Differential Gene Expression Profiles in a Human T-Cell Line Stimulated with a Tumor-Associated Self-Peptide Versus an Enhancer Agonist Peptide

1616 Vol. 9, 1616–1627, May 2003 Clinical Cancer Research

Advances in Brief 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 Schlom,1 and Introduction Kwong-Yok Tsang With the advent of DNA microarray technology, one can Laboratory of Tumor Immunology and Biology, Center for Cancer now better understand the molecular basis of different immu- Research, National Cancer Institute, NIH, Bethesda, Maryland 20892 nological phenomena. Expression of large numbers of genes can be simultaneously monitored using cDNA microarrays to elu- cidate gene products associated with differentiation, activation, Abstract and proliferation of immune cells in response to diverse stimuli. Purpose: Previous studies have shown that a specific Microarray technology has been used to study both B-cell 9-mer amino acid epitope (designated CAP-1) of the human and T-cell populations from apparently normal individuals, as “self” tumor-associated carcinoembryonic antigen can be well as from patients with immune deficiencies and lymphoid used to stimulate CD8؉ T cells from peripheral blood malignancies (1–4). To date, however, cDNA microarrays have mononuclear cells of carcinoma patients vaccinated with been used to study human T-cell activation in response to 2 pox vector-based carcinoembryonic antigen vaccines. A T- mitogens (2, 5, 6) or anti-CD3 Ab (1). Other studies have used cell receptor agonist epitope of CAP-1 (designated CAP1- this technology to explore those gene pathways related to the heat shock response in human T cells (7) or to investigate the 6D) has been shown to enhance the stimulation of T cells effects of HIV infection of CD4ϩ T cells at the gene expression over levels obtained using CAP-1. The purpose of this study level (8). Previous studies have also examined differential gene was to analyze gene expression profiles in T cells stimulated expression profiles between (a) T-helper 1 and T-helper 2 with the native CAP-1 versus the agonist CAP1-6D peptide. ϩ subsets of human CD4 T cells stimulated with mitogens (9, 10) Experimental Design: Microarray analyses were con- ϩ and (b) human memory and naı¨ve CD4 T cells, at rest and after ducted to analyze differential gene expression profiles of a activation with anti-CD3 Ab (11). Despite a rapid incursion of T-cell line stimulated with native versus agonist peptides. array technology in the field of immunology, none of the pub- Results: Numerous genes and gene clusters are identi- lished studies to date have studied responses of T-cell lines fied as differentially expressed as a consequence of stimula- directed against defined antigens or epitopes. tion with the agonist peptide versus the native peptide; two We have demonstrated previously (2) that human CD8ϩ genes, however, stand out in magnitude: the chemokine T-cell lines specific for human CEA can be generated in vitro; lymphotactin and granzyme B. In particular, lymphotactin these T cells were derived from PBMCs of carcinoma patients expression is >12 times more pronounced in agonist-stimu- vaccinated with either vaccinia or avipox recombinant vaccines lated T cells. An ELISA assay was developed that confirmed expressing CEA (12–14). The T-cell lines were established marked lymphotactin secretion in T cells when stimulated using a defined 9-mer epitope of CEA designated CAP-1 (12, with the agonist versus the native peptide. A chemotaxis 15). It was subsequently shown that this peptide can be modified assay also demonstrated the biological activity of the lym- in an amino acid residue that is predicted to contact the T-cell photactin produced. receptor and consequently improve epitope-specific T-cell re- Conclusions: To our knowledge, these are the first stud- sponses (16). The agonist peptide, designated CAP1-6D 3 ies of gene expression profiles of a defined T-cell line in (6N D), has been demonstrated to enhance the levels of T- ␥ response to stimulation with a defined antigen. They are also helper 1 cytokines (GM-CSF and IFN- ) produced by CAP-1- the first to compare, via cDNA microarray, responses of a specific T-cell lines as compared with the native CAP-1 peptide T-cell line to (a) a tumor-associated self-antigen and (b)a (17). In addition, CAP-1-specific CTL lines were able to lyse native epitope versus an agonist epitope. 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 recog- nize and lyse human tumor cells expressing native CEA in a MHC-restricted (class I-A2) manner more efficiently than T Received 8/20/02; revised 11/12/02; accepted 11/13/02. cells derived using CAP-1 (16). 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 2 The abbreviations used are: Ab, antibody; CEA, carcinoembryonic Tumor Immunology and Biology, Center for Cancer Research, National antigen; PBMC, peripheral blood mononuclear cell; GM-CSF, granulo- Cancer Institute, NIH, 10 Center Drive, Room 8B09, MSC 1750, cyte/macrophage colony-stimulating factor; RT-PCR, reverse transcrip- Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; tion-PCR; aRNA, amplified RNA; IL, interleukin; IRF, IFN regulatory E-mail: [email protected]. factor; TNF, tumor necrosis factor. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 1617

Phase I clinical trials have demonstrated the ability of A260 nm reading, and RNA quality was evaluated by the ratio CEA-based vaccination to elicit T-cell immune responses in A260 nm/280 nm. RNA was amplified following the method de- patients with advanced CEA-expressing carcinoma (13, 18–22). scribed by Wang et al. (24). One ␮g of total RNA was used for A Phase I study using dendritic cells pulsed with the native amplification. Briefly, the phage T7 RNA polymerase promoter CAP-1 peptide elicited only modest CAP-1-specific immune was incorporated into cDNA by reverse transcription in the responses in advanced cancer patients (21). It has recently been presence of oligo(dT) (15)-T7 primer (5Ј-AAA-CGA-CGG- shown, however, that vaccination of patients with metastatic CCA-GTG-AAT-TGT-AAT-ACG-ACT-CAC-TAT-AGG-CGC- Ј Ј CEA-expressing carcinoma with dendritic cells loaded with the T15-3 ) and template switch oligonucleotide primer (5 -AAG- agonist epitope of CAP-1 (CAP1-6D), resulted in clinical re- CAG-TGG-TAA-CAA-CGC-AGA-GTA-CGC-GGG-3Ј) using sponses, and these clinical responses correlated with the expan- Superscript II reverse transcriptase (Invitrogen). Full-length sion of CEA-specific CD8ϩ T cells (23). double-stranded cDNA was synthesized using Advantage Po- In the studies reported here, cDNA microarrays have been lymerase (Clontech, Palo Alto, CA) after treatment with RNase used to identify those particular genes that are differentially H (Invitrogen). PCR cycles were 5 min at 37°C for RNA expressed in CEA-specific T cells as a consequence of stimu- digestion, 2 min at 94°C for denaturing, 1 min at 65°C for lation with the agonist CAP1-6D peptide as compared with the specific priming, and 30 min at 75°C for extension. After native CAP-1 peptide. These studies have identified a number of purification, double-stranded cDNA was used as a template for genes that are differentially expressed; among these, granzyme in vitro transcription using a T7 MEGAscript kit (Ambion Inc., B and, in particular, the SCYC1 gene (encoding the C-chemo- Austin, TX). Synthesized RNA was purified from template kine lymphotactin) were highly expressed in CAP1-6D-stimu- DNA by Trizol reagent (Invitrogen). The concentration and lated CEA-specific T cells as compared with T cells stimulated purity of aRNA were determined by measuring A260 nm and with CAP-1. These results were confirmed by RT-PCR. An A260 nm/280 nm. ELISA assay developed for lymphotactin and a chemotaxis cDNA Microarray Hybridization. The method de- biological assay also demonstrated higher levels of lymphotac- scribed by Wang et al. (24) was used for cDNA microarray tin protein released by CEA-specific T cells after stimulation hybridization. Briefly, 3 ␮g of aRNA were labeled by reverse with the agonist peptide. From these results, one can hypothe- transcription using random hexamer primer (Boehringer Mann- size a potential role of the chemokine lymphotactin in enhanc- heim, Indianapolis, IN) in the presence of Cy-3-dUTP or Cy-5- ing antitumor immune responses mediated by tumor antigen- dUTP (New England Nuclear Life Sciences, Boston, MA). For specific T cells stimulated in the presence of agonist peptides. 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 Materials and Methods total of 9130 spotted cDNAs. Fluorescent images of hybridized Cell Cultures. The V8T cell line is a CD8ϩ CTL line microarrays were taken using a GenePix 4000 scanner (Axon directed against the CAP-1 epitope of CEA (12), which was Instruments, Foster City, CA) at various PMT voltages to obtain generated from PBMCs of a patient with metastatic colon car- maximum signal intensities with minimum saturation (Ͻ1%). cinoma who was vaccinated with a recombinant vaccinia-CEA Data were analyzed using GenePixPro 3.0 (Axon Instruments) vaccine (15). The immunophenotyping and other properties of and GeneSpring software (Silicon Genetics, Redwood City, the V8T cell line have been described previously in detail (12, CA). To conduct comparisons between different experiments, 15–17). The V8T cell line was maintained by in vitro restimu- data were normalized by dividing the signal intensity of each lation every 14 days with irradiated (22,000 rads) EBV-transgene by the median intensity of all of the measurements taken in formed autologous B cells pulsed with 25 ␮g/ml CAP-1 peptide. that sample. The ratio between red intensity (Cy-5-dUTP, The effector:APC ratio was 1:2. Cultures in RPMI 1640 (In- CAP1-6D-stimulated V8T cells) and green intensity (Cy-3- vitrogen, Carlsbad, CA) were supplemented with 2 mM gluta- dUTP, CAP-1-stimulated V8T cells) for each gene was calcu- mine, 100 units/ml penicillin, 100 ␮g/ml streptomycin, 10% lated, and the results shown below were the average for at least human AB serum (Gemini Bio-Products, Woodland, CA), and two replicated experiments. Hierarchical clustering (25) was 20 units/ml IL-2. Medium was replenished every 3 days. applied to those genes that showed fluorescence intensities of Peptides. The CEA peptide CAP-1 (YLSGANLNL; Ref. Ͼ100 in both channels and had a normalized intensity ratio of 12) and the CAP-1 agonist peptide CAP1-6D (YLSGADLNL; Ն3orՅ0.33. Pearson correlation was used as distance matrix. Ref. 16) were Ͼ96% pure and manufactured by American Quantification of mRNA Using RT-PCR. One ␮gof Peptide Company, Inc. (Sunnyvale, CA). total RNA was reverse transcribed in 20 ␮lof1ϫ first-strand RNA Isolation and Amplification. For cDNA microar- buffer containing 1 mM deoxynucleotide triphosphate mix, 0.3 6 6 ray studies, V8T cells (2 ϫ 10 cells, 1 ϫ 10 cells/ml) were nM oligo(dT) (20), 0.01 M DTT, 20 units of RNase inhibitor stimulated with irradiated autologous EBV-transformed B cells (Promega, Madison, WI), and 200 units of Superscript II reverse pulsed with 10 ␮g/ml CAP-1 or CAP1-6D peptide or without transcriptase (Invitrogen). Samples were incubated at 42°C for peptide. The effector:APC ratio was 1:2. At various stimulation 90 min. PCR was performed using 5 ␮l of the reverse transcrip- ϩ times (8, 24, and 96 h), cells were collected, and CD8 T cells tion product in 1ϫ PCR buffer, 0.4 mM deoxynucleotide triphos- were separated using anti-CD8-conjugated magnetic beads phate mix, 50 pmol of each forward and reverse primer, and 1 (Miltenyi Biotec, Auburn, CA). Total RNA from T cells was ␮l of Advantage Polymerase enzyme mix (Clontech). Primer isolated using the RNeasy RNA extraction kit (Qiagen sequences (26) were as follows: lymphotactin, 5Ј-TCA-GCC- Inc., Valencia, CA). RNA concentrations were determined by ATG-AGA-CTT-CTC-3Ј (forward) and 5Ј-TAA-TTT-TAT-

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 for8h(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 Ն2-fold change in expression, and the center line indicates equivalent expression.

TCA-TGC-AGT-GCT-TTC-3Ј (reverse); IFN-␥,5Ј-AGC-TCT- Chemotaxis Assay. Chemotactic responses were exam- GCA-TCG-TTT-TGG-GTT-3Ј (forward) and 5Ј-GTT-CCA- ined following a modification of a previously described method TTA-TCC-GCT-ACA-TCT-GAA-3Ј (reverse); granzyme B, (27) using Blind Well Chambers (Neuroprobe, Gaithersburg, 5Ј-GCT-TAT-CTT-ATG-ATC-TGG-GAT-C-3Ј (forward) and MD) with polyvinylpyrrolidone-free 5-␮m-pore polycarbonate 5Ј-AAG-TCA-GAT-TCG-CAC-TTT-CGA-3Ј (reverse); and ␤- filters previously coated on one side with mouse Collagen IV actin, 5Ј-ATC-TGG-CAC-CAC-ACC-TTC-TAC-AAT-GAG-3Ј (Trevigen, Gaithersburg, MD). Supernatants from V8T cells (forward) and 5Ј-CGT-GGT-GGT-GAA-GCT-GTA-GCC-GCG- activated for 24 h with autologous APCs pulsed with 10 ␮g/ml CTC-3Ј (reverse). Amplification consisted of 35 cycles of 1 min at CAP-1 or CAP1-6D peptide or without peptide were added to 4 95°C, 1 min at 55°C, and 1 min at 72°C. PCR products were the lower chambers, and healthy donor PBMCs (7.5 ϫ 10 cells, resolved on 1.5% agarose gel. Gel images were taken, and tran- 75 ␮l in complete RPMI 1640 containing 1% human AB serum) script levels were quantified for normalization to expression of were added to the upper chambers. As negative and positive ␤-actin, using a Kodak 1D 3.0 documentation and analysis system. controls, the lower chambers were loaded with medium alone or Detection of IFN-␥. Supernatants of V8T cells stimu- recombinant lymphotactin (50 ng/ml in complete RPMI 1640 lated with peptide-pulsed autologous EBV-transformed B cells containing 1% human AB serum), respectively. After incubation were collected at 8, 24, and 96 h of stimulation and screened for for4hat37°C, filters were removed from the chambers, fixed, IFN-␥ secretion using an ELISA kit (R&D Systems, Inc., Min- and stained with Diff-Quik stain (Dade Behring Inc., Newark, neapolis, MN). Results were expressed in pg/ml. DE). Blocking assay was performed using anti-lymphotactin Ab ELISA for Lymphotactin. An ELISA was developed (R&D Systems, Inc.). The number of cells associated with the for the detection of lymphotactin. Flat-bottomed 96-well plates lower side of the membranes was evaluated by direct counting (medium binding immunoassay plates; Greiner Bio-One Inc., of at least six ϫ40 objective fields for the standard samples or Longwood, FL) were coated with 2 ␮g/ml capture Ab (goat at least nine ϫ40 objective fields for the experimental samples. antihuman lymphotactin Ab; R&D Systems, Inc.), incubated overnight at room temperature, and then washed with wash Results buffer [0.05% Tween 20 in PBS (pH 7.4)]. Nonspecific binding Gene Expression Profiles of CEA-specific T Cells after was blocked by adding 5% BSA in PBS (pH 7.4) for2hat Stimulation with Either the Native CAP-1 Peptide or Its 37°C. After rinsing with wash buffer, dilutions of the standard Agonist CAP1-6D Peptide. The V8T CD8ϩ T-cell line was (recombinant human lymphotactin; R&D Systems, Inc.) and derived from PBMCs of a patient who had been vaccinated with unknown samples were incubated onto the plates for2hatroom recombinant vaccinia-CEA. It was generated and passaged us- temperature. After washing, 100 ng/ml biotinylated antihuman ing the CEA CAP-1 peptide as described in “Materials and lymphotactin Ab (R&D Systems, Inc.) and a 1:1000 dilution of Methods.” Three time points were selected to carry out gene streptavidin-horseradish peroxidase conjugate were added to the expression studies in V8T cells stimulated with either CAP-1 or wells for1hatroom temperature. Plates were then incubated for CAP1-6D peptide using cDNA microarrays. V8T cells were 15 min with substrate solution, and absorbance in each well was stimulated for 8, 24, and 96 h with each peptide; total RNA was measured at 450 nm. isolated from each sample and amplified as described in “Ma-

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 Number of genes differentially expresseda 2-fold up 2-fold down 3-fold up 3-fold down CAP-1 vs. no peptide 181 432 33 179 CAP1-6D vs. no peptide 25 296 9 89 CAP1-6D vs. CAP-1 210 108 42 12 a Total RNA was isolated from V8T cells stimulated for 24 h with autologous B cells pulsed with 10 ␮g/ml of either CAP-1 or CAP1-6D peptide or without peptide. The differential gene expression was analyzed by cDNA microarray as described in “Materials and Methods.”

terials and Methods.” For each time point, fluorescent cDNA Further analysis was focused on those genes that exhibited probes were made by reverse transcription of aRNA with Cy- at least a 3-fold difference in expression levels between CAP1- 3-dUTP and Cy-5-dUTP for CAP-1- and CAP1-6D-stimulated 6D- and CAP-1-stimulated V8T cells and showed fluorescence samples, respectively. Labeled cDNA probes were incubated intensity units of Ͼ100 in each channel. Using the standard onto cDNA microarrays containing a total of 9130 genes (Hs- correlation coefficient, genes with at least a 3-fold change in UniGEM2; National Cancer Institute). After acquisition of flu- expression clustered into two nodes (Fig. 2). Cluster group A orescent images, data were processed as described in “Materials contains genes whose expression was increased at least 3-fold in and Methods.” CAP1-6D-stimulated V8T cells as compared with CAP-1-stim- To get an overview of the differential gene expression in ulated V8T cells at any time point analyzed. Genes whose V8T cells stimulated with CAP-1 versus CAP1-6D peptide, red expression levels were decreased at least 3-fold in CAP1-6D- fluorescence intensity (signal from CAP1-6D-stimulated sam- stimulated V8T cells (at any time point) were clustered in ple) was plotted against green fluorescence intensity (signal group B. from CAP-1-stimulated sample) for each individual gene at each The normalized relative intensity ratio for each individual time point (Fig. 1). Eight h of stimulation with CAP-1 or gene that had at least a 3-fold difference in expression between CAP1-6D peptide promoted a differential gene expression in V8T cells stimulated for 8 h with CAP1-6D versus CAP-1 V8T cells as shown in Fig. 1A. Most of the genes contained in peptide is shown in Table 2. Of a total of 12 genes differentially the arrays were expressed at equivalent levels in CAP-1- versus expressed, 4 of them encode secreted products of T cells, such CAP1-6D-stimulated V8T cells. As indicated by genes spotted as granzyme B, GM-CSF (denoted as CSF2 in Fig. 2), and two in red, a relatively high number of genes were overexpressed in different T-cell-specific chemokines, SCYC1 (lymphotactin) CAP1-6D-stimulated V8T cells relative to CAP-1-stimulated and SCYA1 (I-309). Other genes differentially expressed en- V8T cells. A lower number of genes were expressed at de- code for membrane proteins, transporters, and genes related to creased levels in CAP1-6D-stimulated V8T cells compared with cell cycle control, transcriptional regulation, and metabolism. CAP-1-stimulated V8T cells, as indicated by the genes spotted Above all, transcripts for lymphotactin were strongly expressed in green. The scatter in the points in Fig. 1B indicates a greater in V8T cells stimulated with CAP1-6D peptide, showing an number of genes differentially expressed in V8T cells stimu- increase of 8.5-fold relative to its expression in CAP-1-stimulated for 24 h with CAP1-6D versus CAP-1 peptide, as com- lated V8T cells. pared with that observed at 8 h. There was a prevalence of Individual genes that were differentially expressed at 24 h, overexpression of genes in CAP1-6D-stimulated V8T cells as compared with CAP-1-stimulated V8T cells. Fig. 1C shows the showing at least a 3-fold change in expression, are listed in comparison of gene expression in V8T cells stimulated with Table 3. Genes were grouped in accordance with their presumed each peptide at 96 h. As expected, most of the genes were functions. A total of 54 genes that had a relative CAP1-6D: Ն Յ equivalently expressed after stimulation with each peptide. CAP-1 intensity ratio of 3or 0.33 were detected at this time Additional microarray analyses were also conducted to point. As also observed at8hofstimulation, some of the genes compare gene expression profiles at 24 h of V8T cells stimu- differentially expressed at 24 h belonged to the category of lated with autologous APCs pulsed with CAP-1 versus no chemokines, growth factors, or effector molecules secreted by T peptide, as well as V8T cells stimulated with autologous APCs cells. Transcripts of all these genes were more abundant in pulsed with CAP1-6D peptide versus no peptide. As seen in CAP1-6D-stimulated V8T cells. Lymphotactin and granzyme B Table 1, over 600 genes were differentially expressed Ͼ2-fold, showed the highest difference in expression (fold changes of and over 200 genes were differentially expressed Ͼ3-fold when 12.9 and 5.6, respectively) when CAP1-6D-stimulated V8T comparing CAP-1-stimulated with non-peptide-stimulated V8T cells were compared with CAP-1-stimulated V8T cells. GM- T cells. When comparing CAP1-6D-stimulated V8T cells with CSF and the chemokines RANTES and Mip-1b were also up- non-peptide-stimulated T cells, Ͼ300 genes were differentially regulated in agonist-stimulated V8T cells. Other genes differ- expressed 2-fold, and approximately 98 genes were differen- entially expressed at 24 h encoded for surface and membrane tially expressed Ͼ3-fold. When comparing CAP1-6D- versus proteins, transporters, and genes involved in cell cycle control, CAP-1-stimulated V8T cells, Ͼ300 genes were differentially cell signaling, transcriptional regulation, and miscellaneous expressed 2-fold, whereas 54 genes were differentially ex- genes. A high number of genes differentially expressed at 24 h pressed 3-fold (Table 1). between agonist- and native peptide-stimulated V8T cells were

Fig. 2 Hierarchical cluster of differentially expressed genes between CAP1-6D- and CAP- 1-stimulated V8T cells. In- cluded in the cluster analysis were genes that differed at least 3-fold in expression. Rows cor- respond 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.

related to processing, synthesis, and turnover of RNA and lymphotactin mRNA in unstimulated (Fig. 3, no peptide), CAP- proteins. 1-stimulated, and CAP1-6D-stimulated V8T cells were 2.3, 4.3, Increase of Lymphotactin and Granzyme B mRNA and 278, respectively. The substantial increase in lymphotactin Levels in CAP1-6D-stimulated V8T Cells Was Confirmed by mRNA observed after stimulation with CAP1-6D peptide con- RT-PCR. To confirm and validate the results obtained by firmed and actually magnified the results obtained by cDNA cDNA microarray studies, RT-PCR was used to analyze expres- microarray studies. sion levels of lymphotactin mRNA in CAP-1- and CAP1-6D- Additional studies were also carried out to quantitate the stimulated V8T cells. Total RNA was prepared from V8T cells level of granzyme B mRNA in V8T cells after 24 h of stimu- stimulated for 24 h with autologous APCs pulsed with 10 ␮g/ml lation with either CAP-1 or CAP1-6D peptide. As shown in Fig. CAP-1 or CAP1-6D peptide or without peptide. After reverse 3B, relative levels of granzyme B mRNA in V8T cells stimu- transcription, cDNA from each sample was amplified using lated with CAP-1 and CAP1-6D peptide were 0.1 and 8.0, PCR primer pairs specific for lymphotactin. Amplification of respectively (note the different scales in Fig. 3, A and B). These ␤-actin in each sample served as the positive control. RT-PCR results were also in agreement with those observed in the cDNA products were quantified and normalized to those of ␤-actin in microarray experiments. each sample. As shown in Fig. 3A, relative expression levels of Unfortunately, the cDNA arrays used in these studies did

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 Fold increasea Gene name and description (CAP1-6D vs. CAP-1) Chemokines, growth factors, effector molecules SCYC1—lymphotactin ϩ8.5 CSF2—colony stimulating factor 2 (granulocyte-macrophage) ϩ4.1 SCYA1—small inducible cytokine A1 (I-309) ϩ3.2 GZMB—granzyme B ϩ3.0 Membrane proteins GNAI1—guanine nucleotide binding protein (G protein) ϩ3.9 Transporters ATP5D—ATP synthase, mitochondrial F1 complex ϩ3.0 Proliferation, cell cycle CUL5—cullin 5 ϩ3.3 Transcriptional regulation ZNF197—zinc finger protein 197 ϩ3.3 Metabolism PTGES—prostaglandin E synthase ϩ3.4 Miscellaneous EST ϩ3.4 ESTs, moderately similar to protein FLJ20378 ϩ3.3 EST ϩ3.1 a Genes were considered to be differentially expressed if a change of at least 3-fold or greater was observed in duplicate experiments. Numbers indicate the fold change in expression for each gene when levels of transcripts in T cells stimulated with CAP1-6D were compared with those in T cells stimulated with CAP-1 peptide. Differentially expressed genes were grouped in accordance with their presumed functions.

not contain IFN-␥ cDNA. To compare the level of IFN-␥ no protein was detected in supernatants of these cells. Detection mRNA with that of lymphotactin and granzyme B, RT-PCR was positive at 8 and 24 h of stimulation with the two peptides, amplification was performed in total RNA obtained from V8T whereas results were negative at 96 h. A marked increase in cells after2hofstimulation with either CAP-1 or CAP1-6D lymphotactin protein levels was observed as a consequence of peptide. Results are shown in Fig. 3C. Relative levels of lym- stimulation with the agonist peptide, which supports the results photactin mRNA were 0.3 and 3.7 in V8T cells stimulated with observed at the RNA level. Maximum protein expression was CAP-1 or CAP1-6D, respectively. IFN-␥ mRNA showed rela- detected at 24 h, with a 389% increase observed in CAP1-6D- tive levels of 0.5 and 5.6 in CAP-1- and CAP1-6D-stimulated stimulated V8T cells as compared with CAP-1-stimulated V8T V8T cells, respectively. Relative levels of granzyme B mRNA cells. were 0.3 and 9.0 in V8T cells stimulated for 2 h with CAP-1 or Levels of lymphotactin protein were then compared with CAP1-6D peptide, respectively. In contrast to the results shown those of IFN-␥ protein. As observed in Fig. 4B, IFN-␥ protein in Fig. 3, A and B, for lymphotactin and granzyme B expression was undetectable in supernatants of unstimulated V8T cells at 24 h, no IFN-␥ mRNA could be detected in the V8T cells (cultured with autologous APCs without peptide), whereas its stimulated for 24 h with each peptide (data not shown). These expression was detectable in supernatants of cells stimulated observations are in accordance with previous reports (28, 29) with each peptide at 8, 24, and 96 h of stimulation. These results and will be discussed below. are not in concordance with those observed at the RNA level, in Lymphotactin mRNA was detectable at higher levels at that IFN-␥ transcripts are not detectable at 24 h of stimulation 24 h of stimulation with both CAP-1 and CAP1-6D peptides, as with each peptide. These observations will be discussed below. compared with2hofstimulation. In contrast, IFN-␥ mRNA At each time point, levels of IFN-␥ protein were higher in levels were higher at2hofstimulation, as compared with 24 h, supernatants from agonist-stimulated V8T cells, with a maxi- with either CAP-1 or CAP1-6D peptide. Finally, granzyme B mum difference observed at 24 h (508% increase). expression was relatively similar at 2 and 24 h of stimulation Supernatants from CAP1-6D-stimulated V8T Cells En- with each peptide. Thus, from the analysis of the relative levels hance in Vitro Migration of Human PBMCs. Lymphotactin of transcripts encoding for IFN-␥, lymphotactin, and granzyme has previously been shown to enhance chemotactic responses of B, three different temporal patterns of expression can be distin- different immune cell populations. A chemotaxis assay was guished. used, as described in “Materials and Methods,” to measure in Detection of Lymphotactin by ELISA Assay. An vitro chemotactic responses of human PBMCs to supernatant ELISA assay was set up to measure lymphotactin-soluble pro- fluids of V8T CEA-specific T cells stimulated with APCs tein in supernatants of cultured cells. Results are shown in Fig. pulsed with CAP-1 peptide or CAP1-6D peptide or without 4A. Lymphotactin protein was analyzed in supernatants of V8T peptide. As seen in Fig. 5A, supernatant fluids from V8T cells cells after 8, 24, and 96 h of stimulation with APCs pulsed with stimulated with CAP1-6D clearly enhanced migration of CAP-1 or CAP1-6D peptide or without peptide (Fig. 4A). Al- PBMCs over that observed in response to supernatant fluids though lymphotactin mRNA was detected by RT-PCR in V8T from CAP-1-stimulated V8T cells. Purified recombinant lym- cells cultured with autologous APCs in the absence of peptide, photactin was also used as a positive control in this assay. To

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 Gene name and description Fold increase or decreasea Chemokines, growth factors, effector molecules SCYC1—lymphotactin ϩ12.9 GZMB—granzyme B ϩ5.6 CSF2—colony-stimulating factor 2 ϩ3.7 SCYA5—small inducible cytokine A5 (RANTES) ϩ3.4 SCYA4—small inducible cytokine A4 (Mip-1b) ϩ3.4 Surface molecules, membrane proteins HLA class II region expressed gene KE4 ϩ3.8 GNAI1—guanine nucleotide binding protein ϩ3.2 LTB—lymphotoxin ␤ (TNF superfamily) ϩ3.2 CSF2RB—colony-stimulating factor 2 receptor B ϩ3.0 PALM—paralemmin Ϫ3.6 ADORA1—adenosine A1 receptor Ϫ5.6 Transporters VDAC1—voltage-dependent anion channel 1 ϩ3.2 ATP5D—ATP synthase, mitochondrial F1 complex ϩ3.1 TAP1—transporter 1, ATP binding cassette ϩ3.1 ATP6V1E1—ATPase, lysosomal ϩ3.0 Proliferation, cell cycle, stress response HSPA8—heat shock 70-kDa protein 8 ϩ4.2 CDKN1A—cyclin dependent kinase inhibitor 1A ϩ3.2 PIM2—pim-2 oncogene ϩ3.1 PPP2R4—protein phosphatase 2A regulator Ϫ3.2 Cell signaling TBL3—transducin (␤)-like 3 ϩ3.1 MAP3K12—mitogen-activated protein kinase Ϫ3.0 Transcriptional regulation IRF7—interferon regulatory factor 7 Ϫ3.8 PAX8—paired box gene 8 Ϫ3.9 Metabolism PTGES—prostaglandin E synthase ϩ3.1 SHMT1—serine hydroxymethyltransferase 1 Ϫ3.2 RNA processing and turnover, protein synthesis, modification, and turnover RPL23—ribosomal protein L23 ϩ4.3 RPS29—ribosomal protein S29 ϩ4.0 PGGT1B—geranylgeranyltransferase type I, ␤ϩ3.7 RPL37—ribosomal protein L37 ϩ3.7 RPL13A—ribosomal protein L13a ϩ3.5 RPL6—ribosomal protein L6 ϩ3.4 RPL41—ribosomal protein L41 ϩ3.3 RPL28—ribosomal protein L28 ϩ3.2 RPS3A—ribosomal protein S3A ϩ3.2 RPL14—ribosomal protein L14 ϩ3.1 MRPL3—mitochondrial ribosomal protein L3 splicing factor, arginine/serine-rich 9 ϩ3.1 EEF2—eukaryotic translation elongation factor ϩ3.1 RPS20—ribosomal protein S20 ϩ3.0 RPL7A—ribosomal protein L7a ϩ3.0 RPS24—ribosomal protein S24 ϩ3.0 RPL10A—ribosomal protein L10a ϩ3.0 U2AF1RS2—U2 small RNP auxiliary factor Ϫ3.1 PRPF8—pre-mRNA processing factor 8 Ϫ3.4 DPH2L1—diptheria toxin resistance protein Ϫ4.2 Miscellaneous Homo sapiens mRNA full-length insert ϩ4.0 EST ϩ3.6 HSU52521—arfaptin 1 ϩ3.4 MDS006—ϫ 006 protein ϩ3.4 EST ϩ3.2 HSPC132—hypothetical protein ϩ3.1 OXT—oxytocin, prepro- (neurophysin I) ϩ3.1 APS—adaptor protein Ϫ3.1 FALZ—fetal Alzheimer antigen Ϫ3.2 a Genes were considered to be differentially expressed if a change of at least 3-fold or greater was observed in duplicate experiments. Numbers indicate the fold change in expression for each gene when levels of transcripts in T cells stimulated with CAP1-6D were compared with those in T cells stimulated with CAP-1 peptide. Positive values indicate that transcripts were more abundant in CAP1-6D-stimulated cells as compared with CAP-1-stimulated cells. Negative values indicate that the transcripts are more abundant in CAP-1-stimulated T cells. Differentially expressed genes were grouped in accordance with their presumed functions.

Fig. 3 Relative expression levels of lymphotactin, IFN-␥, 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 ␤-actin. RT-PCR products were quantified and normalized to those of ␤-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-␥, and granzyme B mRNA in total RNA from V8T cells stimulated for 2 h with native versus agonist CEA peptide.

ascertain that the observed chemotactic responses were due to (34, 35), natural killer cells (36, 37), intraepithelial ␥␦ T cells the presence of lymphotactin, a chemotaxis assay was carried (38), and mast cells (39). Lymphotactin, either in vivo or in out with or without anti-lymphotactin Ab; two concentrations of vitro, is a powerful chemoattractant for CD4ϩ and CD8ϩ T cells Ab were used, and the results are shown in Fig. 5B. The addition and a moderate chemoattractant for natural killer cells (31, 36). of anti-lymphotactin Ab clearly inhibited the PBMC migration Recently, it has also been demonstrated that neutrophils and B induced by purified recombinant lymphotactin. Because there cells expressing the lymphotactin receptor (XCR1) chemotacti- was little if any migration using supernatants of CAP-1-stimu- cally respond to this chemokine (40). The functional properties lated V8T cells, as expected, no effect was observed for super- of lymphotactin suggest an important role in the control of natants from CAP-1-stimulated V8T cells when the Ab was lymphocyte and neutrophil trafficking during inflammatory and added into the assay. Migration of human PBMCs in response to immunological responses. Moreover, an increased expression of supernatant fluids of CAP1-6D peptide-stimulated V8T cells, this chemokine was observed in the lymphocytic infiltrates however, was significantly decreased after the addition of anti- characteristic of Crohn’s disease (41). Recently, it has been lymphotactin Ab, and the inhibition was Ab dose dependent. proposed that lymphotactin mediates the preferential recruitment of antigen-specific CD62Llo T cells over nonspecific Discussion CD62Lhi T cells in vitro as well as in vivo (42). In the present study, we have characterized gene expression Although the “self-antigen” CEA is expected to be poorly profiles in a human CEA-specific T-cell line (V8T) after in vitro immunogenic, the use of this tumor-associated antigen as a stimulation with the native CAP-1 peptide versus its agonist, target for immunologic-based therapies has demonstrated it to CAP1-6D, in an attempt to understand the molecular basis of be potentially applicable in the treatment of CEA-bearing their differences in biological function (16, 17). Our results malignancies, such as colorectal, pancreatic, breast, and lung showed many genes differentially expressed in CEA-specific T carcinomas. Phase I clinical trials have shown measurable cells stimulated with CAP1-6D versus CAP-1 after 8 or 24 h, CEA-specific T-cell responses in patients vaccinated with CEA- whereas expression profiles were quite similar in CAP-1- or expressing recombinant vaccines (13, 18). Slack et al. (22) CAP1-6D-stimulated V8T cells at 96 h of stimulation. recently showed an association between the CEA-specific T-cell Among those genes that showed different levels of tran- response and survival of patients vaccinated with pox vector- scription in T cells activated with the agonist CAP1-6D versus based CEA-expressing recombinant vaccines. Clinical re- CAP-1 peptide, the chemokine lymphotactin showed the highest sponses have also been reported after vaccination of advanced degree of overexpression. The increased level of lymphotactin CEA carcinoma patients with CAP1-6D peptide-loaded den- mRNA was substantiated with an increase at the protein level, as dritic cells; these responses, moreover, were associated with detected by ELISA assay. Lymphotactin constitutes the only increased levels of T-cell responses to the CAP1-6D peptide member of the C-chemokine family cloned from activated pro-T (23). Clinical trials combining vaccinia and avipox recombi- cells (30–33). It is produced by activated CD4ϩ and CD8ϩ cells nants that express the modified sequence of CEA (CAP1-6D

ated. 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 CD4ϩ and CD8ϩ T cells as well as neutrophils (27). A synergistic enhancement of antitumor immunity with adoptively transferred tumor-specific CD4ϩ and CD8ϩ 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-␥ after stimulation with the agonist peptide, as compared with the native peptide. In the studies reported here, a consistent cosecretion of IFN-␥ 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 at2hofstimulation and accumulated up to 24 h postactivation, whereas transcripts encoding IFN-␥ were induced at2hand Fig. 4 Release of lymphotactin and IFN-␥ by CEA-specific T cells then declined to undetectable levels at 24 h postactivation. The (V8T cells) stimulated with CAP-1 or CAP1-6D agonist peptide-pulsed control of IFN-␥ gene expression constitutes a complex process APCs. V8T cells (1 ϫ 106 cells/ml) were stimulated with irradiated autologous B cells pulsed with 10 ␮g/ml of either CAP-1 or CAP1-6D that involves transcriptional and posttranscriptional regulation peptide or without peptide. The effector:APC ratio was 1:2. Superna- (47). Other authors have previously reported that IFN-␥ mRNA tants were collected at 8, 24, and 96 h of stimulation and assayed for levels increase rapidly within 1–2 h of stimulation, whereas ␥ lymphotactin (A) or IFN- (B) by ELISA. Results are expressed in longer periods of activation result in progressively lower levels pg/ml. of transcripts for IFN-␥ (28, 29). Our observations are in accordance with those previous reports. The elevated amounts of IFN-␥ protein detected in supernatants of 24 h-stimulated V8T agonist epitope) and multiple costimulatory molecules (B7-1, cells, when the levels of IFN-␥ mRNA were undetectable, ICAM-1, and LFA-3) are now being analyzed in protocols for indicate that early accumulation of transcripts precedes the later the treatment of advanced CEA-expressing carcinomas (43). accumulation of the secreted protein. This observation is also in The effectiveness of the agonist peptide CAP1-6D in elic- accordance with previous reports that showed an earlier induc- iting specific T-cell responses and some clinical responses, tion of IFN-␥ mRNA followed by a later accumulation of the together with our experimental observations, led us to speculate protein (48). Lower levels of expression of IRF-7 gene were about the role of an increased secretion of lymphotactin when observed in V8T cells stimulated for 24 h with the agonist CEA-specific CTLs are stimulated in vivo with the agonist peptide CAP1-6D as compared with the native CAP-1 peptide. CAP1-6D peptide. To investigate the possible role of this pro- IRF-7 is a transcription factor that participates in IFN gene tein in the enhanced lytic susceptibility of targets in the presence regulation. It has been shown that upon incubation of T cells of the agonist peptide (16), we performed a CTL assay using with virally infected cells (such as EBV-transformed cells), CAP1-6D-pulsed target cells with the addition of Abs directed there is an up-regulation of the transcription factor IRF-3, re- against lymphotactin. No inhibition of lysis was observed in the sulting in higher production of IFN-␣ and IFN-␤. IFN-␣ and presence of the blocking Abs (data not shown), thus discarding IFN-␤ can lead to up-regulation of IRF-7 through IFN-stimu- a probable role in the in vitro killing of targets using this lated gene factor 3 (49, 50). In our microarray experiments, we mechanism. The elevated production of lymphotactin as a con- observed a slight up-regulation of IFN-␣ mRNA in V8T cells sequence of activating CEA-specific T cells with the agonist activated for 24 h with CAP-1 peptide-pulsed B cells, as com- peptide CAP1-6D and its ability to stimulate recruitment of pared with V8T cells incubated with unpulsed B cells. No antigen-specific T cells, however, could facilitate migration of up-regulation of IFN-␣ mRNA was detected in V8T cells stim- more CEA-specific T cells to the site of peptide delivery, ulated for 24 h with CAP1-6D-pulsed B cells, as compared with augmenting their chance to be activated and to proliferate. Thus, V8T cells incubated with unpulsed B cells. Because IRF-7 has antitumor immune response could be accelerated and potenti- been shown to be responsive only to IFN-␣ and IFN-␤, this

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.

might explain why IRF-7 is down-regulated when CAP1-6D- croarray experiments when CAP1-6D-stimulated V8T cells stimulated V8T cells were compared with CAP-1-stimulated were compared with CAP-1-stimulated V8T cells. Moreover, no V8T cells. increase in the levels of IL-2 or TNF-␣ mRNA was detected Due to the complex interplay between cytokines and che- when V8T cells stimulated for 24 h with either CAP-1- or mokines during inflammation and immune responses, several CAP1-6D-pulsed B cells were compared with V8T cells stim- groups have studied the role of IFN-␥ in lymphotactin expres- ulated with unpulsed B cells. There is thus a possibility that an sion. Results are controversial; using IFN-␥ knockout mice, early induction of the IL-2 and TNF-␣ genes (prior to 24 h) some authors have shown that lymphotactin gene expression is might have occurred followed by a decrease in the levels of both induced after infection or during allograft rejection in the ab- transcripts. sence of IFN-␥ (51, 52), whereas others have reported that We have demonstrated previously that the agonist peptide lymphotactin gene expression is impaired in the absence of the CAP1-6D increases ZAP-70 phosphorylation over that observed cytokine (53). We have performed experiments on concanavalin with the CAP-1 peptide. In general, activation of T cells through A stimulation of normal and IFN-␥ deficient mice T cells. Our the T-cell receptor is accompanied by biochemical events such results indicated that in the absence of IFN-␥, the production of as phosphorylation/dephosphorylation of kinases that are al- lymphotactin was not affected. In contrast, Cerdan et al. (54) ready present in the cytoplasm of the T cells. These events occur have conducted studies to analyze the effect of lymphotactin on early after activation of the T cells (in minutes) and do not IFN-␥ production; lymphotactin has been shown to impair the require de novo generation of mRNA. Therefore, the lack of expression of IFN-␥ mRNA in CD4ϩ human T cells but not detection of increased levels of mRNA encoding for signaling CD8ϩ cells. Because our studies were conducted with CD8ϩ T molecules such as ZAP-70 is not surprising in our microarray cells, our observations are not opposed to those reported previ- study. The decrease in the levels of MAP3K12 when CAP1- ously. 6D-stimulated V8T cells were compared with CAP-1-stimulated We have reported previously (17) that CEA-specific CTLs V8 T cells might be due in part to the fact that the signaling secrete higher levels of GM-CSF, TNF-␣, and IL-2 after stim- pathways invoked by the CAP1-6D peptide might be different ulation with the agonist peptide CAP1-6D as compared with the from the ones invoked by the lower affinity peptide CAP-1. native peptide CAP-1. Higher levels of mRNA encoding for In the present studies we have also reported higher levels of GM-CSF were detected by cDNA microarray analysis at 8 and granzyme B mRNA in agonist-activated V8T cells. A substan- 24 h of stimulation with the agonist CAP1-6D peptide as com- tial increase in the levels of granzyme B mRNA was also pared with the native CAP-1 peptide (Tables 2 and 3). These detected by RT-PCR in CAP1-6D-stimulated V8T cells, as results correlate well with the observations at the protein level. compared with CAP-1-stimulated V8T cells. The higher secre- However, no increase in the levels of mRNA encoding for IL-2 tion of granzyme B by agonist-activated T cells may also be one or TNF-␣ were observed at any time point in our cDNA mi- of the principal factors in enhancing the susceptibility of

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Claudia Palena, Jeffrey Schlom and Kwong-Yok Tsang

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