Splenic Antigen-presenting Cells Research Articles

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice.

We investigated the biological function of the mould allergen Alt a 1 as a carrier of micronutrients, such as the vitamin A metabolite retinoic acid (RA) and the influence of RA binding on its allergenicity invitro and invivo. Alt a 1-RA complex formation was analyzed in silico and invitro. PBMCs from Alternaria-allergic donors were stimulated with Alt a 1 complexed with RA (holo-Alt a 1) or empty apo-Alt a 1 and analyzed for cytokine production and CD marker expression. Serum IgE-binding and crosslinking assays to apo- and holo-protein were correlated to B-cell epitope analysis. Female BALB/c mice already sensitized to Alt a 1 were intranasally treated with apo-Alt a 1, holo-Alt a 1 or RA alone before measuring anaphylactic response, serum antibody levels, splenic cytokines and CD marker expression. In silico docking calculations and invitro assays showed that the extent of RA binding depended on the higher quaternary state of Alt a 1. Holo-Alt a 1 loaded with RA reduced IL-13 released from PBMCs and CD3+CD4+CRTh2 cells. Complexing Alt a 1 to RA masked its IgE B-cell epitopes and reduced its IgE-binding capacity. In a therapeutic mouse model of Alternaria allergy nasal application of holo-Alt a 1, but not of apo-Alt a 1, significantly impeded the anaphylactic response, impaired splenic antigen-presenting cells and induced IL-10 production. Holo-Alt a 1 binding to RA was able to alleviate Th2 immunity invitro, modulate an ongoing Th2 response and prevent anaphylactic symptoms invivo, presenting a novel option for improving allergen-specific immunotherapy in Alternaria allergy.

Intravenous Senescent Erythrocyte Vaccination Modulates Adaptive Immunity and Splenic Complement Production.

Targeted delivery of vaccines to the spleen remains a challenge. Inspired by the erythrophagocytotic process in the spleen, we herein report that intravenous administration of senescent erythrocyte-based vaccines profoundly alters their tropism toward splenic antigen-presenting cells (APCs) for imprinting adaptive immune responses. Compared with subcutaneous inoculation, intravenous vaccination significantly upregulated splenic complement expression in vivo and demonstrated synergistic antibody killing in vitro. Consequently, intravenous senescent erythrocyte vaccination produces potent SARS-CoV-2 antibody-neutralizing effects, with potential protective immune responses. Moreover, the proposed senescent erythrocyte can deliver antigens from resected tumors and adjuvants to splenic APCs, thereby inducing a personalized immune reaction against tumor recurrence after surgery. Hence, our findings suggest that senescent erythrocyte-based vaccines can specifically target splenic APCs and evoke adaptive immunity and complement production, broadening the tools for modulating immunity, helping to understand adaptive response mechanisms to senescent erythrocytes better, and developing improved vaccines against cancer and infectious diseases.

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.

Repeated dose treatment of Tinospora cordifolia polysaccharide rich extract activates splenic antigen presenting cells with no associated organ toxicity

Tinospora cordifolia is a medically valuable plant widely used in Ayurveda and has been attributed with immunomodulatory properties. We compared the immunomodulation of repeated doses of the purified polysaccharide G1-4A and polysaccharide-rich extract (PRE) of T. cordifolia in BALB/c mice. Since both had comparable effects, we further compared the repeated dose effects of 50 and 500 mg/kg b.w PRE per dose with particular emphasis on histological changes in different organs, splenic cell death and DNA damage and immunomodulation. In mice treated with both concentrations of PRE, no gross changes were observed in any organ by histopathological analyses along with normal biochemical parameters similar to control. There was no increase in splenic lymphocyte cell death or DNA damage. A significant increase in the number of splenic macrophages with no increase in phagocytosis was observed. Increased expression of phenotypic maturation markers was observed in splenic dendritic cells and not in bone marrow-derived dendritic cells of PRE-treated mice. Increased activation of B cells in response to mitogen with no change in such a response by T cells was also observed. These results from repeated dose experiments indicate that PRE treatment has no associated organ toxicity and activates antigen-presenting cells and thus can be used instead of G1-4A as a promising immunotherapeutic adjuvant.

Co-opting erythrocyte clearance

T Cells Whereas T cell dysfunction contributes to immune evasion in cancer and chronic viral infection, strategies that induce dysfunction in autoreactive T cells may facilitate self-tolerance. Watkins et al. used phage display to identify a human antibody fragment (Fab) that selectively binds erythrocytes, enabling efficient antigen targeting to splenic antigen-presenting cells that rapidly clear apoptotic erythrocytes. Fab-tethered antigen induced antigen-specific T cell dysfunction in mice that was sustained in response to antigen rechallenge for at least 3 months and required Batf3-dependent dendritic cells. In a mouse model of experimental autoimmune encephalomyelitis, erythrocyte-targeted antigen prevented encephalogenic T cells from contributing to autoimmune pathology, demonstrating the therapeutic potential of leveraging erythrocyte clearance pathways to disarm overactive T cells. Sci. Immunol. 6 , eabe1801 (2021).

CD73+ Dendritic Cells in Cascading Th17 Responses of Experimental Autoimmune Uveitis-Induced Mice.

Previous studies have shown that CD73 is pivotal in the conversion of pro-inflammatory adenosine triphosphate into anti-inflammatory adenosine and that immune cells of the same type that express different levels of CD73 are functionally distinct. In this study we show that adenosine enhances the Th17 promoting effect of dendritic cells (DCs), and DCs expressing CD73 critically augment Th17 responses. Bone marrow dendritic cells (BMDCs) do not constantly express CD73; however, a significant portion of the BMDCs expressed CD73 after exposure to Toll-like receptor ligand, leading to stronger Th17 responses by converting adenosine monophosphate to adenosine. We show that the CD73+ BMDCs play a critical role in cascading Th17 responses, and CD73+ BMDCs are functionally augmented after treatment with Toll-like receptor ligand. Splenic antigen presenting cells (DCs) of CD73−/− mouse have a poor Th17-stimulating effect, even after exposure to lipopolysaccharide (LPS) or γδ T cells, indicating that induction of CD73+ DCs is critically involved in augmented Th17 responses. We conclude that CD73+ DCs critically trigger cascading Th17 responses, and the activated Th17 cells that express CD73 further augment Th17 responses, leading to cascading exacerbation. Hence, disabling the CD73 function of DCs should block this cascading response and mitigate Th17 responses.

Galectin-8 Enhances T cell Response by Promotion of Antigen Internalization and Processing.

SummaryGalectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4+ T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface.

Intrapulmonary (i.pulmon.) Pull Immunization With the Tuberculosis Subunit Vaccine Candidate H56/CAF01 After Intramuscular (i.m.) Priming Elicits a Distinct Innate Myeloid Response and Activation of Antigen-Presenting Cells Than i.m. or i.pulmon. Prime Immunization Alone.

Understanding the in vivo fate of vaccine antigens and adjuvants and their safety is crucial for the rational design of mucosal subunit vaccines. Prime and pull vaccination using the T helper 17-inducing adjuvant CAF01 administered parenterally and mucosally, respectively, has previously been suggested as a promising strategy to redirect immunity to mucosal tissues. Recently, we reported a promising tuberculosis (TB) vaccination strategy comprising of parenteral priming followed by intrapulmonary (i.pulmon.) mucosal pull immunization with the TB subunit vaccine candidate H56/CAF01, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses. Here, we investigate the uptake of H56/CAF01 by mucosal and systemic innate myeloid cells, antigen-presenting cells (APCs), lung epithelial cells and endothelial cells in mice after parenteral prime combined with i.pulmon. pull immunization, and after parenteral or i.pulmon. prime immunization alone. We find that i.pulmon. pull immunization of mice with H56/CAF01, which are parenterally primed with H56/CAF01, substantially enhances vaccine uptake and presentation by pulmonary and splenic APCs, pulmonary endothelial cells and type I epithelial cells and induces stronger activation of dendritic cells in the lung-draining lymph nodes, compared with parenteral immunization alone, which suggests activation of both innate and memory responses. Using mass spectrometry imaging of lipid biomarkers, we further show that (i) airway mucosal immunization with H56/CAF01 neither induces apparent local tissue damage nor inflammation in the lungs, and (ii) the presence of CAF01 is accompanied by evidence of an altered phagocytic activity in alveolar macrophages, evident from co-localization of CAF01 with the biomarker bis(monoacylglycero)phosphate, which is expressed in the late endosomes and lysosomes of phagocytosing macrophages. Hence, our data demonstrate that innate myeloid responses differ after one and two immunizations, respectively, and the priming route and boosting route individually affect this outcome. These findings may have important implications for the design of mucosal vaccines intended for safe administration in the airways.

Transfusion with Stored Antigen-Negative Blood Impairs T Cell Cross Priming to Red Cell Alloantigen in a Subsequent Transfusion

BACKGROUND: Donor red blood cell (RBC) alloantigens are efficiently cross-presented by recipient antigen presenting cells (APCs), thereby priming recipient naive CD8 T cells; however, this process and its downstream immunological consequences are poorly understood. RBC antigen cross presentation has been linked to cytotoxic T cell responses driving bone marrow graft rejection, but in other systems it causes tolerance. The conventional dendritic cell (cDC) subset, cDC1, is required for cross-presentation of RBC antigen. Macrophages are insufficient for cross-presentation in the absence of cDC1s, but do promote RBC antigen cross-presentation by cDCs in vitro. After prolonged storage, a portion of transfused RBCs are rapidly cleared by the spleen. In murine models, transfused stored RBCs have multiple effects on splenic APCs, including architectural distortion, dendritic cell activation, and macrophage depletion via ferroptosis due to increased erythrophagocytosis. Thus, the antigen presentation machinery controlling alloimmunization to RBC antigens may be influenced by the storage duration of previously transfused RBCs.AIMS: To better understand how recent transfusions may influence the immunogenicity of future transfusions, we used a mouse model to determine whether prior transfusion of stored, antigen-matched RBCs influences CD8 T cell cross priming to antigen mismatched RBCs in a subsequent transfusion.METHODS: In an in vivo cross-priming assay, OVA-expressing RBCs from transgenic HOD mice (with RBC surface expression of hen egg lysozyme, ovalbumin (OVA), and Duffy antigens) were the transfused antigen source and naïve OT-1 CD8 T cells, which express an MHC class I restricted OVA-specific TCR transgene, were the responders. Magnetically-selected CD8+ OT-1 cells, labeled with CellTrace Far Red (CTFR), were adoptively transferred into MHC identical (H-2Kb) UBC-GFP C57BL6/J recipients. After 16 hours, these mice were transfused with fresh or stored (12 days in CPDA-1), leukoreduced UBC-GFP RBCs or PBS. Five hours after the initial transfusion, recipients were transfused again with fresh leukoreduced HOD RBCs. To determine the effect of stored RBC transfusion on T cell priming to a non-RBC associated antigen, groups of mice carrying adoptively transferred OT-1 T cells were transfused with fresh or stored RBCs and then challenged 5 hours later with soluble OVA protein (10 µg, IV). After 4 days, splenic OT-1 T cells were analyzed for proliferation (by CTFR dye dilution) and activation (by CD44 and CD122 expression).RESULTS: Proliferation and activation of OT-1 T cells in response to challenge with soluble OVA protein were significantly increased in mice that had been previously transfused with stored RBCs compared to those transfused with fresh RBCs (Fig. 1A). In contrast, OT-1 T cell activation in response to HOD RBC transfusion was decreased in mice that had previously received a stored RBC transfusion, as compared to those receiving fresh RBCs or PBS (Fig. 1B).CONCLUSION: The increased T cell priming after soluble OVA challenge in the mice previously transfused with stored vs. fresh RBCs may be related to the increased expression of co-stimulatory molecules on DCs after stored RBC transfusion. However, T cell priming to the RBC-associated antigen requires additional factors, which are disrupted by previous transfusion of stored, but not fresh, RBCs. Splenic red pulp macrophages potentiate OT-1 T cell cross priming by dendritic cells in vitro and become severely depleted after stored RBC transfusion in vivo. Thus, macrophage help may be needed for T cell cross priming to an RBC antigen in vivo, and stored RBC transfusions may disrupt macrophage function, thereby affecting the immunogenicity of subsequent RBC alloantigen exposure.Figure 1. Effect of fresh and stored RBC transfusion on priming after antigenic challenge with soluble OVA or HOD RBCs.Representative contour plots show proliferation-induced dye dilution and CD44 expression on OT-1 cells in the spleen 4 days after transfusion of fresh (left) or stored (right) RBCs followed 5 hours later with IV injection of soluble OVA protein (A) or HOD RBCs (B). Dot plots at the right of representative contour plots show compiled data from two independent experiments quantifying the percentage of activated and proliferated cells (CTFR diluted, CD44hi) in the OT-1 subgate (mean ± SEM, n = 4-9). *P < 0.05, **P < 0.01, unpaired t test. DisclosuresNo relevant conflicts of interest to declare.

Ionizable Amino-Polyesters Synthesized via Ring Opening Polymerization of Tertiary Amino-Alcohols for Tissue Selective mRNA Delivery.

The utility of messenger RNA (mRNA) as a therapy is gaining a broad interest due to its potential for addressing a wide range of diseases, while effective delivery of mRNA molecules to various tissues still poses a challenge. This study reports on the design and characterization of new ionizable amino-polyesters (APEs), synthesized via ring opening polymerization (ROP) of lactones with tertiary amino-alcohols that enable tissue and cell type selective delivery of mRNA. With a diverse library of APEs formulated into lipid nanoparticles (LNP), structure-activity parameters crucial for efficient transfection are established and APE-LNPs are identified that can preferentially home to and elicit effective mRNA expression with low in vivo toxicity in lung endothelium, liver hepatocytes, and splenic antigen presenting cells, including APE-LNP demonstrating nearly tenfold more potent systemic mRNA delivery to the lungs than vivo-jetPEI. Adopting tertiary amino-alcohols to initiate ROP of lactones allows to control polymer molecular weight and obtain amino-polyesters with narrow molecular weight distribution, exhibiting batch-to-batch consistency. All of which highlight the potential for clinical translation of APEs for systemic mRNA delivery and demonstrate the importance of employing controlled polymerization in the design of new polymeric nanomaterials to improve in vivo nucleic acid delivery.

Metabolites Linked to Bariatric Surgery Prolong Allograft Survival in Obese Recipients

Introduction: In transplantation, obesity has become a common co-morbidity linked to inferior transplant outcome through far-reaching effects on the complex network of metabolism, inflammation and immunity. Methods: Fully mismatched skin transplantations were performed in diet-induced obese (DIO) C57/Bl6 mice and lean littermates. Sleeve gastrectomies (SGx) were performed prior to transplantation to induce weight loss. Results: Obese graft recipients showed significantly accelerated allograft rejection when compared to lean littermates (p < 0.001), associated with increased IFN-γ expression in splenic CD4+ T cells. Bariatric surgery, on the other hand, prolonged allograft survival even beyond that of lean animals (p = 0.005) and promoted protective IL-10-skewed conditions with reduced alloreactivity as confirmed by ELISPOT. Of note, sham procedures and dietary weight loss showed inferior results in allograft survival and weight loss when compared to bariatric surgery. Quantitative metabolomic profiling identified a secondary bile acid and a proteinogenic amino acid as upregulated subsequent to bariatric surgery. Strikingly, combined application reduced obesity and resulted in significant prolongation of graft survival (p = 0.005; Fig.1). Metabolic treatment was also associated with reduced pro-inflammatory responses in splenic CD4+ T cells and a significant reduction in splenic M1-like antigen-presenting cells (Fig. 2). Mechanistically, metabolic treatment reduced M1-polarization of monocytes/macrophages through activation of the bile acid receptor TGR5. Activation of TGR5 through a specific agonist blocked M1-polarization in vitro and prolonged allograft survival to levels comparable to metabolic treatment, while disruption of intracellular signaling of TGR5 during metabolic treatment abrogated these effects. Of note, studies in metabolic cages demonstrated a reduced oral intake of treated obese animals while maintaining regular physical activity, indicating that weight loss was not induced through toxic side-effects of metabolic treatment. Conclusion: Obesity augments alloimmune responses resulting in accelerated graft rejection, while bariatric surgery induced a change in cytokine balances with transplant outcome superior even to lean controls. Two metabolic compounds were identified as key mediators skewing the interplay of metabolism and inflammation towards anti-inflammatory conditions, providing unique insights linking obesity, metabolism and immunity.FigureFigure

Mechanism of MOG i. p. for protecting mice from EAE

Objective To investigate mechanisms underlying the prevention of experimental autoimmune encephalomyelitis (EAE) in mice by intraperitoneal infusion of myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) (MOG i. p.). Methods C57BL/6 mice were immunized with MOG35-55 to establish the model of EAE and then were intraperitoneally injected daily with MOG35-55 or ovalbumin (OVA, served as control) from day 6 to day 16. EAE was evaluated daily using a general clinical scoring system and histological analysis. Numbers of lymphocytes in peripheral blood and central nervous system (CNS) were detected at different time points. Effects of MOG i. p. on the migration of MOG-T cells in vivo were analyzed by an adoptive transfer experiment. Maturation of splenic antigen-presenting cells (APCs) and migration of MOG-T cells in vitro were examined by fluorescence activated cell sorting (FACS) and a Transwell system, respectively. Results MOG i. p. protected the mice from development of EAE by blocking the lymphocyte recruitment to CNS. More effector T cells were trapped in the periphery of EAE and naive mice in adoptive transfer experiment after MOG i. p. treatment. MOG i. p. induced the maturation of splenic APCs and enhanced the expression of CD80, CD86 and major histocompatibility complex class Ⅱ (MHCⅡ) molecules. Mature APCs blocked the recruitment of effector T cells to CNS. Conclusion MOG i. p. protects mice from EAE by inducing the maturation of splenic APCs. Key words: Experimental autoimmune encephalomyelitis; Immune tolerance; Antigen-presenting cell; Cell migration

Abstract 527: Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition

Abstract In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Blüml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Günther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [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 527.

Antigen-oriented T cell migration contributes to myelin peptide induced-EAE and immune tolerance

Treatment with soluble myelin peptide can efficiently and specifically induce tolerance to demyelination autoimmune diseases including multiple sclerosis, however the mechanism underlying this therapeutic effect remains to be elucidated. In actively induced mouse model of experimental autoimmune encephalomyelitis (EAE) we analyzed T cell and innate immune cell responses in the central nervous system (CNS) and spleen after intraperitoneal (i.p.) infusion of myelin oligodendrocyte glycoprotein (MOG). We found that i.p. MOG infusion blocked effector T cell recruitment to the CNS and protected mice from EAE and lymphoid organ atrophy. Innate immune CD11b+ cells preferentially recruited MOG-specific effector T cells, particularly when activated to become competent antigen presenting cells (APCs). During EAE development, mature APCs were enriched in the CNS rather than in the spleen, attracting effector T cells to the CNS. Increased myelin antigen exposure induced CNS-APC maturation, recruiting additional effector T cells to the CNS, causing symptoms of disease. MOG triggered functional maturation of splenic APCs. MOG presenting APCs interacted with MOG-specific T cells in the spleen, aggregating to cluster around CD11b+ cells, and were trapped in the periphery. This process was MHC II dependent as an MHC II directed antibody blocked CD4+ T cell cluster formation. These findings highlight the role of myelin peptide-loaded APCs in myelin peptide-induced EAE and immune tolerance.

High-Fat Diet-Induced Obesity Enhances Allograft Rejection.

Obesity promotes a state of low-grade inflammation that exacerbates chronic inflammatory diseases, such as asthma and inflammatory bowel disease. In transplantation, the survival of organs transplanted into obese patients is reduced compared with allografts in lean recipients. However, whether this is due to increased alloimmunity remains to be addressed conclusively. We used a mouse model of high-fat diet (HFD)-induced obesity and assessed immune responses to allogeneic stimulation in vitro, allogeneic splenocyte immunization in vivo, and allogeneic heart transplantation. Our results indicate that HFD altered the composition and phenotype of splenic antigen-presenting cells that led to their enhanced capacity to stimulate T cells. Immunization with allogeneic splenocytes in vivo resulted in increased alloreactivity, as determined by IFNγ production. Moreover, cardiac allograft rejection in HFD mice was modestly accelerated compared to aged-matched control animals fed a low-fat diet, correlating with enhanced alloreactive T cell function. Our results highlight the increased alloresponse triggered by HFD-induced obesity and its negative impact on transplant outcome.

Myeloid PTEN deficiency impairs tumor-immune surveillance via immune-checkpoint inhibition

ABSTRACTTumor–host interaction is determined by constant immune surveillance, characterized by tumor infiltration of myeloid and lymphoid cells. A malfunctioning or diverted immune response promotes tumor growth and metastasis. Recent advances had been made, by treating of certain tumor types, such as melanoma, with T-cell checkpoint inhibitors. This highlights the importance of understanding the molecular mechanisms underlying the crosstalk between tumors and their environment, in particular myeloid and lymphoid cells.Our aim was to study the contribution of the myeloid PI3K/PTEN-signaling pathway in the regulation of tumor-immune surveillance in murine models of cancer. We made use of conditional PTEN-deficient mice, which exhibit sustained activation of the PI3K-signaling axis in a variety of myeloid cell subsets such as macrophages and dendritic cells (DCs).In colitis-associated colon cancer (CAC), mice deficient in myeloid PTEN showed a markedly higher tumor burden and decreased survival. We attributed this observation to the increased presence of immune-modulatory conventional CD8α+ DCs in the spleen, whereas other relevant myeloid cell subsets were largely unaffected. Notably, we detected enhanced surface expression of PD-L1 and PD-L2 on these DCs. As a consequence, tumoricidal T-cell responses were hampered or redirected.Taken together, our findings indicated an unanticipated role for the PI3K/PTEN-signaling axis in the functional regulation of splenic antigen-presenting cells (APCs). Our data pointed at potential, indirect, tumoricidal effects of subclass-specific PI3K inhibitors, which are currently under clinical investigation for treatment of tumors, via myeloid cell activation.

DOCK8-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. The relative role of dendritic cells, B cells and macrophages in the induction of RBC alloimmunization remain unclear. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused RBCs. A particularly useful transgenic "HOD mouse" has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffyb blood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune cells or receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs.Using these models, we recently discovered that mice lacking the GEF (guanine nucleotide exchange factor) DOCK8 fail to develop alloimmunity to transfused RBCs. Dendritic cells in these knockout mice fail to migrate to T cells due to lack of coordinated actin rearrangement governed by this GEF. Both B cell and T cell activation in the spleen to the transgenic transfused RBCs is abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in DOCK8-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. Our preliminary data now suggest that DOCK8-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The need for dendritic cell migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed; these mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion and the specific role of splenic dendritic cell subsets in CD4+ T cell priming to allogeneic RBCs. DisclosuresNo relevant conflicts of interest to declare.

Transfused Stored or Antibody-Coated Red Blood Cells Are Internalized By and Activate Splenic Professional Antigen Presenting Cells

BACKGROUND: Alloimmunization to antigens on transfused red blood cells (RBCs) is a major problem in transfusion medicine. Development of alloantibodies limits the availability of compatible RBC units for chronically transfused patients. In addition, chronic RBC transfusion therapy is an important risk factor for cell-mediated bone marrow transplant rejection. To develop anti-RBC alloantibodies, professional antigen presenting cells (APCs) are required to digest and load RBC antigens into MHC class II for presentation to CD4 helper T cells. Mouse studies suggest that RBC-associated minor histocompatibility antigens can also be cross-presented into recipient MHC class I; thereby contributing to CD8 T-cell mediated immune responses against bone marrow allografts. Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Splenic red pulp macrophages (RPMs) ingest damaged erythrocytes, catabolize heme, and recycle iron but they are weak APCs. Conversely, dendritic cells (DCs) are the most potent of all APCs. A rare and genetically distinct subset of CD8+ DCs can efficiently ingest dead or dying cells and is uniquely capable of activating CD4 and CD8 T cell responses by presenting antigen via MHC class II and by cross-presenting cell-associated antigens via MHC class I. The RBC storage lesion, in addition to promoting phagocytosis by RPMs, may also induce RBC uptake by splenic DCs. Inflammation induced by stored RBC transfusions, or by antibody-mediated RBC clearance, may enhance DC activation and RBC uptake, thereby triggering humoral and cell-mediated immune responses to RBC antigens.AIMS: A mouse model of green fluorescent protein (GFP)+ RBC transfusion and flow cytometry were used to track the uptake of fresh, antibody-coated and stored RBCs into splenic professional APC subsets and the effects of these RBCs on APC activation.METHODS: Cohorts of 8-12 week old C57BL/6 male mice (n=4 per group) were transfused with one mouse-equivalent unit of syngeneic leukoreduced and citrate-phosphate-dextrose-adenine suspended fresh GFP+ RBCs (<24 hours old), stored GFP+ RBCs (11 days old), fresh GFP+ RBCs coated with anti-RBC polyclonal antibodies, or saline. Two hours after transfusion, spleens were dissociated (Liberase™ DL plus DNAse), FcBlocked, and stained with 7-color antibody panels to define CD8+ DCs (CD11c+ MHC-II+ CD8+ DEC205+), CD8- DCs (CD11c+ MHC-II+ CD8-CD11b+), and RPMs (VCAM-1+ CD11blo). Activation markers included MHC class II and CD86. Data were analyzed using FlowJo and Excel.RESULTS: RBC transfusion after prolonged storage markedly increased GFP+ RBC uptake by RPMs (5.8±0.91% fresh vs. 47±2.3% stored) and CD8+ DCs (1.3±0.27% fresh vs. 13±1.0% stored). A smaller increase in RBC uptake was seen in CD8- DCs (0.6±0.068% fresh vs 1.5±0.083% stored). Antibody-coated RBCs showed two-fold increased uptake by CD8+DCs (25±1.5%) compared with stored RBCs and they showed similar uptake into RPMs (50±1.2%) and CD8- DCs (1.1±0.047%). Lack of GFP+ DC staining with the Ter119 RBC marker confirmed that the GFP signal was internalized and not adsorbed onto the DC surface. CD8+ DCs that internalized GFP+ RBCs showed significantly increased CD86 and MHC class II expression consistent with activation.CONCLUSION: Taken together, these results suggest that transfusion of stored or antibody-coated RBCs increases RBC uptake by, and activation of, professional splenic APCs. CD8+ DC uptake of stored or antibody-coated RBCs may contribute to humoral and cellular anti-RBC immune responses via antigen presentation in MHC class II and class I, respectively. Thus, these mechanisms may increase the risk of alloimmunization to RBC antigens. [Display omitted] DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.

Preemptive Tolerogenic Delivery of Donor Antigens for Permanent Allogeneic Islet Graft Protection.

We have previously developed a robust regimen for tolerance induction in murine models of islet cell transplantation using pre- and posttransplant infusions of donor splenocytes (SPs) treated with a chemical cross-linker ethylcarbodiimide (ECDI). However, the requirement for large numbers of fresh donor SPs for ECDI coupling impairs its clinical feasibility, and additionally, the compatibility of this tolerance regimen with commonly used immunosuppressive drugs is largely unknown. In the current study, we demonstrate that equivalent tolerance efficacy for islet cell transplantation can be successfully achieved not only with a significantly lower dose of ECDI-SPs than originally established but also with culture-expanded donor B-cells or with soluble donor antigens in the form of donor cell lysate, which is ECDI coupled to recipient SPs. We further demonstrate that tolerance induced by donor ECDI-SPs is dependent on a favorable apoptotic-to-necrotic cell ratio post-ECDI coupling and is not affected by a transient course of conventional immunosuppressive drugs including tacrolimus and mycophenolate mofetil. While splenic antigen-presenting cells of the recipient play an important role in mediating the tolerogenic effects of donor ECDI-SPs, splenectomized recipients can be readily tolerized and appear to employ liver Kupffer cells for uptaking and processing of the ECDI-SPs. We conclude that infusion of donor ECDI-SPs is a versatile tolerance strategy that has a high potential for adaptation to clinically feasible regimens for tolerance trials for human islet cell transplantation.

Liver sinusoidal endothelial cells (LSECs) isolated ex vivo by a novel MACS-based technology induce proliferation in naïve CD4+ T-cells and Th2 effector cells

The liver has been demonstrated to contribute to the induction of systemic tolerance by trapping, deletion or modulation of T-cells. This property seems to be at least partially mediated by liver sinusoidal endothelial cells (LSECs) since in vivo priming of CD8+ T-cells on LSECs leads to development of anergic T-cell subsets. Studying the function of endothelial cells (ECs) ex vivo usually meets experimental obstacles: isolation protocols are time consuming and require considerable technical equipment. Here we make use of the novel antibody ME-9F1 (murine endothelial cell antibody 9-F1) strongly reacting with mouse endothelium and report a simple antibody mediated, MACS-based ex vivo isolation technique which allows the preparation of LSECs in a short time and with a high purity (90–95%). Compared to the standard isolation protocol by centrifugal elutriation the MACS-based technology leads to comparable numbers and purity of cells. The isolated LSECs express a normal morphology in culture and are capable of antigen presentation in MHC II context. They induce antigen-specific proliferation in naive CD4+ T-cells as well as Th1/Th2 effector cells as shown by co-culture experiments with CFSE-labeled T-cell receptor transgenic T-cells. Compared to splenic antigen-presenting cells, they rather promote in vitro and in vivo survival and expansion of IL-4-expressing Th2 cells than Th1 cells. Taken together, our data support the point of view that LSECs are not only involved in tolerance induction in CD8+ but also CD4+ T-cell populations. Therefore, we speculate that the liver might especially play a role in tolerance induction to exogenous (food) antigens presented in MHC II context.