6B). rescue by PD-L1 blockade, this regimen may induce lethal autoimmunity. In this report, we show that PD-L1 blockade together with CD4 T cell depletion effectively rescued deeply exhausted CD8 T cells and enhanced antiviral control during the late stage of chronic contamination without any associated mortality. These data demonstrate the pleiotropic effects of antiCPD-L1 therapy on both virus-specific CD8 T cells and Tregs, and suggest a novel strategy for effectively rescuing deeply exhausted CD8 T cells. Introduction T cell exhaustion is usually a hallmark of chronic contamination and is characterized by progressive downregulation of T cell function (1C6). In particular, the immunoinhibitory programmed cell death-1 (PD-1) pathway is critical in regulating T cell function during chronic infections and cancers (5, 7C9). PD-1 is usually upregulated on exhausted T cells (9) and ligation with programmed death-ligand 1 (PD-L1) results in reduced signal transduction after TCR triggering (10). In different models of chronic contamination, blockade of the PD-1/PD-L1 pathway results in significant rescue of exhausted CD8 T cell responses (9, 11C16). Until now, all studies with the chronic lymphocytic choriomeningitis virus (LCMV) contamination model have assessed T cell exhaustion at early time points after the establishment of persistent contamination (9, 17C20). These reports have shown that PD-L1 blockade within the first two months of chronic contamination results in substantial rescue of exhausted CD8 T cell responses, but a detailed analysis of the impact of PD-L1 blockade during the later stages of chronic contamination is lacking. In this study, we corroborated that PD-L1 blockade during the early stage of a chronic LCMV contamination (about day 60) results in robust functional rescue of exhausted CD8 T cell responses. However, we observed reduced efficacy of PD-L1 blockade at rescuing exhausted CD8 T cell responses during the late stages of chronic contamination LB42708 (>150 d). Strikingly, the reduction in the efficacy of PD-L1 blockade in nonresponding mice (at late times postinfection) was associated with accumulation of PD-1+ regulatory T cells (Tregs). We also show that treatment with CD4 T cellCdepleting Abs partially re-establishes responsiveness to PD-L1 blockade therapy at the late stage of chronic contamination. These findings demonstrate an effective strategy for improving the efficacy of PD-L1 blockade in the context of advanced chronic diseases and highlight an inverse association between the levels of PD-1+ Tregs and response to LB42708 PD-L1 blockade. Materials and Methods Mice and infections Four- to 8-wk-old C57BL6J mice (Jackson Laboratories) were infected with LCMV Armstrong or Cl-13. Memory T cell responses were generated by i.p. injection with 2 105 PFU LCMV Armstrong (21), which results in an acute contamination that is cleared within 8 d, resulting in the generation of memory immune responses. Lifelong chronic infections with exhausted CD8 T cell responses were generated by CD4 T cell depletion followed by i.v. injection with 2 106 PFU LCMV Cl-13 as described previously (22). Transient systemic LCMV Cl-13 infections were induced by i.v. injection with 2 106 PFU LCMV Cl-13 without prior CD4 T cell depletion. All animal experiments were performed with approval of the Beth Israel LB42708 Deaconess Medical Center Institutional Animal Care and Use Committee. Titration of LCMV was performed on Vero cell monolayers as previously described (23). In brief, serial 10-fold dilutions from serum or homogenized tissues were distributed on Vero cell monolayers in six-well plates. Plates were then incubated for 1 h rocking every 15 min. A 1:1 solution of 1% agarose in 2 199 media Rabbit Polyclonal to NCAM2 was overlaid on top of the monolayers. After 4 d, a 1:1 solution of 1% agarose in 2 199 media with 1:50 neutral red was aliquoted on each well. PFUs were counted at day 5 with the aid of a transluminator. Adenoviral immunizations with various replication incompetent adenoviral vaccine vectors expressing LCMV glycoprotein (GP) were given i.m. at 1010 viral particles.