Anaplastic astrocytoma and glioblastoma are frequent and malignant brain tumors that are infiltrated by T lymphocytes. Whether these cells result from non-specific inflammation following blood-brain barrier disruption or an antigen-driven specific immune response is unknown. In this study, an in-depth characterization of TCR diversity in tumor and blood RNA biopsies was performed in a series of 16 patients with malignant astrocytoma. Whilst there was no obvious restriction of the AV and BV gene segment usage, complementarity-determining region 3 size analysis and sequencing of amplified TCR transcripts revealed multiple T cell oligoclonal expansions in all astrocytomas analyzed. Unique T cell clones were present in different adjacent areas of a given tumor, but never detected in the blood. Quantification of the number of TCR clonal transcripts per microg of tumor RNA indicated that certain T cell clonal expansions may represent at least 300 cells/10(6) tumor cells. Furthermore, we demonstrated that the in vivo expanded clones were almost exclusively confined to the CD8(+) subset. Overall, these data suggest that spontaneous antigen-driven immune responses may be elicited against human astrocytoma despite the immunosuppressive microenvironment generated by the brain and the tumor itself. However, the ultimate failure of the immune system to control tumor growth could be the consequence of a deficient CD4 T(h) component of the response. This observation could have important consequences for the development of immunotherapies for astrocytoma patients.