Receptor-Mediated Antigen Delivery by &Alpha;<Sub>2</Sub>-Macroglobulin: Effect on Cytotoxic T Lymphocyte Immunity and Implications for Vaccine Development

Abstract

<p><p>The receptor-recognized form of &alpha;₂-macroglobulin (&alpha;₂M*) targets antigens (Ag) to professional Ag-presenting cells (APCs) for rapid internalization, processing, and presentation. When employed as an Ag delivery vehicle, &alpha;₂M* amplifies major histocompatibility complex (MHC) class II presentation as demonstrated by increased antibody (Ab) titers. Recent evidence, however, suggests that &alpha;₂M*-encapsulation may also enhance Ag-specific cytotoxic T lymphocyte (CTL) immunity. In these studies, we demonstrate that &alpha;₂M*-delivered Ag (ovalbumin, OVA) enhances the production of specific <italic>in vitro</italic> and <italic>in vivo</italic> CTL responses. <br><p>Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA_257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-&gamma; and IL-2 secretion when treated <italic>in vitro</italic> with &alpha;₂M*-OVA compared to soluble OVA. The frequency of IFN-&gamma; -producing cells was increased ~15-fold as measured by ELISPOT. Expansion of the OVA-specific CD8<super>+</super> T cells, as assayed by tetramer binding and [<super>3</super>H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by &alpha;₂M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone. <br><p>We also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with &alpha;₂M*-OVA. These &alpha;₂M*-OVA-immunized mice displayed increased protection against a subcutaneously implanted OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The anti-tumor response observed with &alpha;₂M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique. <br><p>To further understand the mechanism underlying this enhanced CTL immunity, the subsets of professional APCs capable of cross-presenting &alpha;₂M*-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to &alpha;₂M*-encapsulated Ag, CD8<super>+</super>CD4<super>-</super> and CD8<super>-</super>CD4<super>+</super> DCs appear to do so with the greatest efficiency. The implications of this finding to the ongoing debate regarding the relative contributions of APC subsets to Ag cross-presentation and the determinants of which cells cross-present with high efficiency are discussed. <br><p>These observations demonstrate that &alpha;₂M*-mediated Ag delivery promotes cross-presentation resulting in enhanced Ag-specific CTL immunity. Considered in the context of previous work, these results support &alpha;₂M* as an effective Ag delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.</p>