Expression In Antigen-presenting Cells Research Articles

Regulation of the Function of T Follicular Helper Cells and B Cells in Type 1 Diabetes Mellitus by the OX40/OX40L Axis.

To study the expression of OX40 on T follicular helper (Tfh) cells and the ligand OX40L on antigen-presenting cells (APCs) in peripheral blood of patients with Type 1 diabetes mellitus (T1DM) and the role of OX40 signaling in promoting Tfh cells to assist B-cell differentiation. Cross-sectional study. Endocrinology department of a university hospital. Twenty-five patients with T1DM and 35 with newly diagnosed T2DM from January 2021-December 2021 (39 males, 21 females; mean age: 31.0 ± 4.5, range: 19-46 years). None. The peripheral blood proportion of CD4+CD25-CD127+CXCR5+PD1+ Tfh cells in patients with T1DM or T2DM and the OX40L expression in CD14+ monocytes and CD19+ B cells were analyzed by flow cytometry. The OX40 signal effect on Tfh-cell function was analyzed by co-incubating B cells with Tfh cells under different conditions. Flow cytometry detected the ratio of CD19-CD138+ plasmacytes. The Tfh cells ratio and intracellular IL-21 expression in peripheral blood was significantly higher in patients with T1DM than with T2DM, and the OX40 expression in peripheral Tfh cells and OX40L expression in APC were significantly higher in T1DM. After adding OX40L protein, the CD19-CD138+-plasmacytes percentage was significantly increased and higher in T1DM. Blocking of anti-OX40L monoclonal antibodies significantly reduced the plasmacytes ratio. The peripheral Tfh cells proportion increased and the OX40 expression in peripheral Tfh cells was upregulated in patients with T1DM versus patients with T2DM. OX40/OX40L signaling enhanced the Tfh-cell function to assist B-cell differentiation, which may contribute to the pathogenesis of T1DM.

Abstract 4016: mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment

Abstract Background: Unlike hematologic targets, TMR Chimeric Antigen Receptors with binding restricted to antigen in the tumor microenvironment lack CAR dependent stimulatory signals for proliferation outside the tumor. Increased exposure to CAR-T is associated with improved clinical outcomes and therefore tools to safely and selectively increase proliferation represent potential adjunctive CAR-T therapeutics. Therefore, we developed a surrogate CAR Specific Stimulating Protein (CSSP) approach encoded by synthetic mRNA for expression in antigen presenting cells to boost CAR-T proliferation in vitro and in vivo. Methods: NFAT-Luc reporter lymphocytes were transduced with lentivectors encoding TMR CARs with or without epitopes reactive to CSSP’s in the CAR. Subsequently, PBMCs were transfected with LNPs containing synthetic mRNAs for either GFP or candidate membrane bound CSSPs. Transfection efficiency was confirmed by microscopy and flow cytometry. The activity of CSSP’s expressed in monocytes cultured with T cells expressing CAR variants with NFAT-luc reporter was measured by luciferase expression and compared to CD3 stimulation. Additionally, the kinetics of CAR stimulation with monocytes expressing CSSPs were determined by serial luciferase measurements. Finally, the proliferation of PBMCs transduced with a TMR-HER2 CAR co-cultured with CSSP treated purified monocytes were evaluated. Results: Following exposure of PBMCs expressing CSSPs to TMR-CAR-expressing NFAT T cells, CAR specific luciferase signaling was readily detectable compared to non-specific CD3 stimulation, indicating that the CSSPs were capable of CAR specific stimulation through the CD3ζ chain of the CAR. Two of the CSSP variants demonstrated stimulation in a CAR specific fashion at levels similar to or greater than non-specific CD3 stimulation. In kinetic experiments, stimulation of NFAT could be detected over a period of 8 days, indicating persistence of the CSSP similar to GFP in antigen presenting cell controls. Finally, co-culture of CSSP mRNA expressed in purified monocytes were capable of TMR-HER2-CAR T cells stimulation, which do not proliferate when cultured with HER2 antigen expressing cells under physiologic conditions. Conclusion: TMR-CAR-T cells can achieve proliferative capacity outside the tumor microenvironment using synthetic mRNA encoding CAR Specific Stimulatory Proteins. This novel mRNA approach may provide additional tools to promote the proliferation and/or persistence of solid tumor targeted CAR-T cells outside the tumor environment. Citation Format: Frederic Vigant, Anirban Kundu, Michael Betts, Sid P. Kerkar, Gregory I. Frost. mRNA encoding a CAR specific stimulating protein (CSSP) mediates tumor metabolism regulated (TMR) CAR signaling for CAR-T expansion beyond the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4016.

Prohibitin Expression in Antigen-Presenting Cells: Implications for Inciting Trigger in CNS IgG4-Related Disease.

Immunoglobulin G4-related disease (IgG4-RD) is a rare autoimmune disorder with an unknown etiology. Using orthogonal immune profiling and automated sequential multiplexing, we found an enhanced frequency of activated circulating B cells, antigen-presenting myeloid cells in peripheral blood, and a distinct distribution of immune cells within the CNS lesions. Prohibitin-expressing CD138+ plasma B cells and CD11c+ dendritic cells have been found interacting with T cells resulting in irmnune cell activation within the lesion. The data implicate prohibitin as a potential triggering antigen in the pathogenesis of IgG4-RD and shed light on the cellular dynamics and interactions driving IgG4-RD in the central nervous system, emphasizing the need for further studies corroborating these findings.

Esterase-Labile Quaternium Lipidoid Enabling Improved mRNA-LNP Stability and Spleen-Selective mRNA Transfection.

Ionizable cationic lipids are recognized as an essential component of lipid nanoparticles (LNPs) for messenger RNA (mRNA) delivery but can be confounded by low lipoplex stability with mRNA during storage and in vivo delivery. Herein, the rational design and combinatorial synthesis of esterase-triggered decationizable quaternium lipid-like molecules (lipidoids) are reported to develop new LNPs with high delivery efficiency and improved storage stability. This top lipidoid carries positive charges at the physiological condition but promptly acquires negative charges in the presence of esterase, thus permitting stable mRNA encapsulation during storage and in vivo delivery while balancing efficient mRNA release in the cytosol. An optimal LNP formulation is then identified through orthogonal optimization, which enables efficacious mRNA transfection selectively in the spleen following intravenous administration. LNP-mediated delivery of ovalbumin (OVA)-encoding mRNA induces efficient antigen expression in antigen-presenting cells and elicits robust antigen-specific immune responses against OVA-transduced tumors. The work demonstrates the potential of decationizable quaternium lipidoids for spleen-selective RNA transfection and cancer immunotherapy.

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

Immune-checkpoint expression in antigen-presenting cells (APCs) of cytomegaloviruses infection after transplantation: as a diagnostic biomarker

Immune-checkpoint expression in antigen-presenting cells (APCs) of cytomegaloviruses infection after transplantation: as a diagnostic biomarker

The role of dietary magnesium deficiency in inflammatory hypertension.

Nearly 30% of adults consume less than the estimated average daily requirement of magnesium (Mg2+), and commonly used medications, such as diuretics, promote Mg2+ deficiency. Higher serum Mg2+ levels, increased dietary Mg2+ in-take, and Mg2+ supplementation are each associated with lower blood pressure, suggesting that Mg2+-deficiency contributes to the pathogenesis of hypertension. Antigen-presenting cells, such as monocytes and dendritic cells, are well-known to be involved in the pathogenesis of hypertension. In these cells, processes implicated as necessary for increased blood pressure include activation of the NLRP3 inflammasome, IL-1β production, and oxidative modification of fatty acids such as arachidonic acid, forming isolevuglandins (IsoLGs). We hypothesized that increased blood pressure in response to dietary Mg2+-depletion leads to increased NLRP3, IL-1β, and IsoLG production in antigen presenting cells. We found that a Mg2+-depleted diet (0.01% Mg2+ diet) increased blood pressure in mice compared to mice fed a 0.08% Mg2+ diet. Mg2+-depleted mice did not exhibit an increase in total body fluid, as measured by quantitative magnetic resonance. Plasma IL-1β concentrations were increased (0.13 ± 0.02 pg/mL vs. 0.04 ± 0.02 pg/mL). Using flow cytometry, we observed increased NLRP3 and IL-1β expression in antigen-presenting cells from spleen, kidney, and aorta. We also observed increased IsoLG production in antigen-presenting cells from these organs. Primary culture of CD11c+ dendritic cells confirmed that low extracellular Mg2+ exerts a direct effect on these cells, stimulating IL-1β and IL-18 production. The present findings show that NLRP3 inflammasome activation and IsoLG-adduct formation are stimulated when dietary Mg2+ is depleted. Interventions and increased dietary Mg2+ consumption may prove beneficial in decreasing the prevalence of hypertension and cardiovascular disease.

Immortalized B Cells Transfected with mRNA of Antigen Fused to MITD (IBMAM): An Effective Tool for Antigen-Specific T-Cell Expansion and TCR Validation

Peripheral mononuclear blood cells (PBMCs) are the most widely used study materials for immunomonitoring and antigen-specific T-cell identification. However, limited patient PBMCs and low-frequency antigen-specific T cells remain as significant technical challenges. To address these limitations, we established a novel platform comprised of optimized HLA-matched immortalized B cells transfected with mRNA of a prototype viral or tumor antigen conjugated to MHC class-I trafficking domain protein (MITD) to increase the efficiency of epitope expression in antigen-presenting cells (APCs) essential to expanding antigen-specific T cells. When applied to CMV as a model, the IBMAM platform could successfully expand CMV-specific T cells from low-frequency CMV PBMCs from seropositive donors. Additionally, this platform can be applied to the validation of antigen specific TCRs. Together, compared to using APCs with synthesized peptides, this platform is an unlimited, highly efficient, and cost-effective resource in detecting and expanding antigen-specific T cells and validating antigen-specific TCRs.

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

303-OR: Localized CTLA-4-Ig and Antioxidant Islet Encapsulation Delays Allograft Rejection

Type 1 diabetes is characterized by beta-cell-specific autoimmune destruction, leading to life-long exogenous insulin-dependence. Islet transplantation can restore proper glycemic control, but graft longevity remains low due to immune-mediated rejection. The use of nanothin encapsulation materials consisting of poly (N-vinylpyrrolidone) (PVPON) and tannic acid (TA) , an antioxidant, can delay islet allograft rejection and decrease inflammatory immune responses. However, when (PVPON/TA) -encapsulated C57BL/6 islets were transplanted into streptozocin-treated diabetic NOD mice in the absence of systemic immunosuppression, only 50% of recipients maintained long-term allograft function. Therefore, modification of (PVPON/TA) coatings to include immune inhibitors are needed to prolong islet allograft survival. The adaptability of our (PVPON/TA) layers allow for its conjugation to CTLA-4-Ig, a fusion protein that can block CD28-mediated co-stimulation of T cells. We hypothesize that the addition of CTLA-4-Ig to (PVPON/TA) outer layers, denoted as (PVPON/TA/CTLA-4-Ig) , will extend islet allograft survival by inhibiting proinflammatory effector T cell responses. Transplantation of (PVPON/TA/CTLA-4-Ig) -encapsulated NOD.Rag islets significantly delayed islet allograft rejection in diabetic C57BL/6 mice compared to non- (p<0.0001) and control IgG-containing (PVPON/TA/IgG) -encapsulated islets (p<0.01) . Additionally, (PVPON/TA/CTLA-4-Ig) -encapsulation significantly increased FOXP3+ regulatory and CD73+ FR4+ anergic CD4+ T cell count when compared to both non- and (PVPON/TA/IgG) -encapsulation. Since CTLA-4-Ig can affect the tryptophan catabolism pathway, future studies will investigate the effect of (PVPON/TA/CTLA-4-Ig) -encapsulation on indolamine-2,3-dioxygenase expression in antigen-presenting cells. The success of (PVPON/TA/CTLA-4-Ig) coatings to delay allograft rejection warrant their use in future human translational studies. Disclosure J.Barra: None. K.S.Burnette: None. V.Kozlovskaya: None. E.Kharlampieva: None. H.M.Tse: None. Funding NIH/NIDDK Raward (DK099550) NIH/NIDDK Raward (DK127497) JDRF award (SRA-2016-270-S-B) JDRF award (2-SRA-2019-692-S-B) NIH/NIDDK R56 award (DK126456) NIH/NIDDK Raward (DK126456) NIH/NIGMS (T32GM109780) NIH/NIGMS (T32GM008111)

A novel β-glucan–oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1

Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the β-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG–Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40–SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’RLM-RACE) technique. In vivo activity of siCD40–SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40–SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5’RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Nasal alum-adjuvanted vaccine promotes IL-33 release from alveolar epithelial cells that elicits IgA production via type 2 immune responses.

Aluminum hydroxide salts (alum) have been added to inactivated vaccines as safe and effective adjuvants to increase the effectiveness of vaccination. However, the exact cell types and immunological factors that initiate mucosal immune responses to alum adjuvants are unclear. In this study, the mechanism of action of alum adjuvant in nasal vaccination was investigated. Alum has been shown to act as a powerful and unique adjuvant when added to a nasal influenza split vaccine in mice. Alum is cytotoxic in the alveoli and stimulates the release of damage-associated molecular patterns, such as dsDNA, interleukin (IL)-1α, and IL-33. We found that Ag-specific IgA antibody (Ab) production was markedly reduced in IL-33-deficient mice. However, no decrease was observed in Ag-specific IgA Ab production with DNase I treatment, and no decrease was observed in IL-1α/β or IL-6 production in IL-33-deficient mice. From the experimental results of primary cultured cells and immunofluorescence staining, although IL-1α was secreted by alveolar macrophage necroptosis, IL-33 release was observed in alveolar epithelial cell necroptosis but not in alveolar macrophages. Alum- or IL-33-dependent Ag uptake enhancement and elevation of OX40L expression were not observed. By stimulating the release of IL-33, alum induced Th2 immunity via IL-5 and IL-13 production in group 2 innate lymphoid cells (ILC2s) and increased MHC class II expression in antigen-presenting cells (APCs) in the lung. Our results suggest that IL-33 secretion by epithelial cell necroptosis initiates APC- and ILC2-mediated T cell activation, which is important for the enhancement of Ag-specific IgA Ab production by alum.

Synergistic Immunostimulation through the Dual Activation of Toll-like Receptor 3/9 with Spherical Nucleic Acids.

Toll-like receptors (TLRs) are a family of proteins that modulate the innate immune system and control the initiation of downstream immune responses. Spherical nucleic acids (SNAs) designed to stimulate single members of the TLR family (e.g., TLR7 or TLR9) have shown utility in cancer immunotherapy. We hypothesized that SNAs synthesized with multiple TLR agonists would enable the simultaneous activation of multiple TLR pathways for maximally synergistic immune activation. Here, we describe the synthesis of SNAs that incorporate both a TLR3 agonist (polyinosinic:polycytidylic acid, poly(I:C)) and TLR9 agonist (CpG oligonucleotide) on the same liposomal scaffold. In this design, CpG comprises the SNA oligonucleotide shell, and poly(I:C) is encapsulated in the liposome core. These dual-TLR activating SNAs efficiently codeliver high quantities of both agonists to the same target cell, yielding enhanced immunostimulation in various murine and human antigen-presenting cells (APCs). Moreover, codelivery of TLR agonists using the SNA both synchronizes and prolongs the duration of costimulatory molecule and major histocompatibility complex class II expression in APCs, which has been shown to be important for efficient downstream immune responses. Taken together, this SNA design provides a strategy for potently activating immune cells and increasing the efficiency of their activation, which likely will inform the preparation of nanomaterials for highly potent immunotherapies.

Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction.

Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy.

Encapsulation of LXR ligand by D-Nap-GFFY hydrogel enhances anti-tumorigenic actions of LXR and removes LXR-induced lipogenesis.

Background and purpose: Activation of liver X receptor (LXR) by its ligand T0901317 (T317) enhances interferon-γ (IFNγ) production to inhibit tumor growth. However, induction of severe hypertriglyceridemia and fatty liver by T317 limits its application. The naphthylacetic acid modified D-enantiomeric-glycine-phenylalanine-phenylalanine-tyrosine (D-Nap-GFFY) can form a nanofiber hydrogel which is selectively taken up by antigen-presenting cells (APCs). In this study, we determined if D-Nap-GFFY-encapsulated T317 (D-Nap-GFFY-T317) can potently inhibit tumor growth while having no adverse lipogenic effects on the liver.Methods: We prepared D-Nap-GFFY-T317 nanofiber hydrogel and subcutaneously injected it into IFNγ deficient (IFNγ-/-) and wild-type (WT) mice with lung carcinoma, either inoculated LLC1 cells or urethane-induced carcinoma. Mice received oral T317 administration were used for comparison. Effects of treatment on tumor growth, lipogenesis and involved mechanisms were investigated.Results: Compared with T317 oral administration, injection of D-Nap-GFFY-T317 more potently inhibited LLC1 tumor growth in mice. The inhibition was dependent on LXR-activated IFNγ expression in APCs. D-Nap-GFFY-T317 increased M1 while reducing M2 type macrophages in tumors. Associated with activation of IFNγ expression, D-Nap-GFFY-T317 enhanced dendritic cell maturation and infiltration into tumors, increased CD3+/CD8+ cells in tumors, and inhibited tumor angiogenesis. Similarly, D-Nap-GFFY-T317 more potently inhibited growth of urethane-induced lung carcinomas than T317 oral administration. In these two tumor models, T317 oral administration, but not D-Nap-GFFY-T317 injection, activated hepatic lipogenesis and induced fatty liver.Conclusion: Our study demonstrates that D-Nap-GFFY-T317 inhibits lung tumor growth without adverse effects on the liver, indicating the hydrogel-encapsulated LXR ligand might be a novel therapy for tumor treatment.

Developing a novel thymus organoid as a cell therapy to correct autoimmunities

Background & Aim Complete athymia results in severe immune deficiency, as the thymus is the organ responsible for equipping the body with functional and diverse T cells. Thymus epithelial cells (TECs) are critically important in the selection and education of effective T cells that do not aberrantly attack self-antigen. Thymus transplants are an investigational therapy at Duke for young patients born with athymia, most often seen in complete DiGeorge Anomaly (cDGA). MHC-unmatched donor thymus tissue is collected from infants that undergo elective cardiac surgery for treatment of congenital heart disease, cultured for 12-21 days, then implanted into the quadriceps muscle of a cDGA patient. These transplants save lives in an otherwise untreatable disease. However, the newly made immune system is not perfect, demonstrated by blunted T cell numbers and a high incidence of autoimmunities such as autoimmune hemolytic anemia in these patients. Our lab replicated clinical thymus transplants in an athymic nude mouse system with the goal of first understanding the mechanisms of immune reconstitution in order to improve T cell output and limit post-transplant immune dysregulation. Methods, Results & Conclusion Nude mice transplanted with cultured, neonatal, MHC-mismatched donor thymus achieve CD3+ T cell reconstitution at 4.86% (± 0.59 SEM) of recipient CD45+ ten weeks post-transplant compared to 38.6% (± 2.75 SEM) in wildtype mice and show a phenotypic skew to memory T cells over naive T cells. We also have data showing that recipient antigen presenting cells (APCs) enter donor thymus tissue and are responsible for the education of recipient T cells. Due to the lack of tissue-specific antigen expression in APCs that is normal in thymus epithelial cells, we hypothesized that this abnormal education may be responsible for the T cell abnormalities. We set out to create a transplantable thymus organoid system to replace donor tissue and mimic “normal” T cell thymic education to increase T cell numbers, T cell receptor diversity, and limit autoimmunity. Our data demonstrate T cell reconstitution in athymic mice as early as 2 weeks after transplantation with a microporous annealed particle (MAP) scaffold system seeded with stromal cells. This proposed system of thymus organoids may be therapeutically beneficial for many other disorders that are linked with thymic dysfunction or loss of immune tolerance, such as in aging or multiple sclerosis.

GILT in thymic epithelial cells facilitates central CD4 T cell tolerance to a tissue-restricted, melanoma-associated self antigen

Abstract Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral antigen presenting cells (APCs), gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, while TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T:Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection and increased Treg cells, resulting in a diminished anti-tumor response to a tissue-restricted, melanoma-associated self antigen.

Abstract B98: IGFBP2 reprograms pancreatic cancer immune surveillance at epitranscriptome levels

Abstract In recent years, checkpoint blockade has been used successfully to treat many cancer types including melanoma, non-small cell lung, and colorectal cancers. In stark contrast, the response to these new immunotherapeutics has been poor in patients with pancreatic ductal adenocarcinoma (PDAC), the most common and lethal type of pancreatic cancer. Thus, PDAC is considered a cold tumor for immunotherapy and the mechanism underlying this resistance is poorly understood. Insulin-like growth factor binding protein-2 (IGFBP2), a secreted cytokine, is elevated in the serum, pancreatic juice, and PDAC tissues in many pancreatic cancer patients, and we demonstrated that high levels associate to reduced overall patient survival and promote the immunosuppressive M2 tumor-associated macrophage phenotype in the tumor microenvironment (TME). We recently identified IGFBP2 as a key oncogenic signaling molecule for PDAC metastasis and epithelial-to-mesenchymal transition. Our preliminary microarray analysis showed that major histocompatibility complex class II antigen (MHC class II) genes were significantly downregulated in AsPC-1 pancreatic cancer cells overexpressing IGFBP2. MHC class II molecules are constitutively expressed in antigen-presenting cells (APCs) such as dendritic and Langerhans cells, macrophages, and B lymphocytes. In PDAC, increased MHC class II expression correlates to a higher histologic grade of differentiation. Also, CD4+ T lymphocyte numbers in PDAC tissues positively correlate to MHC class II expression levels. We envision that high IGFBP2 represses MHC class II gene expression in the tumor-associated APCs, resulting in failure of recruitment and activation of CD4+ lymphocytes, augmenting the immunosuppressive TME. The mechanism whereby IGFBP2 regulates MHC class II gene expression, however, is unknown. IGFBP2 was reported to physically interact with Pim-1-associated protein-1 (PAP-1)-associated protin-1 (PAPA-1) in the nucleus. PAPA-1 (or INO80B) is a key component in the ATP-dependent INO80 chromatin-remodeling complex. A recent study shows that INO80 is required for super-enhancer-mediated oncogenic transcription and tumor growth in melanoma. Interestingly, the MHC class II genes HLA-DR and HLA-DQ were found to be regulated by a super-enhancer mediated mechanism in autoimmune disease. We hypothesized that tumor-secreted IGFBP2 directly regulates INO80 function and inhibits the MHC class II expression in APCs in the TME. Our preliminary data show that exogenous IGFBP2 translocates into the cell nucleus within hours, and co-immunoprecipitation confirmed that IGFBP2 interacts with INO80B in vitro. Furthermore, MHC Class II expression was downregulated by IGFBP2 in the murine DC 2.4 dendritic cell line and in mouse bone marrow-derived dendritic cells by RT-PCR and flow cytometry. Our data suggest that PDAC IGFBP2 levels might predict checkpoint blockade therapy readiness, and importantly, serve as a target mechanism for enabling immunotherapy effectiveness. Citation Format: Ping-Chieh Chou, Liang Liu, Elizabeth Forbes, Ashley Ballard, Tao Li, Wei Zhang. IGFBP2 reprograms pancreatic cancer immune surveillance at epitranscriptome levels [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B98.

The Role of Proteasome Activator PA28α in Multiple Myeloma

Multiple myeloma (MM) is a commonly occurring hematologic malignancy in the United States with poor prognosis. Among all treatments, proteasome inhibitor (PI) based regimens have been a major breakthrough for patients' outcomes. Available PIs all target 20S proteasome core complex, and the duration of response is limited by toxicity and resistance development. Until now, the underlying mechanism of drug resistance remains unclear. The proteasome is the major proteolytic machinery in protein homeostasis which is pivotal for myeloma cell survival. A functional proteasome consists of 20S proteasome core particle with regulatory particle on one or both ends. There are 3 types of proteasome regulators that could activate a 20S proteasome, PA700 (19S), 11S REG (PA28) and PA200. The 11S REG (PA28) protein family consists of three members, α, β, and γ. PA28 α/β are IFN-γ inducible and with higher expression in antigen presenting cells. Currently, the function of 11S subunit remains largely unknown. Our analysis of plasma cells from MM patients and healthy donors has demonstrated that expression of 11S proteasome is higher in myeloma cells than normal plasma cells and progressively upregulated with disease progression. To further identify the function of 11S proteasome especially PA28α in MM, we generate PA28α knockdown stable MM cell lines. We have found that knockdown of PA28α inhibits MM cell growth and proliferation, also induces myeloma cell resistance to PIs. The mechanism of PI resistance is different from knocking down of 19S or 20S proteasome subunits. Silencing of PA28α inhibits proteasome activity and decreases proteasome work load concurrently, resulting in a favorable proteasome load vs capacity ratio. Altogether, in this report, we describe the function of PA28α in MM cells, also provide novel insights into regulating PIs sensitivity through modulation of the 11S proteasome subunit PA28α. Disclosures Hofmeister: Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Kaufman:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Winship Cancer Institute of Emory University: Employment; AbbVie: Consultancy; Takeda: Consultancy; TG Therapeutics: Consultancy. Nooka:Amgen: Honoraria, Other: advisory board participation; GSK: Honoraria, Other: advisory board participation; Celgene: Honoraria, Other: advisory board participation; Takeda: Honoraria, Other: advisory board participation; Spectrum pharmaceuticals: Honoraria, Other: advisory board participation; BMS: Honoraria, Other: advisory board participation; Janssen: Honoraria, Other: advisory board participation; Adaptive technologies: Honoraria, Other: advisory board participation. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Lonial:Takeda: Consultancy, Research Funding; Amgen: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; GSK: Consultancy; Karyopharm: Consultancy; Genentech: Consultancy; Celgene Corporation: Consultancy, Research Funding.

Paraimmunobiotic Bifidobacteria Modulate the Expression Patterns of Peptidoglycan Recognition Proteins in Porcine Intestinal Epitheliocytes and Antigen Presenting Cells.

Peptidoglycan recognition proteins (PGLYRPs) are a family of pattern recognition receptors (PRRs) that are able to induce innate immune responses through their binding to peptidoglycan (PGN), lipopolysaccharide, or lipoteichoic acid, or by interacting with other PRR-ligands. Recently, progress has been made in understanding the immunobiology of PGLYRPs in human and mice, however, their functions in livestock animals have been less explored. In this study, we characterized the expression patterns of PGLYRPs in porcine intestinal epithelial (PIE) cells and antigen-presenting cells (APCs) and their modulation by the interactions of host cells with PRR-ligands and non-viable immunomodulatory probiotics referred to as paraimmunobiotics. We demonstrated that PGLYRP-1, -2, -3, and -4 are expressed in PIE cells and APCs from Peyer’s patches, being PGLYPR-3 and -4 levels higher than PGLYRP-1 and -2. We also showed that PGLYRPs expression in APCs and PIE cells can be modulated by different PRR agonists. By using knockdown PIE cells for TLR2, TLR4, NOD1, and NOD2, or the four PGLYRPs, we demonstrated that PGLYRPs expressions would be required for activation and functioning of TLR2, TLR4, NOD1, and NOD2 in porcine epitheliocytes, but PGLYRPs activation would be independent of those PRR expressions. Importantly, we reported for the first time that PGLYRPs expression can be differentially modulated by paraimmunobiotic bifidobacteria in a strain-dependent manner. These results provide evidence for the use of paraimmunobiotic bifidobacteria as an alternative for the improvement of resistance to intestinal infections or as therapeutic tools for the reduction of the severity of inflammatory damage in diseases in which a role of PGLYRPs-microbe interaction has been demonstrated.