Published online before print December 15, 2006
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axis by IFN-
therapy for hepatitis C
,1,3
Departments of
* Medicine and Molecular Microbiology and Immunology and
Pediatrics, Johns Hopkins University, Baltimore, Maryland, USA;
Maryland Medical Research Institute, Baltimore, Maryland, USA;
Departments of Pediatrics and Pathology and Laboratory Medicine, Children’s National Medical Center, George Washington University School of Medicine, Washington, DC, USA;
|| Division of Gastroenterology, Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana, USA;
¶ Department of Pediatrics, University of Colorado Health Sciences Center, and The Pediatric Liver Center, The Children’s Hospital, Denver, Colorado, USA;
# Wyeth Research, Cambridge, Massachusetts, USA; and
** Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, and the University of Cincinnati, Cincinnati, Ohio, USA
4 Correspondence: Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail: chris.karp{at}chmcc.org
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forms the foundation of therapy for chronic hepatitis C, only a minority of patients has a sustained response to IFN- alone. The antiviral activities of IFN- formed the rationale for its use in viral hepatitis. However, IFN- and the other Type I IFNs are also pleiotropic immune regulators. Type I IFNs can promote IFN-
production by activating STAT4 but can also inhibit production of IL-12, a potent activator of STAT4 and IFN- production. The efficacy of IFN- in the treatment of hepatitis C may therefore depend in part on the balance of IFN--inducing and IL-12-suppressing effects. We characterized the effects of pegylated IFN- therapy for hepatitis C on the capacity of patients’ PBMC to produce IL-12 and IFN- ex vivo. Cells from patients with a sustained virological response to therapy had significantly greater levels of IFN--driven IFN- production prior to treatment than those from nonresponding patients. No differences in pretreatment IL-12 productive capacity were seen between patient groups. However, therapy with IFN- led to suppression of inducible IL-12 production throughout the course of therapy in both groups of patients.
Key Words: immunomodulation • cytokine • memory T cell • IFN-ß
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IFN- forms the mainstay of therapy for HCV infection. Although the efficacy of IFN- in acute HCV infection is high [3
], only 10–30% of chronically infected adults have a sustained, virological response to IFN- alone, a rate that is increased by pegylation of IFN- and/or by concomitant therapy with the ribonucleoside analog ribavirin [4
5
6
7
8
]. The antiviral activities of the Type I IFN superfamily of genes formed the rationale for their therapeutic use in viral hepatitis. In addition to antiviral activities, however, these cytokines are pleiotropic immune regulators [9
, 10
]. The complicated immunoregulatory functions of the Type I IFNs are likely to be important determinants of their therapeutic efficacy. Positive and negative effects on the IFN-/IL-12 axis may be of particular importance.
IFN- is a key effector cytokine secreted by the innate (NK cell, NKT cell, /
T cell) and adaptive (CD4+ T cell, CD8+ T cell) immune systems. In turn, IL-12, secreted by APCs after stimulation by microbial and T cell-related stimuli, is a central regulator of cell-mediated immune responses, largely through its ability to drive IFN- secretion. IL-12 is a potent inducer of IFN- from T and NK cells and is necessary for the development of Th1 responses in most systems [11
]. Of note, Type I IFNs can also up-regulate IFN- production: augmenting NK cell IFN- production, promoting IL-12-independent CD8+ T cell production of IFN-, and substituting for IL-12 in some models of the polarization of naive human CD4+ T cells into a Th1 phenotype [12
13
14
15
16
17
18
19
20
]. Conversely, type I IFNs can also negatively regulate IFN- production by NK and T cells, at least during viral infection [21
22
23
]. The effects of type I IFNs on IL-12 production are marked by similar complexity. Although autocrine and/or paracrine IFN-ß is necessary for TLR-driven IL-12 production [24
], potent IL-12 inhibition by type I IFNs has been demonstrated in vivo in virus-infected mice and in patients receiving IFN-ß for multiple sclerosis [25
, 26
].
In acute HCV infection, production of type 1 cytokines by peripheral blood CD4+ T cells in response to viral antigens appears to correlate with self-limited infection, detectable type 2 cytokine responses seen with the development of chronic infection [27
, 28
]. Similarly, chronic HCV infection is marked by elevated type 2 and suppressed type 1 cytokine responses in PBMC [28
29
30
31
32
]. Importantly, increased peripheral type 1 cytokine responses appear to correlate with responsiveness to IFN- therapy [29
, 33
34
35
36
37
38
], and type 2 cytokine responses decrease in parallel with viral load during successful therapy [31
, 39
]. Taken together, these data have suggested an important role for IFN- in successful responses to therapy, something underscored by the fact that control of HCV infection in chimpanzees correlates with the appearance of IFN--producing, intrahepatic CD4+ and CD8+ T cells [40
]. As for IL-12, the immunological phenotype of HCV infection noted above has suggested that it has an important role in successful viral clearance. Indeed, although no baseline defects in stimulatable IL-12 production have been reported in patients with hepatitis C, maintenance of IL-12 production during the course of IFN- therapy appears to correlate with successful virological responses [41
, 42
]. Given the likely importance of the IFN-/IL-12 axis to the outcome of HCV infection, the ability of IFN- to modulate this axis is of particular interest. Augmentation of IFN- production would be expected to be beneficial; suppression of IL-12 production may well hinder successful therapy.
To characterize the effects of therapeutic IFN- on the IL-12/IFN- axis in patients with hepatitis C, we measured the stimulated production of these cytokines by PBMC from patients beginning therapy with IFN-. Notably, pretreatment IFN--driven IFN- production was significantly greater in patients who had a sustained, virological response to therapy than in nonresponding patients. Although the effects of type I IFNs on naive CD4+ T cells have been characterized extensively, effects on memory responses are likely to be more important during the therapy of chronic diseases such as hepatitis C. We demonstrate here that IFN- and IFN-ß augment IFN- secretion by memory CD4+ T cells. We further show that like IL-12, type I IFNs can activate STAT4 in memory as well as naive CD4+ T cells. As for IL-12 itself, no differences in IL-12 production were seen between patient groups. However, therapy with IFN- led to significant, sustained suppression of IL-12 production in both patient groups—suppression that reversed when therapy was stopped.
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Longitudinal peripheral blood samples were also obtained from 11 (out of 14) children, 2–8 years old, with chronic HCV genotype 1 infection enrolled in a pilot, multicenter, open label study of pegylated IFN--2a (40 KD) (PEG-IFN; Roche Pharmaceuticals, Nutley, NJ, USA) [43
]. Major inclusion criteria included age between 2 and 8 years, serologic evidence of chronic HCV infection along with serum HCV-RNA 
600 IU/ml, and compensated liver disease without evidence of cirrhosis (consistent with chronic HCV infection by liver biopsy performed within 15 months of enrollment). Major exclusion criteria included: previous IFN or ribavirin therapy, any recent investigational drug or systemic antiviral therapy, evidence of coinfection with HAV, HBV, or HIV, history of another cause for chronic liver disease, history of immune-mediated disease, or other evidence of severe illness [43
]. All patients received a weekly s.c. injection of PEG-IFN for 48 weeks (starting dose: 180 µg/1.73 m2 body surface area). For adverse events, the dose was reduced or discontinued, per protocol, according to adverse reaction severity. With resolution, a return to initial dosing was permitted, unless the patient had received the reduced dose for more than 4 weeks. Six patients had undetectable (<50 IU/ml) HCV RNA at Week 72 and were considered to have a sustained, virological response (responders); five patients had persistent viremia at Week 72 (nonresponders). Baseline characteristics were similar between responders and nonresponders, except that responders were significantly younger (Table 1
). Responders also tended to have higher serum ALT concentrations than nonresponders. More responders than nonresponders had genotype 1b, thought to be associated with lower responses to IFN- therapy [44
], although this trend lacked significance. Adverse events and PEG-IFN dose were similar between patient groups. Serial blood samples were collected at Weeks 0 (baseline), 4, 8, 24, and 48 of treatment and 24 weeks after discontinuation of treatment (Week 72), for quantitation of HCV-RNA levels (Cobas Amplicor HCV test, Version 2.0, Roche Laboratories) and for harvest of PBMC. Blood samples were shipped overnight to Johns Hopkins for PBMC isolation; blood samples drawn at Johns Hopkins were similarly held at room temperature overnight before PBMC isolation. Written, informed consent was obtained from the parents of all patients. The study was approved by the institutional review boards of all involved institutions.
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Table 1. Patient Characteristics
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Purified memory CD4+ T cells (CD4+/CD45RO+, negative for the noted activation antigens) were isolated from normal volunteers by modifications of a protocol reported previously [45 ]. Briefly, PBMC, depleted of monocytes byadherence to plastic, were incubated with mAb reactive with CD8, CD14, CD19, CD16, and CD25 (all from Caltag, Burlingame, CA, USA), CD69 (from Becton Dickinson, San Jose, CA, USA), and HLA-DR and CD45RA (both from PharMingen, San Diego, CA, USA). After negative selection with magnetic beads (goat antimouse IgG, Dynal, Oslo, Norway), CD4+/CD45RO+ cells were FACS-sorted from the remaining cell population. FITC-conjugated mAb to CD4 was from Becton Dickinson; PE-conjugated mAb to CD45RO was from PharMingen. Purity was consistently >98%. Resting memory CD4+ T cells were plated at a density of 1 x 106 cells/ml in 96-well plates in the media noted above.
Reagents
Staphylococcus aureus Cowan 1 strain (SAC) was from Calbiochem (San Diego, CA, USA). Recombinant human CD40 ligand (rhu-CD40L) trimer was a gift from Immunex/Amgen (Seattle, WA, USA/Thousand Oaks, CA, USA). rhu-IFN- and mAb, reactive with CD3 and CD28, were from PharMingen. PMA was from Sigma Chemical Co. (St. Louis, MO, USA). rhu-IFN-2b [1.8–3.0x108 IU/mg (antiviral activity)] was from Schering (Kenilworth, NJ, USA), and rhu-IFN-ß (1.7x106–2x107 IU/mg) was from Peprotech (Rocky Hill, NJ, USA). We chose to standardize type I r-IFN preparations by mass units instead of antiviral units, as the antiviral activity was reported in a range that varied by up to a log, and we have found a better correlation of mass units (than antiviral units) with effects on cytokine production. rhu-IL-12p70 was from Wyeth (Madison, NJ, USA). Neutralizing antibody for IL-12p70 was from R&D Systems (Minneapolis, MN, USA); that for IL-18 was from Peprotech.
Cytokine assays
Cell-free supernatants were stored at –70°C until assayed. Cytokine concentrations were measured by ELISA: IL-12p40, IFN-, and IL-4 (PharMingen) and IL-12p70 (R&D Systems). All cytokine assays had a sensitivity of 1–10 pg/ml.
Western blot analysis
Lymphocytes were solubilized in a buffer consisting of 1% Triton X-100 (Pierce Chemical Co., Rockford, IL, USA), 0.1% SDS, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 µg/ml aprotinin, 3 µg/ml antipain, 1 mM benzamidine, 1 mM DTT, 1 mM PMSF, 5 mM glycerophosphate, 1 mM orthovanadate, and 1 mM NaF (all from Sigma Chemical Co.), and 3 µg/ml leupeptin and 3 µg/ml pepstatin A (both from Boehringer Mannheim, Germany). Cell lysate proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). After blocking in 5% nonfat dry milk, membranes were probed with affinity-purified, antiphospho-STAT4 antibodies (Zymed, San Francisco, CA, USA), followed by an HRP-conjugated, antirabbit IgG polyclonal antibody (Amersham, Piscataway, NJ, USA), and developed using ECL (Amersham). The blots were subsequently stripped and reprobed using affinity-purified STAT4 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Statistical analysis
Comparisons between stimulation conditions in PBMC from healthy donors were analyzed using the paired t-test. Associations between baseline characteristics and outcome group in the longitudinal study were examined using t-tests and 
2 tests. In the longitudinal study, as a result of the repeated measurements of children (baseline and Weeks 4, 8, 24, 48, and 72), the data were analyzed using mixed models [46
], which also enabled appropriate adjustments for missing data at some time-points [47
]. Cytokine production at Weeks 4, 8, 24, 48, and 72 was compared with that at baseline. Significant differences and estimates with 95% confidence intervals computed via bootstrapping [48
] are displayed in the figures. For all variables except IFN- production, data for responders and nonresponders were combined, as preliminary analyses indicated that these groups did not differ. Statistical analyses were conducted using SAS Version 9.1 (SAS Institute, Cary, NC, USA). The criterion of statistical significance was P < 0.05.
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production by memory CD4+ T cells production from naive CD4+ lymphocytes is well-described: IFN-/ß can substitute for IL-12 in some models of Th1 polarization [15
16
17
, 49
]. During type I IFN therapy of ongoing disease processes, however, effects on IFN- production by memory CT4+ T cells are likely to be at least as important. We therefore characterized the effects of IFN-/ß on human CD4+ memory T cells. Activated CD4+ T cells and resting CD4+ memory T cells express the RO isoform of CD45, widely used as marker for memory T cells [45
]. Our purification procedures thus involved negative selection for a variety of activation antigens (CD25, CD69, HLA-DR) to assure purification of memory, as opposed to recently activated, naive T cells. As the blood volume required for purification of adequate numbers of CD4+ T memory cells (
150 ml) precluded performance of these studies in children, healthy adult volunteers were used.
We first characterized IFN-/ß-mediated effects on IFN- production during initial stimulation. IFN- production, after submaximal mitogen stimulation with PMA + anti-CD28, was increased markedly by IFN- (mean fold increase in IFN- secretion=2.6; P<0.02; n=4) and IFN-ß (mean fold increase=3.2; P<0.02; n=4; Fig. 1
A). Similarly, IFN- secretion, after submaximal stimulation with antibodies to CD3 and CD28, was augmented by the inclusion of IFN- (mean fold increase=2.7; P=0.006; n=5) and IFN-ß (mean fold increase=2.8; P<0.003; n=4; Fig. 1B
). Effects on IL-4 production in the latter conditions were variable, lacking statistical significance (not shown).
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Figure 1. Type I IFN-driven IFN- production by memory CD4+ T cells. Resting memory CD4+ T cells were isolated from normal volunteers and stimulated with (A) PMA (10 ng/ml) + anti-CD28 (1 µg/ml) or (B) anti-CD28 + plate-bound anti-CD3 (5 µg/ml), in the absence (O) or presence of IFN- (5 ng/ml) or IFN-ß (10 ng/ml). IFN- was quantified in supernatants harvested after 24 h. Data shown are means (+SD) of triplicate wells from individual donors; individual bars represent individual donors. ND = Not done.
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production induced by type I IFNs during the initial stimulation of memory CD4+ T cells led to stable hyperpolarization toward a Th1 phenotype. After initial stimulation, as described above, memory cells were washed and replated at the initial density (1x106 viable cells/ml). Despite the reported effects of type I IFNs on T cell proliferation and apoptosis [50
, 51
], we found no significant differences in cell numbers or viability between the different conditions of primary stimulation. Secondary stimulation was carried out with antibodies to CD3 and CD28 alone. As shown in Figure 2
A, only the presence of IL-12 during initial stimulation led to a stable increase in IFN- production by purified memory CD4+ T cells during secondary stimulation. A trend toward an increase in IL-4 production after secondary stimulation was seen in purified memory CD4+ T cells that received costimulation with IL-12 during primary in vitro stimulation (Fig. 2B)
; however, this increase did not reach statistical significance.
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Figure 2. Failure of Type I IFNs to stably hyperpolarize the cytokine responses of memory CD4+ T cells. Resting memory CD4+ T cells, isolated from normal volunteers, were stimulated with anti-CD3 + anti-CD28 in the absence or presence of IFN-, IFN-ß, or IL-12 (10 ng/ml) as indicated. After 72 h, cells were washed, counted, and restimulated with anti-CD3 + anti-CD28 alone. Twenty-four hours later, cell-free supernatants were harvested. Data represent the means (+SE) of five (IFN-ß) to six (IFN-, IL-12) donors. *, P < 0.01. NS, Not significant.
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/ß to promote Th1 development in humans but not mice is thought to be a result of the fact that type I IFNs, such as IL-12, can induce STAT4 phos-phorylation in naive human T cells but not murine T cells [54
55
56
]. Among T cells, type I IFNs have been shown to induce STAT4 phosphorylation and/or transactivation in PHA-activated, bulk PBMC and T cells [54
, 57
], as well as naive cord blood CD4+ T cells and differentiated lines derived from such cells [55
]. Given our observation of the transient augmentation of Th1 responses by IFN-/ß, we assessed adult, memory CD4+ T cells. As shown in Figure 3
, IFN-, IFN-ß, and IL-12 stimulate robust phosphorylation of STAT4 in these cells.
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Figure 3. Type I IFN signaling through STAT4 in memory CD4+ T cells. Resting memory CD4+ T cells were stimulated with anti-CD3 for 40 h, followed by media control (Lane 1), IFN- (Lane 2), IFN-ß (Lane3), or IL-12 (Lane 4) for 20 min. Western transfers of SDS-PAGE-separated lysates were probed with antibodies to phospho-STAT4 or STAT4, as indicated. The cross-reactive, extra band in Lanes 2 and 3 is phospho-STAT1. Data shown are derived from the lymphocytes of a single donor, representative of n = 4.
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production by PBMC in an IL-12-independent manner/ß on IFN- production by bulk PBMC. As shown in Figure 4
, Type I IFNs significantly up-regulated the amount of IFN- secreted by the PBMC of normal adult donors under conditions of submaximal mitogen stimulation. The mean increase in IFN- production effected by IFN- was 3.4-fold (n=10; P=0.01); that due to IFN-ß, was 2.9-fold (n=10; P=0.04). Similar IFN--driven effects have been reported before with Mycobacterium bovis and influenza virus infection [58
, 59
]. FACS analysis indicated that CD3+ cells were responsible for most, if not all, of the IFN- production in these cultures (data not shown). Augmentation of IFN- production by Type I IFNs was IL-12-independent, as the presence of neutralizing antibodies to IL-12 failed to alter the observed increase in IFN- production (Fig. 4)
. Similarly, neutralizing antibodies to IL-18 failed to inhibit IFN-/ß-induced IFN- secretion (data not shown; n=2).
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Figure 4. IL-12-independent up-regulation of PBMC IFN- production by IFN-/ß. PBMC were stimulated for 44 h with PMA + anti-CD28. Parallel stimulations were done with the inclusion of IFN- (5 ng/ml) or IFN-ß (10 ng/ml) in the absence or presence of neutralizing antibody to IL-12 p70 (1 µg/ml), as noted. (A) Mean fold increase (+SE) in IFN- secretion induced by Type I IFN inclusion in 10 normal volunteers (three wells/data point). All comparisons (mitogen vs. mitogen+Type I IFN, with or without neutralizing antibodies to IL-12) were significant (P<0.05; paired t-test). na-IL-12, Neutralizing anti-IL-12. (B) Mean IFN- production (+SE) in response to PMA + anti-CD28 in these donors.
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-driven IFN- production is associated with sustained hepatitis C virus clearance in children treated with PEG-IFN-driven IFN- production in children with chronic hepatitis C enrolled in a multicenter study of PEG-IFN. Children were studied as, in contrast to adults, IFN- monotherapy was standard treatment for children at the time this study was performed. PBMC, isolated from responders at baseline (prior to the onset of therapy), exhibited significantly greater IFN--mediated augmentation of IFN- production than did PBMC isolated at baseline from nonresponders (Fig. 5
). No significant correlations were found between baseline plasma viral load and response to therapy or IFN--driven IFN- production (data not shown). In responders, the magnitude of IFN--driven IFN- production decreased significantly (to the level exhibited by nonresponders) with the onset of therapy.
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Figure 5. Greater pretreatment IFN--augmented IFN- production in patients who respond to IFN- therapy for chronic hepatitis C. PBMC were stimulated for 48 h with PMA and anti-CD28, in the presence or absence of IFN-. (A) Mean (±95% confidence intervals) fold increase in IFN- secretion induced by IFN- stimulation in responders ( , n=6) and nonresponders (•, n=5). Vertical, dotted line, End of therapy. Pretreatment samples were available from all 11 patients; follow-up sample numbers varied from six to 10. *, P < 0.01; **, P < 0.05. (B) Pretreatment mean fold increase in IFN- secretion (+SE) induced by IFN- stimulation in responders (R) and nonresponders (NR).
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therapy on stimulatable IL-12 production, which primes for peak IL-12 secretion by PBMC. No differences in IL-12 productive capacity were seen between patient groups (data not shown), a finding that may be a result of the small numbers of patients in the study. However, as reported previously for patients with multiple sclerosis beginning therapy with IFN-ß, systemic therapy with PEG-IFN led to significant, sustained suppression of IL-12 production by PBMC (Fig. 6
). These data raise several points. First, as seen with IFN-ß therapy [26
], there were concordant effects on the secretion of the functional IL-12p70 heterodimer and the IL-12p40 subunit, consistent with the fact that high doses of exogenous Type I IFNs lead to transcription inhibition of IL-12p40 [60
]. Second, IFN- therapy led to suppression of both SAC- and CD40L-driven IL-12 production. Finally, IL-12 suppression abated with the end of therapy.
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Figure 6. IL-12 secretion is suppressed by IFN- therapy in patients with chronic hepatitis C. PBMC, isolated from patients before, during, and after therapy with IFN-2a, were stimulated with IFN- (300 IU/ml) plus SAC (0.01% wt/vol; A and B) or rhu-CD40L trimer (1 µg/ml; C and D). Data shown represent mean IL-12 concentrations ± 95% confidence intervals. Vertical, dotted line, End of therapy. *, P < 0.05; **, P < 0.01, compared with baseline (Week 0).
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/ß on the IFN-/IL-12 axis in humans. Extending previous studies of naive human CD4+ T cells, we demonstrate that IFN-/ß drives IFN- secretion by memory human CD4+ T cells. The likely biological relevance of such IFN- amplification of IFN- responses is underscored by the finding that hepatitis C patients with a sustained virological response to IFN- therapy were marked by greater IFN--driven IFN- production by PBMC prior to therapy. However, in addition to driving secretion of IFN-, the paradigmatic effector cytokine of cell-mediated immunity, IFN- can inhibit secretion of IL-12, the central regulatory cytokine of this arm of the immune system. Notably, we demonstrate that IFN- therapy leads to significant suppression of the IL-12 productive capacity of PBMC.
IL-12 plays a central role in protection from diverse intracellular pathogens [11
]. As might therefore be expected, IL-12 has, itself, been shown to be targeted by various viruses, including HIV, measles, and HCV [61
62
63
64
65
66
]. Cell-mediated, immune responses have clear potential for causing injury to the host. IL-12 production is thus kept under tight inhibitory control by the host under normal conditions [11
]. IFN-/ß was first shown to inhibit IL-12 production by mouse splenic leukocytes in the context of acute infection with lymphocytic choriomeningitis virus [25
]. Since then, the effects of Type I IFNs on IL-12 have been shown to be quite complex, involving up- and down-regulation, depending on Type I IFN source (autocrine/paracrine vs. systemic or exogenous), dose and timing, and IL-12-producing cell type (immature vs. mature dendritic cells; monocyte/macrophages) [9
, 12
, 24
, 26
, 60
, 67
68
69
70
71
72
73
74
]. On the one hand, TLR-driven induction of IL-12 depends on autocrine/paracrine production of IFN-ß, which acts to up-regulate IL-12p35 mRNA expression [24
]. On the other hand, high, sustained doses of exogenous Type I IFNs inhibit the transcription of IL-12p40 [60
]. It will be noted that this biphasic pattern of regulatory effects is reminiscent of that seen with another innate inflammatory mediator, the anaphylatoxin C5a: Although modest signaling through the C5a receptor is permissive for IL-12 production, high concentrations of C5a inhibit IL-12 production [75
76
77
]. Given this complexity, what is the overall effect of IFN- therapy on IL-12 production? The data presented herein clearly demonstrate that, at least in the accessible peripheral blood compartment, such therapy leads to sustained inhibition of IL-12 productive capacity. Effects on the related cytokine, IL-23, remain to be determined.
Type I IFNs play a similarly complex role in regulating secretion of the principal effector cytokine downstream of IL-12—IFN-. Although numerous studies have shown that IFN-/ß can enhance IFN- secretion by diverse innate and adaptive immune cells [12
13
14
15
16
17
18
19
20
, 58
, 59
, 78
, 79
], studies by Biron and colleagues [21
22
23
] have revealed that, at least in CD8+ T cells and NK cells, IFN-/ß can also suppress IFN- secretion. The outcome (enhancement vs. suppression) depends on variable use of STAT proteins in the signaling cascade downstream of the Type I IFN receptor, something regulated by changes in STAT protein expression and accessibility [21
22
23
]. In the CD4+ T cell compartment, thought to be essential for successful responses to HCV infection and therapy, the ability of IFN-/ß to drive Th1 polarization (and hence, IFN- production) of naive CD4+ T cells in the absence of IL-12 has been of considerable interest [15
, 17
, 49
, 55
]. Our data clearly show that IFN-/ß can also enhance IFN- from memory CD4+ T cells, a finding of likely relevance to IFN- therapy. IL-12 acts via the STAT4 signaling pathway. The ability of Type I IFNs to mimic IL-12 in Th1 development is thought to be a result of the fact that IFN-/ß can also signal through STAT4 in humans [55
]. We show here for the first time that IFN-/ß induces STAT4 phosphorylation in memory CD4+ cells, a significant point, given stage-specific regulation of STAT protein availability in T cells [21
22
23
]. It is interesting that we found that, unlike IL-12, Type I IFNs fail to drive sustained increases in IFN- production by memory CD4+ T cells. This may well be a result of differing kinetics of STAT4 activation by IL-12 and IFN-/ß [80
].
In general, enhanced IFN- production would be predicted to favor HCV clearance. Our finding that the magnitude of pretreatment of IFN--driven IFN- responses correlates with sustained responses to therapy is certainly consistent with this prediction. Intriguingly, functional genomic analysis of unstimulated PBMC from adult patients with chronic HCV undergoing therapy with IFN- and ribavirin has similarly linked a lack of IFN--mediated induction of IFN-stimulated genes (along with baseline-increased expression of such genes) with resistance to such therapy [81
, 82
]. Although we examined the role of IFN- in amplifying IFN- productive capacity in response to mitogenic stimulation, previous studies have looked directly at the effect of IFN- therapy on HCV protein-specific CD4+ T cell proliferation and IFN- production. Notably, in both acute and chronic HCV infection, successful IFN- therapy is associated with the development of robust, multispecific Th1 responses to HCV proteins [35
36
37
38
], findings that are mirrored by the data presented here.
On the other hand, although augmentation of IFN- production is likely beneficial, suppression of IL-12 production would be predicted to hinder HCV clearance, a prediction that has received circumstantial support by recent genetic studies [83
84
85
]. Taken together, the positive and negative immunoregulatory effects of IFN- on IFN- and IL-12 suggest that patients with chronic hepatitis C may benefit from adjunctive targeting of the IFN-/IL-12 axis. Combination therapy which includes IFN- [86
] or IL-12 [87
, 88
], or compounds with similar activities (a relevant, suggested mode of action for ribavirin [35
, 89
90
91
92
93
]), comes to mind. That said, caution is clearly in order. Compartmentalization of the immune response occurs with HCV infection [94
]. Although successful responses to HCV infection appear to correlate with increased Type 1 cytokine responses by peripheral CD4+ T cells, analysis of intrahepatic CD4+ T cell clones and mRNA expression strongly suggests that Type 1 cytokine responses are also associated with the development of hepatic pathology in the absence of HCV clearance [95
96
97
98
].
Increased mechanistic understanding of the complex activities of the Type I IFNs should allow for more rational, therapeutic exploitation of their antiviral and immunoregulatory properties.
2 Current address: Technical Resources International Inc., 6500 Rock Spring Dr., #650, Bethesda, MD 20817, USA.
3 Current address: Children’s Mercy Hospital, 2401 Gillham Rd., #2155, Kansas City, MO 64108, USA.
Received October 9, 2006; revised November 1, 2006; accepted November 8, 2006.
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