Abstract
Problem: Recent clinical reports demonstrate that adoptive immunotherapy (AIT) can mediate objective tumor regressions and positively impact survival in cancer patients. Advances in murine models have identified L-selectin(low) effector T-lymphocytes as the memory population essential for curative outcome in AIT. L-selectin(high) T-lymphocytes can include suppressor cells which can subvert AIT. Preclinical optimization of human culture is essential to apply these advances to AIT of head and neck cancer.
Methods: Appropriate healthy volunteers consented to leukapharesis before and after receiving a tetanus (TET) booster. Unfractionated, L-selectin(low), and L-selectin(high) T-lymphocyte populations were isolated and stimulated with TET, control antigen (Ag), or anti-CD3. Growth curves were measured, proportions of Ag-specific cells were determined by intracellular cytokine assays, and surface immunophenotypic modulations were determined by FACS analysis.
Results: T lymphocytes which shed L-selectin during cryopreservation regenerated their original spectrum of expression during 24-hour preliminary culture, enabling subsequent separation of L-selectin(low) and L-selectin(high) subpopulations. Preliminary data indicate that TET booster results in the appearance of L-selectin(low) TET-specific T cells in the peripheral blood. Regardless of vaccination, however, TET-specific T lymphocytes with an L-selectin(low), CCR7(low), HLA-DR(pos) phenotype resulted from appropriate culture activation and expansion. These T lymphocytes could be enriched to 15% of the total population during the initial round of culture.
Conclusion: Culture optimization allows for the identification, isolation, and expansion of Ag-specific human T lymphocytes with the L-selectin(low) immunophenotype associated with efficient tumor destruction in murine models of AIT. Even after cryopreservation, L-selectin(high) T cells can be effectively removed prior to culture activation.
Significance: Once extended from irrelevant marker Ag to tumor-associated antigens such as HER2-neu, this culture system will enable clonal expansion of anti-tumor T lymphocytes which may improve AIT models for treatment of head and neck cancer.
Support: We gratefully acknowledge the American Academy of Otolaryngology-Head and Neck Surgery Foundation for their Resident Research Award grant support.