MECHANISMS OF ADOPTIVE IMMUNOTHERAPY - IMPROVED METHODS FOR INVIVO TRACKING OF TUMOR-INFILTRATING LYMPHOCYTES AND LYMPHOKINE-ACTIVATED KILLER-CELLS

Adoptive immunotherapy with tumor-infiltrating lymphocytes and lymphok ine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced can cer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor ac tivity and optimize treatment protocols. Traditional cell tracking met hods such as fluorescent protein labeling and radiolabeling using In-1 11, I-125, or Cr-51 are limited by isotope half-life, leakage or trans fer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows l ong-term cell tracking but is laborious to perform and difficult to qu antitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and I-125-PKH95) which stably partition into lipid r egions of the cell membrane to track immune cells in vivo. Concentrati ons of each tracking compound which had no adverse effects were determ ined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up t o 5 muM for PKH95 and 20 muM for PKH26. TIL proliferation was unaltere d by labeling with up to 5 muM PKH95, 20 muM PKH26, or a combination o f 15 muM PKH26 and 5 muM PKH95. In vitro cytotoxic effector function a nd in vivo therapeutic efficacy of lymphokine-activated killer cells a nd TIL were also unimpaired by labeling with 20 muM PKH26 or 1 muM I-1 25-PKH95. Subsequent studies in an adoptive transfer immunotherapy mod el used I-125-PKH95 to track the biodistribution of TIL in tumor and i n non-tumor-bearing animals and PKH26 fluorescence to monitor microdis tribution within tissues and distinguish TIL from host T-cells. The re sults suggest that differential accumulation. selective retention, or proliferation at the tumor site cannot account for the observed patter n of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeu tic effect.