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Clinical Cancer Research Vol. 9, 1616-1627, May 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Differential Gene Expression Profiles in a Human T-cell Line Stimulated with a Tumor-associated Self-peptide versus an Enhancer Agonist Peptide

Claudia Palena, Jeffrey Schlom1 and Kwong-Yok Tsang

Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Purpose: Previous studies have shown that a specific 9-mer amino acid epitope (designated CAP-1) of the human "self" tumor-associated carcinoembryonic antigen can be used to stimulate CD8+ T cells from peripheral blood mononuclear cells of carcinoma patients vaccinated with pox vector-based carcinoembryonic antigen vaccines. A T-cell receptor agonist epitope of CAP-1 (designated CAP1-6D) has been shown to enhance the stimulation of T cells over levels obtained using CAP-1. The purpose of this study was to analyze gene expression profiles in T cells stimulated with the native CAP-1 versus the agonist CAP1-6D peptide.

Experimental Design: Microarray analyses were conducted to analyze differential gene expression profiles of a T-cell line stimulated with native versus agonist peptides.

Results: Numerous genes and gene clusters are identified as differentially expressed as a consequence of stimulation with the agonist peptide versus the native peptide; two genes, however, stand out in magnitude: the chemokine lymphotactin and granzyme B. In particular, lymphotactin expression is >12 times more pronounced in agonist-stimulated T cells. An ELISA assay was developed that confirmed marked lymphotactin secretion in T cells when stimulated with the agonist versus the native peptide. A chemotaxis assay also demonstrated the biological activity of the lymphotactin produced.

Conclusions: To our knowledge, these are the first studies of gene expression profiles of a defined T-cell line in response to stimulation with a defined antigen. They are also the first to compare, via cDNA microarray, responses of a T-cell line to (a) a tumor-associated self-antigen and (b) a native epitope versus an agonist epitope.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
With the advent of DNA microarray technology, one can now better understand the molecular basis of different immunological phenomena. Expression of large numbers of genes can be simultaneously monitored using cDNA microarrays to elucidate gene products associated with differentiation, activation, and proliferation of immune cells in response to diverse stimuli.

Microarray technology has been used to study both B-cell and T-cell populations from apparently normal individuals, as well as from patients with immune deficiencies and lymphoid malignancies (1, 2, 3, 4) . To date, however, cDNA microarrays have been used to study human T-cell activation in response to mitogens (2 , 5 , 6) or anti-CD3 Ab2 (1) . Other studies have used this technology to explore those gene pathways related to the heat shock response in human T cells (7) or to investigate the effects of HIV infection of CD4+ T cells at the gene expression level (8) . Previous studies have also examined differential gene expression profiles between (a) T-helper 1 and T-helper 2 subsets of human CD4+ T cells stimulated with mitogens (9 , 10) and (b) human memory and naïve CD4+ T cells, at rest and after activation with anti-CD3 Ab (11) . Despite a rapid incursion of array technology in the field of immunology, none of the published studies to date have studied responses of T-cell lines directed against defined antigens or epitopes.

We have demonstrated previously (2) that human CD8+ T-cell lines specific for human CEA can be generated in vitro; these T cells were derived from PBMCs of carcinoma patients vaccinated with either vaccinia or avipox recombinant vaccines expressing CEA (12, 13, 14) . The T-cell lines were established using a defined 9-mer epitope of CEA designated CAP-1 (12 , 15) . It was subsequently shown that this peptide can be modified in an amino acid residue that is predicted to contact the T-cell receptor and consequently improve epitope-specific T-cell responses (16) . The agonist peptide, designated CAP1-6D (6N->D), has been demonstrated to enhance the levels of T-helper 1 cytokines (GM-CSF and IFN-{gamma}) produced by CAP-1-specific T-cell lines as compared with the native CAP-1 peptide (17) . In addition, CAP-1-specific CTL lines were able to lyse CAP1-6D-pulsed target cells more efficiently than target cells pulsed with the CAP-1 peptide (16 , 17) . Most importantly, CTL lines generated using the agonist CAP1-6D peptide can recognize and lyse human tumor cells expressing native CEA in a MHC-restricted (class I-A2) manner more efficiently than T cells derived using CAP-1 (16) .

Phase I clinical trials have demonstrated the ability of CEA-based vaccination to elicit T-cell immune responses in patients with advanced CEA-expressing carcinoma (13 , 18, 19, 20, 21, 22) . A Phase I study using dendritic cells pulsed with the native CAP-1 peptide elicited only modest CAP-1-specific immune responses in advanced cancer patients (21) . It has recently been shown, however, that vaccination of patients with metastatic CEA-expressing carcinoma with dendritic cells loaded with the agonist epitope of CAP-1 (CAP1-6D), resulted in clinical responses, and these clinical responses correlated with the expansion of CEA-specific CD8+ T cells (23) .

In the studies reported here, cDNA microarrays have been used to identify those particular genes that are differentially expressed in CEA-specific T cells as a consequence of stimulation with the agonist CAP1-6D peptide as compared with the native CAP-1 peptide. These studies have identified a number of genes that are differentially expressed; among these, granzyme B and, in particular, the SCYC1gene (encoding the C-chemokine lymphotactin) were highly expressed in CAP1-6D-stimulated CEA-specific T cells as compared with T cells stimulated with CAP-1. These results were confirmed by RT-PCR. An ELISA assay developed for lymphotactin and a chemotaxis biological assay also demonstrated higher levels of lymphotactin protein released by CEA-specific T cells after stimulation with the agonist peptide. From these results, one can hypothesize a potential role of the chemokine lymphotactin in enhancing antitumor immune responses mediated by tumor antigen-specific T cells stimulated in the presence of agonist peptides.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Cultures.
The V8T cell line is a CD8+ CTL line directed against the CAP-1 epitope of CEA (12) , which was generated from PBMCs of a patient with metastatic colon carcinoma who was vaccinated with a recombinant vaccinia-CEA vaccine (15) . The immunophenotyping and other properties of the V8T cell line have been described previously in detail (12 , 15, 16, 17) . The V8T cell line was maintained by in vitro restimulation every 14 days with irradiated (22,000 rads) EBV-transformed autologous B cells pulsed with 25 µg/ml CAP-1 peptide. The effector:APC ratio was 1:2. Cultures in RPMI 1640 (Invitrogen, Carlsbad, CA) were supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 10% human AB serum (Gemini Bio-Products, Woodland, CA), and 20 units/ml IL-2. Medium was replenished every 3 days.

Peptides.
The CEA peptide CAP-1 (YLSGANLNL; Ref. 12 ) and the CAP-1 agonist peptide CAP1-6D (YLSGADLNL; Ref. 16 ) were >96% pure and manufactured by American Peptide Company, Inc. (Sunnyvale, CA).

RNA Isolation and Amplification.
For cDNA microarray studies, V8T cells (2 x 106 cells, 1 x 106 cells/ml) were stimulated with irradiated autologous EBV-transformed B cells pulsed with 10 µg/ml CAP-1 or CAP1-6D peptide or without peptide. The effector:APC ratio was 1:2. At various stimulation times (8, 24, and 96 h), cells were collected, and CD8+ T cells were separated using anti-CD8-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA). Total RNA from T cells was isolated using the RNeasy RNA extraction kit (Qiagen Inc., Valencia, CA). RNA concentrations were determined by A260 nm reading, and RNA quality was evaluated by the ratio A260 nm/280 nm. RNA was amplified following the method described by Wang et al. (24) . One µg of total RNA was used for amplification. Briefly, the phage T7 RNA polymerase promoter was incorporated into cDNA by reverse transcription in the presence of oligo(dT) (15) -T7 primer (5'-AAA-CGA-CGG-CCA-GTG-AAT-TGT-AAT-ACG-ACT-CAC-TAT-AGG-CGC-T15-3') and template switch oligonucleotide primer (5'-AAG-CAG-TGG-TAA-CAA-CGC-AGA-GTA-CGC-GGG-3') using Superscript II reverse transcriptase (Invitrogen). Full-length double-stranded cDNA was synthesized using Advantage Polymerase (Clontech, Palo Alto, CA) after treatment with RNase H (Invitrogen). PCR cycles were 5 min at 37°C for RNA digestion, 2 min at 94°C for denaturing, 1 min at 65°C for specific priming, and 30 min at 75°C for extension. After purification, double-stranded cDNA was used as a template for in vitro transcription using a T7 MEGAscript kit (Ambion Inc., Austin, TX). Synthesized RNA was purified from template DNA by Trizol reagent (Invitrogen). The concentration and purity of aRNA were determined by measuring A260 nm and A260 nm/280 nm.

cDNA Microarray Hybridization.
The method described by Wang et al. (24) was used for cDNA microarray hybridization. Briefly, 3 µg of aRNA were labeled by reverse transcription using random hexamer primer (Boehringer Mannheim, Indianapolis, IN) in the presence of Cy-3-dUTP or Cy-5-dUTP (New England Nuclear Life Sciences, Boston, MA). For each experiment, both labeled targets were combined, and the mixture was hybridized overnight at 65°C onto cDNA microarrays (Hs-UniGEM2, National Cancer Institute) containing a total of 9130 spotted cDNAs. Fluorescent images of hybridized microarrays were taken using a GenePix 4000 scanner (Axon Instruments, Foster City, CA) at various PMT voltages to obtain maximum signal intensities with minimum saturation (<1%). Data were analyzed using GenePixPro 3.0 (Axon Instruments) and GeneSpring software (Silicon Genetics, Redwood City, CA). To conduct comparisons between different experiments, data were normalized by dividing the signal intensity of each gene by the median intensity of all of the measurements taken in that sample. The ratio between red intensity (Cy-5-dUTP, CAP1-6D-stimulated V8T cells) and green intensity (Cy-3-dUTP, CAP-1-stimulated V8T cells) for each gene was calculated, and the results shown below were the average for at least two replicated experiments. Hierarchical clustering (25) was applied to those genes that showed fluorescence intensities of >100 in both channels and had a normalized intensity ratio of >=3 or <=0.33. Pearson correlation was used as distance matrix.

Quantification of mRNA Using RT-PCR.
One µg of total RNA was reverse transcribed in 20 µl of 1x first-strand buffer containing 1 mM deoxynucleotide triphosphate mix, 0.3 nM oligo(dT) (20) , 0.01 M DTT, 20 units of RNase inhibitor (Promega, Madison, WI), and 200 units of Superscript II reverse transcriptase (Invitrogen). Samples were incubated at 42°C for 90 min. PCR was performed using 5 µl of the reverse transcription product in 1x PCR buffer, 0.4 mM deoxynucleotide triphosphate mix, 50 pmol of each forward and reverse primer, and 1 µl of Advantage Polymerase enzyme mix (Clontech). Primer sequences (26) were as follows: lymphotactin, 5'-TCA-GCC-ATG-AGA-CTT-CTC-3' (forward) and 5'-TAA-TTT-TAT-TCA-TGC-AGT-GCT-TTC-3' (reverse); IFN-{gamma}, 5'-AGC-TCT-GCA-TCG-TTT-TGG-GTT-3' (forward) and 5'-GTT-CCA-TTA-TCC-GCT-ACA-TCT-GAA-3' (reverse); granzyme B, 5'-GCT-TAT-CTT-ATG-ATC-TGG-GAT-C-3' (forward) and 5'-AAG-TCA-GAT-TCG-CAC-TTT-CGA-3' (reverse); and ß-actin, 5'-ATC-TGG-CAC-CAC-ACC-TTC-TAC-AAT-GAG-3' (forward) and 5'-CGT-GGT-GGT-GAA-GCT-GTA-GCC-GCG-CTC-3' (reverse). Amplification consisted of 35 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. PCR products were resolved on 1.5% agarose gel. Gel images were taken, and transcript levels were quantified for normalization to expression of ß-actin, using a Kodak 1D 3.0 documentation and analysis system.

Detection of IFN-{gamma}.
Supernatants of V8T cells stimulated with peptide-pulsed autologous EBV-transformed B cells were collected at 8, 24, and 96 h of stimulation and screened for IFN-{gamma} secretion using an ELISA kit (R&D Systems, Inc., Minneapolis, MN). Results were expressed in pg/ml.

ELISA for Lymphotactin.
An ELISA was developed for the detection of lymphotactin. Flat-bottomed 96-well plates (medium binding immunoassay plates; Greiner Bio-One Inc., Longwood, FL) were coated with 2 µg/ml capture Ab (goat antihuman lymphotactin Ab; R&D Systems, Inc.), incubated overnight at room temperature, and then washed with wash buffer [0.05% Tween 20 in PBS (pH 7.4)]. Nonspecific binding was blocked by adding 5% BSA in PBS (pH 7.4) for 2 h at 37°C. After rinsing with wash buffer, dilutions of the standard (recombinant human lymphotactin; R&D Systems, Inc.) and unknown samples were incubated onto the plates for 2 h at room temperature. After washing, 100 ng/ml biotinylated antihuman lymphotactin Ab (R&D Systems, Inc.) and a 1:1000 dilution of streptavidin-horseradish peroxidase conjugate were added to the wells for 1 h at room temperature. Plates were then incubated for 15 min with substrate solution, and absorbance in each well was measured at 450 nm.

Chemotaxis Assay.
Chemotactic responses were examined following a modification of a previously described method (27) using Blind Well Chambers (Neuroprobe, Gaithersburg, MD) with polyvinylpyrrolidone-free 5-µm-pore polycarbonate filters previously coated on one side with mouse Collagen IV (Trevigen, Gaithersburg, MD). Supernatants from V8T cells activated for 24 h with autologous APCs pulsed with 10 µg/ml CAP-1 or CAP1-6D peptide or without peptide were added to the lower chambers, and healthy donor PBMCs (7.5 x 104 cells, 75 µl in complete RPMI 1640 containing 1% human AB serum) were added to the upper chambers. As negative and positive controls, the lower chambers were loaded with medium alone or recombinant lymphotactin (50 ng/ml in complete RPMI 1640 containing 1% human AB serum), respectively. After incubation for 4 h at 37°C, filters were removed from the chambers, fixed, and stained with Diff-Quik stain (Dade Behring Inc., Newark, DE). Blocking assay was performed using anti-lymphotactin Ab (R&D Systems, Inc.). The number of cells associated with the lower side of the membranes was evaluated by direct counting of at least six x40 objective fields for the standard samples or at least nine x40 objective fields for the experimental samples.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Gene Expression Profiles of CEA-specific T Cells after Stimulation with Either the Native CAP-1 Peptide or Its Agonist CAP1-6D Peptide.
The V8T CD8+ T-cell line was derived from PBMCs of a patient who had been vaccinated with recombinant vaccinia-CEA. It was generated and passaged using the CEA CAP-1 peptide as described in "Materials and Methods." Three time points were selected to carry out gene expression studies in V8T cells stimulated with either CAP-1 or CAP1-6D peptide using cDNA microarrays. V8T cells were stimulated for 8, 24, and 96 h with each peptide; total RNA was isolated from each sample and amplified as described in "Materials and Methods." For each time point, fluorescent cDNA probes were made by reverse transcription of aRNA with Cy-3-dUTP and Cy-5-dUTP for CAP-1- and CAP1-6D-stimulated samples, respectively. Labeled cDNA probes were incubated onto cDNA microarrays containing a total of 9130 genes (Hs-UniGEM2; National Cancer Institute). After acquisition of fluorescent images, data were processed as described in "Materials and Methods."

To get an overview of the differential gene expression in V8T cells stimulated with CAP-1 versus CAP1-6D peptide, red fluorescence intensity (signal from CAP1-6D-stimulated sample) was plotted against green fluorescence intensity (signal from CAP-1-stimulated sample) for each individual gene at each time point (Fig. 1)Citation . Eight h of stimulation with CAP-1 or CAP1-6D peptide promoted a differential gene expression in V8T cells as shown in Fig. 1ACitation . Most of the genes contained in the arrays were expressed at equivalent levels in CAP-1- versus CAP1-6D-stimulated V8T cells. As indicated by genes spotted in red, a relatively high number of genes were overexpressed in CAP1-6D-stimulated V8T cells relative to CAP-1-stimulated V8T cells. A lower number of genes were expressed at decreased levels in CAP1-6D-stimulated V8T cells compared with CAP-1-stimulated V8T cells, as indicated by the genes spotted in green. The scatter in the points in Fig. 1BCitation indicates a greater number of genes differentially expressed in V8T cells stimulated for 24 h with CAP1-6D versus CAP-1 peptide, as compared with that observed at 8 h. There was a prevalence of overexpression of genes in CAP1-6D-stimulated V8T cells as compared with CAP-1-stimulated V8T cells. Fig. 1CCitation shows the comparison of gene expression in V8T cells stimulated with each peptide at 96 h. As expected, most of the genes were equivalently expressed after stimulation with each peptide.



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  		Fig. 1. Comparison of gene expression profiles in CEA-specific T cells (V8T cells) stimulated with CAP-1 or CAP1-6D agonist peptide-pulsed autologous B cells. RNA from V8T cells stimulated for 8 h (A), 24 h (B), or 96 h (C) was prepared as described in "Materials and Methods." Fluorescent cDNA probes were made by reverse transcription using Cy-3-dUTP and Cy-5-dUTP for CAP-1- and CAP1-6D-stimulated V8T cells, respectively. The plots compare red and green fluorescence intensities for each gene. Outer diagonal lines indicate a >=2-fold change in expression, and the center line indicates equivalent expression.

 
Additional microarray analyses were also conducted to compare gene expression profiles at 24 h of V8T cells stimulated with autologous APCs pulsed with CAP-1 versus no peptide, as well as V8T cells stimulated with autologous APCs pulsed with CAP1-6D peptide versus no peptide. As seen in Table 1Citation , over 600 genes were differentially expressed >2-fold, and over 200 genes were differentially expressed >3-fold when comparing CAP-1-stimulated with non-peptide-stimulated V8T T cells. When comparing CAP1-6D-stimulated V8T cells with non-peptide-stimulated T cells, >300 genes were differentially expressed 2-fold, and approximately 98 genes were differentially expressed >3-fold. When comparing CAP1-6D- versus CAP-1-stimulated V8T cells, >300 genes were differentially expressed 2-fold, whereas 54 genes were differentially expressed 3-fold (Table 1)Citation .


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  		Table 1 Differential gene expression in CEA-specific T cells (V8T) stimulated with autologous B cells pulsed with native peptide, agonist peptide, or no peptide

 
Further analysis was focused on those genes that exhibited at least a 3-fold difference in expression levels between CAP1-6D- and CAP-1-stimulated V8T cells and showed fluorescence intensity units of >100 in each channel. Using the standard correlation coefficient, genes with at least a 3-fold change in expression clustered into two nodes (Fig. 2)Citation . Cluster group A contains genes whose expression was increased at least 3-fold in CAP1-6D-stimulated V8T cells as compared with CAP-1-stimulated V8T cells at any time point analyzed. Genes whose expression levels were decreased at least 3-fold in CAP1-6D-stimulated V8T cells (at any time point) were clustered in group B.



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  		Fig. 2. Hierarchical cluster of differentially expressed genes between CAP1-6D- and CAP-1-stimulated V8T cells. Included in the cluster analysis were genes that differed at least 3-fold in expression. Rows correspond to individual genes; each vertical column represents a particular time point. The results represent normalized average intensity ratios. As indicated, the color scale extends from ratios of 3 (red) to 0.33 (green). Two nodes were identified, designated cluster groups A and B, respectively.

 
The normalized relative intensity ratio for each individual gene that had at least a 3-fold difference in expression between V8T cells stimulated for 8 h with CAP1-6D versus CAP-1 peptide is shown in Table 2Citation . Of a total of 12 genes differentially expressed, 4 of them encode secreted products of T cells, such as granzyme B, GM-CSF (denoted as CSF2 in Fig. 2Citation ), and two different T-cell-specific chemokines, SCYC1 (lymphotactin) and SCYA1 (I-309). Other genes differentially expressed encode for membrane proteins, transporters, and genes related to cell cycle control, transcriptional regulation, and metabolism. Above all, transcripts for lymphotactin were strongly expressed in V8T cells stimulated with CAP1-6D peptide, showing an increase of 8.5-fold relative to its expression in CAP-1-stimulated V8T cells.


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  		Table 2 Overexpression of genes in CEA-specific T cells (V8T) stimulated for 8 h with agonist CAP1-6D peptide-pulsed autologous B cells, as compared with V8T cells stimulated with native CAP-1 peptide

 
Individual genes that were differentially expressed at 24 h, showing at least a 3-fold change in expression, are listed in Table 3Citation . Genes were grouped in accordance with their presumed functions. A total of 54 genes that had a relative CAP1-6D:CAP-1 intensity ratio of >=3 or <=0.33 were detected at this time point. As also observed at 8 h of stimulation, some of the genes differentially expressed at 24 h belonged to the category of chemokines, growth factors, or effector molecules secreted by T cells. Transcripts of all these genes were more abundant in CAP1-6D-stimulated V8T cells. Lymphotactin and granzyme B showed the highest difference in expression (fold changes of 12.9 and 5.6, respectively) when CAP1-6D-stimulated V8T cells were compared with CAP-1-stimulated V8T cells. GM-CSF and the chemokines RANTES and Mip-1bwere also up-regulated in agonist-stimulated V8T cells. Other genes differentially expressed at 24 h encoded for surface and membrane proteins, transporters, and genes involved in cell cycle control, cell signaling, transcriptional regulation, and miscellaneous genes. A high number of genes differentially expressed at 24 h between agonist- and native peptide-stimulated V8T cells were related to processing, synthesis, and turnover of RNA and proteins.


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  		Table 3 Differential gene expression patterns in CEA-specific T cells (V8T) stimulated for 24 h with agonist CAP1-6D versus native CAP-1 peptide-pulsed autologous B cells

 
Increase of Lymphotactin and Granzyme B mRNA Levels in CAP1-6D-stimulated V8T Cells Was Confirmed by RT-PCR.
To confirm and validate the results obtained by cDNA microarray studies, RT-PCR was used to analyze expression levels of lymphotactin mRNA in CAP-1- and CAP1-6D-stimulated V8T cells. Total RNA was prepared from V8T cells stimulated for 24 h with autologous APCs pulsed with 10 µg/ml CAP-1 or CAP1-6D peptide or without peptide. After reverse transcription, cDNA from each sample was amplified using PCR primer pairs specific for lymphotactin. Amplification of ß-actin in each sample served as the positive control. RT-PCR products were quantified and normalized to those of ß-actin in each sample. As shown in Fig. 3ACitation , relative expression levels of lymphotactin mRNA in unstimulated (Fig. 3Citation , no peptide), CAP-1-stimulated, and CAP1-6D-stimulated V8T cells were 2.3, 4.3, and 278, respectively. The substantial increase in lymphotactin mRNA observed after stimulation with CAP1-6D peptide confirmed and actually magnified the results obtained by cDNA microarray studies.



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  		Fig. 3. Relative expression levels of lymphotactin, IFN-{gamma}, and granzyme B mRNA in T cells stimulated with native versus agonist CEA peptide, as measured by RT-PCR. A, total RNA was isolated from V8T cells stimulated for 24 h with autologous B cells pulsed with CAP-1 or CAP1-6D agonist peptide or without peptide. After reverse transcription, PCR amplification was performed using primer pairs specific for both lymphotactin and ß-actin. RT-PCR products were quantified and normalized to those of ß-actin in each sample. B, amplification of granzyme B mRNA in total RNA isolated from V8T cells stimulated for 24 h with CAP-1 or CAP1-6D agonist peptide-pulsed B cells. C, amplification of lymphotactin, IFN-{gamma}, and granzyme B mRNA in total RNA from V8T cells stimulated for 2 h with native versus agonist CEA peptide.

 
Additional studies were also carried out to quantitate the level of granzyme B mRNA in V8T cells after 24 h of stimulation with either CAP-1 or CAP1-6D peptide. As shown in Fig. 3BCitation , relative levels of granzyme B mRNA in V8T cells stimulated with CAP-1 and CAP1-6D peptide were 0.1 and 8.0, respectively (note the different scales in Fig. 3, A and BCitation ). These results were also in agreement with those observed in the cDNA microarray experiments.

Unfortunately, the cDNA arrays used in these studies did not contain IFN-{gamma} cDNA. To compare the level of IFN-{gamma} mRNA with that of lymphotactin and granzyme B, RT-PCR amplification was performed in total RNA obtained from V8T cells after 2 h of stimulation with either CAP-1 or CAP1-6D peptide. Results are shown in Fig. 3CCitation . Relative levels of lymphotactinmRNA were 0.3 and 3.7 in V8T cells stimulated with CAP-1 or CAP1-6D, respectively. IFN-{gamma}mRNA showed relative levels of 0.5 and 5.6 in CAP-1- and CAP1-6D-stimulated V8T cells, respectively. Relative levels of granzyme BmRNA were 0.3 and 9.0 in V8T cells stimulated for 2 h with CAP-1 or CAP1-6D peptide, respectively. In contrast to the results shown in Fig. 3, A and BCitation , for lymphotactin and granzyme Bexpression at 24 h, no IFN-{gamma}mRNA could be detected in the V8T cells stimulated for 24 h with each peptide (data not shown). These observations are in accordance with previous reports (28 , 29) and will be discussed below.

Lymphotactin mRNA was detectable at higher levels at 24 h of stimulation with both CAP-1 and CAP1-6D peptides, as compared with 2 h of stimulation. In contrast, IFN-{gamma} mRNA levels were higher at 2 h of stimulation, as compared with 24 h, with either CAP-1 or CAP1-6D peptide. Finally, granzyme B expression was relatively similar at 2 and 24 h of stimulation with each peptide. Thus, from the analysis of the relative levels of transcripts encoding for IFN-{gamma}, lymphotactin, and granzyme B, three different temporal patterns of expression can be distinguished.

Detection of Lymphotactin by ELISA Assay.
An ELISA assay was set up to measure lymphotactin-soluble protein in supernatants of cultured cells. Results are shown in Fig. 4ACitation . Lymphotactin protein was analyzed in supernatants of V8T cells after 8, 24, and 96 h of stimulation with APCs pulsed with CAP-1 or CAP1-6D peptide or without peptide (Fig. 4A)Citation . Although lymphotactinmRNA was detected by RT-PCR in V8T cells cultured with autologous APCs in the absence of peptide, no protein was detected in supernatants of these cells. Detection was positive at 8 and 24 h of stimulation with the two peptides, whereas results were negative at 96 h. A marked increase in lymphotactin protein levels was observed as a consequence of stimulation with the agonist peptide, which supports the results observed at the RNA level. Maximum protein expression was detected at 24 h, with a 389% increase observed in CAP1-6D-stimulated V8T cells as compared with CAP-1-stimulated V8T cells.



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  		Fig. 4. Release of lymphotactin and IFN-{gamma} by CEA-specific T cells (V8T cells) stimulated with CAP-1 or CAP1-6D agonist peptide-pulsed APCs. V8T cells (1 x 106 cells/ml) were stimulated with irradiated autologous B cells pulsed with 10 µg/ml of either CAP-1 or CAP1-6D peptide or without peptide. The effector:APC ratio was 1:2. Supernatants were collected at 8, 24, and 96 h of stimulation and assayed for lymphotactin (A) or IFN-{gamma} (B) by ELISA. Results are expressed in pg/ml.

 
Levels of lymphotactin protein were then compared with those of IFN-{gamma} protein. As observed in Fig. 4BCitation , IFN-{gamma} protein was undetectable in supernatants of unstimulated V8T cells (cultured with autologous APCs without peptide), whereas its expression was detectable in supernatants of cells stimulated with each peptide at 8, 24, and 96 h of stimulation. These results are not in concordance with those observed at the RNA level, in that IFN-{gamma}transcripts are not detectable at 24 h of stimulation with each peptide. These observations will be discussed below. At each time point, levels of IFN-{gamma} protein were higher in supernatants from agonist-stimulated V8T cells, with a maximum difference observed at 24 h (508% increase).

Supernatants from CAP1-6D-stimulated V8T Cells Enhance in Vitro Migration of Human PBMCs.
Lymphotactin has previously been shown to enhance chemotactic responses of different immune cell populations. A chemotaxis assay was used, as described in "Materials and Methods," to measure in vitro chemotactic responses of human PBMCs to supernatant fluids of V8T CEA-specific T cells stimulated with APCs pulsed with CAP-1 peptide or CAP1-6D peptide or without peptide. As seen in Fig. 5ACitation , supernatant fluids from V8T cells stimulated with CAP1-6D clearly enhanced migration of PBMCs over that observed in response to supernatant fluids from CAP-1-stimulated V8T cells. Purified recombinant lymphotactin was also used as a positive control in this assay. To ascertain that the observed chemotactic responses were due to the presence of lymphotactin, a chemotaxis assay was carried out with or without anti-lymphotactin Ab; two concentrations of Ab were used, and the results are shown in Fig. 5BCitation . The addition of anti-lymphotactin Ab clearly inhibited the PBMC migration induced by purified recombinant lymphotactin. Because there was little if any migration using supernatants of CAP-1-stimulated V8T cells, as expected, no effect was observed for supernatants from CAP-1-stimulated V8T cells when the Ab was added into the assay. Migration of human PBMCs in response to supernatant fluids of CAP1-6D peptide-stimulated V8T cells, however, was significantly decreased after the addition of anti-lymphotactin Ab, and the inhibition was Ab dose dependent.



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  		Fig. 5. Chemotactic responses of human PBMCs to supernatant fluids of CEA-specific V8T cells. A, the assay was performed using supernatant fluids of V8T cells stimulated with autologous B cells pulsed with either CAP-1 or CAP1-6D peptide or without peptide for 24 h. Recombinant lymphotactin was used as positive control, and medium was used as negative control. Each dot in the scatter plot represents the number of cells in an independently counted field. B, the assay was carried out in the presence of two concentrations of anti-lymphotactin Ab. For the control samples, only one concentration of the Ab was tested.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In the present study, we have characterized gene expression profiles in a human CEA-specific T-cell line (V8T) after in vitro stimulation with the native CAP-1 peptide versus its agonist, CAP1-6D, in an attempt to understand the molecular basis of their differences in biological function (16 , 17) . Our results showed many genes differentially expressed in CEA-specific T cells stimulated with CAP1-6D versus CAP-1 after 8 or 24 h, whereas expression profiles were quite similar in CAP-1- or CAP1-6D-stimulated V8T cells at 96 h of stimulation.

Among those genes that showed different levels of transcription in T cells activated with the agonist CAP1-6D versus CAP-1 peptide, the chemokine lymphotactinshowed the highest degree of overexpression. The increased level of lymphotactin mRNA was substantiated with an increase at the protein level, as detected by ELISA assay. Lymphotactin constitutes the only member of the C-chemokine family cloned from activated pro-T cells (30, 31, 32, 33) . It is produced by activated CD4+ and CD8+ cells (34 , 35) , natural killer cells (36 , 37) , intraepithelial {gamma}{delta} T cells (38) , and mast cells (39) . Lymphotactin, either in vivo or in vitro, is a powerful chemoattractant for CD4+ and CD8+ T cells and a moderate chemoattractant for natural killer cells (31 , 36) . Recently, it has also been demonstrated that neutrophils and B cells expressing the lymphotactin receptor (XCR1) chemotactically respond to this chemokine (40) . The functional properties of lymphotactin suggest an important role in the control of lymphocyte and neutrophil trafficking during inflammatory and immunological responses. Moreover, an increased expression of this chemokine was observed in the lymphocytic infiltrates characteristic of Crohn’s disease (41) . Recently, it has been proposed that lymphotactin mediates the preferential recruitment of antigen-specific CD62Llo T cells over nonspecific CD62Lhi T cells in vitro as well as in vivo (42) .

Although the "self-antigen" CEA is expected to be poorly immunogenic, the use of this tumor-associated antigen as a target for immunologic-based therapies has demonstrated it to be potentially applicable in the treatment of CEA-bearing malignancies, such as colorectal, pancreatic, breast, and lung carcinomas. Phase I clinical trials have shown measurable CEA-specific T-cell responses in patients vaccinated with CEA-expressing recombinant vaccines (13 , 18) . Slack et al. (22) recently showed an association between the CEA-specific T-cell response and survival of patients vaccinated with pox vector-based CEA-expressing recombinant vaccines. Clinical responses have also been reported after vaccination of advanced CEA carcinoma patients with CAP1-6D peptide-loaded dendritic cells; these responses, moreover, were associated with increased levels of T-cell responses to the CAP1-6D peptide (23) . Clinical trials combining vaccinia and avipox recombinants that express the modified sequence of CEA (CAP1-6D agonist epitope) and multiple costimulatory molecules (B7-1, ICAM-1, and LFA-3) are now being analyzed in protocols for the treatment of advanced CEA-expressing carcinomas (43) .

The effectiveness of the agonist peptide CAP1-6D in eliciting specific T-cell responses and some clinical responses, together with our experimental observations, led us to speculate about the role of an increased secretion of lymphotactin when CEA-specific CTLs are stimulated in vivo with the agonist CAP1-6D peptide. To investigate the possible role of this protein in the enhanced lytic susceptibility of targets in the presence of the agonist peptide (16) , we performed a CTL assay using CAP1-6D-pulsed target cells with the addition of Abs directed against lymphotactin. No inhibition of lysis was observed in the presence of the blocking Abs (data not shown), thus discarding a probable role in the in vitro killing of targets using this mechanism. The elevated production of lymphotactin as a consequence of activating CEA-specific T cells with the agonist peptide CAP1-6D and its ability to stimulate recruitment of antigen-specific T cells, however, could facilitate migration of more CEA-specific T cells to the site of peptide delivery, augmenting their chance to be activated and to proliferate. Thus, antitumor immune response could be accelerated and potentiated. In the studies reported here, supernatants from V8T cells stimulated with the agonist peptide were able to induce higher chemotactic responses of human PBMCs, as compared with supernatants from CAP-1-stimulated V8T cells. Moreover, this chemotactic response induced by CAP1-6D supernatants was shown to be inhibited by anti-lymphotactin Ab. Previous studies have applied the use of lymphotactin in antitumor immune therapies in mice. In one of these studies, mouse dendritic cells genetically modified with a lymphotactin-expressing adenovirus resulted in the induction of protective and therapeutic antitumor immunity (44 , 45) . Myeloma cells transfected to secrete lymphotactin have been demonstrated to lose their ability to form solid tumor masses in mice; these observations were correlated with a high level of tumor infiltration with CD4+ and CD8+ T cells as well as neutrophils (27) . A synergistic enhancement of antitumor immunity with adoptively transferred tumor-specific CD4+ and CD8+ T cells and intratumoral expression of lymphotactin was reported recently (46) .

We have previously reported (17) that CEA-specific CTLs secrete higher levels of IFN-{gamma} after stimulation with the agonist peptide, as compared with the native peptide. In the studies reported here, a consistent cosecretion of IFN-{gamma} and lymphotactin protein was observed, with maximum levels detected 24 h postactivation with the agonist peptide. The observations at mRNA level, however, did not correlate with those at protein level. Transcripts encoding lymphotactin were rapidly induced at 2 h of stimulation and accumulated up to 24 h postactivation, whereas transcripts encoding IFN-{gamma} were induced at 2 h and then declined to undetectable levels at 24 h postactivation. The control of IFN-{gamma}gene expression constitutes a complex process that involves transcriptional and posttranscriptional regulation (47) . Other authors have previously reported that IFN-{gamma}mRNA levels increase rapidly within 1–2 h of stimulation, whereas longer periods of activation result in progressively lower levels of transcripts for IFN-{gamma}(28 , 29) . Our observations are in accordance with those previous reports. The elevated amounts of IFN-{gamma} protein detected in supernatants of 24 h-stimulated V8T cells, when the levels of IFN-{gamma}mRNA were undetectable, indicate that early accumulation of transcripts precedes the later accumulation of the secreted protein. This observation is also in accordance with previous reports that showed an earlier induction of IFN-{gamma}mRNA followed by a later accumulation of the protein (48) . Lower levels of expression of IRF-7gene were observed in V8T cells stimulated for 24 h with the agonist peptide CAP1-6D as compared with the native CAP-1 peptide. IRF-7 is a transcription factor that participates in IFN gene regulation. It has been shown that upon incubation of T cells with virally infected cells (such as EBV-transformed cells), there is an up-regulation of the transcription factor IRF-3, resulting in higher production of IFN-{alpha} and IFN-ß. IFN-{alpha} and IFN-ß can lead to up-regulation of IRF-7 through IFN-stimulated gene factor 3 (49 , 50) . In our microarray experiments, we observed a slight up-regulation of IFN-{alpha} mRNA in V8T cells activated for 24 h with CAP-1 peptide-pulsed B cells, as compared with V8T cells incubated with unpulsed B cells. No up-regulation of IFN-{alpha} mRNA was detected in V8T cells stimulated for 24 h with CAP1-6D-pulsed B cells, as compared with V8T cells incubated with unpulsed B cells. Because IRF-7 has been shown to be responsive only to IFN-{alpha} and IFN-ß, this might explain why IRF-7 is down-regulated when CAP1-6D-stimulated V8T cells were compared with CAP-1-stimulated V8T cells.

Due to the complex interplay between cytokines and chemokines during inflammation and immune responses, several groups have studied the role of IFN-{gamma} in lymphotactin expression. Results are controversial; using IFN-{gamma} knockout mice, some authors have shown that lymphotactingene expression is induced after infection or during allograft rejection in the absence of IFN-{gamma} (51 , 52) , whereas others have reported that lymphotactin gene expression is impaired in the absence of the cytokine (53) . We have performed experiments on concanavalin A stimulation of normal and IFN-{gamma} deficient mice T cells. Our results indicated that in the absence of IFN-{gamma}, the production of lymphotactin was not affected. In contrast, Cerdan et al. (54) have conducted studies to analyze the effect of lymphotactin on IFN-{gamma} production; lymphotactin has been shown to impair the expression of IFN-{gamma} mRNA in CD4+ human T cells but not CD8+ cells. Because our studies were conducted with CD8+ T cells, our observations are not opposed to those reported previously.

We have reported previously (17) that CEA-specific CTLs secrete higher levels of GM-CSF, TNF-{alpha}, and IL-2 after stimulation with the agonist peptide CAP1-6D as compared with the native peptide CAP-1. Higher levels of mRNA encoding for GM-CSF were detected by cDNA microarray analysis at 8 and 24 h of stimulation with the agonist CAP1-6D peptide as compared with the native CAP-1 peptide (Tables 2Citation and 3)Citation . These results correlate well with the observations at the protein level. However, no increase in the levels of mRNA encoding for IL-2 or TNF-{alpha} were observed at any time point in our cDNA microarray experiments when CAP1-6D-stimulated V8T cells were compared with CAP-1-stimulated V8T cells. Moreover, no increase in the levels of IL-2or TNF-{alpha}mRNA was detected when V8T cells stimulated for 24 h with either CAP-1- or CAP1-6D-pulsed B cells were compared with V8T cells stimulated with unpulsed B cells. There is thus a possibility that an early induction of the IL-2 and TNF-{alpha}genes (prior to 24 h) might have occurred followed by a decrease in the levels of both transcripts.

We have demonstrated previously that the agonist peptide CAP1-6D increases ZAP-70 phosphorylation over that observed with the CAP-1 peptide. In general, activation of T cells through the T-cell receptor is accompanied by biochemical events such as phosphorylation/dephosphorylation of kinases that are already present in the cytoplasm of the T cells. These events occur early after activation of the T cells (in minutes) and do not require de novo generation of mRNA. Therefore, the lack of detection of increased levels of mRNA encoding for signaling molecules such as ZAP-70 is not surprising in our microarray study. The decrease in the levels of MAP3K12 when CAP1-6D-stimulated V8T cells were compared with CAP-1-stimulated V8 T cells might be due in part to the fact that the signaling pathways invoked by the CAP1-6D peptide might be different from the ones invoked by the lower affinity peptide CAP-1.

In the present studies we have also reported higher levels of granzyme B mRNA in agonist-activated V8T cells. A substantial increase in the levels of granzyme B mRNA was also detected by RT-PCR in CAP1-6D-stimulated V8T cells, as compared with CAP-1-stimulated V8T cells. The higher secretion of granzyme B by agonist-activated T cells may also be one of the principal factors in enhancing the susceptibility of agonist-loaded targets to in vitro lysis by CEA-specific cytotoxic T cells. Enhanced secretion of granzyme B may also contribute to the efficiency of agonist-activated T cells in vivo to perform lysis of the CEA-bearing tumor cell.

One of the issues that one must consider in evaluating the differential results using the agonist versus the native peptide reported here is this: are the differences observed in the differential gene expression results qualitative and/or quantitative? Previous studies have demonstrated that clear differences exist in type 1 cytokine production when agonist and native peptide are used at similar peptide concentrations to stimulate T cells (17) . However, if 10–1000 times more native peptide is used, the levels of type 1 cytokines secreted by activated T cells approach but do not always reach the levels seen when the agonist peptide is used. Detailed microarray and signaling studies at multiple concentrations of both agonist and native peptide will need to be conducted to determine which phenomena are qualitative and/or quantitative.

In addition to the studies of human T cells using cDNA microarray analysis described above (see "Introduction"), differential gene expression studies of murine T cells have also been conducted (55, 56, 57) . In all of the previous studies, in both humans and mice, T cells were activated using mitogens, anti-CD3 Ab, or superantigens. To our knowledge, these are the first studies reported to use DNA microarray technology to analyze (a) gene expression profiles in an antigen-specific T-cell line after stimulation with a specific antigen, and (b) gene expression patterns of T cells activated by a tumor-associated self-antigen versus an enhancer agonist peptide. A pronounced overexpression of the chemokine lymphotactin was observed after the activation of human CEA-specific CTLs with the agonist CAP1-6D peptide, as compared with the native CAP-1 peptide. These studies implicate a potential role of the chemokine lymphotactin in the biological activity of T cells stimulated with agonist peptides.


   ACKNOWLEDGMENTS
 
We thank Margarita Lora for excellent technical support and Debra Weingarten for editorial assistance in the preparation of the manuscript.


   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, Center for Cancer Research, National Cancer Institute, NIH, 10 Center Drive, Room 8B09, MSC 1750, Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail: js141c{at}nih.gov Back

2 The abbreviations used are: Ab, antibody; CEA, carcinoembryonic antigen; PBMC, peripheral blood mononuclear cell; GM-CSF, granulocyte/macrophage colony-stimulating factor; RT-PCR, reverse transcription-PCR; aRNA, amplified RNA; IL, interleukin; IRF, IFN regulatory factor; TNF, tumor necrosis factor. Back

Received 8/20/02; revised 11/12/02; accepted 11/13/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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Clin. Cancer Res., September 1, 2005; 11(17): 6342 - 6351.
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K.-Y. Tsang, C. Palena, J. Gulley, P. Arlen, and J. Schlom
A Human Cytotoxic T-Lymphocyte Epitope and Its Agonist Epitope from the Nonvariable Number of Tandem Repeat Sequence of MUC-1
Clin. Cancer Res., March 15, 2004; 10(6): 2139 - 2149.
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