In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8+ T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8+ T cells, IFN-γ, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.

Many strategies for cancer treatment use combinations of immunotherapeutic agents for enhanced anti-tumor responses. However, these approaches are often complicated by a need to overcome tumor-induced immune suppression in the tumor microenvironment. In this regard, T regulatory (Treg) cells and myeloid-derived suppressor cells (MDSC) have been identified as functional suppressor cells within tumors (1, 2). The most effective immunotherapeutic regimens are likely to consist of agents that restructure, within the tumor microenvironment, the composition of tumor-infiltrating leukocytes away from these inhibitory elements in favor of effector cells, such as NK cells and CD8 ϩ T cells.Chemokine expression can regulate the polarization of immune responses (3). For example, CXCR3 and CCR5 are preferentially expressed on Th1 T cells and M1 macrophages and their respective ligands are associated with enhanced cellmediated immune responses (3-5) and favorable prognosis in human RCC (5, 6). Another chemokine, monocyte chemoattractant protein (MCP)-1 activates macrophages for enhanced anti-tumor activities (7), however MCP-1 expression is also associated with the recruitment of mononuclear cells capable of producing tumor promoting factors (8, 9), as well as MDSC that contribute to tumor progression through the inhibition of effector cell functions (8, 10).We reported previously that IL-2 and agonistic antibody to CD40 (␣CD40) synergize for the regression of metastatic tumors in mice (11). Although we identified CD8 ϩ T cells and host IFN␥ expression as critical components of this therapeutic approach (11), the specific mechanisms underlying the IL-2/␣CD40 synergistic anti-tumor responses within the microenvironment remain unclear. We demonstrate in a murine model of metastatic renal cancer that ␣CD40 may be limited by its dependency upon MCP-1 and an inability to remove Tregs and MDSC specifically from within the tumor microenvironment, allowing for eventual tumor progression. In contrast, synergistic anti-tumor responses and protection achieved by IL-2/␣CD40 are associated with the expression of Th1 chemokines that are associated with favorable prognosis in RCC (5, 6), an augmentation of effector leukocytes and concomitant removal of suppressive cells specifically within the tumor microenvironment. Results CCR2 Expression Is Required for ␣CD40, but Not IL-2/␣CD40 MediatedAnti-Tumor Responses. Our previous study showed that IL-2/ ␣CD40 exhibited strong synergy for treatment of established metastatic tumors in mice, as compared to IL-2 or ␣CD40 as single agents (11). Furthermore, we found that ␣CD40 treatment of Renca-bearing mice induced significant reduction in tumors in association with high levels of systemic MCP-1 levels, suggesting a possible role for MCP-1 in leukocyte recruitment into tumors and CD40-dependent anti-tumor effects (12). To determine the relative contribution of MCP-1 to the ␣CD40-and IL-2/␣CD40-mediated anti-tumor responses, we compared tumor outcomes in treated WT and mice deficient in CCR2, the rec...

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.