Conventional Dendritic Cell Subsets Research Articles

GM-CSF-dependent CD301b+ lung dendritic cells confer tolerance to inhaled allergens.

The severity of allergic asthma is driven by the balance between allergen-specific T regulatory (Treg) and T helper (Th)2 cells. However, it is unclear whether specific subsets of conventional dendritic cells (cDCs) promote the differentiation of these two T cell lineaeges. We have identified a subset of lung resident type 2 cDCs (cDC2s) that display high levels of CD301b and have potent Treg-inducing activity ex vivo. Single cell RNA sequencing and adoptive transfer experiments show that during allergic sensitization, many CD301b+ cDC2s transition in a stepwise manner to CD200+ cDC2s that selectively promote Th2 differentiation. GM-CSF augments the development and maintenance of CD301b+ cDC2s in vivo, and also selectively expands Treg-inducing CD301b+ cDC2s derived from bone marrow. Upon their adoptive transfer to recipient mice, lung-derived CD301b+ cDC2s confer immunological tolerance to inhaled allergens. Thus, GM-CSF maintains lung homeostasis by increasing numbers of Treg-inducing CD301b+ cDC2s.

Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Focused ultrasound ablation of melanoma with boiling histotripsy yields abscopal tumor control and antigen-dependent dendritic cell activation.

Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.

In Vivo Evidence of Respiratory Syncytial Virus Persistence in a Subset of Pulmonary Dendritic Cells Following a Primary Infection.

Respiratory syncytial virus (RSV) causes annual epidemics of infections affecting the whole population. In vitro, it has been shown to infect and persist in human dendritic cells (DCs) for prolonged periods. Initially persistence is associated with low levels of replication before the virus becomes dormant. Reactivation of viral replication can be triggered many months later. Infection of DCs is likely to influence the host's ability to generate effective long-term memory responses. A well-established animal was utilized to confirm that RSV both infects and persists in pulmonary DCs in vivo. Mice were infected with a modified strain of RSV expressing red fluorescent protein (RSV-RFP) when replicating. Clinical symptoms of infection were monitored using weight change and inflammatory cell counts from bronchoalveolar lavage, which correlated with the RSV viral titer (quantitative polymerase chain reaction). Lung tissues were collected at 3, 5, 7, and 21 days postinfection (dpi) to assess leukocyte populations by flow cytometry. Clinical symptoms and RSV viral load peaked at 5 dpi. RSV-RFP was most prevalent in macrophages at 3 dpi and also observed in B cells and DCs. At 21 dpi, RSV-RFP remained evident in a subset of conventional DCs (CD103+CD11b+) even though both clinical symptoms and pulmonary inflammation had resolved. These results confirm that in this well-established mouse model, RSV persists in lung conventional DCs following resolution of the acute infection. Further work is required to explore whether the virus continues with low-level replication before becoming dormant in vivo, as has been described in vitro.

Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice.

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.

CD200Fc limits dendritic cell and B-cell activation during chronic allergen exposures.

Allergic asthma is a chronic inflammatory disease characterized by Th2, conventional dendritic cell, and B-cell activation. In addition to excessive inflammation, asthma pathogenesis includes dysregulation of anti-inflammatory pathways, such as the CD200/CD200R pathway. Thus, we investigated whether a CD200R agonist, CD200Fc, could disrupt the inflammatory cascade in chronic allergic asthma pathogenesis using a mice model of experimental asthma. Mice were exposed to house dust mites for 5 wk, and CD200Fc treatment was initiated after chronic inflammation was established (starting on week 4). We demonstrate that chronic house dust mite exposure altered CD200 and CD200R expression on lung immune cell populations, including upregulation of CD200 on alveolar macrophages and reduced expression of CD200 on conventional dendritic cells. CD200Fc treatment does not change bronchoalveolar cellular infiltration, but it attenuates B-cell activation and skews the circulating immunoglobulin profile toward IgG2a. This is accompanied by reduced activation of conventional dendritic cells, including lower expression of CD40, especially on conventional dendritic cell subset 2 CD200R+. Furthermore, we confirm that CD200Fc can directly modulate conventional dendritic cell activation in vitro using bone marrow-derived dendritic cells. Thus, the CD200/CD200R pathway is dysregulated during chronic asthma pathogenesis, and the CD200R agonist modulates B-cell and dendritic cell activation but, in our chronic model, is not sufficient to alter inflammation measured in bronchoalveolar lavage.

Aberrant Dendritic Cell Subsets in Patients with Myasthenia Gravis and Related Clinical Features

Introduction: Dendritic cells (DCs) play critical roles in the pathogenesis of myasthenia gravis (MG), and a series of DC-based experimental strategies for MG have recently been developed. However, the definite roles of different DC subsets in the mechanism of MG have scarcely been covered by previous studies. The present study aimed to investigate the levels of three main DC subsets, plasmacytoid DCs (pDCs) (CD303 positive) and two distinct subsets of conventional DCs (cDCs), namely CD1c⁺ cDCs and CD141⁺ cDCs, in MG patients and analyze related clinical features. Methods: From January 2016 to December 2020, 160 newly diagnosed MG patients and matched healthy controls (n = 160) were included in the study, and their clinical data were collected. The blood samples from MG patients before treatment and controls were collected for flow cytometry analysis. A total of 14 MG thymoma, 24 control thymoma, and 3 thymic cysts were used to immunostain the DC subsets. Results: The flow cytometry analysis showed a significantly higher frequency of circulating pDCs, CD1c⁺ cDCs, and CD141⁺ cDCs in MG patients than in healthy controls (p < 0.001 for all). Patients with early-onset MG (<50 years old) had a lower frequency of circulating pDCs but a higher frequency of circulating CD1c⁺ cDCs than those with late-onset MG (≥50 years old) (p = 0.014 and p = 0.025, respectively). The frequency of circulating pDCs was positively associated with the clinical severity of late-onset MG patients (r = 0.613, p < 0.001). 64.3% (9/14) of MG thymoma is of type B2 under the World Health Organization classification, which is higher than that in control thymoma (33.3%, 8/24) (p = 0.019). For type B2 thymoma, there were significantly more pDCs but fewer CD1c⁺ cDCs in MG thymoma than in the controls. Conclusion: The distribution of aberrant pDCs, CD1c⁺ cDCs, and CD141⁺ cDCs in MG patients displayed age- and thymoma-related differences, which may contribute to the impaired immune tolerance and lead to the onset of MG.

Effects of Oral Exposure to Low-Dose Bisphenol S on Allergic Asthma in Mice.

Bisphenol S (BPS) is increasingly being used as an alternative for bisphenol A; however, its health effects remain unclear. We investigated the effects of oral exposure to low-dose BPS on allergic asthma. C3H/HeJ male mice were intratracheally administered with allergen (ovalbumin (OVA), 1 μg/animal) every 2 weeks from 6 to 11 weeks old. BPS was ingested by drinking water at doses equivalent to 0.04, 0.4, and 4 μg/kg/day. We then examined pulmonary inflammation, airway hyperresponsiveness, serum OVA-specific immunoglobulin (Ig) levels, Th2 cytokine/chemokine production, and mediastinal lymph node (MLN) cell activities. Compared with OVA alone, moderate-dose BPS (BPS-M) with OVA significantly enhanced pulmonary inflammation, airway hyperresponsiveness, and OVA-specific IgE and IgG1. Furthermore, interleukin (IL)-5, IL-13, IL-33, and CCL11/Eotaxin protein levels in the lungs increased. Conversely, these allergic responses were reduced in the high-dose BPS+OVA group. In MLN cells, BPS-M with OVA increased the total cell count and activated antigen-presenting cells including conventional dendritic cell subset (cDC2). After OVA restimulation, cell proliferation and Th2 cytokine production (IL-4, IL-5, and IL-13) in the culture supernatant also increased. Therefore, oral exposure to low-dose BPS may exacerbate allergic asthmatic responses by enhancing Th2-polarized responses and activating the MLN cells.

Inositol Requiring Enzyme 1, a metabolic sensor at the heart of dendritic cell biology

Three endoplasmic reticulum (ER) resident proteins, IRE1, PERK and ATF6 monitor the health status of the ER and initiate the unfolded protein response (UPR) to restore ER homeostasis during stress. IRE1, a conserved endonuclease, cleaves Xbp1 mRNA and generates the key transcription factor XBP1s. My lab studies the role of IRE1 in dendritic cells (DCs), antigen presenting cells that bridge innate and adaptive immune responses and are essential both for the generation of effective immunity and tolerance. We noticed that in vivo one subset of conventional DCs -cDC1s - displays constitutive IRE1 activity, in absence of a canonical UPR response. Despite strong activation of IRE1 and concomitant induction of XBP1 splicing in cDC1s, we never observed any typical XBP1-dependent gene expression and we undertook a RNA sequencing approach to retrieve a DC-specific gene signature for XBP1. To this end, we compared WT, XBP1KO and IRE1/XBP1DKO cDC1s, all isolated from the spleen. This comparison allowed us to dissect genes downregulated due to loss of XBP1 transcriptional activity versus genes downregulated due to the activation of Regulated IRE1 Dependent Decay (RIDD), a process driven by hyperactivation of IRE1 endonuclease activity in the absence of XBP1. Our analysis revealed that many genes in cDC1s appeared regulated in an IRE1 endonuclease-but not XBP1-dependent manner. Furthermore, the data point towards a novel physiological role for IRE1 in DC homeostasis and provide unique cues for IRE1 activation in DCs, which will be the main topic of this talk.

Contributions of the PD-1/PD-L1 pathway to Borrelia persistence and inflammation

Abstract Borrelia burgdorferi, the causative agent of Lyme disease, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. Exhausted T cells are present in many long-term infections and mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Indeed, we have found that CD4+ T cells in the draining inguinal and popliteal lymph nodes, as well as the heart and ankle joint, significantly upregulate the immunoinhibitory molecule PD-1 following B. burgdorferi infection. While PD-1 expression on T cells is a strong indication of CD4+ T cell exhaustion, we have extended our findings by assessing expression of a ligand for PD-1, PD-L1, on antigen presenting cells in vitro and in vivo following B. burgdorferi infection. We have shown that not only is PD-L1 is significantly upregulated on both bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) following B. burgdorferi stimulation, but is also upregulated on macrophages and a subset of conventional dendritic cells (cDC2s) in the draining lymph nodes following in vivo infection. We are currently testing if mice deficient in either PD-1 or PD-L1 exhibit changes in ankle joint or heart inflammation compared to wild-type mice, which would suggest that the PD-1/PD-L1 axis is critical for regulating inflammation during B. burgdorferi infection. Together, these data will allow us to determine how this immune checkpoint regulates inflammation during B. burgdorferi infection. Supported by grants from the NIH (R01 AI150157), the Harvard Medical School Fairbairn Family Lyme Research Initiative, and the Tufts University School of Medicine Natalie Zucker Research Center for Women's Scholars Award.

DNMT1 Deficiency Impacts on Plasmacytoid Dendritic Cells in Homeostasis and Autoimmune Disease.

Dendritic cells (DCs) are heterogeneous immune regulators involved in autoimmune diseases. Epigenomic mechanisms orchestrating DC development and DC subset diversification remain insufficiently understood but could be important to modulate DC fate for clinical purposes. By combining whole-genome methylation assessment with the analysis of mice expressing reduced DNA methyltransferase 1 levels, we show that distinct DNA methylation levels and patterns are required for the development of plasmacytoid DC and conventional DC subsets. We provide clonal in vivo evidence for DC lineage establishment at the stem cell level, and we show that a high DNA methylation threshold level is essential for Flt3-dependent survival of DC precursors. Importantly, reducing methylation predominantly depletes plasmacytoid DC and alleviates systemic lupus erythematosus in an autoimmunity mouse model. This study shows how DNA methylation regulates the production of DC subsets and provides a potential rationale for targeting autoimmune disease using hypomethylating agents.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.

Semaphorin 3E deficiency dysregulates dendritic cell functions: In vitro and in vivo evidence.

Regulation of dendritic cell functions is a complex process in which several mediators play diverse roles as a network in a context-dependent manner. The precise mechanisms underlying dendritic cell functions have remained to be addressed. Semaphorins play crucial roles in regulation of various cell functions. We previously revealed that Semaphorin 3E (Sema3E) contributes to regulation of allergen-induced airway pathology partly mediated by controlling recruitment of conventional dendritic cell subsets in vivo, though the underlying mechanism remained elusive. In this study, we investigate the potential regulatory role of Sema3E in dendritic cells. We demonstrated that bone marrow-derived dendritic cells differentiated from Sema3e-/- progenitors have an enhanced migration capacity both at the baseline and in response to CCL21. The enhanced migration ability of Sema3E dendritic cells was associated with an overexpression of the chemokine receptor (CCR7), elevated Rac1 GTPase activity and F-actin polymerization. Using a mouse model of allergic airway sensitization, we observed that genetic deletion of Sema3E leads to a time dependent upregulation of CCR7 on CD11b+ conventional dendritic cells in the lungs and mediastinal lymph nodes. Furthermore, aeroallergen sensitization of Sema3e-/- mice lead to an enhanced expression of PD-L2 and IRF-4 as well as enhanced allergen uptake in pulmonary CD11b+ DC, compared to wild type littermates. Collectively, these data suggest that Sema3E implicates in regulation of dendritic cell functions which could be considered a basis for novel immunotherapeutic strategies for the diseases associated with defective dendritic cells in the future.

Quantitation of corticosterone-induced splenic dendritic cell loss, recovery and prevention in C57BL/6 mice

Dendritic cells (DCs) link the innate and adaptive immune response by capturing antigen to present to and activate naïve T cells. Subsets of DCs vary based on protein expression, which allows for their identification. This research focused on total DCs (CD11c+), and the conventional DC subsets (cDC1 and cDC2). Corticosterone (CORT), a stress-induce glucocorticoid hormone, reduces DC numbers and alters DC function and subsequent T cell responses. The current study quantifies DC recovery after CORT exposure and demonstrates that the loss of DCs can be prevented. Mice were exposed to exogenous CORT in the drinking water for 24 hours. CORT exposure significantly decreased the numbers of all splenic DCs. Mice were then provided varying recovery times (3, 5, or 10 days) post-CORT exposure. DCs quickly returned to normal levels, and even exceeded the percentages in control untreated mice. Studies have shown that injection of mice with anti-CD40 antibody triggers DC maturation and proliferation. Thus, it was hypothesized that injection of anti-CD40 may prevent the CORT-induced loss of DCs. Mice received two injections of anti-CD40 antibody over two days and were then immediately supplied with CORT. When provided anti-CD40 prior to CORT, the loss of DCs was prevented. These studies contribute to the ongoing exploration of the mechanisms underlying the immunological effects of stress-induced glucocorticoids.

Innate immune cells in the tumor microenvironment.

The tumor immune microenvironment (TIME) is a complex ecosystem that contains adaptive and innate immune cells that have tumor-promoting and anti-tumor effects. There is still much to learn about the diversity, plasticity, and functions of innate immune cells in the TIME and their roles in determining the response to immunotherapies. Experts discuss recent advances in our understanding of their biology in cancer as well as outstanding questions and potential therapeutic avenues.

Revisiting Current Concepts on the Tolerogenicity of Steady-State Dendritic Cell Subsets and Their Maturation Stages

The original concept stated that immature dendritic cells (DC) act tolerogenically whereas mature DC behave strictly immunogenically. Meanwhile, it is also accepted that phenotypically mature stages of all conventional DC subsets can promote tolerance as steady-state migratory DC by transporting self-antigens to lymph nodes to exert unique functions on regulatory T cells. We propose that in vivo 1) there is little evidence for a tolerogenic function of immature DC during steady state such as CD4 T cell anergy induction, 2) all tolerance as steady-state migratory DC undergo common as well as subset-specific molecular changes, and 3) these changes differ by quantitative and qualitative markers from immunogenic DC, which allows one to clearly distinguish tolerogenic from immunogenic migratory DC.

Th17 Cell-Mediated Colitis Is Positively Regulated by Interferon Regulatory Factor 4 in a T Cell-Extrinsic Manner.

Inflammatory bowel diseases (IBDs) are characterized by chronic, inflammatory gastrointestinal lesions and often require life-long treatment with immunosuppressants and repetitive surgical interventions. Despite progress in respect to the characterization of molecular mechanisms e.g. exerted by TNF-alpha, currently clinically approved therapeutics fail to provide long-term disease control for most patients. The transcription factor interferon regulatory factor 4 (IRF4) has been shown to play important developmental as well as functional roles within multiple immune cells. In the context of colitis, a T cell-intrinsic role of IRF4 in driving immune-mediated gut pathology is established. Here, we conversely addressed the impact of IRF4 inactivation in non-T cells on T cell driven colitis in vivo. Employing the CD4+CD25− naïve T cell transfer model, we found that T cells fail to elicit colitis in IRF4-deficient compared to IRF4-proficient Rag1 −/− mice. Reduced colitis activity in the absence of IRF4 was accompanied by hampered T cell expansion both within the mesenteric lymph node (MLN) and colonic lamina propria (cLP). Furthermore, the influx of various myeloids, presumably inflammation-promoting cells was abrogated overall leading to a less disrupted intestinal barrier. Mechanistically, gene profiling experiments revealed a Th17 response dominated molecular expression signature in colon tissues of IRF4-proficient, colitic Rag1 −/− but not in colitis-protected Rag1 −/− Irf4 −/− mice. Colitis mitigation in Rag1 −/− Irf4 −/− T cell recipients resulted in reduced frequencies and absolute numbers of IL-17a-producing T cell subsets in MLN and cLP possibly due to a regulation of conventional dendritic cell subset 2 (cDC2) known to impact Th17 differentiation. Together, extending the T cell-intrinsic role for IRF4 in the context of Th17 cell driven colitis, the provided data demonstrate a Th17-inducing and thereby colitis-promoting role of IRF4 through a T cell-extrinsic mechanism highlighting IRF4 as a putative molecular master switch among transcriptional regulators driving immune-mediated intestinal inflammation through both T cell-intrinsic and T cell-extrinsic mechanisms. Future studies need to further dissect IRF4 controlled pathways within distinct IRF4-expressing myeloid cell types, especially cDC2s, to elucidate the precise mechanisms accounting for hampered Th17 formation and, according to our data, the predominant mechanism of colitis protection in Rag1 −/− Irf4 −/− T cell receiving mice.

Essential role of submandibular lymph node dendritic cells in protective sublingual immunotherapy against murine allergy.

While sublingual immunotherapy (SLIT) is known as an allergen-specific treatment for type-1 allergies, how it controls allergic pathogenesis remains unclear. Here, we show the prerequisite role of conventional dendritic cells in submandibular lymph nodes (ManLNs) in the effectiveness of SLIT for the treatment of allergic disorders in mice. Deficiency of conventional dendritic cells or CD4+Foxp3+ regulatory T (Treg) cells abrogates the protective effect of SLIT against allergic disorders. Furthermore, sublingual antigenic application primarily induces antigen-specific CD4+Foxp3+ Treg cells in draining ManLNs, in which it is severely impaired in the absence of cDCs. In ManLNs, migratory CD11b+ cDCs are superior to other conventional dendritic cell subsets for the generation of antigen-specific CD4+Foxp3+ Treg cells, which is reflected by their dominancy in the tolerogenic features to favor this program. Thus, ManLNs are privileged sites in triggering mucosal tolerance mediating protect effect of SLIT on allergic disorders that requires a tolerogenesis of migratory CD11b+ conventional dendritic cells.

IMMU-26. UNRAVELING ANTIGEN PRESENTATION IN CENTRAL NERVOUS SYSTEM ANTI-TUMOR IMMUNITY

Abstract The antigen presenting cell that primes T cells in the central nervous system (CNS) remains unknown. Outside the CNS, the conventional dendritic cell 1 (cDC1) subset presents antigen to and primes CD8 T cells. However, the steady-state CNS parenchyma is relatively devoid of all dendritic cell subsets, including cDC1. cDC1 are required for anti-tumor immunity a variety of other tumor types, but their role CNS tumors remains undefined. Using the orthotopic preclinical glioblastoma models, GL261 and CT2A, we characterized the role of cDC1 in the CNS anti-tumor immune response. While cDC1 are absent in the steady state brain, tumor presence drove recruitment of cDC1 into extravascular spaces within the tumor and adjacent brain parenchyma. We further found that while GL261-bearing wildtype mice experienced survival benefit following anti-PDL1 checkpoint blockade treatment, mice with cDC1 genetically deleted experienced no survival benefit. cDC1-deficient mice completely lacked neoantigen-specific CD8 T cells against the endogenously-primed GL261-neoantigen mutant Imp3, and possessed broad CD8 effector T cell defects compared to wild type mice. Furthermore, using a fluorescent tumor-associated reporter, we detected tumor-derived material within dendritic cells from the tumor, the lymphatic vessel-containing dura, and the cervical lymph nodes. We observed the human cDC1-equivalent CD141+ cDC within human brain tumors (not limited to GBM) and dura as well. We used the GBM-specific reporter, 5-aminoilevulinic acid/protoporphyrin IX (PPIX) fluorescent metabolite to resect the tumor, and observed PPIX specifically in conventional dendritic cell subsets that had infiltrated the resected tumor, but not within those same cell subsets in the periphery, nor in T cells within the tumor. These findings comport with the canonical understanding that cDC1 uptake antigen at the effector site and migrate to draining lymph nodes to prime effector CD8 T cells, and highlight the significant role that cDC1 play in CNS anti-tumor immunity in mice and humans.

Isotype-specific outcomes in Fc gamma receptor targeting of PspA using fusion proteins as a vaccination strategy against Streptococcus pneumoniae infection

Streptococcus pneumoniae (Spn) remains a considerable threat to public health despite the availability of antibiotics and polysaccharide conjugate vaccines. The lack of mucosal immunity in addition to capsular polysaccharide diversity, has proved to be problematic in developing a universal vaccine against Spn. Targeting antigen to Fc receptors is an attractive way to augment both innate and adaptive immunity against mucosal pathogens, by promoting interactions with activating Fcγ receptors (FcγR) that mediate diverse immunomodulatory functions. The effect of targeting FcγR is highly influenced by the IgG subclass, which bares differential affinities for activating and inhibitory FcγR. In the current study we demonstrate targeting activating FcγR with fusion proteins consisting of PspA and IgG2a Fc enhance PspA-specific immune responses, and effectively protect against mucosal Spn challenge. Specifically, targeting PspA to FcγR polarized alveolar macrophage to the AM1 phenotype and increased conventional dendritic cell subsets in the lung in addition to augmenting Th1 cytokines and PspA-specific IgG and IgA. In contrast, fusion proteins consisting of PspA fused to the IgG1 Fc provided minimal benefit over administration of PspA alone, as a result of interaction with the inhibitory FcγRIIB. Protective efficacy of the IgG1 fusion protein was significantly enhanced in animals deficient for FcγRIIB accompanied by increased B cell maturation and proliferation levels in these animals. These studies demonstrate FcγR targeting is an effective strategy for inducing potent cellular and humoral responses via mucosal immunization with Fc fusion proteins, however, careful consideration of the Fc region utilized is required since Fc isotype subclass heavily influenced immunization induced effector functions and survival against lethal Spn challenge. Fc-engineering with specific attention to FcγRIIB engagement presents a valuable vaccine strategy for protecting against Spn infection.