Characterization of CD8 T cell responses after immunotherapy: The role of antigen specificity in anti-tumor effects

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.