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

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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 micro- arrays (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).
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