Induction of functional empty class I major histocompatibility complex glycoproteins by photoactivated...

CD8+ cytotoxic T lymphocytes (CTLs) bind to and selectively lyse tumor cells via T-cell receptor recognition of distinctive peptide antigens presented in the context of surface major histocompatibility complex class I (MHC class I) glycoproteins. Several human and experimental animal tumors express distinctive MHC class I-associated peptides, which can be selectively targeted by specific CD8+ CTLs. Malignant cells expressing low quantities of these peptides are poor inducers of CTL responses. Therefore, we have developed a method of externally loading increased amounts of antigenic peptides onto MHC class I molecules. In order to induce "empty" fillable MHC class I molecules capable of binding antigenic peptides, we exposed transformed murine T cells (RMA) to low dose (3 joules/cm2) ultraviolet A energy and 8-methoxypsoralen (100 ng per ml). Presence of "empty" class I molecules was ascertained by "meltdown" or loss of the thermodynamically unstable cold-induced "empty" molecules as identified by cytofluorography at 37 degrees C. Retained function of "empty" molecules was determined by their stabilization through addition of peptides of the correct size and sequence motif, prior to exposure to physiologic temperature.