233 Human PD-L2 triggers a unique T cell inhibitory program through PD-1 engagement distinct from that of PD-L1

BackgroundPD-1/PD-L1 blockade is responsible for the majority of the success of cancer immunotherapy.1 However, only 14% of patients eligible to receive checkpoint blockade achieve objected clinical responses.2 3 The reason for the failure of PD-L1 blockade may be attributed to the recently appreciated widespread expression of PD-L2 across human cancers and its immunosuppresive stromal cells.4 PD-L2 expression was shown to be as or more predictive of response to PD-1 blockade than PD-L14. PD-L2 traditionally was dismissed as functionally redundant to PD-L1 varying only in pattern of expression. We hypothesize that PD-L2 engages PD-1 to generate a distinct inhibitory signal from that of PD-L1, and antibody mediated blockade and depletion of PD-L2+ cells may promote anti-tumor immunity that is superior to PD-L1 blockade alone.MethodsCell based bioluminescent assay demonstrated the nature of regulation mediated by human PD-L2 through the PD-1 co-receptor. RNA-sequencing identified key differences in the signaling pathways generated in Jurkat T cells by PD-1 binding to PD-L1 or PD-L2. Multidimensional flow cytometry determined the differential effects of PD-L1 and PD-L2 on human T cell proliferation and effector function. Western blot elucidated the temporal kinetics of inhibition mediated by PD-L1 and PD-L2. Survival studies in murine syngeneic lymphoma model evaluated the efficacy of antibody mediated blockade and depletion of PD-L2+ cells.ResultsWe validated that human PD-L2, unlike murine PD-L2, generates a purely co-inhibitory signal in human T cells, albeit with a reduced inhibitory potential relative to PD-L1. We discovered significant differences in downstream T cell signaling pathways generated by PD-L1 versus PD-L2 through PD-1 engagement. Human PD-L1 and PD-L2 differentially modulated T cell effector function and proliferation with PD-L2 preferentially arresting T cells in S-phase of cell cycle. PD-L1 and PD-L2 also differed in the temporal kinetics of dephosphorylation of the membrane proximal proteins in the TCR-CD3 signaling complex. We observed that combination blockade of PD-L1 and PD-L2 improves on blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our data in a syngeneic murine model of EL4 showed that effector-function capable PD-L2 blocking antibodies are therapeutically superior to PD-L1 or PD-L2 blockade alone.ConclusionsWe are the first to report on T cell immunoregulatory functions of PD-L2 which are distinct from those of PD-L1, and demonstrate that the more tumor-selective expression pattern of PD-L2 relative to PD-L1 provides a therapeutic advantage to effector-function capable PD-L2 antibodies.AcknowledgementsAS was supported by the CPRIT Research Training Grant(RP170067)ReferencesRibas A, Wolchok JD (2018). Cancer immunotherapy using checkpoint blockade. Science 359:1350–1355.Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Tomassini JE, Loboda A, Kaufman D (2018). Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science 362.Haslam A, Prasad V (2019). Estimation of the percentage of US patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs. JAMA Netw Open 2:e192535.Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T (2017). PD-L2 expression in human tumors: relevance to anti-PD-1 therapy in cancer. Clin Cancer Res 23:3158–3167.

Kaempferol Alleviates Oxidative Stress and Apoptosis Through Mitochondria-dependent Pathway During Lung Ischemia-Reperfusion Injury

In previous study, we reported that kaempferol ameliorates significantly lung ischemia-reperfusion injury (LIRI), and may be achieved by targeting the SIRT 1 pathway. This study further explored the anti-LIRI mechanism of kaempferol. In vitro, the rat alveolar epithelial cells L2 was cultured and subjected to anoxia/reoxygenation (A/R) insult. In vivo, SD rats were operated to establish LIRI model. The related indicators of oxidative stress and apoptosis in L2 cells and rats lung tissues were detected. Results showed that kaempferol pre-treatment significantly increased the cell viability, improved mitochondrial membrane potential, inhibited the opening of mitochondrial permeability transition pores, reduced the levels of oxidative stress and apoptosis, increased the expressions of Bcl-2 and mitochondrial cytochrome c, and decreased the expressions of Bax and cytoplasmic cytochrome c in L2 cells after A/R insult. In vivo, kaempferol improved the pathological injury, inhibited the levels of oxidative stress and apoptosis, increased the expressions of Bcl-2 and mitochondrial cytochrome c, and decreased the expressions of Bax and cytoplasmic cytochrome c in rats lung tissues after I/R. However, the aforementioned effects of kaempferol were significantly attenuated by the SIRT 1 inhibitor EX527 or the PGC-1α inhibitor SR-18292. What’s more, SR-18292 has not reversed the effect of kaempferol on increasing the protein activity of SIRT 1. Above results suggest that kaempferol ameliorates LIRI by improving mitochondrial function, reducing oxidative stress and inhibiting cell apoptosis. Its molecular mechanism of action includes the SIRT 1/PGC-1α/mitochondria signaling pathway.

Inhibition of the Alpha/Beta Interferon Response by Mouse Hepatitis Virus at Multiple Levels

ABSTRACT Mouse hepatitis virus (MHV) was used as a model to study the interaction of coronaviruses with the alpha/beta interferon (IFN-α/β) response. While MHV strain A59 appeared to induce IFN-β gene transcription and low levels of nuclear translocation of the IFN-β transcription factor interferon regulatory factor 3 (IRF-3), MHV did not induce IFN-β protein production during the course of infection in L2 mouse fibroblast cells. In addition, MHV was able to significantly decrease the level of IFN-β protein induced by both Newcastle disease virus (NDV) and Sendai virus infections, without targeting it for proteasomal degradation and without altering the nuclear translocation of IRF-3 or IFN-β mRNA production or stability. These results indicate that MHV infection causes an inhibition of IFN-β production at a posttranscriptional level, without altering RNA or protein stability. In contrast, MHV induced IFN-β mRNA and protein production in the brains of infected animals, suggesting that the inhibitory mechanisms observed in vitro are not enough to prevent IFN-α/β production in vivo. Furthermore, MHV replication is highly resistant to IFN-α/β action, as indicated by unimpaired MHV replication in L2 cells pretreated with IFN-β. However, when L2 cells were coinfected with MHV and NDV in the presence of IFN-β, NDV, but not MHV, replication was inhibited. Thus, rather than disarming the antiviral activity induced by IFN-β pretreatment completely, MHV may be inherently resistant to some aspects of the antiviral state induced by IFN-β. These findings show that MHV employs unique strategies to circumvent the IFN-α/β response at multiple steps.

Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL. Figure Figure