Increasing infiltration and activation of CD8⁺ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

Abstract

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon γ (IFNγ) plus 10 U tumor necrosis factor α (TNFα) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFNγ or TNFα alone, but IFNγ/TNFα therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFNγ/TNFα treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFNγ/TNFα treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFNγ/TNFα treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFNγ/TNFα plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFNγ/TNFα, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFNγ plus TNFα therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.