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Keywords:

  • HCV;
  • IFN-α;
  • NK cells;
  • NKp30;
  • Treatment

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment-naïve patients who underwent treatment with pegylated IFN-α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56brightCD16+/− cell populations, increased CD56dullCD16+ NK cell proportions, and lower expression of NKp30, DNAM-1, and CD85j. Similarly, reduced NK cell IFN-γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56brightCD16+/− NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL-2 and IFN-α confirmed upregulation of NKp30 and also of NKp46 and DNAM-1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV-1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Chronic HCV infection may lead to cirrhosis, liver failure, and hepatocarcinoma in a proportion of patients 1 and accounts for significant disease burden worldwide. Persistent viraemia has been attributed to immunogenetic aspects of host defences that determine the outcome of virus/host interaction 2–5. Innate immune mechanisms are involved in the establishment chronic persistent replication of intracellular pathogens 6, 7. Their depletion in animal models results in impairment of virus-specific T-cell responses 8. Dysfunction of NK cells, in particular a reduction in activating receptor expression or function, occurs during HIV 9, 10, CMV 11, 12, and Mycobacterium tuberculosis hominis (Mth) 13, as well as during HCV, infection 14. Reports on the levels of natural cytotoxicity receptor (NCR) expression during chronic persistent HCV infection are inconsistent 15, 16.

Although direct exposure of NK cells to HCV in vitro does not substantially impair their function 17, acute HCV infection increases NKG2D expression on NK cells 18. NK cell infiltration of the liver occurs during chronic HCV infection 7, 19. During chronic infection, an imbalance occurs in the ratio of CD56dullCD16+ to CD56brightCD16+/− peripheral NK cell populations 15, 20. At the same time, NK cells are functionally inactivated by inhibitory receptor ligands on hepatocytes, by increased expression of inhibitory receptors such as NKG2A/CD94 on NK cells, and by production of immunomodulatory cytokines (e.g. IL-10) 14, 15. Circulating DC function is also attenuated during chronic HCV 21 similar to other persistent/latent infections 22–25. NK cell defects are thus likely to influence modulation/shaping of adaptive T-cell responses through DC editing 24, 26.

Successful viral clearance after acute HCV infection is associated with the carriage of KIR2DL3 in HLA-C1+ patients 2. The same HLA:KIR combination is associated with resistance to HCV infection 27. Decreased ligand-inhibitory receptor affinity results in reduced inhibition of natural cytotoxic functions induced by NK cell activating receptors 15, 28, 29. Inhibitory KIR:HLA haplotypes with a lower intrinsic inhibitory activity (such as KIR2DL3:HLAC1) might result in more efficient clearance of infected cells upon NK cell activating receptor:ligand interaction. This would also contribute to NKp30- and DNAM1-mediated DC maturation/editing 12, 30, resulting in optimal shaping of downstream adaptive responses 31.

The role of the innate immune response has been increasingly scrutinised in the treatment of chronically infected HCV patients with pegylated IFN-α and ribavirin (PEGIFNα+RBV). Involvement of immunogenetic parameters and of inhibitory NK receptors has been recently pinpointed to contribute to virus clearance. Increased frequency of HLA-C1:KIR2DL3 homozygosity is indeed detected among patients with sustained virological response (SVR) to combined treatment 32. To date, there is limited data available regarding NK-activating receptor expression in HCV-infected patients undergoing standard treatment 15, 16. For this reason, we undertook a prospective observational study of treatment-naïve patients infected with HCV genotype 1, in order to evaluate whether NK cell activating receptors, in particular NCR, could also be involved in generating SVR.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Analysis of peripheral NK cell subsets before IFN-α-RBV treatment

We first studied peripheral NK cell subsets as defined by expression of CD16 and CD56 on CD3CD19CD14 cells (Fig. 1A). At baseline, patients exhibiting SVR had significantly lower proportions of CD56brightCD16+/− NK cells compared with both nonresponding (NR) patients and healthy age-matched donors (HDs) (*p<0.05, ***p<0.0001; Fig. 1B). Higher proportions of CD56dullCD16+ NK cells were detected in HCV-1 NR and SVR patients when compared with HDs (**p<0.01 and ***p<0.0001, respectively). In addition, the CD56dullCD16+ subset was significantly increased in SVR when compared with NR patients (*p<0.05) (Fig. 1B). The proportion of CD56CD16+ NK cells was not increased compared with HDs. No difference in this subset proportions was detected between SVR and NR patients at baseline or at subsequent time points (data not shown).

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Figure 1. Flow cytometric analysis of peripheral NK cells from HCV-infected patients with different responses to treatment. PBMCs were separated by density gradient. CD3CD14CD19 were gated and analyzed by flow cytometry as described. (A) Flow cytometric analysis of NK cells in chronically HCV-infected patients with no response (NR), SVR to standard treatment and in healthy donors (HDs). Dot plots show CD3CD14CD19-gated PBMCs expressing CD16 and CD56. Data are representative of >40 experiments. Lines indicate the gate used to identify CD16+/−CD56bright CD16+CD56dull NK cells. (B) Distribution of NK cell subsets in HCV NR (n=11) and SVR (n=17) patients, and HDs (n=15). Lines in the box plot analysis indicate median expression values; boxes indicate 25th and 75th percentiles; vertical lines express SD *p<0.05, **p<0.01, and ***p<0.0001, Mann–Whitney U-test. (C) Representative flow cytometric comparisons of activating and inhibitory receptors on peripheral NK cells from NR and SVR patients. CD3CD14CD19-gated PBMCs were analyzed for the expression of indicated NK cell molecules on CD56+NK cells. A total of 10 000 gated events were acquired. Data are representative of 28 experiments. (D) Activating NK cell receptor expression on NK cells isolated from NR and SVR patients, and HDs. Expression presented as percent positive (left) and marker MFI (right). Molecular densities are expressed as the ratio of the sample MFI to the control MFI to account for intersample variability (right). Data are representative of all patients and HDs. *p<0.05, **p<0.01, ***p<0.0001, Mann–Whitney U-test. (E) Analysis of inhibitory NK cell receptor expression on NK cells isolated from NR and SVR patients, and HDs. Expression presented as percent positive (left) and marker MFI (right). Data are representative of all patients and HDs. *p<0.05, **p<0.01, Mann–Whitney U-test. (F) Plasma HCV viremia is correlated to the proportion of CD16+/−CD56bright NK cells and NKp30 expression. The relationship between NK cell marker expression and HCV viremia was analyzed among all patients before initiation of standard treatment using Spearman's ρ. **p<0.01 and *p<0.05.

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Expression of activating and inhibitory NK cell receptors in SVR and NR patients at treatment baseline

Next, activating receptor expression on NK cells was studied. A representative panel of flow cytometry NK cell mAb staining in NR and SVR patients (Fig. 1C) shows the proportion of CD56+ NK cells expressing given receptors. In all patients, all CD56brightCD16+/− NK cells were NKp46+ (Fig. 1C). Patients with SVR had significantly decreased proportions of NK cells expressing NKp30 and DNAM-1 at baseline compared with HDs (Fig. 1D). This was also reflected in decreased receptor density on positive cells (as defined by MFI) for both NKp30 and DNAM-1 (Fig. 1D).

NK cell inhibitory receptor analysis revealed a decrease in both the proportion of cells expressing CD85j and CD85j surface density (as measured by MFI) in SVR patients compared with either NR patients or HDs (*p<0.05 and **p<0.01, respectively) (Fig. 1E). In addition, increased surface density of NKG2A was detected on NK cells in both NR and SVR patients, as compared with HDs (*p<0.05 and **p<0.01, respectively), confirming the previous observations.

Since NR patients had higher levels of viraemia (Table 1), we studied whether any of the above NK cell parameters could be correlated with the level of viraemia. The proportion of CD56brightCD16+/− NK cells was directly correlated with the level of viraemia (p<0.01, Spearman's ρ, Fig. 1F). Analysis of the relationship between viraemia and activating receptor expression showed that both the proportion of NKp30+ NK cells and NKp30 surface density (MFI) were directly correlated with viraemia (p<0.05, Spearman's ρ, Fig. 1F). No other significant association was detected.

Table 1. Demographic and clinical characteristics of chronically HCV-infected patients
 Virological NRVirological responder (SVR)Population statistical comparison
Subjects (n)1117n.s.
Gender (n)   
Female27n.s.
Male910n.s.
Age, years (mean±SD)45±15.7643.9±12.11n.s.
ALT I.U./mL (mean±SD)76.14±2693±62n.s.
AST I.U./mL (mean±SD)41.16±18.2466±61.55n.s.
HCVRNA cp/mL (mean±SD)2.57±2.51060.8±1.2106p<0.05

Peripheral NK cell function in patients with a subsequent SVR and NR patients

Cytolytic function was analyzed by the expression of CD107a, a marker of NK cell degranulation correlating with the 51Cr release assay 33. Specific triggering of NCR was obtained by coculturing PBMCs with FcγR+ P815 cells in the presence or absence of an anti-NKp30plus + anti-NKp46 mAb mixture in a redirected killing (reverse-ADCC) assay. Higher proportions of degranulating CD3CD19CD14CD56+ CD107a+ NK cells were detected in NR compared with SVR patients (21.41±4.70 versus 14.07±6.38 mean±SD, respectively, p=0.06) (Fig. 2A). Although not statistically significant, this is in-line with the higher baseline expression of NKp30 on cells from NR patients.

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Figure 2. Functional activity of peripheral NK cells from patients with chronic HCV infection before treatment. Redirected killing/stimulation assay (reverse ADCC) of PBMCs for the analysis of cytotoxicity and IFN-γ production by NK cells (see Methods section). (A) Flow cytometric analysis of CD107a degranulation on peripheral CD56+NK cells in the presence of anti-NCR mAbs and FcγR+ P815 cells. (B) Flow cytometric analysis of IFN-γ production by CD56+ NK cells. Data are representative of 12 experiments.

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We also investigated IFN-γ intracellular production by cytofluorometric analysis of peripheral CD3CD19CD14CD56+ NK cells after NCR-mediated triggering. As shown in Fig. 2B, in both patient groups, decreased proportions of IFN-γ-producing NK cells were detected when compared with HDs. IFN-γ production was similarly defective in SVR compared with NR patients (14.6±5.03 versus 13.63±5.49 mean±SD, respectively, no significant difference in between populations). These findings were confirmed by the assessment of IFN-γ production in the supernatants of the same cultures using a cytofluorometric assay. Production of TNF-α, IL-8, IL-6, IL-10, and IL-1 was studied using the same assay and supernatants of stimulated cultures. No significant differences were observed between NR and SVR patients (data not shown).

Analysis of NK cell receptor expression in patients with SVR and NR during standard HCV treatment

Initial response to treatment was assessed 12 wk after the treatment commenced. NK cell analysis was also performed at this time. After 12 wk of PEGIFN-α+RBV treatment, a significant increase in CD56brightCD16+/− cell numbers was detected in both NR and SVR patients compared with baseline values (*p<0.05 and **p<0.01, respectively) (Fig. 3A). A corresponding decrease in the CD56dullCD16+ NK cell population was observed in both patient groups (*p<0.05 and ***p<0.0001) (Fig. 3A).

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Figure 3. Flow cytometric analysis of activating and inhibitory NK cell receptor expression on peripheral NK cells of HCV-infected patients on treatment (12 wk) and thereafter. Data were obtained as for Fig. 1, at different time points after treatment start (12 wk) and treatment stop (6 months). (A) Analysis of peripheral NK cell subsets by percent positive (top) and marker MFI (bottom) in all NR (n=11) and SVR (n=17) HCV patients. Comparisons are shown for values observed after 12 wk compared with baseline levels. Lines in the box plot analysis indicate median expression values; boxes indicate 25th and 75th percentiles; vertical lines express SD. *p<0.05, **p<0.01, and ***p<0.0001, Mann–Whitney U-test. (B) Activating NK cell receptor expression on peripheral NK cells from NR and SVR patients. Comparisons are shown for the values observed after 12 wk (T12) compared with baseline levels (T0) or with levels observed 6 months after stopping treatment (T6FU.) Data are representative of all HCV patients. *p<0.05, **p<0.01, Mann–Whitney U-test. (C) Activating NK cell receptor expression on peripheral CD56bright and CD56dull NK cells from NR and SVR patients on treatment (T12). Data are representative of all HCV patients. *p<0.05 and **p<0.01, Mann–Whitney U-test. (D) Inhibitory NK cell receptor expression on peripheral NK cells from NR and SVR patients. Comparisons in (B) *p<0.05, **p<0.01, ***p<0.0001, Mann–Whitney U-test. (E) Differences in overall receptor fold-change expression (12 wk versus baseline) in NR and SVR patients. Data represent mean change+SEM for all HCV patients.

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Next, we studied the changes in specific receptors during treatment, stratified by treatment outcome. An increase in the proportion of cells expressing NKp46 and NKp30, with increasing NKp30 molecule density (MFI), was evident in patients with SVR (Fig. 3B). To verify whether the changes in NCR expression at 12 wk of treatment were associated with changes in CD56dullCD16+ or CD56brightCD16+/− cells, we analyzed these two subsets separately. No changes were observed in CD56bright cells. When evaluating CD56dullCD16+ NK cells, we observed that the median frequency of NKp46 expression increased, albeit not significantly. At the same time, a significant increase in the proportion of NKp30+ NK cells was detected on CD56dull NK cells (Fig. 3C). This increase in NCR expression on CD56dull NK cells correlated inversely with the concomitant decrease in the proportion of circulating CD56dull cells, suggesting that the observed NCR changes are not dependent on variations in NK cell subsets but rather that net increased expression is taking place during IFN-α treatment.

Expression of inhibitory receptors other than killer inhibitory receptor (KIR) on NK cells during PEGIFNα+RBV treatment was also affected (Fig. 3D). Treatment resulted in significant reductions in overall KIR expression in both SVR and NR patients, a decrease in CD85j+ NK cells in NR patients with relevant increases in the proportion of NKG2A+ NK cells and surface density of NKG2A on those cells (Fig. 3D). To better assess these changes, we analyzed the change in receptor surface expression before and after treatment for each patient. This analysis confirmed that SVR patients display an overall improvement in NK cell activating receptor balance while on PEGIFNα+RBV, as shown by net increases in NKp46 and NKp30 MFI, decreasing KIR MFI and a steadily low CD85j expression (Fig. 3E).

Differential activating NK cell receptor expression in vitro in patients with SVR and NR patients

In order to confirm that the observed changes in activating NK cell receptor density were associated with treatment (i.e. IFN-α), we studied in vitro the effect of IFN-α supplementation on NK cells isolated from SVR and NR patients before starting treatment. NKp30 expression increased upon NK cell activation by rIL-2 in both groups of patients. Addition of IFN-α to rIL-2-activated NK cells resulted in an additional increase in NKp30 expression in patients with subsequent SVR compared with NR patients (p<0.05) (Fig. 4). Increases in NKp46 surface density compared with baseline expression were observed in cells isolated from patients with SVR, whereas NK cells isolated from NR patients showed decreased expression (Fig. 4). DNAM surface density increased upon activation with rIL-2 in both NR and SVR patients, and did not increase further with the addition of IFN-α. In vitro supplementation with RBV had no effect. Thus, the activating receptor modulation observed in SVR patients can be reproduced in vitro once NK cells are activated. IFN-α supplementation after rIL2 activation induces additional increases NKp30 expression only. Future work using freshly separated NK cell short-term cultures without prior cryopreservation should clarify the contribution of rIL-2 and IFN-α.

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Figure 4. Cytokine-induced in vitro modulation of NK cell receptors before treatment starts. Purified NK cells from SVR and NR HCV patients and HDs were cultured in vitro in the presence of rIL-2 (100 U/mL) and IFN-α (100 UI/mL) before flow cytometric evaluation of NK cell receptor expression. Bars indicate fold change in NK cell molecule density after activation compared with baseline values and are presented as mean+SD. Data represent experiments from seven NR and seven SVR patients. *p<0.05, **p<0.01, Mann–Whitney U-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

In the present study, baseline analysis of HCV-infected patients detected differences in circulating NK cell subsets. Initial NK cell population subset ratios and surface expression of NK cell-activating receptors can prospectively identify patient NK cell response to IFN-α in vivo and to IL-2 activation/IFN-α in vitro. Here, we predominantly address the expression, function, and modulation of activating receptors (NKp46, NKp30, and DNAM), whereas recent work has concentrated on the expression of inhibitory receptors (mainly KIR) 18, 32.

The present findings on baseline NCR expression reconcile previous conflicting observations 15, 16 and suggest that differences in those studies were due to patient selection. Indeed patients with subsequent SVR have depressed NK cell NCR expression compared with HDs, resembling those described by Nattermann et al. 16. NR HCV patients show no such decrease in NCR expression, as described previously by others 15.

SVR patients had lower baseline expression of NKp30 and DNAM-1, which play a pivotal role in the interaction between iDC and NK cells, contributing to DC maturation, editing 34, 35 and downstream adaptive responses. Therefore, SVR patients have a baseline peripheral NK cell phenotype reflecting reduced NK-DC crosstalk. This was confirmed by degranulation experiments showing that CD107a expression, a direct correlate of NK cytotoxicity 15, 33, is decreased in SVR patients. Differences in NK cell function, however, are limited to degranulation. Production of IFN-γ and other cytokines was comparable between NR and SVR patients. IFN-γ production usually is considered to parallel cytotoxicity or CD107a degranulation. Here, IFN-γ production was similarly low across all viraemic HCV patients, irrespective of their subsequent response to treatment, possibly due to HCV-mediated mechanism(s) 36. Variability in CD107a degranulation may more accurately reflect differences in NKp46 and NKp30 expression at baseline.

Twelve wks into continuous dosing, an increase in CD56brightCD16+/− NK cells was detected, in agreement with a previous observation comparing NK cells in HIV/HCV-coinfected patients with monoinfected patients 37. The mechanisms and regulations underlying this imbalance are unclear. Since mobilization of CD56brightCD16+/− NK cells also occurs during IFN administration to multiple sclerosis patients 38, this could reflect the presence of type I IFNs(α/β) with differential homing or compartmentalization, either in the secondary lymphoid organs 39, 40 or in the liver, similar to what suggested during other conditions 13, 41, 42.

When the findings of baseline NK cell receptor expression are considered together with NK cell changes 12 wk into treatment and with the modulation observed in NK cell response in vitro, a picture of differential NCR inducibility emerges. In other words, chronically HCV-1-infected patients displaying low, but still inducible, expression of NCRs, coupled with low levels of baseline CD85j expression, have a higher chance of clearing HCV-1 upon treatment with IFN-α. This would favourably associate with KIR2DL3:HLA-C1 haplotype 2, 27, 32 and could help explain efficient viral clearance even in the absence of such a haplotype. On the contrary, patients with already upregulated NCR expression, possibly following endogenous IFN-α production (e.g. by plasmacytoid DC), fail to control and clear HCV upon additional IFN-α administration.

The present study complements the studies showing increased spontaneous acute infection clearance 2, 27 or HCV clearance with combined treatment 32 in patients carrying specific KIR:HLA haplotypes. For example, Vidal-Castineira et al. show that clearance of infection is not exhaustively explained by KIR:HLA haplotype carriage, as some patients responding to treatment did not carry protective KIR:HLA haplotypes 32. In these cases, one could hypothesize that possession of activating receptors highly responsive to IFN-α treatment may result in HCV clearance even in the presence of strong inhibitory KIR:HLA interactions. Observation of lower inhibitory balance in SVR patients with low CD85j expression supports this hypothesis. CD85j expression has relevant functional consequences in cytotoxic cells 29. Its decreased expression on NK cells from SVR patients suggests that they may be less prone to CD85j-mediated inhibition upon CD85j-HLA class I interaction, which could parallel and add to favourable KIR:HLA interactions.

Our experimental setting does not allow us to determine whether the low expression of NCR and DNAM-1 and their upregulation in SVR patients following treatment with PEGIFNα+RBV may be due to different differentiation/development of NK cells, or rather the consequence of an increased transcription rate. Correlates of this observation have been described in a different setting. HIV-infected, AIDS-free chimpanzees have low/absent NKp30 expression. NKp30 is, however, readily inducible upon NK cell activation 43. Interestingly, a recent report shows that NR patients already have high expression of IFN-stimulated genes before therapy, and that standard IFN-α-based treatment does not upregulate IFN-stimulated genes (ISGs). More importantly, rapid responder patients have low baseline ISG, which are able to be upregulated 44. NCR regulation in our patients seems to parallel this observation, thus opening the possibility that ISG might participate in the regulation of NCR expression at a transcriptional or post-transcriptional level. High-baseline NCR expression together with endogenous IFN type I production in NR HCV patients may account for poor viral clearance. In this case, additional IFN administration does not result in improvement of innate pathways of virus clearance, including increased NCR expression with improved NK-DC crosstalk and with improved lysis of infected cells. In such a case, it remains to be determined whether SVR patients are less sensitive to endogenous type I IFN and therefore benefit of induction through additional exogenous IFN-α, or rather that their endogenous pathways of type I IFN are not active, and therefore exogenous IFN-α simultaneously upregulates ISG and NCR on NK cells with synergic effects.

Additional work is needed to define whether regulation of triggering receptors may be induced during HCV infection, or rather depends on constitutive immunogenotypic regulation of NCR expression, parallel to what has been shown for KIR. Similarly, we have no information on DNAM-1 or NKp30 ligands expressed by DCs during chronic HCV infection nor on hepatocytes. It is possible that future analysis of these ligands could contribute to providing an explanation for poor HCV clearance by those NR patients that already have high-baseline NKp30 and DNAM-1 expression.

Analysis of NK cell phenotype and function before and after treatment could provide additional clues to the role of NCR regulation in the response to standard HCV treatment. However, the different duration of IFN-α treatment between those interrupting treatment soon after the clinical definition of failure and those continuing for at least 48 wk prevents an unbiased comparison. Differences in patient groups could not be excluded in advance in this instance, not only for possible basal background regulation (e.g. NR versus SVR), but also for differences of treatment intensity (e.g. 3–4 months versus 12–14 months of treatment, NR and SVR, respectively).

In conclusion, the present data suggest that, besides KIR:HLA haplotype carriage, differences in CD85j expression and in activating NK cell receptor expression contribute to the differential response of HCV patients to combined PEGIFNα+RBV treatment. NK cell function is balanced by the cooperation of activating and inhibitory functions and thus construction of a response framework consisting of KIR:HLA haplotype, CD85j expression, and NKp30/NKp46/DNAM-1 induction could improve our understanding of the relevant characteristics leading to successful clearance of HCV.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Patients

Between 2006 and 2008, patients chronically infected with HCV genotype 1 undergoing pretreatment evaluation were offered inclusion in a protocol of intensive immune screening. Inclusion criteria were as follows: chronic HCV genotype1 infection and clinical indication for treatment with PEGIFNα+RBV. Exclusion criteria were HIV coinfection or advanced liver involvement, including cirrhosis and hepatocarcinoma. All patients gave full informed consent. Of 35 consecutive patients consenting to the study, 7 opted out and deferred treatment, 28 were administered standard PEGIFN-α RBV. Patients were treated and followed up according to the current guidelines for 48 wk. HCV-RNA was assessed at baseline and after 4 and 12 wk of treatment to identify early virus clearance. SVR was defined as disappearance of viraemia during treatment, remaining persistently negative both at end of treatment and beyond 6 months after stopping treatment. Serum HCV-RNA was measured by Amplicor HCV Monitor (Roche, Milan, Italy. Cutoff limits, quantitative test: 600 IU/mL; qualitative test: 50 IU/mL). HCV genotype was determined before treatment in all patients with the INNO-LiPA HCV II kit (Bayer Diagnostics, Emeryville, CA, USA). PBMCs were separated by density gradient centrifugation from peripheral blood collected before treatment initiation at wk 12 on treatment, and 6 months after treatment interruption, and cryopreserved at −140°C.

Of the 28 patients, 17 exhibited SVR and 11 failed to clear virus (NR). Demographic and clinical data of the patients are summarized in Table 1. Fifteen HDs were used as a negative control. All patients were included after providing informed consent.

Abs

The following panel of mouse anti-human mAb was used: anti-CD3FITC, anti-CD3 allophycocyanin, anti-CD19 allophycocyanin, anti-CD14allophycocyanin, PE- and FITC-conjugated anti-CD16 (BD PharMingen, San Jose, CA, USA), anti-CD56PC7 (Immunotech-Coulter Marseille, France), anti-NKG2C, (R&D Systems, Minneapolis, MN, USA). Anti-NKp46 (IgG1) BAB281, anti-NKp30 (IgG1) AZ20, anti-NKp44 (IgG1) ZIN231, anti-NKG2D (IgG1) BAT, anti-DNAM-1 (IgG1) F22, anti-LIR-1 (CD85j) (IgG1) F278, anti-KIR2DL2/S2 (CD158b1/CD158j), anti-KIR3DL1 (CD158e1), anti-KIR2DL1/S1 (CD158b/CD158 h) (mixture GL183, Z27, 11pb6, IgG1), and anti-NKG2A (IgG2b) Z199. Anti-HLA-DR (IgG2a) D1.12, was kindly provided by Dr. R. S. Accolla (University of Insubria, Varese, Italy). FITC-conjugated (Southern Biotechnology, Birmingham, AL, USA) and PE-conjugated goat anti-mouse anti-isotype Abs (BD PharMingen) were used for controls. For intracellular staining, anti-γ-IFN allophycocyanin and anti-CD107a-PE (BD PharMingen) were used.

Immunofluorescence analysis

PBMC samples were analyzed by four-color cytofluorometry. Purified NK cells populations were analyzed by three-color cytofluorometric assay as previously described (Becton Dickinson, Mountain View, CA, USA) 20. To reduce inter-assay variability, the MFIsample/MFIcontrol ratio was used to compare samples/groups.

NK cell-activating receptor induction assay

NK cells were isolated from PBMCs and cultured as described previously 15. Purified NK cells were cultured in the presence of rIL-2 (100 U/mL; Proleukin, Chiron, Emeryville, CA, USA), irradiated PBMCs (5000 rad) and PHA (1.5 ng/mL; Sigma, Saint Louis, MO, USA) for 12–15 days. Activated NK cell populations were supplemented with IFN-α 100 U/mL (PeproTech, London, UK) for 6 days. Cells were harvested and analyzed by flow cytometry for the expression of NKp46, NKp30, and DNAM-1. Receptor expression changes were determined comparing MFIr to fresh NK cell MFIr: (MFIratioACT−MFIratio baseline)/MFIratio ACT×100.

IFN-γ production assay

PBMCs were stimulated using FcγR+ P815 target cells at 10:1 E/T ratio in complete medium in the presence or absence of an anti-NKp30 and/or anti-NKp46 mAb mixture as described previously 20. We originally ran the first patients/experiments with separate mAbs in the functional tests in addition to the mAb mixture. We realized that very low expression of NKp30 or NKp46 on peripheral NK cells prevented the detection of IFN-γ and CD107a degranulation in some patients (e.g. very low median NKp30 expression in SVR patients). For this reason, we continued the evaluation of the whole cohort only with the mAb mixture, to avoid eliminating patients from the analysis.

CD107a degranulation assay

PBMCs were stimulated as above. Anti-CD107a mAb (BD PharMingen) was added as described previously. Cells were surface-stained using anti-CD3FITC and anti-CD56PC7 mAb.

Statistical analysis

The Mann–Whitney U-test and Kolmogorv–Smirnov, Speraman's Correlation, or χ2 tests were employed. Analysis was performed using StatView 4.2 program (Abacus Concepts, Berkeley, CA, USA). Receptor fold change expression: ex vivo (MFIrratio12 wk–MFIratiobaseline)/MFIratio baseline×100.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

The authors wish to thank Alessandro Moretta, Daniela Pende, Emanuela Marcenaro for mAb gift, Monica Basso MD (Genova) for help with the patients, Mattea Roccatagliata for secretarial assistance. This work was supported by grants awarded by Istituto Superiore di Sanita' (I.S.S.): Programma nazionale di ricerca sull'AIDS, Accordi di collaborazione scientifica n. 40G.41, 40H67 (L. M. and A. D. M.) and 45G.11, 40H69 (A. D. M.), Italian Concerted Action for AIDS vaccine (I.C.A.V.), Accordo di collaborazione scientifica n. 40D61 (A. D. M.); Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), projects L. M.; Ministero dell'Istruzione, dell'Università e della Ricerca: MIUR-FIRB 2003 project RBLA039LSF-001 (L. M.); Ministero della Salute: RF2006 – Ricerca Oncologica-Project of Integrated Program 2006-08, agreements n. RO strategici 3/07 (L. M.).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
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