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Potential conflict of interest: Nothing to report.
Supported by grant We-4675/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany (to J.M.W.) and by the NIDDK NIH intramural research program.
Hepatitis C virus (HCV) infection typically results in chronic disease with HCV outpacing antiviral immune responses. Here we asked whether innate immune responses are induced in healthcare workers who are exposed to small amounts of HCV, but do not develop systemic infection and acute liver disease. Twelve healthcare workers with accidental percutaneous exposure to HCV-infected blood were prospectively studied for up to 6 months for phenotype and function of natural killer T (NKT) and NK cells, kinetics of serum chemokines, and vigor and specificity of HCV-specific T-cell responses. Eleven healthcare workers tested negative for HCV RNA and HCV antibodies. All but one of these aviremic cases displayed NKT cell activation, increased serum chemokines levels, and NK cell responses with increased CD122, NKp44, NKp46, and NKG2A expression, cytotoxicity (as determined by TRAIL and CD107a expression), and interferon-gamma (IFN-γ) production. This multifunctional NK cell response appeared a month earlier than in the one healthcare worker who developed high-level viremia, and it differed from the impaired IFN-γ production, which is typical for NK cells in chronic HCV infection. The magnitude of NKT cell activation and NK cell cytotoxicity correlated with the magnitude of the subsequent HCV-specific T-cell response. T-cell responses targeted nonstructural HCV sequences that require translation of viral RNA, which suggests that transient or locally contained HCV replication occurred without detectable systemic viremia. Conclusion: Exposure to small amounts of HCV induces innate immune responses, which correlate with the subsequent HCV-specific T-cell response and may contribute to antiviral immunity. (Hepatology 2013;58:1621–1631)
Hepatitis C virus (HCV) causes chronic hepatitis in more than 80% of infected subjects. The search for protective immune responses has focused on the ∼20% of patients who spontaneously clear HCV after acute symptomatic infection with high-level viremia and increased liver enzymes. These studies have shown that vigorous CD4 and CD8 T-cell responses correlate with HCV clearance (reviewed) and can mediate protection upon reinfection.[2, 3] In contrast, antibodies do not appear to be required, as evidenced by hypogammaglobulinemic patients who clear HCV.
The role of innate immune cells has not been studied, likely because these cells respond much earlier than T cells, and because blood samples immediately after exposure to HCV are difficult to obtain. Innate immune cells such as natural killer T (NKT) cells and natural killer (NK) cells constitute major cell populations in the liver, and have the capacity to respond rapidly to chemokines and/or to altered cell surface marker expression on infected cells. They may exert direct antiviral effector functions and help priming and modulating the adaptive immune response.[5, 6]
NKT cells are defined by a restricted T-cell receptor repertoire, which in humans consists of the T-cell receptor (TCR) chains Vα24-Ja18 and Vβ11 with a conserved CDR3 region. This invariant TCR recognizes glycolipids that are presented by CD1d, a major histocompatability complex (MHC) class I-like molecule that is up-regulated on hepatocytes in chronic HCV infection. To date, NKT cell responses have not been studied in acute HCV infection.
NK cells are CD3-CD56+ lymphocytes that are controlled by the integration of signals from activating and inhibitory cell surface receptors. These include killer cell immunoglobulin-like receptors (KIRs), lectin-like receptors (NKG2A-F), and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). NKG2C, for example, recognizes the nonclassical MHC I molecule HLA-E, the expression of which is altered in HCV infection, and NKG2D recognizes MICA/B molecules, which are induced in HCV infection. NK cell activation can also be mediated by inflammatory cytokines such as type I interferons and interleukin (IL)-12 that are commonly released in response to viral infections. NK cells are activated during acute hepatitis 8-14 weeks after infection when liver enzymes and viremia reach high levels,[12, 13] but they have not been studied in the very first weeks after exposure when their role as rapid effectors would be most relevant.
Materials and Methods
Twelve healthcare workers were studied prospectively after occupational HCV exposure for HCV RNA using the standard clinical assay at the NIH (Cobas Amplicor, HCV Test 2.0, Roche, Branchburg, NJ), HCV-specific antibodies (Abbott HCV EIA 2.0, Abbott, Princeton, NJ), serum cytokines, and NKT, NK, and T-cell responses. Eleven healthcare workers tested HCV RNA-nonreactive at the assay sensitivity of 100 IU/mL, whereas one developed high-level viremia and started PegIFN/ribavirin treatment 17 weeks after exposure. Peripheral blood mononuclear cells (PBMCs) of the cohort with undetectable HCV RNA were isolated from citrate dextrose-anticoagulated blood on the day of exposure (n = 5 subjects), 2 weeks (n = 11), 4 weeks (n = 11), 6 weeks (n = 11), 13 weeks (n = 10), and more than 24 weeks (n = 11) thereafter, and cryopreserved in liquid nitrogen using previously described techniques. PBMCs of the healthcare worker with high-level viremia were isolated 3, 5, 8, and 14 weeks after exposure. Twenty-nine healthy blood donors were studied as controls at a single timepoint. All gave written informed consent for research testing, according to protocols approved by the participating hospitals' Institutional Review Boards.
NKT Cell Analysis
PBMCs were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-PacificBlue (both from BD Biosciences, San Jose, CA), anti-CD14-PeCy5 (Serotec, Raleigh, NC), and with αGalCer-loaded, streptavidine-PE-conjugated CD1d-tetramers (NIAID Tetramer Facility of the NIH AIDS Research and Reference Reagent Program, Atlanta, GA) to identify NKT cells. Cells were additionally stained with anti-FasL-FITC (Abcam, Cambridge, MA) and anti-NKG2D-PeCy7 (BioLegend, San Diego, CA).
NK Cell Analysis
Frequency and Phenotype
PBMCs were stained with EMA, anti-CD14-PeCy5 (Serotec), anti-CD19-PeCy5, anti-CD3-AlexaFluor700, anti-CD56-PeCy7, and anti-CD16-PacificBlue (all from BD Biosciences) and with either anti-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-PE (BD Biosciences), anti-CD122-FITC, anti-NKp44-PE, anti-NKp46-PE, or anti-NKG2A-PE (all from Beckman Coulter, Brea, CA).
NK cell degranulation was quantitated as an increase in cell surface CD107a expression in response to MHC class I-negative K562 cells (ATCC, Manassas, VA).
PBMCs were cultured at 37°C with or without IL-12 (0.5 ng/mL; R&D Systems) and IL-15 (20 ng/mL R&D Systems) and assessed for interferon-gamma (IFN-γ) production by flow cytometry as described.
Stained cells were analyzed on an LSRII using FacsDiva Version 6.1.3 (BD Biosciences) and FlowJo v. 8.8.6 (Tree Star, Ashland, OR) software.
IFN-γ Enzyme-Linked Immunospot (ELISPOT)
PBMCs were stimulated with seven pools of overlapping 15-mer HCV genotype 1a peptides (1 μg/mL of each peptide) covering the core (38 peptides), NS3 (three pools with 42 peptides each), NS4A pool (12 peptides), and NS4B sequence (two pools with 26 peptides each), phytohemagglutinin (1 μg/mL PHA-M; Invitrogen, Carlsbad, CA), or dimethyl sulfoxide (DMSO) as described. The cutoff for a significant HCV-specific response (spots with antigen minus spots without antigen) was defined as (1) greater than the mean plus two standard deviations of the IFN-γ response of 29 NIH blood donors without a history of HCV infection, and (2) more than 2-fold above the DMSO background.
Serum samples were diluted 1:2 for IFN-α, IFN-γ, TNF-α, IL-10, IL-12(p70) quantitation, and 1:4 for CXCL10 (IP-10), CCL2 (MCP-1), CCL3 (MIP1-β), and CCL5 (RANTES) quantitation using CBA assays (BD Biosciences). Serum IL-18 levels were measured after 1:2 dilution using an enzyme-linked immunosorbent assay (ELISA) (eBioscience).
Nonparametric Wilcoxon matched pairs tests and linear regression analyses were performed with GraphPad Prism v. 5.0a (GraphPad Software, La Jolla, CA). A two-sided P-value of less than 0.05 was considered significant.
Clinical Outcome of Accidental HCV Exposure
The 12 studied healthcare workers were on average 37 ± 4 years old and the majority (83%) were female (Table 1). Ten healthcare workers were accidentally exposed to HCV by needlestick and two by a cutaneous cut. Eleven tested negative for HCV-RNA at the sensitivity level of 100 IU/mL of the standard clinical assay and for HCV antibodies at all study dates, whereas one developed high-level viremia and was successfully treated with PegIFN/ribavirin at week 17 after exposure. The following paragraphs describe first the immune response of the cohort of 11 subjects with undetectable viremia.
Table 1. Characteristics of Studied Healthcare Workers
aDetermined by qualitative RT-PCR (COBAS Amplicor HCV Test 2.0; Roche Diagnostics, Branchburg, NJ). at all study timepoints indicated in Fig. 1. Lower limit of detection 100 IU/mL (270 copies/mL) serum.
bDetermined by Abbott HCV EIA 2.0 (Abbott, Princeton, NJ) at all indicated study timepoints.
cn.a., not applicable.
dThe HCV titer of the source blood was 11.6 x 106 IU/mL (COBAS Ampliprep/COBAS Taqman HCV Test;
Roche Diagnostics, Branchburg, NJ).
Exposed healthcare workers without detectable viremia
NKT Cell Responses of HCV-Exposed Healthcare Workers Without Detectable Viremia
NKT cells were identified in PBMCs by multicolor flow cytometry. After gating on single cells and lymphocytes, and exclusion of CD14+ monocytes, CD19+ B cells, and EMA+ dead cells, NKT cells were identified as CD3+CD1d+ (Fig. 1A). For each subject NKT cell frequency and phenotype were compared in a paired analysis of each study timepoint and the last study timepoint, which was at least 24 weeks (range 24 to 37 weeks) after HCV exposure. The last timepoint was regarded as baseline because week 0 samples were not available from all subjects, because all NKT and NK cell assays yielded lowest values at the final study timepoint, and because none of the healthcare workers reported a second exposure within the study period.
Because NKT cell activation is known to result in apoptosis the expression of apoptosis-inducing transmembrane protein Fas ligand (FasL) was examined on NKT cells. In all but one healthcare worker the frequency of FasL-expressing NKT cells and the FasL expression level per cell (mean fluorescence intensity, MFI) were highest 2 weeks after HCV exposure (paired analysis, Fig. 1B; full time course, Supporting Fig. 1), the timepoint with the lowest mean NKT cell frequency of 0.03% in the lymphocyte gate. Conversely, peak expression of NKG2D at week 6 was associated with recovery of NKT cell frequency (P = 0.008 for the percentage of NKG2D+ NKT cells and P = 0.016 for the NKG2D MFI compared to baseline, respectively, Fig. 1C). Thus, exposure to HCV affected both the frequency and the activation status of peripheral blood NKT cells.
NK Cell Responses of HCV-Exposed Healthcare Workers Without Detectable Viremia
CD3-CD56+ NK cells were identified by flow cytometry after selection of single cells and lymphocytes, exclusion of CD14+ monocytes, CD19+ B cells and EMA+ dead cells, and staining for CD3, CD56, and CD16 (Fig. 2A). Whereas the percentage of circulating NK cells and their CD16+ and CD16− subsets were not altered after HCV exposure (data not shown) several changes in NK cell phenotype were observed. First, the expression of CD122, the subunit of the IL-2 receptor that signals in response to IL-2 and IL-15, was analyzed. In all but one healthcare worker without detectable viremia the frequency of CD122+ NK cells and the CD122 MFI peaked 2 weeks after HCV exposure (Supporting Fig. 2) and was significantly higher than baseline levels in a paired analysis (P = 0.008, Fig. 2B). Increased CD122 expression was followed by peak expression of the activating receptors NKp44 and NKp46 at week 4 (P = 0.039 and P = 0.023 for frequency and MFI of NKp44+ NK cells; P = 0.039 and P = 0.023 for frequency and MFI of NKp46+ NK cells, Fig. 2C,D). Expression of the inhibitory receptor NKG2A peaked later, i.e., at week 6 after HCV exposure (Fig. 2E), and decreased by week 24 (P = 0.023 and P = 0.016 for frequency and MFI of NKG2A+ NK cells). The decrease in NKG2A expression on NK cells in the absence of detectable viremia contrasts with the high NKG2A expression levels that have been reported in chronic HCV infection.
To assess how the observed changes in NK cell phenotype affected NK cell cytotoxicity we studied the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and NK cell degranulation in response to MHC-I negative target cells. As shown in Fig. 3A,B for a paired analysis between peak and baseline expression, there was a significant increase in TRAIL expression and NK cell degranulation at week 4 after HCV exposure in all but one subject (P = 0.039 and P = 0.023 for the percentage and MFI of TRAIL+ NK cells; P = 0.016 and P = 0.04 for the percentage and MFI of CD107a+ NK cells, respectively). This early response was followed by an increase in the percentage of IFN-γ+ NK cells, which peaked at week 6 (P = 0.039, Fig. 3C). The increase in the frequency of IFN-γ+ NK cells correlated with the increase in the frequency of TRAIL+ NK cells in a nonparametric Spearman correlation (rho = 0.81, P = 0.0154, Fig. 3D).
Serum Cytokines of HCV-Exposed Healthcare Workers Without Detectable Viremia
Serial serum samples were tested for IFN-α, IFN-γ, TNF-α, IL-10, IL-12, CCL2 (MCP-1), CCL3 (MIP1-β), CCL5 (RANTES), and CXCL10 (IP-10). Early increases were found for CCL3 (Fig. 4), CXCL10 (Fig. 5), and to a much lesser extent TNF-α (not shown). CCL3 serum levels (Fig. 4) peaked at week 2 after percutaneous exposure in four subjects (subjects 5, 7, 8, 11), at week 4 in four additional subjects (subjects 1, 2, 6, 9) and at week 7 in one subject (subject 4). The peak in this NK cell-recruiting chemokine was related to the peak in NK cell degranulation, TRAIL production, and IFN-γ secretion in most subjects (Fig. 4). In contrast, the increase in CXCL10 levels appeared delayed relative to the increase in CCL3 levels in subjects 2, 6, 7, and 9 (Fig. 5). The timing of CXCL10 responses did not coincide with T-cell responses, which tended to appear earlier (Fig. 5). Only a single healthcare worker tested negative for all chemokine, NKT/NK cell, and T-cell responses (Fig. 4, 5).
T-Cell Responses of HCV-Exposed Healthcare Workers Without Detectable Viremia
The mean T-cell response against both structural (HCV core) and nonstructural (NS3, NS4A, NS4B) HCV proteins peaked 6 weeks after HCV exposure and decreased significantly by week 24 (P = 0.008, Fig. 6A). Its magnitude correlated with the peak expression level of the activating receptor NKG2D on NKT cells (R2 = 0.77, P = 0.004, Fig. 6B) and to peak expression of the degranulation marker CD107a on NK cells (R2 = 0.64, P = 0.016, Fig. 4C), but not to the peak IFN-γ response of NK cells. In contrast, no increased response was observed against pools of EBV and HIV peptides, which were tested as controls.
Innate and Adaptive Immune Responses of an Exposed Healthcare Worker with High-Level Viremia
A single healthcare worker (subject 12, Table 1) developed high-level HCV viremia and was studied up to week 17 after infection, when PegIFN/ribavirin therapy started (Fig. 7A). As shown in Fig. 7A-C, the frequency and MFI of FasL-expressing NKT cells peaked when the frequency of CD1d+ NKT cells in the blood was lowest, which occurred several weeks later than in the healthcare workers with undetectable HCV RNA. Likewise, CD122, NKp44, NKp46, and NKG2A expression on NK cells peaked later (≤week 8), consistent with a later peak in NK cell degranulation, TRAIL, and IFN-γ production (Fig. 7D,E). T-cell responses against HCV core and HCV nonstructural antigens remained undetectable until week 8 but were about 10-fold more vigorous than in healthcare workers with undetectable viremia (Fig. 7F).
Although one HCV virion may suffice to establish HCV transmission and viremia, less than 1% of healthcare workers who are accidentally exposed to low amounts of HCV develop high-level systemic viremia. This may be due to either absence of HCV transmission or to early immune responses that rapidly contain and clear small amounts of transmitted HCV. Here, we demonstrate that even a brief exposure to HCV that did not result in systemic viremia triggered responses of NKT/NK cells, chemokines, and T cells in all but one of the prospectively followed healthcare workers in this study. In contrast, HCV-specific antibodies were not induced in the absence of detectable viremia, consistent with the notion that they require high levels of persisting HCV antigen. Because nonstructural HCV proteins are not part of the viral particle, the detection of T-cell responses against HCV NS3, NS4A, and NS4B peptides suggests transient and anatomically contained HCV RNA translation and/or replication in healthcare workers below the sensitivity of the clinical assay.
The magnitude of the HCV-specific T-cell response correlated with peak NKG2D expression on CD1d+ NKT cells. This is in line with a previous report that NKT cell activation promotes the proliferation of hepatitis B virus-specific CD8 T cells, and that blockade of the NKT-cell-expressed NKG2D molecule prevents T-cell-induced hepatitis in a transgenic mouse model. While little is known of the role of NKT cells in acute HCV infection, NK cells have been a focus of research in recent years. In accordance with many of these studies[15, 25] we observed changes in the expression level of multiple NK markers. Among those, the transient up-regulation of the inhibitory receptor NKG2A on NK cells of HCV-exposed healthcare workers without detectable viremia is of note, because it has been shown to correlate inversely with HCV RNA levels in chronic HCV infection.[25, 26] Furthermore, as in chronic HCV infection,[15, 27] NK cells displayed an activated and cytotoxic phenotype as determined by increased TRAIL expression and NK cell degranulation in response to MHC class I-negative target cells. TRAIL-mediated cytotoxicity appears to be a relevant antiviral mechanism because in vitro activated NK cells have been shown to kill HCV-infected hepatoma cells in a TRAIL-dependent manner, and because HCV infection increases the sensitivity of primary human hepatocytes to TRAIL-mediated killing. Increased IFN-γ production by NK cells has also been described in acute HCV infection[12, 13] but is decreased in chronic HCV infection.[15, 27] This is reminiscent of a mouse model of infection with a hepatotropic virus (lymphocytic choriomeningitis virus) where NK cells produce IFN-γ immediately after infection but lose this capacity when high-level viremia persists. Thus, IFN-γ production may be an important component of an early NK cell response to HCV exposure.
Only a single healthcare worker developed high-level systemic infection and was studied up to week 17, when PegIFN/ribavirin therapy was initiated. Overall, NK/NKT cell responses appeared later (peak week 8) than in the subjects without detectable viremia (peak week 4), and NK cell degranulation and IFN-γ production were weaker.
A limitation of our study is the absence of a negative control group of healthcare workers exposed to HCV-negative blood. However, one exposed healthcare worker without detectable viremia tested negative for cytokine, NKT, NK, and T-cell responses in all assays, which suggests that the needlestick injury was too small to transmit HCV. Conversely, the increase in T-cell responses to HCV, but not to Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV) in the other healthcare workers, and the correlation between NK/NKT cell responses and T-cell responses supports the notion that the observed immune reactions were due to HCV exposure.
While it is possible that innate and adaptive immune responses of the studied healthcare workers are each individually related to a third factor, such as the exposure type or the amount of antigen encountered, they may also support each other. For example, IL-2 from HCV-specific T cells may have contributed to the NK cell response because it occurred earlier (weeks 2-4 after exposure) than in patients who develop high-level systemic viremia (weeks 8-12 after infection). This may indicate memory T-cell responses and would be consistent with the notion that many healthcare workers indicated past HCV exposures. Vice versa, NK cell responses may support T-cell responses by way of their effect on antigen-presenting cells. NK cells preferentially kill immature dendritic cells (DCs)[31, 32] because mature DCs are protected by high MHC class I surface expression. This may result in a relative increase of mature over immature DCs and promote T-cell priming. Furthermore, under conditions where DCs are suboptimally activated by type I IFN, NK cells may license DCs to prime T-cell responses.
In conclusion, these results suggest that low-dose exposure to HCV activates innate and adaptive cellular immune responses, which may contribute to the prevention of high-level systemic viremia and acute liver disease. The multifunctional NK cell response (cytotoxicity and IFN-γ production) in these HCV-exposed healthcare workers differed from the impaired NK cell response (increase in cytotoxicity and decrease in IFN-γ production) in chronic HCV infection.
We thank Dr. Yuji Sobao for performing several of the Elispot assays and the NIAID Tetramer Facility of the NIH AIDS Research and Reference Reagent Program for synthesis of CD1d tetramers.