Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

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5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

SUMMARYThe delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cellsurface expression. Immunization of mice with live recombinant lactobacilli induced signi®cant levels of circulating TTFC-speci®c immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage¯uids, as were antigen-speci®c antibody-secreting cells and antigen-speci®c T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.