Galectin-9 and IL-21 Mediate Cross-regulation between Th17 and Treg Cells during Acute Hepatitis C

<div><p>Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). <i>In vitro</i> supplementation of Tim-3^high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.</p></div

Galectin-9 knockdown limits Treg-mediated inhibition of IFN-γ and IL-21 production by HCV-specific CD4 T cells.

<p>(<b>A</b>) Representative combined CFSE/ICS assay demonstrating reduced Treg-mediated inhibition upon knockdown of Gal-9 by siRNA. Plots depict production of IFN-γ, IL-21 and TNF-α by HCV-specific proliferating (CFSE^low) T cells in response to stimulation with recombinant HCV NS4 protein. PBMC cultures depleted of Tregs (Top panel) or with addition of Tregs (ratio 1∶4, Tregs∶CD25-depleted PBMCs) treated with either control siRNA (Middle panel) or <i>LGALS9</i> siRNA were stimulated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a>. Plots are gated on CD3⁺ CD8^neg lymphocytes. (<b>B</b>) Percentage of Treg-mediated inhibition of IFN-γ and IL-21 production by HCV-specific T cells upon stimulation with recombinant HCV proteins (NS3: black symbols or NS4: grey symbols) in the presence of Treg cells treated with either control or <i>LGALS9</i> siRNA, as described in (A) and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a>. The percentage inhibition of cytokine production upon addition of Tregs to the stimulated PBMC cultures was calculated using the following formula: (% CFSE^lowcytokine⁺CD8^neg T cells in absence of Tregs – % CFSE^lowcytokine⁺CD8^neg T cells in presence of Tregs)/% CFSE^lowcytokine⁺CD8^neg T cells in absence of Tregs×100. P-values were calculated using Wilcoxon signed rank test.</p

Increased frequency of NS3 and NS4-specific CD4 T cells producing Th17 and Th1 cytokines during acute resolving HCV.

<p>CD25-depleted PBMC from patients with acute HCV infection (SR or CI) were stained with CFSE and stimulated with 1 µg/ml of HCV-recombinant proteins (NS3 or NS4). After 6 days of culture, cells were washed and re-stimulated with PMA/ionomycin as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a> to reveal the cytokine profile by ICS. Cells were gated on viable CD3⁺CD8<b>^neg</b> T cells to define the percent of IL-17A-, IL21-, IFN-γ– or TNF-α–producing CFSE^low cells. Data is presented as percent specific cytokine production after subtraction of background corresponding to un-stimulated cells. Samples from SR (n = 8-5) and CI (n = 10-5) patients were studied. Samples were not available for some patients at every time point or exhibited low proliferation. Samples from five SR and five CI patients were examined at all the time points tested. P-values were calculated using a two-tailed Mann Whitney U test.</p

Proposed model for role of Galectin-9 and IL-21 during acute HCV infection.

<p>(<b>A</b>) HCV infection of hepatocytes and antigen uptake and presentation by liver-resident and circulating antigen presenting cells (APCS) prime HCV-specific CTLs and helper CD4 T cells including Th1 and Th17 cells. Function and survival of HCV-specific CTLs are sustained by increased production of IL-21 by Th17 cells during the late acute phase of spontaneously resolving HCV infection. (<b>B</b>) In patients with chronic evolution, multiple inhibitory mechanisms cooperate to induce failure of the immune response and facilitate viral persistence. (1) HCV-specific Th17 cells produce less IL-21 thus compromising CTL survival and function. (2) HCV-specific CTLs become exhausted and up-regulate exhaustion molecules like Tim-3, PD-1 and CTLA-4 and become more susceptible to apoptosis that is aggravated by lack of sufficient IL-21. (3) CD39<b>⁺</b> Tregs are increased in frequency and may directly inhibit Th17 cells and Tim-3-expressing CTLs via Gal-9-mediated apoptosis or through classical mechanisms including cellular contact and immuno-modulatory cytokines like IL-10 and TGF-β.</p

Restoration of HCV-specific IL-17A and IL-21 production by blocking multiple inhibitory pathways.

<p>(<b>A</b>) Representative FACS plot demonstrating enhanced production of Th17 and Th1 cytokines by HCV NS4-specific CD4 T cells upon antibody blockade. CD25^neg PBMCs from acute HCV patients were stained with CFSE and stimulated with 1 µg/ml of HCV NS4 recombinant protein in the presence of control Igs (IgG1, Ig2a) or blocking antibodies against PD-L1, CTLA-4 and Tim-3 (10 µg/ml each). After 6 days of culture, cells were washed and re-stimulated with PMA/ionomycin to detect production of Th17 (IL17A, IL-21) and Th1 (IFN-γ and TNF-α) cytokines by ICS as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a>. Cells are gated on CD3+CD8^neg lymphocytes (left panels). (<b>B, C</b>) Enhanced cytokine expression upon antibody blockade in the SR (open symbols, n = 3–5) and CI patients (closed symbols, n = 3–5). Data is represented as percent cytokine expressing CFSE^low cells within the CD3⁺CD8^neg lymphocyte gate. P-values were calculated using Wilcoxon signed rank test.</p

Expansion of Gal-9⁺ Tregs during acute HCV infection with chronic evolution.

<p>(<b>A</b>) Increased frequency of Tregs in CI patients during late acute HCV. Regulatory T cells were identified <i>ex vivo</i> as CD127^lowCD25^highFoxP3⁺ CD4 T cells in PBMCs from HCV patients at pre-infection (n = 12, grey symbols represent SR and black symbols represent CI), late acute (n = 8 for SR, n = 9 for CI) and long-term chronic HCV patients (n = 22). P-values were calculated using a two-tailed Mann Whitney U test. (<b>B</b>) Increased frequency of CD39⁺ CTLA-4⁺ Tregs in CI patients during late acute HCV. Tregs were defined as CD127^lowCD25^highFoxP3⁺ CD4 T cells in pre-infection samples (n = 12, grey symbols represent SR and black symbols represent CI), late acute phase in SR (n = 8) and CI (n = 8) patients, long-term HCV chronic patients (n = 10) and healthy donors (n = 8). P-values were calculated using a two-tailed Mann Whitney U test. (<b>C</b>) Imbalance between CD39⁺CTLA-4⁺ Tregs and Th17 cells during acute HCV infection. The ratio between the frequencies of CD39⁺CTLA-4⁺ Tregs and CD161^highCCR6⁺CD26⁺ CD4 T cells was calculated during late acute HCV infection in SR and CI patients (n = 8). P-values were calculated using a two-tailed Mann Whitney U test. (<b>D</b>) CD39⁺ Tregs express Galectin-9 in HCV infected patients. Gal-9 mRNA expression in CD39⁺ (grey triangles) and CD39^neg (black triangles) FACS-sorted Tregs from HCV patients was measured by real-time PCR and normalized to 18S mRNA expression. Relative expression was calculated relative to the expression of <i>LGALS9</i> in FACS-sorted CD127^high CD25^neg CD4 T cells (purity>98%). Gal-9 mRNA expression was higher in CD39⁺ than CD39^neg Tregs (p<0.01) (n = 10). P-values were calculated using a two-tailed Mann Whitney U test. (<b>E</b>) CD39⁺ CTLA-4⁺ Tregs express the highest levels of Galectin-9. CD39⁺ (grey triangle) and CD39^neg (black triangle) Tregs were sorted from chronic HCV patients (n = 8) and stained for expression of FoxP3 and CTLA-4. Gal-9 mRNA expression in sorted cells was evaluated by real-time PCR and normalized to 18S mRNA expression. Relative expression was calculated relative to expression of <i>LGALS9</i> in FACS-sorted CD127^high CD25^neg CD4 T cells (purity>98%). Correlation with FoxP3 and CTLA-4 expression was performed using Spearman correlation test.</p

Plasma levels of IL-21 correlate with frequency of HCV-specific CD8 T cells.

<p>(<b>A</b>) Increased plasma levels of IL-21 during acute resolving HCV. Plasma levels of the Th17 cytokines IL-17A and IL-21 were measured by ELISA at the indicated time points in SR (n = 8–12) and CI (n = 8–22) patients or healthy donors (HD) (n = 17). (<b>B</b>) Frequency of HCV-specific CD8 T cells correlates with plasma IL-21 levels. The frequency of HCV-specific CD8 T cells was measured using the following HLA class I tetramers (A1/NS3-1436, A2/NS3-1073, A2/NS3-1406, A2/NS5b-2594, B7/Core-41, B8/NS3-1395 and B27/NS5b-2841) and IL-21 levels were measured in paired plasma samples from 28 acute HCV patients. Correlation was tested using the Spearman correlation test.</p

Characteristics and demographics of HCV patients studied.

a<p>ND: Not determined.</p>b<p>N/A: Not applicable.</p

Selective expansion of high functional avidity memory CD8 T cell clonotypes during hepatitis C virus reinfection and clearance

<div><p>The dynamics of the memory CD8 T cell receptor (TCR) repertoire upon virus re-exposure and factors governing the selection of TCR clonotypes conferring protective immunity in real life settings are poorly understood. Here, we examined the dynamics and functionality of the virus-specific memory CD8 TCR repertoire before, during and after hepatitis C virus (HCV) reinfection in patients who spontaneously resolved two consecutive infections (SR/SR) and patients who resolved a primary but failed to clear a subsequent infection (SR/CI). The TCR repertoire was narrower prior to reinfection in the SR/SR group as compared to the SR/CI group and became more focused upon reinfection. CD8 T cell clonotypes expanding upon re-exposure and associated with protection from viral persistence were recruited from the memory T cell pool. Individual CD8 T cell lines generated from the SR/SR group exhibited higher functional avidity and polyfunctionality as compared to cell lines from the SR/CI group. Our results suggest that protection from viral persistence upon HCV reinfection is associated with focusing of the HCV-specific CD8 memory T cell repertoire from which established cell lines showed high functional avidity. These findings are applicable to vaccination strategies aiming at shaping the protective human T cell repertoire.</p></div

Exhaustion of HCV-specific CD8 T cells and increased Galectin-9 levels during acute HCV with chronic evolution.

<p>(<b>A</b>) Level of expression of Tim-3 defines differential exhaustion statuses of HCV-specific CD8 T cells during acute infection. Expression of PD-1, CD127, CTLA-4 and Tim-3 on HCV tetramer⁺ (dot plot) or total CD8 T cells (contour plot) in SR (at day 22, left) or in CI patients (at days 62 and 51, middle and right, respectively). Data are representative of 11 different patients analyzed corresponding to all 3 categories. (<b>B</b>) Exhaustion of HCV-specific CD8 T cells correlates negatively with plasma IL-21 levels. The co-expression of PD-1 and Tim-3 at the surface of HCV-specific CD8 T cells was measured using the panel of 7 HCV HLA class I tetramers and IL-21 levels were measured in paired plasma samples from 11 acute HCV patients as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#ppat-1003422-g002" target="_blank">Figure 2</a>. Correlation was tested using the Spearman correlation test. (<b>C</b>) Increased Galectin-9 levels in plasma during acute HCV infection with chronic evolution. Gal-9 was quantified in plasma by ELISA at the indicated time points during acute HCV infection in SR (n = 11) and CI (n = 13) patients, long-term chronic HCV patients (n = 8) or healthy donors (HD) (n = 10). P-values were calculated using a two-tailed Mann Whitney U test.</p