[CANCER RESEARCH 59, 3134–3142, July 1, 1999]

Generation of Human T-cell Responses to an HLA-A2.1-restricted Peptide EpitopeDerived from a-Fetoprotein1

Lisa H. Butterfield, Andrew Koh, Wilson Meng, Charles M. Vollmer, Antoni Ribas, Vivian Dissette, Eric Lee,John A. Glaspy, William H. McBride, and James S. Economou2

Divisions of Surgical Oncology [L. H. B., A. K., W. M., C. M. V., A. R., V. D., E. L., J. S. E.], Hematology/Oncology [A. R., J. A. G.], and Experimental Radiation Oncology[W. H. M.], University of California Los Angeles Medical Center, University of California Los Angeles, Los Angeles, California 90095

ABSTRACT

a-Fetoprotein (AFP) is often derepressed in human hepatocellularcarcinoma. Peptide fragments of AFP presented in the context of majorhistocompatibility molecules could serve as potential recognition targetsby CD8 T cells, provided these lymphocytes were not clonally deleted inontogeny. We therefore wished to determine whether the human T-cellrepertoire could recognize AFP-derived peptide epitopes in the context ofa common class I allele, HLA-A2.1. Dendritic cells genetically engineeredto express AFP were capable of generating AFP-specific T-cell responsesin autologous human lymphocyte cultures and in HLA-A2.1/Kb transgenicmice. These T cells recognize a 9-mer peptide derived from the AFPprotein hAFP542–550 (GVALQTMKQ). Identified as a potential A2.1-restricted peptide epitope from a computer analysis of the AFP sequence,hAFP542–550 proved to have low binding affinity to A2.1, but slow off-kinetics. AFP-specific CTL- and IFN-g-producing cells recognizehAFP542–550-pulsed targets. Conversely, hAFP542–550peptide-generated Tcells from both human lymphocyte cultures and A2.1/Kb transgenic micerecognized AFP-transfected targets in both cytotoxicity assays and cyto-kine release assays. These lines of evidence clearly demonstrate thatAFP-reactive clones have not been deleted from the human T-cell reper-toire and identify one immunodominant A2.1-restricted epitope. Thesefindings also clearly establish AFP as a potential target for T-cell-basedimmunotherapy.

INTRODUCTION

HCC3 is one of the most common fatal tumors (1, 2), with anannual global incidence of 1.2 million (3). In the United States,approximately 13,000 new cases are diagnosed each year, and themedian survival is generally less than 6 months (1, 4). Resection,transplantation chemoembolization, alcohol injection, and cryoabla-tion are potentially curative, but only in small, localized tumors (5–7).Unfortunately, most patients have advanced disease at diagnosis, andcurrent systemic therapies are largely ineffective. The development ofnovel treatment strategies is greatly needed. Most gene therapy effortsuse suicide gene (8–12), tumor suppressor gene (13–15), or cytokine-based strategies (16–18).

AFP is expressed during fetal development but transcriptionallyrepressed shortly after birth (19). Certain tumors, principally HCC and

germ cell tumors, express AFP, and its measurement in serum playsan important role in diagnosis and in monitoring responses to treat-ment (20). Human AFP is translated as a 609-aa protein that is cleavedto yield a 590-aa secreted protein (21). The regulation of human andmurine AFP genes has been extensively studied and is largely at thelevel of transcription (22, 23). The normal function of AFP is un-known. It has been hypothesized to play a role in serum componenttransport because AFP has been shown to bind fatty acids, steroids,and heavy metals (24–26). In addition, there have been reports thatAFP may have an immunosuppressive role in fetal development(27–29).

The idea that AFP can serve as a target for immunotherapy is notnew. Efforts were reported in earlier tumor immunology literature thatinvolved attempts to generate humoral responses (30–32). These werepredictably unsuccessful due to high circulating levels of AFP thatneutralized antibody. However, AFP-producing tumors would be ex-pected to process and present AFP-derived peptide fragments on theircell surface in the context of major histocompatibility molecules.These MHC-restricted AFP peptides could potentially be recognizedby the immune system, provided that these T cells were not clonallydeleted during the ontogeny of the immune system. Both murine andhuman T-cell repertoires appear to contain self-reactive T-cell clonesfor such proven and putative tumor rejection antigens as MART-1 (33,34), MAGE (35, 36), gp100 (33, 37), carcinoembyronic antigen(38–41), and others. It would be surprising if potential AFP-reactiveclones could not be marshalled with an appropriate set of activationsignals in an immunostimulatory environment.

Our strategy in examining human T-cell responses to AFP wasguided by our parallel studies of human T-cell responses to thewell-characterized melanoma antigen MART-1 (42). Robust re-sponses could be generatedin vitro by DCs genetically engineered toexpress MART-1. DCs transduced with a recombinant MART-1 AdVexpressed this melanoma antigen at high levels and correctly pro-cessed and presented the immunodominant HLA-A2.1-restrictedMART-127–35 peptide. MART-1 engineered human DCs could beused to generate specific human T-cell responsesin vitro. In addition,we have reported a murine MART-1 model in which potent CTLs,cytokine-producing T cells, and protective immunity are generatedafter immunization with MART-1-engineered DCs (43, 44). We haveexploited these potent antigen-presenting cells to investigate humanT-cell responses to AFP. We report, for the first time, that the humanT-cell repertoire can recognize AFP, and we characterize the responseto an HLA-A2.1-restricted epitope.

MATERIALS AND METHODS

Sequence Analysis and Computer Screening.The University of Wiscon-sin Genetics Computer Group Program “find patterns” was used to screen thehAFP sequence (GenBank accession numbers J00077, J00076, and V01514)and identify 9- and 10-mer peptides that contained (a) two strong bindinganchor residues at positions 2 and 9 (or 10); (b) only one anchor residue; or (c)no anchor residue but other positive binding residues. Of the three groups ofpeptide sequences, those that contained more than one residue thought toabolish binding were eliminated.

Received 3/1/99; accepted 4/30/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH/National Cancer Institute Grants PO1 CA5926, RO1 CA 77623,RO1 CA 79976, T32 CA75956, and K12 CA 79605 and by the Stacy and EvelynKesselman Research Fund (all to J. S. E.); by National Cancer Institute Grant CA 09120;and by the Fondo de Investigacion Sanitaria 97/5458 (to A. R.).

2 To whom requests for reprints should be addressed, at Division of Surgical Oncology,54-140 CHS, University of California Los Angeles Medical Center, 10833 Le ConteAvenue, Los Angeles, CA 90095-1782. Phone: (310) 825-2644; Fax: (310) 825-7575;E-mail: jeconomo@surgery.medsch.ucla.edu.

3 The abbreviations used are: HCC, hepatocellular carcinoma; AFP,a-fetoprotein; aa,amino acid; DC, dendritic cell; MART-1, MART-1/Melan-A; UCLA, University ofCalifornia Los Angeles; hAFP, human AFP; IL, interleukin; RT-PCR, reverse transcrip-tion-PCR; AdV, adenovirus; CMV, cytomegalovirus; RMT, room temperature; LCL,lymphoblastoid cell line; PBMC, peripheral blood mononuclear cell; IMDM, Iscove’smodified Dulbecco’s medium; FBS, fetal bovine serum; tg, transgenic; GM-CSF, gran-ulocyte/macrophage colony-stimulating factor; MOI, multiplicity of infection; CFA, com-plete Freund’s adjuvant.

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Cells, Antibodies, Cytokines, and Viruses.HLA-A2.1 donors and celllines were screened with the BB7.2 (HLA-A2)-specific antibody and a goat-antimouse-FITC secondary antibody (Caltag, South San Francisco, CA) andsubtyped by PCR and direct sequence analysis by the UCLA Tissue TypingLaboratory. The K562, HepG2, Hep3B, B95-8, BB7.2, and W6/32 cell lineswere obtained from American Type Culture Collection (Rockville, MD). TheM202 human melanoma cell line was generated in our laboratory from asurgical specimen and has been described previously (45). T2 cells wereprovided by Peter Cresswell (Yale University School of Medicine). JY cells(HLA-A2.1 homozygous) were provided by Martin Kast (Loyola UniversityCancer Center). Jurkat/A2Kb cells were provided by Linda Sherman (ScrippsResearch Institute). EBV-transformed LCLs were generated by incubatingPBMCs from HLA-A2.11 donors with supernatant from B95-8 cells. Celllines were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD)or IMDM (JY cells; Life Technologies, Inc.) with 10% FBS (Omega Scientific,Tarzana, CA) and pennicillin/streptomycin/fungizone (Life Technologies,Inc.). Anti-b2-microglobulin and the anti-CD4, -CD14, -CD19, and -CD56NA/LE depletion antibodies were obtained from PharMingen (San Diego,CA), anti-pan class I antibody was prepared from concentrated supernatant ofthe W6/32 hybridoma, anti-HLA-A2 antibody was prepared from concentratedsupernatant of the BB7.2 hybridoma, and CD4-FITC, CD8-PE, and CD16-PEantibodies were obtained from Caltag.

The stable transfectant cell lines LCL/hAFP, M202/hAFP, Jurkat A2/Kb/hAFP (Jurkat/AFP), and Jurkat A2/Kb/MART1 (Jurkat/MART) wereprepared by lipofection of parental cells with an expression plasmid(VR1012hAFP and VR1012 from Vical) expressing hAFP and cotransfec-tion with the hygromycin-expressing plasmid pCEP4 (Invitrogen, Carlsbad,CA) or pRcCMVMART1neo (44), followed by selection in hygromycin(Boehringer Mannheim) at 50mg/ml or in G418 at 500mg/ml. Expressionof AFP was confirmed by RT-PCR (primers 59-GCAACCATGAAGT-GGGT and 39-CTCTCTCTCTCTAGAAACTCCCAAAGCAGCACGAGT)and AFP radioimmune assay (UCLA Medical Center Clinical Labs), andexpression of MART-1 was confirmed by RT-PCR as described previously(45). IL-2 was provided by Hoffman-LaRoche (Nutley, NJ), IL-7 wasobtained from Biosource (Camarillo, CA), IFN-g was provided by Dr.Steven Dubinett (UCLA), and keyhole limpet hemocyanin andb2-micro-globulin were obtained from Sigma (St. Louis, MO).

AdVhAFP contains the 1.9-kb hAFP cDNA originally cloned by RT-PCRusing the primers listed above and driven by the CMV promoter/enhancer in apAC-CMVpLpA AdV type 5 backbone. The virus was prepared by recombi-nation of this plasmid with pJM17, which contains the 35-kb AdV genome,deleted in the E1 region, in 293 cells that provide the E1 genes intrans.Recombinant viruses were released into the medium, purified by limitingdilution, and amplified on 293 cells. The empty AdV vector, AdVRR5, hasbeen described previously (46) and served as a control. All viruses used havebeen purified on CsC1 gradients as described previously (46).

Peptide Synthesis.Peptides were synthesized by Chiron (Victoria, Aus-tralia) and at the UCLA Peptide Synthesis Facility using standard f-moctechnology.

T2 Binding Assay. Each peptide was tested for concentration-dependentbinding to T2 cells in a HLA-A2.1 stabilization assay (47, 48). T2 (TAP-deficient) cells that had been incubated at RMT the previous night to increasecell surface MHC class I molecule expression were then incubated overnightwith each peptide over a range of peptide concentrations from 0.1–100mM. Inthe T2 cell line, only MHC molecules that are filled with 8-10-mer peptides arestable on the cell surface. Stability of HLA-A2.1 was assayed by flow cytom-etry after staining the cells with an anti-HLA-A2 antibody (BB7.2) and goatantimouse-FITC. The HLA-A2.1 strongly binding Flu matrix peptide (aa58-66; GILGFVFTL; Flu) was used as a positive control.

JY Peptide Binding Assay. Peptide binding to HLA-A2.1 was determinedusing HLA-A2.11JY cells. Cells were washed twice in PBS before strippingoff peptides with 2 ml of citrate-phosphate buffer [0.131M citric acid and0.066M Na2HPO4 (pH 3.2)] for 90 s (49). After washing once in serum-freemedium (IMDM), cells were resuspended and seeded on a 96-well plate(Costar) at 13 105 cells/well. Different concentrations of each peptide (50mM–50 nM), along with 100 nM humanb2-microglobulin (Sigma), were addedto each well and incubated for 4 h at RMT. Negative controls were either nopeptides or the HLA-A1 binding MAGE-3 peptide. Flu was used as a positivecontrol. After incubation, cells were washed twice before adding 5mg/ml

W6/32 antibody and incubated on ice for 30 min. Surface HLA-A2.1 expres-sion was detected by using a microtiter plate reader quantifying the hydrolysisof the b-galactosidase substrate chlorophenol red-b-D-galactopyranosidemonosodium salt (Boehringer Mannheim) after staining the cells with goatantimouse IgG F(ab9)2 antibody conjugated withb-galactosidase (SouthernBiotechnology Associates, Birmingham, AL). The relative binding strength ofeach peptide is expressed in absorbance of wells containing the peptide overabsorbance in wells without peptides.

Fig. 1. AFP expression and AFP-specific human T-cell generation by AdVhAFP-transduced DCsin vitro. A, RT-PCR of AdVhAFP in human DCs. DCs were prepared asdescribed and transduced with different MOIs of AdV, either empty AdVRR5 orAdVhAFP. RNA was prepared and assayed by RT-PCR for both hAFP expression andb-actin. The AFP1 hepatoma cell line HepG2 was used as a positive control for hAFPexpression.B, cytotoxicity of AdVhAFP/DC-stimulated T-cell cultures against an AFP1target. M202 HLA-A2.11melanoma cells stably transfected with hAFP (M202/hAFP) orparental M202 cells were used as targets in a standard 5-h cytotoxicity assay. T cellsstimulated with AdVhAFP/DC for 3 weeks were used as effectors at the ratios shown.C,ELISPOT analysis of AdVhAFP/DC-stimulated T-cell recognition of AFP1 cells. T cellswere cultured with LCLs and M202 cells (stably transfected with hAFP or parental cells)for 48 h. Cells were subsequently plated for ELISPOT analysis of the frequency ofIFN-g-secreting cells. After 24 h of cytokine secretion, plates were developed, and thecolored spots corresponding to IFN-g-secreting cells were counted.

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MHC-Peptide Complex Stability. HLA-A2.1 LCLs were stripped of sur-face class I peptides andb2-microglobulin with a mild pH 3.2 citrate-phos-phate acid buffer that makes MHC molecules unstable (50). Each peptide wasimmediately pulsed onto stripped cells at 200mM for 1 h in thepresence ofb2-microglobulin at 3mg/ml at RMT. Excess peptide was washed off, and thecells were incubated at 37°C for 0, 2, 4, and 6 h. Cells were washed at the endof each time point and stained for cell surface HLA-A2 expression and thenanalyzed by flow cytometry. The peptide-MHC class I complex was consid-ered stable if the mean fluorescence intensity increased at least 1.5-fold fromcells that were stripped but not pulsed with peptide.

CTL Generation from Peptide-pulsed PBMCs. Peptide-specific CTLswere generated as described previously (42) to various peptides. Briefly,normal donor HLA-A2.1 PBMCs were pulsed with 50mg/ml peptide for 90min at RMT in serum-free IMDM (Life Technologies, Inc.), washed, andcultured on day 0 with IL-7 (10 ng/ml) and keyhole limpet hemocyanin (5mg/ml) in RPMI 1640 10% autologous serum at 33 106 cells/1.5 ml per well.Cells were restimulated weekly by removing the nonadherent cells from theculture and adding them to fresh, autologous, peptide-pulsed, washed, andirradiated PBMCs at a 1:1 ratio. IL-2 was added twice weekly at 10 units/ml.

After 3 weeks of culture, the cultures were tested for cytotoxicity and/orcytokine release.

CTL Generation from AdV-transduced DCs. DCs [prepared as de-scribed from PBMCs incubated with GM-CSF and IL-4 (51, 52)] were trans-duced with AdVhAFP or AdVMART1 at a MOI of 1000 for 2 h. TransducedDCs were washed, irradiated, and plated at 2–53 105 cells/well in a 24-wellplate to serve as stimulators for CTL generation. Autologous nonadherent cellswere depleted of CD4, CD14, CD19, and CD561 cells by magnetic beaddepletion (Dynal, Lake Success, NY) to prepare CD81 enriched respondercells (population generally at least 80% CD81; data not shown). The CD81cells were plated with the transduced DCs at 23 106 cells/well in 5%autologous medium plus IL-7 at 10 ng/ml to generate CTLs. Cultures weresupplemented with IL-2 at 10 units/ml every 3–4 days. The CD81 CTLs wererestimulated weekly with fresh, autologous, AdV-transduced DCs at a ratio of1 DCs: 5-10 CD81 CTLs. Most cultures were phenotyped for CD41 andCD81 cells on a weekly basis.

Human and Murine Cytotoxicity Assay. Target cells were harvested,washed, counted (T2 cells were peptide-pulsed at 50mg/ml) and chromatedwith 100mCi of Na2Cr51O4 (Amersham, Arlington Heights, IL), with shaking

Fig. 2. Cytotoxicity and cytokine release by AdV/DC human T-cell cultures.A, CD81 enriched lymphocyte cultures were stimulated with AdVhAFP/DC for 2 weeks and assessedfor peptide-specific cytotoxicity against AFP542–550- or MART-127–35-pulsed T2 cell targets.B, CD81enriched lymphocyte cultures were stimulated with AdVMART1/DC for 2 weeksand assessed for peptide-specific cytotoxicity against AFP542–550- or MART-127–35-pulsed T2 cell targets.C, AdVhAFP/DC T-cell cultures were assayed for frequency ofIFN-g-secreting cells by ELISPOT after restimulation for 48 h with peptide-pulsed autologous LCLs.D, AdVMART1/DC T-cell cultures were assayed for frequency of IFN-g-secretingcells by ELISPOT after restimulation for 48 h with peptide-pulsed autologous LCL.

Table 1 Properties of peptidesa

Peptide Location Sequenceb AnchorsRelative JY

bindingcRelative off-

kineticsd

hAFP542–550 542–550 GVALQTMKQ 1 0.143 6 hFlu M1 58–66 GILGFVFTL 2 0.211 6 hMART-1 27–35 AAGIGILTV 1 0.077 2 h

a The properties of each peptide used, including the peptide name, the location in the protein, the amino acid sequence, the number of anchor residues for HLA-A2.1, the resultsof the JY concentration-dependent binding assay, and the results of the off-kinetics assay, where the last timepoint of detectable peptide binding are shown.

b Anchor residues are shown in bold.c JY binding assay gives a higher optical density number as a read-out of greater binding affinity.d Off-kinetics assay detects binding at 2-h intervals, beginning with time5 0 and ending with time5 6 h.

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incubation at 37°C for 1.5–2 h. CTLs were washed, counted, and diluted to thedesired concentrations in RPMI 1640/10% AB (or RPMI 1640/10% FBS formurine T cells), and plated in triplicate wells in a V-bottomed 96-well plate(Costar). Target cells were washed three times, diluted to 53 104 (or 1 3 105

for murine assay) cells/ml, and plated with CTLs. To control for nonspecificlysis, a 10–50-fold excess of unchromated (cold) K562 cells was added to mosttarget populations before adding to CTLs (in human assay). The plates werespun briefly at 1000 rpm and incubated for 4–5 h. Supernatants were harvestedand counted in a gamma counter. Triplicate wells were averaged, and thepercentage of specific lysis was calculated as follows: [sample2 spontaneousrelease]/[maximum release2 spontaneous release].

Human ELISPOT Assay. To determine the frequency of antigen-inducedcytokine-producing T cells, the ELISPOT technique was used (53, 54). T-cellrestimulation was performed with 3–53 106 CTLs incubated with 13 105

autologous LCLs pulsed with specific or nonspecific peptides or tumor celllines in 1 ml of RPMI 1640/10% autologous serum medium/well of a 24-wellplate. LCLs were peptide-pulsed in 1 ml of serum-free IMDM (Life Technol-ogies, Inc.) at RMT for 1–2 h with 50mg/ml peptide. The cells were rinsedbefore plating with CTLs. An additional well was prepared with restimulatorcells without CTLs as a negative control. The 24-well plates were incubated ina humidified incubator at 37°C for 48 h. The ELISPOT microtiter plates(Millipore, Bedford, MA) were coated with purified IFN-g-, GM-CSF, ortumor necrosis factora (PharMingen) antibody in coating buffer [0.1MNaHCO3 (pH 8.2)] at 4mg/ml and stored at 4°C overnight. The next day, therestimulated CTLs were rinsed and set to identical cell concentrations inserum-free X-Vivo-10 (Life Technologies, Inc.). The blocked (PBS/10% FBS)plates were then washed with PBS, followed by one wash with X-Vivo-10. Therestimulated CTLs were plated in duplicate wells, in each of three dilutions,and then incubated at 37°C for 24 h. The plates were washed 103 withPBS/Tween, and secondary biotinylated antibody was added at 3mg/ml andincubated at 4°C overnight.

Finally, the plates were washed 103 with RMT PBS/Tween, and a 1:2000dilution of avidin-peroxidase (Vector Laboratories) was added, and the plateswere incubated in the dark at RMT for 1–2 h. The color substrate 3-amino-9-ethylcarbazole (Sigma) was prepared in formamide/0.05M NaOAc buffer (pH5.0) and filtered. The plates were then washed again in PBS/Tween. H2O2

solution was added to the color substrate solution, and the substrate solution

was added to the washed plate. The reaction was stopped by rinsing in tapwater. The spots were counted under a dissecting microscope.

HLA-A2.1/K b tg Mice. HLA-A2.1/Kb tg female mice (created by Dr.Linda Sherman, Scripps Research Institute, La Jolla, CA) were originallypurchased from Harlan-Sprague Dawley (Indianapolis, IN) and are currentlybred by the animal facility of the Department of Radiation Oncology at UCLAand handled in accordance with the animal care policy of the UCLA. Forpeptide immunizations, mice received 100mg of AFP or control peptideemulsified 1:1 in CFA (Difco, Detroit, MI) s.c.

Preparation of Murine DCs and Adenoviral Transduction. DCs weredifferentiated from murine bone marrow progenitor cells following the Inabamethod (55), with modifications (44). Day18 nonadherent and loosely ad-herent cells contained DC aggregates with a high level of MHC class I and II,B7.1 (CD80), B7.2 (CD86), CD1d, CD18, and CD44-positive cells that weresuperior stimulators of a mixed lymphocyte reaction (data not shown).In vitrocultured DCs were transduced in 15-ml conical tubes (Costar) in a final volumeof 1 ml of RPMI 1640/2% FCS to which the virus stock was added at a moiof 100 viral plaque-forming units/DC. Transduction was carried out for 2 h at37°C, and the DCs were then washed extensively and resuspended at 53 105

DCs/0.2 ml PBS/animal for injection into mice. Cell counts were determinedusing a hemocytometer, with viability assessed by trypan blue exclusion. In allcases, viability exceeded 95%.

Murine CTL Generation. Two weeks after priming (by AdV/DC orpeptides), splenocytes (33 106/cells well) were activatedex vivowith irradi-ated, mitomycin C-treated Jurkat/hAFP or Jurkat/MART (53 105/cells well)in 2 ml of RPMI 1640/10% FBS and 50 units/ml IL-2 in 24-well plates for 6days.

Murine ELISPOT Assay. Groups of HLA-A2.1/Kb tg mice were primedby AdV-transduced DC immunizations, and 2 weeks later, splenocytes(4 3 106/well) were activatedex vivowith an optimal concentration of peptidein 1 ml of complete medium (RPMI 1640/10% FBS) in 24-well plates. For tgmice primed through peptide immunizations, the draining popliteal and ingui-nal lymph nodes were removed 10 days after immunization, and a single cellsuspension was prepared. Lymph node cells (53 106 cells/ml) were culturedin T-25 flasks in an equal volume with irradiated Jurkat-A2/Kb cells (stablytransfected with either MART-1 or hAFP at 13 105 cells/ml) plus 50 units/mlIL-2.

Fig. 3. Peptide specificity of human hAFP542–550or MART-127–35lymphocyte cultures.A, PBMCs were stimulated weekly with hAFP542–550and assayed for cytotoxicity againstpeptide-pulsed T2 cells after 4–5 weeks of expansion to confirm peptide specificity.B, PBMCs were stimulated weekly with MART-127–35 and assayed for cytotoxicity againstpeptide-pulsed T2 cells after 4–5 weeks of expansion to confirm peptide specificity.C, hAFP542–550cell cultures were assayed for frequency of peptide-specific IFN-g-secreting cellsby ELISPOT after restimulation for 48 h with peptide-pulsed autologous LCL.D, MART-127–35cell cultures were assayed for frequency of peptide-specific IFN-g-secreting cells byELISPOT after restimulation for 48 h with peptide-pulsed autologous LCLs.

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After culture for 48 h for both splenocyte and lymph node cell cultures, cellswere washed and transferred by serial dilution (from 23 105 to 2 3 104

cells/well) to 96-well microtiter plates precoated with capture antibodies(IFN-g or IL-4; PharMingen) at 2mg/ml in serum-free medium (X-Vivo).After 24 h, cells were removed, and spots were visualized using biotinylatedsecondary antibodies and avidin-D-peroxidase in conjunction with 3-amino-9-ethylcarbazole substrate, as described above. The frequency of antigen-specific cells was determined from the difference between the number of spotsseen with and without antigen during restimulation.

RESULTS

We demonstrate that the human T-cell repertoire can recognize aHLA-A2.1-restricted peptide epitope derived from AFP using thefollowing lines of evidence.

AdVhAFP/DC-stimulated Human T Cells Recognize AFP-ex-pressing Cells.DCs genetically engineered to express tumor antigensby adenoviral transduction are potent inducers of T-cell responsesinvitro and in vivo. We first used DCs transduced with an AdV-expressing human AFP (AdVhAFP) to generate AFP-specific, HLA-A2.1-restricted T-cell responses using CD81 enriched in vitro Tlymphocyte cultures.

Human DCs differentiated from loosely adherent peripheral bloodprogenitors in GM-CSF and IL-4 express hAFP mRNA in a viraldose-dependent manner when transduced by AdVhAFP (Fig. 1A).T-cell cultures stimulated weekly with AdVhAFP/DC generated AFP-specific T cells that recognized AFP-expressing cells by both cyto-

toxicity assay (Fig. 1B) and IFN-g ELISPOT analysis (Fig. 1C). TheseT cells lysed M202/hAFP transfectants but not parental M202 cells(Fig. 1B), and ELISPOT analysis shows a greater frequency of IFN-g-secreting cells responding to AFP-transfected cells compared toparental cells (Fig. 1C).

Identification of Immunogenic AFP Peptides. We next sought toidentify potential HLA-A2.1-restricted hAFP peptide epitopes recog-nized by these AFP-specific T cells. A computer-based analysis of thepublished human AFP coding sequence was performed to identify 9-and 10-mer peptides whose sequences conformed to the well-charac-terized binding motif for HLA-A2.1 (56–59). Here, we report thecomplete analysis of hAFP542–550(GVALQTMKQ). This 9-aa pep-tide has one HLA-A2.1 anchor residue in position 2 and binds withlow affinity but forms a stable complex. Table 1 presents the HLA-A2.1 binding properties of hAFP542–550and those of two well-char-acterized HLA-A2.1 immunodominant peptides, Flu M158–66 andMART-127–35.

AdVhAFP/DC-stimulated T-cell Cultures Recognize AFP542–550.Because AdVhAFP/DC-stimulated T cells from human lymphocytecultures specifically recognized hAFP-transfected targets in both CTLand ELISPOT assays, we wished to determine whether these T cellswould also recognize hAFP542–550 in the context of HLA-A2.1.Therefore, after 7–21 days of culture, AdVhAFP/DC T cells weretested for both cytotoxicity and the frequency of hAFP542–550-specificIFN-g cytokine-producing cells by ELISPOT (Fig. 2AandC). As aninternal control, we prepared AdVMART1/DC-stimulated cell cul-

Fig. 4. AFP specificity of human hAFP542–550lymphocyte cultures.A, PBMCs were stimulated weekly with hAFP542–550and assayed for cytotoxicity against the two knownAFP-expressing HCC cell lines, HepG2 (HLA-A2.11) and Hep3B (A22). HepG2 cells were also treated with IFN-g to up-regulate class I expression treated or with anti-b2-microblobulin to block class I.B, cytotoxicity of hAFP542–550cell cultures against M202 and M202/hAFP.C, hAFP542–550cell cultures were assayed for frequency of AFP-specificIFN-g-secreting cells by ELISPOT after restimulation for 48 h with AFP stable transfectant or untransfected parental cells.

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tures, which, as we have reported previously, will generate T cells thatrecognize the A2.1-restricted MART-127–35immunodominant peptide(Ref. 42); (Fig. 2Band D). AdVhAFP/DC T cell cultures werespecifically cytotoxic for T2 cells pulsed with AFP542–550and, whenrestimulated with autologous peptide-pulsed LCLs, contained overfour times as many IFN-g-secreting cells specific for AFP542–550

compared to MART-127–35.hAFP542–550 Peptide-specific Human T-cell Culture. The

hAFP542–550synthetic peptide was then used to stimulate human Tcellsin vitro. Bulk T-cell cultures were generated from PBMCs pulsedwith hAFP542–550and tested between weeks 3 and 9 of expansion forthe ability to kill both peptide-pulsed (Fig. 3) and AFP-expressingtargets (Fig. 4). These cultures generated hAFP542–550 pep-tide-specific CTLs (Fig. 3A) and an increased frequency ofhAFP542–550-specific T cells (Fig. 3C) compared to controls forboth IFN-g (Fig. 3C) and GM-CSF cytokines (data not shown).Results for MART-127–35peptide internal control cultures are alsoshown (Fig. 3,B and D).

hAFP542–550T-cell cultures were tested for cytotoxicity and cyto-kine release against both hAFP-expressing HCC lines (HepG2 and

Hep3B) and stably transfected targets M202 (M202/hAFP) and LCL(LCL/hAFP). The hAFP542–550 peptide-specific lymphocytes werespecifically cytotoxic for the HLA-A2.1-positive cell line HepG2and not for the A2.1-negative cell line Hep3B (Fig. 4A). Thekilling of HepG2 was increased with IFN-g treatment (whichup-regulates class I) and decreased withb2-microglobulin blockingantibody. The low level of Hep3B killing was eliminated by theaddition of unchromated K562 cells (a natural killer cell target),whereas this did not alter the killing of HepG2 cells (data notshown). Similarly, hAFP transfectants were recognized byhAFP542–550-specific lymphocytes to a much greater extent thanparental cells in both cytotoxicity (Fig. 4B) and IFN-g synthesis(Fig. 4C), indicating that this peptide is naturally processed andpresented in these HLA-A2.1-positive cell lines.

AFP542–550Peptide-specific Responses in HLA-A2.1/Kb tg MiceImmunized with AdVhAFP/DC. HLA-A2.1/Kb tg mice were usedto determine whether the murine T-cell repertoire could recognizehAFP542–550 in the context of HLA-A2.1. Splenocytes from miceimmunized with DCs transduced with AdVhAFP were restimulatedinvitro with hAFP542–550and assayed for both cytotoxicity and IFN-g

Fig. 5. hAFP542–550-specific CTL activity and cytokine release from AdVhAFP/DC immunization in A2.1/Kb tg mice.A andB, 2 weeks after immunization, splenocytes from micewere restimulated with Jurkat cells stably tranfected with hAFP (J-AFP) for 6 days. Spleen cells were assayed against T2 target cells pulsed with hAFP542–550or MART-127–35in a5-h 51Cr release assay. In the same assay, minimal lysis of hAFP542–550-pulsed targets was observed when targets were incubated with effectors from AdVMART-1/DC-immunizedmice restimulated with Jurkat cells stably transfected with MART-1 (J-MART).C andD, increased frequency of IFN-g production in splenocytes from A2.1/Kb tg mice immunizedwith AdVhAFP/DC. Two weeks after immunization, splenocytes from mice were restimulated with 30mg of hAFP542–550or MART-127–35or no peptide for 48 h before assayingcytokine secretion in an ELISPOT assay. Lower frequency of spots was observed in splenocytes from AdVMART-1/DC-immunized mice, identically restimulated, in the sameexperiment.

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production by ELISPOT. AdVhAFP/DC immunization induced astrong AFP542–550-specific response (Fig. 5A andC), whereas naivesplenocytes showed no cytotoxicity and had few IFN-g-producingcells (data not shown). Conversely, AdVMART1/DC-immunizedmice specifically recognized the immunodominant MART-127–35

peptide (Fig. 5B andD).AFP-specific Responses in HLA-A2.1/Kb tg Mice Immunized

with Peptide in CFA. To confirm thatin vivo immunization withhAFP542–550 peptide results in AFP-specific responses, tg micewere immunized s.c. in the foot pads with 100mg of hAFP542–550

or MART-127–35peptides in CFA. Cytotoxicity and IFN-g-specificELISPOT assays were performed with recovered splenocytes andlymph node cells restimulatedin vitro with Jurkat/AFP- or Jurkat/MART-transfected cell lines. Immunization with hAFP542–550andsubsequent restimulation with Jurkat/AFP induced both peptide-specific (Fig. 6A) and AFP-specific (Fig. 6C) cytotoxicity as wellas large numbers of IFN-g-producing cells (Fig. 6E). Lymphocytesfrom PBS-injected mice showed neither cytotoxicity nor IFN-gproduction, regardless of restimulation (data not shown). Mice

immunized with MART-127–35peptide produced MART-1-specificresponses.

DISCUSSION

This is the first report that the human T-cell repertoire can recog-nize AFP in the context of MHC. One of the HLA-A2.1-restrictedpeptide epitopes recognized by these T cells is the 9-mer hAFP542–550

(GVALQTMKQ). This result was obtained using two basic strategies.In the first strategy, DCs genetically engineered to express hAFP wereshown to generate AFP-specific T-cell responses in CD8-enrichedhuman lymphocyte cultures. These AFP-specific T cells also recog-nized AFP542–550-pulsed cells in both cytotoxicity and cytokine re-lease assays. In addition, we demonstrated that AFP542–550peptide-generated T cells recognized both hAFP-expressing HCC lines andhAFP-transfected targets in human lymphocyte cultures and in HLA-A2.1/Kb tg mice. Thus, we provide compelling evidence that: (a) thehuman T-cell repertoire recognizes AFP in the context of HLA-A2.1;and (b) AFP542–550 is naturally processed and presented by AFP-

Fig. 6. hAFP542–550-specific CTI and cytokine release activity from hAFP542–550/CFA immunization in tg mice.A andB, 2 weeks after immunization, splenocytes from mice wererestimulated with Jurkat/AFP (J-AFP) for 6 days. Spleen cells were assayed against T2 target cells pulsed with or without hAFP542–550in a 5-h51Cr release assay. In the same assay,minimal lysis of hAFP542–550-pulsed targets was observed when targets were incubated with effectors from MART-127–35/CFA-immunized mice restimulated with Jurkat/MART.CandD, Jurkat/AFP-specific CTL activity from hAFP542–550/CFA immunization in tg mice. Two weeks after immunization, splenocytes from mice were restimulated with Jurkat/AFPfor 6 days. Spleen cells were assayed against Jurkat/AFP or Jurkat/MART target cells for specific lysis in a51Cr release assay.E andF, recognition of AFP by hAFP542–550/CFA-immunized murine splenocytes. Two weeks after immunization, splenocytes from mice were restimulated with J-AFP or J-MART for 48 h before assaying IFN-g secretion in anELISPOT assay. Frequency of AFP- or MART-1-reactive cells is shown for both hAFP542–550- and MART27–35/CFA-immunized mice.

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engineered DCs and AFP-transfected targets serving as a class I-re-stricted immunogen and antigen, respectively.

The findings from this human AFP model support the importantconcept that certain “self” proteins overexpressed by tumor cells canbe recognized by CD8 T cells in a MHC class I-restricted fashion. Forproperly selected antigens, this autoimmune response can be an ef-fective antitumor immune response (42, 60, 61).

It is clear that the T-cell repertoire is capable of recognizing these“self” peptides. For MART-1 and gp100 immunodominant peptides,the HLA-A2 binding affinities are intermediate due to the absence ofoptimal residues at either the second or ninth anchor positions (59,62–66). One hypothesis that has been forwarded is that high affinitypeptides expressed at high levels on the cell surface may generatetolerance due to strong negative selection in the thymus during initialT-cell education (50, 67). Lower binding or “subdominant” determi-nants may be capable of stimulating peptide-responsive T cells notdeleted from the T-cell repertoire. Work by the Melief group hasprovided evidence that the off-kinetics, or dissociation rate, of apeptide bound to class I is highly predictive of the immunogenicity ofthat peptide (50). This group analyzed known self, immunogenicepitopes from melanoma antigens (gp100 and MART-1) and foundthat their stable binding affinity in a soluble class I reconstitutionassay was low, but they had very slow off-kinetics. Like many tumorantigen peptide epitopes (68–73), hAFP542–550has one anchor residueand intermediate binding affinity to HLA-A2.1. Molecular modelingof GVALQTMKQ in the HLA-A2.1 binding groove showed that thevaline side chain at P2 was buried in the B pocket of the MHCmolecule, whereas the side chains in the central part of the peptide(L4–T6) were mostly exposed. The lack of a hydrophobic side chain(V or L) at P9 may explain the weak affinity of this peptide forHLA-A2.1. We are currently investigating single and double aminoacid substitutions in this peptide to potentially increase binding andimmunogenicity while retaining AFP specificity.

The HLA-A2.1/Kb tg mice allow anin vivoanalysis of AFP epitopeprocessing and presentation in the context of HLA-A2.1. Severalreports have already shown the comparable range of epitopes pre-sented by these mice and humans (74, 75). In these mice, AFP geneticimmunization generated hAFP542–550-specific responses, and peptidehAFP542–550immunization generated AFP-specific responses to cellsendogenously expressing the gene. Together, these data confirm thatAFP can serve as a tumor-specific antigen in HCC and is a suitabletarget for T-cell-mediated immunotherapy strategies in this disease.

ACKNOWLEDGMENTS

We thank Dr. Eli Sercarz and Dr. Mitchell Kronenberg for helpful discus-sions, Dr. C. J. Melief and Dr. S. Van der Burg for suggestions on the use ofthe off-kinetics assay, and Dr. Linda Sherman for suggestions on the use of theHLA-A2.1/Kb tg mice.

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T-CELL RESPONSES TO ANa-FETOPROTEIN EPITOPE

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1999;59:3134-3142. Cancer Res   Lisa H. Butterfield, Andrew Koh, Wilson Meng, et al.   -Fetoprotein

αHLA-A2.1-restricted Peptide Epitope Derived from Generation of Human T-cell Responses to an

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