Key Antigen-presenting Cells Research Articles

Langerhans cells and skin immune diseases.

Langerhans cells (LCs) are the key antigen-presenting cells in the epidermis in normal conditions and respond differentially to environmental and/or endogenous stimuli, exerting either proinflammatory or anti-inflammatory effects. Current knowledge about LCs mainly originates from studies utilizing mouse models, whereas with the development of single-cell techniques, there has been significant progress for human LCs, which has updated our understanding of the phenotype, ontogeny, differentiation regulation, and function of LCs. In this review, we delineated the progress of human LCs and summarized LCs' function in inflammatory skin diseases, providing new ideas for precise regulation of LC function in the prevention and treatment of skin diseases.

Activated B-Cells enhance epitope spreading to support successful cancer immunotherapy.

Immune checkpoint therapies (ICT) have transformed the treatment of cancer over the past decade. However, many patients do not respond or suffer relapses. Successful immunotherapy requires epitope spreading, but the slow or inefficient induction of functional antitumoral immunity delays the benefit to patients or causes resistances. Therefore, understanding the key mechanisms that support epitope spreading is essential to improve immunotherapy. In this review, we highlight the major role played by B-cells in breaking immune tolerance by epitope spreading. Activated B-cells are key Antigen-Presenting Cells (APC) that diversify the T-cell response against self-antigens, such as ribonucleoproteins, in autoimmunity but also during successful cancer immunotherapy. This has important implications for the design of future cancer vaccines.

The reproducibility of dendritic cell and T cell counts to a 30-min high-intensity cycling protocol as a tool to highlight overtraining.

Heavy training has been reported to be immunosuppressive in athletes and lead to blunted cortisol responses to exercise. Cortisol elevates the number of dendritic cells (DCs), key antigen-presenting cells that interact with T cells to initiate an immune response. Reproducible cortisol responses to a 30-min cycle test have been identified but were based on percentage of work rate maximum. To ensure physiological consistency, submaximal anchors, that is, ventilatory threshold (VT1 ) should prescribe intensity. This study aims to assess the reproducibility of the DC and T cell responses to an adapted stress test to assess its usefulness in assessing DC dysfunction with intensified training. Twelve males cycled for 1min at 20% below VT1 and 4min at 50% between VT1 and , for 30min (20/50), with blood samples pre-, post- and 30min post-exercise. This was repeated twice, 2-7days apart. Flow cytometry assessed total DCs, plasmacytoid DCs, myeloid DCs, total T cells, T helper cells and T cytotoxic cells. No significant trial or interaction effects were found for any variable. A significant main effect of time for all variables was found; immune cells increased from pre- to post-exercise and decreased to baseline 30min post-exercise, apart from plasmacytoid DCs, which remained elevated 30min post-exercise. Intraclass correlation coefficients showed overall good-to-excellent reliability for all immune cells, with smallest real difference and Bland-Altman analysis verifying high reproducibility between trials. These results suggest that the 20/50 exercise test induces reproducible DC and T cell count changes, which, implemented before and after a period of intensified training, may highlight the negative states of overtraining.

Regulation of pDC fate determination by histone deacetylase 3.

Dendritic cells (DCs), the key antigen-presenting cells, are primary regulators of immune responses. Transcriptional regulation of DC development had been one of the major research interests in DC biology; however, the epigenetic regulatory mechanisms during DC development remains unclear. Here, we report that Histone deacetylase 3 (Hdac3), an important epigenetic regulator, is highly expressed in pDCs, and its deficiency profoundly impaired the development of pDCs. Significant disturbance of homeostasis of hematopoietic progenitors was also observed in HDAC3-deficient mice, manifested by altered cell numbers of these progenitors and defective differentiation potentials for pDCs. Using the in vitro Flt3L supplemented DC culture system, we further demonstrated that HDAC3 was required for the differentiation of pDCs from progenitors at all developmental stages. Mechanistically, HDAC3 deficiency resulted in enhanced expression of cDC1-associated genes, owing to markedly elevated H3K27 acetylation (H3K27ac) at these gene sites in BM pDCs. In contrast, the expression of pDC-associated genes was significantly downregulated, leading to defective pDC differentiation.

Charactering the immune signature of antigen presenting cells in the immune response to factor VIII in a murine model of hemophilia A using single-cell RNA sequencing

Abstract Deficiency of the procoagulant protein factor VIII (FVIII) is the defining characteristic for individuals with the inherited X-linked bleeding disorder hemophilia A. These individuals require intravenous infusions of FVIII to treat bleeding events, however ~30% develop neutralizing alloantibodies (i.e., inhibitors) against exogenous FVIII. Transcriptome analysis of FVIII-stimulated peripheral blood mononuclear cells in patients with hemophilia A and active inhibitors showed increased expression of innate immune modulators and proinflammatory pathways. Murine models of hemophilia A (FVIII KO) similarly develop anti-FVIII antibodies with FVIII exposure. Although the antibody response to FVIII is CD4-T cell dependent, the key antigen presenting cells (APCs) that mediate inhibitor formation is unclear. Bone marrow chimeras of transgenic mice with depleted dendritic cell (DC) subsets in FVIII KO suggest that type 2 classical DCs (cDC2), but not cDC1 or plasmacytoid DCs, mediate FVIII immunity. We then evaluated the evolution of splenic APCs and their immune cell signatures in FVIII KO mice injected with 0, 1, 3, or 5 doses of FVIII using flow cytometry and single-cell RNA sequencing (scRNAseq). FVIII KO injected with 5 doses of FVIII had similar frequencies of pDC, cDC1, and cDC2 subsets compared to mice injected with normal saline as a control. However, preliminary scRNAseq analysis demonstrated increased expression of phagocytic and interferon-related genes among DC and macrophage clusters, particularly CD8a −CD4 +Itgax −DCs. Further scRNAseq analyses of APC subclusters and differentially expressed genes will be assessed. Overall, these findings suggest that cDCs, specifically cDC2, are critical in FVIII immunity. Supported by NIH K99HL150595 and Hemophilia of Georgia Clinical Scientist Development Grant.

A Balance between Pro-Inflammatory and Pro-Reparative Macrophages is Observed in Regenerative D-MAPS.

Microporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio-active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L- to D-amino acids led to significant tissue regeneration and functional recovery in D-MAPS-treated cutaneous wounds. In this study, the immunomodulatory effect of D-MAPS in a subcutaneous implantation model is investigated. How macrophages are the key antigen-presenting cells to uptake and present these biomaterials to the adaptive immune systemisuncovered. A robust linker-specific IgG2b/IgG1 response to D-MAPS is detected as early as 14 days post-implantation. The fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes is observed in D-MAPS as an indicator for regenerative scaffolds. The work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes.

A44 PGC1-α INHIBITION WORSENS DSS-INDUCED COLITIS IN MICE

BackgroundInflammatory bowel diseases (IBD) represent a wide range of gastrointestinal diseases that are characterized by chronic inflammation of the gastrointestinal tract (GIT). Affecting more than 6 million individuals globally, the most prevalent forms of IBD are Ulcerative colitis (UC), characterized by inflammation and ulceration of the colon and rectum, and Crohn’s disease (CD), characterized by inflammation of several areas of the GIT. Due to the morbidity and occasionally mortality arising from IBD, as well as its increasing global prevalence, the search for novel treatments and cures for IBD presents great importance. Loss of immune tolerance significantly contributes to the inflammatory patterns observed in IBD patients. Dendritic Cells (DCs), key antigen-presenting cells, are important mediators of tolerance in the gut when polarized to tolerogenic DCs (tolDCs). The phenotype of tolDCs has been shown to directly depend on their intracellular metabolism, mostly by adopting mitochondrial oxidative phosphorylation. Peroxisome proliferator-activated receptor-gamma coactivator (PGC)1-α is a transcription coactivator that plays a crucial role in mediating cellular energy metabolism. Evidence shows that activation of PGC1-α stimulates mitochondrial biogenesis and promotes oxidative metabolism in a variety of biological processes.AimsThe aim of this project was to evaluate whether activation or inhibition of PGC1-α impacts inflammatory response in DSS-induced colitic in mice. We hypothesize that the PGC1-α pharmacological activator ZLN005 would ameliorate colitis as a result of tolDC polarization. On other hand, the PGC1-α pharmacological inhibitor SR18292 would increase inflammatory responses and decrease tolerance.MethodsC57/b6 mice received DSS in drinking water for 5 days, followed by 3 days of tap water. Mice were treated with vehicle, ZLN005 (10 mg/kg), or SR18292 treatment (25 mg/kg) for the 3 days of drinking tap water. Under necropsy, colitis was assessed by disease activity score (DAS), colon length, weight change, myeloperoxidase (MPO) activity assay, histopathology score, and cytokines by ELISA. Intestinal tolerance was accessed by evaluating T cells polarization to Treg, TH1, TH2, or TH17 by qPCR of the transcription factors Foxp3 (Treg), Tbx21 (TH1), Gata3 (TH2), and Rorγt (TH17) expressed in the colon.ResultsOur results demonstrated that ZLN005 did not alter the inflammatory response induced by DSS, gauged by MPO assay, DAS, and histopathology, suggesting that ZLN005 was ineffective in polarizing tolDCs. However, we observed a worsening of inflammatory conditions with treatment with SR18292 with a decrease in IL-10 levels and increased MPO values, potentially stemming from the loss of tolDCs and intestinal tolerance.ConclusionsPGC1-α inhibition worsens inflammatory response in DSS-induced colitis potentially by inhibiting tolDC polarization and loss of intestinal tolerance.Funding AgenciesNone

Dual RNA sequencing reveals dendritic cell reprogramming in response to typhoidal Salmonella invasion

Salmonella enterica represent a major disease burden worldwide. S. enterica serovar Typhi (S. Typhi) is responsible for potentially life-threatening Typhoid fever affecting 10.9 million people annually. While non-typhoidal Salmonella (NTS) serovars usually trigger self-limiting diarrhoea, invasive NTS bacteraemia is a growing public health challenge. Dendritic cells (DCs) are key professional antigen presenting cells of the human immune system. The ability of pathogenic bacteria to subvert DC functions and prevent T cell recognition contributes to their survival and dissemination within the host. Here, we adapted dual RNA-sequencing to define how different Salmonella pathovariants remodel their gene expression in tandem with that of infected DCs. We find DCs harness iron handling pathways to defend against invading Salmonellas, which S. Typhi is able to circumvent by mounting a robust response to nitrosative stress. In parallel, we uncover the alternative strategies invasive NTS employ to impair DC functions.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb +/+ and Csf2rb -/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

Protection against autoimmunity is driven by thymic epithelial cell-mediated regulation of Treg development.

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote the development of thymic regulatory T cells (tTregs) that mediate peripheral tolerance. The relative importance of these two mechanisms of thymic education to prevent autoimmunity is unclear. We generated a mouse model to specifically target the development and function of mTECs by conditional ablation of the NF-κB–inducing kinase (NIK) in the TEC compartment. In contrast to germline-deficient NIK−/− mice, Foxn1CreNIKfl/fl mice rapidly developed fatal T cell–dependent multiorgan autoimmunity shortly after birth. Thymic transplantation and adoptive transfer experiments demonstrated that autoimmunity arises specifically from the emergence of dysfunctional tTregs. Thus, Treg function, rather than negative selection, enforces the protection of peripheral tissues from autoimmune attack.

Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

Japanese Encephalitis Virus Infected Human Monocyte-Derived Dendritic Cells Activate a Transcriptional Network Leading to an Antiviral Inflammatory Response.

A comprehensive understanding of the human immune response to virus infection is imperative for developing effective therapies, antivirals, and vaccines. Dendritic cells (DCs) are among the first cells to encounter the virus and are also key antigen-presenting cells that link the innate and adaptive immune system. In this study, we focus on the human immune response to the mosquito-borne Japanese encephalitis virus (JEV), which is the leading cause of virus-induced encephalitis in south-east Asia and has the potential to become a global pathogen. We describe the gene regulatory circuit of JEV infection in human monocyte-derived DCs (moDCs) along with its functional validation. We observe that JEV can productively infect human moDCs leading to robust transcriptional activation of the interferon and NF-κB-mediated antiviral and inflammatory pathways. This is accompanied with DC maturation and release of pro-inflammatory cytokines and chemokines TNFα, IL-6, IL-8, IL-12, MCP-1. and RANTES. JEV-infected moDCs activated T-regulatory cells (Tregs) in allogenic mixed lymphocyte reactions (MLR) as seen by upregulated FOXP3 mRNA expression, suggestive of a host response to reduce virus-induced immunopathology. The virus also downregulated transcripts involved in Peroxisome Proliferator Activated Receptor (PPAR) signalling and fatty acid metabolism pathways suggesting that changes in cellular metabolism play a crucial role in driving the DC maturation and antiviral responses. Collectively, our data describe and corroborate the human DC transcriptional network that is engaged upon JEV sensing.

INKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

The alarmin cytokine IL-1a drives a feedback-amplification loop between dying tumor cells and inflammatory monocyte-lineage dendritic cells

Abstract Successful immunotherapy must transform the normally suppressive tumor microenvironment into a pro-inflammatory, immunogenic milieu. We show that this critical transformation depends on a previously unrecognized initiating signal delivered by the damage-associated “alarmin” cytokine interleukin-1a (IL-1a). Initially, tumor-intrinsic IL-1a was mobilized from dying tumor cells. This drove rapid local differentiation of a population of monocyte-lineage inflammatory dendritic cells (DCs). We have previously described these inflammation-inducible myeloid DCs in tumors (Sharma et. al, Immunity 48:91–106, 2018), where they serve as key antigen-presenting cells during multiple forms of immunotherapy and chemo-immuno-therapy. Once these inflammatory DCs were elicited by tumor-derived IL-1a, they rapidly re-activated anergic/unresponsive tumor-associated CD8+ T cells. Then DCs and T cells together produced reciprocal host-derived IL-1a, thus completing a potent self-amplifying feedback loop. If this IL-1a-driven feedback-amplification loop was disrupted – either by silencing IL-1a in the tumor cells; or ablating IL-1a in the host cells; or disrupting the IL-1-receptor on the inflammatory dendritic cells – then anti-tumor immune activation was lost across multiple forms of immunotherapy and chemotherapy. To restore the ability to initiate immune activation, the only cells that needed to respond to IL-1a were the specific population of inflammatory monocyte-lineage DCs.

A case of juvenile lupus with an extremely rare dendritic cell malignancy: Interdigitating dendritic cell sarcoma – coexistence or paraneoplastic manifestation?

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease, characterized by circulating autoantibodies, widespread inflammation, and tissue destruction. The association between cancer and SLE is intriguing in view of their common pathophysiological pathways. It is established that there is an increased risk for overall malignancies in SLE, with standardized incidence ratios ranging from 1.14 to 3.6. Hematological malignancies are more common than solid organ malignancies in SLE. Malignancy when associated with lupus usually follows the diagnosis, but can occur concurrently or even predate the diagnosis of SLE. Neoplasms of dendritic cells (DCs), the key antigen-presenting cells, are rare, and they account for

Dimethyl fumarate abrogates dust mite-induced allergic asthma by altering dendritic cell function.

IntroductionAllergic asthma is the most common inflammatory disease of upper airways. Airway dendritic cells (DCs) are key antigen presenting cells that regulate T helper 2 (Th2)‐dependent allergic inflammation. Recent studies have shown critical role of airway DCs in the induction of Th2‐mediated allergic inflammation and are attractive therapeutic targets in asthma. However, molecular signaling mechanism that regulate DCs function to Th2 immune responses are poorly understood. Here we aim to evaluate the immunomodulatory effect of dimethyl fumarate (DMF), an FDA approved small molecule drug, in the house dust mite (HDM)‐induced experimental model of allergic asthma.MethodsDMF was administered intranasally in the challenge period of HDM‐induced murine model of experimental asthma. Airway inflammation, airway hyperreactivity, Th2/Th1 cytokine were assessed. The effect of DMF on DC function was further evaluated by adoptive transfer of HDM‐pulsed DMF treated DCs to wild‐type naïve mice.ResultsDMF treatment significantly reduced HDM‐induced airway inflammation, mucous cell metaplasia, and airway hyperactivity to inhaled methacholine. Mechanistically, DMF interferes with the migration of lung DCs to draining mediastinal lymph nodes, thereby attenuates the induction of allergic sensitization and Th2 immune response. Notably, adoptive transfer of DMF treated DCs to naïve mice with HDM challenge similarly reduces the features of allergic asthma.ConclusionThis identifies a novel function of DMF on DC‐mediated adaptive immune responses in the setting of HDM‐induced airway inflammation. Taken together, our results offer a mechanistic rationale for DMF use to target DCs in local lung environment as antiasthmatic therapy.

Dendritic cells, T cells and their interaction in rheumatoid arthritis.

Dendritic cells (DCs) are the key professional antigen-presenting cells which bridge innate and adaptive immune responses, inducing the priming and differentiation of naive to effector CD4+ T cells, the cross-priming of CD8+ T cells and the promotion of B cell antibody responses. DCs also play a critical role in the maintenance of immune homeostasis and tolerance. DC-T cell interactions underpin the generation of an autoimmune response in rheumatoid arthritis (RA). Here we describe the function of DCs and review evidence for DC and T cell involvement in RA pathogenesis, in particular through the presentation of self-peptide by DCs that triggers differentiation and activation of autoreactive T cells. Finally, we discuss the emerging field of targeting the DC-T cell interaction for antigen-specific immunotherapy of RA.

Glucocorticoid-Induced Leucine Zipper: Fine-Tuning of Dendritic Cells Function.

Dendritic cells (DCs) are key antigen-presenting cells that control the induction of both tolerance and immunity. Understanding the molecular mechanisms regulating DCs commitment toward a regulatory- or effector-inducing profile is critical for better designing prophylactic and therapeutic approaches. Initially identified in dexamethasone-treated thymocytes, the glucocorticoid-induced leucine zipper (GILZ) protein has emerged as a critical factor mediating most, but not all, glucocorticoids effects in both non-immune and immune cells. This intracellular protein exerts pleiotropic effects through interactions with transcription factors and signaling proteins, thus modulating signal transduction and gene expression. GILZ has been reported to control the proliferation, survival, and differentiation of lymphocytes, while its expression confers anti-inflammatory phenotype to monocytes and macrophages. In the past twelve years, a growing set of data has also established that GILZ expression in DCs is a molecular switch controlling their T-cell-priming capacity. Here, after a brief presentation of GILZ isoforms and functions, we summarize current knowledge regarding GILZ expression and regulation in DCs, in both health and disease. We further present the functional consequences of GILZ expression on DCs capacity to prime effector or regulatory T-cell responses and highlight recent findings pointing to a broader role of GILZ in the fine tuning of antigen capture, processing, and presentation by DCs. Finally, we discuss future prospects regarding the possible roles for GILZ in the control of DCs function in the steady state and in the context of infections and chronic pathologies.

Restoration of the type I IFN-IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice.

Type I IFN (IFN-I) dysregulation contributes to type 1 diabetes (T1D) development, and although increased IFN-I signals are pathogenic at the initiation of autoimmune diabetes, IFN-I dysregulation at later pathogenic stages more relevant for therapeutic intervention is not well understood. We discovered that 5 key antigen-presenting cell subsets from adult prediabetic NOD mice have reduced responsiveness to IFN-I that is dominated by a decrease in the tonic-sensitive subset of IFN-I response genes. Blockade of IFNAR1 in prediabetic NOD mice accelerated diabetes and increased Th1 responses. Therefore, IFN-I responses shift from pathogenic to protective as autoimmunity progresses, consistent with chronic IFN-I exposure. In contrast, IL-1-associated inflammatory pathways were elevated in prediabetic mice. These changes correlated with human T1D onset-associated gene expression. Prostaglandin E2 (PGE2) and prostaglandin receptor 4 (PTGER4), a receptor for PGE2 that mediates both inflammatory and regulatory eicosanoid signaling, were higher in NOD mice and drive innate immune dysregulation. Treating prediabetic NOD mice with a PTGER4 antagonist restored IFNAR signaling, decreased IL-1 signaling, and decreased infiltration of leukocytes into the islets. Therefore, innate cytokine alterations contribute to both T1D-associated inflammation and autoimmune pathogenesis. Modulating innate immune balance via signals such as PTGER4 may contribute to treatments for autoimmunity.

Immunostimulatory Properties of Lipid Modified CpG Oligonucleotides

Innate immune responses recognizing pathogen associated molecular patterns play important roles in adaptive immunity. As such, ligands which mimic the conserved products of microbial and activate innate immunity are widely used as adjuvants for vaccines. Synthetic single strand oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs which bind Toll-like receptor 9 (TLR9) are powerful molecular adjuvants, potentiating both humoral and cellular responses. However, CpG ODN's in vitro potency has not been translated to in vivo settings primarily due to issues associated with delivery and toxicity. A major challenge in clinical application of CpG ODN is the efficient delivery to lymph nodes, the anatomic sites where all the immune responses are initiated. Targeting CpG to the key antigen presenting cells (APC) is essential for its application as a vaccine adjuvant, as it not only enhances CpG's efficacy, but also greatly reduces the systemic toxicity. We recently discovered an "albumin-hitchhiking" approach by which CpG ODNs were conjugated to a lipophilic lipid tail and follow subcutaneous injection, accumulated in lymph nodes by binding and transporting with endogenous albumin. This molecular approach targets CpG to antigen presenting cells in the draining lymph nodes via an endogenous albumin-mediated mechanism and simultaneously improves both the efficacy and safety of CpG as a vaccine adjuvant. Since CpG ODNs can be divided into structurally distinct classes, and each class of CpG ODN activates different types of immune cells and triggers different types of immunostimulatory activities, it is important to thoroughly evaluate the efficacy of this "albumin-hitchhiking" strategy in each class of CpG. Here we compare the immunostimulatory activities of three classes of lipid conjugated CpG ODNs in vitro and in vivo. Three representative sequences of lipid modified CpG ODNs were synthesized and their stimulatory effects as a vaccine adjuvant were evaluated. Our results showed that in vitro, lipid modified class A CpG exhibited enhanced stimulatory activities toward TLR transfected reporter cells or bone-marrow derived dendritic cells, whereas lipid-modification of class B or C CpG reduces the activation of TLR9 by 2-3 fold, as compared with unmodified class B and class C CpG, respectively. However, in vivo coadministration of ovalbumin (OVA) protein antigen mixed with lipid-conjugated class B or C CpG ODNs, but not class A CpGs induced dramatically increased OVA-specific humoral and cytotoxic CD8+ T cells responses compared with OVA mixed with unmodified CpGs. Further, lipid-modification greatly reduces the toxicity associated with CpG by minimizing the systemic dissemination. Taken together, these results demonstrated that amphiphilic modification of three classes of CpG motifs differentially affected and modulated the immunostimulatory activities in vitro and in vivo. Our study highlights the importance of in vivo lymph node targeting of CpG ODNs in fulfilling their use as vaccine adjuvants, providing implications for the rational design of molecular adjuvant for subunit vaccines.