Vector-driven hyperexpression of a triad of costimulatory molecules confers enhanced T-cell stimulatory capacity to DC precursors.

Activation of T cells requires at least two signals: signal 1, via the T-cell receptor, and signal 2, in which a costimulatory molecule on the antigen presenting cell (APC) interacts with a ligand on the T cell. Dendritic cells (DCs) are the most potent APCs in part due to their expression of costimulatory molecules. DCs, however, constitute only a minor percentage of APCs in the body, and the in vitro preparation of DCs is both costly and time consuming. The studies reported here demonstrate that one can utilize other APCs, such as bone marrow progenitor cells (BMPCs) and make them markedly more effective as APCs; this was accomplished by their infection with recombinant poxviruses (either the replication-defective avipox or vaccinia), which contain transgenes for a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3, designated TRICOM). APCs infected with TRICOM vectors are shown to significantly enhance the activation of both naive and effector CD4(+) and CD8(+) T-cell populations. The use of TRICOM vectors in vaccine strategies is discussed.

Enhancing the potency of peptide-pulsed antigen presenting cells by vector-driven hyperexpression of a triad of costimulatory molecules.

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 costimulatory 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.

Enhanced activation of T cells by dendritic cells engineered to hyperexpress a triad of costimulatory molecules.

BACKGROUND: Activation and proliferation of T cells are essential for a successful cellular immune response to an antigen. Antigen-presenting cells (APCs) activate T cells through a two-signal mechanism. The first signal is antigen specific and causes T cells to enter the cell cycle. The second signal involves a costimulatory molecule that interacts with a ligand on the T-cell surface and leads to T-cell cytokine production and their proliferation. Dendritic cells express several costimulatory molecules and are believed to be the most potent APCs. Two recombinant poxvirus vectors (replication-defective avipox [fowlpox; rF] and a replication-competent vaccinia [rV]) have been engineered to express a triad of costimulatory molecules (B7-1, intercellular adhesion molecule-1, and leukocyte function-associated antigen-3; designated TRICOM). This study was designed to determine if dendritic cells infected with these vectors would have an enhanced capacity to stimulate T-cell responses. METHODS: Murine dendritic cells (of both intermediate maturity and full maturity) were infected with rF-TRICOM or rV-TRICOM and were used in vitro to stimulate naive T cells with the use of a pharmacologic agent as signal 1, to stimulate T cells in allospecific mixed lymphocyte cultures, and to stimulate CD8(+) T cells specific for a peptide from the ovalbumin (OVA) protein. In addition, dendritic cells infected with TRICOM vectors were pulsed with OVA peptide and used to vaccinate mice to examine T-cell responses in vivo. All statistical tests were two-sided. RESULTS: Dendritic cells infected with either rF-TRICOM or rV-TRICOM were found to greatly enhance naive T-cell activation (P<.001), allogeneic responses of T cells (P<.001), and peptide-specific T-cell stimulation in vitro (P<.001). Peptide-pulsed dendritic cells infected with rF-TRICOM or rV-TRICOM induced cytotoxic T-lymphocyte activity in vivo to a markedly greater extent than peptide-pulsed dendritic cells (P =.001 in both). CONCLUSIONS: The ability of dendritic cells to activate both naive and effector T cells in vitro and in vivo can be enhanced with the use of poxvirus vectors that potentiate the hyperexpression of a triad of costimulatory molecules. Use of either rF-TRICOM or rV-TRICOM vectors significantly improved the efficacy of dendritic cells in priming specific immune responses. These studies have implications in vaccine strategies for both cancer and infectious diseases.