Resident DCs Research Articles

Self-adjuvanted L-arginine-modified dextran-based nanogels for sustained local antigenic protein delivery to antigen-presenting cells and enhanced cellular and humoral immune responses.

In the development of cancer vaccines, antigens are delivered to elicit potent and specific T-cell responses to eradicate tumour cells. Nonetheless, successful vaccines are often hampered by the poor immunogenicity of tumour antigens, rapid clearance by the innate immunity, and limited cross-presentation on MHC-I to activate CD8+ T-cells arm. To address these issues, we developed dextran-based nanogels to promote antigen uptake, storage, and cross-presentation on MHC-I, while directing immunogenic maturation of the antigen-presenting cells (APCs). To promote the nanocarriers interaction with cells, we modified DX with L-arginine (Arg), whose immunomodulatory activities have been well documented. The ArgDX nanogel performance was compared with the nanogel modified with L-histidine (His) and L-glutamate (Glut). Moreover, we introduced pH-sensitive hydrazone crosslinking during the nanogel formation for the conjugation and controlled release of antigen ovalbumin (OVA). The OVA-laden nanogels have an average size of 325 nm. We demonstrated that the nanogels could rapidly release cargoes upon a pH change from 7 to 5 within 8 days, indicating the controlled release of antigens in the acidic cellular compartments upon internalization. Our results revealed that the ArgDX nanogel could promote greater antigen uptake and storage in DCs in vitro and promoted a stronger immunogenic maturation of DCs and M1 polarization of the macrophages. The OVA signals were co-localized with lysosomal compartments up till 96 hours post-treatment and washing, suggesting the nanogels could facilitate prolonged antigen storage and supply from endo-lysosomal compartments. Furthermore, all the tested nanogel formulations retained antigens at the skin injection sites until day 21. Such delayed clearance could be due to the formation of micron-sized aggregates of OVA-laden nanogels, extending the interactions with the resident DCs. Amongst the amino acid modifications, ArgDX nanogels promoted the highest level of lymph node homing signal CCR7 on DCs. The nanogels also showed higher antigen presentation on both MHC-I and II than DX in vitro. In the in vivo immune studies, ArgDX nanogels were more superior in inducing cellular and humoral immunity than the other treatment groups on day 21 post-treatment. These results suggested that ArgDX nanogel is a promising self-adjuvanted nanocarrier for vaccine delivery.

Elucidating the role of heat shock protein receptor CD91 on antigen presenting cells during cancer immunosurveillance

Abstract The premise of immunosurveillance is that the immune system, particularly by T cells, allows detection and elimination of emerging tumor cells. However, the mechanisms that allow efficient T cell priming against nascent tumors despite limited antigen availability remain unclear. We have shown that immunogenic heat shock proteins (HSPs) such as gp96 and their receptor CD91 are one such mechanism. HSPs efficiently chaperone minute levels of peptides from tumors to elicit antigen-specific immunity. Cross presentation of HSP-chaperoned peptides by CD91 +dendritic cells is a key mechanism for priming T cell responses. Diverse DC subsets vary in their capacity to modulate such anti-tumor immune responses. In this project, we aim to understand which CD91 +DC subset(s) is/are important for HSP-mediated cancer immunosurveillance. We generated mouse strains with deletion of CD91 on specific DC subsets. cDC1s are thought to be superior cross-presenters that excel in activation of cytotoxic lymphocytes. CD91 deletion in cDC1 render mice highly susceptible to chemically induced tumors. To better understand the CD91-HSP pathway, we tracked the dispersal of tumor-derived gp96 among myeloid cells using tumor cells that express EGFP-tagged gp96. We show that migratory DC subpopulations, such as CD103 +(cDC1) dendritic cells acquire gp96 from the tumor microenvironment, while a different DC subset (CD11b +CD11c +) transfers protective antitumor immunity. The hand-off of tumor-derived gp96 between migratory and lymph node resident DCs is currently under investigation. These results advance our understanding of the HSP-CD91 pathway and its role in DC function which can have significant implications for cancer prognosis and therapy. 5R01CA233803-04

Type 1 interferon mediated signaling is indispensable for eliciting anti-tumor responses by Mycobacterium indicus pranii.

The evolving tumor secretes various immunosuppressive factors that reprogram the tumor microenvironment (TME) to become immunologically cold. Consequently, various immunosuppressive cells like Tregs are recruited into the TME which in turn subverts the anti-tumor response of dendritic cells and T cells.Tumor immunotherapy is a popular means to rejuvenate the immunologically cold TME into hot. Mycobacterium indicus pranii (MIP) has shown strong immunomodulatory activity in different animal and human tumor models and has been approved for treatment of lung cancer (NSCLC) patients as an adjunct therapy. Previously, MIP has shown TLR2/9 mediated activation of antigen presenting cells/Th1 cells and their enhanced infiltration in mouse melanoma but the underlying mechanism by which it is modulating these immune cells is not yet known. This study reports for the first time that MIP immunotherapy involves type 1 interferon (IFN) signaling as one of the major signaling pathways to mediate the antitumor responses. Further, it was observed that MIP therapy significantly influenced frequency and activation of different subsets of T cells like regulatory T cells (Tregs) and CD8+ T cells in the TME. It reduces the migration of Tregs into the TME by suppressing the expression of CCL22, a Treg recruiting chemokine on DCs and this process is dependent on type 1 IFN. Simultaneously, in a type 1 IFN dependent pathway, it enhances the activation and effector function of the immunosuppressive tumor resident DCs which in turn effectively induce the proliferation and effector function of the CD8+ T cells. This study also provides evidence that MIP induced pro-inflammatory responses including induction of effector function of conventional dendritic cells and CD8+ T cells along with reduction of intratumoral Treg frequency are essentially mediated in a type 1 IFN-dependent pathway.

Dysfunction of Dendritic Cells in Tumor-Draining Lymph Nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific 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 (TDLN) and to identify the mechanisms that lead to defective tumor antigen presentation. We transplanted tumors into the flank of mice and analyzed DCs from TDLN and contralateral non-draining lymph nodes (NDLN). Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN either in vivo or 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 DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake by DCs from TDLN. Interestingly, DCs in TDLN also showed poor removal of the Invariant-chain CLIP peptide from the MHC-II peptide binding groove. This is likely to be due to a functional defect of the peptide-editor H2-M. Using Invariant-chain mutant mice and H2-M knock-out mice we confirmed that defective peptide exchange from MHC-II molecules hampered the anti-tumor responses and lead to enhanced tumor growth. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis could allow us to “correct” these DC defects, thereby allowing CD4 T cell priming to provide the necessary help for antitumor CD8 T cells.

Dendritic cells regulate innate cell trafficking into lymph nodes during inflammation

Abstract Dendritic cells (DCs) have well-established roles in antigen presentation, and we recently showed that within several hours of immunization, lymph node (LN) resident DCs undergo activation and reposition into the T cell zone (TZ) to drive early T cell activation. Concurrently, monocytes are also recruited into LNs via high endothelial venules (HEVs) and cooperate with DCs to enhance the generation of adaptive responses. However, the mechanisms driving monocyte recruitment are unknown. Here, we used mouse models of immunization, quantitative imaging, and RNA sequencing to study early innate responses in draining LNs. We find that during inflammation, monocytes as well as neutrophils rapidly infiltrated the LN parenchyma and the TZ, with the timing of cell influx being concordant with that of DC activation and repositioning. As the DCs migrated towards the TZ, they passed through regions highly enriched in HEVs. DCs also expressed multiple inflammatory chemokines involved in monocyte and neutrophil trafficking, as well as high levels of the integrin ligand, ICAM-1, thus generating chemokine- and adhesion molecule-rich perivascular microenvironments. Depletion of DCs or ablation of ICAM-1 in DCs significantly decreased innate cell trafficking into LNs, with the recruited cells being largely trapped near HEVs and less able to infiltrate the TZ. Disruption of DC repositioning with conditional CCR7 ablation also resulted in the mislocalization of neutrophils and monocytes within the LNs. Thus, in addition to their classical roles as antigen presenting cells, during inflammation, DCs facilitate the recruitment of several innate cell types into LNs and guide them into the TZ to promote the generation of functional immune responses. Supported by NIH grants R01AI134713, R21AI142667, T32GM007270 (J.H.), and F31AI161316 (J.H.).

The effect of topical decorin on temporal changes to corneal immune cells after epithelial abrasion

BackgroundCorneal immune cells interact with corneal sensory nerves during both homeostasis and inflammation. This study sought to evaluate temporal changes to corneal immune cell density in a mouse model of epithelial abrasion and nerve injury, and to investigate the immunomodulatory effects of topical decorin, which we have shown previously to promote corneal nerve regeneration.MethodsBilateral corneal epithelial abrasions (2 mm) were performed on C57BL/6J mice. Topical decorin or saline eye drops were applied three times daily for 12 h, 24 h, 3 days or 5 days. Optical coherence tomography imaging was performed to measure the abrasion area. The densities of corneal sensory nerves (β-tubulin III) and immune cells, including dendritic cells (DCs; CD11c+), macrophages (Iba-1+) and neutrophils (NIMP-R14+) were measured. Cx3cr1gfp/gfp mice that spontaneously lack resident corneal intraepithelial DCs were used to investigate the specific contribution of epithelial DCs. Neuropeptide and cytokine gene expression was evaluated using qRT-PCR at 12 h post-injury.ResultsIn decorin-treated corneas, higher intraepithelial DC densities and lower neutrophil densities were observed at 24 h after injury, compared to saline controls. At 12 h post-injury, topical decorin application was associated with greater re-epithelialisation. At 5 days post-injury, corneal stromal macrophage density in the decorin-treated and contralateral eyes was lower, and nerve density was higher, compared to eyes treated with saline only. Lower expression of transforming growth factor beta (TGF-β) and higher expression of CSPG4 mRNA was detected in corneas treated with topical decorin. There was no difference in corneal neutrophil density in Cx3cr1gfp/gfp mice treated with or without decorin at 12 h.ConclusionsTopical decorin regulates immune cell dynamics after corneal injury, by inhibiting neutrophils and recruiting intraepithelial DCs during the acute phase (< 24 h), and inhibiting macrophage density at the study endpoint (5 days). These immunomodulatory effects were associated with faster re-epithelialisation and likely contribute to promoting sensory nerve regeneration. The findings suggest a potential interaction between DCs and neutrophils with topical decorin treatment, as the decorin-induced neutrophil inhibition was absent in Cx3cr1gfp/gfp mice that lack corneal epithelial DCs. TGF-β and CSPG4 proteoglycan likely regulate decorin-mediated innate immune cell responses and nerve regeneration after injury.

Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus

ABSTRACT Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses and constitute a global public health threat. Since USUV and WNV are transmitted by mosquitoes, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively. Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism: WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV than WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favours its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific 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 presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo or 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 DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.

Contribution of a Hybrid Insulin Peptide-reactive T cell to Diabetes progression in the NOD mouse.

Abstract Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the immune destruction of pancreatic β cells. The search for natural equivalents to the mimetopes that can stimulate diabetogenic T cell clones from the Non-Obese Diabetic (NOD) mouse model of T1D led to the realization that hybrid peptides (HP) formed by the joining of β cell granule protein-derived peptide fragments are highly stimulatory to some clones. These hybrid peptides were later identified in vivo and proposed as a new class of post-translationally modified autoantigens mediating this disease. Recent data suggest high concentrations of these proteins in β cell lysosomes during granule turnover can lead to the hybridization of peptide fragments which may then be presented on MHC molecules. In the thymus, medullary thymic epithelial cells (mTECs) mediate T cell tolerance through promiscuous gene expression, but may not mediate HP-reactive T cell tolerance. We hypothesize that HP-reactive T cells preferentially escape the thymus and contribute significantly to T1D progression. We attempted to enhance T cell central tolerance to 2.5HIP, a hybrid insulin peptide formed from Proinsulin and Chromogranin A, the only two antigens known to be critical for T1D progression in the NOD mice, to better understand its impact on T1D incidence. Using an antibody-peptide conjugation system, we targeted 2.5HIP to a subset of thymic resident DCs to enhance presentation of 2.5HIP in early neonatal thymus. In neonatal BDC2.5 TCR-transgenic mice we found that a single dose of antibody-2.5HIP enhanced antigen-specific thymocyte apoptosis and regulatory T cell development. Importantly, a single dose in wild-type neonates resulted in a significant delay of diabetes onset.

IL-12 from endogenous cDC1, and not vaccine DC, is required for Th1 induction.

Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node–resident DCs for Th1 induction. While migration of the injected DCs was essential for antigen delivery to the lymph node, the injected DCs contributed only partially to Th0 priming and were unable to instruct Th1 generation. Instead, we provide evidence that the lymph node–resident XCR1+ DCs are activated by the injected DCs to present the cognate antigen and release IL-12 for Th1 polarization. The timing of interactions in the draining lymph nodes appeared step-wise as (a) injected DCs with cognate T cells, (b) injected DCs with bystander DCs, and (c) bystander DCs with T cells. The transcriptome of the bystander DCs showed a downregulation of Treg- and Th2/Th9-inducing genes and self-antigen presentation, as well as upregulation of MHC class II and genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory DCs direct T cell priming and bystander DC activation, but not Th1 polarization, which is mediated by endogenous IL-12p70+XCR1+ resident bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II molecules) and CD8 cytotoxic T cells (through MHC-I molecules). Activation of tumor-specific 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 presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to 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 TDLN was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.

Protein kinase C-eta is required for Treg-mediated suppression of anti-tumor and viral immunity

Abstract We previously reported that protein kinase C-eta plays an important role in the contact-dependent suppressive activity of Tregs via its association with CTLA4, and that PKC-eta-deficient (Prkch−/−) Tregs fail to suppress anti-melanoma tumor immunity. Here we extend this study to a genetically engineered mouse model of HCC driven by CRISPR-Cas9-driven deletion of Pten and p53. In the Pten-p53 HCC model, Prkch Treg-specific conditional knockout (cKO) mice developed a lower tumor incidence, fewer tumors, lower degree of steatosis phenotype, and showed higher intratumoral CD4+ T cells than WT mice. In addition, B cells and resident DCs displayed higher levels of the costimulatory ligand CD86 in dLNs of cKO mice. Increased CD4+ T cells, memory T lymphocytes, and cytokines production were observed in spleen of cKO mice. These results indicate that Treg-expressed PKC-eta is required for Treg-mediated suppression of anti-HCC tumor immunity. To further explore the importance of PKC-eta in Teff cells, the LCMVArm acute infection model, as well as the in vitro activation of murine or human CD8+ T cells were used. We found that purified Prkch−/− mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact T cell activation in vitro as measured by proliferation and expression of GzmB and IFNg. Interestingly, Treg-specific cKO mice showed improved viral clearance and displayed enhanced expression of GzmB and IFNg by virus-specific CD8+ T cells. Thus, global PKC-eta deletion does not impair overall CD8+ T cell-mediated immunity implying that selective pharmacological PKC-eta inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor- and viral-specific immunity.

Dendritic Cells of Mesenteric and Regional Lymph Nodes Contribute to Yersinia enterocolitica O:3-Induced Reactive Arthritis in TNFRp55-/- Mice.

Dendritic cells (DCs) participate in the pathogenesis of several diseases. We investigated DCs and the connection between mucosa and joints in a murine model of Yersinia enterocolitica O:3-induced reactive arthritis (ReA) in TNFRp55-/- mice. DCs of mesenteric lymph nodes (MLN) and joint regional lymph nodes (RLN) were analyzed in TNFRp55-/- and wild-type mice. On day 14 after Y. enterocolitica infection (arthritis onset), we found that under TNFRp55 deficiency, migratory (MHChighCD11c+) DCs increased significantly in RLN. Within these RLN, resident (MHCintCD11c+) DCs increased on days 14 and 21. Similar changes in both migratory and resident DCs were also detected on day 14 in MLN of TNFRp55-/- mice. In vitro, LPS-stimulated migratory TNFRp55-/- DCs of MLN increased IL-12/23p40 compared with wild-type mice. In addition, TNFRp55-/- bone marrow-derived DCs in a TNFRp55-/- MLN microenvironment exhibited higher expression of CCR7 after Y. enterocolitica infection. The major intestinal DC subsets (CD103+CD11b-, CD103-CD11b+, and CD103+CD11b+) were found in the RLN of Y. enterocolitica-infected TNFRp55-/- mice. Fingolimod (FTY720) treatment of Y. enterocolitica-infected mice reduced the CD11b- subset of migratory DCs in RLN of TNFRp55-/- mice and significantly suppressed the severity of ReA in these mice. This result was associated with decreased articular IL-12/23p40 and IFN-γ levels. In vitro FTY720 treatment downregulated CCR7 on Y. enterocolitica-infected bone marrow-derived DCs and purified MLN DCs, which may explain the mechanism underlying the impairment of DCs in RLN induced by FTY720. Taken together, data indicate the migration of intestinal DCs to RLN and the contribution of these cells in the immunopathogenesis of ReA, which may provide evidence for controlling this disease.

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.

Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy.

Skin is protected by a tough but flexible multilayered barrier and is a front line for immune responses against invading particles. For many years now, skin has been a tissue where certain vaccines are injected for the prevention of infectious disease, however, the detailed mechanisms of the skin immune response are not yet well understood. Using thin and small injection needles, we carefully injected OVA into a restricted region of mouse skin, i.e., intradermal (ID), and examined the antibody response in comparison with subcutaneous (SC) injection or epicutaneous patch administration of OVA. Epicutaneous patches induced a high IgE response against OVA, but IgG production was low. High IgG production was induced by both ID and SC injection, moreover, ID injection induced higher IgG production without any adjutants. Furthermore, OVA-specific IgE production was diminished by ID injection. We found that ID injection could efficiently stimulate skin resident DCs, drive Th1-biased conditions and diminish IgE production. The ID injection response was regulated by Langerin+ dermal DCs, because OVA was taken up mainly by these cells and, after transiently deleting them, the IgE response was no longer diminished and IgG1 production was enhanced. We also tested whether ID injection might be an effective allergy treatment by attempting to inhibit ongoing IgE production in mice with experimentally induced high serum IgE levels. Multiple ID injections of OVA were shown to prevent elevation of serum OVA-specific IgE after repeated allergen challenge. In contrast, SC OVA injection could only transiently inhibit the OVA-specific IgE production. These findings indicated that ID injection results in higher induction of antigen-specific IgG, and thus may be useful for vaccine delivery with little or no adjuvant components. Moreover, the observed diminishment of IgE and induction of Th1-biased immune responses suggest that ID may be a useful injection route for allergy immunotherapy.

Monocyte-Derived Dendritic Cells Promote Th Polarization, whereas Conventional Dendritic Cells Promote Th Proliferation.

Monocyte-derived dendritic cells (moDCs) dramatically increase in numbers upon infection and inflammation; accordingly, we found that this also occurs during allogeneic responses. Despite their prominence, how emergent moDCs and resident conventional DCs (cDCs) divide their labor as APCs remain undefined. Hence, we compared both direct and indirect presentation by murine moDCs versus cDCs. We found that, despite having equivalent MHC class II expression and in vitro survival, moDCs were 20-fold less efficient than cDCs at inducing CD4(+) T cell proliferation through both direct and indirect Ag presentation. Despite this, moDCs were more potent at inducing Th1 and Th17 differentiation (e.g., 8-fold higher IFN-γ and 2-fold higher IL-17A in T cell cocultures), whereas cDCs induced 10-fold higher IL-2 production. Intriguingly, moDCs potently reduced the ability of cDCs to stimulate T cell proliferation in vitro and in vivo, partially through NO production. We surmise that such division of labor between moDCs and cDCs has implications for their respective roles in the immune response.

Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.

Malaria infection begins when a female Anopheles mosquito injects Plasmodium sporozoites into the skin of its host during blood feeding. Skin-deposited sporozoites may enter the bloodstream and infect the liver, reside and develop in the skin, or migrate to the draining lymph nodes (DLNs). Importantly, the DLN is where protective CD8+ T cell responses against malaria liver stages are induced after a dermal route of infection. However, the significance of parasites in the skin and DLN to CD8+ T cell activation is largely unknown. In this study, we used genetically modified parasites, as well as antibody-mediated immobilization of sporozoites, to determine that active sporozoite migration to the DLNs is required for robust CD8+ T cell responses. Through dynamic in vivo and static imaging, we show the direct uptake of parasites by lymph-node resident DCs followed by CD8+ T cell-DC cluster formation, a surrogate for antigen presentation, in the DLNs. A few hours after sporozoite arrival to the DLNs, CD8+ T cells are primed by resident CD8α+ DCs with no apparent role for skin-derived DCs. Together, these results establish a critical role for lymph node resident CD8α+ DCs in CD8+ T cell priming to sporozoite antigens while emphasizing a requirement for motile sporozoites in the induction of CD8+ T cell-mediated immunity.

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC-derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady-state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α⁻CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α-galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α⁻ and CD8α⁺ splenic DC subsets and enhances cross-presentation of exogenous antigens. Based on genome-wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN-γ-mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN-γR and are much less responsive to IFN-γ. Under inflammatory conditions, particularly in the absence of IFN-γ signaling in IFN-γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM-CSF in concert with IL-4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.

Soluble antigen traffics rapidly and selectively from the corneal surface to the eye draining lymph node and activates T cells when codelivered with CpG oligonucleotides

The transport of antigen to the secondary lymphoid tissue is a central component in the initiation of the adaptive immune response. The mechanism of antigen delivery to the DLN from the avascular cornea has not been fully explored. Previous studies in the mouse have shown that cell-associated corneal antigen is delivered within 6 h to the eye draining SM DLN via DCs and macrophages. In this study, we used a system in which antigen and the processed p-MHCII complexes derived from the antigen could be tracked in vivo. We report that soluble antigen applied to an abraded cornea in the mouse is transported rapidly (within 30 min) to the SM DLN, where a proportion is taken up by resident DCs and presented as p-MHCII complexes, while the larger part is cleared by 8 h. At a later time, a second wave of antigen transport in migratory DCs enters the DLN and participates in further continued antigen presentation. With the use of an antigen-specific TCR transgenic mouse system, we demonstrate that T cell activation does not occur during the early stages of soluble antigen delivery to LN, even though p-MHCII complexes are generated. Antigen-specific T cell activation occurs in the later, presumed cell-associated phase but requires codelivery of a "danger" signal, such as the TLR ligand CpG. We suggest that the early delivery of soluble antigen is more likely to induce T cell nonresponsiveness (anergy) unless presented in the context of an innate-immune cell activation (danger) signal.

Effects of sublingual immunotherapy on a murine model of Japanese cedar pollinosis (P4198)

Abstract Japanese cedar (Cryptomeria japonica) pollinosis (JCP) is one of the most common allergic diseases in Japan. Sublingual immunotherapy (SLIT) has been performed as a safer and more beneficial treatment to induce allergen-specific immune tolerance. However, immunological mechanisms and therapeutic biomarkers for SLIT remain unclear. In this study, we have established a new murine model of JCP that resembles human JCP with crude pollen grains and without the use of adjuvant and examined the effects of SLIT. SLIT was performed by the graded doses of cedar pollen extract painting onto the ventral surface of tongue for 28 days. [Results] The frequency of sneezing and the duration of rubbing for 10 min after the final challenge, serum IgE titer, pollen-specific IgG1, and the ratio of eosinophils in BALF and nasal mucosae were significantly elevated in the pollinosis mice. SLIT efficiently reduced the above manifestations. SLIT significantly decreased pollen-specific IL-4, IL-5, IL-13 and IL-10 production by splenic T cells and DC activation status such as MHC class II, CD86, and B7-H1 expression in the regional lymph nodes. Histological examination revealed that repeated pollen extract painting onto the sublingual mucosae replaced sublingual resident DCs with newly recruited CD11b+ DCs. These results suggest that our new pollinosis model is beneficial to evaluate and develop a new immunotherapy and therapeutic biomarkers.