Tumor Infiltrating CD8+ T Cells Mediate Non-Phthisical Intraocular Tumor Rejection by a TNF- Dependent Mechanism

Abstract

Purpose: : CD4⁺ T cells mediate a pristine, non-phthisical rejection of adenovirus type 5 early region 1-transformed embryonic tumors (Ad5E1) in an IFN-γ dependent antitumor response. Interestingly, CD8⁺ T cells also infiltrate intraocular Ad5E1 tumors. In this study, we analyzed CD8⁺ T cells in the Ad5E1 tumor model to determine their role in mediating non-phthisical intraocular tumor rejection.

Methods: : Mice were injected in the anterior chamber with the Ad5E1 tumor cells and tumor growth was recorded for 4 weeks. Pure CD8⁺ T cell populations were obtained by MACS magnetic bead separation followed by anti-CD4 antibody treatment and rabbit complement. Tumor infiltrating T cells were identified by FACS using T cell-specific antibodies. CTL activity was assessed using splenocytes co-cultured with Mitomycin C treated tumor cells in standard 4h ⁵¹Cr CTL assays. Ad5E1 MHC class I expression was analyzed by FACS analysis using MHC class I specific antibodies. Cytokine quantitation was assessed by ELISA. Apoptosis was evaluated using Annexin V staining.

Results: : Adoptive transfer of purified CD8⁺ T cells from B6 mice that previously rejected an intraocular tumor into SCID mice conferred protective immunity against an intraocular Ad5E1 tumor challenge. Interestingly, CD8⁺ mediated tumor cytotoxicity was not due to conventional CTL lysis even though Ad5E1 cells expressed MHC class I molecules. CD8⁺ T cells did not utilize perforin, IFN-γ, FasL, or TRAIL to mediate tumor rejection. Intraocular Ad5E1 tumors were rejected in TNF-α KO mice. However, SCID mice that received an adoptive transfer of purified CD8⁺ T cells from TNF-α KO rejector mice failed to reject intraocular Ad5E1 tumors.

Conclusions: : CD8⁺ T cells from B6 mice that reject intraocular Ad5E1 tumors have the capacity to elicit antitumor immunity. However, the CD8⁺ T cells do not eliminate tumor by conventional CTL mediated lysis, but employ a TNF-α mediated pathway to promote tumor rejection. Together, the data suggests that pristine, non-phthisical Ad5E1 tumor rejection involves a primary IFN-γ dependent CD4⁺ T cell pathway and a secondary TNF-α dependent CD8⁺ T cell pathway to mediate rejection of Ad5E1 ocular tumors without sustaining damage to the surrounding ocular tissues.

Keywords: immune tolerance/privilege • tumors