Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon γ, interleukin (IL)-2, tumor necrosis factor α, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive antitumor activity. Patients with tumors expressing PD-L1 are most likely to respond to this treatment. The aim of our study was to develop a noninvasive imaging technique to determine tumor PD-L1 expression in vivo. This could allow selection of patients that are most likely to benefit from anti–PD-1/PD-L1 treatment and to monitor PD-L1 expression during therapy. The monoclonal antibody PD-L1.3.1 was radiolabeled with Indium-111 (111In) and characterized using PD-L1–expressing MDA-MB-231 cells. Subsequently, the optimal antibody dose and time point for imaging was determined in mice with MDA-MB-231 xenografts. Finally, SPECT/CT imaging was performed in xenograft models with different PD-L1 expression levels and tumor sections were analyzed for PD-L1 expression using IHC. The optimal antibody dose of 111In-PD-L1.3.1 (Kd = 1 nmol/L) for SPECT/CT imaging was ≤1 μg. Highest tumor-to-normal tissue contrast was obtained at days 3 and 7 after injection. 111In-PD-L1.3.1 SPECT/CT showed efficient accumulation in high PD-L1–expressing tumors (MDA-MB-231 and SK-Br-3), whereas no specific uptake was observed in tumors with low or no detectable levels of PD-L1 (SUM149, BT474, and MCF-7). SPECT/CT and autoradiography showed a very heterogeneous distribution of 111In-PD-L1.3.1 within the tumor. In conclusion, this is the first study showing the feasibility of noninvasive in vivo imaging of PD-L1 expression in tumors. 111In-PD-L1.3.1 showed efficient and specific uptake in PD-L1 expressing xenografts. This technique may enable patient selection for PD-1 and PD-L1–targeted therapy. Cancer Res; 75(14); 2928–36. ©2015 AACR.

Tumor relapses remain a serious problem after allogeneic stem cell transplantation (alloSCT), despite the long-term persistence of minor histocompatibility antigen (MiHA)-specific memory CD8⁺ T cells specific for the tumor. We hypothesized that these memory T cells may lose their function over time in transplanted patients. Here, we offer functional and mechanistic support for this hypothesis, based on immune inhibition by programmed death-1 (PD-1) expressed on MiHA-specific CD8⁺ T cells and the associated role of the PD-1 ligand PD-L1 on myeloid leukemia cells, especially under inflammatory conditions. PD-L1 was highly upregulated on immature human leukemic progenitor cells, whereas costimulatory molecules such as CD80 and CD86 were not expressed. Thus, immature leukemic progenitor cells seemed to evade the immune system by inhibiting T-cell function via the PD-1/PD-L1 pathway. Blocking PD-1 signaling using human antibodies led to elevated proliferation and IFN-γ production of MiHA-specific T cells cocultured with PD-L1–expressing leukemia cells. Moreover, patients with relapsed leukemia after initial MiHA-specific T-cell responses displayed high PD-L1 expression on CD34⁺ leukemia cells and increased PD-1 levels on MiHA-specific CD8⁺ T cells. Importantly, blocking PD-1/PD-L1 interactions augment proliferation of MiHA-specific CD8⁺ memory T cells from relapsed patients. Taken together, our findings indicate that the PD-1/PD-L pathway can be hijacked as an immune escape mechanism in hematological malignancies. Furthermore, they suggest that blocking the PD-1 immune checkpoint offers an appealing immunotherapeutic strategy following alloSCT in patients with recurrent or relapsed disease. Cancer Res; 71(15); 5111–22. ©2011 AACR.

Tumor relapse after human leukocyte antigen–matched allogeneic stem cell transplantation (SCT) remains a serious problem, despite the long-term presence of minor histocompatibility antigen (MiHA)–specific memory T cells. Dendritic cell (DC)–based vaccination boosting MiHA-specific T-cell immunity is an appealing strategy to prevent or counteract tumor recurrence, but improvement is necessary to increase the clinical benefit. Here, we investigated whether knockdown of programmed death ligand 1 (PD-L1) and PD-L2 on monocyte-derived DCs results in improved T-cell activation. Electroporation of single siRNA sequences into immature DCs resulted in efficient, specific, and long-lasting knockdown of PD-L1 and PD-L2 expression. PD-L knockdown DCs strongly augmented interferon-γ and interleukin-2 production by stimulated T cells in an allogeneic mixed lymphocyte reaction, whereas no effect was observed on T-cell proliferation. Moreover, we demonstrated that PD-L gene silencing, especially combined PD-L1 and PD-L2 knockdown, resulted in improved proliferation and cytokine production of keyhole limpet hemocyanin–specific CD4⁺ T cells. Most importantly, PD-L knockdown DCs showed superior potential to expand MiHA-specific CD8⁺ effector and memory T cells from leukemia patients early after donor lymphocyte infusion and later during relapse. These data demonstrate that PD-L siRNA electroporated DCs are highly effective in enhancing T-cell proliferation and cytokine production, and are therefore attractive cells for improving the efficacy of DC vaccines in cancer patients.