Melanoma vaccines: the paradox of T cell activation without clinical response

In recent years significant progress in the understanding of the immune bio logy of melanoma has evolved from the identification of melanoma antigens ( MAs) recognized by T cells. MAs consist of intracellular proteins that are expressed on the surface of cancer cells in association with human leukocyt e antigen (HLA) class I molecules and therefore are suitable targets for cy totoxic T lymphocytes (CTLs). Several new monitoring strategies have been i mplemented to evaluate the status of activation and localization of vaccine -induced T cells in the peripheral circulation as well as the tumor site, i ncluding limiting dilution, in vitro sensitization, and ELISPOT. Previous s tudies aimed at monitoring patients receiving vaccination have utilized mai nly those three methods. These methods have demonstrated that antigen-speci fic vaccination can elicit immune responses detectable in the peripheral bl ood of immunized patients. These assays, however, have been faulted by thei r requirement for in vitro expansion of T cells (limiting dilution or in vi tro sensitization) or for limited sensitivity (ELISPOT). More recently, the use of soluble HLA/peptide complex tetramers, intracellular fluorescence-a ctivated cell sorting (FACS) analysis, and real-time polymerase chain react ion (PCR) has been proposed for the monitoring of vaccine trials. These met hods have the appeal of allowing direct enumeration of T cells specific for a particular epitope within relevant samples such as peripheral blood lymp hocytes, lymph nodes, and tumors. We are evaluating whether utilizing a com bination of HLA/peptide tetramer (tHLA) together with Taqman-based real-tim e reverse-transcription (RT)-PCR and intracellular FACS analysis could esta blish a direct and comprehensive strategy for the assessment of epitope-spe cific immune response in vivo. In conditions close to those of the tumor mi croenviroment or in peripheral blood lymphocytes, however, a different stat us of T cell activation call be expected due to a direct stimulation of T c ells by tumor or antigen-presenting cells. We observed that activated T cel ls can easily be detected ill the peripheral blood of patients who have rec eived MA-specific vaccines. However, when T cells are stimulated with their relevant epitope, a high level of T cell receptor downregulation occurs th at does not allow identification of vaccine-specific T cells directly with tHLA. Thus evaluation of epitope-specific T cells at the tumor site, where they might be exposed to stimulation by interaction with tumor cells and/or in bulk peripheral blood mononuclear cells, might be more efficiently anal yzed with functional methods such as intracellular FACS and Taqman-based re al time RT-PCR.