We compared peripheral and mucosal primary CD8 T cell responses to inf lammatory and noninf lammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inf lammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inf lammatory signals in the mucosa.

Abstract. The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules and lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathfin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal [3 lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathfin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases can be incorporated into MIICs simultaneously by a Man-6-P-independent process. M AJOR histocompatibility complex (MHC) 1 molecules mediate the presentation of antigenic peptides by antigen presenting cells (APCs) to T lymphocytes. Almost all cell types express MHC class I molecules, which display a variety of cytoplasmically derived peptides for presentation to CD8-positive T lymphocytes. MHC class II molecules are found on a restricted set of cell types such as B lymphocytes, macrophages, dendritic cells including epidermal Langerhans cells, and thymic epithelial cells. MHC class II molecules bind peptides generated by the proteolysis of exogenous antigens for presentation to CD4-positive T lymphocytes (reviewed in Cresswell, 1994). The MHC class II complex consists of ct and 13 chains that associate in the endoplasmic reticulum (ER) with the 31-33-kD invariant (I) chain (Marks et al., 1990). It is generally accepted that the association of I-chain with MHC class II prevents the binding of endoge-

Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8+ IFN-γ and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-γ in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30–40 residues (3–4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-γ responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8+ responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8+ T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8+ IFN-γ and HTL responses.

CD8 T-cell (T CD8؉)