In previous studies, carried out with long term cultures of CD8 T cell clones, we concentrated on their TCR, identified and characterized some of the naturally occurring peptides they recognize in association with self and non-self MHC proteins, analyzed the affinity, kinetics, and specificity of peptide binding to MHC proteins and of the resulting pep-MHC complexes to the TCR on live T cells, and evaluated how pep-MHC complex density on target cells and TCR affinity for these complexes influence target cell destruction by CTL.
Recently we have begun to the study of T cells in mice. In making this transition, we are collaborating with Professors Jianzhu Chen and Richard Young by focusing on the problem of "CD8 vaccines", i.e., on antigens and immunization strategies aimed at stimulating resting ("naïve") CD8 T cells to develop into effector "killer" cells (CTL) and into long-lived memory T cells. Vaccines of this type have not so far been successfully developed for use in human populations — in contrast to the many highly effective vaccines currently used, all of which owe their potency to the antibody molecules and activated CD4 T cells they elicit. It may be, however, that for several major infectious diseases for which effective vaccines have not been developed — for example, AIDS, malaria, and tuberculosis — CD8 vaccines have more to offer. To address this issue we are taking advantage of T cells in mice that express a transgenic TCR from a CTL clone known as 2C. This TCR has been extensively characterized with respect to the affinity and specificity of its interactions with diverse pep-MHC complexes.
A major obstacle to the development of CD8 vaccines arises from the special process by which antigens for CD8 T cells (pep-MHC complexes) are generated. The peptide components are normally generated intracellularly from cytosolic proteins by proteolytic degradation and they are then displayed in association with class I MHC proteins on the surface of antigen-presenting cells (APC). In conventional vaccines, however, the initially injected antigenic proteins are taken up from extracellular space into cell lysosomes, degraded proteolytically, and the resulting peptides displayed in association with class II MHC proteins for CD4 T cells; the extracellular antigenic proteins are, however, generally not translocated across cell membranes into the cell cytosol, the site of the class I antigen processing pathway: hence their failure to give rise to the pep-MHC complexes needed for CD8 T cell activation.
To overcome this barrier there is currently much interest in the use of strategies that introduce genes for antigens of interest into cells for cytosolic expression. Some examples of these "genetic vaccines" are DNA vectors engineered to contain sequences for protein antigens, injected as naked DNA molecules or incorporated into obligate avirulent intracellular microbes (pox viruses, adenovirus, listeria, etc.) In an alternative approach, we are exploring the use of heat shock fusion proteins. Injected into mice, these proteins have elicited the production of CD8 CTL that respond specifically to peptides from the fusion partner, suggesting that these fusion proteins might penetrate into the cytosolic class I MHC antigen-processing pathway of antigen-presenting cells (e.g. dendritic cells). We are examining this and the alternative possibility that the fusion protein enters dendritic cells by receptor mediated endocytosis and peptides generated in lysomes or late endosomes are loaded onto MHC-I proteins.
In parallel studies, we have in collaboration with Jianzhu Chen defined some of the prominent functional differences between naive and memory CD8 T cells, including differences in the cytokines and TCR ligands required for their growth, differentiation, and survival.
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