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Cytotoxic lymphocytes trigger apoptosis by releasing perforin and granzymes (Grn). GrnB activates the caspase apoptotic pathway, but little is known about GrnA-induced cell death. Perforin was used to load recombinant GrnA and GrnB and enzymatically inactive variants into target cells. GrnA induces single-strand DNA breaks that can be labeled with Klenow polymerase and visualized on alkaline gels. GrnA-induced DNA damage but not cytolysis requires GrnA proteolysis. GrnA-induced membrane perturbation, nuclear condensation, and DNA damage are unimpaired by caspase blockade. GrnA fails to induce cleavage of caspase-3, lamin B, rho-GTPase, or PARP. GrnA-induced cytotoxicity and cleavage of PHAP II, a previously identified GrnA substrate, are unimpaired in Jurkat cells that overexpress bcl-2. Therefore, GrnA activates a novel apoptotic pathway.

Purpose: High-level expression of the telomerase reverse transcriptase (hTERT) in >85% of human cancers, in contrast with its restricted expression in normal adult tissues, points to hTERT as a broadly applicable molecular target for anticancer immunotherapy. CTLs recognize peptides derived from hTERT and kill hTERT؉ tumor cells of multiple histologies in vitro. Moreover, because survival of hTERT؉ tumor cells requires functionally active telomerase, hTERT mutation or loss as a means of escape may be incompatible with sustained tumor growth.Experimental Design: A Phase I clinical trial was performed to evaluate the clinical and immunological impact of vaccinating advanced cancer patients with the HLA-A2-restricted hTERT I540 peptide presented with keyhole limpet hemocyanin by ex vivo generated autologous dendritic cells.Results: As measured by peptide/MHC tetramer, enzyme-linked immunospot, and cytotoxicity assays, hTERTspecific T lymphocytes were induced in 4 of 7 patients with advanced breast or prostate carcinoma after vaccination with dendritic cells pulsed with hTERT peptide. Tetramerguided high-speed sorting and polyclonal expansion achieved highly enriched populations of hTERT-specific cells that killed tumor cells in an MHC-restricted fashion. Despite concerns of telomerase activity in rare normal cells, no significant toxicity was observed. Partial tumor regression in 1 patient was associated with the induction of CD8؉ tumor infiltrating lymphocytes.Conclusions: These results demonstrate the immunological feasibility of vaccinating patients against telomerase and provide rationale for targeting self-antigens with critical roles in oncogenesis.