Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopenia

Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopenia Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic changes within MDSCs elucidate a direct influence on immunologic consequences of their suppressive activity. Liver kinase B1 (LKB1) is a tumor suppressor gene of STK11/LKB1 coding serine/Sue, and LKB1 signaling pathway plays an important role as a "bridge" between metabolic balance and functional homeostasis of immune cells. Our previous studies demonstrated that low dose decitabine, a hypomethylating agent, significantly increased the number of mature polyploidy megakaryocytes and exhibited long-term clinical efficacy. Besides, it also increased the production of Treg and enhanced their immunosuppressive function in ITP. However, whether decitabine could regulate the metabolic and suppressive activity of MDSCs in ITP is unknown. The percentage of MDSCs in peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry, which was shown to be significantly lower in ITP compared with that in healthy controls. We then investigated the effect of low-dose decitabine in patients with active ITP, where decitabine induced a significant expansion of MDSCs in line with an impressive platelet response. In the in vitro experiments, MDSCs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of decitabine (0/10nM/50nM/100 nM/1uM/10μM) for 7 days. A concentration gradient from 50nM to 1uM stimulated MDSCs amplification in a dose-dependent manner, and we chose an optimal concentration of 100 nM. Moreover, we found the mRNA expression level of LKB1, AMPKα1, AMPKα2, AMPKβ1, AMPKβ2, AMPKγ1, and AMPKγ2 was significantly lower in ITP patients than that in healthy control subjects. After incubation with decitabine (100nM), the relative expression of the above molecules were significantly increased compared to untreated levels. We also analyzed oxygen consumption rate (OCR) and key parameters of mitochondrial function within MDSCs. Overall, the OCR curve of ITP patients was lower than that of the healthy control subjects, and the OCR curve of ITP patients significantly improved after treatment with decitabine. We sorted the cultured MDSCs and co-cultured them with CFSE-labeled CD4 +CD25 - T cells to evaluate the suppressive activity of MDSCs. Results indicated that the inhibitory function of decitabine-modulated MDSCs was corrected in line with metabolic rewriting. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. MDSCs were sorted from the bone marrow of wild-type mice and incubated with PBS or decitabine, respectively. SCID mice were divided into three groups and received the same numbers of splenocyte transfer, two groups were given additional transfer of PBS-treated or decitabine-treated MDSCs. Our data showed that the decitaine-treated MDSCs group had significantly higher platelet counts compared with control group and PBS-treated MDSCs group. In summary, our findings suggest that the immune function and metabolic characteristics of MDSCs in ITP patients are impaired. These data shed new light on the molecular mechanism of decitabine action by regulating immune function and aerobic metabolism via LKB1, which supervises the immunosuppressive functions of MDSCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

794 Long-term anti-tumor preclinical efficacy of an optimized anti PD-1/IL-7 bifunctional antibody sustaining activation of progenitor stem-like CD8 TILs and disarming Treg suppressive activity

BackgroundDespite the PD-(L)1 therapy success, a majority of patients remain resistant. PD-1+IL7R+ progenitors CD8 TILs is a key T-cell subset associated with durable PD-(L)1 therapy response. However, this subset may rapidly undergo apoptosis and/or being fully exhausted after PD-(L)1 blockade. Some cytokines have the potential to strengthen PD-(L)1 therapy by promoting T cell survival, however, their clinical developments are limited by a shortened half-life and systemic toxicity. To redirect immunotherapy to tumor-specific T cells, expressing PD1, we propose to selectively deliver the pro-survival IL-7 to PD-1+ T cells using a bifunctional anti-PD1/IL-7 mutein antibody. We previously described that the anti-PD1/IL-7v abrogated suppressive activity of human Treg. Here we evaluated its preclinical anti-tumor efficacy and how it promotes the response of PD1+IL7R+ tumor-specific T cells.MethodsProliferation, IFN-γ, IL-7R signaling, and NFAT assays were tested to determine the mechanism of this antibody. For the suppressive assay, CD4 Treg and autologous CD8 Teff were co-cultured. In vivo experiments were performed in hPD-1 KI immunocompetent or humanized immunodeficient mice.ResultsThe anti-PD1/IL-7v antibody design has been optimized with a monovalent approach to enhance its biological activity: (1) preserved PD-1 antagonist activity, (2) improved pSTAT5 IL7R signaling, and (3) enhanced in vivo drug exposure and antitumor efficacy. An IL7 mutein has been designed to improve activity on PD1+ T cells while sparing PD1neg T cells.Using a chronic antigen stimulation model, anti-PD1/IL-7v restores the proliferation & survival of both early and fully exhausted CD8+ or CD4+ T cells. Similarly, anti-PD1/IL-7v, but not anti-PD1 alone, reactivates exhausted TILs isolated from human resected tumors. Gene expression analysis by Nanostring showed increase cytotoxicity, antigen presentation, and chemokines signatures. In vivo, anti-PD1/IL-7v demonstrated high monotherapy efficacy (90%) in a PD-1 sensitive orthotopic immunocompetent mouse tumor model as well as in a PD-1 refractory tumor model with 70% of CR vs 15% for anti-PD-1 alone. A selective higher expansion of stem-like/progenitors CD8 TILs was observed after therapy with anti-PD1/IL-7v compared to anti-PD1. Memory immune response was demonstrated in 100% of cured mice after tumor rechallenge in the absence of new treatment in 3 different tumor models. Finally, using two different humanized mouse models implanted with human tumors (A549 or MDA-MB231), we confirmed significant preclinical monotherapy efficacy of the anti-PD-1/IL7v.ConclusionsThese data highlight the potential of anti-PD1/IL-7 bifunctional drug to overcome immunotherapy resistance and to promote durable anti-tumor efficacy by preferentially reinvigorating PD-1+IL7R+ stem-like progenitors CD8 T cells.

Two Philippine Photorhabdus luminescens strains inhibit the in vitro growth of Lasiodiplodia theobromae, Fusarium oxysporum f. sp. lycopersici, and Colletotrichum spp.

Background Fungal phytopathogens are one of the leading causes of loss in global food production. Chemical fungicides have always been used to control the phytopathogens to mitigate losses. However, it is widely known that this approach is not sustainable. Thus, it is essential to develop alternative control methods, such as the use of biological control agents. Results This study provided a preliminary data on the efficacy of 2 local Photorhabdus strains, associated with Heterorhabditis indica BSDS and H. indica MAP, against selected post-harvest fungal phytopathogens, Fusarium oxysporum f. sp. lycopersici, Lasiodiplodia theobromae, Colletotrichum musae, and another Colletotrichum sp., by measuring their in vitro inhibitory activity. The Photorhabdus strains were isolated from the hemolymph of Ostrinia furnicalis infected with H. indica BSDS and H. indica MAP and grown selectively on NBTA media. Firstly, the bacterial endosymbionts' generic identity was confirmed through colony PCR based on a Photorhabdus Txp40 toxin-specific marker. Species identity was then elucidated through 16s marker-assisted GenBank mining as P. luminescens, sharing 99.51–99.58% similarity with P. luminescens subsp. akhurstii (Accession no. AY278643.1). Anti-fungal activity was observed by the bioassay experiments, using cell-free culture filtrates (CFCs), obtained from P. luminescens tryptic soy broth suspensions (OD600 = 2.0) amended in PDA medium (25%v/v) based on percentage growth inhibition. The CFCs of P. luminescens BSDS showed a significantly higher suppressive activity against Colletotrichum musae, Colletotrichum sp., and Lasiodiplodia theobromae, with 93.18 ± 0.46%, 74.15 ± 0.54%, and 60.51 ± 2.04% growth inhibition, respectively, while the CFC of P. luminescens MAP showed a significantly higher suppressive activity against F. oxysporum f. sp. lycopersici with 21.87 ± 0.71% growth inhibition. Conclusions The results strongly showed that these strains of Photorhabdus can be promising biological control agents against these fungal phytopathogens. Further extensive research is warranted for the development of these promising biofungicides into a practical, economically viable, and environment-friendly control strategy that can be incorporated into any integrated pest management program.

Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field

CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.

Antibodies Enhance the Suppressive Activity of Extracellular Vesicles in Mouse Delayed-Type Hypersensitivity

Previously, we showed that mouse delayed-type hypersensitivity (DTH) can be antigen-specifically downregulated by suppressor T cell-derived miRNA-150 carried by extracellular vesicles (EVs) that target antigen-presenting macrophages. However, the exact mechanism of the suppressive action of miRNA-150-targeted macrophages on effector T cells remained unclear, and our current studies aimed to investigate it. By employing the DTH mouse model, we showed that effector T cells were inhibited by macrophage-released EVs in a miRNA-150-dependent manner. This effect was enhanced by the pre-incubation of EVs with antigen-specific antibodies. Their specific binding to MHC class II-expressing EVs was proved in flow cytometry and ELISA-based experiments. Furthermore, by the use of nanoparticle tracking analysis and transmission electron microscopy, we found that the incubation of macrophage-released EVs with antigen-specific antibodies resulted in EVs’ aggregation, which significantly enhanced their suppressive activity in vivo. Nowadays, it is increasingly evident that EVs play an exceptional role in intercellular communication and selective cargo transfer, and thus are considered promising candidates for therapeutic usage. However, EVs appear to be less effective than their parental cells. In this context, our current studies provide evidence that antigen-specific antibodies can be easily used for increasing EVs’ biological activity, which has great therapeutic potential.