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Unmasking of -Fetoprotein-Specific CD4⁺ T Cell Responses in Hepatocellular Carcinoma Patients Undergoing Embolization¹

Lakshmana Ayaru^*, Stephen P. Pereira^*, Akeel Alisa^*, Ansar A. Pathan^¶, Roger Williams^*, Brian Davidson, Andrew K. Burroughs, Tim Meyer and Shahriar Behboudi^2,*

^* Department of Medicine, University College London Institute of Hepatology, Academic Department of Oncology, and University Department of Surgery and Liver Transplantation, Royal Free and University College London Medical School, University College London, London, United Kingdom; Liver Transplantation and Hepatobiliary Medicine Unit, Royal Free Hospital, London, United Kingdom; ^¶ Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Necrosis of tumor cells can activate both innate and adaptive antitumor immunity. However, there is little information on the effects of necrosis-inducing cancer treatments on tumor-specific T cell immune responses in humans. We studied the effects of a necrosis-inducing treatment (embolization) on anti--fetoprotein (AFP)-specific CD4⁺ T cell responses in hepatocellular carcinoma (HCC) patients and controls using an array of AFP-derived peptides. In this study, we show that AFP-specific CD4⁺ T cell responses to three immunodominant epitopes in HCC patients were significantly expanded during (p < 0.0001) and after embolization (p < 0.002). The development of higher frequencies of AFP-specific CD4⁺ T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). In addition, we identified two novel HLA-DR-restricted AFP-derived CD4⁺ T cell epitopes (AFP_137–145 and AFP_249–258) and showed that the CD4⁺ T cells recognizing these epitopes produce Th1 (IFN- and TNF-) but not Th2 (IL-5)-type cytokines. AFP_137–145-, AFP_249–258-, and AFP_364–373-specific CD4⁺ T cells were detected in HCC patients but not in patients with chronic liver diseases or healthy donors. In conclusion; our study shows that induction of tumor necrosis by a conventional cancer treatment can unmask tumor rejection Ag cell-mediated immunity and provides a rationale for combining embolization with immunotherapy in HCC patients.

	Introduction

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Despite the spontaneous presence and induction by vaccination of tumor-specific T cell immune responses in humans, the clearance of established tumors by endogenous immune mechanisms is rare. It is increasingly recognized that tumors create an immunosuppressive network that promotes tumor growth and attenuates immunotherapeutic efficiency (1). In situ induction of tumor cell necrosis may provide a means of subverting these tolerizing conditions, for example by producing local inflammation and presenting tumor rejection Ags in an immunostimulatory context (2). In vitro and clinical vaccination studies have shown that necrotic tumor cells release danger signals that can prime Ag-specific immune responses (3, 4, 5). However, whether a necrosis-inducing cancer treatment can activate tumor rejection Ag-specific immunity in humans is unclear. If tolerance could be broken in this manner, the combination of in vivo tumor destruction and immune-potentiating strategies may improve the prognosis of patients.

Transarterial chemoembolization (TACE)³ or transarterial embolization (TAE) are the most widely used treatments for unresectable hepatocellular carcinoma (HCC) and improve survival (6). TACE or TAE involves the injection of coils or particles into the hepatic artery with or without a chemotherapeutic agent, respectively, leading to obstruction of the hepatic artery branch supplying the tumor. This produces necrosis of the well-vascularized tumor and a reduction in tumor burden, although the majority of patients eventually die of tumor progression (7). In theory, tumor progression could be reduced or prevented by expanding antitumoral immune responses.

-Fetoprotein (AFP) is an oncofetal Ag with immunoregulatory properties (8) that is re-expressed in the majority of patients with HCC and used in clinical practice as a diagnostic and prognostic serum marker (9). It also serves as a tumor rejection Ag (10) and is therefore an attractive target for immunotherapy. It has been suggested that the rationale behind an effective epitope-based therapeutic vaccine is to enhance both tumor-specific CD8⁺ and CD4⁺ T cell responses, thus controlling tumor growth (11). A number of studies have identified AFP-specific CTLs and demonstrated their presence in patients with hepatocellular carcinoma (12, 13, 14, 15, 16). CD4⁺ helper T cells are also important in generating potent anticancer immunity, as they can prime and expand CD8⁺ memory T cells (17) or induce tumor regression in the absence of CD8⁺ T cells, including MHC class II-negative or MHC class II-positive tumors (18, 19). We have recently reported the presence of CD4⁺ T cells that recognized an AFP-derived epitope in HCC patients, predominantly in an early stage of the disease (20).

We hypothesized that TACE/TAE-induced necrosis could activate and unmask AFP-specific CD4⁺ T cell responses, thereby providing an attractive window for immunotherapy. We also aimed to identify further novel AFP-derived CD4⁺ T cell epitopes that could be used in an epitope-based vaccine for HCC.

	Materials and Methods

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Synthetic peptides

In total 94 peptides spanning the AFP sequence were synthesized by Mimotopes. Sixty-one were soluble in DMSO and were divided into pools of peptides (1–10) (Table I) for analysis of T cell responses.

The study was approved by the Royal Free, Joint University College London/University College London Hospital and Cromwell Hospital ethical committees and all patients gave written informed consent. We first conducted a cross-sectional study of AFP-specific CD4⁺ T cell responses in the blood of HCC patients (Table II) and controls (Table III). The inclusion criteria for HCC patients in the cross-sectional study were as follows: 1) a diagnosis of HCC made according to the European Association for the Study of Liver disease criteria (9); 2) patients who were most likely to have an AFP_364–373-specific CD4⁺ T cell response (Okuda stage 1 and <1000 ng/ml serum AFP) (20); and 3) patients who were either treatment naive or had only received TACE/TAE as treatments. The non-HCC liver disease controls are age matched to the HCC group. All controls in this study had serum values that were <1000 ng/ml and were therefore matched in terms of the likelihood of detecting AFP-specific CD4⁺ T cell responses.

Screening for HLA-A2 positivity was performed by staining the PBMCs of patients with fluorescence-conjugated anti-HLA-A0201 Ab (Serotec) and analysis was done by flow cytometry.

Clinical response to TACE/TAE was assessed by computerized tomography (CT) using a score that correlates with survival (21). For patients whose total tumor size showed a >25% reduction, one point was assigned. For total tumor necrosis of >50%, one point was assigned. If no new hepatic lesions developed, one point was assigned. If the point total was 2 or 3, the patient was defined as a clinical responder, while a score of 0 or 1 was defined as a clinical nonresponder.

HLA-DR typing

Following centrifugation, buffy coats were separated from the red cell pellet and the white cells were frozen in freezing mix (20% FCS, 70% Terasaki Park medium, and 10% DMSO). DNA was extracted by using a modified salting out procedure. HLA-DR typing was done by molecular techniques in line with recent recommendations (22). HLA analysis was done on DNA extracted from a 5-ml sample of blood by a modified salting out technique (23). The typing systems used defined all HLA-DR specificities (24).

In vitro expansion of AFP-specific T cells

PBMCs were separated by Ficoll centrifugation. Short-term T cell lines were generated as described previously (20) to study naturally occurring AFP-specific T cell responses (25). In brief, PBMCs were resuspended in AIM-V medium (Invitrogen Life Technologies) with 10% FCS and were cultured in duplicate with peptide pools or individual peptides (1 µM). rIL-2 (25 IU/ml) was added on days 2 and 3 of culture.

Flow cytometry and intracellular cytokine assays

On days 10–12 of culture the cells were restimulated with the same peptide/pool, irrelevant peptide/pool (a different AFP-derived peptide/pool) (pools 1 and 2 were used as irrelevant peptide pools), or whole protein (13 µg/ml AFP or 13 µg/ml BSA) and incubated for 5 h at 37°C in the presence of brefeldin A.

Cells were surface stained with Cy-chrome-conjugated anti-CD4⁺ or anti-CD8⁺ Abs. The cells were then permeabilized and fixed using Cytofix/Cytoperm (BD Pharmingen). Afterward, the cells were stained for intracellular cytokines with FITC-conjugated anti IFN-, FITC-conjugated anti-TNF-, PE-conjugated anti-IL-5, or isotype controls (R & D Systems) and washed twice, and the frequency of AFP-specific T cell responses was quantified by flow cytometry.

Definitions of immunological responses

An immunological response/responder was defined as a 2-fold increase in the frequency of cytokine-producing cells above control peptides/pools or proteins. An immunological responder to TACE/TAE or surgery was defined as a 2-fold increase in an immunological response during or after treatment compared with pretreatment.

ELISPOT assay

Ex vivo AFP-specific and nontumor memory T cell responses (20 µg/ml tuberculin-purified protein derivative (PPD) (Statens Serum Institut, Copenhagen, Denmark), 250U/ml streptokinase, and 12.5U/ml streptodornase) in HCC patients undergoing TACE/TAE were analyzed in duplicate after overnight stimulation with individual peptides or protein. Briefly, 96-well plates (MultiScreen-IP; Millipore) were coated overnight at 4°C as recommended by the manufacturer with 5 µg/ml capture mouse anti-human IFN- mAb (Protein Data Bank code 1DIK; Mabtech). Plates were then washed three times with PBS plus 0.05% Tween 20 and blocked with RPMI 1640 plus 10% FCS for 2 h at 37°C. PBMCs (2 x 10⁵) were seeded per well, peptides were added at a concentration of 10 µg/ml or recall Ags were introduced. After overnight incubation at 37°C with 5% CO₂, the plates were washed with PBS plus 0.05% Tween 20 and then 50 µl of 1 µg/ml biotinylated secondary mouse anti-human IFN- mAb (clone 7B6-1; Mabtech) was added. After 3 h of incubation at room temperature, plates were washed four times and 100 µl of goat alkaline phosphatase anti-biotin Ab (Vector Laboratories) was added to wells, and the plates were incubated for a further 2 h at room temperature. Plates were then washed four times, and 75 µl of alkaline phosphatase conjugate substrate (5-bromo-4-chloro-3-indolyl phosphate; Bio-Rad) was added. After 4–7 min, the colorimetric reaction was stopped by washing with distilled water. Plates were air dried and spots were counted using an automated ELISPOT reader (EliSpot Reader System; Autoimmune Diagnostika).

The number of specific IFN-–secreting PBMCs was calculated by subtracting the number of spots obtained in the nonstimulated control well from the stimulated sample. A positive control (PBMC stimulated with PHA) was included with each plate to validate the sensitivity of the assay. Wells were considered as positive if they were at least twice above background with at least 10 spots per well.

In some assays, CD8⁺ or CD4⁺ T cells were depleted from PBMCs using CD8⁺ T cell Dynabeads (Dynal) according to the manufacturer’s directions before an ex vivo ELISPOT assay.

Inhibition of T cell responses with anti-MHC class I and II antibodies

The murine mAbs HL-39 and SPVL-3 (which block peptide presentation to CD4⁺ T cells by HLA-DR and HLA-DQ, respectively) were added (5 µg/ml) 90 min before peptide stimulation. All samples were tested in duplicate and peptide-specific intracellular IFN--production was analyzed using flow cytometry. The murine mAb w6/32 (which blocks peptide presentation by HLA class I to CD8⁺ T cells) was used as described above.

Statistical analysis

The ², Mann-Whitney U, and unpaired t tests were used to test for a difference in variables in treatment-naive patients between immunological responders and nonresponders. The changes in frequency of AFP-specific CD4⁺ T cells during and after treatment with TACE/TAE were compared in a pair-wise fashion using the Wilcoxon rank sum test. The Mann-Whitney U test was used to determine whether there was a difference in the frequency of AFP-specific T cells after treatment between clinical responders and nonresponders. All tests were two-tailed and p < 0.05 was considered as being statistically significant.

	Results

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CD4⁺ T cell responses to a panel of AFP-derived epitopes in HCC patients and controls

We have previously reported the presence of AFP_364–373-specific CD4⁺ T cells in HCC patients that were significantly more likely to be present at an early stage of disease and with a serum AFP of <1000 ng/ml (20).

We therefore first selected 20 HCC patients (Okuda I and <1000 ng/ml AFP) (Table II) who were theoretically most likely to have an AFP_364–373 CD4⁺ T cell response and analyzed CD4⁺ T cell responses to a panel of 60 AFP-derived peptides (10 pools of 6 peptides; Table I) and AFP_364–373. Responses to the panel were also assessed in 21 controls (Table III). Short-term T cell lines were generated in vitro and the recognition of peptide pool-specific CD4⁺ T cell responses was analyzed using an intracellular cytokine assay for IFN-. Peptide pool or AFP_364–373-specific T cell responses were detected in six of ten treatment-naive and nine of ten treated HCC patients, and the results are summarized in Table II and Fig. 1. The most potent and frequent responses were directed to pools 3, 4, and AFP_364–373. Responses to pool 4 were only detected in treated patients. No CD4⁺ T cell responses to any of the pools were detected in the controls (Table III).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Analysis of CD4+ T cell responses to 10 AFP-derived peptide pools (60 peptides) and AFP364–373 are shown in patients 08, 09, 15, and 19. T cell lines were generated in the presence of peptide pools or AFP364–373 and cultured for 10–12 days. The cells were restimulated with the same peptide pool or an irrelevant peptide pool and the production of peptide pool-specific IFN- was analyzed using an intracellular cytokine assay.

CD4⁺ T cells recognizing pool 3 (isolated from patients 08 and 09) and pool 4 (from patients 15 and 19) were expanded in vitro for further study. The cells were restimulated with individual peptides (pool 3: AFP_89–98, AFP_125–134, AFP_137–145, AFP_140–148, AFP_158–166, and AFP_164–173; pool 4: AFP_172–181, AFP_179–188, AFP_187–195, AFP_217–226, AFP_235–243, and AFP_249–258) and analyzed for IFN- production using an intracellular cytokine assay. CD4⁺ T cells generated in the presence of pool 3 recognized AFP_137–145 but not other peptides and produced IFN- (Fig. 2, a and b). CD4⁺ T cells generated in the presence of pool 4 recognized AFP_249–258 but not other peptides and produced IFN- (Fig. 3). AFP_137–145- and AFP_249–258-specific CD4⁺ T cells could also be generated by culturing PBMCs with the respective peptide epitopes (data not shown). The ability of AFP_137–145 and AFP_249–258 specific CD4⁺ T cells to produce other cytokines was analyzed using an intracellular cytokine assay. These cells produced peptide-specific TNF- but not IL-5 (Figs. 2c and 3c). To determine the HLA molecules responsible for presenting the identified epitope to CD4⁺ T cells, an Ab-blocking assay was performed. The addition of an anti-HLA-DR Ab blocked recognition of the peptides by CD4⁺ T cells. The responses were not blocked by Abs to HLA-DQ or HLA class I. These results indicated that CD4⁺ T cells recognize AFP_137–145 and AFP_249–258 in an HLA-DR-restricted manner (Figs. 2, d and 3d). AFP_137–145 and AFP_249–258 CD4⁺ T cell lines were incubated with purified AFP and the production of Ag-specific IFN- production was assessed using an intracellular cytokine assay. AFP_137–145- and AFP_249–258-specific CD4⁺ cells recognized purified AFP but not control protein (Figs. 2b and 3b).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Naturally occurring AFP137–145-specific T cells in patients with HCC. CD4+ T cell lines generated in the presence of pool 3 peptides recognized AFP137–145 (a) and purified AFP (b) but not other peptides in the pool (AFP89–98 is shown as a representative control peptide) or the control protein (BSA) and produced Th1 type cytokines (a–c). AFP137–145-specific CD4+ T cells recognized AFP137–145 in a HLA-DR-restricted manner (d). Each experiment were performed in duplicate and the results are representative of two different experiments on different days.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. CD4+ T cells generated in the presence of pool 4 peptides recognized AFP249–258 (a) and purified AFP (b) but not other peptides in pool 4 (AFP172–181 is shown as a representative control peptide) or control protein (BSA) and produced Th1 type cytokines (c). AFP249–258 was recognized by CD4+ T cells in an HLA-DR restricted manner (d). Each experiment was performed in duplicate and the results are representative of two different experiments on different days.

Transarterial chemoembolization/embolization expands and unmasks AFP-specific CD4⁺ T cell responses that are associated with a clinical response to treatment

Because AFP_249–258-specific CD4⁺ T cell responses were only detected in treated patients and the vast majority (nine of ten) had a response to one or more peptide pools, we hypothesized that embolization may induce and activate AFP-specific immunity. To verify this, we analyzed AFP-specific CD4⁺ T cell responses after short-term in vitro expansion longitudinally for 3–6 mo in 10 consecutive treatment-naive patients undergoing TACE or TAE (Table IV). Seven patients received TAE (patients 22, 23, 24, 25, 27, 29, and 30) and three received TACE (patients 21, 26, and 28). Five patients had clinical responses by CT criteria and five did not.

The average in vitro inter-assay and intra-assay variation was <5% as calculated from the coefficients of variation of five treated patients (20 x duplicates) repeated on different days. Analyses of CD4⁺ T cell responses to the 61 AFP-derived peptides (peptide pools) in patients 21, 22, 23, 24, and 25 were performed. The data confirmed our results in patients 1–20, showing that the responses to AFP_364–373, AFP_137–145, and AFP_249–258 were more potent and more frequently detected than other peptides (data not shown); therefore, responses to these epitopes were studied in all 10 patients.

Before treatment, AFP_364–373-specific CD4⁺ T cells were detected in six of the 10 patients, whereas only one patient had a detectable AFP_137–145 CD4⁺ T cell response. No T cell response was detected to AFP_249–258 before treatment. Pre-existing CD4⁺ T cell responses were expanded in six patients and responses to at least one peptide epitope were induced in all 10 (Fig. 4). AFP peptide-specific T cell responses were detected in patients with completely different HLA-DR haplotypes (Table IV), suggesting that CD4⁺ T cells recognize these epitopes in association with different HLA-DR molecules. For example, CD4⁺ T cells isolated from patient 22 (HLA-DR1 and HLA-DR15) and patient 30 (HLA-DR3 and HLA-DR4) recognized AFP_137–145 and AFP_249–258. Statistical analysis of the effect of TACE/TAE on the generation of AFP-specific CD4⁺ T cells indicated that the treatment increased the frequency of AFP-specific T cells over baseline levels both during treatment (p < 0.0001) and after treatment (p < 0.002) (Fig. 5a).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. TACE- or TAE-induced tumor necrosis unmasks AFP-specific CD4+ T cells. Short-term T cell lines generated from PBMCs were isolated before each of three weekly treatments and at 1 and 3 mo after therapy. The arrows indicate the time of treatment. Each experiment was performed in duplicate and inter-assay and intra-assay variation was <5%.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. TACE/TAE-induced necrosis significantly increases AFP-specific CD4+ T cell responses that are associated with clinical outcome. a, Each symbol represents the average percentage of AFP137–145-, AFP364–373-, or AFP249–258-specific CD4+ T cells before, during, and 1 mo after the last treatment. b, Average percentage of CD4+ T cells recognizing AFP137–145, AFP364–373, and AFP249–258 epitopes are shown before and 1 mo after TACE/TAE in clinical responders and nonresponders.

Short-term T cell lines stimulated with PMA/ionomycin and analyzed with an intracellular cytokine assay for IFN- were used to detect changes in the nonspecific T cell response during and after treatment. Four of five patients with a clinical response had an improvement in nonspecific T cell response compared with one of five clinical nonresponders showing an improvement in T cell function (data not shown).

Ex vivo analysis of T cell responses in patients treated with embolization

There were no detectable CD4⁺ T cell responses to AFP_364–373, AFP_137–145, and AFP_249–258 epitopes ex vivo. We detected a CD8⁺ T cell response to AFP_137–145 in one (patient 30) of the four HLA-A2 positive patients. To determine whether embolization could enhance memory T cell responses to nontumor recall Ags, we assayed ex vivo IFN- ELISPOT responses to PPD and streptokinase/streptodornase before and after treatment. Five patients had memory T cell responses to either PPD (n = 3) or streptokinase (n = 4) before treatment that were increased in two patients after therapy. In patient 26 the strength of response to PPD was increased from a mean value of 220 spots per 10⁶ PMBCs before embolization to 420 spots per 10⁶ PBMCs afterward. A response to PPD in patient 27 was not detectable before embolization but was detectable afterward (55 spots per 10⁶ PMBCs). An increase in T cell response to streptokinase/streptodornase was also detected (before: 75 spots per 10⁶ PBMCs; after: 160 spots per 10⁶ PBMCs).

Removal of tumor by resection expands pre-existing AFP-specific CD4⁺ T cell responses

The above experiments did not differentiate the relative importance of the induction of tumor necrosis or a reduction in tumor burden, both of which are produced by TACE/TAE, in the activation of AFP-specific T cell responses. We hypothesized that a reduction in tumor burden on its own could expand pre-existing AFP-specific T cell responses. Therefore, we studied three patients with HCC undergoing curative resection (reduction in tumor burden in the absence of tumor necrosis) and analyzed T cell responses before and after surgery. Two patients (HCC 1 and HCC 2) did not have pre-existing CD4⁺ T cell responses to the AFP-derived peptides and did not develop new AFP-specific T cell responses by 4 wk after resection (Fig. 6, a and b). In the third patient, pre-existing AFP_137–145- and AFP_364–373-specific CD4⁺ T cell responses were expanded (HCC 3) after resection (Fig. 6c).

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Preexisting anti-AFP CD4+ T cell responses were expanded in one of three patients with HCC undergoing curative tumor resection. Short-term T cell lines were generated from PBMCs isolated from the three HCC patients before resection and 4 wk after surgery. The samples were analyzed in duplicate (average) and the results are representative of two different experiments on different days.

	Discussion

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Most studies of T cell responses to tumor-associated Ags to date have analyzed CD8⁺ T cells (25). In theory, however, an effective peptide-based tumor vaccine for HCC may be most effective if both CD8⁺ and CD4⁺ T cell responses against tumor-associated Ags are induced (26, 27). Helper T cells play an important role in promoting CD8⁺ T cell memory development and activating tumor-associated Ag-specific memory CTLs (28, 29). Indeed, studies of patients with advanced breast or ovarian cancer have shown that when Th epitopes are combined with CTL epitopes they provide a more powerful and longer lasting immunity than CTL epitopes alone (30, 31). Although there have been single reports of CD4⁺ responses to both AFP and cancer testis Ags in HCC patients (12, 32), there is a need to characterize the epitopes recognized by Th cells for future use in peptide vaccination studies.

We have recently reported the first CD4⁺ epitope (AFP_364–373) in HCC patients (20). In this study we extend this work with the description of two further AFP-derived class II-restricted epitopes. CD4⁺ responses to these three epitopes were more frequent and potent than those seen to the other 58 peptides, indicating immunodominance in the panel tested. Responses to the immunodominant epitopes were detected in patients with completely different HLA-DR haplotypes, suggesting that CD4⁺ T cell recognition of these epitopes is not restricted to one HLA-DR haplotype (20). In support of the physiologic relevance of the identified epitopes, we show that purified AFP can be recognized by AFP_137–145- and AFP_249–258-specific CD4⁺ T cells. This indicates that processing and presentation of these epitopes by APCs can take place via the exogenous pathway that is the likely mechanism in vivo.

AFP_137–145 has been described previously as an HLA-A2-restricted epitope (13), and in the present study we show that this peptide is capable of stimulating both CD4⁺ and CD8⁺ T cells. Vaccination with dual-specific epitopes may be more efficacious than a mixture of CTL and Th cell epitopes, as they could allow CD4⁺ and CD8⁺ cells to interact with the same APC, thereby improving communication between cells (33).

As our in vitro inter-assay and intra-assay variation was low and the AFP_249–258-specific responses were only detected in treated patients, the changes in the levels of AFP-specific CD4⁺ T cell responses can be attributed to the effects of TACE/TAE. Therefore predictive factors for detecting an AFP-specific CD4⁺ T cell response in HCC patients include a serum AFP of <1000 ng/ml, Okuda stage 1 (20), and treatment with TACE/TAE. Specific CD4⁺ T cell responses were of a low frequency as evidenced by detection after in vitro expansion but not directly ex vivo. Similarly as other groups, we have detected AFP-specific CD8⁺ T cell responses ex vivo in HCC (15, 16).

Importantly, we noted that the frequency of AFP-specific CD4⁺ T cell responses after treatment was significantly higher in clinical responders than in nonresponders. Because patients with clinical responses had larger volumes of necrosis than clinical nonresponders, this indicates a "dose-response" relationship between necrosis and the stimulation of AFP-specific CD4⁺ T cells. Moreover, as CT evidence of a response correlates well with a longer time to progression of disease and an improved survival (21), it is possible, though not proven, that these tumor-specific T cell responses could play a role in protection against progression or recurrence of malignancy. The in vivo function/efficacy of these AFP-specific T cells could only be determined by vaccination or adoptive T cell transfer studies, ideally in HCC patients, and these are planned. The characterized CD4⁺ T cells are more likely to provide specific antitumor immunity in vivo by providing CTL help at the APC level rather than by direct recognition of a tumor, because HCCs do not express HLA-DR molecules on the cell surface (34).

In the present study, TACE/TAE was probably acting as a form of in situ vaccination leading to the presentation of AFP-derived epitopes in an immunostimulatory context and/or providing an Ag source for the induction of antitumor immunity (35). However, the expanded antitumor CD4 T cells were detected only after short-term T cell culture, suggesting that the frequencies of these cells are low. The expansion of these cells in vivo, perhaps by immunotherapy vaccine, may shift the balance in the favor of immune response and thus improve survival. An AFP-based immunotherapy could be applied to boost the response in patients after TACE/TAE but before tumor relapse.

Memory T cell responses to nontumor recall Ags were augmented after therapy, an indication that embolization can provide an adjuvant effect. Preclinical studies have demonstrated that necrotic cells release danger signals such as heat shock proteins and uric acid that activate dendritic cells, a prerequisite for priming naive T cells (3). The identification of a population of AFP-specific CD4⁺ T cells during therapy that are not detectable in treatment-naive patients could be explained by work showing that necrotic cells induce a different set of class II peptides than cells dying via apoptosis (36). In animal tumor models, an in situ vaccination effect has also been proposed to occur after photodynamic therapy, interstitial laser thermotherapy, and radiofrequency ablation, all of which produce necrosis of tumors (35, 37, 38). Those studies demonstrated treatment-induced generation of a protective tumor-specific T cell response to tumor rechallenge. Recent reports of radio frequency thermal ablation in HCC patients complements our findings by showing that the activation of dendritic cells (39) and the expansion of tumor-specific ex vivo T cell responses (40) occur after treatment. Our study differs from these in that the target epitopes/Ags are defined and the development of responses is associated with an improved clinical outcome.

Our data showing that some patients have improved general T cell function after TACE/TAE suggest that general T cell immunosuppression in advanced HCC can be reversed. Our working hypothesis is that a reduction in tumor burden/regulatory factors by resection or embolization may explain in part the observed concomitant expansion of pre-existing tumor immunity.

In conclusion, necrosis produced by TACE/TAE unmasks tumor rejection Ag-specific T cell responses. It represents a method of in situ immune response induction that could be combined with immunotherapy to increase the frequency of AFP-specific T cells with the aim of controlling tumor growth and improving survival.

	Acknowledgments

 
We thank M. Skelton, E. Evans, and M. Valenzuela for helping to recruit and obtain samples from patients.

	Disclosures

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A patent application based on the findings in this paper has been filed by the University College London BioMedica PLC, and S. Behboudi is listed as inventor.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from the Pathological Society of Great Britain and Ireland (to L.A.). The support of the de Laszlo Foundation to the work of S.B. is gratefully acknowledged.

² Address correspondence and reprint requests to Dr. Shahriar Behboudi, University College London Institute of Hepatology, Royal Free and University College London Medical School, 69-75 Chenies Mews, London WC1E 6HX, U.K. E-mail address: s.behboudi{at}ucl.ac.uk

³ Abbreviations used in this paper: TACE, transarterial chemoembolization; AFP, -fetoprotein; CT, computerized tomography; HCC, hepatocellular carcinoma; PPD, purified protein derivative; TAE, transarterial embolization.

Received for publication September 14, 2006. Accepted for publication November 13, 2006.

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