Figures
Abstract
Background
CD8+ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.
Methodology/Principal Findings
Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8+ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8+ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8+ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.
Citation: Zou Q, Yao X, Feng J, Yin Z, Flavell R, Hu Y, et al. (2011) Praziquantel Facilitates IFN-γ-Producing CD8+ T Cells (Tc1) and IL-17-Producing CD8+ T Cells (Tc17) Responses to DNA Vaccination in Mice. PLoS ONE 6(10): e25525. https://doi.org/10.1371/journal.pone.0025525
Editor: R. Lee Mosley, University of Nebraska Medical Center, United States of America
Received: June 20, 2011; Accepted: September 5, 2011; Published: October 5, 2011
Copyright: © 2011 Zou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by National Key Technology Research & Development Program of China (2004BA519A39 and 2006BAD06A06), National Nature Science Foundation (30771602) and National Mega Grand Program on Key Infectious Diseases of China (2008ZX10001-012) to B.Wang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chronic viral hepatitis B, a disease caused by hepatitis B virus (HBV), is a worldwide health problem [1], [2], [3]. The host immune response plays a key role in the outcome of HBV infection [4], [5], [6]. Efficient induction of multi-specific CD8+ T cell responses against the core and surface antigens of this virus can control HBV infections [7], [8], [9]. Particularly, reports have described the involvement of two main subsets of CD8+ T cells, the IFN-γ-producing Tc1 cells [10], [11] and IL-17-secreting Tc17 cells [12], [13], [14]. It becomes increasingly clear that efficient expansion of virus-specific Tc1 cells resolves HBV infection by cytolytic and noncytolytic mechanisms [15], [16], [17], although the mechanism of action by Tc17 cells remains unclear.
HBV DNA immunization is a promising strategy for inducing strong CD8+ T cell-mediated immunity in mice [18], [19], [20], [21], whereas this effect has been only transient and weak in clinical trials [22], [23]. To move beyond various approaches already employed to improve DNA vaccination, we have explored the use of novel adjuvants that would specifically potentiate the Tc1 and/or Tc17 type of T cells as a means to overcome the problem.
Praziquantel (PZQ) has been used for the treatment of Schistoma japonicum infection without severe side effects [24]. It has been found that the humoral and cellular immune responses of the host were enhanced after being treated with this drug [25], [26]. We recently demonstrated that PZQ could act as an adjuvant to enhance cellular responses to HBsAg DNA vaccination in mice [27]. However, it was not clear how PZQ affected CD8+ T cell responses and whether the effect of PZQ was strong enough to break immune tolerance in HBsAg-transgenic mice. We have begun to address these questions in the present report.
Results
PZQ enhanced CD8+ T cell-mediated responses to HBV DNA vaccine
Delayed-type hypersensitivity (DTH) is a well-established readout for T cell responses to vaccination [28], [29]. To determine the immunogenic effect of PZQ on HBV DNA vaccination, DTH responses from C57BL/6 mice immunized with pcD-S2 in the presence or absence of PZQ were compared. Using HBsAg as a rechallenging antigen, we found that PZQ augmented the DTH response to HBsAg (Fig. 1A).
A: Twelve days after the second immunization with indicated the combinations, all groups were challenged with the rHBsAg in the right foot pad as test and with a saline solution in the left foot pad as control. The thickness of footpads was measured at 24, 48 and 72 h. B, Mice were depleted of CD4+ (CD4del) or CD8+ (CD8del) T cells by in vivo injection of specific mAbs. The efficiency of depletion was determined by flow cytometry of splenocytes. The numbers indicate the percentages of CD4+ or CD8+ T cells. C: Ten days after the second immunization of pcD-S2 plus PZQ, mice were injected i.p. with anti-CD4 mAb, anti-CD8 mAb, or rat IgG and challenged 2 days later with rHBsAg. DTH was measured at 24 h. D: The immunization schedule is shown. The data shown summarizes one of three experiments, all of which demonstrated similar results (* p<0.05; ** p<0.01).
To further determine the subpopulations of T cell in the augmented DTH, over 90% of either CD4+ or CD8+ T cells were depleted with anti-CD4 or anti-CD8 mAbs respectively (Fig. 1B). Interestingly, the depletion of CD8+ T cells significantly blunted DTH, whereas the depletion of CD4+ T cells had little impact (Fig. 1C–D), suggesting that PZQ preferentially augmented CD8+ T cell responses to pcD-S2 vaccination.
Induction of Tc1 and Tc17 cells during DNA vaccination with PZQ
Effector cytotoxic CD8+ T cells can be divided into IFN-γ-producing (Tc1) and IL-17-producing (Tc17) subtypes [10], [11], [12], both of which might be involved in DTH [30], [31], [32], [33], [34]. To delineate which was induced by PZQ, we isolated splenic cells from mice immunized with pcD-S2, pcD-S2 plus vehicle, or pcD-S2 plus PZQ and restimulated the cells in vitro with an HBsAg-derived peptide, S208-215. The CD8+ T cells from mice immunized with pcD-S2 and PZQ secreted both IFN-γ and IL-17 at a higher level than those of the controls (Fig. 2A–C), suggesting PZQ induced both Tc1 and Tc17 subtypes. To identify the cytokine-producing cells, restimulated CD8+ T cells were intracellularly stained with anti-IL-17 and IFN-γ mAbs and examined by flow cytometry. As expected, many IFN-γ single-positive (Tc1) and IL-17 single-positive were observed (Tc17), but very few IFN-γ and IL-17 double-positive, CD8+ T cells were shown (Fig. 2D).
A–B, Splenic T cells were isolated on day 14 after the third immunization and were stimulated with the HBsAg-derived peptide S208-215 in the presence of brefeldin A (5 µg/ml) for 6 h in culture and immunostained for surface CD8, and intracellular IFN-γ and IL-17. C, Summary of the percentage of IFN-γ- and IL-17-expressing CD8+ T cells. D, Intracellular staining for IFN-γ and IL-17 in CD8+ T cells. C57BL/6 mice were immunized with pcD-S2 and PZQ and CD8+ T cells isolated on day 7 after third immunization were stimulated in vitro. Data shown are representative of 3 independent experiments (* p<0.05; ** p<0.01).
To determine whether the generation of Tc1 or Tc17 cells was necessary for the augmented HBsAg-specific DTH, pcD-S2 immunization was carried out in IFN-γ KO mice and IL-17 KO mice. Compared to that of wild-type mice, DTH was significantly decreased in the IFN-γ KO or IL-17KO mice (Fig. 3A and 3B). Taken together, PZQ was shown to facilitate both Tc1 and Tc17 cells.
Mice were challenged with rHBsAg 12 days after the second immunization. DTH was measured at 24, 48 and 72 h. A and B, DTH of IFN-γ and IL-17 KO mice, respectively. Data shown are representative of three independent experiments (* p<0.05; ns, p>0.05).
PZQ broke tolerance to HBsAg in HBsAg-transgenic mice
To test if PZQ-induced Tc1 and Tc17 cells in vivo had a potential to clear tolerized viral antigens, HBsAg-Tg mice were immunized with pcD-S2 in the presence and absence of PZQ. We observed that PZQ also increased the levels of Tc1 and Tc17 cells and the HBsAg-specific DTH in these animals (Fig. 4A and 4B). To assess the possible therapeutic effects, we examined lymphocyte infiltration in the liver of immunized HBsAg-Tg mice. As shown in Fig. 5A, no infiltration was detected in nontreated mice. The highest level of infiltration was noted in mice immunized with pcD-S2 and PZQ, as compared to mice immunized with pcD-S2 alone or with vehicle. In addition, the PZQ-treated DNA vaccine group was the only group showing a significant number of CD8+ T cells in the infiltrates, presumably of the Tc1 and Tc17 subtypes (Fig. 5A). No CD4+ T cells were detected (data not shown) in this group. Lastly, no obvious change in liver morphology was noted in this group, suggesting that the induced CD8+ T cells were not overtly pathogenic.
A, Splenic T cells were isolated on day 14 after the third immunization and stimulated with S208-215 in the presence of brefeldin A (5 µg/ml) for 6 h in culture. CD8+ T cells were intracellularly immunostained for IFN-γ and IL-17. B, 12 days after the second immunization, DTH was measured at 24, 48 and 72 h later. Results were representative of three independent experiments (* p<0.05).
A: livers from each group were obtained on day 14 after the third immunization and fixed, sectioned, and stained with H&E. Bar, 50 µm. CD8-specific immunostaining of the liver from HBsAg transgenic mice on day 14 after final immunization. Bar, 20 µm. Specific immunostaining of HBsAg on day 14 after final immunization. Bar, 50 µm. B: Percentage of HBsAg-positive liver cells in indicated groups on day 14 after final immunization. C: HBsAg antigen in the serum on day 14 after final immunization. D: ALT activity in the serum on weeks 0, 1, 2, 3, and 4 after the third immunization. Results were representative of three independent experiments. There were four mice in each group (* p<0.05; ns, p>0.05).
Consistent with the observed CD8+ T cell infiltration, a significant reduction of HBsAg-positive hepatocytes following immunizations with pcD-S2 plus PZQ was observed (Fig. 5A and 5B). However, the level of HBsAg in the blood was not significantly changed (Fig. 5C).
The reduction of HBsAg-positive hepatocytes was likely due to killing of these cells by the infiltrating CD8+ T cells. To confirm this, serum ALT levels were analyzed on weeks 0, 1, 2, 3, and 4 after the final immunization. As depicted in Fig. 5D, serum ALT reached the highest level on week 1 and gradually declined to a basal level on week 4 in mice immunized with pcD-S2 plus PZQ. In contrast, ALT levels remained low throughout the same period in control mice immunized with pcD-S2 alone and pcD-S2 plus vehicle (Fig. 5D). This result thus supported our speculation.
PZQ-induced cytolytic CD8+ T cells are effectors in killing of HBsAg-positive hepatocytes
In order to further identify the particular role of Tc1 and Tc17 cells in elimination of HBsAg-positive hepatocytes, C57BL/6 wild type, IFN-γ KO, or IL-17 KO mice were immunized with pcD-S2 and PZQ, followed by analysis of HBsAg specific CD8+ T cell-mediated killings in vivo. As expected, the highest percentage of antigen-specific killing (∼60%) was noted in immunized wild type mice, as compared to that in IFN-γ KO (∼30%) or IL-17 KO (∼35%) mice (Fig. 6A–B). This result demonstrated that both Tc1 and Tc17 cells were involved in the elimination of HBsAg positive hepatocytes.
A: In vivo cytotoxic lysis was performed in the wild type, IFN-γ KO and IL-17 KO mice on day 7 after the third immunization with pcD-S2 and PZQ. B: The percentage of specific lysis was summarized. C: HBsAg-specific immunostaining of HBsAg of livers from the HBsAg transgenic mice was analyzed on day 14 after adoptive transfer. Bar, 20 µm. D: Percentage of HBsAg-positive liver cells. E, ALT level in the serum was detected on weeks 0, 1 and 2 after adoptive transfer of CD8+ T cells. The data shown are representative of three independent experiments (* p<0.05; ** p<0.01; ns, p>0.05).
To establish the role of these CD8+ T cells as the effectors for killing of HBsAg-positive hepatocytes, CD8+ T cells were isolated from immunized mice and purified to 90–95% purity on day 7 after the final immunization, and adoptively transferred intravenously into normal HBsAg-Tg mice at 5×106 cells per recipient animal. Only the transfer of CD8+ T cells from the wild type mice could significantly reduce HBsAg expression in the liver of the recipients, whereas CD8+ T cells from the IFN-γ KO or IL-17 KO mice were not effective (Fig. 6C–D). Consistently, the ALT level rose after adoptive transfer of the wild type CD8+ T cells and returned to a normal level on week 2 post transfer (Fig. 6E). These results thus established that both Tc1 and Tc17 cells were the effectors.
Discussion
HBV-specific CD8+ T cells with killing of HBV-infected hepatocytes is the most desirable option for the control of chronic HBV infection [35]. In this report, we find the anti-Schistosoma drug praziquantel (PZQ) to be highly effective at eliciting Tc1 and Tc17 responses in both wild type (WT) and HBsAg-Tg mice. Importantly, the induced HBsAg specific CD8+ T cells are potent enough to break established immune tolerance and kill HBsAg positive hepatocytes.
PZQ was previously shown in schistosomiasis patients to significantly alter immune responses [25], [26], [36]. However, it was not explored as an immune adjuvant until its recent demonstration as an augmenting effect on cellular responses to DNA vaccination [27]. As such, we have sought to further characterize this drug in terms of the types of immune responses and subsets of T cells it affects. To assess the effects of PZQ, DTH was utilized. In this study, we demonstrated that PZQ induced a higher DTH response than that of a DNA vaccine alone. Although Th1 and Tc1 cells have been previously studied in detail to show their associations with IFN-γ dependent DTH responses, Th17 cells, Tc17 and macrophages have also been demonstrated to involve the induction of DTH as well [31], [37], [38]. During DNA vaccination alone, the levels of DTH as showed in Fig. 3 had a slight difference between KO and wild type mice, suggesting that DTH could be derived from CMI other than Tc1 and Tc17 cells. In the case of DNA vaccination with PZQ, however, Tc17 and Tc1 were the main contributors for DTH. Therefore, the causes of DTH could be due to a complicated mechanism.
Tc1 cells are the classical cytotoxic T cells that produce IFN-γ and TNF-α and can destroy virally infected cells through the targeted secretion of perforin and granzymes from lytic granules [39], [40], [41]. Tc17 cells, which produce IL-17 [42], [43], [44], [45], [46], [47], are also cytotoxic [12], [42], [48] and have been shown to protect animal against acute influenza virus infection [48]. To date, HBV specific Tc1 cells have been demonstrated to control chronic HBV infection [7], [15] and eliminate HBV infected hepatocytes in transgenic animal model [19], [20]. In contrast, the role of Tc17 cells in HBV protection is not understood. Our data show, for the first time, that Tc17 cells kill HBsAg-positive hepatocytes in vivo. This observation appears to suggest that, similar to Tc1, Tc17 cells are essential effectors.
Although it has been demonstrated that Tc17 cells-mediated cytolytic killing could support the anti-viral immunity through recruiting neutrophils by IL-17 secretion, by secreting IFN-γ, or by expressing FasL [12], [42], [48], our data demonstrated that these IL-17-postive and IFN-γ-negative Tc17 cells were involved in antigen-specific cytotoxic responses that had capacity to kill HBsAg-positive hepatocytes in vivo. These IL-17-producing Tc17 cells mediated the broad CTL mainly through a common cytolytic or Fas-FasL as previous studies indicated [12], [48]. In future experiments, it will be determined if neutrophils are recruited and infiltrated to the sites. As well, the expressions of FasL of Tc17 cells will be analyzed.
The specific role of IL-17 involved in anti-viral immunity remains elusive. It has been shown that blockade of IL-17 resulted in diminishing the survival in response to lethal influenza virus challenge [49], indicating that IL-17 may play some roles in the anti-viral response and protection. IL-17 had been found to participate in host defense against VV infection and recruit neutrophils [50]. Tc17 cells could protect against acute viral infection through recruiting neutrophils or through the secretion of FasL [12], [42], [48]. Perhaps IL-17 could recruit or activate the innate and adaptive immune cells against viral infection. Therefore, its anti-viral role should be further investigated.
T cell-mediated cytolytic killing can result in tissue injury. Liver injury was detected through the elevation of serum ALT, which reached the highest level on week 1 post last immunization in HBsAg-Tg mice and gradually declined to the basal level. However, the extent of liver damage appeared to be limited, as histology of the liver during the same time window revealed no gross change in liver morphology. Therefore, specific killing of the HBsAg positive cells may cause limited damage to the liver, but not result in gross pathology. Further investigation is needed to further clarify this point.
Elimination of HBsAg positive hepatocytes did not significantly change the level of HBsAg in serum. This disconnection has been described in several previous publications. Particularly, it was reported that therapeutic vaccination cleared chronic hepatitis B virus and caused HBeAg seroconversion in chronic carrier chimpanzees, with HBsAg remaining in serum throughout the observation period [51]. Another report showed that the levels of serum HBV DNA and HBeAg were decreased without HBsAg seroconversion in patients [52]. Therefore, one could speculate that a small proportion of HBsAg positive hepatocytes could maintain the high concentration of HBsAg in the serum, resulting in a mild decrease or clearance of HBsAg in the serum, even if a large proportion of HBsAg positive cells are already cleared.
In summary, our results demonstrate ,for the first time, that a HBsAg DNA vaccine, with PZQ, used as adjuvant can induce both Tc1 and Tc17 in vivo and result in elimination of HBsAg positive cells. Since PZQ is already an approved clinic drug, its use for HBsAg DNA vaccination should be safe enough to advance to clinical trials to further examine its potential as a human adjuvant. A more general application of PZQ as a pro-Tc1 and Tc17 adjuvant may also be explored for other immunotherapeutic vaccines to control other chronic viral infections.
Materials and Methods
Animals and reagents
Female C57BL/6 mice of 8–10 weeks of age were purchased from the Animal Institute of Chinese Medical Academy (Beijing, China). HBsAg-transgenic (Tg) mice (C57BL/6J-Tg(Alb1HBV)44Bri/J) and IFN-γ KO (B6.129S7-Ifngtm1Ts/J) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). IL-17 KO mice (C57BL/6 background) were kindly provided by Richard Flavell (Yale University School of Medicine, New Haven, CT). All animal protocols (#28482) were approved by the Animal Welfare Committee of China Agricultural University and housed with pathogen-free food and water under 12 h light-cycle conditions.
Praziquantel (NCPC, Hebei, China) was initially dissolved in ethanol to 6.7% and subsequently diluted to 0.5% with 0.9% saline solution. The vehicle was 7.5% ethanol with 0.9% saline solution. CHO cells expressing recombinant HBsAg (rHBsAg) was kindly provided by China North Pharmaceutical Group Corporation (NCPC, Hebei, China). The HBsAg-derived peptides S208-215 (ILSPFLPL; H-2Kb-restricted) were synthesized by GL BiochemCo., Ltd. (Shanghai, China). Rat anti-mouse CD4 mAb (GK1.5), Rat anti-mouse CD8 mAb (53-6.7) and rat IgG were purchased from eBioscience (San Diego, CA, USA). Fluorescent-labeled anti-mouse mAbs including anti-CD4-FITC, anti-CD8-FITC, anti-CD8-APC, anti-IFN-γ-PE, anti- IL-17-PE and anti-IL-17-APC were purchased from BD PharMingen (San Diego, CA, USA). The anti-HBsAg antibody for immunohistochemical staining was produced in C57BL/6 mice immunized 3 times with rHBsAg and alum as adjuvant.
Plasmid construction and preparation
For mouse studies, the HBV DNA vaccine (pcD-S2) was prepared as described previously [27]. The plasmid was maxi-prepared by the alkaline method, subsequently purified by Qiagen Maxi prep kit (Qiagen Inc., Duesseldorf, Germany), and diluted in saline solution.
Immunization
Mice were randomly divided into six or seven groups (n = 6 each) according to the design of different experiments, and were immunized with 100 µg pcD-S2 per mouse [27]. pcD was the empty vector for pcDNA3. All mice were immunized on day 0 and boosted on day 14.
Elicitation of delayed-type of hypersensitivity (DTH)
Twelve days after the second immunization, all groups were challenged with 10 µg of rHBsAg in the right footpad as test and saline solution at the left footpad as control. The thicknesses of footpads were measured at 24, 48 and 72 h with a micrometer and calculated using the following formula: thickness footpad = thickness of right footpad - thickness of left footpad.
For elimination of CD4+ and CD8+ T cells, anti-CD4 mAb (200 µg/mouse), anti-CD8 mAb (200 µg/mouse), or rat IgG (200 µg/mouse) was injected (i.p.) twice into immunized mice on days 10 and 11 after the second immunization. The mice were challenged on day12 and DTH was measured as described above.
Flow cytometry
Splenic cells were isolated on day 7 after the final immunization and stimulated with 10 µg/ml S208-215 in the presence of brefeldin A (5 µg/ml) for 6 h at 37°C and 5% CO2. Collected cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin (Sigma-Aldrich). For immunostaining of cytoplasmic IL-17 and IFN-γ, and surface CD8, the appropriate concentrations of fluorescently labeled anti-mouse monoclonal antibodies were added to permeabilized cells for 30 min on ice followed by washing twice with cold PBS. Samples were analyzed by a FACSCalibur.
Histopathology
On day 7 after final immunization, liver tissue was paraffin-embedded, sectioned, stained with H&E, and immunostained with anti-HBsAg mAb, anti-CD4 mAb, or anti-CD8 mAb. Analysis was performed under a light microscope for histological changes.
In vivo cytotoxic assay
Splenocytes from naïve C57BL/6 mice were pulsed with 10−6 M HBsAg-derived peptides S208-215 and labeled with a high concentration of CFSE (15 µM, CFSEhigh cells) as target cells. A portion of the same splenocytes was labeled with a low concentration of CFSE (0.5 µM, CFSElow cells) without peptide pulse as a non-target control. The target and control cells were mixed in a 1∶1 ratio and injected into immunized mice at 2×107 total cells per mouse via the tail vein on day 7 after the third immunization. Eight hours later lymphnodes and the spleen of injected mice were removed and the target and control cells were analyzed by their differential CFSE fluorescent intensities using a FACSCalibur (BD Biosciences, USA). Specific lysis was calculated using the following formula: Percentage specific lysis = [1−(ratio unprimed/ratio primed)×100], where ratio = percentage CFSElow/percentage CFSEhigh.
Adoptive transfer of CD8+ T cells
On day 7 after the third immnuzation with pcD-S2 and PZQ, single-splenocyte suspensions were prepared from spleen of wild type, IFN-γ KO or IL-17KO mice. CD8+ T cells were isolated and purified using the MagCellect Mouse CD8+ T Cell Isolation Kit according to the manufacturer's protocol (R&D Systems, Inc., Minneapolis, USA). Purity of each cell preparation was 90–95%. The cells were adoptively transferred intravenously into normal HBsAg transgenic mice at 5×106 per recipient mouse.
Acknowledgments
We are grateful for Dr. Guoxing Zheng's valuable suggestions. We would like to thank Dr. Jane Q.L. Yu, Mr. Qinghong Zhu and Mr. Zhonghuai He for their assistance in this work.
Author Contributions
Conceived and designed the experiments: QZ B.Wang ZY YW. Performed the experiments: QZ XY JF YH JJ YK B.Wu XL CF HL WL XW. Analyzed the data: QZ B.Wang ZY YW. Contributed reagents/materials/analysis tools: RF JF ZY. Wrote the paper: QZ YW GZ B.Wang.
References
- 1. Chisari FV, Ferrari C (1995) Hepatitis B virus immunopathogenesis. Annu Rev Immunol 13: 29–60.
- 2. Rehermann B, Nascimbeni M (2005) Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 5: 215–229.
- 3. Ganem D, Prince AM (2004) Hepatitis B virus infection–natural history and clinical consequences. N Engl J Med 350: 1118–1129.
- 4. Ferrari C, Penna A, Bertoletti A, Valli A, Antoni AD, et al. (1990) Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 145: 3442–3449.
- 5. Penna A, Chisari FV, Bertoletti A, Missale G, Fowler P, et al. (1991) Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen. J Exp Med 174: 1565–1570.
- 6. Penna A, Del Prete G, Cavalli A, Bertoletti A, D'Elios MM, et al. (1997) Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B. Hepatology 25: 1022–1027.
- 7. Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, et al. (2000) The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 191: 1269–1280.
- 8. Maini MK, Boni C, Ogg GS, King AS, Reignat S, et al. (1999) Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology 117: 1386–1396.
- 9. Rehermann B, Fowler P, Sidney J, Person J, Redeker A, et al. (1995) The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med 181: 1047–1058.
- 10. Zhang X, Issagholian A, Berg EA, Fishman JB, Nesburn AB, et al. (2005) Th-cytotoxic T-lymphocyte chimeric epitopes extended by Nepsilon-palmitoyl lysines induce herpes simplex virus type 1-specific effector CD8+ Tc1 responses and protect against ocular infection. J Virol 79: 15289–15301.
- 11. Brignone C, Grygar C, Marcu M, Schakel K, Triebel F (2007) A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells. J Immunol 179: 4202–4211.
- 12. Yeh N, Glosson NL, Wang N, Guindon L, McKinley C, et al. (2010) Tc17 cells are capable of mediating immunity to vaccinia virus by acquisition of a cytotoxic phenotype. J Immunol 185: 2089–2098.
- 13. Ciric B, El-behi M, Cabrera R, Zhang GX, Rostami A (2009) IL-23 drives pathogenic IL-17-producing CD8+ T cells. J Immunol 182: 5296–5305.
- 14. Hinrichs CS, Kaiser A, Paulos CM, Cassard L, Sanchez-Perez L, et al. (2009) Type 17 CD8+ T cells display enhanced antitumor immunity. Blood 114: 596–599.
- 15. Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, et al. (1996) Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 4: 25–36.
- 16. Webster GJ, Reignat S, Maini MK, Whalley SA, Ogg GS, et al. (2000) Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology 32: 1117–1124.
- 17. Phillips S, Chokshi S, Riva A, Evans A, Williams R, et al. (2010) CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions. J Immunol 184: 287–295.
- 18. Riedl P, Wieland A, Lamberth K, Buus S, Lemonnier F, et al. (2009) Elimination of immunodominant epitopes from multispecific DNA-based vaccines allows induction of CD8 T cells that have a striking antiviral potential. J Immunol 183: 370–380.
- 19. Li X, Yang X, Jiang Y, Liu J (2005) A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice. Int Immunol 17: 1293–1302.
- 20. Schirmbeck R, Dikopoulos N, Kwissa M, Leithauser F, Lamberth K, et al. (2003) Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants. Eur J Immunol 33: 3342–3352.
- 21. Yang K, Whalen BJ, Tirabassi RS, Selin LK, Levchenko TS, et al. (2008) A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection. J Immunol 180: 6159–6167.
- 22. Kosinska AD, Zhang E, Lu M, Roggendorf M (2010) Therapeutic vaccination in chronic hepatitis B: preclinical studies in the woodchuck. Hepat Res Treat 2010: 817580.
- 23. Mancini-Bourgine M, Fontaine H, Scott-Algara D, Pol S, Brechot C, et al. (2004) Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers. Hepatology 40: 874–882.
- 24. Cioli D, Pica-Mattoccia L (2003) Praziquantel. Parasitol Res 90 Supp 1: S3–9.
- 25. Brindley PJ, Sher A (1987) The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response. J Immunol 139: 215–220.
- 26. Joseph S, Jones FM, Walter K, Fulford AJ, Kimani G, et al. (2004) Increases in human T helper 2 cytokine responses to Schistosoma mansoni worm and worm-tegument antigens are induced by treatment with praziquantel. J Infect Dis 190: 835–842.
- 27. Zou Q, Zhong Y, Su H, Kang Y, Jin J, et al. (2010) Enhancement of humoral and cellular responses to HBsAg DNA vaccination by immunization with praziquantel through inhibition TGF-beta/Smad2,3 signaling. Vaccine 28: 2032–2038.
- 28. Grabbe S, Schwarz T (1998) Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunol Today 19: 37–44.
- 29. Ishii A, Oboki K, Nambu A, Morita H, Ohno T, et al. (2010) Development of IL-17-mediated delayed-type hypersensitivity is not affected by down-regulation of IL-25 expression. Allergol Int 59: 399–408.
- 30. He D, Wu L, Kim HK, Li H, Elmets CA, et al. (2006) CD8+ IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses. J Immunol 177: 6852–6858.
- 31. He D, Wu L, Kim HK, Li H, Elmets CA, et al. (2009) IL-17 and IFN-gamma mediate the elicitation of contact hypersensitivity responses by different mechanisms and both are required for optimal responses. J Immunol 183: 1463–1470.
- 32. Hirata T, Furie BC, Furie B (2002) P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin. J Immunol 169: 4307–4313.
- 33. Nakae S, Komiyama Y, Nambu A, Sudo K, Iwase M, et al. (2002) Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 17: 375–387.
- 34. Niesner U, Albrecht I, Janke M, Doebis C, Loddenkemper C, et al. (2008) Autoregulation of Th1-mediated inflammation by twist1. J Exp Med 205: 1889–1901.
- 35. Ando K, Guidotti LG, Wirth S, Ishikawa T, Missale G, et al. (1994) Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol 152: 3245–3253.
- 36. Martins-Leite P, Gazzinelli G, Alves-Oliveira LF, Gazzinelli A, Malaquias LC, et al. (2008) Effect of chemotherapy with praziquantel on the production of cytokines and morbidity associated with schistosomiasis mansoni. Antimicrob Agents Chemother 52: 2780–2786.
- 37. Iwakura Y, Nakae S, Saijo S, Ishigame H (2008) The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunol Rev 226: 57–79.
- 38. Cua DJ, Hinton DR, Kirkman L, Stohlman SA (1995) Macrophages regulate induction of delayed-type hypersensitivity and experimental allergic encephalomyelitis in SJL mice. Eur J Immunol 25: 2318–2324.
- 39. Erard F, Wild MT, Garcia-Sanz JA, Le Gros G (1993) Switch of CD8 T cells to noncytolytic CD8−CD4− cells that make TH2 cytokines and help B cells. Science 260: 1802–1805.
- 40. Croft M, Carter L, Swain SL, Dutton RW (1994) Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J Exp Med 180: 1715–1728.
- 41. Kienzle N, Buttigieg K, Groves P, Kawula T, Kelso A (2002) A clonal culture system demonstrates that IL-4 induces a subpopulation of noncytolytic T cells with low CD8, perforin, and granzyme expression. J Immunol 168: 1672–1681.
- 42. Yen HR, Harris TJ, Wada S, Grosso JF, Getnet D, et al. (2009) Tc17 CD8 T cells: functional plasticity and subset diversity. J Immunol 183: 7161–7168.
- 43. Zou Q, Wu B, He X, Zhang Y, Kang Y, et al. (2010) Increasing a robust antigen-specific cytotoxic T lymphocyte response by FMDV DNA vaccination with IL-9 expressing construct. J Biomed Biotechnol 2010: 562356.
- 44. Zhao Y, Balato A, Fishelevich R, Chapoval A, Mann DL, et al. (2009) Th17/Tc17 infiltration and associated cytokine gene expression in elicitation phase of allergic contact dermatitis. Br J Dermatol 161: 1301–1306.
- 45. Nigam P, Kwa S, Velu V, Amara RR (2011) Loss of IL-17-producing CD8 T cells during late chronic stage of pathogenic simian immunodeficiency virus infection. J Immunol 186: 745–753.
- 46. Kondo T, Takata H, Matsuki F, Takiguchi M (2009) Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17. J Immunol 182: 1794–1798.
- 47. Huber M, Heink S, Grothe H, Guralnik A, Reinhard K, et al. (2009) A Th17-like developmental process leads to CD8(+) Tc17 cells with reduced cytotoxic activity. Eur J Immunol 39: 1716–1725.
- 48. Hamada H, Garcia-Hernandez Mde L, Reome JB, Misra SK, Strutt TM, et al. (2009) Tc17, a unique subset of CD8 T cells that can protect against lethal influenza challenge. J Immunol 182: 3469–3481.
- 49. Kohyama S, Ohno S, Isoda A, Moriya O, Belladonna ML, et al. (2007) IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17. J Immunol 179: 3917–3925.
- 50. Oyoshi MK, Elkhal A, Kumar L, Scott JE, Koduru S, et al. (2009) Vaccinia virus inoculation in sites of allergic skin inflammation elicits a vigorous cutaneous IL-17 response. Proc Natl Acad Sci U S A 106: 14954–14959.
- 51. Sallberg M, Hughes J, Javadian A, Ronlov G, Hultgren C, et al. (1998) Genetic immunization of chimpanzees chronically infected with the hepatitis B virus, using a recombinant retroviral vector encoding the hepatitis B virus core antigen. Hum Gene Ther 9: 1719–1729.
- 52. Nair S, Perrillo RP (2001) Serum alanine aminotransferase flares during interferon treatment of chronic hepatitis B: is sustained clearance of HBV DNA dependent on levels of pretreatment viremia? Hepatology 34: 1021–1026.