Potential of immunotherapy for hepatocellular carcinoma

Clinical Application of Basic Science

Potential of immunotherapy for hepatocellular carcinoma Ekaterina Breous, Robert Thimme⇑ Department of Medicine II, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, T cell-based immunotherapy is considered a promising treatment. In this review, we summarize current knowledge of T cell responses against tumour-associated antigens, as well as the mechanisms underlying the poor quality of these responses in patients with HCC. Insights into these important aspects of HCC immunology are crucial for the further development of novel immunotherapies. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

of tumour cells by inducing or boosting the existing tumour-specific immune response. The rationale for immunotherapy for HCC is based, for example, on the finding that patients with tumours containing infiltrating, presumably tumour-specific effector T cells, had a reduced risk of tumour recurrence following liver transplantation [5]. Moreover, anti-CD3 and IL-2 stimulated autologous T lymphocytes infused in HCC patients significantly improved post-surgical recurrence-free survival [6]. These data imply a central role of T cells in modulating tumour progression and provide strong justification for T cell immunotherapy.

TAA-directed T cell responses

The need for immunotherapy Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide with over 600,000 patients dying from this disease annually [1]. Liver transplantation and tumour resection have proven to be the most effective standard therapies and provide 5-year survival rates of 70% for patients within the Milan criteria, i.e., single tumour 65 cm in size or up to three tumours 63 cm in size [2]. These rates reach 50% with radiofrequency ablation and transarterial chemoembolization, which are the next preferred lines of therapy. However, these therapeutic procedures most often do not provide a complete cure, as half of the treated patients experience tumour recurrence within 3 years. A further major problem is that only 30–40% of patients are eligible for the above-described treatments due to the fact that HCC frequently remains undiagnosed until an advanced stage has been reached. These patients have a median survival time ranging from 3 to 16 months [3]. Thus, there is currently ongoing development and testing of alternative drug-based therapies for HCC that target tumour signalling pathways and vasculature. However, so far, the only drug that significantly prolonged survival (by nearly 3 months) in patients with advanced HCC is sorafenib, a multi-targeted tyrosine kinase inhibitor [4]. In view of these facts, new strategies are required. Immunotherapy aims to provide a more efficient and selective targeting

Keywords: Hepatocellular carcinoma (HCC); Tumour antigens; T cell responses; Immune suppressor mechanisms; Immunotherapy. Received 12 October 2010; accepted 29 October 2010 ⇑ Corresponding author. Tel.: +49 761 2703280; fax: +49 761 2703725. E-mail address: [email protected] (R. Thimme).

A prerequisite for the successful development of T cell-based immunotherapeutic approaches is the identification and characterization of immune responses to tumour-associated antigens (TAAs). So far, six HCC-specific TAAs that are targeted by T cells have been identified (summarized in Table 1).

a-Fetoprotein (AFP) AFP is expressed during foetal development but is transcriptionally repressed shortly after birth. However, it is re-expressed in the majority of HCC patients. Since it is a self-protein, it has been argued that AFP-specific T cell responses are suppressed and thus difficult to activate. Nevertheless, AFP is currently the best studied and most promising target antigen for HCC immunotherapy. In pioneering studies, the group of Butterfield and Economou identified an HLA-A2-restricted AFP-specific CD8 T cell epitope and demonstrated its ability to generate CD8+ T cell responses both in human lymphocyte cultures and in HLA-A2 transgenic mice [7]. The authors further identified four dominant and 10 subdominant AFP-specific epitopes [8–10] (Table 1), all of which induced low to moderate CD8+ T cell responses in PBMCs of HCC patients [11,12]. We recently compared AFP-specific CD8+ T cell responses in PBMCs with those in tumour-infiltrating lymphocytes (TILs) and surrounding non-tumour liver tissue by stimulating lymphocytes with 18-mer overlapping peptides spanning the entire AFP protein [13] (Table 1). Interestingly, we found T cell responses against several previously unidentified epitopes without a clear immunodominance, indicating that more AFP-specific epitopes exist and need to be identified. These results also indicate that CD8+ T cells specific for the self-antigen AFP are present in the normal T cell repertoire and are not centrally or peripherally deleted.

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JOURNAL OF HEPATOLOGY Table 1. TAAs triggering T cell responses in HCC patients.

Antigen

Frequency of expression

AFP

<80%

CD8+ T cell responses

T cell detection method

Presenting HLA

Reference

6/6 patients to dominant epitopes AFP137-145, AFP158-166 , AFP325-334 and AFP542-550

Ex vivo ELISPOT and tetramer staining on PBMCs

A0201

Butterfield, 2003 [12]

6/6 patients to AFP137-145, AFP158-166, AFP325-334, AFP542-550 and subdominant epitopes

ELISPOT on blood CD8+ T cells stimulated with epitopes for 3 weeks

A0201

Liu, 2006 [11]

24/40 patients to 18-mer peptides not comprising above-described epitopes

ELISPOT and intracellular cytokine staining (ICS) on blood and TIL CD8+ T cells non-specifically expanded for 2 weeks

Diverse

Thimme, 2008 [13]

GPC3

<70%

11/22 patients to GPC144-152, GPC298-306

Cytotoxicity assay on blood CD8+ T cells stimulated with epitope-pulsed DCs for 2 weeks

A2 and A24

Komori, 2006 [14]

NY-ESO-1

<50%

5/6 patients to NY-ESO-1157-165

ICS on PBMCs stimulated with epitope for 1 week

A2

Korangy, 2004 [16]

10/28 patients to NY-ESO-1157-165

ELISPOT and tetramer staining on blood CD8+ T cells stimulated with epitope for 2-3 weeks

A2

Shang, 2004 [17]

6/10 patients to NY-ESO-1157-165 with alanine substitution at position 165

ICS on PBMCs stimulated with epitope for 2 weeks

A2

Gehring, 2009 [15]

3/6 patients to SSX-241-49

Tetramer staining on + blood and TIL CD8 T cells stimulated with epitope-pulsed DCs for 3-4 weeks and then with PHA prior to analysis

A2

Bricard, 2005 [18]

3/10 patients to SSX-241-49

ICS same as for NY-ESO-1

A2

Gehring, 2009 [15]

1/6 patients to MAGE-1161-169 1/6 patients to MAGE-1168-176

Tetramer staining on PBMCs and TILs stimulated with epitopes for 2 weeks

A1 and A2

Zerbini, 2004 [19]

3/6 patients to

Tetramer staining same as for SSX-2

A2

Bricard, 2005 [18]

Ex vivo ELISPOT on PBMCs and TILs

A24

Mizukoshi, 2006 [20]

SSX-2

MAGE-A

TERT

<50%

<80%

<80%

MAGE-10254-262

44/72 patients to TERT1088-1096, TERT845-853, TERT 167-175, TERT461-469, TERT324-332, TERT637-645

shown to elicit T cell responses in HCC patients after long-term in vitro stimulation of PBMCs [14] (Table 1).

Glypican-3 (GPC3) GPC3 is a foetal oncoprotein. It belongs to a family of heparan sulphate proteoglycans that can bind growth factors (e.g., Wnt, FGF1/2, and VEGF) and thereby promote tumour growth. GPC3 was recently found to be over-expressed in HCC and is considered a promising target antigen. Two HLA-A2- and HLA-24-restricted CD8 T cell epitopes of GPC3 have been mapped in mice and

NY-ESO-1 Expression of NY-ESO-1 is limited to testis in healthy subjects, however, it is often expressed de novo in HCC and other cancers such as melanoma, ovarian, and breast cancer, with no apparent

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Clinical Application of Basic Science gender bias. NY-ESO-1 is one of the most immunogenic TAAs known to date, with several identified epitopes currently being tested as potential vaccine candidates for cancers other than HCC. By using different approaches, three groups reported NYESO-1-specific CD8+ T cell responses in the peripheral blood of HCC patients to an epitope that had already been identified in melanoma patients [15–17] (Table 1). None of the observed T cell responses were, however, detectable ex vivo and required several weeks of in vitro lymphocyte expansion, suggesting a relatively low frequency of these cells in the circulation. SSX-2 SSX-2 is another cancer-testis antigen that has been recently demonstrated to be over-expressed in HCC patients. By using a CD8 T cell epitope that had been previously identified in melanoma patients, two groups detected CD8+ T cell responses in a small fraction of HCC patients after in vitro expansion of PBMCs and TILs [15,18] (Table 1). Melanoma antigen gene-A (MAGE-A) Antigens of the MAGE-A family were originally characterized in melanoma, but they are also widely expressed in other tumours such as breast carcinoma, glioma, colorectal carcinoma, and HCC. Zerbini et al. demonstrated in vitro induction of CD8+ T cell responses against previously identified MAGE-A1 and MAGE-A3 epitopes in tumour-infiltrating but not peripheral blood lymphocytes derived from HCC patients [19] (Table 1). Similarly, CD8+ T cell responses against MAGE-A10 epitope have also been described by Bricard et al. [18]. Telomerase reverse transcriptase (TERT) TERT is a catalytic enzyme required for telomere elongation; its expression correlates with telomerase activity. Tumours must retain the length of their telomeres and telomerase helps them to do so by maintaining telomeric ends of linear chromosomes and protecting them from degradation. It is, therefore, not surprising that telomerase has been reported to be activated in up to 85% of all human cancers, including HCC, whereas TERT has been proposed as a universal TAA for cancer immunotherapy. Interestingly, Mizukoshi and colleagues reported ex vivo measurable weak CD8+ T cell responses to six TRET epitopes in the peripheral blood of HCC patients [20] (Table 1). In summary, these studies show that although TAA-specific T cells are detectable in patients with HCC, the frequency of these responses is low overall and in vitro stimulation is often required for their detection.

Mechanisms responsible for inefficient T cell responses Several mechanisms could contribute to the weak and often inefficient TAA-specific CD8+ T cell responses in HCC patients (summarized in Fig. 1). Regulatory T cells (Tregs) Tregs are the best characterized suppressor cells and have been shown to suppress tumour immunity in numerous studies. In HCC patients, Alexander and co-workers reported increased num832

bers of CD4+CD25+ Tregs in TILs, compared to distant tumour tissue [21]. These findings were reinforced by the group of Korangy and Greten who also reported increased frequencies of CD4+CD25+ Tregs in TILs and PBMCs of HCC patients [22]. When activated with CD3 and CD28 antibodies, these Tregs were found to produce the immunosuppressive cytokine IL-10. Subsequently, increased Tregs in TILs and PBMCs were shown to express Foxp3 and to inhibit CD3/CD28 stimulated CD8+ T cell proliferation, activation, and production of granzymes and perforin [23]. These combined results suggest that Tregs play a major role in the inhibition of tumour-specific T cell responses in HCC. Further, this is supported by the finding that depletion of Tregs resulted in the emergence of tumour-specific CD4+ T cell responses in six out of 16 HCC patients [24]. However, antigen-specificity of Tregs, their functional and phenotypical profiles, as well as mechanisms of suppression remain to be characterized in more detail. Myeloid-derived suppressor cells (MDSCs) MDSCs comprise a mixture of monocytes/macrophages, granulocytes, and dendritic cells (DCs) at different stages of differentiation. In patients with HCC, blood MDSCs have recently been shown to induce Foxp3 and IL-10 expression in CD4+ T cells via arginase activity [25]. There is currently no specific drug or antibody available that selectively targets MDSCs. Since inhibition of arginase activity can cause side effects, due to the critical role of this enzyme in the urea cycle, it will be important to identify additional specific markers to target these cells. Impairment of TAA processing and presentation Several studies have shown that expression of HLA class I molecules and B7 costimulatory molecules is downregulated in HCC tissue [26] and HCC cell lines [27,28]. Such downregulation is likely to lead to impaired processing of TAAs. Moreover, it has been shown that circulating myeloid DCs in HCC patients were decreased in numbers and had reduced cytokine production [29], raising the possibility that TAA presentation by DCs may also be impaired. Further work is required to determine the extent and mechanisms of such impairment. Inhibitory receptors Many human cancers express PD-L1, the ligand for the inhibitory receptor programmed cell death-1 (PD-1). Tumour-associated PD-L1 has been shown to induce apoptosis of effector T cells and is thought to contribute to immune evasion by cancers. In HCC, tumour infiltrating CD8+ T cells are characterized by an increase in PD-1 expression [30]. Interestingly, intratumoural Kupffer cells have been shown to up-regulate PD-L1 and decrease the effector function of PD-1-expressing CD8+ T cells in HCC patients [31,32]. It should be noted that cells with a MDSC phenotype also up-regulated PD-L1 in these studies and exerted a similar inhibitory effect on T cell activation, raising the possibility of MDSC-mediated T cell suppression. Together these data suggest that the inhibition of PD-1 may be a potential strategy in the boosting of HCC-specific immunity. It will be important to determine whether similar findings may be observed for other important inhibitory receptors such as, for instance, CTLA-4, BTLA, or Tim-3, and whether co-expression of these inhibitory receptors is present in HCC, as has been described in other tumour models [33,34].

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JOURNAL OF HEPATOLOGY DC

TAA

Failure to kill

+

Effector CD8

Kupffer cell

TAA

Tumor cell

PD-L1

PD-1

PD-L1

PD-1

MDSC 0 IL-1

Helper CD4+ Treg Fig. 1. Multiple mechanisms may limit TAA-specific CD8+ T cell responses in HCC. Failure of TAA processing and presentation; insufficient levels of CD4 help; suppression of both CD4+ and CD8+ T cells by Tregs; negative regulation by PD-1/PD-L1 pathway.

Lack of CD4+ T cell responses It is known that a lack of CD4+ T cell help may lead to CD8+ T cell exhaustion. In fact, in HCC, AFP-specific CD4+ T cell responses were only present in patients with early stage disease and became exhausted as the disease progressed [35]. Thus, for the activation of fully functional cytotoxic T lymphocytes it will be important to identify TAA-derived CD4 T helper cell epitopes and include them in a vaccine along with CD8 T cell epitopes. It is thus clear that the mechanisms underlying weak tumourspecific T cell responses are complex, and their further in-depth exploration is required for successful vaccine development. However, a few vaccination approaches have already been tested, resulting in the generation of modest TAA-directed T cell responses in HCC patients that will be summarized in the following section. Vaccine strategies The group of Butterfield and Economou was the first to conduct an HCC vaccine clinical trial [12]. In this phase I trial, six HLA-A0201+ patients were immunized with AFP CD8 T cell epitopes, resulting in the generation of measurable AFP-specific T cell responses in the peripheral blood. In the consecutive phase I/II trial, the authors attempted to further enhance these responses by loading autologous DCs with AFP epitopes and administering them to 10 HLA-A0201+ patients [36]. This treatment, however, resulted only in transient CD8+ T cell responses, possibly caused by the lack of CD4 help. To overcome this limitation, a subsequent clinical trial

by the group of Adams used DCs pulsed with a lysate of the hepatoblastoma cell line HepG2 before transfusion into 35 HCC patients with diverse HLA types [37]. Such an approach of using apoptotic tumour cells as a source of multivalent antigens appears to be ideal at the moment due to poor characterization of HCC TAAs, and should allow the activation of both CD8+ and CD4+ T cells. Indeed, the authors reported functional IFN-c-producing T cells to vaccine and/or AFP in five patients, disease stabilization for 6–16 months in six patients and reductions in serum AFP levels in three patients. These positive results suggest that further improvement of DC-based vaccination strategies, such as the use of multiple TAA epitopes stimulating both CD8+ and CD4+ T cells, may translate in more effective clinical results. Perspectives for immunotherapy Although the above-described vaccine approaches yielded the most successful outcomes observed in T cell-based HCC immunotherapies so far, the achieved tumour-specific T cell responses were often not robust enough to induce clinical responses. In addition to HCC vaccines still being in early development, the observed modest clinical efficacy may be explained by the strong tolerogenic environment at the tumour site and different tumour suppressive mechanisms described above. Clearly, treatment strategies combining blockade of immunoregulatory cell types such as Tregs and MDSCs and of inhibitory receptors, with vaccine-induced activation of TAA-specific T cells may be necessary to achieve the most effective therapeutic anti-tumour activity in HCC. Also, a combination with conventional HCC treatments, such

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Clinical Application of Basic Science as transarterial embolization or local tumour ablation may increase HCC immunogenicity and unmask TAA-specific T cell responses [38]. In conclusion, tremendous progress has been made in the field of immunotherapies for HCC in recent years. However, the repertoire of HLA class I and class II presented TAA-specific T cell epitopes needs to be expanded, and the mechanisms of T cell failure require further investigation before immunotherapy of HCC can successfully enter the clinical stage. Conflict of interest

[18]

[19]

[20]

[21]

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors do not have a relationship with the manufacturers of the drug involved either in the past or present and did not receive funding from the manufacturers to carry out their research.

[22]

[23]

[24]

References [1] Schutte K, Bornschein J, Malfertheiner P. Hepatocellular carcinoma – epidemiological trends and risk factors. Dig Dis 2009;27(2):80–92. [2] Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008;48(4):1312–1327. [3] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362(9399):1907–1917. [4] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359(4):378–390. [5] Unitt E, Marshall A, Gelson W, Rushbrook SM, Davies S, Vowler SL, et al. Tumour lymphocytic infiltrate and recurrence of hepatocellular carcinoma following liver transplantation. J Hepatol 2006;45(2):246–253. [6] Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000;356(9232):802–807. [7] Butterfield LH, Koh A, Meng W, Vollmer CM, Ribas A, Dissette V, et al. Generation of human T-cell responses to an HLA-A2.1-restricted peptide epitope derived from alpha-fetoprotein. Cancer Res 1999;59(13):3134–3142. [8] Butterfield LH, Meng WS, Koh A, Vollmer CM, Ribas A, Dissette VB, et al. T cell responses to HLA-A⁄0201-restricted peptides derived from human alpha fetoprotein. J Immunol 2001;166(8):5300–5308. [9] Meng WS, Butterfield LH, Ribas A, Dissette VB, Heller JB, Miranda GA, et al. Alpha-fetoprotein-specific tumor immunity induced by plasmid primeadenovirus boost genetic vaccination. Cancer Res 2001;61(24):8782–8786. [10] Meng WS, Butterfield LH, Ribas A, Heller JB, Dissette VB, Glaspy JA, et al. Fine specificity analysis of an HLA-A2.1-restricted immunodominant T cell epitope derived from human alpha-fetoprotein. Mol Immunol 2000;37(16):943–950. [11] Liu Y, Daley S, Evdokimova VN, Zdobinski DD, Potter DM, Butterfield LH. Hierarchy of alpha fetoprotein (AFP)-specific T cell responses in subjects with AFP-positive hepatocellular cancer. J Immunol 2006;177(1):712–721. [12] Butterfield LH, Ribas A, Meng WS, Dissette VB, Amarnani S, Vu HT, et al. Tcell responses to HLA-A⁄0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer. Clin Cancer Res 2003;9(16 Pt. 1):5902–5908. [13] Thimme R, Neagu M, Boettler T, Neumann-Haefelin C, Kersting N, Geissler M, et al. Comprehensive analysis of the alpha-fetoprotein-specific CD8+ T cell responses in patients with hepatocellular carcinoma. Hepatology 2008; 48(6):1821–1833. [14] Komori H, Nakatsura T, Senju S, Yoshitake Y, Motomura Y, Ikuta Y, et al. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin Cancer Res 2006;12(9):2689–2697. [15] Gehring AJ, Ho ZZ, Tan AT, Aung MO, Lee KH, Tan KC, et al. Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma. Gastroenterology 2009;137(2):682–690. [16] Korangy F, Ormandy LA, Bleck JS, Klempnauer J, Wilkens L, Manns MP, et al. Spontaneous tumor-specific humoral and cellular immune responses to NYESO-1 in hepatocellular carcinoma. Clin Cancer Res 2004;10(13):4332–4341. [17] Shang XY, Chen HS, Zhang HG, Pang XW, Qiao H, Peng JR, et al. The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b

834

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

peptide in hepatocellular carcinoma patients. Clin Cancer Res 2004; 10(20):6946–6955. Bricard G, Bouzourene H, Martinet O, Rimoldi D, Halkic N, Gillet M, et al. Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma. J Immunol 2005;174(3):1709–1716. Zerbini A, Pilli M, Soliani P, Ziegler S, Pelosi G, Orlandini A, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding genespecific CD8+ cells in patients with hepatocellular carcinoma. J Hepatol 2004;40(1):102–109. Mizukoshi E, Nakamoto Y, Marukawa Y, Arai K, Yamashita T, Tsuji H, et al. Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma. Hepatology 2006;43 (6):1284– 1294. Unitt E, Rushbrook SM, Marshall A, Davies S, Gibbs P, Morris LS, et al. Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells. Hepatology 2005;41(4):722–730. Ormandy LA, Hillemann T, Wedemeyer H, Manns MP, Greten TF, Korangy F. Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma. Cancer Res 2005;65(6):2457–2464. Fu J, Xu D, Liu Z, Shi M, Zhao P, Fu B, et al. Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients. Gastroenterology 2007;132(7):2328–2339. Greten TF, Ormandy LA, Fikuart A, Hochst B, Henschen S, Horning M, et al. Low-dose cyclophosphamide treatment impairs regulatory T cells and unmasks AFP-specific CD4+ T-cell responses in patients with advanced HCC. J Immunother 2010;33(2):211–218. Hoechst B, Ormandy LA, Ballmaier M, Lehner F, Kruger C, Manns MP, et al. A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells. Gastroenterology 2008;135(1):234–243. Fujiwara K, Higashi T, Nouso K, Nakatsukasa H, Kobayashi Y, Uemura M, et al. Decreased expression of B7 costimulatory molecules and major histocompatibility complex class-I in human hepatocellular carcinoma. J Gastroenterol Hepatol 2004;19(10):1121–1127. Kurokohchi K, Carrington M, Mann DL, Simonis TB, Alexander-Miller MA, Feinstone SM, et al. Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma. Hepatology 1996;23(5):1181–1188. Tatsumi T, Takehara T, Katayama K, Mochizuki K, Yamamoto M, Kanto T, et al. Expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on human hepatocellular carcinoma. Hepatology 1997;25(5):1108–1114. Ormandy LA, Farber A, Cantz T, Petrykowska S, Wedemeyer H, Horning M, et al. Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma. World J Gastroenterol 2006;12(20):3275–3282. Shi F, Shi M, Zeng Z, Qi RZ, Liu ZW, Zhang JY, et al. PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients. Int J Cancer 2010. April 19 [Epubahead of print]. Wu K, Kryczek I, Chen L, Zou W, Welling TH. Kupffer cell suppression of CD8+ T cells in human hepatocellular carcinoma is mediated by B7-H1/programmed death-1 interactions. Cancer Res 2009;69(20):8067–8075. Kuang DM, Zhao Q, Peng C, Xu J, Zhang JP, Wu C, et al. Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1. J Exp Med 2009;206(6):1327–1337. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207(10):2187–2194. Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescher IF, Sander C, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010;207(10):2175–2186. Behboudi S, Alisa A, Boswell S, Anastassiou J, Pathan AA, Williams R. Expansion of anti-AFP Th1 and Tc1 responses in hepatocellular carcinoma occur in different stages of disease. Br J Cancer 2010;102(4):748–753. Butterfield LH, Ribas A, Dissette VB, Lee Y, Yang JQ, De la Rocha P, et al. A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. Clin Cancer Res 2006;12(9):2817–2825. Palmer DH, Midgley RS, Mirza N, Torr EE, Ahmed F, Steele JC, et al. A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma. Hepatology 2009;49(1): 124–132. Ali MY, Grimm CF, Ritter M, Mohr L, Allgaier HP, Weth R, et al. Activation of dendritic cells by local ablation of hepatocellular carcinoma. J Hepatol 2005;43(5):817–822.

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