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Specific Cytolytic T-Cell Responses to Human CEA from Patients Immunized with Recombinant Avipox-CEA Vaccine

Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. Z., J. S., K. Y. T.]; Medical Oncology Division, Georgetown University Medical Center, Washington, DC 20007 [J. M.]; and Department of Surgery, Medical University of South Carolina, Charleston, South Carolina 29425 [D. C.]

	ABSTRACT

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Avipox viruses are replication-defective members of the poxvirus family. Avipox-derived vectors such as ALVAC (canarypox) and fowlpox have the ability to infect mammalian cells, including human cells, but do not replicate. The first clinical trial of an avipox recombinant vaccine for patients with advanced carcinomas has recently been conducted using the ALVAC vector and the human carcinoembryonic antigen (CEA) transgene (designated ALVAC-CEA; J. L. Marshall et al., J. Clin. Oncol., 17: 332–337, 1999). The T-cell responses elicited by patients before and after vaccination with the ALVAC-CEA recombinants are characterized in this report. Pre- and postvaccination peripheral blood mononuclear cells (PMBCs) of the eight patients positive for HLA-class I A2 allele, were incubated with the HLA-A2-CEA peptide CAP-1 and interleukin 2. In no cases using prevaccination PMBCs could cultures be established that had the ability to lyse C1R-A2 target cells pulsed with the CAP-1 peptide. However, T-cell cultures from seven of eight of these same patients, obtained from PBMCs after ALVAC-CEA vaccination, were shown to lyse C1R-A2 cells only when pulsed with CAP-1. Moreover, all seven of these T-cell cultures were shown to lyse allogeneic human carcinoma cell lines (SW1463 and SW480) that were both A2⁺ and expressed CEA; an allogeneic tumor cell line (LS174T) expressing CEA that was negative for A2 expression was not lysed. HLA-A2⁺ and CEA⁺ autologous tumor cells were also capable of being lysed by CEA-specific T cells from this patient. Analysis of this CTL line also revealed the expression of several homing and adhesion-associated molecules. Fluorescence-activated cell sorter analysis of the T-cell lines established from patients after ALVAC-CEA vaccination revealed that most were CD8⁺/CD4^-, but many also had a CD8⁺/CD4⁺ component. Analyses of T-cell receptor Vß usage of several of the CEA-specific CTL lines showed a relatively diverse Vß pattern. These studies demonstrate for the first time the ability to vaccinate cancer patients with an avipox recombinant and derive T cells that are capable of lysing allogeneic and autologous tumor cells in a MHC-restricted manner. These studies thus form the rationale to use such replication-deficient recombinant vaccines in future cancer vaccine trials.

	INTRODUCTION

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It has now been demonstrated that several human tumor-associated antigens can be recognized by human CTLs in the context of MHC-peptide complex on the surface of human tumor cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . A Phase I clinical trial involving the use of a recombinant vaccinia virus expressing CEA² (rV-CEA) in patients with metastatic carcinoma (gastrointestinal, lung, or breast) has been completed (15) . The only toxicity observed was that seen with the conventional smallpox vaccine. Cytotoxic T-cell lines specific for a human CEA epitope designated CAP-1 (a 9-mer immunodominant class I HLA-A2 CEA peptide) have been generated from patients immunized with the rV-CEA vaccine (16) .

Poxvirus family members, such as vaccinia and avipox, have been used as poxvirus-based vaccines against viral pathogens (17) . Replication of avipox viruses, which include fowlpox and canarypox, is highly restricted in avian species (18) . It has been reported that fowlpox and canarypox virus-rabies recombinants express the rabies glycoprotein in non-avian tissue culture cells, and the level of expression is sufficient to induce rabies-specific neutralizing antibodies and to protect against a lethal rabies virus challenge (19 , 20) . Potency tests in mice have shown that a canarypox virus vector is highly efficient in expressing rabies glycoprotein (20) . Additional data indicated that the avipox vectors are effective as immunizing agents in non-avian species using other viral immunogens (21 , 22) .

Preclinical evaluation of an ALVAC-human cytomegalovirus gB vaccine in mice indicated that it can induce both humoral and cell-mediated immune responses to human cytomegalovirus in hosts both with and without immunity to vaccinia (23) . Furthermore, results from preclinical studies in nonhuman primates indicate that prior exposure to ALVAC recombinants should not preclude subsequent vaccination with novel ALVAC recombinants (24) . Those findings were supported by Hodge et al. (25) , who demonstrated that mice immunized with rV-CEA and then ALVAC-CEA elicit CEA-specific T-cell responses to levels greater than those found with the use of either vector alone. Phase I clinical trials with ALVAC-rabies glycoprotein recombinant and HIV-I glycoprotein 160 recombinant demonstrated that the experimental vaccines were well tolerated and induced humoral and cellular immune responses in humans (26 , 27) .

A Phase I clinical trial in advanced carcinoma patients using a replication-defective avipox vector containing the human CEA gene (ALVAC-CEA) as vaccine has been completed (28) . The vaccine was administered i.m. three times at 28-day intervals. The vaccine was well tolerated at all dose levels (2.5 x 10⁵, 2.5 x 10⁶, and 2.5 x 10⁷ pfu) with no significant toxicity (28) . No objective antitumor responses were observed during the clinical trial in advanced carcinoma patients with measurable disease. The generation and characterization of CEA-specific cytotoxic T-cell lines from patients immunized with ALVAC-CEA is reported here. These results demonstrate that an ALVAC-CEA recombinant expressing CEA can induce specific anti-CEA CTLs capable of lysing, in a MHC-restricted manner, allogeneic and autologous tumor cells, thereby demonstrating the potential of the ALVAC vector in human anticancer vaccine development.

	MATERIALS AND METHODS

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Cell Cultures
Colorectal carcinoma cell lines SW480 (HLA-A2, 24), SW1463 (HLA-A1, 2) and LS174T (HLA-A2, -) were purchased from American Type Culture Collection (Manassas, VA). The cultures were free of Mycoplasma and were maintained in complete medium (DMEM; Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, 2 nM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). The C1R cell line is a human plasma leukemia cell line that does not express endogenous HLA-A or -B antigens (29) . C1R-A2 cells are C1R cells that express a transfected genomic clone of HLA-A2.1 (30) ; these cells were obtained from Dr. William E. Biddison (National Institute of Neurological Disorder and Stroke, NIH, Bethesda, MD). C1R-A2 cultures were free of Mycoplasma and were maintained in RPMI 1640 complete medium (Life Technologies, Inc.).

Peptides
The peptides CAP-1 (CEA amino acid positions 571–579; YLSGANLNL; Ref. 16 ) and NCA-1 (YRPGENLNL) were generated on a peptide synthesizer (model 432A; Applied Biosystem, Inc., Foster City, CA); and products were dissolved in aqueous solution, sterile filtered, and frozen at -70°C at a concentration of 2 mg/ml. The purity of the peptides was >90%, as analyzed by high-performance liquid chromatography.

Generation of T-Cell Lines
PBMCs were obtained from heparinized blood of patients with metastatic colon carcinoma who enrolled in a Phase I trial using ALVAC-CEA (28) . All experiments involving patient materials were conducted according to NIH guidelines, and written, informed consent was obtained from all individuals. The protocol described by Tsang et al. (16) was used to generate the T-cell line. PBMCs were obtained prior to and after ALVAC-CEA injections given i.m. on days 1, 29, and 58 at 2.5 x 10⁵ pfu (level I), 2.5 x 10⁶ pfu (level II) or 2.5 x 10⁷ pfu (level III). PBMCs from the patient were separated using lymphocyte separation medium gradient (Organon Teknika, Durham, NC) as described previously (31) . Washed PBMCs were resuspended in complete medium (RPMI 1640; Life Technologies, Inc.) supplemented with 10% pooled human AB serum (Valley Biomedical, Winchester, VA), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml of streptomycin (Life Technologies, Inc.). Cells (2 x 10⁵) in complete medium in a volume of 100 µl were put into each well of a 96-well flat-bottomed assay plate (Corning Costar Corp., Cambridge, MA). CAP-1 peptide was added to cultures at a final concentration of 25 µg/ml. Cultures were incubated for 5 days at 37°C in a humidified atmosphere containing 5% CO₂. After the peptide-containing medium was removed, the cultures were supplemented with recombinant human IL-2 (provided by the National Cancer Institute-Frederick Cancer Research and Development Center; 10 units/ml) for 11 days. Medium containing IL-2 was replenished every 3 days. The 5-day incubation with peptide and 11-day treatment with IL-2 constituted one IVS cycle. Primary cultures were restimulated with CAP-1 peptide (25 µg/ml) on day 16 to begin the next IVS cycle. Irradiated (4000 rads) autologous PBMCs (5 x 10⁵) were added in 50 µl of complete medium as APCs.

Cytotoxic Assays
Target cells were labeled with 50 µCi of ¹¹¹In oxyquinoline (Medi-Physics, Inc., Arlington, IL) for 15 min at room temperature. Target cells (0.5 x 10⁴) in 100 µl of complete medium were added to each of 96 wells in U-bottomed assay plates (Corning Costar Corp.). The labeled target cells were incubated with peptides at various concentrations for 60 min at 37°C in 5% CO₂ before adding effector cells. Effector cells were suspended in 100 µl of complete medium supplemented with 10% pooled human AB serum and were added to target cells. The plates were then incubated at 37°C in 5% CO₂ for 4 or 16 h. Supernatant was harvested for gamma counting with the use of harvester frames (Skatron, Inc., Sterling, VA). Determinations were carried out in triplicate, and SDs were calculated. All experiments were carried out three times. Specific lysis was calculated with the following formula:

Spontaneous release was determined from wells to which 100 µl of complete medium were added. Total releasable radioactivity was obtained after targets were treated with 2.5% Triton X-100.

Flow Cytometry
Single-Color Flow Cytometric Analysis.
The method for single-color flow cytometric analysis has been described (32) . Briefly, cells were washed three times with cold Ca²⁺ and Mg²⁺-free DPBS, and then stained for 1 h with a MAb against HLA-A2 (A2, 69, 131HA-1; One Lambda, Inc., Canoga Park, CA) using 10 µl of the 1x working dilution/10⁶ cells. MOPC-104E (Cappel/Organon Teknika Corp., West Chester, PA) was used as isotype control. The cells were then washed three times and incubated with 1:100 dilution of FITC-labeled goat antimouse IgG (Kirkegaard & Perry Labs, Gaithersburg, MD). Anti-CEA MAb COL-1 was used as 100 µl of culture supernatant. The cells were then washed three times with cold DPBS and incubated for 1 h more in the presence of 1:100 dilution (volume of 100 µl PBS containing 1% BSA) of FITC-conjugated goat antimouse immunoglobulin (Kirkegaard & Perry Labs). The cells were again washed three times with DPBS and resuspended in DPBS at a concentration of 1 x 10⁶ cells/ml. The cells were immediately analyzed using a Becton Dickinson FACScan equipped with a blue laser with an excitation of 15 nW at 488 nm. Data were gathered from 10,000 live cells, stored, and used to generate results.

Dual-Color Flow Cytometric Analysis.
The procedure for dual-color flow cytometric analysis was similar to that for single-color analysis, except for the following: the antibodies used were anti-CD4 FITC/anti-CD8 PE conjugate, anti-CD2 FITC/anti-CD54 (Intercellular adhesion molecule-1) PE, anti-CD45 FITC/anti-CD49d PE, anti-CD11a (LFA-1) FITC/anti-CD58 (LFA-3) PE, anti-CD3 FITC/anti-CD62L PE, and anti-IgG1 FITC/anti-IgG2a PE (isotype controls). All of the antibodies listed above were purchased from Becton Dickinson. Staining was done simultaneously for 1 h, after which cells were washed three times, resuspended as above, and immediately analyzed using a Becton Dickinson FACSort equipped with a blue laser (excitation, 15 nW at 488 nm) and the CellQuest program.

	RNA Extraction and cDNA Synthesis

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Total cellular RNA was isolated from 5 x 10⁶ T cells using the standard guanidine isothiocyanate/acid phenol method (33) . First-strand cDNA was then synthesized from 1 µg of total RNA and RT Superscript II (Life Technologies, Inc., Gaithersburg, MD).

	Oligonucleotides

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Vß subfamily-specific primers were used as described by Nishimura et al. (34) . The previously fluorescent runoff Cß-specific primer (5'-X-CACAGCGACCTCGGGTGGG-3'; Ref. 35 ) was synthesized by the manufacturer (Perkin-Elmer, ABI Division, Custom Oligo Synthesis Service, Foster City, CA).

	PCR Amplification

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Aliquots of the cDNA synthesis reaction (corresponding to 200 ng of total RNA) were amplified in 50-µl reactions with 1 of the 25 Vß oligonucleotides and the Cß oligonucleotide. The final concentration of each primer was 20 pM/reaction. 0.2 mM of deoxynucleoside triphosphate and 1.5 mM MgCl₂ were in the Taq polymerase buffer (PE Applied Biosystems, Branchburg, NJ). The PCR cycle was as follows: denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, extension at 72°C for 4 min, and a final, 10-min polymerization at 72°C. A blank control without cDNA was included for each of the 24 Vß-Cß PCR reactions.

	Electrophoresis and Fragment Analysis

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The labeled PCR products were loaded on a 6% acrylamide sequencing gel with the PCR reaction diluted 1:10 prior to loading. Samples were then run on an ABI 373 sequencer to determine their size and fluorescence intensity (35) . The intensities of the peaks present in the labeled PCR products were measured at the end of the electrophoresis run, and the different peaks present in all Vß subfamilies were added together. The relative percentages of each Vß subfamily were then calculated and represented as histograms. This analysis was performed using the GeneScan collection and analysis software.

	Cytokine Detection

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T-cell supernatants incubated with peptide-pulsed APCs for 24 h in IL-2-free medium at a responder:stimulator ratio of 1:3 (10⁶ to 3 x 10⁶ cells/ml) were screened for the secretion of IFN-, tumor necrosis factor-, and IL-4 using an ELISA kit (Genzyme, Cambridge, MA). The results were expressed in pg/ml.

	RNase Protection Assays

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RNA from CAP-1-peptide-stimulated and unstimulated cells were analyzed by multiprobe RNase protection assay. Defined riboprobes for human cytokines were purchased from PharMingen (San Diego, CA). Assays were performed as described previously (36) . Radioactivity contained in bands on dried polyacrylamide gels was quantified with a Storm System PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The net cpm for a given band was calculated by the following formula (cpm of cytokine gene - cpm of background) and was expressed as a percentage of the housekeeping gene transcript L32.

	Statistical Analysis

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Statistical analysis of differences between pre- and postvaccination precursor frequencies was done using a paired t test. Statistical analysis of differences between mean values was performed with a two-tailed t test.

	RESULTS

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Generation of T-Cell Lines Specific for CAP-1.
A Phase I clinical trial was carried out using the replication-deficient avipox vector (ALVAC) containing the CEA gene (ALVAC-CEA) in patients with metastatic tumors expressing CEA. Three dose levels (2.5 x 10⁵, 2.5 x 10⁶, and 2.5 x 10⁷) were used. At each dose level, patients received three injections of vaccine i.m. at 28-day intervals via the Bioject system. In an attempt to establish CEA-specific CTL lines from patients, PBMCs were obtained prior to vaccination, as well as 1 month after the third vaccination. Of the 20 patients enrolled in the study, eight were shown to possess the HLA class I A2 allele. Because an HLA class I A2 binding peptide, designated CAP-1, has been identified (16) , the PBMCs of these HLA-A2-positive patients were chosen for further study.

PBMCs obtained from these patients pre- and postvaccination were incubated with the CAP-1 peptide and IL-2 using the protocol noted in "Materials and Methods." As described, each five-day incubation with CAP-1 was followed by an 11-day incubation with IL-2, which constitutes an IVS cycle. Using prevaccination PBMCs, T-cell cultures could be obtained from five of eight patients, but none of these cultures could be expanded past IVS-5. These cultures were assayed for CTL activity versus both C1R-A2 cells alone and those pulsed with CAP-1 peptide. CAP-1-specific lysis was not observed in any of the cultures from the prevaccination samples (Table 1) .

Human Tumor Cell Cytotoxicity.
The cultures from those patients that showed specificity for lysis of cells pulsed with the CAP-1 peptide were then assayed for their ability to lyse human carcinoma cells endogenously expressing CEA. Three carcinoma cell lines were used as targets: SW1463 and SW480, both of which express HLA-A2 and CEA; and LS174, which expresses CEA but little, if any, HLA-A2 (Table 6) . Cell lines derived from the seven patients that showed CAP-1-specific lysis all showed lysis to both the allogeneic SW1463 and SW480 carcinoma cell lines but not to the LS174 line (Table 2) . The difference in percentage of specific lysis between the A2⁺ lines and A2^- LS174 line was statistically significant (P < 0.05), as determined by the two-tailed t test.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Cytokine RNA expression of a CEA-specific T-cell line established from a vaccinated patient (no. 11). A, T cells from patient 11 were cultured with CAP-1-pulsed autologous EBV-transformed B cells for 24 h in IL-2-free medium at a responder:APC ratio of 1:3 (Lane 1) or without CAP-1 peptide (Lane 2). After stimulation with peptide for 24 h, RNA from T cells was analyzed by multiprobe RNase protection assay. The quantitative representation of results from the autoradiograph is normalized for expression of the housekeeping gene L32 (B). , T cells stimulated with CAP-1-peptide-pulsed autologous B cells; , T cells stimulated with autologous B cells only.

View larger version (21K): [in this window] [in a new window]   Fig. 2. TCR analyses of CEA-specific T-cell lines. The relative percentage of each Vß subfamily is presented by histograms. A, T-cell line from patient 11; B, T-cell line from patient 16; C, T-cell line from patient 18. Total cellular RNA was isolated from 5 x 106 T cells. First-strand cDNA was then synthesized from 1 µg of total RNA. cDNA was applied with 25 Vß oligonucleotides and FITC-labeled Cß oligonucleotide. Labeled PCR products were loaded on a 6% acrylamide sequencing gel, and the samples were then run on an ABI 373 sequencer for size and fluorescence intensity determination.

View larger version (62K): [in this window] [in a new window]   Fig. 3. CDR3 patterns for selected Vß subfamilies of CEA-specific T-cell lines. A, the CDR3 pattern for selected Vß subfamilies of T-cell line 11; B, line 16; C, line 18. See legend to Fig. 2 and "Materials and Methods" for details.

	DISCUSSION

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It has been reported that the frequency of CAP-1-specific CTL precursors was higher in the postvaccination PBMCs than in the prevaccination PBMCs in a rV-CEA Phase I clinical trial (16) . Similarly, in seven of nine patients, the precursor frequencies of CAP-1-specific CTLs were higher in postvaccination PBMCs (28) . The increase in CTL precursor frequency to CAP-1 may be interpreted as the ability of ALVAC-CEA to elicit CEA-specific T-cell responses. The studies reported here demonstrate CTL response to only one CEA peptide (i.e., CAP-1). However, it has been shown (38) that patients can elicit T-cell responses to other A2 CEA peptides after vaccination with rV-CEA. Moreover, human T-cell responses to other CEA peptides (A3 and A24 alleles) have been demonstrated in vitro (39 , 40) .

The Vß gene usage of T-cell lines from each of three patients (nos. 11, 16, and 18) at IVS-6 was analyzed using 25 Vß oligonucleotides. A relatively restricted TCR Vß usage was noted in T-cell line 18 but not in lines 11 and 16. TCR Vß 5, 7, 8, 13, 14, and 21 were detected at a higher frequency in all three T-cell lines. Vß 5, 6, 7, 8, 13, 14, and 21 represented most of the population of T-cell line 18. The CDR3 analysis of all three T-cell lines suggests that these Vß subfamilies consist of relatively polyclonal patterns. This result is in contrast to our previous report that the V8T cell line (41) established from a patient vaccinated with rV-CEA showed oligoclonal patterns in Vß gene usage. The polyclonal pattern may be attributable to the fact that the Vß subfamily analysis was performed using T-cell lines, not clones, at a relatively low IVS (IVS-6). Vß subfamily analysis of the V8T cell line was performed at IVS-10 and IVS-20. It is conceivable that oligoclonal patterns in Vß subfamilies may be observed in the higher IVS in T-cell lines 11, 16, and 18. Furthermore, these results suggest that the TCR repertoire determined in T-cell lines established from patients vaccinated with ALVAC-CEA was diverse. Similar results were obtained from T-cell lines established from patients vaccinated with rV-CEA (41) . Diversity in the TCR Vß usage has been reported in CTLs specific to other tumor-associated antigens (42, 43, 44) .

Expression of homing-associated adhesion molecules and cytotoxic activity to autologous tumor cells was also investigated to ascertain the effectiveness of the ALVAC-CEA vaccine in eliciting specific T-cell responses. T-cell line 11 was used for the detailed analysis. The expression of cell adhesion molecules on T-cell lines has been implicated in CTL function. Molecules such as LFA-1 (CD11a) and CD49d have been shown to be involved in lymphocyte homing in vivo (45) . In this study, CD11a and CD49d were expressed in 98% of the population of T-cell line 11. The expression of these molecules may be important for CTL function and for the development of potential adoptive transfer immunotherapy protocols. Furthermore, T-cell line 11 has a cytokine profile of a CD8 Tc1 effector cell. Analysis of the cytolytic activity of T-cell line 11 indicated that CTLs generated from a patient vaccinated with ALVAC-CEA using CAP-1 peptide can kill not only CAP-1-pulsed C1R-A2 cells but also HLA-A2- and CEA-expressing autologous and allogeneic tumor cells.

Phase I clinical trials using CEA as immunogen have demonstrated that CEA is immunogenic in humans (16 , 28 , 46 , 47) . Analysis of patient sera for antivaccinia antibodies before and after each rV-CEA vaccination indicated an increase in antivaccinia antibodies after the first vaccination (16) . For this reason, CEA-specific immune response to subsequent vaccination with rV-CEA was most likely limited because of inhibition of virus replication. Preclinical studies in mice have shown that prior exposure to vaccinia virus did not diminish the development of human cytomegalovirus gB-specific immune responses after a single dose of ALVAC-gB injected i.p. or s.c. (23) . New strategies involving diversified prime and boost protocols with rV-CEA, followed by boosting with ALVAC-CEA, may be more superior in the induction of CEA-specific immune responses (25) . DCs are potent APCs that have been shown to stimulate both memory and naive T-cell responses in vitro (48) . CEA-specific CTLs can be generated in vitro using peptide-pulsed DCs (47 , 49) . In addition, DCs infected with a poxvirus encoding MART-1/Melan A have been shown to sensitize T-cells in vitro (50) . The use of DCs infected with ALVAC-CEA, perhaps in combination with costimulatory molecules and cytokines, may augment the CEA-specific immune responses and can be used to develop future immunization protocols.

The studies reported here demonstrate for the first time that a replication-defective avipox recombinant can be used to vaccinate cancer patients and elicit T-cell responses specific for a given tumor-associated antigen and epitope (CAP-1), which are capable of lysing human tumor cells expressing that antigen.

	FOOTNOTES

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

¹ To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, 10 Center Drive, Building 10, Room 8B07, Bethesda, MD 20892-1750.

² The abbreviations used are: CEA, carcinoembryonic antigen; rV-CEA, recombinant vaccinia-CEA; gB, glycoprotein B; pfu, plaque-forming unit(s); PBMC, peripheral blood mononuclear cell; DC, dendritic cell; APC, antigen-presenting cell; MAb, monoclonal antibody; IL, interleukin; IVS, in vitro stimulation; TCR, T-cell receptor; DPBS, Dulbecco’s phosphate-buffer saline; PE, phycoerythrin; LFA, leukocyte function antigen.

Received 3/26/99; revised 10/ 1/99; accepted 10/18/99.

	REFERENCES

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