Receptor-mediated antigen delivery by alpha2-macroglobulin: Effect on cytotoxic T lymphocyte immunity and implications for vaccine development

The receptor-recognized form of alpha2-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, alpha2M* 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 in vitro and in vivo CTL responses.;Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-gamma and IL-2 secretion when treated in vitro with alpha2M*-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+ T cells, as assayed by tetramer binding and [3H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by alpha2M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone.;We also observed enhanced humoral and CTL responses by naive mice following intradermal immunization with alpha2M*-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 alpha2M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique.;To further understand the mechanism underlying this enhanced CTL immunity, subsets of professional APCs capable of cross-presenting alpha2M *-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to alpha2M*-encapsulated Ag, CD8 +CD4- and CD8- CD4+ 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.;These observations demonstrate that alpha2M*-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.