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[Display omitted] • sPD1 targeted antigen to DCs. • Flt3L enhanced the cross-presentation function of sPD1 vaccines. • Flt3L can simultaneously utilize the PDL1-inhibitor function of sPD1. • In 4T1 model, cyclophosphamide reduced the Tregs increase caused by Flt3L. • In Panc02 model, aCD40 activated the DCs expanded by Flt3L. DNA vaccines are prospective for their efficient manufacturing process, but their immunogenicity is limited as they cannot efficiently induce CD8+ T cell responses. A promising approach is to induce cross-presentation by targeting antigens to DCs. Flt3L can expand the number of type 1 conventional DCs and thereby improve cross-presentation. In this study, we first constructed a DNA vaccine expressing soluble PD1 and found that the therapeutic effect of targeting DCs with only the sPD1 vaccine was limited. When combined the vaccine with Flt3L, the anti-tumor effect was significantly enhanced. Considering the complexity of tumors and that a single method may not be able to activate a large number of effective CD8+ T cells, we combined different drugs and the vaccine with Flt3L based on the characteristics of different tumors. In 4T1 model, we reduced Tregs through cyclophosphamide. In Panc02 model, we increased activated DCs by using aCD40. Both strategies triggered strong CD8+ T cell responses and significantly improved the therapeutic effect. Our study provides important support for the clinical exploration of DC-targeted DNA vaccines in combination with Flt3L. [ABSTRACT FROM AUTHOR]

• FLT3 + DC are decreased after liver transplantation (LT) in peripheral blood and liver tissue human and mouse. • After liver transplantation, proportion of FLT3 + DC changed dynamically in LT patients and mouse model. • There appears to be a restoration of the immune equilibrium as resulting from the feedback loop between Tregs and FLT3 + DCs in LT. Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (RTK) primarily expressed in hematopoietic stem cells and dendritic cells (DCs). While FLT3 plays a critical role in the proliferation, development and maintenance of DCs, thus influencing immune responses under both normal and pathological conditions, there also exists some evidence that FLT3+DC may be involved with immune responses in liver transplantation (LT). In this study, results from single-cell sequencing data analysis revealed a clear relationship between FLT3+DCs and Regulatory T cells (Tregs) in liver tissue of LT recipients. In peripheral blood samples of LT patients, levels of FLT3+DCs were decreased post-LT-surgery, while Tregs were increased. In a LT mouse model, levels of FLT3+DCs in the liver and bone marrow exhibited an initial time-dependent decrease followed by an increase after LT surgery. Results as obtained with co-culture experiments using mature BMDCs and CD4+ T cells revealed fluctuations in Tregs in response to FLT3 inhibitors and the FLT3 ligand. These findings suggest that FLT3+DCs could emerge as a novel target for mitigating immune rejection in LT. [ABSTRACT FROM AUTHOR]

Image, graphical abstract A higher number of donor plasmacytoid dendritic cells (pDCs) is associated with increased survival and reduced graft-versus-host disease (GVHD) in human recipients of unrelated donor bone marrow (BM) grafts, but not granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood grafts. We show that in murine models, donor BM pDCs are associated with increased survival and decreased GVHD compared with G-CSF-mobilized pDCs. To increase the content of pDCs in BM grafts, we studied the effect of FMS-like tyrosine kinase 3 ligand (Flt3L) treatment of murine BM donors on transplantation outcomes. Flt3L treatment (300 μg/kg/day) resulted in a schedule-dependent increase in the content of pDCs in the BM. Mice treated on days -4 and -1 had a >5-fold increase in pDC content without significant changes in numbers of HSCs, T cells, B cells, and natural killer cells in the BM graft. In an MHC-mismatched murine transplant model, recipients of Flt3L-treated T cell-depleted (TCD) BM (TCD F-BM) and cytokine-untreated T cells had increased survival and decreased GVHD scores with fewer Th1 and Th17 polarized T cells post-transplantation compared with recipients of equivalent numbers of untreated donor TCD BM and T cells. Gene array analyses of pDCs from Flt3L-treated human and murine donors showed up-regulation of adaptive immune pathways and immunoregulatory checkpoints compared with pDCs from untreated BM donors. Transplantation of TCD F-BM plus T cells resulted in no loss of the graft-versus-leukemia (GVL) effect compared with grafts from untreated donors in 2 murine GVL models. Thus, Flt3L treatment of BM donors is a novel method for increasing the pDC content in allografts, improving survival, and decreasing GVHD without diminishing the GVL effect. [ABSTRACT FROM AUTHOR]

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8 + T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer. [ABSTRACT FROM AUTHOR]

The contribution of cross-presenting XCR1+ dendritic cells (DCs) and SIRPα+ DCs in maintaining T cell function during exhaustion and immunotherapeutic interventions of chronic infections remains poorly characterized. Using the mouse model of chronic LCMV infection, we found that XCR1+ DCs are more resistant to infection and highly activated compared with SIRPα+ DCs. Exploiting XCR1+ DCs via Flt3L-mediated expansion or XCR1-targeted vaccination notably reinvigorates CD8+ T cells and improves virus control. Upon PD-L1 blockade, XCR1+ DCs are not required for the proliferative burst of progenitor exhausted CD8+ T (T PEX) cells but are indispensable to sustain the functionality of exhausted CD8+ T (T EX) cells. Combining anti-PD-L1 therapy with increased frequency of XCR1+ DCs improves functionality of T PEX and T EX subsets, while increase of SIRPα+ DCs dampened their proliferation. Together, this demonstrates that XCR1+ DCs are crucial for the success of checkpoint inhibitor-based therapies through differential activation of exhausted CD8+ T cell subsets. [Display omitted] • XCR1+ DCs are more functional and less prone to LCMV infection than SIRPα+ DCs • Expanding XCR1+ DCs via Flt3L or delivering XCR1-targeting Ag improve virus control • Anti-PD-L1 treatment increases XCR1+ DC numbers and IL-12 production • XCR1+ DCs promote T EX functionality but are dispensable for T PEX proliferation burst Domenjo-Vila et al. show that XCR1+ DCs are crucial in augmenting exhausted CD8+ T cell effector functions during immunotherapeutic interventions in chronic virus infections. Increased T cell functionality leads to reduced virus loads. XCR1+ DCs should be among the preferential targets in immunotherapeutic cure strategies. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) that can orchestrate immune responses and maintain host homeostasis, are indispensable components of the immune system. Although distributed widely in many lymphoid and non-lymphoid tissues, their rarity in number has become a limiting factor for DC related research and therapies. Therefore, methods for efficiently generating large numbers of DC resembling their in vivo counterparts are urgently needed for DC related research and therapies. Herein we summarize the current methods for generating mouse and human DC in vitro and hope that these will facilitate both studies of DC biology and their clinical applications. [ABSTRACT FROM AUTHOR]

Purpose: Malaria remains the most deadly human parasitic disease, mostly because of the mosquito-born protozoan parasite Plasmodium falciparum with ~ 627,000 deaths reported in 2012. Unfortunately, there is resistance to most drugs, and successful vaccines are still not developed. The role of the immune system is critical but poorly understood. Methods: One specific publication that reported a new way through which the immune system may promote malaria pathogenesis is discussed. Findings: Kenyan children with mild and severe malaria had increased plasma levels of the Flt3 ligand, a soluble cytokine released from the surface of mast cells (MCs). A positive correlation was found between disease severity and frequencies of circulating BD CA3+ dendritic cells. These human equivalents of the rodent CD8+ T cells migrate to tissues with a heavy parasite load and cause damage primarily through cytolysis. Implications: Malaria parasites may promote malaria pathogenesis by triggering MCs, which expand a unique class of dendritic cells with the subsequent activation of pathogenic CD8+ T cells. However, MCs may have additional regulatory functions. Selective inhibition of MC activation may serve as an adjuvant treatment. [ABSTRACT FROM AUTHOR]

Abstract: Hematopoietic stem cell (HSC) transplantation has curative potential for patients with hematological malignancies. Clinically, HSCs derived from mobilized peripheral blood are used more frequently than bone marrow. However, current standard mobilizing agents yield grafts that may not contain sufficient HSCs. Here, using murine models, we discovered that FLT3L synergized with plerixafor to mobilize phenotypically defined HSCs and their combination (FP) was superior to granulocyte colony-stimulating factor (G-CSF) alone or in combination with plerixafor (GP). Additionally, FP mobilized more regulatory T cells, natural killer cells, and plasmacytoid dendritic cells compared with G-CSF alone or GP. Both syngeneic and allogeneic grafts mobilized by FP led to long-term survival in transplanted mice. Collectively, FP represents a promising novel and potent mobilization regimen with potential clinical application in both the autologous and allogeneic transplantation settings. [Copyright &y& Elsevier]

Highlights: [•] Dendritic cell-based vaccination strategy to protect from autoimmune diseases. [•] Flt3L induces maturation of CD8α+ DC. [•] CD8α+ DC vaccination promotes protective CD4+ Th1 cells. [•] Protection conferred by CD8α+ DC vaccination is IFN-γ dependent. [ABSTRACT FROM AUTHOR]

Abstract: Glioblastoma multiforme (GBM) is the most common and deadliest of adult primary brain tumors. Due to its invasive nature and sensitive location, complete resection remains virtually impossible. The resistance of GBM against chemotherapy and radiotherapy necessitate the development of novel therapies. Gene therapy is proposed for the treatment of brain tumors and has demonstrated pre-clinical efficacy in animal models. Here we review the various experimental therapies that have been developed for GBM including both cytotoxic and immune stimulatory approaches. We also review the combined conditional cytotoxic immune stimulatory therapy that our lab has developed which is dependent on the adenovirus mediated expression of the conditional cytotoxic gene, Herpes Simplex Type 1 Thymidine Kinase (TK) and the powerful DC growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Combined delivery of these vectors elicits tumor cell death and an anti-tumor adaptive immune response that requires TLR2 activation. The implications of our studies indicate that the combined cytotoxic and immunotherapeutic strategies are effective strategies to combat deadly brain tumors and warrant their implementation in human Phase I clinical trials for GBM. [Copyright &y& Elsevier]

Abstract: Granulocyte–macrophage colony stimulating factor (GM-CSF) is a cytokine that functions as a hematopoietic growth factor for the generation of white blood cells and is used clinically to stimulate hematopoiesis following chemotherapy. Apart from stimulating production of granulocytes and monocytes/macrophages, GM-CSF has also long been used for in vitro survival/generation of dendritic cells (DCs) from monocytes and bone marrow cells. Evidence has emerged pointing to an additional role for GM-CSF in regulating the function and differential development of several DC subsets. These newly ascribed functions of GM-CSF may underscore its importance in immunity against pathogens as well as initiating/mediating immunopathology in chronic inflammation. Here we summarize recent advances on the role of GM-CSF in regulating the development and function of DC subsets and discuss the biological significance of these new findings. [Copyright &y& Elsevier]

Abstract: The purpose of this work was to assess the immune response against plasmid DNA encoding hepatitis B virus (HBV) HBsAg in C57BL/6 mice inoculated by micro-needle. Fms-like tyrosine kinase 3 ligand (Flt3L), a hematopoietic growth factor, was used as an adjuvant. Immune response was determined by analysis of cytokine production. In addition, cytotoxic lymphocyte (CTL) activity was measured 7 days after in vitro addition of tumor cells (CT26/S) stabling expressing HBsAg. The efficacy of immunoprotection against CT26/S was determined by antibody response to this challenge. A strong antibody response was induced by inoculation with the DNA vaccine via micro-needles assisted with Flt3L, Administration of the vaccine to splenocytes induced significantly higher levels of interleukin-12 and gamma interferon. CTL response to the vaccine was stronger than that stimulated by intramuscular or micro-needle injections alone. Our study suggests useful strategies for improving the efficacy of vaccines against HBV and other pathogens. [Copyright &y& Elsevier]

Abstract: Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8+ T cell responses. When E6Co and E7Co were fused (E67Co), CD8+ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8+ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8+ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67Co further enhances HPV-specific CD8+ T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67Co delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer. [Copyright &y& Elsevier]

Abstract: Glioblastoma multiforme is an intracranial tumor that has very poor prognosis. Patients usually succumb to their disease 6 to 12 months after they are diagnosed despite very aggressive treatment modalities. We tested the efficacy of a potent differentiation and proliferation factor for the professional antigen-presenting dendritic cells (DCs), i.e., Flt3L, for its potential role as a novel therapy for gliomas. We investigated the ability of recombinant adenoviral vectors encoding human soluble Flt3L (hsFlt3L) to improve the survival of Lewis rats bearing intracranial syngeneic CNS-1 gliomas. We show that RAdhsFlt3L can improve survival in a dose-dependent manner. Seventy percent of rats survive when treated with 8 × 107 pfu RAdhsFlt3L (P < 0.0005). In addition we demonstrate in both naïve Lewis rats and C57BL/6 mice the presence of increased numbers of cells bearing DC markers (OX62 and MHCII, in rats, or CD11C, 33D1, MHCII, and F4/80, but not DEC205, in mice) in sites of brain delivery of RAdhsFlt3L. These results show that expression of hsFlt3L in the brain leads to the presence of cells displaying DC markers. We demonstrate that treatment with hsFlt3L leads to inhibition of tumor growth and significantly increased life span of animals implanted with syngeneic CNS-1 glioma cells. Animals that had survived for long periods, i.e., 6 months, had eliminated the implanted tumors after neuropathological analysis; on the other hand, some of the 3-month survivors still appeared to harbor brain tumors. Our results have profound implications for immune-mediated brain tumor therapy and also suggest the ability to recruit DC-like cells within the brain parenchyma in response to the local expression of Flt3L from adenoviral vectors. [Copyright &y& Elsevier]

Plasmacytoid dendritic cells (PDCs) or natural interferon-producing cells, function as the body’s innate defense against viral infections. As discussed here, they may play additional roles in response to bacterial pathogens and may have the capacity to induce different type of T-cell responses depending on what signals they receive. The discovery of murine PDCs will allow for the design of models to study viral immunobiology in vivo and to determine their function in various diseases that involve plasmacytoid dendritic cells, such as selected leukemias, lymphomas, allergies, different autoimmune conditions, and their possible role in inducing and maintaining tolerance. [Copyright &y& Elsevier]

Summary: The phenotype and function of liver dendritic cells (LDCs) are poorly understood. This Snapshot summarizes our current knowledge on LDCs in the healthy and injured liver, and their role in fibrosis progression and reversal. It also draws attention to various pitfalls in the current experimental design and conclusions based on available data. [Copyright &y& Elsevier]

Plasmacytoid dendritic cells (plasmacytoid DC, pDC) are major IFN-I producers and have been shown to be affected by HIV through ill-defined mechanisms. In this study, we directly assess the role of pDC in early infection, evaluating whether modulating their abundance can alter viral replication. First, HIV infection of humanized mice induces systemic depletion of pDC, and in the presence of soluble FMS-like tyrosine kinase 3 ligand (Flt3L), pDC levels remain elevated. Flt3L significantly delays the onset of viremia and reduces viral replication via a process that is dependent on pDC and mediated through an enhanced early IFN-I response. pDC from Flt3L-treated mice are more prone to express IFN-α following TLR7 stimulation, but this propensity is gradually decreased during infection. In conclusion, maintaining pDC levels and function is key to effective early viral control, and in this context, these findings provide practical insights for anti-HIV strategies and vaccine design. • HIV infection of hu-mice depletes pDC globally and impairs IFN-α production by pDC • DC expansion by Flt3L suppresses HIV infection in a pDC-dependent manner • pDC from Flt3L-treated mice are more responsive to TLR7 stimulation • Early blocking of IFN-I signaling abolishes Flt3L-mediated suppression of viremia Plasmacytoid DC (pDC) are vital for a host's early antiviral responses. Pham et al. demonstrate that HIV infection of hu-mice induces widespread depletion of pDC and impairs their ability to make IFN-α. Flt3L treatment expands pDC and suppresses HIV replication through early enhancement of IFN-I responses. [ABSTRACT FROM AUTHOR]