[CANCER RESEARCH 62, 2043–2051, April 1, 2002] Synergistic Enhancement of Antitumor Immunity with Adoptively Transferred Tumor-specific CD4  and CD8  T Cells and Intratumoral Lymphotactin Transgene Expression 1 Hui Huang, Fang Li, John R. Gordon, and Jim Xiang 2 Research Unit, Saskatchewan Cancer Agency, Departments of Oncology [H. H., J. X.] and Veterinary Microbiology [F. L., J. R. G.] University of Saskatchewan, Saskatoon, Saskatchewan, S7N 4H4 Canada ABSTRACT The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have shown that transplanted SP2/0 myeloma tumors that have been engineered to express lymphotactin (Lptn) invariably regress under the influence of infiltrating XCR1  T cells and neutrophils. Herein, we characterize these T cells and investigate their therapeutic efficacy, either alone or with Lptn gene therapy. After stim- ulation with SP2/0 cells, these T cells were CD25  FasL  L-selectin  , expressed XCR-1, and were chemoattracted by Lptn in vitro. They com- prised 66% CD4  Th1 and 33% CD8  Tc1 cells, both of which expressed significant amounts of IFN-, perforin, and tumor necrosis factor-, but not interleukin-4. The CD4  Th1 and CD8  Tc1 cells, which were inhib- ited and stimulated, respectively, for proliferation with Lptn signaling, displayed 38 and 84% specific killing, respectively, for Ia d /H-2K d -express- ing SP2/0 tumor cells (E:T ratio, 100). In vivo, combined intratumoral Lptn gene transfer and adoptive immunotherapy with these CD4  and CD8  T cells eradicated well-established SP2/0 tumors in six of eight mice, and dramatically slowed tumor growth in the other two mice. Cell track- ing using labeled T cells confirmed that these cells infiltrated better into the Lptn-expressing tumors than non-Lptn-expressing ones. Control or Lptn adenoviral treatments by themselves did not alter the lethal outcome for tumor-bearing mice, nor did T-cell therapy by itself, although the latter two treatments did slow its time frame. Combined Lptn gene trans- fer and adoptive CD4  or CD8  cell transfers were not nearly as effica- cious as the combined Lptn gene and unfractionated T-cell transfers. Taken together, our data provide solid evidence of a potent synergy between adoptive CD4  and CD8  T-cell therapy and Lptn gene transfer into tumor tissues, which culminated in the eradication of well-established tumor masses. INTRODUCTION CTLs play a crucial role in the host immune response to cancer. Effective adoptive cancer immunotherapy with tumor-sensitized CTLs has been well documented in animal models (1– 4), where transfer of such tumor-specific cells into mice bearing established tumors has resulted in tumor eradication. However, even in such model systems, this therapeutic approach is limited to early-stage tumors (e.g., 3- or 10-day lung metastases) or established s.c. tumors that have been previously irradiated (to facilitate T-cell infiltration; Refs. 3– 6). In clinical settings, only limited numbers of patients have responded to CTL therapy (7–9), with an objective response rate of 30% (10). In general, it is assumed that the antitumor efficacy of the transferred T cells is, to a large extent, determined by their ability to leave the vasculature and infiltrate the tumor (5, 6), but the overall fraction of transferred T cells that accumulate in tumors is rather small (11, 12). The resistance of tumors to T-cell infiltration may be attributable in part to limited expression on the neovasculature within these growths of the adhesion molecules that are essential for T-cell adherence and transendothelial migration, e.g., vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 (13). The trafficking of lymphocytes from the systemic circulation into tissues is a dynamic, multistep process. It requires selectin-mediated rolling and tethering, lymphokine-induced activation of integrins, firm adhesion of the lymphocytes to endothelial cells and their diapedesis through the endothelium, as well as migration within the connective tissues along established chemoattractant gradients (14). Although adhesion to endothelial cells and various extracellular matrix proteins is critical for successful T-cell recruitment into inflammatory sites, the signals that regulate these processes have not been fully elucidated. It is believed that essential steps are mediated by chemokines produced at the sites of inflammation (15). Chemokines are a superfamily of cytokines that attract and activate leukocytes (16). They are produced by multiple cell types (e.g., leukocytes, endothelial cells, fibroblasts, and tumor cells) in response to viruses, bacteria, lipopolysaccharide, and proinflammatory cyto- kines (e.g., IL-1 3 and TNF-; Ref. 17). The superfamily’s four major branches are defined by the spacing of the first two cysteines in a conserved four-cysteine motif. The two cysteines of the  subfamily (C-X-C) are separated by another residue and those of the  subfamily (C-C) are adjacent, whereas the  subfamily (C) has only one cysteine at its NH 2 terminus (18). The newly identified C-X 3 -C subfamily has two cysteines separated by three other residues (19). Generally, C- X-C chemokines are potent activators and chemoattractants for neu- trophils, whereas the C-C chemokines have the potential to chemoat- tract monocytes and T lymphocytes (17). The C chemokine Lptn was originally reported to induce T and NK cell, but not monocyte, migration in vitro (20) through its interactions with the G-protein- coupled, seven-transmembrane domain receptor XCR-1 (21). Re- cently, it has been reported that Lptn also chemoattracts neutrophils and B cells expressing XCR-1 (22) and acts as an innate mucosal adjuvant (23). More recently, we engineered a mouse myeloma cell line, SP2/0, to express a Lptn transgene. We found that such engineered SP2/0-Lptn tumor cells invariably regressed when implanted into syngeneic BALB/c mice, after Lptn-dependent infiltration of the tumors by CD4 + and CD8 + T cells and neutrophils (24). Furthermore, the wild-type SP2/0 cells induced very little in the way of effector immune responses after tumor inoculation, such that neither IFN- nor significant tumor-specific CTL responses could be discerned in these animals. Because Lptn can induce T-cell migration in vitro (20) and in vivo (24, 25), we reasoned that Lptn expression within tumors, induced by adenovirus-mediated Lptn gene transfer, should enhance T-cell infiltration and thus the therapeutic efficiency of adoptively transferred tumor-specific T cells. In the present study we tested this Received 8/27/01; accepted 1/31/02. 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 This study was supported by Research Grants ROP-15151 and MT11861 of the Canadian Institute of Health Research and Medical Research Council of Canada. 2 To whom requests for reprints should be addressed, at Research Unit, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, Saskatchewan, S7N 4H4 Canada. E-mail: JXiang@SCF.SK.CA. 3 The abbreviations used are: IL, interleukin; TNF-, tumor necrosis factor-; Lptn, lymphotactin; NK, natural killer, FasL, Fas ligand; PE, phycoerythrin; pfu, plaque- forming unit(s); APC, antigen-presenting cell; RT-PCR, reverse transcription-PCR; TRITC, tetramethylrhodamine B isothiocyanate. 2043 Research. on November 8, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from