Human tumor-infiltrating lymphocytes (TIL) include a minor population of tumor-specific T cells, but the nature of the majority of TIL remains unknown. Recently, it has been suggested that T cells that recognize stressed cells may play an important role in immune surveillance. We have examined the proliferative response of anti-CD3-activated TIL cultures from human tumors against heat-stressed (hs) (42.8 degrees C, 25 min) B cell lines (TK6 (HLA-DR7+, -DRw13+, -DRw52+, and -DRw53+) and JY (HLA-DR4+ and -DRw52+)) and the mutant cell line (T2 (no HLA-DR)) by measuring [3H]thymidine incorporation. TIL lines from three of four ovarian cancers, two of four lung cancers, one of two renal cell cancers, one of two melanomas, and one of one breast cancer showed a positive proliferative response against hs-TK6 and/or hs-JY, but not against hs-T2. These TIL did not respond to autologous tumor cells. The response to hs-B cells was mediated by CD4+ TIL and inhibited by anti-HLA-DR Ab, but not by anti-HLA class I. Protein analysis revealed a significant increase of heat shock protein 70 (hsp70) expression in hs-TK6 and hs-JY. In addition, the CD4+ TIL responded to TK6 that had been pulsed with hsp70. This response could be blocked by anti-HLA-DR Ab. The CD4+ TIL produced IFN-gamma, but not IL-4, in response to hs-TK6. From these results, we conclude that hsp70-reactive CD4+ T cells exist in tumor tissues. Furthermore, these TIL recognize stressed cells and seem to play a Th1-like role that may support antitumor T cell responses at local tumor sites.