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Originally published online as doi:10.1189/jlb.0507268 on September 19, 2007

Published online before print September 19, 2007
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(Journal of Leukocyte Biology. 2007;82:1407-1419.)
© 2007 by Society for Leukocyte Biology

HCV core protein interaction with gC1q receptor inhibits Th1 differentiation of CD4+ T cells via suppression of dendritic cell IL-12 production

Stephen N. Waggoner*,1, Caroline H. T. Hall* and Young S. Hahn*,{dagger},2

* Departments of Microbiology, Beirne Carter Center for Immunology Research, and
{dagger} Pathology, University of Virginia, Charlottesville, Virginia, USA

2Correspondence: Department of Microbiology and Beirne B. Carter Center for Immunology Research, University of Virginia, HSC, Box 801386, Charlottesville, VA 22903, USA. E-mail: ysh5e{at}virginia.edu

ABSTRACT

Dendritic cells (DCs) isolated from patients with chronic hepatitis C virus (HCV) infection display an impaired capacity to generate type 1 CD4+ T cell immunity. Several reports have described an immunomodulatory function for the HCV core protein, and circulating core has been shown to associate with the putative gC1q receptor, gC1qR, expressed on host immune cells. However, the molecular mechanism(s) of HCV core-mediated DC dysfunction has not been defined. Herein, ligation of gC1qR on human monocyte-derived DCs (MDDCs) with HCV core or anti-gC1qR agonist antibody was shown to inhibit TLR-induced IL-12 production but not the production of other TLR-stimulated cytokines. Furthermore, engagement of gC1qR on MDDCs resulted in reduced IFN-{gamma} secretion by allogeneic CD4+ T lymphocytes during mixed lymphocyte culture. Differentiation of CD4+ T cells cocultured with HCV core- or anti-gC1qR antibody-treated MDDCs was also skewed toward production of Th2 cytokines, including IL-4. Importantly, that addition of IL-12 rescued IFN-{gamma} production and Th1 differentiation by CD4+ T cells. Therefore, engagement of gC1qR on DCs by HCV core limits the induction of Th1 responses and may contribute to viral persistence.

Key Words: human • DC • Th2 cells • viral • inflammation

INTRODUCTION

Hepatitis C virus (HCV) is a blood-borne pathogen infecting roughly 4% of the world’s population. This virus establishes persistent infection in as many as 80% of infected individuals, leading to chronic liver diseases, including cirrhosis and hepatocellular carcinoma [1 ]. Viral clearance is associated with strong and broadly targeted T cell responses, and significant production of type 1 cytokines includes IFN-{gamma} [2 ]. Antiviral CD4+ T cell responses are critical for the resolution of HCV infection by providing for CD8+ T cell priming and generation of antibody responses [3 4 5 6 ]. However, the levels of IFN-{gamma} in the serum are markedly diminished in chronically infected individuals, wherein cytokine expression by peripheral blood lymphocytes is skewed toward Th2 cytokines such as IL-4 [7 8 9 ]. Furthermore, HCV-specific T and B cells are not detected until 1–3 months postinfection [6 , 10 , 11 ]. This suggests that there is a significant delay in the generation of adaptive immunity during HCV infection compared with the rapid induction of antiviral T cell and antibody responses, which occur within 3 weeks of infection with another persistent virus such as HIV.

The defective expansion and differentiation of HCV-specific T cells observed in chronic infection suggest that the early stages of an antiviral immune response against HCV may be impaired. Dendritic cells (DCs) play an important role in linking innate and adaptive immunity [12 , 13 ]. A number of bacterial and viral gene products signal DC activation via TLRs expressed on the DC surface. Microbial TLR ligands stimulate up-regulation of costimulatory receptor expression and the secretion of numerous proinflammatory cytokines, including IL-12. These DC-associated, activation-induced inflammatory signals are crucial for CD4+ T cell differentiation, particularly the production of the Th1 cytokine IFN-{gamma}. Multiple independent studies have shown that DCs isolated from patients chronically infected with HCV display a reduced capacity to induce T lymphocyte activation [9 , 14 15 16 17 18 ]. These monocyte-derived DCs (MDDCs) also displayed a severely suppressed capacity to produce the Th1-inducing cytokine IL-12 in response to TLR or CD40 ligand (CD40L) stimulation [9 , 17 , 19 ]. In addition, myeloid DC (MDC) and plasmacytoid DC (PDC), purified from the blood of chronically infected individuals, also made less IL-12 (MDC) and IFN-{alpha} (PDC) in vitro, resulting in reduced IFN-{gamma} production by allogeneic CD4+ T cells [20 21 22 23 24 ].

The HCV core protein has been shown to play an immunomodulatory role in experimental models of viral infection as well as in chronically infected HCV patients [1 , 25 ]. Two independent groups used recombinant adenovirus constructs expressing the HCV core or core and envelope proteins (Ad-CE1) to demonstrate functional suppression of DCs by viral proteins [26 , 27 ]. Infection of DCs from several strains of mice (C57BL/6, BALB/c, and A/J) with Ad-CE1 yielded DCs with greatly decreased T cell stimulatory capacity in vitro and in vivo [27 , 28 ]. The lymphocytes stimulated by these DCs also displayed a "stunned" or exhausted phenotype, similar to that observed in T cells isolated form chronically infected patients [27 ]. However, the molecular mechanism for HCV core-mediated DC dysfunction has yet to be identified. Moreover, although HCV core protein is secreted by infected cells into patient sera [29 , 30 ], the role of circulating core protein in the observed induction of DC dysfunction is not known.

Based on our previous observation that HCV core interaction with gC1q receptor (gC1qR) results in a suppression of monocyte production of IL-12p70 in response to TLR stimulation [31 , 32 ], we sought to determine the effect of HCV core/gC1qR interaction on DC function and examine the impact of HCV core treatment of DCs on CD4+ T cell dysfunction. The production of IL-12p70 was suppressed exclusively and significantly in response to LPS or polyinosinic:polycytidylic acid [poly(I:C)], and maturation and additional cytokine expression was normal. Furthermore, the inhibition of IL-12 following gC1qR ligation on human DCs strongly correlated with reduced expression of IFN-{gamma} by allogeneic CD4+ T cells in favor of increased Th2 differentiation. These results establish a potential role for HCV core interaction with gC1qR in virus-induced DC dysfunction and reduced Th1 cytokine production during chronic HCV infection.

MATERIALS AND METHODS

Reagents
Recombinant β-galactosidase-fused core protein was obtained from Virogen (Watertown, MA, USA). Recombinant human (rh)GM-CSF and rhIL-4 were obtained from R&D Systems (Minneapolis, MN, USA). Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was obtained from Molecular Probes (Eugene, OR, USA). 3H-Thymidine was obtained from Perkin Elmer (Wellesley, MA, USA). rhIL-12p70 was obtained from PeproTech (Rocky Hill, NJ, USA). Salmonella typhimurium LPS, poly(I:C), and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Generation of gC1qR-specific antibodies and recombinant proteins
Mouse mAb specific for gC1qR were generated as described previously [32 ]. Briefly, mice were immunized with recombinant full-length GST-gC1qR and were screened for specificity using recombinant full-length His6-gC1qR (University of Virginia Hybridoma Center, Charlottesville, VA, USA). One anti-gC1qR IgG1 mAb clone (3A4) was purified and used for flow cytometric staining as described previously [31 32 33 ]. An anti-gC1qR IgM mAb clone (5F4) was characterized by its ability to ligate or cross-link gC1qR and was concentrated using Centricon Ultra-20 columns (Millipore, Bedford, MA, USA). As a control, mouse isotype IgM {kappa} (BD PharMingen, San Diego, CA, USA; clone C48-6) was resuspended in hybridoma culture medium concentrated in the same manner as the anti-gC1qR IgM clone (5F4).

Recombinant His6-gC1qR and His6-dihydrofolate reductase (DHFR) were produced and purified in our laboratory as described previously [34 ]. Where indicated, these proteins were added at 50 µg/mL to MDDC or MDDC-T cell cultures to compete with cellular gC1qR for binding to anti-gC1qR antibodies. A protein concentration of ≥20 µg/mL His6-gC1qR was determined previously to be minimally necessary for blocking the binding of anti-gC1qR and resultant suppression of IL-12 [32 ].

Preparation of PBMC, isolation of monocytes and CD4+ T cells, and generation of MDDCs
Human PBMC were isolated from healthy blood donors (Virginia Blood Services) by Lympholyte gradient centrifugation (Cedarlane Laboratories, Burlington, NC, USA). Monocytes were purified by positive selection with CD14 microbeads (StemSep) to ≥95% purity. Purified monocytes were differentiated into DCs by culture in RPMI 1640 supplemented with 10% (v/v) FBS (HyClone, Logan, UT, USA), penicillin/streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (10 mM), and 2-ME (55 µM) with 800 IU/ml rhGM-CSF and 800 IU/ml rhIL-4 at 37°C with 7% CO2 in a humidified atmosphere for a total of 6 days. Fresh media and cytokines were added at Days 2 and 4 of culture. For mature DCs (mDCs), 100 ng/mL LPS or 50 µg/mL poly(I:C) was added for the final 48 h of culture. Immature MDDCs were routinely >95% HLA-DR+CD11c+CD14–. Human CD4+ T cells were purified from CD14+ monocyte-depleted, normal human PBMC using CD4 microbeads (Miltenyi Biotech, Auburn, CA, USA) to ≥90% purity.

Analysis of gC1qR expression and determination of DC maturation phenotype by flow cytometry
Total PBMC or immature MDDCs (Day 5 of culture) were analyzed for gC1qR expression by flow cytometry. Cells (5x105) were stained initially with 5 µg anti-gC1qR IgG1 (3A4) in 10% normal goat serum for 30 min on ice. Following three washes, cells were stained with 0.2 µg PE-conjugated goat anti-mouse Ig (#550589, BD Biosciences, San Jose, CA, USA) in 10% goat serum for 30 min on ice. Cells were washed five times prior to staining with 10 µL each of one or more directly conjugated mouse antibodies or isotype controls (eBioscience, San Diego, CA, USA), including allophycocyanin-CD3 (UCHT1), allophycocyanin-CD14 (61D3), allophycocyanin-HLA-DR (LN3), and FITC-CD11c (3.9). Stained cells were then fixed, collected on a FACSCanto (BD Biosciences), and analyzed using Flowjo (TreeStar, Ashland, OR, USA).

Maturation of immature DCs (iDCs) was induced on Day 5 of culture by addition of 100 ng/mL LPS or 50 µg/mL poly(I:C). The agonistic anti-gC1qR mAb (5F4) or isotype control IgM (1 µg/mL) was added to iDCs, alone or in conjunction with maturation stimuli. After 24 h of additional culture in the presence or absence of maturation stimuli, Day 6 DCs were stained with FITC-CD80 (B7.1; 2D10.4), FITC-CD83 (HB15e), FITC-CD40 (5C3), PE-CD86 (B7.2; IT2.2), allophycocyanin-HLA-DR, PE-programmed death ligand-1 (PD-L1; B7-H1; MIH1), PE-PD-L2 (B7-DC; MIH18), FITC-CD11c, PE-CD14 (61D3), or isotype controls (eBioscience). Stained DCs were then fixed, collected on a FACSCalibur (BD Biosciences), and analyzed using Flowjo.

DC stimulation and MLR
Day 5 iDCs were stimulated with 100 ng/mL LPS, 50 µg/mL poly(I:C), or PBS (immature) for a total of 48 h. Concurrently, cells were treated with 1 µg/mL anti-gC1qR mAb agonist (5F4), murine isotype IgM, or 5 µg/mL HCV core protein. In some instances, 50 µg/mL His6-gC1qR or His6-DHFR was added at time of antibody treatment to compete with DC cell-surface gC1qR for binding to anti-gC1qR mAb. Following 8 h of stimulation, MDDCs were washed thoroughly prior to an additional 40 h of culture in fresh medium. At 48 h of DC stimulation, half of the culture supernatant was collected and stored at –80°C for analysis of cytokine production by ELISA.

The 48-h MDDCs (2x105) were then cocultured with 1 x 106 freshly isolated, allogeneic CD4+ T cells in a final volume of 1 mL in a 12-well plate, except during proliferation assays as described below. In some instances, 50 µg/mL His6-gC1qR or His6-DHFR was added at time of DC/T cell mixing to compete with T cell surface gC1qR for binding to anti-gC1qR antibody, possibly presented on bound DCs. Furthermore, in some experiments, 1 ng/mL rhIL-12p70 or PBS was added at time of DC/T cell mixing to restore levels of IL-12. At Day 3 of DC/T cell coculture, two-thirds of the culture supernatant was collected and frozen at –80°C for analysis of cytokine production by ELISA. Following 6 days of allogeneic DC/T cell coculture, T cells were then restimulated for intracellular cytokine analysis as described below.

CFDA-SE labeling and dilution
Purified CD4+ T cells were labeled with CFDA-SE as described by the manufacturer (Molecular Probes). Briefly, cells were incubated with a final concentration of 5 µM CFDA-SE in PBS containing 0.1% BSA for 10 min at 37°C. Following this incubation, cells were washed three times in PBS and incubated an additional 10 min at 37°C in plain PBS. CFDA-SE-labeled T cells were cocultured with immature or LPS-mature, allogeneic MDDCs (treated with anti-gC1qR or isotype antibody as described above) at a 1:10 ratio of DCs:T cells for 5 days. Conversely, CFSE-labeled T cells were stimulated with 1 µg/mL anti-CD3 (OKT3) and anti-CD28 (CD28.2; eBioscience). At Days 0, 3, 4, and 5 of DC/T cell coculture, cells were stained with allophycocyanin-CD4 (OKT-4, eBioscience), fixed, and collected on a FACSCalibur (BD Biosciences).

Assay of T cell proliferation by 3H-thymidine incorporation
MDDCs were treated with LPS or poly(I:C) and anti-gC1qR agonist (5F4) or isotype IgM as described above. After 48 h, various doses of DCs (200–40000 cells) were {gamma}-irradiated and washed extensively to remove TLR and gC1qR ligands. These DCs were then mixed with purified, allogeneic CD4+ T cells (2x105 cells) in 96-well, flat-bottom culture plates for 5 days. During the final 18 h of DC/T cell coculture, 1 µCi 3H-thymidine was added to each well. Cells were harvested onto filter paper and analyzed by a Microbeta TriLux harvester (Perkin Elmer).

Determination of cytokine production by ELISA
MDDC culture (48 h) or DC/T cell MLR coculture (Day 3) supernatants were stored at –80°C. Cytokine production was assessed by specific ELISA for IL-12p70 and TGF-β (BD PharMingen) as well as IL-2, IL-10, IFN-{gamma}, IL-17, and TNF-{alpha} (eBioscience), according to the manufacturer’s instructions. IL-5 and IL-13 ELISAs were performed according to the manufacturer’s specifications (Pierce Endogen, Rockford, IL, USA).

Intracellular cytokine staining
Following 6 days of DC/T cell coculture, T cells were restimulated with 50 ng/mL PMA and 1 µg/mL ionomycin for a total of 5 h. Brefeldin A (1 µg/mL, BD PharMingen) was added to the culture during the final 4 h of restimulation. At the end of this incubation, T cells were stained for CD3 and intracellular cytokines according to the manufacturer’s specification (BD PharMingen). Briefly, following surface staining with allophycocyanin-CD3, as described above, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences, #555028). Permeabilized cells were then stained with FITC-IFN-{gamma}/PE-IL-4 Fastimmune reagent (#340456) or Fastimmune isotype (#340458), as indicated by the manufacturer (BD Biosciences). The stained cells were then collected on a FACSCanto and analyzed using FlowJo.

Statistical analysis
Results are expressed as mean ± SEM, except where noted. Statistical analysis was performed using Wilcoxon signed-rank test for paired data or Mann-Whitney U-test for unpaired data. A P value of <0.05 was considered to be significant. Correlation was determined using Pearson’s coefficient of correlation (r).

RESULTS

The expression of gC1qR is detectable at the cell surface of human PBMC and MDDCs
HCV core protein is secreted by infected cells and is detectable in the circulation of HCV-infected individuals [29 , 30 ]. The interaction of HCV core with gC1qR has been reported to modulate T lymphocyte responses [25 , 35 , 36 ]. However, expression of gC1qR on various subsets of human PBMC has not been analyzed in detail. To determine the role of gC1qR in the regulation of DC function as well as subsequent shaping of T lymphocyte responses, we first analyzed the cell surface expression of gC1qR in immune cells, including monocytes, lymphocytes, and MDDCs, with a focus on possible variation of gC1qR expression in different blood donors. To this end, we analyzed receptor expression simultaneously on PBMC from 14 healthy blood donors by flow cytometry (Fig. 1 A and 1B ). Staining of gC1qR on CD14+ monocytes (99.1±0.8% gC1qR+) and CD3+ lymphocytes (99.8±0.2% gC1qR+) revealed ubiquitous expression of this receptor on these cell populations. Monocytes (mean relative gC1qR MFI=6.5±1.1) and T lymphocytes (mean relative gC1qR MFI=3.0±1.0) displayed some variation in gC1qR expression levels, although all of the gated (CD14+ and CD3+) events within this pool of donors expressed substantial levels of gC1qR (Fig. 1B) . In addition, expression of gC1qR was evident on iDCs from several human blood donors (mean relative gC1qR MFI=7.4±2.8; Fig. 1C ). These data suggest that gC1qR expression is detectable on peripheral immune cells from multiple healthy blood donors but that expression levels may vary slightly and would possibly affect the ability of HCV core or other gC1qR ligands to activate gC1qR-mediated intracellular signaling.


Figure 1
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  		Figure 1. The cell surface expression of gC1qR on human PBMC. (A) Representative flow cytometry of gC1qR expression on human PBMCs, CD14+ monocytes, and CD3+ lymphocytes. Electronic gating of CD14+ and CD3+ events is shown in upper middle and right panels. Staining of gC1qR (line histograms) with 3A4 monoclonal IgG1 and PE-conjugated secondary is shown in comparison with isotype control staining (solid histograms). SSC, Side-scatter; FSC, forward-scatter. (B) The mean fluorescence intensity (MFI) of gC1qR staining on total PBMC or gated populations for multiple healthy blood donors (n=14) was normalized to isotype controls (MFI gC1qR divided by MFI isotype control). Monocytes (CD14+) and T lymphocytes (CD3+) from these blood donors were uniformly gC1qR+, compared with isotype controls (≥97% gC1qR+). Small, horizontal bars indicate mean value of relative gC1qR expression; each dot represents one individual donor. Horizontal, dashed line represents level of background staining. (C) Representative flow cytometry of the expression of gC1qR on human MDDCs. Live event gating, HLA-DR and CD11c staining, and gC1qR staining of HLA-DR+CD11c+-gated cells are shown.

Ligation of gC1qR by HCV core or anti-gC1qR antibody on MDDCs results in suppression of IL-12 production
Although DCs from chronically infected HCV patients display a reduced capacity to secrete IL-12 [9 , 20 ], the role of HCV core/gC1qR interaction in this suppression is not known. Therefore, we sought to ascertain the effect of gC1qR ligation on IL-12 production by MDDCs from healthy blood donors. Immature MDDCs from normal, healthy blood donors were collected and stimulated with TLR-4 or TLR-3 ligands [LPS and poly(I:C), respectively] for 48 h in the presence of an anti-gC1qR agonistic mAb (5F4), HCV core protein, or isotype mouse IgM control antibody (Fig. 2 ). It is important that core- or anti-gC1qR antibody-mediated engagement of gC1qR on MDDCs strongly suppressed the production of IL-12p70 in response to TLR stimulation (Fig. 2A) . Conversely, LPS- and poly(I:C)-induced production of additional pro- or anti-inflammatory cytokines, including IL-10, TGF-β, and TNF-{alpha}, was not affected by ligation of gC1qR (Fig. 2 A and 2B ). The suppression of IL-12 was observed consistently in LPS (P=0.001)- and poly(I:C)-stimulated (P=0.021) MDDCs from every blood donor examined, although the degree of suppression was variable (Fig. 2B) . Trypan blue exclusion analysis following gC1qR ligation revealed no change in MDDC viability (data not shown). These results suggest that gC1qR ligation specifically suppresses TLR-induced IL-12p70 production by MDDCs.


Figure 2
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  		Figure 2. Ligation of gC1qR on human MDDCs mediates specific suppression of TLR-induced IL-12p70 production. Human MDDCs (2x105) were stimulated for 8 h with 100 ng/mL LPS or 50 µg/mL poly(I:C) to induce DC maturation or cultured in the absence of stimulatory agents to maintain immature status (Imm). Concurrently, immature and mature MDDCs were treated with 1 µg/mL anti-gC1qR IgM agonist mAb (open bars), isotype mouse IgM control antibody (black bars), or 5 µg/mL recombinant HCV core protein (gray bars). After 8 h of stimulation, MDDCs were washed thoroughly to remove unbound, stimulatory reagents and returned to culture in fresh medium for an additional 40 h. Supernatants from 48 h cultures were analyzed for production of cytokines by specific ELISA. (A) Cytokine levels for MDDCs from a representative blood donor are presented as mean of triplicate ± SD. (B) The mean values of LPS- or poly(I:C)-induced cytokine production for MDDCs from different healthy blood donors (n=10–19) are shown as a pair-wise comparison of the effects of isotype IgM (Iso) versus anti-gC1qR agonist ({alpha}-G), wherein each line represents an individual donor. Statistically significant differences between treatments were determined by two-tailed Mann-Whitney U-test.

gC1qR ligation does not interfere with TLR-induced MDDC maturation
A hallmark of DC biology is the maturation-induced up-regulation of costimulatory molecules critical for activation of naïve T lymphocytes following stimulation with TLR ligands [12 , 13 ]. High-level expression of CD80 (B7.1) and CD86 (B7.2) on mDCs ensures potent stimulation of T lymphocytes via CD28 [37 ], and expression of CD40 by DCs enables amplification of immune activation through interaction with CD40L (CD154) on T lymphocytes [38 ].

We sought to ascertain whether ligation of gC1qR may affect MDDC maturation following TLR-4 ligand-induced activation of iDCs. Stimulation of iDCs with anti-gC1qR agonistic antibody or mouse isotype IgM for 24 h had no effect on steady-state expression of the costimulatory markers CD80 (B7.1), CD86 (B7.2), CD83, CD40, and MHC class II (HLA-DR; Fig. 3 A and 3C ). Furthermore, up-regulation of cell surface expression of these costimulatory markers following LPS-induced MDDC maturation was not abrogated following gC1qR ligation on a representative blood donor (Fig. 3A) or among all blood donors analyzed (Fig. 3C) . Similar results were obtained using HCV core protein in place of anti-gC1qR antibody (data not shown).


Figure 3
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  		Figure 3. DC maturation is normal following gC1qR ligation. (A and B) Human immature MDDCs were stimulated with 1 µg/mL anti-gC1qR agonist (lower row) or isotype IgM control (upper row) antibody in the presence (LPS mature; heavy line histograms) or absence (immature; line histograms) of 100 ng/mL LPS. After 24 h, cells were stained using a panel of antibodies and analyzed by flow cytometry for surface expression of costimulatory (A) and coinhibitory (B) receptors. Isotype control staining is shown (solid histograms). (C) The expression levels of these various receptors on MDDCs from multiple healthy blood donors (n=5) are displayed as mean ± SEM of the MFI of receptor staining normalized to isotype control MFI (nMFI=MFI staining/MFI isotype) and to the basal expression (horizontal, dashed line) of each receptor on immature MDDCs (nMFI LPS mature/nMFI immature).

PD-L1 (B7-H1) and PD-L2 (B7-DC) are inhibitory receptors, which have been shown to play a role in tolerance and suppression of T cell activation [39 , 40 ]. It is intriguing that these receptor pairs have been strongly implicated in T cell exhaustion during chronic viral infection, including HCV [41 42 43 ]. Examination of expression levels of PD-L1 and PD-L2 on immature as well as mature MDDCs revealed that gC1qR ligation had no effect on expression of these inhibitory receptors (Fig. 3 B and 3C) . These results suggest that gC1qR ligation has no effect on MDDC expression of costimulatory or inhibitory receptors and that TLR-induced MDDC maturation occurs normally following engagement of gC1qR.

gC1qR ligation on human MDDCs results in skewing of T cell cytokines from a Th1 phenotype to increased expression of type 2 cytokines
As a result of the diminished IL-12p70 production characteristic of gC1qR-stimulated MDDCs (Fig. 2) , we hypothesized that CD4+ T cell-mediated IFN-{gamma} production may be inhibited following stimulation with these gC1qR-treated MDDCs in an allogeneic MLR. Ligation of gC1qR on iDCs did not influence cytokine expression in allogeneic DC/T cell coculture supernatants (Fig. 4 and Table 1 ), in part, as iDCs do not demonstrate detectable IL-12p70 expression, regardless of gC1qR engagement. As expected, LPS-induced maturation of MDDCs resulted in increased T cell IFN-{gamma} production as compared with iDCs. This increase in IFN-{gamma} was markedly inhibited when gC1qR on MDDCs was engaged by anti-gC1qR agonist antibody or HCV core protein prior to coculture with T cells (Fig. 4A) . The suppression of IFN-{gamma} production in allogeneic DC/T cell coculture supernatants was observed consistently in all blood donor pairings tested (Fig. 4B) . In contrast, the production of other cytokines, including IL-2 and IL-10, was markedly unaffected by gC1qR engagement, suggesting that suppression following gC1qR ligation is specific (Fig. 4B and Table 1 ).


Figure 4
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  		Figure 4. Ligation of gC1qR during MDDC maturation results in poor capacity to induce IFN-{gamma} production by allogeneic T cells. Human MDDCs (2x105) from multiple healthy blood donors were stimulated for 8 h with LPS, poly(I:C), or PBS (Imm) in the presence of 1 µg/mL anti-gC1qR agonist (open bars), isotype IgM (black bars), or 5 µg/mL HCV core protein (gray bars), as described in Figure 2 . The MDDCs were washed thoroughly prior to extended culture in fresh medium for a total of 48 h. MDDCs were then cocultured with 1 x 106 freshly isolated, allogeneic CD4+ T cells. Coculture supernatants were collected, and cytokine production was analyzed by ELISA. (A) Cytokine production in a representative, allogeneic pairing is displayed as mean of triplicate ± SD. (B) The mean production of IFN-{gamma} (n=17) and IL-10 (n=15) in multiple independent, allogeneic mixtures. Each line represents the comparison of mean cytokine expression with isotype IgM (Iso) versus anti-gC1qR agonist ({alpha}-G) antibody for an individual donor pairing. Statistical significance was analyzed using the two-tailed Mann-Whitney U-test.


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  		Table 1. Changes in Cytokine Expression During Allogeneic DC/T Cell MLR (n = 8–17)

The differentiation of CD4+ T cells into Th1 IFN-{gamma}-producing or Th2 IL-4-producing cells is controlled by DC-mediated cytokine production [44 , 45 ]. Given the importance of IL-12 to Th1 differentiation and the strong suppression of IL-12p70 by gC1qR ligands (Fig. 2) , we hypothesized that ligation of gC1qR on DCs may inhibit the Th1 differentiation of allogeneic CD4+ T cells and be involved in promoting Th2 differentiation. Upon restimulation with PMA and ionomycin, CD4+ T cells cocultured with HCV core- or anti-gC1qR antibody-treated MDDCs were less capable of producing IFN-{gamma} compared with CD4+ T cells cocultured with isotype antibody-treated MDDCs (Fig. 5 A and 5B ). This effect was observed as a decrease in the proportion of IFN-{gamma}+ T cells (28–35% decrease, P<0.01, Fig. 5A ) and in the MFI of IFN-{gamma} staining (29–34% decrease, P<0.01, Fig. 5B ) in T cells cultured with anti-gC1qR antibody-treated compared with isotype antibody-treated MDDCs. It is striking that we observed a parallel increase (1.6- to 2.2-fold, P<0.01) in the proportion of IL-4+ T cells (Fig. 5A) . The skewing from Th1 to Th2 during the differentiation of these T cells suggests that gC1qR ligation on MDDCs may favor Th2 cytokine production during primary stimulation.


Figure 5
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  		Figure 5. Engagement of gC1qR on MDDCs limits Th1 differentiation by allogeneic CD4+ T cells. (A and B) Human MDDCs from multiple healthy blood donors (n=10) were stimulated with LPS (LPS DC), poly(I:C) [p(I:C) DC], or without maturation stimuli (Imm) in the presence of 1 µg/mL anti-gC1qR agonist antibody, IgM isotype, or 5 µg/mL HCV core protein for a total of 48 h (with washing) as described in Figure 4 . These MDDCs were then combined with freshly isolated, allogeneic CD4+ T cells and cocultured as described in Materials and Methods. At the end of DC/T cell coculture, the T cells were restimulated with PMA/ionomycin and stained for flow cytometric analysis of cytokine production as described in Materials and Methods. The analyzed populations were routinely ≥95% CD3+ (top left histogram), and intracellular cytokines were undetectable in the absence of stimulatory reagents (top right dot plot). (A) Representative cytokine staining of live, gated CD3+ T cells from an individual blood donor pairing is shown with quadrants drawn based on isotype control staining. The mean percent of IFN-{gamma}+ (lower right quadrants) and IL-4+ (upper left quadrants) T cells ± SEM is shown (n=10). Significant differences between paired frequencies for anti-gC1qR- or HCV core- and isotype-treated MDDCs were determined by two-tailed Wilcoxon signed-rank test (*, P<0.05; **, P<0.01). (B) The {Delta}MFI (MFI stain–MFI isotype control) of intracellular IFN-{gamma} staining is shown (mean±SEM, n=10) for isotype IgM (black bars)-, anti-gC1qR (open bars)-, and HCV core-treated (gray bars) DCs. (C) Cytokine production (mean±SEM, n=8) in MLR supernatants was analyzed by ELISA, as described for Figure 4 . Significant differences in paired data were analyzed by Wilcoxon signed-rank test.

In addition to IL-4, IL-5 and IL-13 are prototypical cytokines produced by Th2 cells [46 ]. Analysis of IFN-{gamma}, IL-5, and IL-13 expression in Day 3 MLR supernatants from multiple DC/T cell allogeneic coculture donor pairings (n=8) revealed that a substantial decrease in IFN-{gamma} production accompanied a significantly increased release of IL-5 and IL-13 in T cells cocultured with HCV core- or anti-gC1qR antibody-treated MDDCs (Fig. 5C) . These results together suggest that the lack of a potent Th1-inducing cytokine such as IL-12 results in a skewing toward production of Th2 cytokines, similar to that observed in the periphery of patients chronically infected with HCV [7 , 8 ].

gC1qR-induced cytokine dysfunction by T cells is not a result of diminished T cell proliferation and is independent of gC1qR on the T cell surface
DCs purified and derived from patients with chronic HCV infection display a reduced capacity to induce allogeneic T cell proliferation [9 , 14 15 16 17 18 ]. Incomplete or dysfunctional activation of T cell proliferation by anti-gC1qR antibody-treated MDDCs could result in a reduced expansion of IFN-{gamma}-producing CD4+ T cells, which may account for the decreased level of IFN-{gamma} expression seen in the MLR culture supernatants. To test this possibility, we examined the ability of anti-gC1qR antibody-treated MDDCs to induce T cell proliferation.

The effect of gC1qR ligation on immature or mature MDDC-induced T cell proliferation, as measured by 3H-thymidine incorporation, was highly variable among a multitude of blood donor pairings (n=14–20, Fig. 6A ), possibly reflecting the heterogeneous population of CD4+ T cell responders used in this analysis. Overall, there was no statistically significant effect of gC1qR ligation on MDDC stimulatory capacity in terms of T cell proliferation (P>0.3, Fig. 6A ). Analysis of CFSE dilution in CD4+ T cells following coculture with allogeneic (Fig. 6B) and autologous (data not shown) MDDCs confirms the lack of suppression of T cell proliferation following gC1qR ligation on MDDCs. These results suggest that gC1qR engagement on MDDCs does not affect their ability to induce allogeneic T cell proliferation and imply that core/gC1qR interaction is not a major cause of the reduced capacity of HCV patient-derived DCs to induce T cell proliferation [9 , 14 15 16 17 18 ].


Figure 6
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  		Figure 6. Normal, allogeneic T cell proliferation following gC1qR ligation on MDDCs. Human MDDCs were matured with LPS, poly(I:C), or PBS (Immature) in the presence of isotype IgM (Iso) or anti-gC1qR agonist ({alpha}-G) antibodies as described in Figure 4 . (A) MDDCs (4x103) were lethally irradiated and washed thoroughly prior to coculture with allogeneic CD4+ T cells (2x105), as described in Materials and Methods. For the final 18-h of DC/T cell coculture, 1 µCi 3H-thymidine was added to each well. Radioactive incorporation was determined using Trilux instrumentation. Each line represents the mean incorporation (cpm) for an individual donor pairing. Statistical significance was analyzed by Mann-Whitney U-test. (B) Dilution of CFSE in CD4+ T cells following coculture of 2 x 104 MDDCs, treated as described above, with 2 x 105 freshly isolated, CFSE-labeled, allogeneic CD4+ T cells. Proliferation following direct stimulation of CD4+ T cells with 1 µg/mL (each) anti-CD3 and anti-CD28 antibodies is shown in the top row. Comparison of isotype- or anti-gC1qR-treated immature MDDC (Rows 2 and 3) or LPS mature MDDC (Rows 4 and 5) induction of proliferation in live, gated CD4+ T cells is shown, where numbers represent the proportion of CFSElo cells.

As MDDCs treated with anti-gC1qR agonist antibody were cocultured with CD4+ T cells, we cannot rule out the possibility that the anti-gC1qR antibody might interact directly with gC1qR on T cells (Fig. 1 A and 1B) and inhibit their function. To this end, we attempted to remove unbound anti-gC1qR antibody prior to MDDC mixing with T cells and block any presentation of MDDC-bound antibody to gC1qR on the surface of T cells. Soluble recombinant His6-gC1qR (sgC1qR) was used as a competitor for anti-gC1qR antibody binding to cell surface gC1qR at the time of DC stimulation (Fig. 7A ) or coculture of DCs and T cells (Fig. 7B) . Addition of sgC1qR, but not a control protein DHFR (sDHFR), during MDDC stimulation with anti-gC1qR agonist antibody, abrogated suppression of IL-12 completely (Fig. 7A , left bar graphs). Furthermore, this sgC1qR-mediated abrogation of IL-12 inhibition resulted in normalization of T cell-mediated IFN-{gamma} production and Th1 differentiation (Fig. 7A) . These results confirm that anti-gC1qR antibody-mediated suppression of MDDC IL-12 production and subsequent expression of IFN-{gamma} by T cells are mediated through anti-gC1qR antibody interaction with gC1qR on the MDDC surface. However, the addition of sgC1qR at the onset of MDDC-T cell coculture did not reverse anti-gC1qR-treated, MDDC-mediated suppression of IFN-{gamma} production or Th1 differentiation (Fig. 7B) . Similar results to those described in Figure 7 were obtained using poly(I:C) as a maturation stimulus in place of LPS and when HCV core protein was substituted in place of anti-gC1qR agonist antibody (data not shown). Together, these results suggest that ligation of gC1qR on the surface of MDDCs exerts potent suppression, which is completely independent of gC1qR expression on the T cell surface.


Figure 7
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  		Figure 7. Suppression of IFN-{gamma} production by CD4+ T cells is dependent on MDDC gC1qR ligation but independent of T cell gC1qR. MDDCs (2x105) were stimulated with 100 ng/mL LPS in the presence of 1 µg/mL isotype IgM or anti-gC1qR agonist antibody, washed, and cocultured with freshly isolated, allogeneic CD4+ T cells (1:5 ratio of MDDC:T cells), as described in Figure 4 . (A) At time of antibody addition to MDDCs, cultures also received 50 µg/mL sgC1qR (DC+sgC1qR, lower row) or irrelevant control protein, His6-DHFR (DC+sDHFR, upper row) as a competitor for anti-gC1qR agonist antibody binding to gC1qR on MDDCs. (B) Conversely, 50 µg/mL His6-gC1qR (DC/T+sgC1qR) or His6-DHFR (DC/T+sDHFR) was added to DC/T cell cocultures after DC stimulation and washed to compete with MDDC-bound, anti-gC1qR antibody interaction with gC1qR on T cells. IL-12p70 production was analyzed in MDDC monoculture supernatants, and IFN-{gamma} was analyzed in MLR supernatants by ELISA, as described in Materials and Methods. Cytokine ELISA results are presented as mean ± SD. Intracellular cytokine staining of cocultured T cells is shown. Numbers in the upper left and lower right quadrants represent frequencies of IL-4+ and IFN-{gamma}+, respectively, in live, gated CD3+ T cell populations. Numbers in parentheses indicate {Delta}MFI (MFI stain–MFI isotype) of IFN-{gamma} staining. (A and B) Results are representative of those obtained using three different donor pairings.

Dysfunctional IFN-{gamma} production by CD4+ T cells correlates with reduced IL-12 and is rescued following reconstitution with rIL-12
The cytokine IL-12 plays a crucial role in the induction of IFN-{gamma} production by CD4+ and CD8+ T cells as well as NK and NKT cells [44 ]. However, several related (e.g., IL-23) and unrelated (e.g., IL-18 and type I IFN) cytokines are postulated to play important roles in Th1 differentiation, in the presence or absence of IL-12 [47 , 48 ]. To ascertain the relationship between IL-12 and IFN-{gamma} in our system, we first attempted to show a correlation between the degree of IL-12 suppression on MDDCs and that of IFN-{gamma} by CD4+ T cells. The percent decrease in IL-12p70 and IFN-{gamma} expression, in MDDC culture-monoculture supernatants and DC/T cell coculture supernatants, respectively, was calculated for multiple, healthy blood donor pairs (Fig. 8A , n=18). These results were paired and ranked to determine Pearson’s coefficient of correlation, which reveals a strong, positive coefficient of correlation (r=0.74) between the suppression of IL-12p70 and the inhibition of IFN-{gamma} in these donor pairings.


Figure 8
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  		Figure 8. Addition of rIL-12 rescues deficient Th1 differentiation of allogeneic T cells. (A) Correlation between suppression of MDDC-derived IL-12p70 and inhibition of IFN-{gamma} produced by CD4+ T cells. MDDCs were stimulated and cocultured with allogeneic CD4+ T cells as described in Figure 4 . The production of IL-12p70 and IFN-{gamma} was determined by ELISA analysis of MDDC monoculture supernatants or DC/T cell coculture supernatants, respectively. The percent suppression of IL-12p70 and IFN-{gamma} was then calculated {% suppression=100–[(mean anti-gC1qR-DC/mean isotype-DC)x100]} for each allogeneic, healthy blood donor pairing (n=18). The coefficient of correlation between suppression of IL-12 and suppression of IFN-{gamma} was assessed using Pearson’s method. (B and C) MDDCs were stimulated and cocultured with allogeneic CD4+ T cells as described in Figure 4 , except that 1 ng/mL rhIL-12p70 (IL-12) or PBS was added to cocultures at time of DC-T cell mixing. (B) Intracellular staining of IL-4 and IFN-{gamma} within live, gated CD3+ T cells is shown for a representative donor pairing. Numbers in the upper left and lower right quadrants indicate frequency of IL-4+ and IFN-{gamma}+ cells, respectively. Numbers in the upper right quadrants represent {Delta}MFI (MFI stain–MFI isotype control) of IFN-{gamma} staining (mean±SEM, n=3). (C) IFN-{gamma} production in DC/T cell coculture supernatants is shown for mixtures, which received PBS or IL-12, as described above (mean±SEM, n=6). Significant differences between isotype (black bars) and anti-gC1qR (open bars) antibody-stimulated MDDC sample groups were analyzed by two-tailed Wilcoxon signed-rank test.

We next sought to determine if reconstitution of IL-12 in the DC/T cell MLR coculture would restore the decreased IFN-{gamma} production as well as dysfunctional Th1 differentiation observed following ligation of gC1qR on MDDCs. Addition of 1 ng/mL IL-12p70 to these cocultures restored Th1 differentiation of T cells stimulated with anti-gC1qR-treated MDDCs (38% reduction in IFN-{gamma} MFI) to levels similar to those seen using isotype IgM-treated MDDCs (2% reduction in IFN-{gamma} MFI; Fig. 8B ). Moreover, addition of IL-12 to cocultures increased the overall level of IFN-{gamma} production and abrogated the suppression of IFN-{gamma} seen following gC1qR ligation on LPS- or poly(I:C)-matured MDDCs (Fig. 8C) . Suppression of IFN-{gamma} production (data not shown) and Th1 differentiation (Fig. 8B) by HCV core protein were also reversed by addition of IL-12 to the DC/T cell coculture. These results strongly suggest that gC1qR ligation on MDDCs results in a potent suppression of TLR-induced IL-12p70 production, which mediates a powerful inhibition of subsequent IFN-{gamma} production and Th1 differentiation by T cells.

DISCUSSION

Herein, we describe an immunomodulatory role for HCV core/gC1qR interaction on MDDCs involving the inhibition of IL-12p70 production in response to prototypical bacterial (LPS, TLR-4) and viral [poly(I:C), TLR-3] stimuli. Importantly, HCV core/gC1qR-mediated suppression of IL-12 production by MDDCs resulted in an inefficient induction of IFN-{gamma} production by CD4+ T lymphocytes. This decrease in type 1 cytokine expression correlated with an increased secretion of the Th2 mediators, IL-5 and IL-13. Restimulation of CD4+ T cells, previously activated by HCV core or gC1qR agonist, mAb-treated MDDCs, revealed a clear skewing or selective expansion of Th2 rather than Th1 effectors. It is remarkable that reconstitution of IL-12 in the DC/T cell coculture abrogated gC1qR-mediated inhibition of IFN-{gamma} production during the allogeneic MLR and restored Th1 differentiation to levels similar to those observed in CD4+ T cells stimulated with isotype antibody-treated MDDCs. These results suggest that HCV core/gC1qR interaction is involved in the negative regulation of MDDC-induced Th1 differentiation through inhibition of IL-12 production.

Of interest, the selective inhibition of TLR-induced IL-12 production, following treatment of MDDCs with HCV core protein or anti-gC1qR antibody, resulted in suppression of IFN-{gamma} production by CD4+ T cells but did not affect T cell proliferative responses. This effect could be explained by findings concerning the regulation of costimulatory molecule expression and induction of IL-2 production by gC1qR ligand-treated MDDCs. First, cross-linking of gC1qR on MDDCs did not influence maturation-induced up-regulation of costimulatory receptors (e.g., CD80) or the expression of inhibitory PD-L1 and PD-L2. This suggests that ligation of gC1qR does not modulate the antigenic or costimulatory signals provided during T lymphocyte activation. Second, IL-2 production during coculture of allogeneic CD4+ T cells with anti-gC1qR antibody-treated MDDCs was not inhibited. As IL-2 production by activated CD4+ T cells is an important characteristic of T cell activation and a crucial factor in T cell proliferation, the unabated induction of IL-2 following gC1qR cross-linking may result in the relatively normal induction of CD4+ T cell proliferative expansion. Therefore, HCV core-mediated engagement of gC1qR on the surface of DCs may not be responsible for the defective T cell proliferation observed in HCV core-treated PBMC cultures [35 , 49 ].

Based on the previous finding that treatment of PBMC with HCV core led to the inhibition of IL-2 and IFN-{gamma} production as well as T cell proliferation [34 , 35 ], it is tempting to speculate that the suppression of adaptive immune responses by HCV core may occur at multiple steps during the development of effective cell-mediated immunity. HCV core-mediated immune regulation in vivo may involve a dual targeting of gC1qR on the surface of APCs as well as T cells. Although direct binding of HCV core on T cells results in reduced proliferation, IL-2 secretion, and IFN-{gamma} production, further inhibition of CD4+ T cell function and differentiation occurs following contact with gC1qR-ligated DCs. We are currently investigating these possibilities.

Despite profound suppression of IL-12 expression following gC1qR ligation, it remains to be determined what effect gC1qR signaling has on the production of other Th1-inducing cytokines. Additional members of the IL-12 (e.g., IL-23), IL-1 (e.g., IL-18), and type I IFN (e.g., IFN-{alpha}) families are also capable of inducing IFN-{gamma} production by CD4+ T cells. Based on our previous observation that gC1qR-mediated inhibition of IL-12p70 targets transcription of the IL-12p40 subunit [31 , 32 ], which is shared by the IL-12p70 and IL-23 heterodimers [50 ], we can hypothesize that production of both cytokines is suppressed following gC1qR ligation. Indeed, IL-23 production by MDDCs was inhibited by anti-gC1qR antibody treatment (C. H. T. Hall, unpublished observations). Given the proposed role for IL-23 in the induction and maintenance of IL-17-producing CD4+ T cells [51 , 52 ], diminished IL-23 production by gC1qR-cross-linked MDDCs might lead to inhibition of IL-17 production. It is surprising that IL-17 levels were increased slightly in allogeneic DC/T cell coculture supernatants following ligation of gC1qR on MDDCs. Recent evidence suggests that IL-6 and TGF-β also play critical roles in the generation of IL-17-producing Th17 cells [53 ]. Although expression of these cytokines by mature MDDCs is relatively unchanged following gC1qR ligation (Fig. 2 ; C. H. T. Hall, unpublished observations), it is possible that unabated secretion of IL-6 and TGF-β by HCV core- or anti-gC1qR antibody-treated MDDCs, in the absence of potent Th1-inducing cytokines, may be sufficient to elicit increased Th17 differentiation. Such a mechanism may allow for expansion of pathogenic Th17 cells, which potentially cause liver damage rather than mediate viral clearance.

The pivotal role of DCs in the generation and shaping of adaptive immune responses suggests that their dysfunction may lead to profound immune suppression. However, such global immune suppression is not thought to be a characteristic of chronic HCV infection [2 ]. Despite the critical function of T cell-derived IFN-{gamma} in immune defense against intracellular pathogens, including HCV, the importance of IL-12 to resolution of infectious diseases in humans is less clear [54 ]. Individuals chronically infected with HCV were more likely to be homozygous for an IL12B (IL-12p40 subunit) gene polymorphism, profoundly associated with reduced IL-12 production than those who resolved infection spontaneously [55 56 57 ]. This suggests that HCV core-induced loss of IL-12 production, despite normal induction of additional DC-derived cytokines, may have direct implications for immune defense against HCV infection without mediating global immune suppression.

Based on the observation that ligation of gC1qR on DCs alone is sufficient to impair T lymphocyte activation and cytokine production, it is tempting to speculate that HCV core-induced immune suppression via gC1qR is likely to occur at an early phase of infection prior to the appearance of HCV-specific antibody responses. Indeed, the significant delay (4–12 weeks) in the appearance of antiviral T and B cell responses following HCV infection, compared with infection with HIV (within 3 weeks), suggests that the induction of the adaptive response is impaired in some manner [6 , 10 , 11 , 58 , 59 ]. As HCV core protein is secreted from infected cells and present in the serum of infected patients [29 , 30 ], this viral protein may interact with gC1qR on DCs and restrain effective priming of HCV-specific T cells. Furthermore, cross-talk between gC1qR and receptors, which induce DC activation, particularly TLRs, during antigen uptake by DCs, may inhibit the signaling responsible for processing and presentation of viral antigens [60 , 61 ]. However, the levels of soluble HCV core protein available in the periphery, within secondary lymphoid organs (e.g., draining lymph node) and at the site of viral replication (e.g., the liver), may vary dramatically. The high level of viral replication occurring within the liver may constitute an ample source of core protein mediating local suppression of intrahepatic DC function, and the levels of circulating core protein present within secondary lymphoid structures might be insufficient to elicit suppression of T cell priming by HCV antigen-bearing DCs. It is likely that gC1qR-mediated inhibition of DC IL-12 production suppresses an early step in the T cell response to HCV, resulting in impaired expansion and poor acquisition of effector functions by virus-specific lymphocytes, despite massive viral expansion in the liver.

The mechanism of suppression of IL-12 production following gC1qR engagement on DCs has yet to be determined. A role for the PI-3K pathway in gC1qR-mediated suppression of IL-12 secretion in monocytes was established recently [32 ]. However, attempts to extend this observation to MDDCs were inconclusive (data not shown). The suppressors of cytokine signaling (SOCS) family of inhibitory signaling mediators are known to suppress signaling through cytokine and TLRs [62 63 64 ]. Silencing of SOCS-1 expression in human DCs was shown recently to result in dramatically increased IL-12 production and improved stimulation of antitumor T cell responses, without changes in DC costimulatory marker expression [65 , 66 ]. It is interesting that mRNA expression for SOCS-1 is up-regulated in monocytes following treatment with anti-gC1qR antibody (S. N. Waggoner, unpublished observations). The similar, suppressive effects of SOCS-1 and gC1qR on IL-12 production by DCs suggest a functional, biological activity for the SOCS-1 up-regulation, observed following gC1qR engagement. A detailed analysis of the role of SOCS-1 in gC1qR-mediated suppression of IL-12 production by TLR ligand-stimulated DCs is currently under investigation in our laboratory.

In conclusion, HCV core/gC1qR interaction on MDDCs induces a potent suppression of TLR-induced IL-12p70 production. The suppression of IL-12 correlated strongly with reduced expression of IFN-{gamma} by allogeneic CD4+ T cells and resulted in a skewed differentiation favoring Th2 cytokines, including IL-4, IL-5, and IL-13. These results establish a potential role for HCV core/gC1qR interaction in the observed dysfunction of DCs isolated from chronically infected individuals. Therefore, HCV core/gC1qR-mediated immune modulation may impair the development of a strong type 1, virus-specific T cell response and augment viral persistence. Future analysis of HCV core/gC1qR interaction will be aimed at determining the role of gC1qR-induced changes in the host cytokine environment in the etiology of HCV-induced liver disease.

ACKNOWLEDGEMENTS

This work was supported by grants AI057591 (to Y. S. H.) and training fellowship 5T32AI10749608 from the National Institutes of Health. We thank Barbara Small, Dr. Kara Cummings, and Dr. Heesik Yoon for excellent technical support for use of 3H-thymidine proliferation assays, cell separations from PBMC, and CFSE labeling of T cells, respectively. We thank Dr. Judith Woodfolk for her gracious gift of IL-5 and IL-13 ELISA reagents. We are grateful to Drs. John Steinke and Anjeanette Roberts for advice on statistical analysis.

FOOTNOTES

Current address: Department of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Back

Received May 1, 2007; revised July 19, 2007; accepted August 26, 2007.

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