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Clinical Cancer Research Vol. 11, 6686-6694, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Analysis of CD4+ T-Cell Responses to a Novel {alpha}-Fetoprotein-Derived Epitope in Hepatocellular Carcinoma Patients

Akeel Alisa1,2, Annette Ives1, Ansar A. Pathan5, Cristina V. Navarrete3,4, Roger Williams1,2, Antonio Bertoletti1 and Shahriar Behboudi1

Authors' Affiliations: 1 Institute of Hepatology, University College London; 2 Liver Unit, Cromwell Hospital; 3 Department of Histocompatibility and Immunogenetics, NBS-London and SE Zone, North London Blood Centre; 4 Department of Immunology and Molecular Pathology, Royal Free and University College Medical School, University College London, London, United Kingdom; and 5 Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom

Requests for reprints: Shahriar Behboudi, Institute of Hepatology, University College London, 69-75 Chenies Mews, London, WC1E 6HX, United Kingdom. Phone: 44-20-7679-6517; Fax: 44-20-7380-0405; E-mail: s.behboudi{at}ucl.ac.uk.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: {alpha}-Fetoprotein (AFP) is a tumor-associated antigen in hepatocellular carcinoma and is a target for the development of cancer vaccine. Four immunodominant AFP-derived HLA-A*0201-restricted peptides have been identified and the administration of these peptides with an adjuvant has stimulated AFP-specific CTL responses in hepatocellular carcinoma patients. However, no AFP-derived CD4 T-cell epitope has yet been reported and the status of AFP-specific CD4+ T-cell responses in hepatocellular carcinoma patients is not fully understood. The aim of this study was to analyze naturally occurring CD4+ T-cell responses to AFP.

Experimental Design: We analyzed the ability of CD4+ T cells to recognize an HLA-DR-restricted AFP-derived epitope in 41 hepatocellular carcinoma patients and 24 non-hepatocellular carcinoma control patients using intracellular cytokine assays for IFN-{gamma}.

Results: Here, for the first time, we report the identification of an AFP-derived CD4+ T-cell epitope that is recognized by circulating lymphocytes from hepatocellular carcinoma patients in association with HLA-DR. The absence of detectable responses in healthy donors and patients with chronic liver disease suggests that AFP-specific CD4+ T cells in the responder patients had been previously expanded in vivo in response to the tumor. The anti-AFP CD4+ T-cell response was only detected in hepatocellular carcinoma patients with normal or mildly elevated serum AFP levels who were in the early stage of disease.

Conclusion: Our data will be instrumental in the development of cancer vaccine using AFP-derived immunogens.

Several studies in patients with malignant tumors have shown the existence of immune responses to antigens expressed by both malignant and normal cells. However, this naturally occurring antitumor immune response fails to induce regression of established tumors in some patients. It has been suggested that the rationale behind an epitope-based therapeutic vaccine is to enhance preexisting CD4+ and CD8+ T-cell responses thus controlling tumor growth. The identification and characterization of T-cell epitopes on tumor-associated antigens is a crucial prerequisite in the design of epitope-based therapeutic vaccines (1).

{alpha}-Fetoprotein (AFP) is a tumor-associated antigen in hepatocellular carcinoma and testicular carcinoma and is a target for immunotherapy (2–5). In patients with hepatocellular carcinoma, the AFP gene is reactivated and the level of serum AFP is increased. The measurement of serum AFP plays an important role in the diagnosis of hepatocellular carcinoma and in monitoring the response to various treatment modalities (5). One of the biological properties of AFP is its regulatory effects on immune response (6–9). We have shown that APCs of hepatocellular carcinoma patients with high levels of AFP, not those with normal or mildly elevated AFP levels, are dysfunctional and in vitro AFP impairs dendritic cell function and induce their apoptosis (6). Naturally occurring immune response against AFP can be mounted in patients with hepatocellular carcinoma (10–12), but there is limited information about its association with hepatocellular carcinoma staging and serum AFP levels. Furthermore, no AFP-derived CD4+ T-cell epitope has yet been identified (4, 13–15), and the effects of AFP expression levels on antigen-specific T-cell response are unknown.

In this study, we identified the first AFP-derived T-cell epitope recognized by circulating CD4+ T cells from hepatocellular carcinoma patients in association with HLA-DR. The response was only detected in hepatocellular carcinoma patients with normal or mildly elevated AFP levels who were in the early stage of disease. However, the lack of response in patients with high serum AFP maybe due to a nonspecific CD4+ T-cell suppression and hence a reduction in IFN-{gamma} levels. Our data indicate that the 364-373 sequence plays a role in the induction of CD4+ T-cell responses to AFP, and the identification and detection of this response might be important in the development and application of AFP-based vaccines.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. All patients were reviewed at the Liver Unit of Cromwell Hospital in London with the ethical committee's approval. Peripheral blood was collected from the patients with their informed consent and peripheral blood mononuclear cells (PBMC) were isolated. The clinical staging of patients with hepatocellular carcinoma was determined using Okuda classifications (16).

HLA typing. Following centrifugation, buffy coats were separated from the red cell pellet and the white cells frozen in freezing mix (FCS 20%, Terasaki Park Medium 70%, and DMSO 10%). DNA was extracted by using a modified salting-out procedure. HLA-A, HLA-B, and HLA-DR typing was done by molecular techniques in line with recent recommendations (17). HLA analysis was done on DNA extracted from a ~5-mL sample of blood by a modified salting out technique (18). The typing systems used defined all HLA-A, HLA-B, and HLA-DR specificities (19) with the exception of A203 and A210 (included in A2), A2403 (included in A24), B703 (included in B7), B2708 (included in B27), B3901 and B3902 (included in B39), B5101 and B5103 (included in B51), B64 and B65 (included in B14), and DR1403 and DR1404 (included in DR14), and additionally HLA-B82 was defined.

Synthetic peptides. Peptides corresponding to the sequence of AFP were purchased from Mimotopes Pty, Ltd. (Clayton, Victoria, Australia). ProPed MHC class II binding prediction server (http://www.imtech.res.in/raghava/propred) was used to predict HLA-DR13-binding region of AFP.

Cells and cell lines. RPMI 1640, MEM, penicillin and streptomycin, and 10% heat-inactivated FCS were purchased from Life Technologies (Grand Island, NY). Purified human cord blood AFP (purity, >95% SDS-PAGE) and purified bovine serum albumin (purity >97%) were obtained from Calbiochem (La Jolla, CA) and Sigma-Aldrich (St. Louis, MO), respectively. EBV-immortalized B cells were generated from PBMCs by culturing 5 x 106 cells with 1 mL of EBV B95/8 supernatant and 0.4 µg/mL cyclosporine A (Sigma-Aldrich).

Generation of T-cell lines. T-cell lines were generated as described previously (20). In brief, PBMCs were resuspended at a concentration of 1.5 x 106/mL in MEM, 10% FCS (Life Technologies). PBMCs were stimulated with individual peptides (1 µmol/L) or peptide pools in 96-well plates. Recombinant interleukin-2 (rIL-2, 25 IU/mL) was added on day 3 of culture and cells were analyzed after a total of 10 to 12 days of culture.

Intracellular cytokine staining. AFP-specific T-cell lines were incubated for 5 hours at 37°C at 1 x 106 cells/mL in the cell culture medium with the AFP-derived peptides (1 µmol/L) and in the presence of Brefeldin A (10 µg/mL, Sigma-Aldrich). Cells were surface stained with Cychrome-conjugated anti-CD4 or anti-CD8 antibodies (BD PharMingen, Cowley, United Kingdom). The cells were then permeabilized and fixed using Cytofix/Cytoperm (BD PharMingen). Afterwards, the cells were stained for intracellular cytokines with FITC-conjugated anti IFN-{gamma}. FITC-conjugated anti-IL-2 PE-conjugated anti-tumor necrosis factor-{alpha}, PE-conjugated anti-IL-5, or isotype controls (R&D Systems, Abingdon, United Kingdom); washed twice; and analyzed by flow cytometry.

Inhibition of T-cell responses with anti-MHC class I and II antibodies. To determine the HLA molecules responsible for presenting AFP-derived epitopes to CD4 T cells, antibody-blocking assays were done. The murine monoclonal antibodies HL-39 and SPVL-3 which block peptide presentation to CD4+ T cells by HLA-DR and HLA-DQ, respectively, were added at 5 µg/mL to two separate wells containing T-cell lines followed by 1 µmol/L peptide 90 minutes later. Two controls were used, a positive control using only the peptide and a negative control that did not contain any peptide or antibody. All samples were tested in duplicates and peptide-specific intracellular IFN-{gamma} production was analyzed using flow cytometry. The murine monoclonal antibody w6/32, which blocks peptide presentation by HLA class I to CD8+ T cells, was used as described above.

CD4+ T-cell proliferation assay. AFP364-373-specific T-cell lines were labeled with carboxyfluorescein diacetate succinimidyl ester (1 µmol/L) and the cells were washed twice in PBS and resuspended in medium supplemented with 10% human serum (Sigma-Aldrich). Carboxyfluorescein diacetate succinimidyl ester–labeled cells (3 x 106 cells/mL) were incubated in 96-well plates (Nunc, Naperville, IL) in the presence of 1 µmol/L AFP364-373 peptide or an irrelevant peptide. Phytohemagglutinin (1 µg/mL) and rIL-2 (20 units/mL) were used as positive controls. After 5 days of in vitro incubation, cells were stained with PE-Cy5-labeled anti-CD4- and peptide-specific CD4+ T-cell proliferation on gated CD4+ T cells was analyzed using flow cytometry.

{alpha}-Fetoprotein measurement. Levels of serum AFP were measured using microparticle enzyme immunoassay kit obtained from Abbott Laboratories (Abbott Park, IL) and done according to the manufacturer's instruction. In brief, anti-AFP microparticles were incubated with the blood specimen, and an aliquot of the reaction mixture was transferred to the matrix cell. The matrix cell was washed, removing unbound materials, and the anti-AFP conjugate was dispensed onto the matrix cell. The substrate was added to the matrix cell and the fluorescent product is measured by the microparticle enzyme immunoassay optical assembly.

Statistical analysis. The Mann-Whitney U test was used to compare the levels of serum AFP in hepatocellular carcinoma patients with or without CD4+ T-cell response to the identified epitope (responder and nonresponder). The {chi}2 was used to evaluate the significant difference in the prevalence of Okuda grade 2 and 3 in responders and nonresponders. The Spearman rank coefficient was used to evaluate correlations between serum AFP levels and the percentages of CD4+ IFN-{gamma}-producing cells. Statistical significance was defined at P ≤ 0.05 for all analyses done.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Isolation of {alpha}-fetoprotein-specific CD4+ T cells from circulating lymphocytes of a hepatocellular carcinoma patient. The region of 369 to 379 within AFP sequence was predicted to bind HLA-DR13 molecules using ProPed MHC class II binding prediction server. Nine to 14 amino-acid-long peptides were synthesized to cover the region (Fig. 1). PBMCs were isolated from an HLA-DR13+ hepatocellular carcinoma patient (HCC041) and stimulated with the AFP-derived peptides. Two weeks later, cultures were tested using a pool containing AFP-derived peptides (AFP364-373, AFP372-381, and AFP379-387) or individual peptides. The presence of specific CD4+ T cells was analyzed by intracellular staining with IFN-{gamma} and CD4-specific monoclonal antibodies (Fig. 2). The peptide pool and AFP364-373 peptide stimulated significant levels of IFN-{gamma}-producing CD4+ T cells as compared with samples with no peptide or irrelevant peptides (AFP379-387 and AFP372-381; Fig. 2). AFP364-373-specific CD4+ T cells recognized AFP365-373 (9 amino acid long), AFP 362-373 (12 amino acid long), and AFP 360-373 (14 amino acid long) peptides and produced peptide-specific IFN-{gamma} but did not recognize irrelevant peptides (AFP379-387 and AFP372-381; Fig. 1B). T-cell lines expanded in the presence of shorter (9 amino acid long) or longer peptides (12 and 14 amino acid long) recognized AFP364-373 peptide (data not shown). The frequency of IFN-{gamma}-producing cells among CD4+ T cells was lower in cells stimulated with AFP365-373 peptide, but there was no difference in the ability of other peptides (i.e., AFP364-373, AFP 362-373, and AFP 360-373; Fig. 1).



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  		Fig. 1. A, amino acid sequence of 360-387 region of AFP and predicted epitope (bold and underlined) within this region for different HLA-DR13 alleles (ProPed class II binding prediction server was used, http://www.imtech.res.in/raghava/propred). B, positions and sequences of AFP-derived peptides analyzed in this study for their activity. No response (–), peptide-specific response (+), and potent peptide-specific response (++).

 


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  		Fig. 2. Detection of AFP364-373-specific CD4+ T cells among circulating T lymphocytes from patient HCC041 after stimulation with a pool containing AFP-derived peptides. The presence of peptide-specific CD4+ T cells in the culture was assessed by staining with anti-IFN-{gamma} monoclonal antibody after incubation in the absence of added peptide or after stimulation with a pool containing AFP-derived peptides or individual peptides. An example of two individual peptides with no activity. Numbers in the top right quadrants are the percentage of cytokine-producing cells among CD4+ T cells.

 
The AFP364-373-specific CD4+ T cells were expanded in vitro to further define the epitope. The obtained CD4+ T cells recognized the AFP364-373 peptide in a dose-dependent manner and produced IFN-{gamma} but did not recognize an irrelevant peptide (Fig. 3A). Furthermore, AFP364-373-specific CD4+ T cells recognized AFP364-373 peptide and produced peptide-specific tumor necrosis factor-{alpha} and IL-2 (Th1-type cytokine) and expanded in vitro but did not recognize an irrelevant peptide (Fig. 3B-C). The AFP364-373-specific CD4+ T cells did not produce IL-5 (Fig. 3B).



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  		Fig. 3. A, AFP364-373 peptide activity is shown in peptide titration experiment. AFP364-373-specific CD4+ T-cell line (isolated from PBMCs of patient HCC041) was stimulated with different concentration of AFP364-373 peptide or an irrelevant peptide and the production of peptide-specific IFN-{gamma} by CD4+ T cells was analyzed using intracellular cytokine assay. B, production of tumor necrosis factor-{alpha} (TNF-{alpha}), IL-2 (Th1-type cytokines), and IL-5 (Th2-type cytokine) by AFP364-373-specific CD4+ T cells was assessed by staining with anti-tumor necrosis factor-{alpha}, anti-IL-2 or anti-IL-5 monoclonal antibodies after incubation in the absence of peptide or after stimulation with AFP364-373 or an irrelevant peptide. Numbers in the top right quadrants are the percentage of cytokine-producing cells among CD4+ T cells. C, carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled T-cell lines were incubated in vitro with AFP364-373 peptide, an irrelevant peptide or stimulated with Phytohemagglutinin and rIL-2 as positive control. After 5 days of incubation, cells were stained with PE-Cy5-labeled- anti-CD4 antibody and the peptide-specific CD4+ T-cell proliferation was analyzed using flow cytometry. The numbers indicated in each quadrant are the percentage of CD4+ CFSElow T cells. Representative of two different experiments.

 
AFP364-373 is recognized by peptide-specific CD4+ T cells from patient HCC041 in the context of HLA-DR13. To determine the HLA molecules responsible for presenting the identified epitope to CD4+ T cells, an antibody-blocking assay was done. The T-cell line was incubated with the relevant peptides in the presence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA class I antibodies. T-cell reactivity was measured using an intracellular cytokine assay for IFN-{gamma}. The addition of anti-HLA-DR antibody blocked recognition of peptide AFP364-373 by a peptide-specific CD4+ T cells. This response was not blocked by antibodies to HLA-DQ or HLA class I. This result indicates that CD4+ T cells recognize AFP364-373 in a HLA-DR-restricted manner (Fig. 4A).



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  		Fig. 4. HLA class II restriction of AFP364-373-specific CD4+ T cells. A, peptide recognition was assessed either in the absence or in the presence of anti-HLA-DR, DQ, or anti-MHC class I antibodies. B, AFP364-373-specific CD4+ T-cell line generated from PBMCs from patient HCC041 (HLA-DR12, HLA-DR13, and HLA-DR52) was incubated with partially histocompatible EBV-B cells pulsed with the relevant or control peptide. HLA-DR class II alleles of the target cells matched with patient HCC041 are indicated. Percentage of peptide-specific IFN-{gamma}-producing cells among CD4+ T cells in duplicate. Representative of two individual experiments.

 
To establish the presenting allele(s), we first determined the HLA-DR alleles of the patient. Patient HCC041 expressed HLA-DR12, HLA-DR13, and HLA-DR52. Then, we assessed presentation by partially matched APCs from other individuals. Partially histocompatible EBV-B cells pulsed with peptide AFP364-373 or a control peptide were used to find matching HLA-DR class II alleles. In the case of two EBV-B cells expressing HLA-DR13 and HLA-DR52, we obtained efficient presentation of the peptide to the CD4+ T cells from patient HCC041, whereas in the case of two EBV-B cells expressing HLA-DR12 and/or HLA-DR52 but not HLA-DR13, no presentation was observed. This suggest that peptide AFP364-373 was recognized by an HLA-DR13-restricted CD4+ T cells in the case of patient HCC041 (Fig. 4B). However, we cannot rule out that other DRB gene products can also present the identified epitope to specific CD4+ T cells. This was confirmed when some HLA-DR13-negative hepatocellular carcinoma patients (HCC157, HCC159, HCC028, and HCC018) exhibited CD4 T-cell responses to the identified epitope (Table 1).


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  		Table 1. List of patients with hepatocellular cancer tested in this study for the presence of circulating AFP364-373-specific CD4+ T-cell response

 
Recognition of purified {alpha}-fetoprotein by AFP365-373-specific CD4 T cells. PBMCs isolated from patient HCC041 were stimulated with purified AFP (13 µg/mL) and rIL-2. After 2 weeks, the resulting cells were restimulated with AFP364-373 or an irrelevant peptide (AFP379-387) or cultured in medium only (no peptide) and the presence of AFP-reactive CD4+ T cells were analyzed using intracellular cytokine assay for IFN-{gamma}. The AFP-specific CD4+ T cells recognized AFP364-373 peptide but not the irrelevant peptide and produced IFN-{gamma} (Fig. 5).



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  		Fig. 5. A, the recognition of purified AFP by AFP364-373-specific CD4+ T cells. PBMCs were stimulated with AFP364-373 peptide and 2 weeks later, the culture was restimulated with purified AFP, control protein (bovine serum albumin), or AFP364-373 peptide. The production of antigen-specific IFN-{gamma} by CD4+ T cells was assessed using intracellular cytokine assay. Percentage of antigen-specific CD4+ IFN-{gamma}+ cells. Representative of two individual experiments. B, detection of AFP-specific CD4+ T cells among circulating lymphocytes isolated from patient HCC041 after stimulation with purified AFP antigen. The presence of AFP364-373-specific CD4+ T cells was assessed using intracellular cytokine assay for IFN-{gamma} after incubation of the culture in the absence of added peptide, irrelevant peptide, or after stimulation with AFP364-373 peptide, as indicated. Numbers in the top right quadrants are the percentage of cytokine-producing cells among CD4+ T cells.

 
To establish the physiologic relevance of the identified epitope, AFP364-373-specific CD4+ T-cell line was incubated with purified AFP (13 µg/mL) and the production of antigen-specific IFN-{gamma} production was assessed using intracellular cytokine assay. AFP-specific CD4+ T cells recognized purified AFP but not control protein (bovine serum albumin) and produced IFN-{gamma} (Fig. 5B). To stimulate AFP364-373-specific CD4+ T-cell line, we used purified AFP at concentrations ranging from 1 to 20 µg/mL. The T-cell lines restimulated with purified AFP at concentration <8 µg/mL did not produce detectable IFN-{gamma} and 13 µg/mL was the optimal concentration in our system. The activation of PBMCs or control T-cell lines with purified AFP (13 µg/mL) did not induce nonspecific IFN-{gamma} production.

Assessment of AFP364-37-specific CD4+ T cells in hepatocellular carcinoma patients and control groups. To further analyze the spontaneous immunogenicity of the identified epitope, we assessed the responsiveness to AFP364-373 using PBMCs from 40 additional hepatocellular carcinoma patients as well as seven healthy donors, 13 patients with liver cirrhosis, and four patients with non-hepatocellular carcinoma secondary liver cancer (Tables 1 and 2). Short-term T-cell lines were tested against AFP364-373 peptide and two irrelevant peptides (AFP372-381 and AFP379-387). AFP364-373-specific CD4+ T cells were detected in 11 of 41 patients and seven responder patients expressed HLA-DR13 molecules (Table 1). AFP364-373-specific CD4+ T cells recognized AFP364-373 peptide but did not recognize AFP372-381 and AFP379-387 peptides (data not shown). No CD4+ T-cell response to the tested AFP-derived peptides (AFP364-373, AFP372-381, and AFP379-387) was detected in the control groups. AFP364-37-specific IFN-{gamma} production by CD4+ T cells isolated from six hepatocellular carcinoma patients is shown (Fig. 6). The results obtained from antibody blocking assay show that CD4+ T cells isolated from the responders recognize AFP364-373 in association with HLA-DR molecules (data not shown).


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  		Table 2. List of control groups (liver cirrhosis patients, healthy controls, and patients with secondary liver non-hepatocellular cancer tumors) analyzed in this study

 


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  		Fig. 6. Detection of AFP364-373-specific CD4+ T cells in circulating lymphocytes of different patients with hepatocellular cancer. PBMCs were stimulated with AFP364-373 peptide and the presence of AFP364-373-specific CD4+ T cells was assessed 2 weeks later after incubation of the culture with AFP364-373 peptide. Numbers in the top right quadrants are the percentage of IFN-{gamma}-producing cells among CD4+ T cells.

 
Association between the presence of a detectable {alpha}-fetoprotein-specific CD4+ T-cell response and low serum {alpha}-fetoprotein level. In this study, we have shown that the AFP364-373-specific CD4+ T-cell response was only detected in hepatocellular carcinoma patients with normal or mildly elevated serum AFP. In addition, median serum AFP levels were 7.2-fold higher in nonresponders than that in responders (P = 0.0044, Mann-Whitney U test; Fig. 7A). There is a significant difference in the prevalence of Okuda stage II and III in responders and nonresponders ({chi}2, P = 0.002; 95% confidence intervals; Fig. 7B). This indicates that patients in stage II or III are significantly less likely to have CD4+ T-cell response to the identified epitope. However, it was unknown whether the lack of AFP364-373-specific CD4+ T-cell response in hepatocellular carcinoma patients with high levels of serum AFP was due to a general immunosuppression of CD4+ T cells or anergy of AFP-specific CD4+ T cells. To address this question, eight hepatocellular carcinoma patients with different levels of serum AFP were chosen and PBMCs isolated from these patients were stimulated with phorbol 12-myristate 13-acetate and ionomycin ex vivo and the frequency of CD4+ T cells producing nonspecific IFN-{gamma} was analyzed. High levels of serum AFP and IFN-{gamma} production by CD4+ T cells are inversely correlated ({rho} = –0.738; P = 0.037, Spearman rank-order test). As shown in Fig. 7C, hepatocellular carcinoma patients with high levels of serum AFP produced significantly lower levels of IFN-{gamma}, suggesting that there is a general CD4+ T-cell dysfunction in terms of IFN-{gamma} production in this group of patients. CD4+ T cells isolated from this group of patients did not proliferated in the presence of rIL-2 (data not shown), suggesting that their CD4+ T cells are dysfunctional. Although, it has been shown that AFP has immunoregulatory properties (6, 9), this does not prove that the impairment of CD4+ T cells in hepatocellular carcinoma patients with high levels of AFP is solely induced by AFP. Other regulatory factors released by the tumor (such as transforming growth factor-ß) may be involved. We are currently studying the role of AFP, transforming growth factor-ß and tumor-specific regulatory T cells in the induction of CD4+ T-cell dysfunction.



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  		Fig. 7. Serum AFP levels, disease stage, and the presence of AFP-specific CD4 T-cell response in hepatocellular cancer patients. A, AFP364-373-specific CD4+ T cells were only detected in hepatocellular cancer patients with normal or mildly elevated serum AFP. Levels of serum AFP in patients with and without AFP364-373-specific CD4+ T-cell response. Median of serum AFP levels for the responders and nonresponders (P = 0.044). Closed dots are responders and open dots are nonresponders. B, the majority of hepatocellular cancer patients with AFP364-373-specific CD4+ T-cell response were in their early disease stage. Disease stage was determined using Okuda classification (stages I, II, and III). Closed dots are responders (n = 11) and open dots are nonresponders (n = 30). {chi}2 was used to evaluate the significant difference in the prevalence of grade 2 and 3 in responders and nonresponders (P = 0.002; difference in proportions, –0.6; 95% confidence interval, –0.26 to –0.76). C, CD4+ T cells isolated from patients with hepatocellular cancer and high levels of serum AFP produced significantly lower IFN-{gamma}. PBMCs were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 5 hours, harvested, and stained with Cychrome-conjugated anti-CD4 monoclonal antibody for surface molecules and FITC-conjugated IFN-{gamma} for intracellular cytokine. Cells were analyzed using flow cytometry. Percentages of CD4+ T cells that express IFN-{gamma} were analyzed. Results obtained from HCC patients ({blacksquare}, n = 8), with the y-axis representing percentage of IFN-{gamma}-producing cells among CD4+ T cells and the x-axis representing the levels of serum AFP. The correlation coefficients were calculated by using the Spearman rank-order ({rho} = –0.738; P = 0.037 for serum AFP levels compared with percentage of CD4+ IFN-{gamma}-producing cells) *, statistically significant.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The AFP-derived peptide described here, QLAVSVILRV (AFP364-373), was recognized by CD4+ T cells isolated from 11 different patients with hepatocellular carcinoma. The CD4+ T-cell lines also recognized AFP360-373, AFP 362-373, and AFP365-373. We initially concentrated our analysis on patient HCC041, who expressed HLA-DR13, who had an elevated serum AFP. After a single in vitro stimulation, we assessed the presence of specific CD4+ T cells among circulating lymphocytes from this patient using 9 to 14 amino-acid-long peptides from the 362 to 387 region. As revealed by the analysis of AFP-derived peptides, the peptide corresponding to the epitope recognized by AFP-specific CD4+ T cells is located between AFP residues 64 and 73, a region that partially overlaps the predicted epitope for HLA-DR13 molecules. In support of the physiologic relevance of the identified epitope, we showed that APCs cultured with purified AFP can stimulate IFN-{gamma} production by AFP364-373-specific CD4+ T cells. This indicates that processing and presentation of tumor-derived AFP antigen by APCs, through the exogenous pathway, was the mechanism through which spontaneous responses to AFP occurred in vivo. In addition, we detected AFP364-373-specific CD4+ T cells from PBMCs isolated from patient HCC041 that had been cultured in the presence of purified AFP. This shows that APCs can take up purified AFP, process, and present the epitope to specific CD4+ T cells.

AFP364-373 epitope also seemed recognized by CD4+ T cells from 11 of 41 hepatocellular carcinoma patients analyzed. However, not all the responders expressed HLA-DR13 molecules suggesting that other HLA-DR molecules may be able to present the identified epitope. This is not surprising as many CD4+ T-cell epitopes can be presented by different HLA class II molecules. Six of 11 hepatocellular carcinoma patients with the circulating AFP-specific CD4+ T cells had a mildly elevated serum AFP and five patients showed a normal serum AFP level (normal serum AFP level is between 10 and 20 ng/mL). Various frequencies of AFP-specific CD4+ T cells were recorded in the responder group, which could not be explained by the difference in their clinical data (age, gender, serum AFP level, and the presence of different viral hepatitis) or their T-cell ability to expand in vitro. It is noteworthy that AFP staining can be shown in hepatocellular carcinoma tissues of hepatocellular carcinoma patients with serum AFP level of <20 ng/mL (21). The CD4+ T-cell response was not detected in patients with high levels of serum AFP (>1,000 ng/mL) and the majority of patients with a response were in the early stage of disease.

CD4+ T cells from hepatocellular carcinoma patients with high levels of serum AFP did not exhibited anti-AFP CD4+ T cells responses (cytokine production and antigen-specific T-cell proliferation). Furthermore, CD4+ T cells isolated from this group of patients did not responded to activation by rIL-2 as measured by T-cell proliferation (data not shown) or nonspecific cytokine production, suggesting that CD4+ T cells from this group of patients are dysfunctional. It should be emphasized that the identified AFP-derived epitope did not exhibit any regulatory property and the regulatory ability of AFP should be attributed to other sections of AFP sequence. The lack of anti-AFP CD4+ T-cell response in patients with high levels of AFP could be due to immunoregulatory effects of AFP in high levels (6, 9), T-cell exhaustion or anergy caused by high levels of antigen (22), other regulatory cytokines released by tumor (such as transforming growth factor-ß), and/or tumor-specific regulatory T cells. Further studies using peptide-specific MHC class II tetramers are required to determine whether AFP364-373-specific CD4+ T cells are dysfunctional or deleted in hepatocellular carcinoma patients with high levels of serum AFP. Before one can proceed with this experiment, peptide-specific class II tetramer must be constructed and fully characterized, which is beyond the scope of the present study. Whichever mechanism, the results obtained from this study imply that only hepatocellular carcinoma patients with normal or mildly elevated serum AFP (up to 1,000 ng/mL) who are in the early stage of disease exhibit CD4+ T-cell response to the identified epitope and thus might benefit from an anti-AFP immunotherapy vaccination. A recent report by Ritter et al. (23) has also shown the presence of AFP-specific CD4+ and CD8+ T cells directly ex vivo in the peripheral blood of hepatocellular carcinoma patients. CD4 T-cell responses to AFP (the whole protein) were detected in higher percentages of hepatocellular carcinoma patients (59%) than that in our study (about 20% responded to the identified epitope), suggesting that CD4 T cells from some hepatocellular carcinoma patients may recognize not yet unidentified CD4 epitopes. We are currently examining this possibility and will determine whether CD8 T-cell responses are also dysfunctional in hepatocellular carcinoma patients with high levels of AFP (>1,000 ng/mL).

The control groups (n = 24) tested in this study did not exhibit any response to the AFP-derived peptides and no AFP-specific CD4+ T-cell response was detected in this group suggesting that AFP-specific CD4+ T cells in the responder patients had been previously expanded in vivo in response to the autologous tumor. Other authors have succeeded in showing a specific B- and T-cell response to AFP both in hepatocellular carcinoma and patients with liver cirrhosis (10, 12). One explanation for this discrepancy could be that the CD4+ T cells of cirrhotic patients may recognize yet unidentified AFP-derived epitopes. Identification of a larger number of AFP-derived CD4+ T-cell epitopes and analysis of the CD4+ T-cell response to these epitopes in cirrhotic patients and hepatocellular carcinoma patients may cast a light on the role of the AFP-specific CD4+ T-cell response in disease progression and may be used as a prognostic/diagnostic marker.

In conclusion, we report a dominant role of 364-373 region of AFP antigen in the induction of specific CD4+ T-cell responses in hepatocellular carcinoma patients. The identified epitope is presented to specific CD4+ T cells by HLA-DR molecules. This finding is likely to contribute significantly to the development of AFP-based vaccines.


   Footnotes
 
Grant support: Central Research and Development Commitee project grant, de Laszlo Foundation, Citrina Foundation, and Alex and Madelaine von Nolcken.

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.

Received 3/ 7/05; revised 5/31/05; accepted 6/ 7/05.


   References
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 References
 

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