Vaccine 19 (2001) 3552–3567 Enhancing the potency of peptide-pulsed antigen presenting cells by vector-driven hyperexpression of a triad of costimulatory molecules James W. Hodge a , Douglas W. Grosenbach a , Ariel N. Rad b , Mariateresa Giuliano a , Helen Sabzevari a , Jeffrey Schlom a, * a Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Building 10, Room 8B07, 10 Center Drie, Bethesda, MD 20892 -1750, USA b Howard Hughes Medical Institute, Research Scholar’s Program at the NIH, Bethesda, MD 20892, USA Received 11 June 2000; accepted 2 January 2001 Abstract Recombinant orthopox vectors (both replication-defective fowlpox [rF], and replication competent vaccinia [rV] have been developed that simultaneously express three T-cell costimulatory molecule transgenes. The constituents of this triad of costimula- tory molecules (designated TRICOM) are B7-1, ICAM-1, and LFA-3. We have previously shown that infection of murine dendritic cells (DCs) with TRICOM vectors increases their level of expression of the triad of costimulatory molecules and enhances the efficacy of DCs to activate T cells. While DCs are arguably the most potent antigen presenting cell (APC), limitations clearly exist in their use due to the level of effort and cost for their generation. The studies reported here demonstrate that a generic APC population, murine splenocytes, can be made markedly more efficient as APCs by infection with either rF-TRICOM or rV-TRICOM vectors. Infection of splenocytes with either TRICOM vector led to significant improvement of APC capabilities in terms of: (a) enhancement of mixed lymphocyte reactions; (b) a reduction in the amount of signal 1 to activate naive T cells; and (c) a reduction in the amount of APCs required to activate T cells using a constant amount of signal 1. TRICOM-enhanced T-cell activation was shown to correspond to increases in type-1 cytokines and a reduced level of apoptosis, compared with T cells activated with uninfected or control vector-infected splenocytes. In vitro and in vivo experiments compared DCs with TRICOM-infected splenocytes. Infection of splenocytes with TRICOM vectors markedly enhanced their ability to activate T cells to levels approaching that of DCs. These studies thus demonstrate for the first time that an abundant and accessible population of APCs obtainable without lengthy culture or the use of costly exogenous cytokines (in contrast to that of DCs) can be made more potent as APCs with the use of vectors that express a triad of costimulatory molecules. Published by Elsevier Science Ltd. Keywords: Splenocytes; Costimulation; Fowlpox; Vaccinia; Vaccine; T cells www.elsevier.com/locate/vaccine 1. Introduction Effective activation of either CD4 + or CD8 + T cells requires two or more signals [1–3]. The first signal is mediated through a peptide/MHC molecule complex on the surface of the APC, which interacts with the T-cell receptor on the surface of the T cell. The second signal and any subsequent signals are mediated through one or more costimulatory molecules on the APC surface, which can interact with the same or different ligands on the surface of the T cell. Several distinct molecules normally found on the surface of APCs have been reported as capable of providing the second signals critical for T-cell activation. These molecules include B7-1 (CD80), intercellular adhesion molecule-1 (ICAM- 1; CD54), and leukocyte function-associated antigen-3 (LFA-3; human CD58; murine CD48) [1,4 – 7]. Profes- sional APCs (i.e. macrophages, monocytes, B cells and dendritic cells [DCs]) are usually defined as those cells that express high levels of both MHC and costimula- tory molecules. DCs are believed to be the most potent * Corresponding author. Tel.: +1-301-4964343; fax: +1-301- 4962756. 0264-410X/01/$ - see front matter Published by Elsevier Science Ltd. PII: S0264-410X(01)00062-7