Direct T cell activation via CD40 ligand generates high avidity CD8 ⁺ T cells capable of breaking immunological tolerance for the control of tumors

CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Primarily expressed by activated CD4⁺ T cells, CD40L binds to CD40 on antigen presenting cells (APCs), thereby inducing APC activation. APCs, in turn, prime cytotoxic T lymphocytes (CTLs). Here, two tumor-associated antigen (TAA) animal models, p53-based and GP100-based, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8 ⁺ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another construct encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses as well as the potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8⁺ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the functional avidity of antigen-specific CD8⁺ T cells. Thus, our data show that vaccination with TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of better functioning antigen-specific CD8 ⁺ T cells. Our study serves as an important foundation for future clinical translation.