DIFFERENTIAL INSITU EXPANSION AND GENE-EXPRESSION OF CD4+ AND CD8+ TUMOR-INFILTRATING LYMPHOCYTES FOLLOWING ADOPTIVE IMMUNOTHERAPY IN A MURINE TUMOR-MODEL SYSTEM

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene-induced rhabdomyosarcoma (MCA/76-9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy-1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy-1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three- to sixfold more interleukin (IL)2 mRNA and six- to eightfold more IL 6 mRNA than CD8+ cells, while CD8+ TIL expressed three- to sixfold more IL 2 receptor (IL 2R) mRNA and four- to sixfold more interferon-gamma mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL 2 over a 24-h period, whereas both IL 6 and interferon-gamma activities were demonstrable. The level of IL 2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL 2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL 2 supplemented with irradiated spleen cells and "spent" 76-9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of > 90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor-associated antigen.