Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8+ T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire alpha-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8+ T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon-γ (IFN-γ)-producing CD8+ T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8+ T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8+ T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8+ T-cell proliferation but did not restore IFN-γ-production. Conclusion: Naturally occurring TAA-specific CD8+ T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8+ T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8+ T-cell responses. (Hepatology 2014;59:1415-1426)
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite its growing incidence, however, therapeutic options remain limited. Consequently, HCC patients suffer from a high mortality rate. New therapies for HCC are therefore urgently required. Immunotherapy is a promising approach for the treatment of HCC. The rationale for immunological intervention is based on the presence of high numbers of tumor-infiltrating T cells in HCC tissue, the correlation between the density of lymphocytic infiltrates in HCC lesions and prognosis, and, most importantly, the finding that adoptive immunotherapy with interleukin (IL)−2/anti-CD3-stimulated autologous lymphocytes lowers postsurgical recurrence rates in humans. The central effectors in this scenario are CD8+ T cells that recognize tumor-associated antigens (TAA) and kill tumor cells. TAA comprise a range of self-derived proteins rendered immunogenic in tumors either by mutation or aberrant expression. A variety of different TAA can spontaneously induce CD8+ T-cell responses in HCC patients; these include α-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1), and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1).[7-10] In addition, TAA-specific responses can also be boosted in vivo in HCC patients by dendritic cell-based vaccination with tumor lysate.
The identification of TAA that are frequently recognized by CD8+ T cells in HCC patients could provide important insights into the choice of appropriate targets for immunotherapy. However, most previous studies focused on single TAA, thus precluding within-patient comparisons. Indeed, to our knowledge, only two previous studies have compared CD8+ T-cell responses to different TAA in HCC patients.[12, 13] Moreover, these studies were limited to analyses of previously described epitopes restricted by human leukocyte antigen (HLA)-A*02 and HLA-A*24, respectively.
In this study we used overlapping peptides spanning the entire sequences of AFP, GPC-3, MAGE-A1, and NY-ESO-1 in a cohort of 96 HCC patients to evaluate naturally occurring CD8+ T-cell responses against four major HCC-associated TAA irrespective of HLA restriction. Our results provide the first comprehensive view of TAA-specific CD8+ T-cell responses in this setting with attendant implications for therapeutic vaccine design.
In this study we used overlapping peptides spanning the entirety of the AFP, GPC-3, MAGE-A1, and NY-ESO-1 proteins to conduct a comprehensive and unbiased analysis of immunodominant TAA-specific CD8+ T-cell responses in patients with HCC. Several important findings emerged from our investigation.
First, antigen-specific CD8+ T-cell responses directed against all four TAA analyzed were readily observed in more than 50% of HCC patients, significantly greater than the corresponding frequency of detection in healthy donors or patients with viral hepatitis. This is in agreement with our earlier study focused on AFP. In addition, these TAA-specific CD8+ T-cell responses were more frequently detectable in patients with very early tumor stage. It is important to note that some of these patients had undergone prior locoregional therapy. In this setting it has been shown that especially RFTA enhances and/or induces TAA-specific CD8+ T-cell responses in individual patients.[15, 16] We show that this effect is also detectable for patients treated with TACE. Indeed, it may contribute to the observed enrichment of CD8+ T-cell responses in patients with early stage HCC and supports the view that the release of autologous TAA can induce and/or boost TAA-specific CD8+ T-cell responses.[15, 16]
Second, the occurrence of TAA-specific CD8+ T-cell responses in HCC patients was associated with improved PFS. Our results suggest that TAA-specific CD8+ T-cell responses may indeed be a prognostic factor of patient survival that appears to be independent of the potentially confounding factors of BCLC stage and prior treatment. The mentioned association of strong TAA-specific CD8+ T-cell responses with PFS in patients undergoing locoregional therapy further supports a possible protective role of such responses.[16, 19] Especially the occurrence of CD8+ T-cell responses specific for multiple TAA may be beneficial. Even though this aspect requires confirmation in a larger cohort, we found significantly superior PFS in patients with CD8+ T-cell responses to one or two TAA and a similar trend for those with responses to three or four TAA. Furthermore, we observed only one case of tumor progression among the four patients with responses against all four TAA. Based on these data, it is tempting to speculate that TAA-specific CD8+ T-cell responses may benefit patient survival by hindering tumor growth and thus maintaining early stage disease for longer periods of time.
Third, CD8+ T-cell responses specific for individual TAA displayed clear immunodominance patterns. Immunodominant regions are preferential targets for T-cell responses because they are immunogenic in most individuals within a cohort. Indeed, even in our HLA-independent assay, we observed that most CD8+ T-cell responses targeted single immunodominant regions within a given TAA. Despite our inability to establish a clear immunodominance profile for AFP in a previous study, we were able to identify an immunodominant region at the N-terminus by using the larger cohort studied here. In addition, we could identify immunodominant regions in the middle of MAGE-A1 and at the C-termini of GPC-3 and NY-ESO-1. It is important to note, however, that the protein regions identified by overlapping peptides in our approach may contain several CD8+ T-cell epitopes restricted by different HLA-molecules. In this study, almost a third of the overlapping peptides that triggered responses contained previously described CD8+ T-cell epitopes. For example, the immunodominant region of NY-ESO-1 contains the epitope NY-ESO-1157-165 which is frequently recognized by HLA-A*02+ HCC patients. However, given that only 58% of patients with a response to the corresponding NY-ESO-1-peptide were HLA-A*02+, it is likely that this region of NY-ESO-1 contains additional, unknown CD8+ T-cell epitopes restricted by other HLA-molecules. Of note, we were able to characterize three novel CD8+ T-cell epitopes within the immunodominant region of GPC-3 and within subdominant regions of GPC-3 and NY-ESO-1, respectively. Previous studies comparing different TAA focused on epitopes restricted by single HLA-molecules. For HLA-A*02, the NY-ESO-1157-165 epitope is thought to be immunodominant compared to similarly restricted epitopes derived from other TAA. However, for HLA-A*24, no single epitope or TAA was found to be immunodominant. These results underline the importance of HLA-independent analyses. Indeed, to our knowledge, this is the first study that compares TAA-specific CD8+ T-cell responses in HCC patients irrespective of HLA-restriction. Accordingly, our results have important implications for immunotherapy and tumor vaccine design.
Fourth, functional TAA-specific CD8+ T cells of HCC patients cannot readily be expanded in vitro. Indeed, we could not detect peptide-specific production of IFN-γ after antigen-specific culture of CD8+ T cells with epitopes derived from MAGE-A1 and NY-ESO-1. These results are in line with previous studies showing that CD8+ T-cell lines specific for either AFP or GPC-3 fail to produce IFN-γ.[7, 8] However, when we used tetramers representing epitopes derived from MAGE-A1 and NY-ESO-1 to detect TAA-specific CD8+ T cells independent of their functionality, we were able to find those cells in more than 50% of HCC patients. The presence of tetramer-binding TAA-specific CD8+ T cells after in vitro expansion, but not ex vivo (data not shown), indicates proliferation of a very low frequency precursor population. Thus, cytokine production rather than proliferation may be defective in HCC-specific CD8+ T cells. This is consistent with other studies reporting dysfunctional TAA-specific CD8+ T cells.[8, 9, 12] It is also a particularly important observation given that the induction of TAA-specific IFN-γ production by T cells has been proposed as a major determinant of success in therapeutic tumor vaccine trials targeting other malignancies. Furthermore, we found a limited expression of cytotoxic mediators by the expanded TAA-specific CD8+ T cells. While FasL was produced, expression of perforin or granzyme B was often not detectable. Indeed, a reduced ex vivo expression of perforin and granzyme B among CD8+ TIL in HCC patients has been described. However, in contrast to HCV-specific CD8+ T cells that also lack expression of perforin ex vivo, TAA-specific CD8+ T cells were mostly incapable of up-regulating perforin after antigen-specific expansion.
Several mechanisms may explain this dysfunctional phenotype of TAA-specific CD8+ T cells. First, the immunosuppressive tumor microenvironment may be responsible, for example, by the production of immunoregulatory cytokines or by the local action of inhibitory receptors such as PD-1, as described recently in a mouse model.[22, 23] This is in line with our observation of significantly reduced magnitudes of TAA-specific CD8+ T-cell responses at the tumor site compared to the periphery. Furthermore, we detected heterogeneous expression of the inhibitory receptors PD-1 and Tim-3 on antigen-specifically expanded TAA-specific CD8+ T cells and effects of PD-L1 blockade on individual cell lines that were in one case even sufficient to restore the production of IFN-γ. Second, TAA-specific T-cell receptors (TCRs) may have very low affinities for peptide-HLA complexes relative to virus-specific TCRs.[24, 25] Consequently, physiological stimulation of TAA-specific CD8+ T cells with the cognate peptide may deliver a signal that is too weak to restore functionality. This may also explain why IFN-γ production was preserved after antigen-independent expansion, which relies on direct crosslinking of CD3ζ rather than TCR engagement. Third, a lack of CD4+ T cell help and the action of Treg may also contribute to the observed functional defects.[18, 26] Indeed, the important role of Treg in the immunobiology of HCC is suggested by the enrichment of these cells in HCC patients, a negative association with patient survival, and the unmasking of AFP-specific CD4+ T-cell responses after in vivo depletion of Treg in HCC patients.[18, 27] Of note, we also observed Treg enrichment in tumor and liver tissue in HCC patients. In addition, the depletion of Treg prior to antigen-specific stimulation increased the proliferation of TAA-specific CD8+ T cells in 60% of cases. However, these expanded TAA-specific CD8+ T cells still failed to produce IFN-γ, suggesting that different or additional mechanisms beyond Treg-mediated effects may contribute to the lack of cytokine production observed in our study and that addressing single pathways contributing to T-cell failure may be insufficient to restore CD8+ T-cell functionality.
In summary, we have shown that immunodominant TAA-specific CD8+ T-cell responses in HCC patients are spread across multiple antigens and associated with patient survival. However, the expansion of functional TAA-specific CD8+ T cells from patients with HCC is difficult due to functional impairments. A better understanding of the mechanisms that underlie this dysfunctionality is required to expedite the development of effective immunotherapies for HCC.
The authors thank all patients for participating in the study, Katja Nitschke and Julia Schmidt for revision of the article, and Melanie Frank for help with statistical analysis. Recombinant human IL-2 was kindly provided by the NIH AIDS reagent program. DAP is a Wellcome Trust Senior Investigator.