Inhibition Of Dendritic Cells Maturation Research Articles

Human Dendritic Cell Maturation Is Modulated by Leishmania mexicana through Akt Signaling Pathway.

Dendritic cells (DC) along with macrophages are the main host cells of the intracellular parasite Leishmania. DC traverse a process of maturation, passing through an immature state with phagocytic ability to a mature one where they can modulate the immune response through the secretion of cytokines. Several studies have demonstrated that Leishmania inhibits DC maturation. Nevertheless, when cells are subjected to a second stimulus such as LPS/IFN-γ, they manage to mature. In the maturation process of DC, several signaling pathways have been implicated, importantly MAPK. On the other hand, Akt is a signaling pathway deeply involved in cell survival. Some Leishmania species have shown to activate MAPK and Akt in different cells. The aim of this work was to investigate the role of ERK and Akt in the maturation of monocyte-derived DC (moDC) infected with L. mexicana. moDC were infected with L. mexicana metacyclic promastigotes, and the phosphorylation of ERK and Akt, the expression of MHCII and CD86 and IL-12 transcript, and secretion were determined in the presence or absence of an Akt inhibitor. We showed that L. mexicana induces a sustained Akt and ERK phosphorylation, while the Akt inhibitor inhibits it. Moreover, the infection of moDC downregulates CD86 expression but not MHCII, and the Akt inhibitor reestablishes CD86 expression and 12p40 production. Thus, L. mexicana can modulate DC maturation though Akt signaling.

Oxymatrine combined with rapamycin to attenuate acute cardiac allograft rejection

Background and aimSolid organ transplantation remains a life-saving therapeutic option for patients with end-stage organ dysfunction. Acute cellular rejection (ACR), dominated by dendritic cells (DCs) and CD4+ T cells, is a major cause of post-transplant mortality. Inhibiting DC maturation and directing the differentiation of CD4+ T cells toward immunosuppression are keys to inhibiting ACR. We propose that oxymatrine (OMT), a quinolizidine alkaloid, either alone or in combination with rapamycin (RAPA), attenuates ACR by inhibiting the mTOR–HIF–1α pathway. MethodsGraft damage was assessed using haematoxylin and eosin staining. Intragraft CD11c+ and CD4+ cell infiltrations were detected using immunohistochemical staining. The proportions of mature DCs, T helper (Th) 1, Th17, and Treg cells in the spleen; donor-specific antibody (DSA) secretion in the serum; mTOR–HIF–1α expression in the grafts; and CD4+ cells and bone marrow-derived DCs (BMDCs) were evaluated using flow cytometry. ResultsOMT, either alone or in combination with RAPA, significantly alleviated pathological damage; decreased CD4+ and CD11c+ cell infiltration in cardiac allografts; reduced the proportion of mature DCs, Th1 and Th17 cells; increased the proportion of Tregs in recipient spleens; downregulated DSA production; and inhibited mTOR and HIF-1α expression in the grafts. OMT suppresses mTOR and HIF-1α expression in BMDCs and CD4+ T cells in vitro. ConclusionsOur study suggests that OMT-based therapy can significantly attenuate acute cardiac allograft rejection by inhibiting DC maturation and CD4+ T cell responses. This process may be related to the inhibition of the mTOR–HIF–1α signaling pathway by OMT.

In vitro Production of Dendritic Cells as Cancer Immunotherapy: Highlights on Sample Source, Culture Period, Differentiation and Maturation Cytokines

Dendritic cells (DC) are antigen-presenting cells between innate and adaptive immune cells and commonly used as immunotherapy. Despite this promising potential, protocols detailing the specifics of the DC production are varied, affecting the potency of dendritic as immunotherapy. There are various factors affecting the production and DC potency, such as sample source, culture period, differentiation and maturation cytokines. Due to the limited number and quality of DC in humans, the monocyte could be isolated and differentiated to mature DC. The purity and viability monocytes shall be maintained to produce a high yield of DC. Negative sorting maintains the potency of DC as a therapeutic agent. Monocytes from umbilical cord blood (UCB) are naïve and can be differentiated to DC easily. Meanwhile, the tumor microenvironment (TME) may inhibit DC maturation from monocyte-derived peripheral blood. Without pro-inflammatory cytokines and a short maturation period, DC remain immature and fails to activate T cells. Long-period culture correlates with decreased DC viability and function. This review outlines several factors which can produce higher cytotoxic T cells and pro-inflammatory cytokines that might help each facility in developing its protocol to ensure the best procedure in DC production. Increasing purity and yield through close and automatic system under GMP production are mandatory to decrease risk of contamination during DC production.Keywords: differentiation cytokines, maturation cytokines, culture period, sample source, isolation technique

Long-term hypoxic hUCMSCs-derived extracellular vesicles alleviates allergic rhinitis through triggering immunotolerance of their VEGF-mediated inhibition of dendritic cells maturation

BackgroundExtensions of mesenchymal stem cells (MSCs) in vitro may lead to the loss of their biological functions. However, hypoxic culturation has been shown to enhance the proliferation, survival, and immunomodulatory capacity of MSCs. ObjectiveWe aimed to investigate the effects of long-term hypoxic cultivation on the properties of human umbilical cord-derived MSCs (hUCMSCs) and the therapeutic effects of their extracellular vesicles (EVs) in allergic rhinitis (AR). MethodsProliferation, senescence, telomerase activity and multipotent properties of hUCMSCs were analyzed under long-term culturation of hypoxia (1%) or normoxia (21%), and the therapeutic effects of their conditional medium (CM) and EVs were evaluated in OVA-induced AR mice. Effects of hypoxia-EVs (Hy-EVs) or normoxia-EVs (No-EVs) on human monocyte-derived dendritic cells (DCs) were investigated, and the possible mechanisms of Hy-EVs in induction of immunotolerance were further explored. ResultsLong-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs. Hy-CM and Hy-EVs showed better therapeutic effects in AR mice compared to No-EVs, seen as improvement of AR-related behaviors such as rubbing and sneezing, and attenuation of inflammation in nasal tissues. In addition, Hy-EVs significantly reduced the expressions of HLA-DR, CD80, CD40, and CD83 induced by OVA plus LPS in DCs, inhibiting the maturation of DCs. Furthermore, we observed that VEGF was remarkably enriched in Hy-EVs, but not in No-EVs, and the inhibition of DCs maturation was markedly neutralized by VEGF antibodies, suggesting that VEGF derived from Hy-EVs was responsible for the inhibition of DCs maturation. ConclusionOur results demonstrated that long-term hypoxia significantly promoted the proliferation, inhibited cell senescence, maintained the multipotent status of hUCMSCs, and hypoxia treated hUCMSCs-derived EVs enhanced their therapeutic effects in AR mice through VEGF-mediated inhibition of DCs maturation.

TIPE2 deficiency prolongs mouse heart allograft survival by facilitating immature DCs-induced Treg generation

It has been reported that deletion of tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2, TIPE2) facilitates the activation of T-cell receptors. However, the role of TIPE2 in T-cell-mediated acute transplant rejection remains unclear. To illustrate the underlying cellular mechanisms, we transplanted BALB/c hearts into C57BL/6 wild-type (WT) or C57BL/6 mice deficient for TIPE2 (TIPE2−/−) and found that TIPE2−/− recipient mice showed significantly prolonged survival of heart allografts and suppressed maturation of CD11c+ dendritic cells (DCs), which largely abolished the activation and proliferation of alloreactive T cells and their cytotoxic activity. TIPE2−/− DCs increased CD4+CD25+Foxp3+CD127− regulatory T cells (Tregs)generation, likely by inhibiting DCs maturation and CD80 and CD86 expression. Administration of anti-CD25 abolished the allograft survival induced by TIPE2 deficiency. Moreover, TIPE2 deficiency increased IL-10 production in T cells and in recipient serum and allografts. Mechanistic studies revealed that TIPE2−/− restrained the maturation of DCs via inhibition of PI3K/AKT phosphorylation during alloantigen stimulation. Taken together, TIPE2 deficiency in recipient mice inhibited acute rejection by increasing Tregs generated by immature DCs. Thus, TIPE2 could be a therapeutic target for suppressing rejection in organ transplantation.

Long Non Coding RNA FOXD3‑AS1 Alleviates Allergic Rhinitis by Elevating the Th1/Th2 Ratio via the Regulation of Dendritic Cells

ABSTRACT This article aimed to explore whether the regulation of Th1/Th2 immune responses by FOXD3-AS1 is associated with dendritic cells (DCs) in allergic rhinitis (AR). HE staining was performed to assess the pathological changes in the nasal mucosa; ELISA was performed to measure the levels of Th1/Th2-related cytokines; flow cytometry was performed to analyze Th1/Th2 cells and MHC-II-, CD80-, and CD86-positive DCs; and qRT‒PCR and western blotting were performed to measure mRNA and protein expression levels, respectively. Our data revealed that LV-FOXD3-AS1 improved AR and increased the Th1/Th2 cell ratio in AR model mice. LV-FOXD3-AS1 further inhibited DC maturation both in vivo and in vitro. Moreover, the coculture system of DCs and CD4+ T cells demonstrated that LV-FOXD3-AS1 increased the Th1/Th2 cell ratio by inhibiting the maturation of DCs. In addition, LV-FOXD3-AS1 reduced the level of phosphorylated STAT6 in DCs derived from healthy mice, and STAT6 overexpression eliminated the inhibitory effect of LV-FOXD3-AS1 on the maturation of DCs. In summary, LV-FOXD3-AS1 ameliorated AR by increasing the Th1/Th2 cell ratio by inhibiting DC maturation via the inhibition of STAT6 phosphorylation. Our data confirmed the protective effect of FOXD3-AS1 in AR and provided a novel idea for the treatment of this disease.

Abstract 4159: The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy

Abstract Background: We previously found upregulation of the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway in the tumor microenvironment (TME) of cancer types that respond poorly to anti-PD-1 therapy, and showed that TME-resident cytokines such as IL-1B induce COX-2 expression in human tumor and myeloid cells. To investigate a potential role for COX-2/PGE2 in anti-PD-1 resistance, we studied PGE2-mediated effects on human myeloid and T cells and their dependence on prostaglandin (EP) receptor signaling. Methods: Monocytes (Monos) and T cells were enriched from normal donor peripheral blood by negative selection. PGE2 effects were tested on Monos activated with IFN-g (PD-L1 expression; FACS) or LPS (cytokine secretion; ELISA). PGE2 effects on monocytic dendritic cells (DCs) generated with GM-CSF+IL-4 and matured with CD40L were assessed by phenotyping for costimulatory molecules (CD80, CD86) and subpopulation markers (CD14, CD16, CD83). T cells activated with anti-CD3/CD28 +/- PGE2 for 2-6 days were assessed for expression of CD25, CD69, CD107a, CD137, OX40, GITR, LAG-3, PD-1, PD-1, PD-L1 and TIM3 (FACS); proliferation markers (Ki67, CFSE; FACS); cytokine secretion (IFN-g, IL-2, IL-10, TGF-b; ELISA); and metabolic function (Seahorse assay). In some experiments, cells were pre-incubated with inhibitors of EP2 (PF-04418948), EP4 (ONO-AE3-208), or the dual inhibitor TPST-1495 (Tempest Therapeutics). Results: Myeloid cell functions were suppressed by PGE2, which reduced IFNg-induced PD-L1 expression and modulated LPS-induced cytokine secretion. Furthermore, PGE2 inhibited DC maturation, evidenced by persistent CD14 and CD16 expression and a decreased CD83+ subset. In CD4 and CD8 T cells activated with anti-CD3/CD28, PGE2 reduced expression of CD25 and CD69 (early activation markers), CD107a (cytolytic potential), Ki67 (cell proliferation), co-stimulatory molecules (CD137, OX40, GITR) and checkpoint molecules (LAG-3, PD-1, PD-L1, TIM3) in a dose-dependent manner. PGE2 also reduced the CFSE-low (proliferating) population in activated CD4, CD8 and Treg cells. Secretion of proinflammatory cytokines IFN-g and IL-2, and inhibitory cytokines IL-10 and TGF-b from activated T cells was also suppressed by PGE2. These effects were specifically reversed by chemical inhibitors of EP2 and/or EP4. PGE2 exposure at the time of T cell activation was associated with depressed mitochondrial oxidation and cellular glycolysis, suggesting global effects on T cell metabolism. Conclusion: Understanding and preventing anti-PD-1 resistance is a critical goal in oncology. Our results suggest that the COX-2/PGE2 pathway exerts immunosuppressive effects on myeloid and T cells via the EP2 and EP4 receptors. COX-2 and PD-L1 are distinct immune checkpoints, providing a rationale to test anti-PD-1 in conjunction with COX-2 pathway inhibitors such as IL-1B antagonists and highly specific EP2 and EP4 inhibitors in patients with cancer. Citation Format: Shuming Chen, Seoho Lee, Tracee L. McMiller, Isaac Morales, Preethi Sankaran, Brian Francica, Robert D. Leone, Tom W. Dubensky, Suzanne L. Topalian. The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4159.

Majoon chobchini reinstates PDL-1 expression and blocks dendritic cell -T helper 17 pathogenic axis in rheumatoid arthritis animal model.

Dendritic cells (DCs) are the critical players in the puzzle of rheumatoid arthritis (RA) disease pathogenesis. Blockade of DC activation has been shown to curtail Th17 cell differentiation and its aberrant function in RA. Recent studies have pointed to the role of the PI3K/AKT signaling axis in the maturation and activation of DCs. However, it is yet to be established how PI3K/AKT inhibition would lead to the abolishment of DC activation and Th17 cell plasticity in RA. Herein, our study decoded whether and how majoon chobchini, an unani compound, abated dendritic cell maturation and regulated the Th17/Treg paradigm in RA. Given our results, majoon chobchini conspicuously restrained MHC II, CD86 expression and, subsequently elevated PDL-1 levels in DCs in-vivo. Of note, inhibition of DC maturation by majoon chobchini, in turn, favoured suppression of the Th17 cell population while driving Treg cell development in adjuvant induced arthritic (AA) rats. Concurrently, majoon chobchini decreased the catabolic effects of IL-17 (Th17 associated cytokine) via a reciprocal increase in IL-10 (Treg associated cytokine) levels in AA rats. Mechanistically, majoon chobchini sustained FoxO1 nuclear localization signaled through dampened PI3K/AKT phosphorylation in-vitro. In concert, PDL-1 expression was heightened in majoon chobchini treated activated DCs that provides a framework for ablation of the DC-Th17 cell pathogenic axis in RA. Notwithstanding, the PI3K inhibitor LY294002 exhibited similar inhibitory effects. In essence, majoon chobchini enhanced PDL-1 expression that abolished DC maturation via regulation of the PI3K/AKT/FoxO1 axis, thereby hindering Th17 differentiation in an animal model of RA. This further warrants a clinical investigation that could validate majoon chobchini as a prospective therapeutic drug in the treatment of RA.

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases.

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an 'eat-me' signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.

Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

PA-X protein of H1N1 subtype influenza virus disables the nasal mucosal dendritic cells for strengthening virulence

ABSTRACT PA-X protein arises from a ribosomal frameshift in the PA of influenza A virus (IAV). However, the immune regulatory effect of the PA-X protein of H1N1 viruses on the nasal mucosal system remains unclear. Here, a PA-X deficient H1N1 rPR8 viral strain (rPR8-△PAX) was generated and its pathogenicity was determined. The results showed that PA-X was a pro-virulence factor in mice. Furthermore, it reduced the ability of H1N1 viruses to infect dendritic cells (DCs), the regulator of the mucosal immune system, but not non-immune cells (DF-1 and Calu-3). Following intranasal infection of mice, CCL20, a chemokine that monitors the recruitment of submucosal DCs, was downregulated by PA-X, resulting in an inhibition of the recruitment of CD11b+ DCs to submucosa. It also attenuated the migration of CCR7+ DCs to cervical lymph nodes and inhibited DC maturation with low MHC II and CD40 expression. Moreover, PA-X suppressed the maturation of phenotypic markers (CD80, CD86, CD40, and MHC II) and the levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhancing endocytosis and levels of anti-inflammatory IL-10 in vitro, suggesting an impaired maturation of DCs that the key step for the activation of downstream immune responses. These findings suggested that the PA-X protein played a critical role in escaping the immune response of nasal mucosal DCs for increasing the virulence of H1N1 viruses.

Immunosuppressive effects of vascular endothelial growth factor (Review)

Vascular endothelial growth factor (VEGF) serves a critical role in vasculogenesis, angiogenesis, tumor, inflammatory angiogenesis and lymphangiogenesis. Since 2004, bevacizumab (Avastin), a humanized anti-VEGFA monoclonal antibody, has been approved for the treatment of non-small cell lung, breast, kidney and ovarian cancer in combination with standard chemotherapy. VEGF has been demonstrated to be important in the clinic as a therapeutic target in the anti-angiogenic approach to cancer therapy. The targeting of VEGF, together with immunotherapy, has been reported to be able to reverse the immunosuppressive effects of VEGF. A positive correlation between VEGF expression and the reduced survival rates of patients with cancer has also been demonstrated. Furthermore, increased VEGF expression can lead to immune suppression via the inhibition of dendritic cell maturation, the reduction of T-cell tumor infiltration and the promotion of inhibitory cell types in the tumor microenvironment.

Effect of Xijiao Dihuang Combined Prescription on Human Dendritic Cell Function Induced by Lipopolysaccharide

To observe the effects of drug-containing serum of Xijiao Dihuang combined prescription(XJDH) on the related functions of dendritic cells(DCs) induced in vitro, and to explore the mechanisms underlying the effectiveness of XJDH treatment on primary immune thrombocytopenia(ITP). Peripheral blood samples were colle-ted from 6 healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation, and CD14+ mononuclear cells were collected by the magnetic separation technique. CD14+ mononuclear cells were induced into immature DCs by recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin 4 (IL-4). Immature DCs were divided into three groups: control group, model group and XJDH group. CCK-8 assay was used to determine the intervention concentration and time of drug-containing serum. Lipopolysaccharide(LPS) with the final concentration of 1 μg/ml was added to model group and XJDH group respectively for 24 h to induce DCs maturation. Normal rat serum was added to control group and model group, and XJDH was added to XJDH group for 24 h. Flow cytometry was used to detect the levels of CD80, CD83 and HLA-DR on the surface of DCs. Western blot was used to detect the expression of TLR4 and NF-κB, and levels of IL-6, IL-12 and TNF-α in cell supernatant was detected by ELISA. Compared with the control group, LPS stimulation increased the expression of CD80, CD83 and HLA-DR, with subsequent increasing expression of TLR4 and NF-κB, as well as IL-6, IL-12 and TNF-α increased(P<0.05). In comparison with model group, the expression of DCs surface molecules CD80, CD83 and HLA-DR, DCs' expression of TLR4 and NF-κB protein, and the levels of IL-6, IL-12 and TNF-α in the cell supernatant of XJDH group decreased after the intervention of XJDH (P<0.05). Drug containing serum of Xijiao Dihuang combined prescription can down-regulate TLR4/NF-κB signaling pathway related protein expression, inhibit DCs maturation, and reduce proinflammatory factor secretion, which may be one of the mechanisms of drug-containing serum of Xijiao Dihuang combined prescription in the treatment of immune thrombocytopenia.

Characterization of a small molecule modulator of inflammatory cytokine production

In the present study, the effect(s) of the immunomodulatory drug GLS-1027 on various cell types involved in inflammation were investigated. GLS-1027 reduced LPS-stimulated secretion of pro-inflammatory cytokines by macrophage or monocytic cells and cell lines. This reduction was likely due in part to decreased activation of NF-κB family transcription factors and inhibition of p38 MAPK signaling in GLS-1027-treated cells. Independent from its effects on macrophages, GLS-1027 inhibited dendritic cell maturation and differentiation of naïve CD4+ T cells into Th17 cells, reducing the production of typical pro-inflammatory cytokines associated with both processes. In vivo administration of GLS-1027 prevented the development of type 1 diabetes in NOD mice which correlated with reduced serum levels of IL17A in GLS-1027 treated animals and reduced ex vivo production of IL17A from both spleen and lymph-node cells. Overall, our data show that GLS-1027 can reduce inflammation through multiple actions, including the reduction of pro-inflammatory cytokine production by innate immune cells, the inhibition of dendritic cells maturation, and the inhibition of Th17 cells polarization.

Inhibition of miR-let-7i Induces DC Immature Cells and Improves Skin Graft Tolerance.

Dendritic cells (DC) initiate the immune response in the body. They can stimulate T cell activation, proliferation, and differentiation and ultimately participate in the immune response and the immune tolerance response. The purpose of this study was to coculture DCs and T cells and subcutaneously inject DCs transfected with miR-let-7i into rhesus monkey transplantations to verify the role of miR-let-7i in allograft immune tolerance. In vitro studies found that the expression of miR-let-7i was upregulated after inducing the maturation of DCs. The low expression of miR-let-7i inhibited the maturation of DCs, promoted the differentiation of T cells into T helper T cells 2 (Th2), and inhibited T helper T cell 1- (Th1-) driven rejection. In vivo studies also obtained similar results, and subcutaneous injection of DCs transfected with miR-let-7i inhibitor prolonged the survival time of allogeneic skin transplantation. Therefore, we conclude that inhibition of miR-let-7i inhibits DC maturation and improves the tolerance of grafted skin.

A pilot study of lenvatinib plus pembrolizumab in patients with advanced sarcoma.

TPS11588 Background: New treatment options are needed for sarcomas. Pazopanib is the only targeted agent approved for multiple soft tissue sarcoma (STS) subtypes with a response rate of 6% and a PFS of 4.6 months. Immunotherapy has a limited role in STS, as the SARC028 study of pembrolizumab demonstrated an overall response rate of 18%, with the highest response rate seen in the undifferentiated pleomorphic sarcoma (UPS) cohort at 23%. Lenvatinib is an oral, multi-tyrosine kinase inhibitor approved for the treatment of multiple cancer types including progressive, radioiodine-refractory thyroid cancer and unresectable hepatocellular carcinoma with inhibitory activity against the receptor tyrosine kinases VEGFR 1-3, FGFR 1-3, KIT, PDGFR alpha/beta, and RET. Early outcomes with the combination of lenvatinib and pembrolizumab suggest that this regimen could be broadly superior to PD-1 targeting alone for several tumor types as high rates of objective response have been noted. The rationale for this study is based on preclinical work demonstrating the immunosuppressive effects of VEGF in the tumor immune microenvironment including inhibition of dendritic cell maturation, recruitment of immunosuppressive Tregs, MDSCs and TAMs and up-regulation of PD-1 on CD8+ cells. Methods: This is a pilot study evaluating the efficacy of lenvatinib and pembrolizumab in the treatment of select metastatic and/or unresectable sarcomas. Patients will be enrolled in one of five cohorts: Cohort A: leiomyosarcoma; Cohort B: UPS; Cohort C: vascular sarcomas (including angiosarcoma and epithelioid hemangioendothelioma); Cohort D: synovial sarcoma and malignant peripheral nerve sheath tumor; and Cohort E: bone sarcomas (limited to osteosarcoma and chondrosarcoma). Eligible patients should have had at least one prior therapy for unresectable and/or metastatic disease, but no more than three prior lines of therapy. Prior treatment with angiogenesis inhibitors or immunotherapy is excluded. Archival tissue is required for eligibility. Patients enrolled in the study will be treated initially with a 2 week run-in of lenvatinib 20 mg orally daily which will be continued daily thereafter. Subsequently, they will start pembrolizumab 200 mg intravenously every 21 days. The primary endpoint for each cohort is best overall response rate documented by RECIST v1.1 Criteria at 27 weeks. A sample size of 10 patients is planned for each of the five histological cohorts. If 2 or more confirmed responses are observed among the 10 patients in an arm, the drug combination will be considered positive and worthy of further investigation for that arm. Secondary endpoints are PFS, OS, duration of response and safety/tolerability of the combination. On-treatment biopsy and blood samples will be required for correlative assessments. Accrual in all cohorts is ongoing. Clinical trial information: NCT04784247.

A phase II/III trial of chemotherapy plus cetuximab versus chemotherapy plus bevacizumab versus atezolizumab plus bevacizumab following progression on immune checkpoint inhibition in recurrent/metastatic head and neck cancers: ECOG-ACRIN EA3202.

TPS6098 Background: There is no standard treatment beyond progression on first-line pembrolizumab (P) in patients with R/M HNSCC. Vascular endothelial growth factor (VEGF) modulates anti-tumor immunity via proliferation of inhibitory T regulatory and myeloid-derived suppressor cells, inhibition of dendritic cell maturation and suppression of effector T cell responses. Efficacy data from other disease sites and from early-phase trials done in HNSCC suggests promising clinical activity of VEGF and PD-1 inhibitor combination therapy. We have thus designed a Phase II/III, randomized, multi-arm trial to evaluate the efficacy of atezolizumab (A) and bevacizumab (B) beyond progression on P in patients with R/M HNSCC. Methods: EA3202 is enrolling patients with CPS ≥1% R/M HNSCC previously treated with P either alone or in combination with other checkpoint inhibitors without progression for at least 12 weeks. In phase II, patients will be randomized to one of three treatment arms: platinum doublet chemotherapy (C) plus cetuximab (E) for six cycles followed by E maintenance (control arm), C + B for six cycles followed by B maintenance, or A + B. Therapy in each arm will continue until progression, toxicity, or for a total period of two years. Patients will be stratified by p16 status, CPS score (≥ 20 vs. &lt; 20), distant metastases (M0 vs M1), and disease progression pattern with first-line P (while on P vs. after discontinuation). 216 patients will be enrolled. The primary endpoint for phase II is progression-free survival (PFS). Each experimental arm will be compared to the control arm at a one-sided alpha level of 0.10. Pre-specified rules will be used to pick a winner among the two experimental arms, which will then advance to phase III against the control arm. 214 patients will be enrolled in phase III for a total sample size of 430 patients. The primary endpoint for phase III is overall survival (OS) which will be compared using a stratified log-rank test. Secondary endpoints include OS in the two phase III arms among the CPS ≥ 20 cohort and comparison of grade 3 or higher treatment-related adverse events between the two arms. A total of 277 OS events will be required for full information for phase III evaluation to give a nominal ̃90% power at one-sided alpha level 0.0125. 144 patients enrolled in phase II (from the control and selected experimental arms) will be included in the phase III analysis. With this design the overall study power is at least 80% under a true 40% hazard reduction for PFS and 35% hazard reduction for OS. Blood and tumor tissue will be banked. EA3202 was activated in December 2021. It is the first randomized study to compare systemic treatments for R/M HNSCC in the immunotherapy era and will define the best second-line treatment approach beyond P. Clinical trial information: NCT05063552.

Trichostatin A inhibits dendritic cell maturation through down-regulating NF-κ B (p65) pathway.

Recent evidence suggested that histone deacetylase inhibitor (HDACi) could inhibit dendritic cell (DC) maturation. However, the mechanism is unclear. Here, we aimed to study whether Trichostatin A (TSA), the most widely studied HDACi,inhibits the maturation of DCs by down-regulating NF-κB (p65) pathway. Mouse bone marrow-derived DCs were cultured. Lipopolysaccharide (LPS) was applied as stimulation for maturation. Triptolide (TTL) was applied as p65 inhibitor. Microphotography and flow cytometry showed that TSA and p65 inhibitor separately inhibited the maturation of DCs stimulated by LPS from the aspects of cell morphology and cell phenotype. Mixed lymphocyte reaction test and ELISA showed that TSA and p65 inhibitor synergistically inhibited the proliferation of T lymphocytes stimulated by DCs, reduced the secretion of pro-inflammatory cytokine IL-12 and elevated the secretion of anti-inflammatory cytokine IL-10. Western blot and RT-qPCR showed that TSA down-regulated the expression of phosphorylated IκBα, phosphorylated-p65, Ikkβ and Ikkγ, suggesting TSA down-regulates NF-κB (p65) pathway. TSA inhibits DC maturation through down-regulating NF-κB (p65) pathway.

Poly-γ-Glutamic Acid Encapsulation of Bacillus anthracis Inhibits Human Dendritic Cell Responses.

The capsule of Bacillus anthracis is composed of a d isomer poly-γ-glutamic acid polymer, which is especially nonstimulatory to dendritic cells, even more so than similar mixed d, l isomer polymers from nonpathogenic Bacillus species. Capsule is an essential virulence factor for B. anthracis, protecting the bacilli from phagocytosis by innate immune cells. In this study, we demonstrate that encapsulation provides a further pathogenic advantage by shielding more inflammatory Ags on the bacillus surface, thereby reducing dendritic cell responses. We exposed human immature dendritic cells (DCs) to increasing multiplicities of infection (MOIs) of killed B. anthracis bacilli from the fully encapsulated wild-type Ames strain (WT) and an isogenic capsule-deficient strain (capA mutant). Both strains elicited robust cytokine responses, but IL-23, TNF-α, and IL-10 were significantly reduced in response to the encapsulated WT compared with capA mutant up to an MOI of 15. capA mutant bacilli could induce phenotypic maturation of immature DCs with upregulation of MHC classes I and II, CD83, and CCR7 at an MOI of 3.75, whereas encapsulated WT bacilli still did not induce significant upregulation of MHC classes I and II at an MOI of 15. DCs exposed to capA mutant bacilli (MOI 3.75) exhibited CCR7-dependent chemotaxis that was comparable to that of LPS-stimulated controls, whereas DCs exposed to encapsulated WT bacilli exhibited significantly less chemotaxis. We conclude that capsule shields more inflammatory surface Ags, delaying development of an adaptive immune response by reducing TNF-α, thereby inhibiting DC maturation.

Indoleamine 2,3-dioxygenase 1 limits hepatic inflammatory cells recruitment and promotes bile duct ligation-induced liver fibrosis

Liver fibrosis is a course of chronic liver dysfunction, can develop into cirrhosis and hepatocellular carcinoma. Inflammatory insult owing to pathogenic factors plays a crucial role in the pathogenesis of liver fibrosis. Indoleamine 2,3-dioxygenase 1 (IDO1) can affect the infiltration of immune cells in many pathology processes of diseases, but its role in liver fibrosis has not been elucidated completely. Here, the markedly elevated protein IDO1 in livers was identified, and dendritic cells (DCs) immune-phenotypes were significantly altered after BDL challenge. A distinct hepatic population of CD11c+DCs was decreased and presented an immature immune-phenotype, reflected by lower expression levels of co-stimulatory molecules (CD40, MHCII). Frequencies of CD11c+CD80+, CD11c+CD86+, CD11c+MHCII+, and CD11c+CD40+ cells in splenic leukocytes were reduced significantly. Notably, IDO1 overexpression inhibited hepatic, splenic CD11c+DCs maturation, mature DCs-mediated T-cell proliferation and worsened liver fibrosis, whereas above pathological phenomena were reversed in IDO1−/− mice. Our data demonstrate that IDO1 affects the process of immune cells recruitment via inhibiting DCs maturation and subsequent T cells proliferation, resulting in the promotion of hepatic fibrosis. Thus, amelioration of immune responses in hepatic and splenic microenvironment by targeting IDO1 might be essential for the therapeutic effects on liver fibrosis.