Host Dendritic Cells Research Articles

An Update from the United States National Heart, Lung, and Blood Institute‐funded Production Assistance for Cellular Therapies (PACT) Program: A Decade of Cell Therapy

Recognizing the challenges faced by researchers and clinicians working in the field of cellular therapy, the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, established the Production Assistance for Cellular Therapies (PACT) program in 2003 and expanded it in 2010. The PACT program provides both clinical product manufacturing support that furthers the mission of NHLBI in the areas of cardiac, lung, and blood diseases and broad support of translational development across all disease areas to serve the entire cell therapy community. The program also provides access to expertise in project management, regulatory affairs, and quality assurance and control. Education initiatives include webinars, cell processing facility-hosted workshops, national workshops, and active participation and leadership within the cell therapy community through collaboration with other cell therapy organizations and academia. So far, over 650 PACT-manufactured cell therapy products have been administered in 32 clinical trials for a range of illnesses and diseases such as acute myocardial infarction, sickle cell disease, and graft-versus-host disease.

Intravital imaging of donor allogeneic effector and regulatory T cells with host dendritic cells during GVHD

AbstractGraft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon–DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.

Sphingosine Analog AAL-R Promotes Activation of LCMV-Infected Dendritic Cells

Sphingosine analogs display diverse immunoregulatory activities with curative potential in autoimmune diseases and viral infections. Recently, the sphingosine analog AAL-R was shown to increase DC activation upon TLR7 stimulation. Here, we investigated the effect of AAL-R on activation of dendritic cells (DCs) infected by lymphocytic choriomeningitis virus (LCMV). Concomitant treatment of LCMV-infected DCs with AAL-R enhanced DC maturation and DC ability to stimulate and expand antiviral CD8(+) T cells. Importantly, AAL-R's stimulatory activity was abrogated in type I interferon (IFN) receptor-deficient DCs following LCMV infection. In support of this observation, AAL-R increased type I IFN production from DCs infected with LCMV. Taken together, the sphingosine analog could directly act on DCs to promote defensive host DC responses to the viral invasion via type I IFN signaling.

TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin–miltefosine combination therapy in visceral leishmaniasis

The combination of paromomycin-miltefosine is a successful anti-leishmanial therapy in visceral leishmaniasis (VL). This encouraged us to study its effect on Toll-like receptor (TLR)-mediated immunomodulation of dendritic cells (DC), as DC maturation and activation is crucial for anti-leishmanial activity. In silico protein-ligand interaction and biophysical characterization of TLR9-drug interaction was performed. Interaction assays of HEK293 cells with different concentrations of miltefosine and/or paromomycin were performed, and NF-κB promoter activity measured. The role of TLR9 and MyD88 in paromomycin/miltefosine-induced maturation and activation of DCs was evaluated through RNA interference techniques. The effect of drugs on DCs was measured in terms of counter-regulatory production of IL-12 over IL-10, and characterized by chromatin immunoprecipitation assay at the molecular level. Computational and biophysical studies revealed that paromomycin/miltefosine interact with TLR9. Both drugs, as a monotherapy/combination, induced TLR9-dependent NF-κB promoter activity through MyD88. Moreover, the drug combination induced TLR9/MyD88-dependent functional maturation of DCs, evident as an up-regulation of co-stimulatory markers, enhanced antigen presentation by increasing MHC II expression, and increased stimulation of naive T-cells to produce IFN-γ. Both drugs, by modifying histone H3 at the promoter level, increased the release of IL-12, but down-regulated IL-10 in a TLR9-dependent manner. These results provide the first evidence that the combination of paromomycin-miltefosine critically modifies the maturation, activation and development of host DCs through a mechanism dependent on TLR9 and MyD88. This has implications for evaluating the success of other combination anti-leishmanial therapies that act by targeting host DCs.

The Role of Dendritic Cells in Graft-Versus-Tumor Effect

Dendritic cells (DCs) are the most potent antigen presenting cells. DCs play a pivotal role in determining the character and magnitude of immune responses to tumors. Host and donor hematopoietic-derived DCs play a critical role in the development of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation. GVHD is tightly linked with the graft-versus-tumor (GVT) effect. Although both host and donor DCs are important regulators of GVHD, the role of DCs in GVT is poorly understood. GVT is caused by donor T cells that attack recipient tumor cells. The donor T cells recognize alloantigens, and tumor specific antigens (TSAs) are mediating GVHD. The process of presentation of these antigens, especially TSAs remains unknown. Recent data suggested that DC may be essential role for inducing GVT. The mechanisms that DCs possess may include direct presentation, cross-presentation, cross-dressing. The role they play in GVT will be reviewed.

Host Genetic Factors and Dendritic Cell Responses Associated with the Outcome of Interferon/Ribavirin Treatment in HIV-1/HCV Co-Infected Individuals.

HIV-1/HCV co-infection is a significant health problem. Highly active antiretroviral treatment (HAART) against HIV-1 has proved to be fairly successful. On the other hand, direct acting antiviral drugs against HCV have improved cure rates but high cost and development of drug resistance are important concerns. Therefore PEGylated interferon (PEG-IFN) and ribavirin (RBV) still remain essential components of HCV treatment, and identification of host factors that predict IFN/RBV treatment response is necessary for effective clinical management of HCV infection. Impaired dendritic cell (DC) and T cell responses are associated with HCV persistence. It has been shown that IFN/RBV treatment enhances HCV-specific T cell functions and it is likely that functional restoration of DCs is the underlying cause. To test this hypothesis, we utilized an antibody cocktail (consisting of DC maturation, adhesion and other surface markers) to perform comprehensive phenotypic characterization of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in a cohort of HIV-1/HCV co-infected individuals undergoing IFN/RBV treatment. Our results show that pre-treatment frequencies of mDCs are lower in non-responders (NRs) compared to responders (SVRs) and healthy controls. Although, the treatment was able to restore the frequency of mDCs in NRs, it downregulated the frequency of CCR7+, CD54+ and CD62L+ mDCs. Pre-treatment frequencies of pDCs were lower in NRs and decreased further upon treatment. Compared to SVRs, NRs exhibited higher ratio of PD-L1+/CD86+ pDCs prior to treatment; and this ratio remained high even after treatment. These findings demonstrate that enumeration and phenotypic assessment of DCs before/during therapy can help predict the treatment outcome. We also show that before treatment, PBMCs from SVRs secrete higher amounts of IFN-γ compared to controls and NRs. Upon genotyping IFNL3 polymorphisms rs12979860, rs4803217 and ss469415590, we found rs12979860 to be a better predictor of treatment outcome. Collectively, our study led to identification of important correlates of IFN/RBV treatment response in HIV-1/HCV co-infected individuals.

Allogeneic Cytokine-Induced Killer Cells Eliminate Host Dendritic Cells Due To The Enhanced Killing Activity By IFN-Gamma, Leading To Less Graft-Versus-Host Disease

Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. It was reported previously that IFN-gamma was a critical cytokine for preventing GVHD because CIK cells generated from IFN-gamma knock out mice caused lethal GVHD. One possible mechanism of protective effect against GVHD was that a large amount of IFN-gamma secreted by CIK cells could promote Fas-mediated apoptosis of recipient antigen activated donor T cells. However the mechanism of suppression of GVHD with CIK cells is not fully understood. Host residual dendritic cells (DCs) are most important cells in initiating GVHD reaction. Therefore, we hypothesize that CIK cells eliminate host DCs as same as kill tumor cells, and then reduce GVHD severity. To test this, 51Cr release assay was performed using BM derived DCs as target cells. We showed that allogeneic CIK cells had cytolytic activity against DCs, in marked contrast killing activity of IFN-gamma deficient CIK cells was much less compared to that of WT CIK cells (p< 0.05). Expression level of surface markers including NKG2D and CD8/CD4 ratio was not different between WT and IFN-gamma KO CIK cells. To further explore the mechanism why IFN-gamma deficient CIK cells reduced killing activity against DCs, IFN-gamma neutralizing antibody was added into killing mixture with WT CIK cells. However killing activity of WT CIK cells against DCs with IFN-gamma antibody did not changed compared to that of WT CIK alone. These results suggested that IFN-gamma itself did not affect killing activity directly, but IFN-gamma was essential cytokine to enhance killing activity during the culture of CIK cells. Next concern was whether CIK cells eliminated host DCs in vivo or not, we compared the residual number of host-type splenic DCs between the mice receiving bone marrow (BM) cells plus WT CIK cells and IFN-gamma deficient CIK cells. As shown in the figure below, the number of host DCs in the spleen in the mice receiving WT CIK cells was significantly lower compared to those of the mice receiving IFN-gamma KO CIK cells (p<0.05). In conclusion, allogeneic CIK cells eliminated host DCs due to the enhanced killing activity by IFN-gamma, leading to much less GVHD. Disclosures: No relevant conflicts of interest to declare.

CD8α+ dendritic cells dictate immune responses against murine AML

Spontaneous T cell responses generated against a variety of solid malignancies are often subverted by immune evasion mechanisms active in the tumor microenvironment. In contrast, the mechanisms that regulate T cell activation versus tolerance to hematopoietic malignancies, such as acute myeloid leukemia (AML), have not been well-characterized. Our recent work in a murine AML model has demonstrated that following a systemic introduction of leukemia cells, T cells specific for leukemia-derived antigens underwent abortive proliferation and were deleted from the host. This deletional tolerance in mice with established AML was reversible upon administration of an agonistic anti-CD40 antibody to activate host dendritic cells (DCs), and argued that these cells may play a dominant role in tolerance induction to AML. Investigation of the DCs populations which engulfed AML cells in vivo, and which were likely promoting T cell tolerance, led to the critical observation that AML cells were phagocytosed exclusively by CD11c+CD8α+ DCs (CD8α+ DCs). CD8α+, but not CD8α- DCs purified from mice following an intravenous inoculation of AML cells, were able to cross-present leukemia-derived antigens to T cells in vitro, providing strong evidence that CD8α+ DC generate T cell tolerance to AML. Ongoing work utilizing mice deficient in particular DC subsets is focused on identifying a functional link between CD8α+ DCs and T cell tolerance. Additionally, the receptors expressed selectively on CD8α+ DCs which facilitate phagocytosis and cross-presentation of leukemia derived antigens are under investigation.

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Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR genes recognize polymorphic HLA class I including HLA-A, B & C allotypes. The KIR genes after binding specific ligands may transmit inhibitory or activating signals. NKC sense & kill target cells lacking MHC class I molecules & release various cytokines on activation. UCB is an increasingly used source of HSC for pts without matched adult donor for Tx. The goal of the study was to attain frequencies (freq) of HLA-L in UCBU aimed at providing information (info) for finding UCB with NK benefit for HSC Tx pts. Total of 21,002 UCBU from Cord Bank at MD Anderson was included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4 alleles, C1 and C2 HLA-C alleles were obtained. KIR3DL1 recognizes allotypes with HLA-Bw4 serological motif; KIR2DL1 recognizes HLA-C allotypes with lysine at codon 80 (group 2 - C2); & KIR2DL2/KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1 - C1). Among the 21,002 UCBU, 70.7% carry Bw4 alleles. UCB bearing C1/C2 heterozygous genotype (GT) accounted for 45.6%, C1/C1 homozygous GT for 39.6%, & C2/C2 GT for 14.7% only. Incompatibility of KIR HLA-L between pt & donor has been shown to impact graft outcome (GO) in HSC Tx. NKC from the donor greatly contribute to eradication of leukemia blasts escaping the preparative regimen & to clearance of residual host dendritic cells & T lymphocytes thus preventing graft-versus-host disease & graft rejection. This study showed high freq of Bw4-bearing GT, increased freq of C1/C2, & C1/C1 but not C2/C2 GT in UCBU. These freq info could be useful for prediction of the possibility for HSC Tx pts, especially those with Bw6 or C2/C2 GT, to find suitable UCB donor(s) with beneficial NK reactivity, thereby improving GO. Further study of diversification of KIR HLA-ligands in pts would be warranted to assess the importance of these findings.

Pathogenesis of graft-versus-host disease: innate immunity amplifying acute alloimmune responses

In addition to reduced-intensity conditioning, which has expanded the eligibility for hematopoietic cell transplantation (HCT) to older patients, increased availability of alternative donors, including HLA-mismatched unrelated donors, has increased access to allogeneic HCT for more patients. However, acute graft-versus-host disease (GVHD) remains a lethal complication, even in HLA-matched donor-recipient pairs. The pathophysiology of GVHD depends on aspects of adaptive immunity and interactions between donor T-cells and host dendritic cells (DCs). Recent work has revealed that the role of other immune cells and endothelial cells and components of the innate immune response are also important. Tissue damage caused by the conditioning regimen leads to the release of exogenous and endogenous "danger signals". Exogenous danger signals called pathogen-associated molecular patterns and endogenous noninfectious molecules known as damage-associated molecular patterns (DAMPs) are responsible for initiating or amplifying acute GVHD by enhancing DC maturation and alloreactive T-cell responses. A significant association of innate immune receptor polymorphisms with outcomes, including GVHD severity, was observed in patients receiving allogeneic HCT. Understanding of the role of innate immunity in acute GVHD might offer new therapeutic approaches.

Receptor-mediated recognition of mycobacterial pathogens

Mycobacteria are a genus of bacteria that range from the non-pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen-associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non-opsonically recognize mycobacterial PAMPs, including membrane-bound C-type lectin receptors, membrane-bound and cytosolic Toll-like receptors and cytosolic NOD-like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor-mediated recognition of mycobacterial pathogens by macrophages and dendritic cells.

Suppression of acute graft-versus-host response by TCDD is independent of the CTLA-4-IFN-γ-IDO pathway.

Activation of the aryl hydrocarbon receptor (AhR) by its prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces potent suppression of an acute graft-versus-host (GVH) response and prevents GVH disease (GVHD). Suppression is associated with development of a regulatory population of donor CD4(+) CD25(+)T-cells that express high levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, a direct link between these AhR-induced Tregs (AhR-Tregs) and suppression of GVHD remains to be shown. CTLA-4 is a negative regulator of T-cell responses and is associated with the induction of tolerogenic dendritic cells (DCs) that produce indoleamine 2,3-dioxygenase (IDO). We hypothesized that AhR-Tregs mediate suppression via their enhanced expression of CTLA-4, which, in turn, induces IFN-γ and IDO in host DCs. Subsequent depletion of tryptophan by IDO leads to termination of the donor T-cell response prior to development of effector CTL. Here, we show that despite increased expression of Ifng, Irf3, Irf7, Ido1, and Ido2 in the lymph nodes of TCDD-treated host mice, inhibition of IDO enzyme activity by 1-methyl-tryptophan was unable to relieve TCDD-mediated suppression of the GVH response. Furthermore, treatment with an anti-CTLA-4 antibody that blocks CTLA-4 signaling was also unable to alleviate TCDD-mediated suppression. Alternatively, we investigated the possibility that donor-derived AhR-Tregs produce IFN-γ to suppress effector CTL development. However, suppression of GVHD by TCDD was not affected by the use of Ifng-deficient donor cells. Together, these results indicate that neither overexpression of CTLA-4 nor production of IFN-γ by AhR-Tregs plays a major role in the manifestation of their immunosuppressive function in vivo.

Copresentation of Intact and Processed MHC Alloantigen by Recipient Dendritic Cells Enables Delivery of Linked Help to Alloreactive CD8 T Cells by Indirect-Pathway CD4 T Cells

In transplantation, direct-pathway CD8 T cells that recognize alloantigen on donor cells require CD4 help for activation and cytolytic function. The ability of indirect-pathway CD4 T cells to provide this help remains unexplained, because a fundamental requirement for epitope linkage is seemingly broken. The simultaneous presentation, by host dendritic cells (DCs), of both intact MHC class I alloantigen and processed alloantigen would deliver linked help, but has not been demonstrated definitively. In this study, we report that following in vitro coculture with BALB/c DCs, small numbers (~1.5%) of C57BL/6 (B6) DCs presented acquired H-2(d) alloantigen both as processed allopeptide and as unprocessed Ag. This represented class I alloantigen provides a conformational epitope for direct-pathway allorecognition, because B6 DCs isolated from cocultures and transferred to naive B6 mice provoked cytotoxic CD8 T cell alloimmunity. Crucially, this response was dependent upon simultaneous presentation of class II-restricted allopeptide, because despite acquiring similar amounts of H-2(d) alloantigen upon coculture, MHC class II-deficient B6 DCs failed to elicit cytotoxic alloimmunity. The relevance of this pathway to solid-organ transplantation was then confirmed by the demonstration that CD8 T cell cytotoxicity was provoked in secondary recipients by transfer of DCs purified from wild-type, but not from MHC class II-deficient, C57BL/6 recipients of BALB/c heart transplants. These experiments demonstrate that representation of conformationally intact MHC alloantigen by recipient APC can induce cytotoxic alloimmunity, but simultaneous copresentation of processed allopeptide is essential, presumably because this facilitates linked recognition by indirect-pathway CD4 Th cells.

AB0104 Modulation of immunity through cigarette smoke induced changes in dendritic cell functions - potential relevance to autoimmunity

BackgroundEpidemiological studies implicate cigarette smoking as a major risk factor predisposing to the development of seropositive rheumatoid arthritis (RA). The mechanisms by which cigarette smoking promotes RA are largely unknown,...

Direct and indirect antigen presentation lead to deletion of donor-specific T cells after in utero hematopoietic cell transplantation in mice

AbstractIn utero hematopoietic cell transplantation (IUHCTx) is a promising method to induce donor-specific tolerance but the mechanisms of antigen presentation that educate host T cells and the relative importance of deletion vs regulation in this setting are unknown. We studied the roles of direct and indirect antigen presentation (mediated by donor- and host-derived antigen-presenting cells [APCs], respectively) in a mouse model of IUHCTx. We found that IUHCTx leads to precocious maturation of neonatal host dendritic cells (DCs) and that there is early differentiation of donor-derived DCs, even after transplantation of a stem cell source without mature APCs. We next performed allogeneic IUHCTx into donor-specific T-cell receptor transgenic mice and confirmed that both direct and indirect antigen presentation lead to clonal deletion of effector T cells in chimeras. Deletion did not persist when chimerism was lost. Importantly, although the percentage of regulatory T cells (Tregs) after IUHCTx increased, there was no expansion in Treg numbers. In wild-type mice, there was a similar deletion of effector cells without expansion of donor-specific Tregs. Thus, tolerance induction after IUHCTx depends on both direct and indirect antigen presentation and is secondary to thymic deletion, without de novo Treg induction.

Tumor cell-derived exosome-targeted dendritic cells stimulate stronger CD8+ CTL responses and antitumor immunities

Tumor cell-derived exosomes (TEX) have been widely used to induce antitumor immune responses in animal models and clinical trials. However, the efficiency of the antitumor immunity that is induced by TEX is still relatively weak. In this study, we compared the antitumor immunities between EG7 tumor cell-derived exosomes (EXOEG7) and EXOEG7-targeted dendritic cells (DCEXO). We found that EXOEG7 harbored OVA and peptide major histocompatibility complex I (pMHC-I), which were expressed on its parental EG7 tumor cells, and they could transfer OVA and pMHC-I to dendritic cells (DCs) in vitro. DCEXO could more efficiently induce antitumor immunity than EXOEG7. In addition, we showed that the immune stimulatory effects of EXOEG7 were dependent on the host DCs and, whereas those of DCEXO were not, indicating the important role of the host DCs in TEX vaccines. Taken together, TEX-targeted DCs may be more effective for EXO-based vaccines for the induction of antitumor immunity.

Neurokinin-1 receptor stimulated dendritic cell vaccines downregulate IL-10 and license host inflammatory dendritic cells to bias Th1 immunity (P4236)

Abstract Substance-P (SP) is a neuropeptide that stimulates immunity by binding to the neurokinin-1 receptor (NK1R). As professional Ag presenting cells, dendritic cells (DC) are unique activators of naïve T cells. DC express functional NK1R, indicating that NK1R agonists may influence their T cell-stimulatory function. Relevant studies analyzing the relationship between neuropeptides and DC function are lacking. Here, we demonstrate that conditioning bone marrow derived DC (BMDC) with NK1R agonists leads to maturation and abrogation of IL-10 without inducing secretion of IL-12p70. Mechanistic analyses in vitro demonstrated that NK1R signaling blocks the CREB1/TORC2 pathway to inhibit the Il-10 promoter. Following subcutaneous administration as a cellular vaccine, Ag-loaded and NK1R-signaled BMDC homed to skin draining lymph nodes (sDLN), initiating inflammation and Th1 and Tc1 immunity in vivo. This type-1 immunity required IL-12p70 secreted in sDLN by host DC, including LN-resident, and recruited inflammatory DC. Utilizing CCR7KO BMDC and IL-12p35KO mice, we elucidated that endogenous DC subsets were activated in situ by NK1R-signaled-BMDC homing to sDLN. Moreover, reduced levels of IL-10 by NK1R signaled BMDC licensed IL-12 secretion by host DC and mediated type-1 immunity. Our data demonstrate that NK1R-signaling promotes immunostimulatory DC and provide a relevant insight into the mechanisms employed by neuromediators to regulate the outcome of cellular immune responses.

Immunologically active cryogels for breast cancer therapy (P4329)

Abstract Passive immunotherapy has become an effective adjunct for the treatment of HER2/neu-overexpressing breast cancers, as patients can respond well to monoclonal antibodies such as trastuzumab (anti-HER-2/neu antibody therapy). However, patients with late-stage disease, who often become immunosuppressed are unlikely to respond, motivating the development of new prophylactic vaccines. To this end, we have developed an injectable, polymer-based cryogel vaccine containing living, attenuated HER-2/neu-overexpressing breast cancer cells. The cryogel-based vaccine mimics key aspects of bacterial infection and directly controls immune-cell trafficking and activation in the body. This system provides a sustained release of GM-CSF to recruit host dendritic cells (DCs) to the porous material, and subsequently presents breast cancer antigens and TLR9 ligand CpG oligonucleotides to activate the resident DCs. Subcutaneous injection of the cryogel vaccines provide potent prophylactic protection against mammary cancer in mice, consistent with significantly higher titers of Her-2/neu-specific antibodies compared to the humoral responses induced by bolus injection of irradiated GM-CSF-secreting breast tumor cells. The cryogel-based vaccines induced a 70-fold increase in antibody production compared to untreated mice and 100% survival was achieved in vaccinated mice. The cryogel-based vaccines represent a promising tool for the development of novel active immunotherapeutic approaches to cancer.

Novel mechanisms of anti-tumor action for therapeutic T-bet gene-modified dendritic cells (P4228)

Abstract Clinical implementation of Type-1 polarized dendritic cell (DC) in vaccine formulations holds significant promise in the cancer therapy setting. We have recently demonstrated that DC engineered to express the T cell Type-1 transactivator protein T-bet (TBX21) promote robust Type-1 T cell-mediated immunity via a unique mechanism that is largely interleukin (IL)-12p70-independent. In the current study, we investigated the anti-tumor mechanisms of action associated with intratumoral administration of DC.Tbet developed from syngenic IL-12p35 -/-, IL-12p40 -/-, IFNγ -/- or β2m -/- mice. We observed that Type-1 anti-tumor immunity associated with slowed tumor growth was effectively developed in animals receiving DC.Tbet deficient in their ability to produce IL-12 family cytokines (i.e. IL-12, IL-23, IL-35) or IFN-γ. Surprisingly, we observed that the ability of DC.Tbet to present MHC class I/peptide complexes (i.e. lost in DC developed from β2m -/- mice) was not important for early priming of anti-tumor CD8+ T cells, but was essential for the sustained therapeutic efficacy of DC.Tbet (and treatment-induced anti-tumor CD8+ T cells) within the tumor microenvironment (TME). In order to best translate this cancer treatment modality into the clinic, we are currently evaluating how DC.Tbet conditions the TME for improved function of Ag-primed Type-1 T effector cells and how these APC collaborate with host DC to diversify the therapeutic T cell repertoire.

Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12

AbstractSubstance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.