Dendritic Cells In Lymph Nodes Research Articles

Phenotypically distinct neutrophils patrol uninfected human and mouse lymph nodes

Neutrophils play a key role in innate immunity. As the dominant circulating phagocyte, they are rapidly recruited from the bloodstream to sites of infection or injury to internalize and destroy microbes. More recently, neutrophils have been identified in uninfected organs, challenging the classical view of their function. Here we show that neutrophils were present in lymph nodes (LNs) in homeostasis. Using flow cytometry and confocal imaging, we identified neutrophils within LNs in naive, unchallenged mice, including LNs draining the skin, lungs, and gastrointestinal tract. Neutrophils were enriched within specific anatomical regions, in the interfollicular zone, a site of T cell activation. Intravital two-photon microscopy demonstrated that LN neutrophils were motile, trafficked into LNs from both blood and tissues via high endothelial venules and afferent lymphatics, respectively, and formed interactions with dendritic cells in LNs. Murine and human LN neutrophils had a distinct phenotype compared with circulating neutrophils, with higher major histocompatibility complex II (MHCII) expression, suggesting a potential role in CD4 T cell activation. Upon ex vivo stimulation with IgG immune complex (IC), neutrophils up-regulated expression of MHCII and costimulatory molecules and increased T cell activation. In vivo, neutrophils were capable of delivering circulating IC to LNs, suggesting a broader functional remit. Overall, our data challenge the perception that neutrophil patrol is limited to the circulation in homeostasis, adding LNs to their routine surveillance territory.

Differential Roles of Dendritic Cells in Expanding CD4 T Cells in Sepsis.

Sepsis is a systemically dysregulated inflammatory syndrome, in which dendritic cells (DCs) play a critical role in coordinating aberrant immunity. The aim of this study is to shed light on the differential roles played by systemic versus mucosal DCs in regulating immune responses in sepsis. We identified a differential impact of the systemic and mucosal DCs on proliferating allogenic CD4 T cells in a mouse model of sepsis. Despite the fact that the frequency of CD4 T cells was reduced in septic mice, septic mesenteric lymph node (MLN) DCs proved superior to septic spleen (SP) DCs in expanding allogeneic CD4 T cells. Moreover, septic MLN DCs markedly augmented the surface expression of MHC class II and CD40, as well as the messaging of interleukin-1β (IL-1β). Interestingly, IL-1β-treated CD4 T cells expanded in a dose-dependent manner, suggesting that this cytokine acts as a key mediator of MLN DCs in promoting septic inflammation. Thus, mucosal and systemic DCs were found to be functionally different in the way CD4 T cells respond during sepsis. Our study provides a molecular basis for DC activity, which can be differential in nature depending on location, whereby it induces septic inflammation or immune-paralysis.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-β treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Analysis of hypoxia-associated dendritic cells in colitic mice and effects of probiotics on IL-10 production in inflammatory dendritic-cells under hypoxia.

The aim of this study was to analyse hypoxia-associated dendritic cells (DCs) in colitic mice and the effects of probiotics on interleukin (IL)-10 production in inflammatory DCs under hypoxic conditions. Extensive hypoxia was observed in the colonic mucosa of dextran sodium sulphate-induced colitic mice. Flow cytometric analysis demonstrated that hypoxia-inducible factor-1α+ DCs in colonic lamina propria (CLP) lymphocytes and mesenteric lymph nodes (MLN) were more abundant in colitic mice than those in controls. Among three subsets of DCs, i.e. plasmacytoid DCs, conventional DCs (cDCs), and monocyte-derived DCs (mDCs), cDCs and mDCs were more abundant in CLP of colitic mice. Bone marrow-derived Flt-3L-induced DCs (Flt-DCs) but not bone marrow-derived GM-CSF-induced DCs (GM-DCs), incubated with 1% O2 exhibited an inflammatory phenotype, with higher CD86, IL-6, and tumour necrosis factor-α expression, and lower IL-10 levels than those in Flt-DCs incubated with 21% O2. The hypoxia-induced decrease in IL-10 expression in Flt-DCs was restored by Bifidobacterium bifidum JCM 1255T promoted IL-10 expression through the p38 pathway under normoxic conditions. The anti-inflammatory effects of B. bifidum JCM 1255T in Flt-DCs were mediated through different cellular mechanisms under hypoxic and normoxic conditions. B. bifidum JCM 1255T could be used therapeutically for its anti-inflammatory effects.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC class II) and CD8 cytotoxic T cells (through MHC class I). Activation of tumor-specific CD4 and CD8 T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective MHC class II presentation of tumor antigens. We injected either B16-F10 melanoma or MB49 bladder cancer adenocarcinoma cells into the flank of mice and isolated DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to OVA-specific CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo and ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident CD8-positive and CD8-negative DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Examination of antigen uptake and processing also showed defects in DCs in TDLN. RNA-seq revealed many differences in gene expression between both resident and migratory DCs isolated from TDLN and NDLN. Characterized phenotypical and functional differences, confirmed by gene expression studies, may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow proper CD4 T cell priming to provide help to anti-tumor CD8 T cells.

PARP-1 Is Critical for Recruitment of Dendritic Cells to the Lung in a Mouse Model of Asthma but Dispensable for Their Differentiation and Function.

Dendritic cells (DCs) are critical in asthma and many other immune diseases. We previously demonstrated a role for PARP-1 in asthma. Evidence on PARP-1 playing a role in Th2-associated DC function is not clear. In this study, we examined whether PARP-1 is critical for DC differentiation and function using bone marrow progenitors and their migration to the lung in an ovalbumin-based mouse model of asthma. Results show that changes in PARP-1 levels during GM-CSF-induced DC differentiation from bone marrow progenitors were cyclic and appear to be part of an array of changes that included STAT3/STAT5/STAT6/GRAIL/RAD51. Interestingly, PARP-1 gene deletion affected primarily STAT6 and γH2AX. PARP-1 inhibition significantly reduced the migration of DCs to the lungs of ovalbumin-challenged mice, which was associated with a concomitant reduction in lung levels of the adhesion molecule VCAM-1. The requirement of PARP-1 for VCAM-1 expression was confirmed using endothelial and lung smooth muscle cells. PARP-1 expression and activity were also required for VCAM-1 in differentiated DCs. An assessment of CD11b+/CD11c+/MHCIIhigh DCs in spleens and lymph nodes of OVA-sensitized mice revealed that PARP-1 inhibition genetically or by olaparib exerted little to no effect on DC differentiation, percentage of CD80+/CD86+/CD40+-expressing cells, or their capacity to promote proliferation of ovalbumin-primed (OTII) CD4+ T cells. These findings were corroborated using GM-CSF-induced differentiation of DCs from the bone marrow. Surprisingly, the PARP-1−/− DCs exhibited a higher intrinsic capacity to induce OTII CD4+ T cell proliferation in the absence of ovalbumin. Overall, our results show that PARP-1 plays little to no role in DC differentiation and function and that the protective effect of PARP-1 inhibition against asthma is associated with a prevention of DC migration to the lung through a reduction in VCAM-1 expression. Given the current use of PARP inhibitors (e.g., olaparib) in the clinic, the present results may be of interest for the relevant therapies.

Immunomodulatory effects of sleep deprivation at different timing of psoriasiform process on skin inflammation

Sleep deprivation affects the pathophysiology of immune-inflammatory skin diseases such as psoriasis. Increasing efforts are directed toward exploring potential mechanisms involving sleep deprivation and immune responses. However, studies focusing on the effects of different timing of sleep deprivation on skin inflammation are lacking. This study thus investigated the immunomodulatory effects of paradoxical sleep (PS) deprivation at different timing of psoriasiform process on skin inflammation in the psoriasis-like mouse model. Male adult C57BL/6 mice were divided into 5 groups (each n = 5): control group (CON), IMQ-S2 group [treating with 31.25 mg imiquimod (IMQ) per day for 5 days with 48 h of PS deprivation on the fourth and fifth day], S2-IMQ group (treating with 31.25 mg IMQ per day for 5 days with 48 h of PS deprivation on the first and second day), 31.25 group (treating with 31.25 mg IMQ per day for 5 days) and 62.5 group (treating with 62.5 mg IMQ per day for 5 days). Compared with IMQ-S2 group and 31.25 group, S2-IMQ group mice had significant increase of IL-17A mRNA level in skin lesions and lymph nodes, and more severe cutaneous inflammation. However, IMQ-S2 group had the highest IL-1β mRNA level in skin lesions and the highest IL-6 mRNA level in lymph nodes among the three groups. Results of flow cytometry showed that frequencies of γδT cell, IL-17A+γδT cell, dendritic cell (DC) and MHCⅡ+ DC in lymph nodes of S2-IMQ group were significantly higher than IMQ-S2 group and 31.25 group, so was the frequency of γδT cells in skin lesions. However, the frequency of DCs in skin lesions of S2-IMQ group was significantly lower than IMQ-S2 group. These data suggest that PS deprivation at the early stage rather than the late stage of psoriasiform process exacerbates the skin inflammation through modulation of the immune system, which may involve that IL-1β and IL-6 stimulated by PS deprivation in turn increasing IL-17 expression through activation and proliferation of γδT cells and DCs migration.

CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes.

Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell-cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22-CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity.

In Situ Dendritic Cell Vaccine for Effective Cancer Immunotherapy

A cancer vaccine is an important form of immunotherapy. Given their effectiveness for antigen processing and presentation, dendritic cells (DCs) have been exploited in the development of a therapeutic vaccine. Herein, a versatile polymersomal nanoformulation that enables generation of tumor-associated antigens (TAAs) and simultaneously serves as adjuvant for an in situ DC vaccine is reported. The chimeric cross-linked polymersome (CCPS) is acquired from self-assembly of a triblock copolymer, polyethylene glycol-poly(methyl methyacrylate- co-2-amino ethyl methacrylate (thiol/amine))-poly 2-(dimethylamino)ethyl methacrylate (PEG-P(MMA- co-AEMA (SH/NH2)-PDMA). CCPS can encapsulate low-dose doxorubicin hydrochloride (DOX) to induce immunogenic cell death (ICD) and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), a photosensitizer to facilitate photodynamic therapy (PDT) for reactive oxygen species (ROS) generation. This combination is able to enhance the population of TAAs and DC recruitment, eliciting an immune response cascade. In addition, CCPS with primary and tertiary amines act as adjuvant, both of which can stimulate DCs recruited to form an in situ DC vaccine after combination with TAAs for MC38 colorectal cancer treatment. In vivo results indicate that the all-in-one polymersomal nanoformulation (CCPS/HPPH/DOX) increases mature DCs in tumor-draining lymph nodes (tdLNs) and CD8+ T cells in tumor tissues to inhibit primary and distant MC38 tumor growth following a single intravenous injection with a low dose of DOX and HPPH.

Abstract IA37: Enhancing the function of CAR T cells via a universal vaccine strategy

Abstract We recently developed a strategy to target peptide vaccines to lymph nodes, by linking peptide antigens to albumin-binding phospholipid-polymers. Small peptides are normally rapidly dispersed in the bloodstream following parenteral injection, but binding of amphiphile-peptides to endogenous albumin, which constitutively traffics from blood to lymph, retargeted these molecules to lymph nodes. However, these lipid-polymer conjugates bind to albumin with a relatively low affinity, and these molecules can also partition into cell membranes. We hypothesized that by attaching a small molecule, peptide, or protein ligand for a chimeric antigen receptor (CAR) to the same polymer-lipid tail (forming an “amph-vax” molecule), CAR ligands could be delivered efficiently to lymph nodes by albumin and subsequently partition into membranes of resident antigen presenting cells (APCs), thereby co-displaying a CAR T cell ligand from the cell surface together with native cytokine/receptor costimulation. In syngeneic mouse models of adoptive cell therapy, we demonstrated that this approach effectively concentrates CAR T ligands on the surfaces of dendritic cells in lymph nodes, leading to profound expansion of amph-vax-boosted CAR T cells in vivo. Amph-vax boosting safely increased the polyfunctionality of CAR T cells in parallel with T cell expansion. In a syngeneic model of melanoma, this converted a CAR T treatment that had no impact on tumor progression to a therapy that strongly delayed tumor outgrowth and enhanced survival. We have reduced to practice three different strategies to generalize this approach to any CAR of interest. This concept provides a strategy to regulate the expansion and function of CAR T cells directly in vivo to enhance adoptive cell therapy of cancer. Citation Format: Darrell J. Irvine. Enhancing the function of CAR T cells via a universal vaccine strategy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA37.

P156 PSYCHOLOGICAL STRESS INDUCES ALTERATIONS IN BEHAVIOR AND THE MUCOSAL IMMUNE SYSTEM IN A SPONTANEOUS MOUSE MODEL OF ILEITIS

Patients with Crohn’s disease (CD) suffer from abnormally high rates of depression and anxiety. Depression among patients with CD are higher than other debilitating chronic medical conditions, such as cancer. Behavioral co-morbidities are associated with increased rates of flares, more severe disease course, and increased rate of corticosteroid prescription. Psychological stress, even among CD patients in remission, is recognized as a risk factor for flare-ups. Despite the well-established relationship between stress and symptom relapse, a rigorous mechanistic explanation remains elusive. Here we demonstrate alterations in the behavioral profile and the mucosal immune system in the SAMP1/YitFc (SAMP1) mouse, a spontaneous model of CD-like ileitis, following exposure to acute and chronic psychological stress. SAMP1 littermates were sex matched and divided into two groups (n=8). The first group was subjected to restraint stress (RS) for seven days. Mice were restrained for 180 minutes per day in a 50mL conical tube with air holes drilled for adequate ventilation. Stool samples were collected each day. Subsequently, each group was subjected to behavioral testing to determine anxiety-like behavior (open field and elevated plus maze), depressive-like behavior (tail suspension), motor deficits (line crossings and rota-rod), and cognitive deficits (Y-maze). Immediately after, mice were sacrificed and tissue samples were collected for immunological analysis. Mice subjected to RS displayed increased immobility time during tail suspension indicating a depressive-like phenotype (p < 0.05). All other behavioral characteristics remained unchanged compared to control. Interestingly, FACS analysis revealed that RS mice had a marked increase in mesenteric lymph node (MLN) dendritic cells (p < 0.01) despite similar T-cell populations. Further analysis, including histopathology, serum cytokine ELISA, fecal corticosterone, 16s rRNA analysis of the microbiome, and RT-PCR analysis of ileum and spleen are currently underway and will be ready by the end of the year. The marked difference in the MLN dendritic cell (DC) population suggests increased luminal sampling of intestinal bacteria. Determining how the microbiome affects or is affected by the altered DC population is of particular interest to us. Is the DC population altered because of the microbiome or is the DC population altering the microbiome? Our preliminary data has suggested that depressive states are associated with alterations in the microbiome. Again, whether the changes are a cause or an effect of depression is yet to be answered but is our immediate goal.

Not only anti-inflammation, etanercept abrogates collagen-induced arthritis by inhibiting dendritic cell migration and maturation.

The application of tumor necrosis factor inhibitors (TNFi) is a major breakthrough in the treatment of rheumatoid arthritis (RA). While the anti-inflammatory nature of TNFi is thought to contribute to the therapeutic effects, recent data show that the pharmacology of TNF-α blockade is probably more complex than previously thought. This study investigates whether etanercept (ETN), one of the TNF antagonists, suppresses arthritis development through modulation of dendritic cell (DC) functions. Bone marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide (LPS) and treated with ETN for 24 hrs. DC functions, including maturation and migration, were determined. DCs from the lymph nodes (LNs) of ETN-treated collagen-induced arthritis (CIA) mice were analyzed for phenotypes and subsets. ETN efficiently inhibited the phenotypic maturation both in vitro and in vivo. ETN treatment delayed the onset and reduced the severity of arthritis in CIA mice. Moreover, ETN treatment strongly down regulated the number of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in LNs, possibly due to the depressive effect on the expression of CXCR4 on DCs in peripheral blood. The impaired DC migration to local LNs by ETN down regulated the number of T cells and B cells, and changed the LN cellular composition. The data show that TNF-α blockade has profound effects on DC maturation and migration, which may contribute to its immune regulatory effects in RA patients.

The Regulatory Status Adopted by Lymph Node Dendritic Cells and T Cells During Healthy Aging Is Maintained During Cancer and May Contribute to Reduced Responses to Immunotherapy.

Aging is associated with an increased incidence of cancer. One contributing factor could be modulation of immune cells responsible for anti-tumor responses, such as dendritic cells (DCs) and T cells. These immunological changes may also impact the efficacy of cancer immunotherapies in the elderly. The effects of healthy aging on DCs and T cells, and their impact on anti-mesothelioma immune responses, had not been reported. This study examined DCs and T cells in young (2–5 months; equivalent to 16–26 human years) and elderly (20–24 months; equivalent to 60–70 human years) healthy and mesothelioma-bearing C57BL/6J mice. During healthy aging, elderly lymph nodes adopted a regulatory profile, characterized by: (i) increased plasmacytoid DCs, (ii) increased expression of the adenosine-producing enzyme CD73 on CD11c+ cells, and (iii) increased expression of multiple regulatory markers (including CD73, the adenosine A2B receptor, CTLA-4, PD-1, ICOS, LAG-3, and IL-10) on CD8+ and CD4+ T cells, compared to lymph nodes from young mice. Although mesotheliomas grew faster in elderly mice, the increased regulatory status observed in healthy elderly lymph node DCs and T cells was not further exacerbated. However, elderly tumor-bearing mice demonstrated reduced MHC-I, MHC-II and CD80 on CD11c+ cells, and decreased IFN-γ by CD8+ and CD4+ T cells within tumors, compared to young counterparts, implying loss of function. An agonist CD40 antibody based immunotherapy was less efficient at promoting tumor regression in elderly mice, which may be due to: (i) failure of elderly CD8+ T cells to up-regulate perforin, and (ii) increased expression of multiple regulatory markers on CD11c+ cells and T cells in elderly tumor-draining lymph nodes (including CD73, PD-1, ICOS, LAG-3, and TGF-β). Our findings suggest that checkpoint blockade may improve responses to immunotherapy in elderly hosts with mesothelioma, and warrants further investigation.

Nanoparticle-Conjugate TLR7/8 Agonist Localized Immunotherapy Provokes Safe Antitumoral Responses.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.

Rehmannia glutinosa polysaccharide functions as a mucosal adjuvant to induce dendritic cell activation in mediastinal lymph node

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced activation of dendritic cells (DCs) in human and mouse subjects. In this study, we evaluated the effect of RGP as a mucosal adjuvant for inducting activation of DCs in the mediastinal lymph node (mLN) in the mouse. The C57BL/6 mice were intranasally (i.n.) treated with RGP and activation of DC in the mLN was analyzed. The treatment with RGP induced a substantial increase in the number of DCs in the mLN due to the up-regulation of C-C motif chemokine receptor 7 (CCR7) in the DCs. Moreover, the expression of co-stimulatory molecules in the mLN DCs and the concentration of pro-inflammatory cytokines in the lung were up-regulated by RGP treatment. Also, RGP treatment induced interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production in the mLN T cells. The combination treatment of RGP and ovalbumin (OVA) induced OVA-specific TCR transgenic I (OT-I) and OT-II cell proliferation in the mLN. Finally, the combination treatment of RGP and tyrosinase-related protein 2 (TRP2) peptide, a melanoma self-antigen, protected mice from melanoma challenge. Thus, these data demonstrated that RGP can be used as a mucosal adjuvant for inducing activation of immune responses in the lung.

Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo

Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP). The iTEP was able to solubilize CTL epitopes. CTL epitopes were connected to the carrier, iTEP-sMMP, through sMMP so that the epitopes can be cleaved from the carrier by MMP-9. iTEP-sMMP was found to release its epitope payloads in the DC culture media, which contained MMP-9 released from DCs. iTEP-sMMP allowed for the direct loading of CTL epitopes onto the surface MHC class I complexes of DCs. Importantly, iTEP-sMMP resulted in greater epitope presentation by DCs both in vitro and in vivo than a control carrier that cannot directly load epitopes. iTEP-sMMP also induced 2-fold stronger immune responses than the control carrier. To further enhance the direct epitope-loading strategy, we furnished iTEP-sMMP with an albumin-binding domain (ABD) and found the new carrier, ABD-iTEP-sMMP, had greater lymph node (LN) accumulation than iTEP-sMMP. ABD-iTEP-sMMP also resulted in greater immune responses than iTEP-sMMP by 1.5-fold. Importantly, ABD-iTEP-sMMP-delivered CTL epitope vaccine induced stronger immune responses than free CTL epitope vaccine. Taken together, these carriers utilized two physiological features of DCs to realize direct epitope-loading in vivo: the accumulation of DCs in LNs and MMP-9 released from DCs. These carriers are a potential substitute for DC-based CTL epitope vaccines.

Improved Induction of Anti-Melanoma T Cells by Adenovirus-5/3 Fiber Modification to Target Human DCs.

To mount a strong anti-tumor immune response, non T cell inflamed (cold) tumors may require combination treatment encompassing vaccine strategies preceding checkpoint inhibition. In vivo targeted delivery of tumor-associated antigens (TAA) to dendritic cells (DCs), relying on the natural functions of primary DCs in situ, represents an attractive vaccination strategy. In this study we made use of a full-length MART-1 expressing C/B-chimeric adenoviral vector, consisting of the Ad5 capsid and the Ad3 knob (Ad5/3), which we previously showed to selectively transduce DCs in human skin and lymph nodes. Our data demonstrate that chimeric Ad5/3 vectors encoding TAA, and able to target human DCs in situ, can be used to efficiently induce expansion of functional tumor-specific CD8+ effector T cells, either from a naïve T cell pool or from previously primed T cells residing in the melanoma-draining sentinel lymph nodes (SLN). These data support the use of Ad3-knob containing viruses as vaccine vehicles for in vivo delivery. “Off-the-shelf” DC-targeted Ad vaccines encoding TAA could clearly benefit future immunotherapeutic approaches.

Activation of Dendritic Cells in Dry Eye Mouse Model.

The immune system plays a major role in the pathogenesis of dry eye diseases (DED), and dendritic cells (DCs) are known to be important initiators of acquired immunity. Thus, the purpose of this study was to determine the contribution of DCs to the development of DED. Mouse dry eye model was induced by subcutaneous injections of scopolamine and was euthanized at the baseline, and 2, 4, and 7 days postinjection. The activation of the DCs was determined by the mixed leukocyte reaction (MLR), and the number of activated CD86+ DCs in the lymph nodes was determined by flow cytometry. Upregulation of cytokines in the culture supernatant of MLR was determined by ELISA. Significantly increased superficial corneal punctate lesions and decreased number of goblet cells in the conjunctiva were observed in scopolamine-injected mice. The number of activated CD86+ DCs was significantly increased in the cervical lymph nodes but not in the inguinal lymph nodes of the dry eye mice. The stimulatory activity of the DCs derived from the cervical lymph nodes of dry eye mice was significantly higher than that of control mice, and upregulations of IL-17, IL-2, and IL-4 were observed in the culture supernatant of MLR. These results indicate that the DCs of the cervical lymph nodes were activated by the scopolamine injections. Our results indicate that DCs in our dry eye model were sufficiently activated to stimulate the T cells that participate in the onset and progression of DED.

Rapid loss of early antigen-presenting activity of lymph node dendritic cells against Ag85A protein following Mycobacterium bovis BCG infection

BackgroundControl of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood.ResultsIn the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette–Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo.ConclusionsOur results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.

Dendritic cell intrinsic IRF4 expression regulates production of regulatory cytokines during influenza virus infection

Abstract Lung resident dendritic cells (DCs) are a heterogeneous population with diverse roles in respiratory infection. The development and function of DC subsets are controlled by distinct transcription factors including IRF4, which regulates CD11b+ cDC2 development, migration, and function. CD11c-cre Irf4fl/fl mice (KO) with a deletion of Irf4 in DCs lack a subset (P1) of lung CD11b+ DCs while other DCs are present but lack IRF4. To test the hypothesis that DC intrinsic IRF4 regulates anti-viral responses, wild type (WT) and KO mice were infected with a sublethal dose of influenza A/PuertoRico/8/34. On days 3–8 post-infection, a novel flow cytometry scheme was used to assess numbers and functional responses of lung and mediastinal lymph node (mLN) DCs, including tissue DC subsets CD103+ and CD11b+ (P1, P2), and inflammatory monocyte-derived Ly6Chi DCs. In addition to lacking CD11b+P1 DCs, KO mice had increased numbers of lung CD103+DCs, a defect in migration of CD11b+P2 DCs to the mLN and reduced numbers of Ly6Chi DCs in the mLN. In WT mice, CD11b+P1 DCs mice produced IL-10, and CD103+DCs preferentially displayed a latent form of TGFβ (LAP) and expressed Itgb8 needed to retain surface LAP for TGFβ activation. KO CD103+DCs showed reduced expression of TGFβ-LAP and Itgb8. Thus, DC production of both IL-10 and TGFβ is deficient in infected KO mice. The reduction of regulatory cytokines is consistent with our earlier finding that infected KO mice harbor decreased numbers of Foxp3+ T regulatory and CD8+ T memory cells, yet greater numbers of effector IFNg+ influenza-specific CD8+ T cells. In sum, IRF4 differentially regulates lung DC subsets to promote regulatory cytokines, and this diversity balances the anti-viral immune response to influenza infection.