Characterization of CD8 T cell responses after immunotherapy: The role of antigen specificity in anti-tumor effects
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We have previously demonstrated that immunotherapy with an agonist CD40 antibody in combination with IL-2 results in synergistic CD4-indenpendent anti-tumor effects but actually impairs the ability to generate an antigen-specific response. In the present study, our goal was to examine the role of antigen specificity in the efficacy of cytokine-based immunotherapy. Due to the massive CD8 T cell expansion that occurs after these regimens in both normal and tumor-bearing mice, we hypothesized that the anti-tumor effects resulting from immunotherapy are due not only to the induction of antigen specific T cells, but also to the increased activation and non-specific killing capability of CD8 T cells. This hypothesis was supported by the observations that CD8 T cells did not upregulate surface molecules that are indicative of recent TCR ligation following immunotherapy, and that these cells were highly lytic. To specifically determine if TCR engagement was necessary for the expansion of CD8 T cells following immunotherapy, we performed adoptive transfer studies with T Cell Receptor (TCR) transgenic (Tg) OT-1 mice and observed an increase in proliferation following anti-CD40 and IL-2 immunotherapy in the absence of antigen. Direct treatment of OT-1 mice with immunotherapy resulted in the increased functional activation and lytic capability of CD8 T cells against irrelevant tumor targets. These effects were observed despite a surprising lack of proliferation after immunotherapy. After determining that CD8 T cells were mediating anti-tumor responses in the absence of TCR ligation, we wanted to determine a possible tumor recognition mechanism for CD8 T cells following immunotherapy. NKG2D ligands are upregulated on several tumor types, and we observed an increase in the expression of NKG2D on CD8 T cells isolated from immunotherapy treated OT-1 and wild type mice. In light of the increased NKG2D expression with therapy, we further hypothesized that signaling through NKG2D on CD8+ T cells could be one mechanism by which non-specific recognition and killing occurs. We found that when treated with an NKG2D blocking antibody, CD8 T cells isolated from immunotherapy treated mice exhibited decreased lysis of NKG2D sensitive targets. The role of NKG2D in CD8 T cell killing following immunotherapy was further established by the observation that anti-tumor effects were diminished in mice that were treated with immunotherapy in the presence of an NKG2D blocking antibody. These data demonstrate that immunotherapy with anti-CD40 and IL-2 results in the expansion of antigen specific CD8 T cells, despite its deleterious effects on CD4+ T cells, and that anti-tumor responses may be generated through the increased lytic function of CD8+ T cells in an NKG2D assisted manner. Furthermore, these mechanisms may play an important role in the anti-tumor responses observed with this therapy.