HCV core protein interaction with gc1q receptor inhibits Th1 differentiation of CD4 T cells via suppression of dendritic cell IL-12 production

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1 HCV core protein interaction with gcq receptor inhibits Th differentiation of CD T cells via suppression of dendritic cell IL- production Stephen N. Waggoner,*, Caroline H. T. Hall,* and Young S. Hahn*,, Departments of *Microbiology, Beirne Carter Center for Immunology Research, and Pathology, University of Virginia, Charlottesville, Virginia, USA Abstract: Dendritic cells (DCs) isolated from patients with chronic hepatitis C virus (HCV) infection display an impaired capacity to generate type CD 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 gcq receptor, gcqr, expressed on host immune cells. However, the molecular mechanism(s) of HCV coremediated DC dysfunction has not been defined. Herein, ligation of gcqr on human monocytederived DCs (MDDCs) with HCV core or antigcqr agonist antibody was shown to inhibit TLRinduced IL- production but not the production of other TLR-stimulated cytokines. Furthermore, engagement of gcqr on MDDCs resulted in reduced IFN- secretion by allogeneic CD T lymphocytes during mixed lymphocyte culture. Differentiation of CD T cells cocultured with HCV core- or anti-gcqr antibody-treated MDDCs was also skewed toward production of Th cytokines, including IL-. Importantly, that addition of IL- rescued IFN- production and Th differentiation by CD T cells. Therefore, engagement of gcqr on DCs by HCV core limits the induction of Th responses and may contribute to viral persistence. J. Leukoc. Biol. 8: 7 9; 7. Key Words: human DC Th cells viral inflammation INTRODUCTION Hepatitis C virus (HCV) is a blood-borne pathogen infecting roughly % of the world s population. This virus establishes persistent infection in as many as 8% of infected individuals, leading to chronic liver diseases, including cirrhosis and hepatocellular carcinoma []. Viral clearance is associated with strong and broadly targeted T cell responses, and significant production of type cytokines includes IFN- []. Antiviral CD T cell responses are critical for the resolution of HCV infection by providing for CD8 T cell priming and generation of antibody responses [ ]. However, the levels of IFN- in the serum are markedly diminished in chronically infected individuals, wherein cytokine expression by peripheral blood lymphocytes is skewed toward Th cytokines such as IL- [7 9]. Furthermore, HCV-specific T and B cells are not detected until months postinfection [,, ]. 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 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 [, ]. 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-. These DC-associated, activation-induced inflammatory signals are crucial for CD T cell differentiation, particularly the production of the Th cytokine IFN-. Multiple independent studies have shown that DCs isolated from patients chronically infected with HCV display a reduced capacity to induce T lymphocyte activation [9, 8]. These monocyte-derived DCs (MDDCs) also displayed a severely suppressed capacity to produce the Th-inducing cytokine IL- in response to TLR or CD ligand (CDL) stimulation [9, 7, 9]. In addition, myeloid DC (MDC) and plasmacytoid DC (PDC), purified from the blood of chronically infected individuals, also made less IL- (MDC) and IFN- (PDC) in vitro, resulting in reduced IFN- production by allogeneic CD T cells [ ]. 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 [, 5]. Two independent groups used recombinant adenovirus constructs expressing the HCV core or core and envelope proteins (Ad- Current address: Department of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 55, USA. Correspondence: Department of Microbiology and Beirne B. Carter Center for Immunology Research, University of Virginia, HSC, Box 88, Charlottesville, VA 9, USA. ysh5e@virginia.edu Received May, 7; revised July 9, 7; accepted August, 7. doi:.89/jlb /7/8-7 Society for Leukocyte Biology Journal of Leukocyte Biology Volume 8, December 7 7

2 CE) to demonstrate functional suppression of DCs by viral proteins [, 7]. Infection of DCs from several strains of mice (C57BL/, BALB/c, and A/J) with Ad-CE yielded DCs with greatly decreased T cell stimulatory capacity in vitro and in vivo [7, 8]. 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 [7]. 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 [9, ], 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 gcq receptor (gcqr) results in a suppression of monocyte production of IL-p7 in response to TLR stimulation [, ], we sought to determine the effect of HCV core/gcqr interaction on DC function and examine the impact of HCV core treatment of DCs on CD T cell dysfunction. The production of IL-p7 was suppressed exclusively and significantly in response to or polyinosinic:polycytidylic acid [poly(i:c)], and maturation and additional cytokine expression was normal. Furthermore, the inhibition of IL- following gcqr ligation on human DCs strongly correlated with reduced expression of IFN- by allogeneic CD T cells in favor of increased Th differentiation. These results establish a potential role for HCV core interaction with gcqr in virus-induced DC dysfunction and reduced Th cytokine production during chronic HCV infection. MATERIALS AND METHODS Reagents Recombinant -galactosidase-fused core protein was obtained from Virogen (Watertown, MA, USA). Recombinant human (rh)gm-csf and rhil- were obtained from R&D Systems (Minneapolis, MN, USA). Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was obtained from Molecular Probes (Eugene, OR, USA). H-Thymidine was obtained from Perkin Elmer (Wellesley, MA, USA). rhil-p7 was obtained from PeproTech (Rocky Hill, NJ, USA). Salmonella typhimurium, poly(i:c), and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Generation of gcqr-specific antibodies and recombinant proteins Mouse mab specific for gcqr were generated as described previously []. Briefly, mice were immunized with recombinant full-length GST-gCqR and were screened for specificity using recombinant full-length His -gcqr (University of Virginia Hybridoma Center, Charlottesville, VA, USA). One antigcqr IgG mab clone (A) was purified and used for flow cytometric staining as described previously [ ]. An anti-gcqr IgM mab clone (5F) was characterized by its ability to ligate or cross-link gcqr and was concentrated using Centricon Ultra- columns (Millipore, Bedford, MA, USA). As a control, mouse isotype IgM (BD PharMingen, San Diego, CA, USA; clone C8-) was resuspended in hybridoma culture medium concentrated in the same manner as the anti-gcqr IgM clone (5F). Recombinant His -gcqr and His -dihydrofolate reductase (DHFR) were produced and purified in our laboratory as described previously []. Where indicated, these proteins were added at 5 g/ml to MDDC or MDDC-T cell cultures to compete with cellular gcqr for binding to anti-gcqr antibodies. A protein concentration of g/ml His -gcqr was determined previously to be minimally necessary for blocking the binding of anti-gcqr and resultant suppression of IL- []. Preparation of PBMC, isolation of monocytes and CD 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 CD microbeads (StemSep) to 95% purity. Purified monocytes were differentiated into DCs by culture in RPMI supplemented with % (v/v) FBS (Hy- Clone, Logan, UT, USA), penicillin/streptomycin ( g/ml), L-glutamine ( mm), HEPES ( mm), and -ME (55 M) with 8 IU/ml rhgm-csf and 8 IU/ml rhil- at 7 C with 7% CO in a humidified atmosphere for a total of days. Fresh media and cytokines were added at Days and of culture. For mature DCs (mdcs), ng/ml or 5 g/ml poly(i:c) was added for the final 8 h of culture. Immature MDDCs were routinely 95% HLA- DR CDc CD. Human CD T cells were purified from CD monocyte-depleted, normal human PBMC using CD microbeads (Miltenyi Biotech, Auburn, CA, USA) to 9% purity. Analysis of gcqr expression and determination of DC maturation phenotype by flow cytometry Total PBMC or immature MDDCs (Day 5 of culture) were analyzed for gcqr expression by flow cytometry. Cells (5 5 ) were stained initially with 5 g anti-gcqr IgG (A) in % normal goat serum for min on ice. Following three washes, cells were stained with. g PE-conjugated goat anti-mouse Ig (#55589, BD Biosciences, San Jose, CA, USA) in % goat serum for min on ice. Cells were washed five times prior to staining with L each of one or more directly conjugated mouse antibodies or isotype controls (ebioscience, San Diego, CA, USA), including allophycocyanin-cd (UCHT), allophycocyanin-cd (D), allophycocyanin-hla-dr (LN), and FITC-CDc (.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 ng/ml or 5 g/ml poly(i:c). The agonistic anti-gcqr mab (5F) or isotype control IgM ( g/ml) was added to idcs, alone or in conjunction with maturation stimuli. After h of additional culture in the presence or absence of maturation stimuli, Day DCs were stained with FITC-CD8 (B7.; D.), FITC-CD8 (HB5e), FITC-CD (5C), PE- CD8 (B7.; IT.), allophycocyanin-hla-dr, PE-programmed death ligand- (PD-L; B7-H; MIH), PE-PD-L (B7-DC; MIH8), FITC-CDc, PE-CD (D), 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 ng/ml, 5 g/ml poly(i:c), or PBS (immature) for a total of 8 h. Concurrently, cells were treated with g/ml anti-gcqr mab agonist (5F), murine isotype IgM, or 5 g/ml HCV core protein. In some instances, 5 g/ml His -gcqr or His -DHFR was added at time of antibody treatment to compete with DC cell-surface gcqr for binding to anti-gcqr mab. Following 8 h of stimulation, MDDCs were washed thoroughly prior to an additional h of culture in fresh medium. At 8 h of DC stimulation, half of the culture supernatant was collected and stored at 8 C for analysis of cytokine production by ELISA. The 8-h MDDCs ( 5 ) were then cocultured with freshly isolated, allogeneic CD T cells in a final volume of ml in a -well plate, except during proliferation assays as described below. In some instances, 5 g/ml His -gcqr or His -DHFR was added at time of DC/T cell mixing to compete with T cell surface gcqr for binding to anti-gcqr antibody, possibly presented on bound DCs. Furthermore, in some experiments, ng/ml rhil-p7 or PBS was added at time of DC/T cell mixing to restore levels of IL-. At Day of DC/T cell coculture, two-thirds of the culture supernatant was collected and frozen at 8 C for analysis of cytokine production by ELISA. Following 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 CD T cells were labeled with CFDA-SE as described by the manufacturer (Molecular Probes). Briefly, cells were incubated with a final 8 Journal of Leukocyte Biology Volume 8, December 7

3 concentration of 5 M CFDA-SE in PBS containing.% BSA for min at 7 C. Following this incubation, cells were washed three times in PBS and incubated an additional min at 7 C in plain PBS. CFDA-SE-labeled T cells were cocultured with immature or -mature, allogeneic MDDCs (treated with anti-gcqr or isotype antibody as described above) at a : ratio of DCs:T cells for 5 days. Conversely, CFSE-labeled T cells were stimulated with g/ml anti-cd (OKT) and anti-cd8 (CD8.; ebioscience). At Days,,, and 5 of DC/T cell coculture, cells were stained with allophycocyanin-cd (OKT-, ebioscience), fixed, and collected on a FACSCalibur (BD Biosciences). lymphocytes, and MDDCs, with a focus on possible variation of gcqr expression in different blood donors. To this end, we analyzed receptor expression simultaneously on PBMC from healthy blood donors by flow cytometry (Fig., A and B). Assay of T cell proliferation by H-thymidine incorporation MDDCs were treated with or poly(i:c) and anti-gcqr agonist (5F) or isotype IgM as described above. After 8 h, various doses of DCs ( cells) were -irradiated and washed extensively to remove TLR and gcqr ligands. These DCs were then mixed with purified, allogeneic CD T cells ( 5 cells) in 9-well, flat-bottom culture plates for 5 days. During the final 8 h of DC/T cell coculture, Ci H-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 (8 h) or DC/T cell MLR coculture (Day ) supernatants were stored at 8 C. Cytokine production was assessed by specific ELISA for IL-p7 and TGF- (BD PharMingen) as well as IL-, IL-, IFN-, IL-7, and TNF- (ebioscience), according to the manufacturer s instructions. IL-5 and IL- ELISAs were performed according to the manufacturer s specifications (Pierce Endogen, Rockford, IL, USA). Intracellular cytokine staining Following days of DC/T cell coculture, T cells were restimulated with 5 ng/ml PMA and g/ml ionomycin for a total of 5 h. Brefeldin A ( g/ml, BD PharMingen) was added to the culture during the finalhofrestimulation. At the end of this incubation, T cells were stained for CD and intracellular cytokines according to the manufacturer s specification (BD PharMingen). Briefly, following surface staining with allophycocyanin-cd, as described above, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences, #5558). Permeabilized cells were then stained with FITC-IFN- /PE-IL- Fastimmune reagent (#5) or Fastimmune isotype (#58), 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.5 was considered to be significant. Correlation was determined using Pearson s coefficient of correlation (r). RESULTS The expression of gcqr 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 [9, ]. The interaction of HCV core with gcqr has been reported to modulate T lymphocyte responses [5, 5, ]. However, expression of gcqr on various subsets of human PBMC has not been analyzed in detail. To determine the role of gcqr in the regulation of DC function as well as subsequent shaping of T lymphocyte responses, we first analyzed the cell surface expression of gcqr in immune cells, including monocytes, Fig.. The cell surface expression of gcqr on human PBMC. (A) Representative flow cytometry of gcqr expression on human PBMCs, CD monocytes, and CD lymphocytes. Electronic gating of CD and CD events is shown in upper middle and right panels. Staining of gcqr (line histograms) with A monoclonal IgG and PE-conjugated secondary is shown in comparison with isotype control staining (solid histograms). SSC, Sidescatter; FSC, forward-scatter. (B) The mean fluorescence intensity (MFI) of gcqr staining on total PBMC or gated populations for multiple healthy blood donors (n ) was normalized to isotype controls (MFI gcqr divided by MFI isotype control). Monocytes (CD ) and T lymphocytes (CD ) from these blood donors were uniformly gcqr, compared with isotype controls ( 97% gcqr ). Small, horizontal bars indicate mean value of relative gcqr expression; each dot represents one individual donor. Horizontal, dashed line represents level of background staining. (C) Representative flow cytometry of the expression of gcqr on human MDDCs. Live event gating, HLA-DR and CDc staining, and gcqr staining of HLA-DR CDc -gated cells are shown. Waggoner et al. Suppression of DC IL- and Th induction by gcqr 9

4 Staining of gcqr on CD monocytes (99..8% gcqr ) and CD lymphocytes (99.8.% gcqr )revealed ubiquitous expression of this receptor on these cell populations. Monocytes (mean relative gcqr MFI.5.) and T lymphocytes (mean relative gcqr MFI..) displayed some variation in gcqr expression levels, although all of the gated (CD and CD ) events within this pool of donors expressed substantial levels of gcqr (Fig. B). In addition, expression of gcqr was evident on idcs from several human blood donors (mean relative gcqr MFI 7..8; Fig. C). These data suggest that gcqr 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 gcqr ligands to activate gcqr-mediated intracellular signaling. Ligation of gcqr by HCV core or anti-gcqr antibody on MDDCs results in suppression of IL- production Although DCs from chronically infected HCV patients display a reduced capacity to secrete IL- [9, ], the role of HCV core/gcqr interaction in this suppression is not known. Therefore, we sought to ascertain the effect of gcqr ligation on IL- production by MDDCs from healthy blood donors. Immature MDDCs from normal, healthy blood donors were collected and stimulated with TLR- or TLR- ligands [ and poly(i:c), respectively] for 8 h in the presence of an anti-gcqr agonistic mab (5F), HCV core protein, or isotype mouse IgM control antibody (Fig. ). It is important that coreor anti-gcqr antibody-mediated engagement of gcqr on MDDCs strongly suppressed the production of IL-p7 in response to TLR stimulation (Fig. A). Conversely, - and poly(i:c)-induced production of additional pro- or anti-inflammatory cytokines, including IL-, TGF-, and TNF-, was not affected by ligation of gcqr (Fig., A and B). The suppression of IL- was observed consistently in (P.)- and poly(i:c)-stimulated (P.) MDDCs from every blood donor examined, although the degree of suppression was variable (Fig. B). Trypan blue exclusion analysis following gcqr ligation revealed no change in MDDC viability (data not shown). These results suggest that gcqr ligation specifically suppresses TLR-induced IL-p7 production by MDDCs. gcqr ligation does not interfere with TLRinduced 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 [, ]. High-level expression of CD8 (B7.) and CD8 (B7.) on mdcs ensures potent stimulation of T lymphocytes via CD8 [7], and expression of CD by DCs enables amplification of immune activation through interaction with CDL (CD5) on T lymphocytes [8]. A. α-gcqr HCV core B. p =. p =. p =.9 p =.8 IL-p7 (pg/ml) Imm TNF-α (pg/ml) Imm IL-p7 (pg/ml) Iso α-g Iso α-g TNF-α (pg/ml) Iso α-g Iso α-g 75 5 p =.9 p =.8 p =.78 p =.99 IL- (pg/ml) 5 5 TGF-β (pg/ml) 5 IL- (pg/ml) TGF-β (pg/ml) Imm Imm Iso α-g Iso α-g Iso α-g Iso α-g Fig.. Ligation of gcqr on human MDDCs mediates specific suppression of TLR-induced IL-p7 production. Human MDDCs ( 5 ) were stimulated for 8 h with ng/ml or 5 g/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 g/ml anti-gcqr IgM agonist mab (open bars), isotype mouse IgM control antibody (black bars), or 5 g/ml recombinant HCV core protein (gray bars). After 8hofstimulation, MDDCs were washed thoroughly to remove unbound, stimulatory reagents and returned to culture in fresh medium for an additional h. Supernatants from 8 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 - or poly(i:c)-induced cytokine production for MDDCs from different healthy blood donors (n 9) are shown as a pair-wise comparison of the effects of isotype IgM (Iso) versus anti-gcqr agonist ( -G), wherein each line represents an individual donor. Statistically significant differences between treatments were determined by two-tailed Mann-Whitney U-test. Journal of Leukocyte Biology Volume 8, December 7

5 We sought to ascertain whether ligation of gcqr may affect MDDC maturation following TLR- ligand-induced activation of idcs. Stimulation of idcs with anti-gcqr agonistic antibody or mouse isotype IgM for h had no effect on steady-state expression of the costimulatory markers CD8 (B7.), CD8 (B7.), CD8, CD, and MHC class II (HLA-DR; Fig., A and C). Furthermore, up-regulation of cell surface expression of these costimulatory markers following -induced MDDC maturation was not abrogated following gcqr ligation on a representative blood donor (Fig. A) or among all blood donors analyzed (Fig. C). Similar results were obtained using HCV core protein in place of anti-gcqr antibody (data not shown). PD-L (B7-H) and PD-L (B7-DC) are inhibitory receptors, which have been shown to play a role in tolerance and suppression of T cell activation [9, ]. It is intriguing that these receptor pairs have been strongly implicated in T cell exhaustion during chronic viral infection, including HCV [ ]. Examination of expression levels of PD-L and PD-L on immature as well as mature MDDCs revealed that gcqr ligation had no effect on expression of these inhibitory receptors (Fig., B and C). These results suggest that gcqr ligation has no effect on MDDC expression of costimulatory or inhibitory receptors and that TLR-induced MDDC maturation occurs normally following engagement of gcqr. % maximum A. Immature mature B % maximum α-gcqr CD8 CD8 CD8 CD HLA-DR C. PD-L PD-L 5 8 Relative CD8 Relative CD8 Relative CD8 5 Relative CD Immature mature Immature mature Immature mature Immature mature α-gcqr Relative HLA-DR Relative PD-L 8 Relative PD-L Immature mature Immature mature Immature mature Fig.. DC maturation is normal following gcqr ligation. (A and B) Human immature MDDCs were stimulated with g/ml anti-gcqr agonist (lower row) or isotype IgM control (upper row) antibody in the presence ( mature; heavy line histograms) or absence (immature; line histograms) of ng/ml. After h, cells were stained using a panel of antibodies and analyzed by flow cytometry for surface expression of costimulatory (A) and coinhibitory (B) receptors. 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 mature/nmfi immature). Waggoner et al. Suppression of DC IL- and Th induction by gcqr

6 gcqr ligation on human MDDCs results in skewing of T cell cytokines from a Th phenotype to increased expression of type cytokines As a result of the diminished IL-p7 production characteristic of gcqr-stimulated MDDCs (Fig. ), we hypothesized that CD T cell-mediated IFN- production may be inhibited following stimulation with these gcqr-treated MDDCs in an allogeneic MLR. Ligation of gcqr on idcs did not influence cytokine expression in allogeneic DC/T cell coculture supernatants (Fig. and Table ), in part, as idcs do not demonstrate detectable IL-p7 expression, regardless of gcqr engagement. As expected, -induced maturation of MDDCs resulted in increased T cell IFN- production as compared with idcs. This increase in IFN- was markedly inhibited when gcqr on MDDCs was engaged by anti-gcqr agonist antibody or HCV core protein prior to coculture with T cells (Fig. A). The suppression of IFN- production in allogeneic DC/T cell coculture supernatants was observed consistently in all blood donor pairings tested (Fig. B). In contrast, the production of other cytokines, including IL- and IL-, was markedly unaffected by gcqr engagement, suggesting that suppression following gcqr ligation is specific (Fig. B and Table ). The differentiation of CD T cells into Th IFN- -producing or Th IL--producing cells is controlled by DC-mediated cytokine production [, 5]. Given the importance of IL- to Th differentiation and the strong suppression of IL-p7 by gcqr ligands (Fig. ), we hypothesized that ligation of gcqr on DCs may inhibit the Th differentiation of allogeneic CD T cells and be involved in promoting Th differentiation. Upon restimulation with PMA and ionomycin, CD T cells cocultured with HCV core- or anti-gcqr antibody-treated MDDCs were less capable of producing IFN- compared with CD T cells cocultured with isotype antibody-treated MDDCs (Fig. 5, A and B). This effect was observed as a decrease in the proportion of IFN- T cells (8 5% decrease, P., Fig. 5A) and in the MFI of IFN- staining (9 % decrease, P., Fig. 5B) in T cells cultured with anti-gcqr antibody-treated compared with isotype antibody-treated MDDCs. It is striking that we observed a parallel increase (.- to.-fold, P.) in the proportion of IL- T cells (Fig. 5A). The skewing from Th to Th during the differentiation of these T cells suggests that gcqr ligation on MDDCs may favor Th cytokine production during primary stimulation. In addition to IL-, IL-5 and IL- are prototypical cytokines produced by Th cells []. Analysis of IFN-, IL-5, and IL- expression in Day MLR supernatants from multiple DC/T cell allogeneic coculture donor pairings (n 8) revealed that a substantial decrease in IFN- production accompanied a significantly increased release of IL-5 and IL- in T cells cocultured with HCV core- or anti-gcqr antibody-treated MDDCs (Fig. 5C). These results together suggest that the lack of a potent Th-inducing cytokine such as IL- results in a skewing toward production of Th cytokines, similar to that observed in the periphery of patients chronically infected with HCV [7, 8]. A. IFN-γ (ng/ml) IL-7 (pg/ml) B. IFN-γ (pg/ml) IL- (pg/ml) Imm p =.97 α-gcqr IL- (pg/ml) IL- (ng/ml) Imm Imm Imm p =.5 p =. Iso α-g Iso α-g Iso HCV core Immature DC DC p =. p =.7 p =.88 α-g Iso α-g Iso α-g Iso α-g Immature DC DC Fig.. Ligation of gcqr during MDDC maturation results in poor capacity to induce IFN- production by allogeneic T cells. Human MDDCs ( 5 ) from multiple healthy blood donors were stimulated for 8 h with, poly(i:c), or PBS (Imm) in the presence of g/ml anti-gcqr agonist (open bars), isotype IgM (black bars), or 5 g/ml HCV core protein (gray bars), as described in Figure. The MDDCs were washed thoroughly prior to extended culture in fresh medium for a total of 8 h. MDDCs were then cocultured with freshly isolated, allogeneic CD 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- (n 7) and IL- (n 5) in multiple independent, allogeneic mixtures. Each line represents the comparison of mean cytokine expression with isotype IgM (Iso) versus anti-gcqr agonist ( -G) antibody for an individual donor pairing. Statistical significance was analyzed using the two-tailed Mann-Whitney U-test. Journal of Leukocyte Biology Volume 8, December 7

7 TABLE. Changes in Cytokine Expression During Allogeneic DC/T Cell MLR (n 8 7) Immature DC mature DC poly(i:c) mature DC Antibody Mean SEM Mean SEM Mean SEM (pg/ml) P value a (pg/ml) P value a (pg/ml) P value a IFN gclqr IL gclqr IL gclqr TGF gclqr IL gclqr a Analyzed by Mann-Whitney U-test (two-tailed). A P value of.5 was considered to be significant. gcqr-induced cytokine dysfunction by T cells is not a result of diminished T cell proliferation and is independent of gcqr 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, 8]. Incomplete or dysfunctional activation of T cell proliferation by anti-gcqr antibody-treated MDDCs could result in a reduced expansion of IFN- -producing CD T cells, which may account for the decreased level of IFN- expression seen in the MLR culture supernatants. To test this possibility, we examined the ability of anti-gcqr antibodytreated MDDCs to induce T cell proliferation. The effect of gcqr ligation on immature or mature MDDCinduced T cell proliferation, as measured by H-thymidine incorporation, was highly variable among a multitude of blood donor pairings (n, Fig. A), possibly reflecting the heterogeneous population of CD T cell responders used in this analysis. Overall, there was no statistically significant effect of gcqr ligation on MDDC stimulatory capacity in terms of T cell proliferation (P., Fig. A). Analysis of CFSE dilution in CD T cells following coculture with allogeneic (Fig. B) and autologous (data not shown) MDDCs confirms the lack of suppression of T cell proliferation following gcqr ligation on MDDCs. These results suggest that gcqr engagement on MDDCs does not affect their ability to induce allogeneic T cell proliferation and imply that core/ gcqr interaction is not a major cause of the reduced capacity of HCV patient-derived DCs to induce T cell proliferation [9, 8]. As MDDCs treated with anti-gcqr agonist antibody were cocultured with CD T cells, we cannot rule out the possibility that the anti-gcqr antibody might interact directly with gcqr on T cells (Fig., A and B) and inhibit their function. To this end, we attempted to remove unbound anti-gcqr antibody prior to MDDC mixing with T cells and block any presentation of MDDC-bound antibody to gcqr on the surface of T cells. Soluble recombinant His -gcqr (sgcqr) was used as a competitor for anti-gcqr antibody binding to cell surface gcqr at the time of DC stimulation (Fig. 7A) or coculture of DCs and T cells (Fig. 7B). Addition of sgcqr, but not a control protein DHFR (sdhfr), during MDDC stimulation with anti-gcqr agonist antibody, abrogated suppression of IL- completely (Fig. 7A, left bar graphs). Furthermore, this sgcqr-mediated abrogation of IL- inhibition resulted in normalization of T cell-mediated IFN- production and Th differentiation (Fig. 7A). These results confirm that anti-gcqr antibody-mediated suppression of MDDC IL- production and subsequent expression of IFN- by T cells are mediated through anti-gcqr antibody interaction with gcqr on the MDDC surface. However, the addition of sgcqr at the onset of MDDC-T cell coculture did not reverse anti-gcqr-treated, MDDC-mediated suppression of IFN- production or Th differentiation (Fig. 7B). Similar results to those described in Figure 7 were obtained using poly(i:c) as a maturation stimulus in place of and when HCV core protein was substituted in place of anti-gcqr agonist antibody (data not shown). Together, these results suggest that ligation of gcqr on the surface of MDDCs exerts potent suppression, which is completely independent of gcqr expression on the T cell surface. Dysfunctional IFN- production by CD T cells correlates with reduced IL- and is rescued following reconstitution with ril- The cytokine IL- plays a crucial role in the induction of IFN- production by CD and CD8 T cells as well as NK and NKT cells []. However, several related (e.g., IL-) and unrelated (e.g., IL-8 and type I IFN) cytokines are postulated to play important roles in Th differentiation, in the presence or absence of IL- [7, 8]. To ascertain the relationship between IL- and IFN- in our system, we first attempted to show a correlation between the degree of IL- suppression on MDDCs and that of IFN- by CD T cells. The percent decrease in IL-p7 and IFN- expression, in MDDC culture-monoculture supernatants and DC/T cell coculture supernatants, respectively, was calculated for multiple, healthy blood donor pairs (Fig. 8A, n 8). These results were paired and ranked to determine Pearson s coefficient of correlation, which reveals a strong, positive coefficient of correlation (r.7) between the suppression of IL-p7 and the inhibition of IFN- in these donor pairings. Waggoner et al. Suppression of DC IL- and Th induction by gcqr

8 A. % Maximum 8 IL- 5 Unstimulated C..5. p =. p =. p =.5 p =. α-gcqr HCV core 5 5 CD 5 IFN-γ α-gcqr HCV Core IFN-γ (ng/ml).5. Imm ** **. --. DC p =. DC p =.5 IL- DC 5 p =. p =. 5 B. IFN-γ MFI ** IFN-γ p =. p =.5 ** ** 5 5 **. --. p =.7 5 p =. * * DC α-gcqr HCV core IL-5 (pg/ml) IL- (ng/ml) 75 5 DC p =. p =. DC p =. p =. 5 Immature DC DC DC DC Fig. 5. Engagement of gcqr on MDDCs limits Th differentiation by allogeneic CD T cells. (A and B) Human MDDCs from multiple healthy blood donors (n ) were stimulated with ( DC), poly(i:c) [ DC], or without maturation stimuli (Imm) in the presence of g/ml anti-gcqr agonist antibody, IgM isotype, or 5 g/ml HCV core protein for a total of 8 h (with washing) as described in Figure. These MDDCs were then combined with freshly isolated, allogeneic CD 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% CD (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 CD T cells from an individual blood donor pairing is shown with quadrants drawn based on isotype control staining. The mean percent of IFN- (lower right quadrants) and IL- (upper left quadrants) T cells SEM is shown (n ). Significant differences between paired frequencies for anti-gcqr- or HCV core- and isotype-treated MDDCs were determined by two-tailed Wilcoxon signed-rank test (*, P.5; **, P.). (B) The MFI (MFI stain MFI isotype control) of intracellular IFN- staining is shown (mean SEM, n ) for isotype IgM (black bars)-, anti-gcqr (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. Significant differences in paired data were analyzed by Wilcoxon signed-rank test. We next sought to determine if reconstitution of IL- in the DC/T cell MLR coculture would restore the decreased IFN- production as well as dysfunctional Th differentiation observed following ligation of gcqr on MDDCs. Addition of ng/ml IL-p7 to these cocultures restored Th differentiation of T cells stimulated with anti-gcqr-treated MDDCs Journal of Leukocyte Biology Volume 8, December 7

9 A. H-Thy incorporation (x cpm) B. CD 9 p =.8 p =.5 p =. 9 9 Iso α-g Iso α-g Iso α-g Immature DC DC Day Day Day Day DC Immature CFSE α-cd α-cd8 Fig.. Normal, allogeneic T cell proliferation following gcqr ligation on MDDCs. Human MDDCs were matured with, poly(i:c), or PBS (Immature) in the presence of isotype IgM (Iso) or anti-gcqr agonist ( -G) antibodies as described in Figure. (A) MDDCs ( ) were lethally irradiated and washed thoroughly prior to coculture with allogeneic CD T cells ( 5 ), as described in Materials and Methods. For the final 8-h of DC/T cell coculture, Ci H-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 CD T cells following coculture of MDDCs, treated as described above, with 5 freshly isolated, CFSE-labeled, allogeneic CD T cells. Proliferation following direct stimulation of CD T cells with g/ml (each) anti-cd and anti-cd8 antibodies is shown in the top row. Comparison of isotype- or anti-gcqr-treated immature MDDC (Rows and ) or mature MDDC (Rows and 5) induction of proliferation in live, gated CD T cells is shown, where numbers represent the proportion of CFSE lo cells. Iso α-g Iso α-g (8% reduction in IFN- MFI) to levels similar to those seen using isotype IgM-treated MDDCs (% reduction in IFN- MFI; Fig. 8B). Moreover, addition of IL- to cocultures increased the overall level of IFN- production and abrogated the suppression of IFN- seen following gcqr ligation on - or poly(i:c)-matured MDDCs (Fig. 8C). Suppression of IFN- production (data not shown) and Th differentiation (Fig. 8B) by HCV core protein were also reversed by addition of IL- to the DC/T cell coculture. These results strongly suggest that gcqr ligation on MDDCs results in a potent suppression of TLR-induced IL-p7 production, which mediates a powerful inhibition of subsequent IFN- production and Th differentiation by T cells. DISCUSSION Herein, we describe an immunomodulatory role for HCV core/ gcqr interaction on MDDCs involving the inhibition of IL- p7 production in response to prototypical bacterial (, TLR-) and viral [poly(i:c), TLR-] stimuli. Importantly, HCV core/gcqr-mediated suppression of IL- production by MDDCs resulted in an inefficient induction of IFN- production by CD T lymphocytes. This decrease in type cytokine expression correlated with an increased secretion of the Th mediators, IL-5 and IL-. Restimulation of CD T cells, previously activated by HCV core or gcqr agonist, mabtreated MDDCs, revealed a clear skewing or selective expansion of Th rather than Th effectors. It is remarkable that reconstitution of IL- in the DC/T cell coculture abrogated gcqr-mediated inhibition of IFN- production during the allogeneic MLR and restored Th differentiation to levels similar to those observed in CD T cells stimulated with isotype antibody-treated MDDCs. These results suggest that HCV core/gcqr interaction is involved in the negative regulation of MDDC-induced Th differentiation through inhibition of IL- production. Of interest, the selective inhibition of TLR-induced IL- production, following treatment of MDDCs with HCV core protein or anti-gcqr antibody, resulted in suppression of IFN- production by CD 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- production by gcqr ligandtreated MDDCs. First, cross-linking of gcqr on MDDCs did not influence maturation-induced up-regulation of costimulatory receptors (e.g., CD8) or the expression of inhibitory PD-L and PD-L. This suggests that ligation of gcqr does not modulate the antigenic or costimulatory signals provided during T lymphocyte activation. Second, IL- production during coculture of allogeneic CD T cells with anti-gcqr antibody-treated MDDCs was not inhibited. As IL- production by activated CD T cells is an important characteristic of T cell activation and a crucial factor in T cell proliferation, the unabated induction of IL- following gcqr cross-linking may result in the relatively normal induction of CD T cell proliferative expansion. Therefore, HCV core-mediated engagement of gcqr on the surface of DCs may not be responsible for the defective T cell proliferation observed in HCV coretreated PBMC cultures [5, 9]. Based on the previous finding that treatment of PBMC with HCV core led to the inhibition of IL- and IFN- production as well as T cell proliferation [, 5], 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 gcqr on the surface of APCs as well as T cells. Although direct binding of HCV core on T cells results in reduced proliferation, IL- secretion, and IFN- production, further inhibition of CD T Waggoner et al. Suppression of DC IL- and Th induction by gcqr 5

10 A. B. Wash sdhfr or α-g or Iso sgcqr Wash sdhfr or α-g or Iso sgcqr DCs sdhfr DCs sgcqr 5 5 IL- (pg/ml) Iso α-g IFN-γ (ng/ml) Iso α-g IL- 5 5 α-gcqr.9 5. () (8) (7) (7) IFN-γ DC/T sgcqr DC/T sdhfr IFN-γ (ng/ml)..5 Iso α-g.. Iso α-g IL- 5 5 α-gcqr (88) (5) (87) (5) IFN-γ Fig. 7. Suppression of IFN- production by CD T cells is dependent on MDDC gcqr ligation but independent of T cell gcqr. MDDCs ( 5 ) were stimulated with ng/ml in the presence of g/ml isotype IgM or anti-gcqr agonist antibody, washed, and cocultured with freshly isolated, allogeneic CD T cells (:5 ratio of MDDC:T cells), as described in Figure. (A) At time of antibody addition to MDDCs, cultures also received 5 g/ml sgcqr (DC sgcqr, lower row) or irrelevant control protein, His -DHFR (DC sdhfr, upper row) as a competitor for anti-gcqr agonist antibody binding to gcqr on MDDCs. (B) Conversely, 5 g/ml His -gcqr (DC/T sgcqr) or His -DHFR (DC/T sdhfr) was added to DC/T cell cocultures after DC stimulation and washed to compete with MDDC-bound, anti-gcqr antibody interaction with gcqr on T cells. IL-p7 production was analyzed in MDDC monoculture supernatants, and IFN- 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- and IFN-, respectively, in live, gated CD T cell populations. Numbers in parentheses indicate MFI (MFI stain MFI isotype) of IFN- staining. (A and B) Results are representative of those obtained using three different donor pairings. cell function and differentiation occurs following contact with gcqr-ligated DCs. We are currently investigating these possibilities. Despite profound suppression of IL- expression following gcqr ligation, it remains to be determined what effect gcqr signaling has on the production of other Th-inducing cytokines. Additional members of the IL- (e.g., IL-), IL- (e.g., IL-8), and type I IFN (e.g., IFN- ) families are also capable of inducing IFN- production by CD T cells. Based on our previous observation that gcqr-mediated inhibition of IL- p7 targets transcription of the IL-p subunit [, ], which is shared by the IL-p7 and IL- heterodimers [5], we can hypothesize that production of both cytokines is suppressed following gcqr ligation. Indeed, IL- production by MDDCs was inhibited by anti-gcqr antibody treatment (C. H. T. Hall, unpublished observations). Given the proposed role for IL- in the induction and maintenance of IL-7- producing CD T cells [5, 5], diminished IL- production by gcqr-cross-linked MDDCs might lead to inhibition of IL-7 production. It is surprising that IL-7 levels were increased slightly in allogeneic DC/T cell coculture supernatants following ligation of gcqr on MDDCs. Recent evidence suggests that IL- and TGF- also play critical roles in the generation of IL-7-producing Th7 cells [5]. Although expression of these cytokines by mature MDDCs is relatively unchanged following gcqr ligation (Fig. ; C. H. T. Hall, unpublished observations), it is possible that unabated secretion of IL- and TGF- by HCV core- or anti-gcqr antibodytreated MDDCs, in the absence of potent Th-inducing cytokines, may be sufficient to elicit increased Th7 differentiation. Such a mechanism may allow for expansion of pathogenic Th7 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 []. Despite the critical function of T cell-derived IFN- in immune defense against intracellular pathogens, including HCV, the importance of IL- to resolution of infectious diseases in humans is less clear [5]. Individuals chronically infected with HCV were more likely to be homozygous for an ILB (IL-p subunit) gene polymorphism, profoundly associated with reduced IL- production than those who resolved infection spontaneously [55 57]. This suggests that HCV core-induced loss of IL- 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 gcqr on DCs alone is sufficient to impair T lymphocyte activation and cytokine production, it is tempting to speculate that HCV coreinduced immune suppression via gcqr is likely to occur at an early phase of infection prior to the appearance of HCVspecific antibody responses. Indeed, the significant delay ( weeks) in the appearance of antiviral T and B cell Journal of Leukocyte Biology Volume 8, December 7

11 IL- A. % Suppression of IFN-γ production B. 5 5 C. IFN-γ (ng/ml) MFI 5 MFI 5 MFI MFI 5 MFI 5 MFI α-gcqr IFN-γ % Suppression of IL-p7 production α-gcqr PBS r =.7 p = p =. p = HCV Core p =. p =.75 DC DC DC DC IL- PBS IL- Fig. 8. Addition of ril- rescues deficient Th differentiation of allogeneic T cells. (A) Correlation between suppression of MDDC-derived IL-p7 and inhibition of IFN- produced by CD T cells. MDDCs were stimulated and cocultured with allogeneic CD T cells as described in Figure. The production of IL-p7 and IFN- was determined by ELISA analysis of MDDC monoculture supernatants or DC/T cell coculture supernatants, respectively. The percent suppression of IL-p7 and IFN- was then calculated {% suppression [(mean anti-gcqr-dc/mean isotype-dc) ]} for each allogeneic, healthy blood donor pairing (n 8). The coefficient of correlation between suppression of IL- and suppression of IFN- was assessed using Pearson s method. (B and C) MDDCs were stimulated and cocultured with allogeneic CD T cells as described in Figure, except that ng/ml rhil-p7 (IL-) or PBS was added to cocultures at time of DC-T cell mixing. (B) Intracellular staining of IL- and IFN- within live, gated CD T cells is shown for a representative donor pairing. Numbers in the upper left and lower right quadrants indicate frequency of IL- and IFN- cells, respectively. Numbers in the upper right quadrants represent MFI (MFI stain MFI isotype control) of IFN- staining (mean SEM, n ). (C) IFN- production in DC/T cell coculture supernatants is shown for mixtures, which received PBS or IL-, as described above (mean SEM, n ). Significant differences between isotype (black bars) and anti-gcqr (open bars) antibody-stimulated MDDC sample groups were analyzed by two-tailed Wilcoxon signed-rank test responses following HCV infection, compared with infection with HIV (within weeks), suggests that the induction of the adaptive response is impaired in some manner [,,, 58, 59]. As HCV core protein is secreted from infected cells and present in the serum of infected patients [9, ], this viral protein may interact with gcqr on DCs and restrain effective priming of HCV-specific T cells. Furthermore, cross-talk between gcqr 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 [, ]. 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 gcqr-mediated inhibition of DC IL- 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- production following gcqr engagement on DCs has yet to be determined. A role for the PI-K pathway in gcqr-mediated suppression of IL- secretion in monocytes was established recently []. 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 [ ]. Silencing of SOCS- expression in human DCs was shown recently to result in dramatically increased IL- production and improved stimulation of antitumor T cell responses, without changes in DC costimulatory marker expression [5, ]. It is interesting that mrna expression for SOCS- is up-regulated in monocytes following treatment with anti-gcqr antibody (S. N. Waggoner, unpublished observations). The similar, suppressive effects of SOCS- and gcqr on IL- production by DCs suggest a functional, biological activity for the SOCS- up-regulation, observed following gcqr engagement. A detailed analysis of the role of SOCS- in gcqr-mediated suppression of IL- production by TLR ligand-stimulated DCs is currently under investigation in our laboratory. In conclusion, HCV core/gcqr interaction on MDDCs induces a potent suppression of TLR-induced IL-p7 production. The suppression of IL- correlated strongly with reduced expression of IFN- by allogeneic CD T cells and resulted in a skewed differentiation favoring Th cytokines, including IL-, IL-5, and IL-. These results establish a potential role for HCV core/gcqr interaction in the observed dysfunction of DCs isolated from chronically infected individuals. Therefore, HCV core/gcqr-mediated immune modulation may impair the development of a strong type, virusspecific T cell response and augment viral persistence. Future analysis of HCV core/gcqr interaction will be aimed at determining the role of gcqr-induced changes in the host Waggoner et al. Suppression of DC IL- and Th induction by gcqr 7