To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.

Rat monoclonal antibodies specific for mouse kappa light chains and mouse gamma heavy chains have been generated. These rat monoclonal antibodies have been biosynthetically labelled with 35S methionine. The free label was dialyzed from the medium and, without further purification, the medium containing the radioactive monoclonal antibody was used in a radioimmunoassay to screen the sera of the immunized animals and hybridomas for specific mouse antibodies of the IgG class.

In the present work, we explored the cytokine-dependent regulation of bone marrow-derived mast cell (BMMC) antigen-presenting cell (APC) function, and co-stimulation requirements, and analyzed the nature of antigens presented to T cells. We observed an up-regulation of the APC function of mast cells induced by granulocyte/macrophage-colony-stimulating factor (GM-CSF) and a complete abrogation by interferon (IFN)-gamma. Expression of co-stimulatory molecules CD80 and CD86 was suggested by the ability of mast cells to activate purified lymph node-derived T cells. Indeed, addition of the fusion protein mCTLA4-Ig strongly inhibited antigen presentation by mast cells to normal T cells and to the T cell hybridoma 3DO-54.8. The regulatory mechanisms of APC function by GM-CSF and IFN-gamma were investigated by measuring CD80 and CD86 transcripts in mast cells. GM-CSF-treated must cells showed a strong increase in the expression of both CD80 and CD86 transcripts, whereas in IFN-gamma-treated mast cells, this expression was completely abrogated. Thus, up- and down-regulation of CD80 and CD86 expression by GM-CSF and IFN-gamma is directly correlated to the APC function. In addition, we analyzed antigen presentation by mast cells of endogenous self-antigens. Mast cells failed to activate anti-I-A or anti-I-E-specific T cell hybridomas and alloreactive T cells in primary mixed lymphocyte reactions (MLR). Furthermore, mast cells did not present the mouse beta 2-microglobulin (m beta 2-m) peptide 25-40, constitutively expressed on B cells. However, mast cells, especially those treated with GM-CSF, activated an anti-m beta 2-m-specific T cell hybridoma in the presence of exogenous peptide. The minor lymphocyte-stimulating antigen-1 Mls-1a is a viral superantigen (vSAG) encoded by the the mouse mammary tumor provirus-7 (MMTV-7). Mast cells, despite a reasonable amount of major histocompatibility complex class II on the cell surface and the presence of MMTV transcripts predicted to encode the vSAG, cannot stimulate in vivo or in vitro V beta 6+ T cells specific for Mls-1a. In contrast, mast cells could present the exogenous bacterial SAG, staphylococcal enterotoxin B (SEB), to specific V beta 8+ T cells. The selective ability of mast cells to present exogenous antigens may have physiological relevance in that mast cells could participate in immune response regulatory mechanisms by discriminating self from nonself.