Differential in situ 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 IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three‐ to sixfold more IL 2 receptor (IL 2R) mRNA and four‐to sixfold more interferon‐γ mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24‐h period, whereas both IL6 and interferon‐γ activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 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 IL2 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.