γ, IL-17 and IL-10 was analyzed by ELISpot. Serum cytokines and chemokines were analyzed by LUMINEX. Recipients with recurrent HCV induced allograft inflammation and fibrosis/cirrhosis demonstrated a significant increase in frequency of HCV specific CD4+Th17 cells. Increased pro-inflammatory mediators (IL-17, IL-1β, IL-6, IL-8 and MCP-1), decreased IFN-γ, and increased IL-4, IL-5 and IL-10 levels were identified. OLT recipients with allograft inflammation and fibrosis/cirrhosis demonstrated increased frequency of Foxp3+ regulatory T cells (Tregs) that inhibited HCV specific CD4+Th1 but not Th17 cells. This suggests that recurrent HCV infection in OLT recipients induces an inflammatory milieu characterized by increased IL-6, IL-1β and decreased IFN-γ which facilitates induction of HCV specific CD4+Th17 cells. These cells are resistant to suppression by Tregs and may mediate an inflammatory cascade leading to cirrhosis in OLT recipients following HCV recurrence.

Figure 1. Increased HCV specific IL17 and IL10 and decreased INF-γ CD4+ Tcell responses in HCV OLT recipients with recurrent HCV induce allograft fibrosis and high grade inflammation

ELISpot comparing CD4+Tcell IL17(A), INF-γ(B) and IL10(C) response to HCV antigens (NS3, NS4, NS5, CORE) and non specific peptide (HIV Gp120) among four groups - Controls (Normal healthy patients and Non HCV OLT recipients), Group 1(Scheuer G 0-4, S 0-2), Group 2 (Scheuer G0-2, S3-4), Group 3 (Scheuer G3-4, S3-4). Values represented as mean ± SEM spots per million (spm) and significance calculated using Mann Whitney test. 3×10⁵ CD4+ T cells were used per well and each antigen was cultured in triplicate.

Figure 2. Increased frequency of CD4+CD25+Foxp3+ Regulatory T cells in OLT recipients who developed allograft fibrosis/cirrhosis following HCV recurrence

(A) Schematic gating for CD4+CD25+Foxp3+ T cells. PBMCs were gated for leukocyte population(i) CD4(FITC) CD25(APC) double positive cells were gated(ii) Foxp3(PE)+ cells in the gated population were enumerated(iii) (B) Increased frequency of CD4+CD25+Foxp3+ T cells in OLT recipients with HCV recurrence compared to non-HCV OLT and controls. OLT recipients with HCV induced allograft fibrosis (Group 2 – Scheuer G0-2 S3-4, Group 3 –Scheuer G3-4 S3-4) demonstrated increased frequencies of Tregs compared to OLT recipients with no/minimal allograft fibrosis (Group 1) (p<0.05). No statistically significant difference in the frequency of Tregs was observed between Groups 2 and 3.

Figure 3. HCV specific CD4+ Th17 cells but not CD4+ Th1 cells demonstrate resistance to Treg mediated suppression

Five patients each (who had no confounding factors of co-existent alcohol intake and were both donor and recipient CMV negative) were selected from Group 1 (Scheuer G0-4 S0-2), Group 2(Scheuer G0-2, S3-4), Group 3 (Scheuer G3-4, S3-4). CD25 depleted CD4+ effector T cells (Teff) (3×10⁵ cells/well) were co cultured with activated CD25+ CD4+ regulatory T cells(Treg) at varying ratios in the presence of HCV CORE antigen and autologous irradiated APCs and IL17 and INF-γ response measured using ELISpot. (A) - High Foxp3 expression(normalized with GADPH) in the isolated CD4+CD25+ T regulatory compared to CD4+CD25- T effector subsets using real time PCR before co-culture experiments. (B, C) - Tregs at increasing concentrations suppress the frequency of IFN-γ but not IL-17 secreting HCV CORE specific Teff in all the three groups analyzed. (B ii, C ii) – Results of Linear regression analysis and ANOVA to determine Treg number dependant change in responses. Decrease in INF-γ responses significant. (D) Tregs co cultured with Teff cells do not suppress HCV specific IL17 response in non transplant chronic HCV patients. Values in B (i), C (i), D represented as mean ± SEM in spots per million (spm)

Figure 4. In contrast to CD4+ Th1 cells, HCV specific CD4+ Th17 cells demonstrate resistance to IL-10 mediated suppression

Frequencies of CD4+ T cells that secrete either IL-17(A) or INF-γ (B), in response to HCV CORE antigen were evaluated by ELISpot following addition of either anti-IL-10 neutralizing Abs (10μg/mL) or matched isotype control Abs. Anti-IL-10 neutralizing increase the frequency of HCV CORE specific IFN-γ but not IL-17 secreting CD4+ T cells in all the three groups analyzed. (An ii, B ii) Results of linear regression analysis and ANOVA to determine anti-IL-10 concentration dependant change in responses. Increasing anti-IL10 increased INF-γ responses. Values in A (i), B (i) represented as mean ± SEM spots per million cells.

Figure 5. Induction of HCV CORE specific Th17 cells in vitro using sera derived from Group 3 patients containing high levels of pro-Th17 factors

HCV CORE specific Th17 cells stimulated in vitro with HCV CORE antigen for 8–10 days in 25% pooled sera derived from Group 3 patients (Scheuer G3-4, S3-4; filled boxes) or 25% normal AB negative or autologous sera used as a control medium (open boxes) were measured by ELISpot using PBMCs derived from patients of Group 1(Scheuer G0-4 S0-2;n=5), Group 2 (Scheuer G0-2, S3-4; n=5) and control individuals (non HCV). Values represented as mean ± SEM spots per million cells (A) Increased HCV CORE specific Th17 response in Group 1 and Group 2 cultured in Group 3 sera when compared to those cultured in autologous serum or control individuals (B) IL-17 secreting HCV CORE specific Th17 cells induced in vitro using Group 3 sera were enriched in the memory T cell compartment (CD45ROhighCD45RAlow) when assessed by flow cytometry. (C) CD45ROhighCD45RAneg (Memory) and CD45ROnegCD45RAhigh (Naïve) T cells were sorted from HCV CORE specific CD4+ T cells from Group 1 and 2 patients incubated with either control medium or Group 3 sera. ROR-C expression was quantified using real time PCR (values represented normalized to GADPH expression). Memory cells when incubated with Group 3 sera were noted to express significantly higher ROR- C transcripts. * p value <0.05, ** p<0.01

Figure 6. IFN-γ down modulates and IL-6, IL-1β synergistically promote the induction of HCV specific Th17 cells in OLT recipients with HCV recurrence

Frequency of HCV CORE specific CD4+ T cells secreting IL-17 induced in vitro was evaluated using ELISpot following neutralization of IFN-γ or IL-6 or IL-1β bioactivity by addition of appropriate mAbs (10μg/mL) to cultures incubated with pooled Group 3 sera (filled boxes) or 25% autologous or normal AB negative sera was used as a control (open boxes). Values represented as mean ± SEM. Neutralization of IL-6 or -IL1β reduced the frequency of HCV CORE specific Th17 cells by 15.6±3.2% and 16.2±2.9% respectively. Combined blockade of IL-6 and IL-1β decreased the frequency of HCV CORE specific Th17 cells by 37.4±4.6% (p<0.01) than either alone. Neutralization of IFN-γ demonstrated a 30.5±4.2% (p<0.01) increase in the frequency of HCV CORE specific Th17 cells compared to cultures treated with isotype control Abs. ** p value <0.01