Microenvironment and Immunology Peptide Vaccination after T-Cell Transfer Causes Massive Clonal Expansion, Tumor Eradication, and Manageable Cytokine Storm Long V. Ly 1 , Marjolein Sluijter 2 , Mieke Versluis 1 , Gre P.M. Luyten 1 , Sjoerd H. van der Burg 2 , Cornelis J.M. Melief 3 , Martine J. Jager 1 , and Thorbald van Hall 2 Abstract Adoptive T-cell transfer (ACT) is successfully applied as a cancer treatment that is based on the activation and effector functions of tumor-specific T cells. Here, we present results from a mouse model in which ACT is combined with a long peptide–based vaccine comprising gp100 T-cell epitopes. Transferred CD8 + T cells expanded up to 1,000-fold after peptide vaccination, leading to a 3-fold increase in white blood cell count and a very high frequency in the generation of antigen-specific memory T cells, the generation of which tended to correlate with effective antitumor responses. An enormous pool of effector T cells spread widely to different tissues, including the skin and the immune-privileged eye, where they mediate tumor eradication. Importantly, these striking T-cell dynamics occurred in immunocompetent mice without prior hematologic conditioning. Continued activation of the specific T-cell pool by vaccination led to strong T-cell–mediated cytokine storm and lethality due to multi- organ failure. However, this immunopathology could be prevented by controlling the rapid biodistribution of the peptide or by using a weakly agonistic peptide. Together, these results identify a peptide vaccination strategy that can potently accentuate effective ACT in non-lymphodepleted hosts. Cancer Res; 70(21); 8339–46. ©2010 AACR. Introduction Adoptive cell transfer (ACT) with tumor-specific T cells is successfully applied in patients with post-transplant lympho- proliferative disease, leukemia, and melanoma (1–3). Adoptively transferred T lymphocytes are frequently obtained from stem cell donors in the case of hematopoietic tumors, but also autol- ogous T cells have the capacity to control the outgrowth of ma- lignant cells. In melanoma, transfer of tumor-infiltrating lymphocytes that have been activated and expanded in vitro is capable of inducing the regression of large metastasized lesions and results in long-term survival in approximately half of the stage IV melanoma patients (3). The function and maintenance of the transferred melanoma-reactive T cells were greatly im- proved when the recipients were pretreated with a lymphode- pleting or even myeloablative regimen (3–6). In the absence of host conditioning, the transferred T cells were hardly detectable after a few days, despite the fact that high doses of the T-cell growth factor interleukin (IL)-2 were provided. The “empty” host apparently promotes the proliferation and survival of the transferred T cells, leading to a more efficient tumor regression. Furthermore, lymphodepletion may help to overcome immune- suppressive mechanisms orchestrated by tumors (6–8). These clinical findings on melanoma nicely illustrate that the patients' own T-cell repertoire does comprise functional tumor- specific lymphocytes, although their contribution to the natural tumor resistance seems to be limited. Therapeutic peptide vac- cination aims at the recruitment of such endogenous tumor- specific T cells, but its clinical success has thus far been limited. Recently, we have observed much more robust immune and clinical responses following therapeutic vaccination when using longer peptides comprising T-cell epitopes (9, 10). The efficient activation of tumor-specific T cells that was observed in these patients did not depend on any kind of hematologic condition- ing of the host, in contrast to the findings for ACT. We here report on the combination treatment of ACT and pep- tide vaccination. Long peptides comprising the melanoma gp100 25–33 epitope were applied to activate adoptively transferred T cells in vivo. Extreme clonal expansions up to 1,000-fold and ef- fective antimelanoma responses were observed in the absence of lymphodepletion. Our results imply that the field of T-cell –based immunotherapy reaches an era in which, next to clinical efficacy, also adverse immunopathology needs to be carefully evaluated. Materials and Methods Mice C57BL/6jico mice, 8 weeks old, were obtained from Charles River (France). T-cell receptor (TCR) transgenic mice containing Authors' Affiliations: Departments of 1 Ophthalmology, 2 Clinical Oncology, and 3 Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Thorbald van Hall, Department of Clinical Oncology, K1-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, the Netherlands. Phone: 31-71-5266945; Fax: 31-71- 5266760; E-mail: T.van_Hall@lumc.nl. doi: 10.1158/0008-5472.CAN-10-2288 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 8339