InVivoMAb anti-human PD-L1 (B7-H1)

Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1(pos)) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8(pos)) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.

Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.

Antigen persistence in chronic infections and cancer upregulates inhibitory networks, such as the PD-1 and interleukin-10 (IL-10) pathways, that impair immunity and lead to disease progression. These pathways are attractive targets for immunotherapy, as demonstrated by recent clinical trials of PD-1/PD-L1 blockade in cancer patients. However, in HIV-1 infection not all subjects respond to inhibition of either pathway and the mechanistic interactions between these two networks remain to be better defined. Here we demonstrate that in vitro blockade of PD-L1 and/or IL-10Ralpha results in markedly different profiles of HIV-1-specific CD4 T cell restoration. Whereas PD-L1 blockade leads to balanced increase in gamma interferon (IFN-gamma), IL-2, and IL-13 secretion, IL-10Ralpha blockade preferentially restores IFN-gamma production. In viremic subjects, combined PD-L1/IL-10Ralpha blockade results in a striking 10-fold increase in IFN-gamma secretion by HIV-1-specific CD4 T cells that is not observed in subjects with spontaneous (elite controllers) or therapy-induced control of viral replication. In contrast to the dramatic increase in IFN-gamma production, concurrent blockade has a marginal additive effect on IL-2 production, IL-13 secretion, and HIV-1-specific CD4 T cell proliferation. IFN-gamma produced by Thelper cells upregulates PD-L1, HLA I/II, and IL-12 expression by monocytes. The effect of combined blockade on IFN-gamma was dependent on reciprocal reinforcement through IL-12. These studies provide crucial information on the different immunoregulatory qualities of PD-1 and IL-10 in progressive disease and link exhausted virus-specific CD4 T cells and monocytes in the regulation of IFN-gamma and IL-12 secretion. IMPORTANCE: Infection with HIV results in most people in uncontrolled viral replication and progressive weakening of the body defenses. In the absence of antiviral therapy, this process results in clinical disease, or AIDS. An important reason why HIV continues to multiply is that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection.

NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.