Mouse Antigen-presenting Cells Research Articles

Loss of Acid Ceramidase in Myeloid Cells Impairs MHC II Expression on Dendritic Cells and Macrophages to Alleviate Chronic Colitis in IL10−/− Mice

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the colon and drastically increases the risk in the development of colorectal cancer (CRC). Over expression of acid ceramidase (AC) resulting in accumulation of sphingosine-1-phosphate (S1P), which amplify inflammatory pathways, has been implicated in patients with IBD and CRC. The conditional loss of AC in myeloid cells (ACMYE) has demonstrated promise as a therapeutic target in an acute colitis model. We sought to expand the investigation of AC loss in a physiologically relevant model of IBD using IL-10 deficient mice. IL10−/− mice were crossed with AC conditional knockout mice where AC is deleted in myeloid cells to generate ACMYE/IL10−/−, with ACfl/fl/IL10−/− mice used as control. Male and female animals were collected at 8, 12, and 24 weeks of age and assessed for intestinal inflammation. Upon examination of basic parameters of colitis, ACMYE mice exhibited reduced body weight and enlarged spleens at 12 weeks of age. Histologic assessment revealed no difference between genotypes in colitis severity at 8 or 12 weeks of age. Flow cytometry analysis was utilized to define the role of AC in immune cell recruitment. ACMYE mice exhibit reduced MHC II surface expression on antigen presenting cells (APCs) as well as reductions in neutrophil activation markers in peripheral tissues and the colon at 8 weeks of age. Interestingly, at 12 weeks of age ACfl/fl mice demonstrate significant immunosuppression across various cell types in the colon compared to ACMYE mice, whose colonic immune cells demonstrate little change over time. Furthermore, CD8+ cytotoxic T cell populations were significantly elevated in peripheral tissues despite continued reductions of MHC II surface expression on APCs in ACMYE mice at 12 weeks of age. These data indicate that loss of AC may provide protection in early age while resulting in altered inflammation in later age; however, loss of AC may also disrupt MHC II expression as seen consistently in APCs, and may indicate interference with cell-to-cell cross talk. Realtime qPCR revealed that ACMYE mice exhibited significant ablation in the expression of several pro-inflammatory cytokines at 24 weeks of age. Together these data implicate AC as a potential therapeutic target in reducing inflammation in chronic IBD. This work is supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK132079 (AJS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity

Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive Tcells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.

The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice.

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.

A distinct human cell type expressing MHCII and RORγt with dual characteristics of dendritic cells and type 3 innate lymphoid cells.

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.

NOD2/RIG-I Activating Inarigivir Adjuvant Enhances the Efficacy of BCG Vaccine Against Tuberculosis in Mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) kills about 1.5 million people each year and the widely used Bacille Calmette-Guérin (BCG) vaccine provides a partial protection against TB in children and adults. Because BCG vaccine evades lysosomal fusion in antigen presenting cells (APCs), leading to an inefficient production of peptides and antigen presentation required to activate CD4 T cells, we sought to boost its efficacy using novel agonists of RIG-I and NOD2 as adjuvants. We recently reported that the dinucleotide SB 9200 (Inarigivir) derived from our small molecule nucleic acid hybrid (SMNH)® platform, activated RIG-I and NOD2 receptors and exhibited a broad-spectrum antiviral activity against hepatitis B and C, Norovirus, RSV, influenza and parainfluenza. Inarigivir increased the ability of BCG-infected mouse APCs to secrete elevated levels of IL-12, TNF-α, and IFN-β, and Caspase-1 dependent IL-1β cytokine. Inarigivir also increased the ability of macrophages to kill MTB in a Caspase-1-, and autophagy-dependent manner. Furthermore, Inarigivir led to a Capsase-1 and NOD2- dependent increase in the ability of BCG-infected APCs to present an Ag85B-p25 epitope to CD4 T cells in vitro. Consistent with an increase in immunogenicity of adjuvant treated APCs, the Inarigivir-BCG vaccine combination induced robust protection against tuberculosis in a mouse model of MTB infection, decreasing the lung burden of MTB by 1-log10 more than that afforded by BCG vaccine alone. The Inarigivir-BCG combination was also more efficacious than a muramyl-dipeptide-BCG vaccine combination against tuberculosis in mice, generating better memory T cell responses supporting its novel adjuvant potential for the BCG vaccine.

Protamine sulphate coated poly (lactide-co-glycolide) nanoparticles of MUC-1 peptide improved cellular uptake and cytokine release in mouse antigen presenting cells

Aim MUC-1-peptide (M-1-pep) loaded poly (lactide-co-glycolide) nanoparticles were coated with protamine sulphate (PS), M-1-pep-PS-P-NPs for targeting antigen presenting cells (APCs) to evoke cytokine release. Methods and results M-1-pep-PS-P-NPs were tailored by emulsion-diffusion evaporation method and characterised in vitro under a set of rigorous parameters. The average particle size and zeta potential of optimised M-1-pep-PS-P-B-NPs was measured to be 132.21 ± 30.71 nm and 6.29 ± 0.71 mV, significantly (p < 0.01) higher than 71.24 ± 17.76-nm and −43.41 ± 3.37 mV of M-1-pep-P-NPs. Further, 50-μg/ml concentration of M-1-pep-PS-P-B-NPs displayed 82.4% cellular uptake in RAW 264.7 cells calculated in setting of fluorescence intensity significantly (p < 0.05) elevated than 63.1% of M-1-pep-P-NPs. Consistent to quantitative results, M-1-pep-PS-P-B-NPs also confirmed advanced cellular uptake (CU) in RAW 264.7 cells in contrast to M-1-pep-P-NPs suppose to be through multiple mechanisms including phagocytosis and clathrin mediated endocytosis. Conclusion M-1-pep-PS-P-B-NPs must be evaluated in vivo through inhalation route of administration for antitumor prospective in lung cancer xenograft model.

The Wnt-β-catenin-IL-10 signaling axis in intestinal antigen-presenting cells protects mice from colitis-associated colon cancer in response to gut microbiota

Abstract Loss of immune tolerance to gut microflora is inextricably linked to inflammatory bowel disease (IBD) and colitis-associated colon cancer (CAC). Intestinal antigen presenting cells (APCs) play a pivotal role in regulating intestinal inflammation and CAC, but underlying signaling pathways and molecular mechanisms remain largely unknown. In the present study, using a mouse model of CAC, we show that the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6)-β-catenin-IL-10 signaling axis in CD11c+ APCs suppresses CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of Wnt co-receptors LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of tumor-promoting pro-inflammatory factors and decreased expression of the immunosuppressive cytokine, IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor promoting inflammatory factors in CD11c+ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin in CD11c+ APCs or in vivo administration of IL-10 in LRP5/6ΔCD11c mice markedly reduced intestinal inflammation and CAC by suppressing the expression of inflammatory factors. In summary, here we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in regulating CAC in response to the commensal flora and suggest potential means for therapeutic intervention in inflammatory intestinal diseases.

Prevention of Type 1 Diabetes via a lipid Nanoparticle-Based Oral Immunotherapy that Targets Selective Lymphoid Tissues

Abstract Type 1 diabetes (T1D) is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons contribute to T1D, and the inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of the disease. However, maintenance of a therapeutic concentration of these drugs is challenging and their prolonged use leads to significant side effects. Our objective was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess if, via this strategy, a short-term administration would impact T1D development. We identified a formulation of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have the unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Importantly, short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promoted a significant delay of T1D onset. Cell profiling and in vivo challenges indicated that this therapeutic effect is likely due to localized inhibition of APCs maturation and promotion of anergy in diabetogenic T cells. These results highlight the versatility of this lipid nanoparticle-based drug delivery strategy.

Removal of Mannose-Ending Glycan at Asn2118 Abrogates FVIII Presentation by Human Monocyte-Derived Dendritic Cells.

The development of an immune response against therapeutic factor VIII is the major complication in hemophilia A patients. Oligomannose carbohydrates at N239 and/or N2118 on factor VIII allow its binding to the macrophage mannose receptor expressed on human dendritic cells, thereby leading to factor VIII endocytosis and presentation to CD4+ T lymphocytes. Here, we investigated whether altering the interaction of factor VIII with mannose-sensitive receptors on antigen-presenting cells may be a strategy to reduce factor VIII immunogenicity. Gene transfer experiments in factor VIII-deficient mice indicated that N239Q and/or N2118Q factor VIII mutants have similar specific activities as compared to non-mutated factor VIII; N239Q/N2118Q mutant corrected blood loss upon tail clip. Production of the corresponding recombinant FVIII mutants or light chains indicated that removal of the N-linked glycosylation site at N2118 is sufficient to abrogate in vitro the activation of FVIII-specific CD4+ T cells by human monocyte-derived dendritic cells. However, removal of mannose-ending glycans at N2118 did not alter factor VIII endocytosis and presentation to CD4+ T cells by mouse antigen-presenting cells. In agreement with this, the N2118Q mutation did not reduce factor VIII immunogenicity in factor VIII-deficient mice. Our results highlight differences in the endocytic pathways between human and mouse dendritic cell subsets, and dissimilarities in tissue distribution and function of endocytic receptors such as CD206 in both species. Further investigations in preclinical models of hemophilia A closer to humans are needed to decipher the exact role of mannose-ending glycans in factor VIII immunogenicity.

94-OR: Lipid Nanoparticle-Mediated Delivery of Enhanced Costimulation Blockade to Prevent Type 1 Diabetes

Type 1 diabetes (T1D) remains an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Because of its complex etiology, many immunotherapy strategies have been investigated, but with disappointing results. Costimulation blockade, blocking the CD28 pathway via administration of CTLA4-Ig, is a promising approach, but recent observations suggest its efficacy is antagonized by inflammatory factors. As antigen presenting cells malfunctions and aberrant accumulation of type 1 interferons are associated with T1D, we pose that inhibiting the signaling of inflammatory cytokines via Tofacitinib (Tofa), a JAK inhibitor, would enhance the efficacy of CTLA4-Ig to prevent T1D development. The objective of this study was to design the controlled and localized delivery of Tofa via implementation of biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess the immunomodulatory impact of this strategy. We identified a specific composition of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging using fluorescently-labeled NLC confirmed that these particles have the unique property of accumulating in lymphoid tissues. Moreover, when administrated via oral gavage, they bypassed first-pass metabolism and accumulated in spleen, pancreatic and mesenteric lymph nodes. Ex-vivo, Tofa- NLC rapidly delivered Tofa to mouse antigen presenting cells preventing their maturation and the release of inflammatory cytokines. Ongoing experiments show that short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in NOD promotes a significant reduction of T1D onset. Overall, Tofa-NLC represent a promising strategy to complement CTLA4-Ig (currently investigated) and we envision this combination strategy could enhance the efficacy of antigen specific immunotherapy. Disclosure Y. Zhang: None. J. Wang: None. X. Calderon-Colon: None. O. Tiburzi: None. M. Iglesias Lozano: None. J. Patrone: None. G. Raimondi: None.

Lipid Nanoparticle-Mediated Delivery of Enhanced Costimulation Blockade to Prevent Type 1 Diabetes

Abstract Type 1 diabetes (T1D) remains an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Costimulation blockade, blocking the CD28 pathway via CTLA4-Ig, is one promising immunotherapeutic, but recent observations suggest its efficacy is antagonized by inflammatory factors. As antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons are associated with T1D, we pose that inhibiting the signaling of inflammatory cytokines via Tofacitinib (Tofa), a JAK inhibitor, would enhance the efficacy of CTLA4-Ig in preventing T1D development. The objective of this study was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess the immunomodulatory impact of this strategy. We identified a specific composition of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Ongoing experiments show that short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promotes a significant reduction of T1D onset. Overall, Tofa-NLC represent a promising strategy to complement CTLA4-Ig and we envision this combined strategy could enhance the efficacy of antigen specific immunotherapy.

Updated insights into the mechanism of action and clinical profile of the immunoadjuvant QS-21: A review

BackgroundVaccine adjuvants are compounds that significantly enhance/prolong the immune response to a co-administered antigen. The limitations of the use of aluminium salts that are unable to elicite cell responses against intracellular pathogens such as those causing malaria, tuberculosis, or AIDS, have driven the development of new alternative adjuvants such as QS-21, a triterpene saponin purified from Quillaja saponaria. PurposeThe aim of this review is to attempt to clarify the mechanism of action of QS-21 through either receptors or signaling pathways in vitro and in vivo with special emphasis on the co-administration with other immunostimulants in new adjuvant formulations, called adjuvant systems (AS). Furthermore, the most relevant clinical applications will be presented. MethodsA literature search covering the period 2014–2018 was performed using electronic databases from Sci finder, Science direct, Medline/Pubmed, Scopus, Google scholar. ResultsInsights into the mechanism of action of QS-21 can be summarized as follows: 1) in vivo stimulation of Th2 humoral and Th1 cell-mediated immune responses through action on antigen presenting cells (APCs) and T cells, leading to release of Th1 cytokines participating in the elimination of intracellular pathogens. 2) activation of the NLRP3 inflammasome in mouse APCs with subsequent release of caspase-1 dependent cytokines, Il-1β and Il-18, important for Th1 responses. 3) synthesis of nearly 50 QS-21 analogs, allowing structure/activity relationships and mechanistic studies. 4) unique synergy mechanism between monophosphoryl lipid A (MPL A) and QS-21, formulated in a liposome (AS01) in the early IFN-γ response, promoting vaccine immunogenicity. The second part of the review is related to phase I-III clinical trials of QS-21, mostly formulated in ASs, to evaluate efficacy, immunogenicity and safety of adjuvanted prophylactic vaccines against infectious diseases, e.g. malaria, herpes zoster, tuberculosis, AIDS and therapeutic vaccines against cancer and Alzheimer's disease. ConclusionThe most advanced phase III clinical applications led to the development of two vaccines containing QS-21 as part of the AS, the Herpes Zoster vaccine (HZ/su) (Shingrix™) which received a license in 2017 from the FDA and a marketing authorization in the EU in 2018 and the RTS,S/AS01 vaccine (Mosquirix™) against malaria, which was approved by the EMA in 2015 for further implementation in Sub-Saharan countries for routine use.

Activation of CD8⁺ T Cell Responses after Melanoma Antigen Targeting to CD169⁺ Antigen Presenting Cells in Mice and Humans.

The lack of tumor-reactive T cells is one reason why immune checkpoint inhibitor therapies still fail in a significant proportion of melanoma patients. A vaccination that induces melanoma-specific T cells could potentially enhance the efficacy of immune checkpoint inhibitors. Here, we describe a vaccination strategy in which melanoma antigens are targeted to mouse and human CD169 and thereby induce strong melanoma antigen-specific T cell responses. CD169 is a sialic acid receptor expressed on a subset of mouse splenic macrophages that captures antigen from the blood and transfers it to dendritic cells (DCs). In human and mouse spleen, we detected CD169+ cells at an equivalent location using immunofluorescence microscopy. Immunization with melanoma antigens conjugated to antibodies (Abs) specific for mouse CD169 efficiently induced gp100 and Trp2-specific T cell responses in mice. In HLA-A2.1 transgenic mice targeting of the human MART-1 peptide to CD169 induced strong MART-1-specific HLA-A2.1-restricted T cell responses. Human gp100 peptide conjugated to Abs specific for human CD169 bound to CD169-expressing monocyte-derived DCs (MoDCs) and resulted in activation of gp100-specific T cells. Together, these data indicate that Ab-mediated antigen targeting to CD169 is a potential strategy for the induction of melanoma-specific T cell responses in mice and in humans.

Abstract 130: ATP Release Drives the Immune Responses Associated With Hypertension

Inflammation is critical in progression of hypertension. However, how an elevated blood pressure (BP) initiates inflammation is unknown, as are the precise effects of high BP on the immune responses. To study this, we first challenged mice with antigen ovalbumin (OVA). Two-fold more OVA-specific CD8 + T cells were present in the blood (2.7 ± 0.23% vs. 1.2 ± 0.47%) and spleen (2.62 ± 0.31% vs. 1.31 ± 0.17%) of angiotensin (Ang) II induced hypertensive mice as compared to those of normotensive controls. To address whether the over-activation of T cell-mediated immune responses is pathogenic, two models of autoimmune disease were studied. RIP-mOVA is a transgenic mouse line that expresses OVA in pancreatic islet β cells. When OVA-specific OT-I T cells were infused into Rip-mOVA mice, the animals with hypertension induced by either Ang II or L-NAME developed more severe diabetes (blood glucose (mg/dl): Ang II 331, L-NAME 315 vs. Control 168). Concanavalin A-induced hepatitis was also significantly worsened by hypertension as compared to the pathology observed in normotensive mice. After examining the characteristics of T cells and antigen presenting cells (APCs), we found that the most distinguishable difference in hypertensive mice was the upregulation of CD86 on APCs. Blocking CD86 by antibody completely abrogated the elevated immune responses in hypertensive mice. To understand what causes CD86 upregulation in APCs of hypertensive mice, we studied damage-associated molecular patterns and found that plasma ATP levels rose as early as 3 days after the induction of hypertension and reached from about 1μM of baseline to a peak level of 3 μM after 2 weeks of hypertension, which exactly parallels the kinetics of CD86 elevation on APCs. Hydrolyzing ATP or blocking its P2X 7 receptor normalized APC CD86 expression and eliminated hypertension-induced T cell over-activation. Further, untreated human hypertensive patients have substantially elevated plasma ATP levels (2.21 ± 0.99 μM, n = 27) compared to treated hypertensive patients (0.94 ± 0.34 μM, n = 17) or normotensive controls (0.63 ± 0.38 μM, n =30). A linear trend was observed between ATP levels and BP among all subjects. These studies indicate ATP is critical in initiating hypertension-associated inflammation.

Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State.

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

Canonical Wnt Signaling in CD11c+ antigen presenting cells controls inflammation-associated colon carcinogenesis and commensal homeostasis

Abstract The intestine contains high levels of the Wnt family of ligands, and the Wnt-signaling pathway is essential for intestinal homeostasis. Thus, aberrant Wnt/β-catenin-signaling may result in inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. Although past studies have focused on this signaling pathway’s role in intestinal stem cell proliferation and epithelial cell maintenance, its role in shaping mucosal immune responses and commensal homeostasis in the gut remains unknown. In the present study, using a mouse model of colitis-associated colorectal cancer, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation and suppresses inflammation-driven colon carcinogenesis by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6), in CD11c+ APCs in mice (LRP5/6ΔAPC mice) caused enhanced intestinal inflammation with increased tumor numbers in the colon due to increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors resulting from commensal dysbiosis. Mechanistically, loss of LRP5/6-mediated signaling in intestinal APCs resulted in enhanced effector T cell differentiation and decreased regulatory T cell differentiation. Furthermore, our study demonstrates that in LRP5/6ΔAPC mice conditional activation of β-catenin in intestinal APCs or in vivo blockade of IL-6 results in reduced intestinal inflammation with decreased colon tumor burden. These results reveal a mechanism by which intestinal APCs control gut inflammation and commensal homeostasis via canonical Wnt signaling.

Gap Junction Mediated Antigen Transport in Epithelium Promotes Amplification of CD8 T Cell Mediated Bystander Pathology Following Chlamydial Infection

Abstract Chlamydia-specific CD8 T cells play a significant role in oviduct pathology, not bacterial clearance. We hypothesized that gap junction mediated antigen transport (GMAT) from infected to uninfected cells could explain the apparent targeting of uninfected epithelial cells by Chlamydia-specific CD8 T cells. Since gap junction protein connexin 43 is highly expressed in oviduct epithelium, we generated Foxj1Cre-Cx43flox mice with a conditional deficiency of CX43 only in ciliated columnar epithelia in oviducts. Foxj1Cre-Cx43flox mice displayed similar chlamydial infection, but reduced splenic Ag-specific CD8 T cell response and oviduct pathology when compared to WT mice, suggesting that CX43-mediates chlamydial pathogenesis. To evaluate the role of GMAT via CX43 in pathogenesis, HeLa S3 cells were engineered to express functional CX43 or a closed channel point-mutant CX43 (T154A). Chlamydial peptides were observed in neighboring uninfected cells at mid-developmental cycle upon infection of HeLa-CX43, not HeLa S3 or HeLa-CX43-T154A cells. Naïve mouse antigen-presenting cells (APC) optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with Chlamydia-infected HeLa-CX43, when compared to HeLa S3 or HeLa-CX43-T154A cells, demonstrating that GMAT is involved in activation of CD8 T cells. Furthermore, naïve mouse APC optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with a 1:4 mixture of Chlamydia-infected HeLa-CX43 and uninfected HeLa-CX43 cells, when compared to mixtures of infected HeLa-CX43 with uninfected HeLa-S3 or HeLa-CX43-T154A cells. These results demonstrate that GMAT via uninfected epithelial cells amplifies pathogenic Chlamydia-specific CD8 T cell response.

Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-β2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1β, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-β2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

Systemic Expression of Notch Ligand Delta-Like 4 during Mycobacterial Infection Alters the T Cell Immune Response.

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

Abstract LB-089: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy

Abstract Recognition of PAMPs via pattern recognition receptors is central to immune recognition of foreign threats and to the generation of a coordinated innate and adaptive immune response. Cancers lack PAMPs and are poorly immunogenic. Consequently, the immune system often fails to mount an effective, coordinated anti-cancer immune attack. Though immunotherapies (e.g. checkpoint inhibitors) are effective in some cancer patients, the majority of patients fail to achieve substantial therapeutic benefit. To fully realize the potential of immune checkpoint inhibitors, there is substantial interest in developing therapeutically-viable PAMPs capable of enabling the maturation and function of professional antigen presenting cells (e.g. dendritic cells, DCs). Bacterial and viral PAMPs (i.e. TLR and STING agonists) can elicit DC maturation but are poorly tolerated systemically and are thereby limited to intra-tumoral delivery. The soluble yeast β-1,3/1,6 glucan, Imprime PGG (Imprime), is a PAMP that has been successfully administered intravenously (IV), is well-tolerated and shows promising efficacy in a series of clinical trials in &amp;gt; 400 total subjects. We sought to determine whether Imprime could drive maturation and enhanced function of antigen presenting cells in vivo. We now show that Imprime binds in vivo to various dendritic cell (DC) subsets in both human and mouse. In mice dosed IV, Imprime also elicits DC maturation as indicated by CD86 upregulation and successfully induces a type I interferon transcriptional profile. In C57Bl/6 mice immunized with the OVA257-264 model antigen, Imprime treatment elicits the specific expansion of adoptively transferred OT-I CD8 T cells (transgenic T cells engineered to recognize the OVA antigen). These OT-I T cells are functional effector cells, showing enhanced degranulation and increased capacity to produce IFN-γ and IL-2 when compared to OT-I cells isolated from mice challenged with OVA peptide alone. In ex vivo human whole blood, Imprime also enables DC maturation (enhanced expression of CD86, CD83, MHC class II), T cell expansion and production of the potent anti-tumor cytokine IFN-γ. Importantly, we now show preliminary data that peripheral blood monocytes and classical DCs from Imprime-treated cancer patients show elevated CD86 expression. Collectively, these data show that Imprime, a novel, systemically-administered, well-tolerated PAMP, can effectively elicit the maturation and function of antigen presenting cells in vivo and may thereby enhance T cell priming and anti-tumor immune response elicited by checkpoint inhibitors. Citation Format: Ross B. Fulton, Steven M. Leonardo, Adria B. Jonas, Kathryn A. Fraser, Anissa S.H. Chan, Nadine R. Ottoson, Michael E. Danielson, Nandita Bose, Jeremy R. Graff, Keith Gorden. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-089.