The in vitro stimulation of T lymphocytes is frequently used as a technique to expand specific cells present at low precursor frequency in vivo. However, cells analysed after such procedures may no longer reflect those originally present in vivo because of the variable efficiency of outgrowth of different T cell subpopulations. To systematically assess this and to complement functional assays, we have analysed the TCR repertoire using a new high resolution RT-PCR method to determine TCR β chain

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... CDR3 transcript length. In the ex vivo analysis of tumor infiltrating lymphocytes (TIL) of renal cell carcinoma and glioblastoma patients, we observed and quantified oligoclonally expanded populations of T cells that were very susceptible to repertoire modification upon subsequent in vitro culture with autologous tumor cells. This in vitro repertoire skewing occurred preferentially with TIL rather than peripheral blood lymphocytes and we noted that tumor cells rather than normal cells of the same tissue type were the most potent inducers of the effect. It was striking that this selection was sometimes negative: certain prominent T cell populations that were highly represented in vivo disappeared after in vitro re-stimulation. This suggests that the presentation of tumor associated antigens during culture may eliminate rather than enrich for in vivo primed T cells. It is clear that in vitro functional tests cannot adequately describe all T cells with tumor specificity. Approaches that allow the assessment of potentially antigen-reactive T cell populations ex vivo are thus an important advance in the global appraisal of anti-tumor T cell immune responses.