Abstract Tumor cells employ various methods of immune suppression to overcome anti-tumor immunity. One such method is that of the integral membrane protein programmed death ligand-1 (PDL1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD1) on T cells. Treatment of PDL1+ tumor cells with a soluble form of the costimulatory molecule CD80 (CD80-Fc) prevented PDL1-mediated immune suppression by binding PDL1 and blocking interaction with PD1, thus restoring T cell activation. Additionally, CD80-Fc maintained activation of T cells in vitro more effectively than either PD1 or PDL1 antagonist monoclonal antibodies. Because CTLA-4 is also a receptor for CD80 and CD80-Fc has the potential to bind and deliver inhibitory signals into T cells via CTLA-4, it is important to assess CD80-Fc and CTLA-4 interactions. Healthy donor human PBMC stimulated with PHA for three days express CD28, PDL1, and CTLA-4 at the cell surface. To determine which of these receptors CD80-Fc binds to, we have blocked subsets of the receptors using very high concentrations of specific antibodies, and monitored CD80-Fc binding by flow cytometry. CD80-Fc binds to activated T cells blocked for CD28 and PDL1, PDL1 and CTLA-4, or CTLA-4 and CD28, and did not bind when all three receptors were blocked. These findings indicate that CD80-Fc binds to CTLA-4. However, inclusion of CTLA-4 antagonist antibodies L3D10 or Ipilimumab in co-cultures of human PBMC and PDL1+ C8161 human melanoma cells did not increase T cell production of IFNγ, suggesting that CD80-Fc is not suppressing through CTLA-4. Historically, CTLA-4 has been considered as a negative regulator of T cell function through its ability to deliver suppressive signals to T cells. Recent re-evaluations of CD80-CTLA-4 interactions suggest that CTLA-4 inhibits T cell function not by delivering intracellular signals, but by serving as a decoy receptor for CD80. Our results support this alternative functional mechanism and are consistent with the concept that CD80-Fc maintains T cell activation by saturating CTLA-4 receptors while simultaneously blocking PDL1 suppression and providing CD28 costimulation. Citation Format: Lucas Horn, Virginia Clements, Suzanne Ostrand-Rosenberg. A soluble form of CD80 enhances anti-tumor immunity by inhibiting PDL1 immune suppression and does not suppress via CTLA-4. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B013.