The absence of PD-L1 signaling did not significantly alter IL-10 expression in the spleen (Fig. lose their ability to proliferate, to lyse virally infected cells, and to produce multiple effector molecules (1C4). The loss of these polyfunctional T-cell responses (termed exhaustion) directly leads to the failure to eliminate the virus and terminate the infection. Sustained T-cell activity prevents persistent viral infection. Prolonged T-cell responses strongly correlate with acute clearance of hepatitis C virus (HCV) infection and transfer of functional virus-specific CD4 and CD8 T cells completely eliminates an established persistent viral infection (5C10). In addition to viral components MW-150 dihydrochloride dihydrate (11), the host-derived suppressive cytokine IL-10 and the programmed death-1 (PD-1)/PD-ligand (L) 1 inhibitory pathways actively suppress T-cell responses, allowing MW-150 dihydrochloride dihydrate viral persistence (9, 12, 13). Antibody blockade to relieve IL-10 or PD-L1-mediated inhibition rapidly enhances T-cell function and reduces viral replication(9, 12, 13). Thus, the re-establishment of functional T-cell responses leads to increased control of persistent viral infection, but in many cases an even further enhancement of immunity is required to completely eliminate infection (5, 9, 12C14). The suppressive environment maintained by IL-10 and PD-L1 not only limits T-cell responses to infection, but also inhibits the ability to stimulate immunity through vaccination (15, 16). Yet, despite the profound impact IL-10 and PD-L1 have on T-cell immunity, little is known about the actual mechanisms used to suppress the immune response. Consequently, MW-150 dihydrochloride dihydrate whether these suppressive factors induce each other and function through a linear pathway or whether they Rabbit Polyclonal to NT are invoked and function through distinct mechanisms is unclear. Defining these mechanisms of suppression is necessary to determine whether simultaneous blockade of IL-10 and PD-L1 will further enhance T-cell immunity and more effectively control persistent viral replication. Here, we demonstrate that IL-10 and PD-L1 suppress antiviral T-cell activity via separate pathways and consequently, simultaneous blockade of IL-10 and PD-L1 dramatically increases T-cell responses over that seen by neutralizing either molecule alone. The combinatorial blockade of both IL-10 and PD-L1 rapidly eliminates persistent virus infection. Results Distinct Mechanisms of IL-10 and PD-1/PD-L1 Immunosuppression During Persistent Viral Infection. To determine the functional relationship between IL-10 and the PD-1/PD-L1 pathways during persistent viral infection, WT C57BL/6 mice or C57BL/6 mice genetically deficient in IL-10 (IL-10 KO) or PD-L1 (PD-L1 KO) were infected with lymphocytic choriomeningitis virus (LCMV) Clone 13 (Cl 13). Infection with LCMV-Cl 13 generates a persistent infection that rapidly aborts T-cell function by inducing the expression of host-immunosuppressive factors (9, MW-150 dihydrochloride dihydrate 12, 13). To evaluate if the increased expression of IL-10 triggered the up-regulation of PD-1 on virus-specific CD8 T cells or PD-L1 on antigen-presenting cells (APC) (i.e., dendritic cells, B cells, macrophages), WT C57BL/6 and IL-10-deficient mice were infected with LCMV. Because IL-10-deficient mice rapidly clear the otherwise persistent LCMV-Cl 13 infection, PD-1/PD-L1 levels were analyzed 5 days after LCMV-Cl 13 infection at a time when T-cell responses and viral titers are similarly high in WT mice and IL-10-deficient mice (serum viral titers in WT: 2.5 104 2.2 104 versus IL-10 KO: 7.1 103 4.9 103; = four mice per group; = 0.22). The similar virus titers enable differentiation of factors induced by IL-10 versus those triggered in response to heightened virus replication despite the acute clearance of LCMV-Cl 13 in IL-10 KO mice (9, 13). Five days after infection, PD-1 expression was similar on virus-specific CD8 T cells in WT and IL-10-deficient mice (Fig. 1< 0.05. (< 0.05 compared to LCMV-Cl 13.