bluuebunny/crossref_metadata_2025 huggingface dataset:
H3K4me3 Profiles of LPS and Immune Complex-Activated Macrophages
Authors: Palaga Tanapat , Ruenjaiman Vichaya , Leu Yu-Wei
Published: January 2023

Macrophages play an important role in immune response such as inflammation, phagocytosis, metabolism and tissue homeostasis. Lipopolysaccharide (LPS)-stimulated macrophages produces pro-inflammatory cytokines such as TNFα, IL-6 and IL-12 while repeated stimulation with LPS can lead to unresponsive state called LPS tolerance. Stimulation with LPS in the presence of immune complex (IC) drastically changes gene expression profiles of macrophages. LPS/IC-stimulated macrophages produce high level of IL-10 and low level of IL-12. In this study, we investigated the plasticity between LPS-stimulated and LPS/IC-stimulated macrophages and compared active histone marks (trimethylation on lysine 4 on histone 3 (H3K4me3)) in LPS-stimulated macrophages and LPS/IC-stimulated macrophages using bone marrow derived murine macrophages by the ChIP-seq approach. Stimulation with LPS that was followed immediately by LPS/IC did not result in IL-10 production. Defects in activation of p38, p44/42 Erk and NF-kB p65 were observed. In contrast, the resting of 2 days between LPS and LPS/IC yielded IL-10hiIL-12lo phenotype. Our ChIP-seq data suggested that the global H3K4me3 profiles differed between these LPS- and LPS/IC-stimulated macrophages. Some key genes that are uniquely upregulated in LPS/IC-stimulated macrophages were found to contain H3K4me3 in the regulatory region. Taken together, LPS/IC-stimulated macrophages exhibits unique H3K4me3 profiles and our results may help explain the plasticity of LPS vs. LPS/IC-stimulated macrophages.
Cross-talk between MDP/NOD2 and GPIbα/Mac-1 pathways controls production of PGE2 and of IL-1b and IL-6 cytokines in human monocytes activated with MDP adjuvant
Authors: Zaitseva Marina , Liu Fengjie , Endo Yukinori , Romantseva Tatiana , Wu Wells W , Shen Rongfong , Golding Hana
Published: January 2023

Adjuvants are included in vaccine formulations to activate antigen presenting cells (APC). In some cases, strong activation of APC by adjuvants may induce excessive release of inflammatory substances causing adverse reactions in vaccine recipients. Here we investigate the mechanism of production of prostaglandin E2 (PGE2), a proximal mediator of fever, and of pyrogenic cytokines IL-1β and IL-6 in human monocytes activated with muramyl dipeptide (MDP) adjuvant. MDP was associated with fever and reactogenicity in rabbits and humans.

In monocytes, MDP alone induced nuclear NFkB but did not induce PGE2. Addition of conditioned medium obtained from CD3 bead-purified human T cells (TCM) to MDP resulted in production of PGE2, IL-1β and IL-6, suggesting a need for a second signal. The co-stimulatory factor in TCM was identified as Glycoprotein Ib alpha (GPIbα) protein by mass spectrometry. Western blotting confirmed increased GPIbα in TCM due to shedding of GPIbα from CD3 bead-purified T cells. Antibodies against GPIbα and against the GPIbα receptor, Mac-1 integrin (aMβ2), inhibited secretion of PGE2 and IL-1β/IL-6 in monocytes activated with MDP and TCM. The contribution of Mac-1 to inflammatory response to MDP in vivo was further confirmed by showing reduced expression of COX-2 mRNA in the liver and spleen in β2 KO and aM KO mice following administration of MDP compared with wild type mice. In summary, we describe for the first-time production of GPIbα by T cells and a two-signal model of PGE2 production by monocytes: MDP-induced activation of NFkB represent Signal 1 and Signal 2 is transmitted from GPIbα-activated Mac-1. Further study of T cell-monocyte nexus might help in the assessment/prediction of inflammatory potential of novel adjuvants.
Identification of age-related changes in chromatin accessibility and gene expression in T cells from thymus to periphery
Authors: achour achouak , Chen Guobing , Sharov Alexei , Nguyen Thomas , Li Xiang , Patrick Michael , Wood William , De Supriyo , Becker Kevin , Peng Weiqun , Weng Nan-Ping
Published: January 2023

Aging of immune system is characterized by progressive decline of physiological and cellular function of T cells, leading to a reduced immune function in the old. Recent studies of naïve T cells from young and old mice have identified age-related altered gene expressions, but the mechanisms underlying age associated changes of naïve T cells remain poorly understood. Here, we compared chromatin accessibility and the transcriptome in mature thymocytes and in naïve T cells of spleen between young (6–8 weeks) and old (95–114 weeks) C57BL/6 mice, using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and Agilent microarray methods, respectively. We identified age-related changes of chromatin accessibility that are correlated with the gene expression changes, providing a chromatin basis of age-related changes in gene expression. Interestingly, some age-related changes of CD4 and CD8 T cells in thymus did not retain in the spleen, and others appeared later only in the spleen, suggesting different tissue environment may contribute to the age-related changes of T cells. To directly address environmental effects, we currently examine changes of naïve T cells from old mouse before and after hosted in young mouse for 21 days in chromatin accessibility, transcriptomes, and protein expression. Collectively, these approaches will allow us to dissect the time and environmental influences in naïve T cell function in old mice. Our findings will have important implications both for better understanding the mechanisms underlying these age-related changes as well as opening potential new targets to mitigate the age-related reduced T cell functions.
Dose-Dependent Requirements for ETS1 in Invariant Natural Killer T (iNKT) Cell Development
Authors: Morgan Roxroy C , Kee Barbara L
Published: January 2023

iNKT cells are innate-like T cells that use an invariant T cell receptor to recognize glycolipids presented by the non-classical major histocompatibility protein CD1d. Upon recognition, iNKTs rapidly produce cytokines that stimulate innate cells to produce cytokines and prime adaptive cells for activation upon antigenic stimulation. Studies in Ets1 germline-deficient mice showed that the transcription factor ETS1 is required for the development of CD4+ NK1.1+ NKT cells. Development of CD1d tetramers and the recent elucidation of multiple effector iNKT cell subsets has improved our understanding of NKT cell development and function, and revealed that CD4+ NK1.1+ iNKT cells are a minor subset of total NKTs. Using conditional deletion for Ets1 in thymocytes, we show that Ets1 is a dose dependent regulator of all iNKT cells and appears to be particularly important for development of interferon-g-producing iNKT1 cells. Deletion of Ets1 in developing iNKT1 cells using Tbx21Cre demonstrated the critical role of ETS1 in this iNKT cell effector subset. Our findings set the stage for future studies focused on the molecular mechanism by which ETS1 controls iNKT cell development and function.
Peroxisome Proliferator-activated Receptor gamma (PPARγ) suppresses inflammation and bacterial control during influenza bacterial super-infection
Authors: Gopal Radha , Mendy Angelico , Marinelli Michael A , Richwalls Lacee J , Seger Philip J , Patel Shivani , McHugh Kevin J , Rich Helen E , Grousd Jennifer A , Forno Erick , Alcorn John F
Published: January 2023

Influenza is one of the most common causes of respiratory illness worldwide and can be complicated by secondary bacterial pneumonia, which is a common cause of mortality. Influenza-induced “cytokine storm” is thought to play a role in lung pathogenesis during influenza infection. PPARγ is a member of nuclear hormone receptor superfamily that is known to enhance lipid and glucose metabolism, cellular differentiation and suppress the inflammatory immune response. Synthetic PPARγ agonists (thiazolidinedione, rosiglitazone) have been used clinically in the treatment of type II diabetes. In this study, we determined the effect of rosiglitazone in mice infected with influenza or influenza, methicillin-resistant Staphylococcus aureus (MRSA) super-infection. We found decreased influenza viral burden, decreased numbers of neutrophils and macrophages in bronchoalveolar lavage (BAL), and decreased production of IL-6, IL-12, CCL2, and CXCL10 in influenza-infected, rosiglitazone-treated mice when compared to influenza-infected control mice. However, rosiglitazone treatment compromised bacterial control during influenza-bacterial super-infection. Further, we found decreased numbers of neutrophils and decreased gene expression of neutrophil elastase and cathepsin G during super-infection in rosiglitazone-treated mice. Similarly, using data from the National Health and Nutrition Examination Survey (NHANES), we found increased influenza/pneumonia mortality in rosiglitazone-treated patients compared to other diabetic patients (adjusted hazard ratio=3.4, 95% confidence interval=1.1–10.7). Both human and mouse data suggest that rosiglitazone treatment worsens the outcome of influenza-associated pneumonia.
T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium
Authors: Ramirez Valerie Trinidad , Brust-Mascher Ingrid , Gardner Mariana Barboza , Sladek Jessica A. , Gareau Melanie G. , Reardon Colin
Published: January 2023

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.
Analysis of cholinergic pathway-induced alterations at the intestinal mucosa and their association with enhanced resistance to lethal bacterial infection
Authors: Fernandez-Cabezudo Maria J , Al-Mansouri Alreem A , Bashir Ghada , Al-Sbiei Achraf , Venkatachalam Karthikkumar , al-Ramadi Basel K
Published: January 2023

The mucosal innate immune system represents the first line of defense against microorganisms invading the intestinal tract. Increasing evidence suggests that mucosal immune defenses are tightly regulated by immunological and neural pathways. We previously demonstrated that activation of the cholinergic pathway through the administration of a highly specific acetylcholinesterase inhibitor (AChEI), paraoxon, confers resistance to an oral infection by virulent S. typhimurium. This protection is mediated by enhanced innate anti-microbial mucosal defenses in the GI tract. In the present study, we investigated the involvement of different immune cell populations in the GI tract in the modulation of AChEI-mediated anti-Salmonella resistance. Morphological evaluation of intestinal tissue from control- or AChEI-treated mice revealed a significant thickening of the mucin layer and increased B and T lymphocytic infiltration into intestinal mucosa in the AChEI group. Multi-color flow cytometric analysis of iliac intraepithelial lymphocytes (IELs) showed that cholinergic stimulation increased the percentage of CD3+CD8+gamma/delta TCR+ cells. No significant alterations were observed in lamina propria cells. Our findings indicate that cholinergic pathway is able to modulate not only mucin-secreting cells but more importantly gamma/delta T cells, the cell population known to be involved in tissue homeostasis and repair as well as surveillance of the epithelial barrier.
Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation
Authors: Rivas Magali Noval , Wakita Daiko , Franklin Michael , Chen Shuang , Yamashita Michifumi , Crother Timothy R. , Shimada Kenichi , Arditi Moshe
Published: January 2023

Kawasaki Disease (KD), a systemic vasculitis of unknown etiology is the leading cause of acquired heart disease among children in the US. Subsets of KD children display gastrointestinal abnormalities during the acute phase of the disease and may have increased serum levels of secretory IgA (SIgA), suggestive of a “leaky gut” phenotype. Additionally, KD patients have higher frequencies of IgA producing B cells in the inflamed coronary artery and in the respiratory and gastrointestinal tracts. How intestinal permeability participates in cardiovascular lesions of KD remains unknown. We observed increased intestinal permeability and elevated circulating sIgA in KD patients as well as elevated sIgA and IgA deposition in vascular tissues in the Lactobacillus casei-cell wall extract murine model of KD vasculitis. Pharmacological blocking of intestinal permeability prevented gut permeability, vascular tissues IgA deposition, and ameliorated cardiovascular pathology. By using genetic and pharmacologic inhibition of IL-1β signaling, we demonstrated that IL-1β lies upstream of disrupted intestinal barrier function, subsequent IgA vasculitis development, and cardiac inflammation. Thus, targeting mucosal barrier dysfunction and the IL-1β pathway may be applicable to other IgA-related diseases including IgA vasculitis and IgA nephropathy. These observations enhance our current understanding of KD pathogenesis and may provide new diagnostic and therapeutic strategies for affected patients.
Vitamin A signaling regulates pathological epithelial cell shedding via IL-18
Authors: Iyer Namrata R , Vaishnava Shipra
Published: January 2023

The intestinal epithelium is under continuous renewal via a tightly controlled process of cell proliferation and cell shedding. During infection and sepsis, the rate of cell shedding increases dramatically as a protective response. However, the mechanisms through which epithelial turnover rate is controlled in the gut remain elusive. Here we identify diet-mediated control of epithelial cell shedding as an important mechanism in defense against infectious disease. We show that abrogating vitamin A signaling by expressing a dominant negative retinoic acid receptor exclusively in intestinal epithelial cells (dnRARVillin-Cre) makes mice resistant to pathological epithelial cell shedding and cell death in an LPS model of sepsis. In a Salmonella infection model, dnRARVillin-Cremice had increased intracellular loads of the pathogen in the intestine as compared to control mice. This trend was observed even at early time points of the infection and was associated with drastically reduced epithelial cell shedding compared to dnRARfl/fl mice. Intestinal epithelium RNA-seq and protein analysis uncovered that levels of the pro-inflammatory cytokine IL-18 are severely reduced in epithelia of dnRARVillin-Cre mice. LPS and pathogen induced cell death were both rescued upon IL-18 reconstitution in dnRARVillin-Cre mice. Furthermore, even dietary vitamin A could modulate epithelial IL-18 expression. Our results thus reveal a novel mechanism whereby dietary vitamin A modulates cell shedding response in the epithelium via IL-18, affecting barrier function and infection susceptibility in the gut.
The Cd8 locus Controls the Functional Conversion of CD4 Th cells to CD4 CTL in the Intestine
Authors: Iwaya Hitoshi , Larange Alexandre , Kakugawa Kiyokazu , Cheroutre Hilde
Published: January 2023

CD4 T cells adapt to their surroundings by differentiating into various T helper (Th) subsets. At steady state, regulatory CD4 T cells (Treg) and interleukin (IL)-17 producing CD4 T cells (Th17 cells) are present in the intestine and localize mainly to the lamina propria, whereas CD4 cytotoxic T lymphocytes (CTL) reside in the epithelium. A hallmark of CD4 CTL is the reactivation of the Cd8 locus and the re-expression of CD8a controlled by the Cd8 enhancer I (E8I). We showed before that CD4 Th cells can reprogram to CTL. Here, we elucidated a mechanism that drives the CTL reprogramming of CD4 Th cells. We identified a long noncoding RNA transcribed from the Cd8 locus, as the critical regulator of the reciprocal expression of T-BET and RUNX combined with the suppression of Foxp3 and Rorc in CD4 CTL precursors. We further found that the E8I region functions as is central coordinator of the RUNX3-controlled cytotoxic gene expression program marked by the re-expression of CD8a. Adoptive transfer of naïve E8I deletion mutant CD4 T cells to Rag1−/− recipient mice led to accelerated weight loss, and severe small and large intestine inflammation with excessive accumulation of pathogenic IL-17A and IL-17A/IFNg double producing CD4 T cells. Altogether, these findings indicate that the Cd8 locus controls the functional polarization of CD4 Th cells and furthermore, that the conversion to CTL not only enhances the direct protective capacity but also regulate the immune response and prevents excessive inflammation and immune pathology at the mucosal border of the intestine.
Mechanisms of antigen transport from the female reproductive tract to the gastrointestinal tract
Authors: Howe Savannah E , Shillova Nita , Konjufca Vjollca H
Published: January 2023

One of the major functions of the female reproductive tract (FRT) is the establishment and maintenance of pregnancy, resulting in healthy offspring. Although many pathogens (e.g. Chlamydia spp.) infect the FRT, no vaccines against these pathogens have been licensed to date. This lack of success has been in part attributed to the inability of the FRT to efficiently internalize antigens. We showed that FRT epithelium of mice can internalize nanoparticles (NPs) < 40 nm and that protein coated 20 nm NPs prime the intestinal mucosa for secretion of IgA. This finding led us to hypothesize that NPs reach the GI tract following per-vaginal (PV) instillation. Using multiphoton and fluorescence microscopy, we show that PV-instilled antigens (DAPI, Dextran, NPs) reach the stomach antrum and the small intestine within 1–3 h. It is proposed that Chlamydia spp. colonizes the GI tract following PV instillation by entering the circulation. We show that colonization of the GI tract by C. muridarum is dependent on cell transport. While within 7 days of PV infection Chlamydia colonizes the GI tract of controls, while no Chlamydia can be detected in iliac lymph nodes (ILNs) or GI tracts of CCR7−/− mice. Moreover, treatment of mice with FTY720 inhibits colonization of the GI tract, but it does not preclude infection of ILNs by Chlamydia. This finding indicates that Chlamydia transport from the ILNs to the GI tract depends on sphingosine 1-phosphate-mediated cell egress. Elucidation of mechanisms by which C. muridarum disseminates systemically to colonize the lymphoid tissues and the GI tract will be important for understanding both the immunity to and pathogenesis of Chlamydia spp.
Human CD39 overexpression or APT102 recombinant apyrase administration enhances Treg and Tr1 cell immunity in inflammatory bowel disease
Authors: Vuerich Marta , Robles Rene’ J. , Mukherjee Samiran , Xie Anyan , Harshe Rasika , Moss Alan C. , Robson Simon C. , Longhi Maria Serena
Published: January 2023

CD39/ENTPD1 is an ectonucleotidase that scavenges pro-inflammatory nucleotides to ultimately generate adenosine. Single nucleotide polymorphisms within the promoter region of the ENTPD1 gene are linked to higher rates of inflammatory bowel disease (IBD) in humans. Furthermore, decreased levels of CD39 expression are noted in Th17 cells purified from patients with Crohn’s disease, because of aberrant signaling by aryl-hydrocarbon-receptor (AhR) in response to endogenous ligands e.g. unconjugated bilirubin (UCB). Deletion of CD39 increases susceptibility to disease in experimental colitis while overexpression of human CD39 (hCD39) in transgenic mice results in protection from ischemia reperfusion injury and thromboembolism.

Here, we show that overexpression of hCD39 results in amelioration of dextran-sulfate-sodium (DSS)-induced experimental colitis and protects mice from the deleterious effects of systemic hypoxia, pharmacologically induced by deferoxamine. We further report that treatment with APT102, soluble recombinant apyrase, boosts Foxp3 expression in wild type Treg and renders Crohn’s-derived Treg and Tr1 cells more responsive to UCB-AhR stimulation in vitro. Further, APT102 enhances the beneficial regulatory effects of UCB in DSS colitic mice, in vivo.

We conclude that transgenic overexpression of hCD39 is beneficial in experimental colitis and ameliorates systemic hypoxic injury in vivo. Exogenous administration of APT102 promotes AhR-mediated regulatory effects in vivo while boosting the regulatory T-cell pool in Crohn’s disease ex vivo. Boosting endogenous CD39 or augmenting ectonucleotidase activity, as with pharmacological treatment with APT102, might represent therapeutic options in IBD.
B-cell specific duo-conjugates for therapy of autoimmune myasthenia gravis
Authors: Huda Ruksana , Bahauddin Afrin , Fang Xiang , Wu Xiaorong
Published: January 2023

We have prepared a unique set of monoclonal antibody-siRNA conjugates which will target two different subsets of pathogenic B cells in a mouse model of autoimmune myasthenia gravis (EAMG). At the optimum dose, our duo-conjugates significantly reduced two crucial receptors, CD268 and -269, of pathogenic B220 positive cells in PBMCs, lymph node cells and splenocytes in EAMG mice. Modifying the base-composition of the conjugate components further reduced adverse effects of the high dose and significantly decreased pathogenic autoantibody levels, which corresponded with improved clinical symptoms. Our ongoing works suggest a potential for further developing target specific conjugates as highly effective therapeutics for myasthenia gravis.
Blocking the Pro-Inflammatory Response Associated with Inflammatory Bowel Disease with 7-Ketocholesterol
Authors: Bamezai Anil K , Mallon Francine , Lawrence Audrey
Published: January 2023

Inflammatory bowel disease (IBD) affects more than one million people in the United States alone, and currently has no cure. IBD is a chronic inflammatory condition, perpetrated by dysregulation of the adaptive immune system’s response to nonpathogenic flora in the gastrointestinal tract. CD4+ T cells, critical components of the adaptive immune response, misperceive commensal microbiota in the gut as threats and persistent stimulators of CD4+ T cells, resulting in uncontrolled and episodic responses. Regulatory T cells, through secretion of interleukin 10 (IL-10) cytokine, suppress the pro-inflammatory environment in the gut. In the absence of this suppression a chronic inflamed state can be reached. Previous studies have shown that 7-ketocholesterol (7-KC), a natural metabolite of cholesterol, can successfully disrupt lipid raft-based membrane order in vitro, promoting a disordered state. This forces the question of whether pro-inflammatory responses can be disrupted through this method. We investigated the potential of 7-KC to suppress gut inflammation using the IL-10 KO animal model. Mice deficient in IL-10 have inflamed guts and symptoms of IBD as observed in humans. IL-10 knockout mice were orally gavaged twice daily, 3 days a week for 3 weeks to deliver 7-KC. Treated and control mice were observed for 4 weeks and necropsies were performed on day 27 to dissect their colons for immunohistochemistry. We observed a change in tissue constitution, stool quality, and fatty pockets in the treatment group in comparison with the controls receiving saline. These preliminary findings with IL-10 knockout mouse model reflect the potential of 7-KC as immunomodulatory agent for suppressing the inflammatory environment in the gut.
Development of a Novel Antigen-Specific Therapy for Autoimmune Diabetes Using an Insulin-ChgA Hybrid Peptide Neoantigen
Authors: Jamison Braxton , Beard K. Scott , Neef Tobias , Bradley Brenda , Baker Rocky , Gill Ronald G. , Miller Stephen D. , Haskins Kathryn
Published: January 2023

In Type 1 Diabetes (T1D) patients and non-obese diabetic (NOD) mice, autoreactive CD4 T cells contribute to destruction of insulin-producing β-cells in the pancreatic islets. Our lab has discovered that in NOD mice the endogenous ligand for the diabetogenic CD4 T cell clone BDC-2.5 is a hybrid insulin peptide (2.5HIP), consisting of a fragment from proinsulin fused to a peptide from chromogranin A (ChgA). The purpose of this study was to determine if tolerogenic poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) coupled with 2.5HIP (2.5HIP-PLG) could be used to induce antigen-specific T cell tolerance. We have found that treatment with 2.5HIP-PLG NPs synergizes with a short course of low-dose recombinant IL-2, leading to robust induction of 2.5HIP-specific Foxp3+ regulatory T cells (Tregs) and prevention of spontaneous diabetes in 50% of NOD mice. In a disease reversal model, spontaneously diabetic NOD mice were transplanted with 500 syngeneic islets and recipients were infused with HEL-PLG (control) or 2.5HIP-PLG NPs pre- and post-transplant. In contrast to HEL-PLG treated mice that showed aggressive disease recurrence in around 2 weeks, graft destruction in 2.5HIP-PLG treated mice was significantly delayed up to an average of 8 weeks, indicating that targeting graft-infiltrating 2.5HIP-specific T cells dampens ongoing autoimmunity. Our current goal is to determine whether the therapeutic effects observed in the transplantation model are due to expansion of antigen-specific Tregs, induction of anergy in effector T cells, or both. This novel antigen-specific therapy using a disease-relevant neoantigen may have implications for development of an immune-modifying treatment for T1D patients.
Transplantation of MHC-mismatched Mouse Embryonic Stem Cell-Derived Thymic Epithelial Progenitors Prevents Autoimmune Diabetes
Authors: Su Min , Lin Yujun , Lai Laijun
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease results from the destruction of insulin-secreting islet β-cells by autoreactive T cells. NOD mice are the widely used animal model for human T1D. Autoimmunity in NOD mice is associated with particular MHC loci and impaired islet autoantigen expression and/or presentation in the thymus, which results in defects in both central and peripheral tolerance. It has been reported that induction of mixed chimerism with MHC-mismatched, but not MHC-matched donor bone marrow (BM) transplants prevents the development T1D in NOD mice. We have reported that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into thymic epithelial cells (TECs) in vivo to support T cell development. We show here that transplantation of MHC-mismatched mESC-TEPs and MHC-matched donor BM prevents insulitis and T1D development in NOD mice. This is associated with higher expression of proinsulin 2, a key islet autoantigen, in the mESC-TECs, and an increased number of regulatory T cells. Our results suggest that embryonic stem cell-derived TEPs may offer a new approach to control T1D.
Neutralization of IL-2 prevents acute GVHD while preserving GVL activity via regulating PD-L1/PD-1 signaling
Authors: Song Qingxiao , Wang Xiaoning , Martin Paul J. , Chen Yuanzhong , Zeng Defu
Published: January 2023

Acute gut graft versus host disease (GVHD) has a critical impact on the outcome of allogeneic hematopoietic cell transplantation (allo-HCT). Our recent studies indicate that IL-2 from donor CD4+ T cells make infiltrating T cells resistant to host-tissue PD-L1-mediated tolerance (Ni & Song et al: JCI 2017), but the mechanisms remain unclear. To tested whether in vivo neutralization of IL-2 early after allo-HCT differentially regulates PD-L1/PD-1 signaling in GVHD target tissues and lymphoid tissues, spleen and BM cells from C57BL/6 (H-2b) donors were transplanted into lethally irradiated wild-type (WT) or PD-L1−/− BALB/c (H-2d) recipients in the presence or absence of BCL1 leukemia/lymphoma cells. Recipients were treated with neutralizing anti-IL-2 mAb or control IgG. Administration of anti-IL-2 completely prevented gut GVHD while preserving GVL activity in WT recipients but not in PD-L1−/− recipients. This effect was associated with expansion of donor T cells in the lymphoid tissues (i.e. spleen and MLN) but reduction of donor T cells in the GVHD target tissues such as the gut. Prevention of GVHD resulted from increased donor T apoptosis, with increased expression of Fas but reduced expression of Bcl-XL. Residual T cells showed evidence of enhanced anergy/exhaustion and upregulated expression of FR4, CD73, KLRG1 and Eomes. Finally, prevention of GVHD was associated with inhibition of pAKT/mTOR pathway in a donor T cell PD-1-dependent and host tissue PD-L1-dependent manner. These results indicate that neutralizing IL-2 early after allo-HCT augments tolerance-induction through PD-1 signaling mediated by host PD-L1 in GVHD target tissues but not in lymphoid tissues, thereby preventing gut GVHD while preserving GVL effects.
Ex vivo fucosylation promotes the GVL effect of NK cells after Allo-HSCT
Authors: Gong Huanle , Ma Shoubao , Wu Depei
Published: January 2023

Recent studies demonstrated that ex vivo fucosylation can increase the persistence and anti-GVHD potency of Treg cells. Fucosylation also improves human cord blood engraftment. However, the role of fucosylation in graftversus- leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) remains unknown.

Here, we established a murine GVL model by intravenously injecting luciferase (lus+)/yfp-expressing B-cell lymphoma (A20) cells and founded that adoptive transfer of fucosylated NK cells markedly prolonged the survival than control NK cells. Ex vivo fucosylation significantly upregulated IFN-γ production in NK cells and enhanced their cytotoxic activity. Flow cytometry analysis revealed that fucosylation promoted the infiltration of NK cells, which may be due to the elevated binding to CD62E. Further studies are needed to confirm the contribution of IFN-γ by using IFN-γ KO mice and investigate the detailed mechanisms of fucosylated NK cells in the regulation of GVL responses.
Profiling pre-existing autoantibodies for prediction of Primary Graft Dysfunction after lung transplantation (LT)
Authors: Li Quanzhen , Zhu Chengsong , Kaza Vaidehi
Published: January 2023

Background

Primary graft dysfunction (PGD) is acute allograft injury that results in increasing morbidity and mortality after LT. Pre-existing autoantibodies (autoAbs) targeting to self-antigens may play a major role for PGD.

Method

In this study, the effects of autoAbs and their specific patterns were comprehensively evaluated on serum samples collected from 51 patients before and after LT. The pre-existing AutoAbs in sera were measured using proteomic arrays bearing 375 autoantigens developed by UT Southwestern Microarray core. The score of each autoAb was compared between the patients who developed PGD (n=21) with those without PGD (n=30). Survival analysis was performed to investigate the effects of differential autoAbs upon the survival proportion. A machine learning program combined with lasso and elastic-net model was used to select the panel of AutoAbs with the best predict value for PGD.

Results

The patients who developed PGD had a lower survival rate compared with patients without PGD. The autoAbs against AchR3, AGTR1, FUCA1 and aldolase showed significant higher expression in patients with PGD compared with those without PGD (fold change >1.5, p<0.05). The levels of anti-AchR3 and anti-AGTR1 were significantly correlated with the survival rate in PGD group. A panel of 19 AutoAbs were selected by a machine learning program with the best predictive value for PGD. A receive operative curve was generated using the 19 autoAb panel with AUC 0.96 and CI 0.99–1.0.

Conclusion

Our study identified AutoAbs associated with PGD in LT patients. A prediction model developed with a panel of AutoAbs with high accuracy may make a significant impact in the management of LT recipients.
Expression of the Butyrate/niacin Receptor, GPR109a on T cells Plays an Important Role in a Mouse Model of Graft Versus Host Disease
Authors: Docampo Melissa D , Stein-Thoeringer Christoph , Lazrak Amina , da Silva Marina Burgos , van den Brink Marcel R.M.
Published: January 2023

The intestinal microbiota is essential for the fermentation of dietary fibers into metabolites, notably the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors such as GPR109a to activate downstream anti-inflammatory effects. GPR109a is the main butyrate receptor and we have previously demonstrated that butyrate can ameliorate gastrointestinal injury during graft versus host disease (GVHD). Therefore, we hypothesized that GPR109a is important in the pathophysiology of intestinal GVHD. Using a MHC-mismatched model of GVHD, we demonstrated a lower incidence of disease when mice received wild type (WT) bone marrow (BM) and T cells from a GPR109a−/− mouse. It is known that GPR109a is highly expressed on myeloid cell populations such as dendritic cells and macrophages; however, expression on T cells is not entirely resolved. At baseline, we observed low expression of GPR109a on T cells, which increases upon T-cell receptor stimulation. T cell development in GPR109a−/− mice appears normal, however, 7 days after transplant, we found significantly fewer CD4+ and CD8+ T cells in the intestines of mice that received WT BM and GPR109a−/− T cells. While frequency of Th1 and Th17 cells were similar between the groups, we saw significantly more regulatory T cells in the intestines and spleen of GPR109a−/−T cell recipients. This reduction of GVHD was accompanied by maintenance of the gut commensal Clostridium and increased butyrate, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Thus, we propose a novel role for GPR109a in the expansion and activation of T cells during GVHD.
Role of Akt Isoforms in Hyperlipidemia induced changes to Regulatory T cells
Authors: Hyde Michael , Bagley Jessamyn , Iacomini John
Published: January 2023

Hyperlipidemia is a common health condition in the transplant patient population. This condition causes end-stage heart disease in approximately 40% of all patients requiring a heart transplant, developing in 50% of heart transplant recipients after the first year and 95% of patients within 5 years of transplant. A history of ischemic heart disease resulting from coronary artery disease due to hyperlipidemia is also a major predictor of heart transplant rejection. In spite of its prevalence in the transplant population and the observation that it appears to be a significant risk factor in rejection, the role of hyperlipidemia in transplant rejection remains poorly understood. We have recently shown that hyperlipidemia promotes an aggressive rejection response that results in accelerated rejection of allogeneic heart transplants and resistance to tolerance induction. Hyperlipidemia causes a variety of systemic immune system changes including increased production of IL-17, and regulatory T cell dysfunction. Here we examined how activation of Akt alters regulatory T cells phenotype and metabolism. In mice, Akt has three isoforms, two of which play a critical role in regulatory T cells, Akt1 and Akt2 which have non-overlapping roles in cellular functions. Here we examine the mechanism of Akt activation, activation of downstream targets of Akt1 and Akt2, and modification of metabolic pathways in regulatory T cells derived from hyperlipidemic mice.
The Ocular Immune Response in a Rodent Model of Whole Eye Transplantation
Authors: Li Bing , Zhang Wensheng , Wang Yong , Banaee Touka , Chen Wendy , Su An-Jey , Owens Charles , Miller Maxine , Komatsu Chiaki , Washington Kia
Published: January 2023

Introduction

Approximately 36 million people are totally blind globally. Using a rodent whole eye orthotopic transplant model (WET), we are investigating a potential clinical treatment to restore vision. Typically, clinical transplantation involves allogeneic individuals and immune rejection is the first obstacle after surgery. Her--e, we use an MHC class I and II mismatched transplant model without immunosuppression to describe for the first time the features of the immune reaction in WET.

Methods

14-wk old male Brown Norway (BN) rats served as donor and recipient in the syngeneic group. 14-wk old male BN rats served as donors with age/sex matched Lewis rats used as recipient in the allogeneic group. Both groups underwent WET, subsequently the transplanted eye blood supply and structure were evaluated on post-operative day (POD) 2 by optical coherence tomography (OCT). Skin and cornea rejection at POD 2,4,5,6,8 were assessed separately by a scoring system. The progressive infiltration of immune cells into the eye was evaluated by flow cytometry and histology.

Results

At POD5 allogeneic corneas had transparency changes that coincided with skin rejection and Cornea thickness dramatically increased by 150% over syngeneic eyes. Also, the allogeneic group had significant CD4+, CD8+ T cells and NK cell infiltration in aqueous humor, iris, retina and choroid with no difference in cornea.

Conclusion

These preliminary results indicate that acute rejection in WET is T cell mediated and the blood ocular barrier is compromised post-operatively, where immune cells can access the eye. Rejection after WET can be potentially monitored by noninvasive measurement of the cornea thickness or easily accessible aqueous humor.
A longitudinal analysis of TCRβ repertoire diversity in immune tolerance of pediatric patients post-UCBT: T cell clonal deletion over clonal anergy
Authors: Chen Xiaohua , Szabolcs Paul
Published: January 2023

The mechanisms of long term immune tolerance after successful hematopoietic stem cell transplantation are not fully elucidated. We set up serial experiments to test central and peripheral mechanisms sustaining tolerance after HLA-mismatched unrelated cord blood transplant (UCBT) in the GvH direction. TCRβ ImmunoSEQ® (Adaptive Biotechnology®) was applied for tracking and characterizing host-reactive T cell clones from graft or patient samples.

Seven UCBT recipients with non-malignant diseases were studied. All patients enrolled on the same reduced intensity conditioning trial (NCT01852370). Patients achieved tolerance if discontinued immunosuppression, exhibited immunocompetence without GvHD or graft rejection. Purified T cells responses to host APCs were measured by mixed lymphocyte reaction, cytokine bioplex, and micro CTL assays. T cells clones expanding >0.1% frequency in response to host APC were tracked and characterized with TCRβ ImmunoSEQ®.

ImmunoSEQ® revealed the disappearance of host-reactive clones that expanded from an aliquot of the infused cord blood graft by 7 days of stimulation with purified host APC. Most of these allo-reactive T cell clones were not identifiable even in the presence of IL2 supplementation to break anergy. Serial experiments post-UCBT revealed novel host-reactive clones that were not identifiable within the alloreactive pool of the graft pre-UCBT.

In summary, the acquisition of immune tolerance in the GvH direction post-UCBT is characterized by hypo-reactivity in all assays employed towards host APC. The disappearances of most host-specific clones in the graft indicate that deletion is a major mechanism of long term tolerance with possibly a small degree of anergy in contribution.
Inducible nitric oxide synthase (iNOS) mediated inhibition of T cell receptor (TCR) signaling is a mechanism for eosinophil dependent amelioration of alloimmunity in lung grafts
Authors: Onyema Oscar Okwudiri , Guo Yizhan , Mahgoub Bayan , Manafi Amir , Criswell Stacey , Lannigan Joanne , Jacobsen Elizabeth A , Gelman Andrew E , Kreisel Daniel , Krupnick Alexander S.
Published: January 2023

Eosinophils play a surprising role in downregulating lung graft-specific alloimmunity, even without immunosuppression. We demonstrate here, a mechanism used by eosinophils to downregulate alloimmune responses.

To study the mechanism of eosinophil dependent downregulation of alloimmune responses, we used a model of diphtheria toxin (DT) dependent ablation of eosinophils in iPHIL, a mouse model expressing DT receptor on the eosinophil-peroxidase gene locus. Fully MHC-mismatched Balb/c (H2Kd) lungs were engrafted to iPHIL (H2Kb) recipients without immunosuppression. We modeled alloreactivity in vitro, with mixed leukocyte cultures, comprising Th1-polarized eosinophils as modulators of T cells from C57BL/6J or Nur77-GFP mice stimulated by either allogeneic dendritic cells or anti-CD3/CD28 agonistic antibodies.

Ablation of eosinophils in non-immunosuppressed lung graft recipients leads to enhanced T cell activation and graft rejection. Similarly, in vitro, we observed a dose and contact dependent downregulation of T cell activation by eosinophils. While contact between eosinophils and T cells is mediated through the upregulation of PD-L1 on both wild-type and iNOS−/− eosinophils after TH-1 polarization, the inhibition of T cell activation is iNOS dependent and is directly linked to the iNOS dependent inhibition of the TCR signaling early gene, Nr4a1, monitored in Nur77-GFP mice.

Thus, following contact with T cells, iNOS-expressing eosinophils downregulate lung graft alloimmunity through a direct effect on TCR engagement early genes, leading to the inhibition of T cell activation. This provides an explanation for the surprising observation of lung-graft specific alloimmune downregulation by eosinophils.
Selenocompounds mediated upregulation of HLA class I expression enhanced mammaglobin-A peptide-specific cytotoxic T lymphocyte responses against breast cancer cells
Authors: Tiriveedhi Venkataswarup , Khandekar Durga , Amara Suneetha
Published: January 2023

Recent phase I DNA vaccine clinical trials with Mammaglobin-A (Mam-A), a human breast tumor-associated antigen (TAA), have shown to be safe and efficient. However, the success of cancer vaccines is limited by the diminished expression of HLA class I molecules on cancer cells. In our current communication, we studied the impact of various selenocompounds towards the expression of HLA class I molecules on breast cancer cells and their impact on the cytotoxicity of MamA2.1 (HLA-A2 immunodominant epitope of Mam-A) activated human CD8+T lymphocytes (hCTLs). We noted an enhanced HLA-A2 expression along with upregulation of components involved in antigen presentation machinery in all four breast cancer cell lines, namely AU565, UACC-812, MCF-7, and MDA-MB-231, following treatment with methyselenol producing methylseninic acid (MSA) and dimethylselenide (DMDSe). Furthermore, we have demonstrated enhanced cytotoxicity of MamA2.1 activated CTLs on HLA-A2+/Mam-A+ AU565 and UACC-812 cell lines following pre-treatment with MSA and DMDSe, while no significant toxicity was noted under similar conditions on HLA-A2+/Mam-A− MCF-7 and MDA-MB-231 breast cancer cell lines. Taken together, our data demonstrated that MSA and DMDSe potentiate effector cytotoxic responses following TAA vaccine specific activation of CD8+T lymphocytes, and thus suggesting their futuristic role as vaccine adjuvants in cancer immunotherapy.
Monoclonal antibodies for reducing opioid use disorders and overdose
Authors: Baehr Carly Anne , Pravetoni Marco
Published: January 2023

Vaccines and monoclonal antibodies (mAb) have been proposed as a therapeutic option for substance use disorders by selectively sequestering the target drug in serum and preventing drug distribution to the brain and drug-induced toxicity. Although there are multiple pharmacological interventions available to treat opioid use disorder, issues such as access, side effects, and compliance limit their clinical use. As an advantage over opioid receptor antagonists, opioid-specific antibodies do not interfere with endogenous opioid signaling. Whereas clinical efficacy of vaccines for substance use disorder is limited, as up to 70% of subjects do not achieve antibody levels sufficient for efficacy, passive immunization with opioid-specific mAb circumvents this limitation. We have developed a method to rapidly generate hybridomas expressing opioid-specific mAb by magnetic enrichment of opioid-specific B cells prior to fusion. Using this strategy, we identified murine hybridomas expressing mAb specific for oxycodone, morphine and fentanyl. Passive immunization with purified opioid-specific mAb was effective in reducing opioid distribution to the brain in mice. In this study, mAb consisting of different IgG subclasses were compared for efficacy against oxycodone. Sequencing of opioid-specific mAb showed limited variability in antigen-binding regions. Integration of functional, structural and sequencing characterization can aid development of next-generation mAbs for treatment of opioid use disorder, or to counteract other toxic chemicals.
Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma
Authors: Dolina Joseph S , Lee Joey , Peters Bjoern , Schoenberger Stephen P
Published: January 2023

The comparative resistance of some cancers including head and neck squamous cell carcinoma (HNSCC) to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens (NeoAg). SCC VII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity in syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation containing overlapping minimal epitopes during in vitro recall assays. NeoAgs eliciting CD4+ T cell responses alone were not protective in vivo. However, the more immunogenic Cltc NeoAg prophylactically protected hosts from tumor challenge and caused regression of established tumors as an immunotherapy. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection by boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.