Abstract
The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8+ T cell immune response to MAGE-A3 p271–279 peptide (M3271) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-γ producing M3271 specific CD8+ T cells were detected in 30.8% (8/26) of HLA-A2+MAGE-A3+ HCC patients. The effector CD8+ T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2+MAGE-A3+ HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3271 peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3271-specific CD8+ T cells in HCC patients makes M3271 a potential target for immunotherapy in these patients. The responsive CD8+ T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens.
Keywords: Antigens/Peptides/Epitopes, Cytotoxic T cells, Tumor immunity
Introduction
The MAGE-A3 protein, encoded by a cancer-germ line gene, is one of the common and frequent cancer-testis (CT) antigens expressed in many tumors of various histological origins, but not expressed in normal tissues except testis [2]. The capacity of this antigen to elicit both CD4+ and CD8+ T cell responses makes it a good target for immunotherapy of cancer patients [1, 5, 12, 21, 23, 26]. Many CD4+ and CD8+ T cell epitopes have been identified including the HLA-A2 restricted epitope p271–279, hereafter referred to as M3271 [3, 11, 22]. Clinical trials involving the immunization of cancer patients using MAGE-A3 peptides and whole MAGE-A3 protein have been undertaken and tumor regression associated with elevated T cell responses has been reported in a minority of metastatic melanoma patients [5, 7, 10, 14, 17]. To date, a variety of CT antigens have been found to be expressed with a high percentage in human hepatocellular carcinoma (HCC) and their products are promising targets for antigen-specific immunotherapy of this tumor [8]. Among the tumor antigens, MAGE-A3 is one of the CT antigens highly expressed in HCC. Even though the M3271-specific CD8+ T cell responses could be detected after vaccination in cancer patients, the argument implies that the natural tumor cells may not be able to generate and present M3271 peptide on their cell surfaces due to the inaccurate cleavage by proteosome of endogenously produced MAGE-A3 protein in melanoma cells [20]. To this end, we have performed a study by identification of the peptides directly eluted from tumor cells. We have demonstrated through mass spectrometry analysis that the M3271 peptide could be eluted from the surface of HCC cells freshly isolated from resected cancer tissue of a patient [27]. The amount of M3271 peptide was estimated at 38–39 copies per HCC cell, which is sufficient to allow recognition by peptide specific CD8+ T cells as well as their subsequent activation [6]. This result provides direct evidence that the HCC cells are indeed to be able to generate and present this peptide. In fact, Zerbini and his colleagues have reported that HLA-A2/M3271 tetramer positive cells were detectable in tumor infiltrating CD8+ T cells in HCC patients, but the functional status of these cells was not illustrated [24]. For this reason, we have investigated the specific CD8+ T cell responses and their functional status in MAGE-A3+HCC patients. We found that around 30% of MAGE-A3+HLA-A2+HCC patients possess functional M3271 specific CD8+ T cells present in the peripheral blood. In addition, a substantial proportion of responsive CD8+ T cells specific to NY-ESO-1 and MAGE-A3 were found in patients bearing HCC co-expressing both antigens.
Materials and methods
Patients
The surgically resected tumor samples and peripheral blood mononuclear cells (PBMCs) were obtained from 41 HLA-A2+ HCC patients. Collection of all the samples was agreed by the patients with written consent and approved by Hospital Ethic Review Committee. Tumor expression of the MAGE-A3 and NY-ESO-1 mRNA was assessed by RT-PCR using oligonucleotide primers under the same conditions as reported previously [4]. The primers for MAGE-A3 were: 5′-TGGAGGACCAGAGGCCCCC-3′ and 5′-GGACGATTATCAGGAGGCCTGC-3′, and primers for NY-ESO-1 were: 5′-ATGGATGCTGCAGATGCGG-3′ and 5′-GGCTTAGCGCCTCTGCCCTG-3′, respectively. The HLA-A2 subtypes of each patient were defined using PCR-sequence specific primer (PCR-SSP) and sequence-based typing (SBT) as described previously [25].
Peptides and tetramers
The HLA-A2-restricted MAGE-A3 peptide p271–279 (FLWGPRALV) was used for analysis of the CD8+ T cell response to MAGE-A3. The HLA-A2-restricted NY-ESO-1b peptide, p157–165 (SLLMWITQC) was used as a control. Tetramers of each peptide were prepared as described previously [9].
Target cells
There were four cell lines used as target cells to test the function of effector CD8+ T cells. The mutant TAP-deficient cell line T2 was cultured in RPMI 1640 medium supplemented with 10% FCS (Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml). The primary HCC cell lines Ch-hep-3, Ch-hep-4 and Ch-hep-6 were kindly provided by Dr. Ya-Jun Guo (Institute of Oncology, Second Military Medical University, Shanghai, China) and cultured in a mixture of RPMI-1640 and DMEM (1:1, v/v) media.
Expansion of peptide specific CD8+ T cells
CD8+ T cells isolated from PBMCs with a purity of >95% were seeded into 48-well plates at a concentration of 5 × 105 cells/well in 10% human AB serum-RPMI 1640 medium. For antigen-presentation, autologous dendritic cells (DCs) were incubated with 2.5 μg/ml β2 microglobulin (Sigma, St. Louis, MO, USA) and 10μg/ml peptide for 2 h and then irradiated at 30 Gy. After washing, DCs were added to the plates at a ratio of 1:5. IL-7 (10 ng/ml, Sigma) was added to the initiation of the cultures for 48 h, then, IL-2 (10 units/ml, R&D System, Minneapolis, MN, USA) was added. The CD8+ T cells were restimulated at day 8 and in some samples at day 15, with peptide-loaded, irradiated autologous DCs.
Tetramer, perforin and CD45 Staining
To optimize and control the specificity of staining, tetramers were first used to stain a M3271 specific CD8+ T cell clone (a kindly gift from Dr. Thierry Boon, Ludwig Institute for Cancer Research, Brussels, Belgium) using a series of titrations from 1:100 to 1:4,000. The best titration of 1:2,000 was selected as the percentage of control tetramer+ (HLA-A*0201/NY-ESO-1p157–165) cells was below 0.01% while the percentage of HLA-A*0201/MAGE-A3 p271–279 tetramer+ cells remained unchanged. The detailed procedure for staining was as follows: CD8+ T cells were presensitized with peptide as described above. Cells were first stained with phycoerythrin (PE)-labeled M3271 tetramer and control tetramer for 15 min at room temperature, then, the fluorescein isothiocyanate (FITC)-conjugated CD8 mAb (BD Pharmingen, San Diego, CA, USA) was added for further 15 min staining on ice. After washing, stained cells were analyzed by flow cytometry (FACSCalibur; Becton Dickinson, San Diego, CA, USA).
For perforin staining, cells were first stained with tetramer as described above, then the cells were permeabilized using Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) and stained with either FITC-conjugated anti-perforin or FITC-conjugated mouse IgG2b (BD Pharmingen, San Diego, CA, USA) for 30 min at room temperature. After washing, the stained cells were analyzed by flow cytometry.
For the anti-CD45 Antibody staining, the effector CD8+ T cells were first stained with PE-labeled M3271 tetramer for 15 min at room temperature, then, the FITC-CD45RA or FITC-CD45RO mAb and perCp-CD8 mAb (BD Pharmingen, San Diego, CA, USA) were added for further 15 min staining on ice. After washing, the stained cells were analyzed by flow cytometry.
Cytotoxicity assay
The cytotoxicity assay was performed using lactate dehydrogenase (LDH) release assay with CytoTox96 Non-Radioactive Cytotoxicity Kit (Promega, Madison, WI, USA) and the assay was done according to the manufacture’s protocol. After in vitro expansion for 14 days, the generated M3271-specfic effector CD8+ T cells were mixed with M3271 peptide pulsed T2 cells at a series of effector/target ratio and incubated for 4 h at 37°C. The culture supernatants were collected and the released LDH was measured at 490 nm of ELISA reader. The spontaneous release of LDH from effector and target cells was measured in parallel. The percentage of cytotoxicity was calculated by the following formula:
 |
Interferon-γ and Granzyme B enzyme-linked immunospot assay (ELISPOT assay)
The IFN-γ release ELISPOT assay was performed as described by Jager et al. [9], with slight modifications [19], using a commercial kit (MABTECH, Stockholm, Sweden). Plates (Millipore MAHA S45; Millipore, Bedford, MA, USA) were coated overnight with anti-human IFN-γ monoclonal antibody (mAb) and washed six times. After blocking with 10% human AB serum RPMI 1640, peptide pulsed T2 cells (5 × 104 cells per well) were added together with effector T cells (5 × 104 cells per well). After incubation for 20 h at 37°C, the cells were removed, and the plates were developed with a second (biotinylated) antibody (Ab) to human IFN-γ and streptavidin-alkaline phosphatase. Plates were developed for 10 min at room temperature in the dark, and the reaction was stopped by rinsing the plates with distilled water. The membranes were air dried, and spots were counted using the Champ Spot II ELISOT reader system (Sage Creation, Beijing, China).
The granzyme B (GrB) release ELISPOT assay was performed in a similar manner to the IFN-γ ELISPOT assay as described previously [18].
Intracellular interferon-γ staining for fluorescence-activated cell-sorting analysis (CytoSpot Assay)
The IFN-γ release CytoSpot assay was performed as described previously [19] using the Cytofix/Cytoperm with GolgiStop kit (BD PharMingen, San Diego, CA, USA). Briefly, the effector CD8+ T cells were stimulated with peptide-loaded DCs for 1 h at an effector to target cell ratio of 10:1, followed by the addition of GolgiStop (monesin) for 5 h. Cells were stained with FITC-conjugated anti-CD8 mAb, and then fixed and permeabilized using Cytoperm for 20 min on ice. After washing, the cells were stained with PE-labeled anti-human IFN-γ mAb. The stained cells were washed and then analyzed by flow cytometry.
Results
Patient characteristics
The characteristics of the 41 HLA-A2+ HCC patients initially enrolled in the study are summarized in Table 1. The MAGE-A3 mRNA expression and HLA-A2 subtypes of each patient are shown in Table 2. Of those patients studied, 26 had the HCC expressing MAGE-A3 mRNA detected in the resected tumor tissues. The HLA-A2 subtyping was performed in 32 patients and the percentages of A2 subtypes were: A*0201, 25% (8/32); A*0203, 28% (9/32); A*0206, 28% (9/32) and A*0207, 28% (9/32) (patients CL019, CL039 and CL070 were found to be heterozygotes for A2 subtypes and have been counted twice).
Table 1.
Patient demographics (n = 41)
| Parameter | n (%) |
|---|---|
| Age (years) | |
| Median (range) | 50 (22–73) |
| <40 | 9 (21.9%) |
| 40–60 | 20 (48.8%) |
| >60 | 12 (29.3%) |
| Gender | |
| Male | 38 (92.7% ) |
| Female | 3 (7.3% ) |
| Etiology | |
| HBV | 36 (87.8%) |
| HCV | 0 |
| α-fetoprotein (ng/ml) | |
| Median (range) | 373 (0.932–1210) |
| Tumor size (cm) | |
| Median (range) | 6 (1.5–12) |
| TNM stage | |
| I | 1 (2.4%) |
| II | 15 (36.6%) |
| III | 13 (31.7%) |
| IV | 12 (29.3%) |
Table 2.
Specific CD8+ T cell responses to HLA-A2-restricted M3271 peptide: summary of data from IFN-γ ELISPOT, IFN-γ CytoSpot and Granzyme B ELISPOT assays in 41 HCC patients with different HLA-A2 subtypes
| Patient | MAGE-A3 mRNA | HLA-A2 subtype | Magnitude of specific CD8+ T-cell responses to M3271 | ||
|---|---|---|---|---|---|
| IFN-γ ELISPOTa | IFN-γ CytoSpotb | Granzyme B ELISPOTa | |||
| BW01 | + | A*0207 | 66 (60) | ND | ND |
| BW02 | + | A*0206 | 256 (100) | ND | ND |
| BW03 | + | A*0206 | 150 (84) | ND | ND |
| BW04 | + | A*0201 | 40 (30) | ND | ND |
| BW05 | + | A*0201 | 144 (90) | ND | ND |
| BW06 | + | A*0206 | 16 (22) | ND | ND |
| BW07 | + | A*0206 | 24 (30) | ND | ND |
| BW08 | + | A*0206 | 16 (14) | ND | ND |
| BW09 | + | A*0201 | 18 (20) | ND | ND |
| BW11 | + | A*0201 | 164 (4) | ND | ND |
| BW14 | + | ND | 242 (48) | 2.96% | ND |
| BW15 | + | A*0201 | 720 (80) | ND | ND |
| BW16 | + | ND | 10 (8) | ND | ND |
| BW42 | + | A*0203 | 66 (63) | 0.04% | 70 (41) |
| CL005 | + | A*0201 | ND | 0.01% | ND |
| CL008 | + | A*0203 | ND | 0.57% | ND |
| CL015 | + | A*0207 | ND | 0.02% | ND |
| CL019 | + | A*0206/0207 | 34 (28) | 0.04% | ND |
| CL020 | + | A*0206 | ND | 2.69% | ND |
| CL021 | + | ND | ND | 0.02% | ND |
| CL039 | + | A*0206/0207 | 19 (38) | ND | 75 (41) |
| CL067 | + | A*0207 | 155 (250) | ND | 277 (222) |
| CL070 | + | A*0201/0203 | 116 (8) | ND | 42 (5) |
| CL080 | + | A*0203 | 5 (9) | ND | 6 (5) |
| CL082 | + | A*0203 | 10 (7) | ND | 7 (7) |
| CL085 | + | A*0207 | 105 (7) | ND | 48 (1) |
| BW10 | − | A*0206 | 18 (8) | ND | ND |
| BW12 | − | A*0201 | 82 (52) | ND | ND |
| BW13 | − | A*0207 | 2 (2) | ND | ND |
| BW17 | − | A*0207 | 48 (42) | ND | ND |
| BW18 | − | ND | 46 (60) | ND | ND |
| BW19 | − | ND | 24 (31) | ND | ND |
| BW20 | − | ND | 15 (10) | ND | ND |
| BW21 | − | ND | 53 (65) | ND | ND |
| BW43 | − | ND | 48 (36) | ND | 48 (39) |
| BW44 | − | ND | 73 (66) | ND | 88 (75) |
| CL007 | − | A*0203 | ND | 0.02% | ND |
| CL014 | − | A*0203 | ND | 0.00% | ND |
| CL017 | − | A*0203 | ND | 0.01% | ND |
| CL030 | − | A*0203 | ND | 0.01% | ND |
| CL031 | − | A*0207 | ND | 0.01% | ND |
ND not determined
aA number of 50,000 CD8+ T cells pre-sensitized with M3271 peptide were tested for IFN-γ and/or Granzyme B release after 20 h of incubation with 50,000 T2 cells pulsed with M3271. The results shown are averages of duplicate wells. Values in parentheses indicate average numbers of spots in T2 cells without peptide pulsing. Figures in bold represent significant numbers (three times over background values) obtained in the presence of M3271 peptide in comparison with values obtained in the presence of T2 cells alone
bResults of IFN-γ-producing Cytospot assay are presented as net percentage of CD8+IFN-γ+ T cells by subtracting the background reactivity (no peptide) from the specific response to the stimulated peptide (M3271). Figures in bold are determined as positive
Identification of M3271 specific CD8+ T cells in peripheral blood of HCC patients
To identify the M3271 specific CD8+ T cells, we initially directly stained the ex vivo isolated PBMCs from peripheral blood of ten HCC patients by HLA-A*0201/M3271 tetramer. There were no significant numbers of these peptide specific CD8+ T cells detected in the PBMCs (all <0.01%, Fig. 1c). This result may be attributed to the very low frequency of the memory CD8+ T cells in the peripheral blood, which was below the detection limit. For this reason, we expanded the M3271 specific CD8+ T cells using a rapid expansion protocol, which could amplify the CD8+ T cells (see Materials and methods). After 7 day’s expansion, among four samples tested, a low percentage of tetramer+CD8+ T cells (0.01%) was found in patient BW04 (Fig. 1a). Following 14 days’ expansion, among ten samples tested, relatively high numbers of tetramer+CD8+ T cells were shown in two samples (CL070 (0.11%) and CL085 (0.20%)); low numbers of tetramer+CD8+ T cells were shown in three samples (BW04 (0.01%), CL080 (0.01%) and CL082 (0.06%)); but the tetramer positive cells were still undetectable in the other five samples. After 21 day’s expansion, among the 6 samples tested, the number of tetramer+CD8+ T cells was further increased in the two samples of CL070 (0.31%) and CL085 (0.45%), the figures being higher than those after 14 day’s expansion (Fig. 1b); but being unchanged in the other four samples of CL080, BW03, BW04 and CL067. Thus, the tetramer+CD8+ T cells can be expanded in vitro in the samples that had relatively higher tetramer+ cells after 14-day’s stimulation in vitro.
Fig. 1.
M3271 tetramer+CD8+ T cells in HCC patients. M3271 specific CD8+ T cells were stained with HLA-A*0201/M3271 tetramer and control tetramer HLA-A*0201/NY-ESO-1b after 7- (a), 14- and 21-day expansion (b). Percentages of M3271 tetramer+ versus NY-ESO-1b tetramer+ CD8+ T cells from 10 HCC patients after expansion are listed (c)
Cytotoxicity of M3271 specific effector CD8+ T cells to target HCC cells
The M3271 specific CD8+ T cells from patient CL085 were further tested for their capacity to generate perforin, a cytotoxic molecule to execute cytotoxicity to the target cells, after restimulation with HLA-A2 matched MAGE-A3 mRNA+ HCC cell lines. We demonstrated that most of the M3271 specific tetramer+CD8+ T cells expressed high levels of perforin in their cytosol (Fig. 2a) and were also functional in the release of granzyme B (GrB) after restimulation with HLA-A2+ MAGE-A3+ primary HCC cell lines (Ch-hep-3 and Ch-hep-6, Fig. 2b). Moreover, the effector CD8+ T cells were also cytotoxic to the targets of peptide pulsed T2 cells (Fig. 2c). Such cytotoxic function was specific and MHC class I restricted, as these M3271 specific CD8+ T cells could not release GrB after being co-cultured with HLA-A2+ MAGE-A3− HCC cell line (Ch-hep-4), and the release of cytotoxic molecules could be blocked by anti-MHC class I antibody W6/32 (data not shown, see Ref. [19]). These results suggest that the M3271 specific CD8+ T cells have cytotoxic activity to the targets of the MAGE-A3 expressing HCC cells. The phonotype of the M3271 tetramer+ specific effector CD8+ T cells in sample GL085 was CD45RO+CD45RA−, a memory cell phenotype (Fig. 2d).
Fig. 2.
Cytotoxic function of M3271 specific CD8+ T cells against MAGE-A3 expressing HLA-A2+ HCC cells. The effector CD8+ T cells from CL085, the sample contains potent CD8+ T cells responsive to M3271 as assessed by IFN-γ release ELISPOT and tetramer assays, were tested for their intracellular perforin expression (a), the Granzyme B- and IFN-γ release after restimulation with MAGE-A3+HLA-A2+ primary HCC cell lines (b, Ch-hep-3, Ch-hep-4 and Ch-hep-6), and the cytotoxicity to the targets of peptide pulsed T2 cells assessed by LDH release assay (c). The CD45 phenotype of effector CD8+ T cells was identified (d). The effector CD8+ T cells of patient GL085 were divided into two groups, cells of group one were stained with PE-tetramer, FITC-anti-CD45RA and perCp-anti-CD8 mAbs, while the cells of group two were stained with PE-tetramer, FITC-anti-CD45RO and perCp-anti-CD8 mAbs. Cells were gated on CD8+ T cells
The frequency and magnitude of specific CD8+ T cell responses to the M3271 peptide
To further evaluate the function of the M3271 specific T cells in a larger population, a total of 41 HCC patients were enrolled in this study to monitor their specific CD8+ T cell response to this peptide. These patients expressed different HLA-A2 subtypes and 26 of them were found to have MAGE-A3 mRNA+ tumors (see Table 2). After antigen-driven cell expansion for 14–21 days in vitro, the cultures from these patients were assayed by either IFN-γ release ELISPOT, IFN-γ producing CytoSpot or GrB release ELISPOT assay. Representative data for each assay are shown in Fig. 3.
Fig. 3.
Specificity of CD8+ T cell responses to the HLA-A2 restricted M3271 peptide. Representative data of specific CD8+ T cell responses to M3271 peptide are shown in patients CL070, CL080 (a) and CL020 (b). IFN-γ ELISPOT and IFN-γ CytoSpot assays were performed after 2-week in vitro culture stimulated by M3271 pulsed autologous DCs. Tetramer analysis of M3271 specific CD8+ T cells in the same patients was performed on day 21. Numbers in the top right quadrant indicate the percentage of HLA-A2/M3271 tetramer+ cells. The control experiments were set up using T2 cells without antigen pulse and T2 cells loaded with an irrelevant peptide NY-ESO-1b. There was no positive response in the controls
Of 26 patients bearing MAGE-A3 mRNA+ tumors, 8 were found to have a specific T cell response to M3271 in their peripheral blood after antigen-driven cell expansion in vitro. The IFN-γ release ELISPOT, IFN-γ producing CytoSpot and GrB release ELISPOT assays were performed in cultures from 21, 8 and 7 patients, respectively (Table 2). Among these patients, eight were assessed by two assays and one by three assays. The results from these three assays were comparable with each other indicating our CD8+ T cells functional assays were reproducible and reliable. The average frequency of specific IFN-γ-producing cells were 0.53 ± 0.46 % (267 ± 230) and 2.07 ± 1.30% as counted by ELISPOT and CytoSpot assay, respectively. The frequencies of the effector T cells were similar in both IFN-γ and GrB release ELISPOT assays suggesting that the IFN-γ secreting CD8+ T cells can also release GrB in the samples we tested. In contrast, antigen-specific IFN-γ producing CD8+ T cells were not detected in the cultures from the other 15 patients bearing MAGE-A3 mRNA− tumors by any of these three assays.
More interestingly, the specific CD8+ T cell responses were also observed in patients bearing HLA-A2 subtypes of A*0203 (CL008), A*0206 (BW02, CL020) and A*0207 (CL085).
Responsive CD8+ T cell responses to MAGE-3 and NY-ESO-1 antigenic peptides in HCC patients bearing tumors co-expressing both antigens
The specific CD8+ T cell responses to NY-ESO-1b peptide (p157–165) and M3271 were assessed in the same cohort of HCC patients bearing tumors expressing NY-ESO-1 mRNA, MAGE-3 mRNA, or both. Among the 41 HCC patients studied, 34.2% (14/41) were found to co-express NY-ESO-1 and MAGE-A3 mRNA (assigned as group A); 51.2% (21/41) expressed either NY-ESO-1 or MAGE-A3 mRNA (group B); and 14.6% (6/41) expressed neither NY-ESO-1 nor MAGE-A3 mRNA (group C). As shown in Table 3, of the 14 samples in group A, the responsive CD8+ T cells were detected in 5 samples specific to both NY-ESO-1 and MAGE-3 peptides and in one sample specific to NY-ESO-1b peptide only. The positive rate of responsive CD8+ T cells was 35.7% (5/14) to both antigenic peptides in this group. In group B, the positive rate of responsive CD8+ T cells was 25.0% (3/12) and 33.3% (3/9) specific to MAGE-A3 peptide and NY-ESO-1b peptide, respectively. In group C, there was no specific CD8+ T response detected (0.0%). Therefore, the specific CD8+ T cell responses to both antigenic peptides were detected in a substantial proportion of HCC patients bearing NY-ESO-1 and MAGE-A3 mRNA positive tumors.
Table 3.
State of specific CD8+ T-cell responses in HCC patients with tumors co-expressing NY-ESO-1 and MAGE-3
| Group | mRNA expression in HCC tissuea | Samples | Patterns of specific CD8+ T-cell responses (n = 41)b | Positive response (%)c | |||||
|---|---|---|---|---|---|---|---|---|---|
| M3 | ESO-1 | M3271 | + | + | − | − | |||
| NY-ESO-1b | + | − | + | − | |||||
| A | + | + | 14 | 5 | 0 | 1 | 8 | 42.9% (6/14) | |
| B | + | − | 12 | 0 | 3 | 0 | 9 | 25% (3/12) | |
| − | + | 9 | 0 | 0 | 3 | 6 | 33.3% (3/9) | ||
| C | − | − | 6 | 0 | 0 | 0 | 6 | 0% (0/6) | |
aDetected by RT-PCR
bAssessed by IFN-γ ELISPOT, CytoSpot and/or GrB ELISPOT assay
cPercentage of positive CD8+ T cell responses to either M3271 or NY-ESO-1b
Discussion
In this study, to analyze anti-M3271 specific CD8+ T cell responses in HCC patients without antigen vaccination, we have confirmed the presence of M3271 specific T cells in around 50% (5/10) of HCC patients detected by HLA-A2/M3271 tetramer assay firstly reported by Zerbini and colleagues [24]. We have further demonstrated that these M3271/HLA-A2 restricted CD8+ T cells in HCC patients having relatively high M3271/HLA-A2 tetramer+CD8+ T cells (>0.1%) were functional in terms of antigen-driven cell expansion in vitro culture and IFN-γ and GrB release as well as the production of perforin after restimulation with HLA-A2+MAGE-A3+HCC cells. Of the two samples, a sufficient number of granzyme B releasing effector CD8+ T cells permitted us to perform cytotoxicity assays, where they exhibited a killing capacity to the M3271 peptide-pulsed T2 cells (Fig. 2c). Thus, the generated effector CD8+ T cells were cytotoxic to the target cells. Collectively, in HCC cells the M3271 peptide could be generated from endogenously produced MAGE-A3 protein and presented in the context of HLA-A2 on cell surfaces; the functional anti-M3271 specific CD8+ T cells were present in the tumor-bearing HCC patients. Therefore, the M3271 is apparently a good vaccine candidate for the immunotherapy of HCC patients bearing MAGE-A3+ tumors.
The frequency of anti-M3271 specific CD8+ T cell precursors freshly isolated from peripheral blood of HLA-A2+MAGE-A3 mRNA+HCC patients was very low, which was <0.01% in M3271/HLA-A2 tetramer assay and hence unable to analyze their functional states directly. Only after in vitro culture under the stimulation of the peptide presented by autologous DCs for 14–21 days, could the antigen-specific CD8+cells be expanded and their functional states estimated. These results were somehow different from those observed in non-small cell lung cancer patients, where the anti-M3271 CD8+ T cells could not be detected prior to MAGE-A3 protein immunization even after the PBMCs had been activated with antigen pulsed-DC and cultured in vitro for 10 days, whereas anti-M3271 CD8+ T cells were readily detected 3 weeks post-vaccination [1]. The frequency of pre-existing anti-M3271 CD8+ T cells in HCC patients may be higher than that in the non-small cell lung cancer patients, and hence are functionally detectable after in vitro antigen-driven expansion.
In our assays, we were unable to unambiguously determine whether MAGE-A3 mRNA+HCC patients had spontaneous anti-M3271 CD8+ T cell responses. We have tested the phonotype of specific effector CD8+ T cells in two samples after 14 days in vitro expansion driven by antigen stimulation; they displayed a memory phenotype of CD45RO+CD45RA−. However, due to the limited volume of blood obtained from the patients, the T cells have to be expanded in vitro, which did not permit us to determine if they were derived from naïve or memory T cells. According to our previous study using PBMCs from healthy donors, the antigen specific effector T cells could be detected only after four to five rounds of antigen-pulsed autologous DC stimulation [13]. The functional effector T cells generated after two rounds of antigen stimulation may preferentially be derived from in vivo antigen primed T cells. Thereby, the HCC patients may have spontaneous CD8+ T cell responses without antigen vaccination.
The consequences of specific CD8+ T cell responses in clinical outcomes are not definitely clear. We performed the follow up survey in those patients where the CD8+ T cell responses had been assessed. We found that one patient (BW02) with potent CD8+ T cell response to M3271 has been surviving for over 53 months since surgery. Whether this prolonged survival time was due to the existence of natural T cell immunity is still uncertain at present. More patients need to be followed to clarify such a correlation.
The NY-ESO-1 p157–165 and MAGE-A3 p271–279 peptides have been frequently applied as CT antigen peptide vaccines in tumor immunotherapy for Caucasian patients. Both peptides are presented by HLA-A*0201, this subtype is positive in 95% of HLA-A2+ Caucasians and in 40–50% of HLA-A2+ ethnic Han Chinese [15, 16]. The augment is that whether this HLA-A*0201 restricted tumor antigen peptide vaccine is applicable in Chinese patients. In our previous studies, we have reported the functional subtypes of HLA-A2 for the presentation of HLA-A*0201 restricted influenza virus matrix p58–66 and NY-ESO-1 p157–165 peptides to activate the antigen-specific CD8+ T cells [19, 25]. We found that a number of HLA-A2 subtypes could present the HLA-A*0201 restricted peptides in ethnic Han Chinese. In this study, we have observed that the functional supertype of M3271 peptide comprised four HLA-A2 subtypes (A*0201, A*0203, A*0206 and A*0207) in Chinese HCC patients. These four HLA-A2 subtypes constituted 96% of the HLA-A2 in an ethnic Han Chinese population. Such functional supertype of HLA-A2 alleles has widely expanded the potential applicability of MAGE-A3 p271–279 peptide vaccine for immunotherapy of Chinese patients.
An important observation in this study was that the responsive CD8+ T cells specific to both NY-ESO-1p157–165 and MAGE-A3 p271–279 were detected in a substantial proportion (35.7%) of HCC patients bearing tumors co-expressing both antigens. This finding provides a rationale for designing bivalent vaccine of NY-ESO-1 and MAGE-A3 for immunotherapy of the HCC patients with the tumor expressing both antigens.
Another critical event was observed in our assays. Among the 5 HCC patients having M3271-responsive CD8+ T cells detected by HLA-A2/M3271 tetramer, three HCC patients had tetramer+ cells with frequencies between 0.01 and 0.06%; these M3271-specific CD8+ T cells could not expand and differentiate into functional effectors after antigen stimulation and being cultured for 2–3 weeks in vitro. These cells are apparently functionally defective. In order to improve tumor vaccine efficacy, the T cell responsive defect must be surmounted.
In conclusion, our work has demonstrated that around 30% of HLA-A2+ MAGE-A3 mRNA+ HCC patients exhibited functionally specific CD8+ T cell responses to M3271/HLA-A2 molecules. The M3271 peptide is a potential vaccine candidate for the immunotherapy of HCC patients. A bivalent vaccine targeting NY-ESO-1 and MAGE-A3 would be amendable for immunotherapy of HCC patients with the tumors co-expressing both antigens.
Acknowledgments
The work was undertaken within the Beijing Municipal Government Foundation for Natural Sciences (7071006), the James R. Kerr Program of the Ludwig Institute for Cancer Research (KSP003), and the China National 863 High Technology Program (2006AA02Z486).
Footnotes
H-G Zhang, H-S Chen, and J-R Peng are contributed equally to this paper.
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