TCR ANALYSIS REVEALS SIGNIFICANT REPERTOIRE SELECTION DURING IN-VITROLYMPHOCYTE CULTURE

The in vitro stimulation of T lymphocytes is frequently used as a tech nique to expand specific cells present at low precursor frequency in v ivo. However, cells analysed after such procedures may no longer refle ct those originally present in vivo because of the variable efficiency of outgrowth of different T cell subpopulations. To systematically as sess this and to complement functional assays, we have analysed the TC R repertoire using a new high resolution RT-PCR method to determine TC R beta chain CDR3 transcript length, In the ex vivo analysis of tumor infiltrating lymphocytes (TIL) of renal cell carcinoma and glioblastom a patients, we observed and quantified oligoclonally expanded populati ons of T cells that were very susceptible to repertoire modification u pon subsequent in vitro culture with autologous tumor cells. This in v itro repertoire skewing occurred preferentially with TIL rather than p eripheral 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 th e presentation of tumor associated antigens during culture may elimina te rather than enrich for in vivo primed T cells, It is clear that in vitro functional tests cannot adequately describe all T cells with tum or specificity, Approaches that allow the assessment of potentially an tigen-reactive T cell populations ex vivo are thus an important advanc e in the global appraisal of anti-tumor T cell immune responses.