Purpose: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO_119-143 region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO_119-143 tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1).

Experimental Design: We generated tetramers of DRB1*0101 incorporating peptide ESO_119-143 using a previously described strategy. We assessed ESO_119-143-specific CD4 T cells in peptide-stimulated postvaccine cultures using the tetramers. We isolated DR1/ESO_119-143 tetramer⁺ cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4⁺ DR1/ESO_119-143 tetramer⁺ T cells ex vivo and characterized them phenotypically.

Results: Staining of cultures from vaccinated patients with DR1/ESO_119-143 tetramers identified vaccine-induced CD4 T cells. Tetramer⁺ cells isolated by cell sorting were of T_H1 type and efficiently recognized full-length ESO. We identified ESO_123-137 as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO_119-143 tetramer⁺ cells using T cell receptor (TCR) β chain variable region (Vβ)-specific antibodies, we identified several frequently used Vβ. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells.

Conclusions: The development of DR1/ESO_119-143 tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients. Clin Cancer Res; 16(18); 4607–15. ©2010 AACR.