Intratumor gene expression after adoptive immunotherapy in a murine tumor model. Regulation of messenger RNA levels associated with the differential expansion of tumor-infiltrating lymphocytes.

Abstract

The aim of this paper was to show that the adoptive immunotherapy (AIT) of established tumors resulted in the activation of defined lymphocyte-associated genes at the site of rejection. C57BL/6J mice bearing the moderately immunogenic syngeneic MCA/76-9 sarcoma received combination therapy 10 days after tumor cell implantation. This consisted of a single i.p. injection of cyclophosphamide (CY) followed by the i.v. injection of tumor-sensitized T cells (CY/AIT). The previously observed in situ differential expansion of tumor-infiltrating lymphocytes (TIL) was associated with a parallel modulation of CD4 (L3T4) and CD8 Ag expression. Flow cytometric analysis indicated that the CD8+ TIL appeared to contain two subpopulations during the reported proliferative phase, but by day 8 after CY/AIT the cells were composed of a single bright population. The CD4+ TIL similarly appeared to show two subpopulations, but in contrast to the CD8+ TIL there was a shift to a predominantly less bright single population by day 8. The expression of lymphocyte genes (Ly-2 and Ly-4 encoding the CD8 and CD4 Ag, respectively, IL-2, IL-2R, IFN-gamma, and IL-6) was analyzed by Northern hybridization using RNA extracted from whole tumor tissue. Progressing tumors expressed only low and relatively constant levels of mRNA for all of the genes except Ly-2 over a 19-day period. In contrast, there were considerable temporal fluctuations in mRNA levels depending on whether the mice had received CY or CY/AIT, but it was apparent that CY/AIT induced the more dramatic changes as far as Ly-2, Ly-4, IL-2, and IFN-gamma mRNA levels were concerned. A preliminary survey of cytokine bioactivity released by tumor-associated cells isolated 9 days after CY/AIT indicated that both IFN-gamma and IL-6 activities were released by the cells, but not IL-2, despite the relatively high levels of IL-2 mRNA. These data provide evidence that the differential expansion of TIL after CY/AIT is accompanied by well defined changes in the levels of mRNA-encoding TIL membrane Ag and lymphokine genes and are concordant with the view that amplified anti-tumor immune responses occur after AIT.