bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8aa TCRab intraepithelial lymphocytes MHC restriction (P1462)
Authors: Lambolez Florence , Mayans Sofia , Stepniak Dariusz , Palida Sakina , Larange Alexandre , Cheroutre Hilde
Published: January 2023

T lymphocytes develop in the thymus into two major lineages characterized by expression of ab and gd TCR. Each lineage can be further subdivided into distinct subsets that differ in TCR specificity, phenotype and function. CD8aa TCRab intraepithelial lymphocytes (T-IEL) located in the epithelium of the small intestine is phenotypically different from conventional T cells. CD8aa T-IEL have been studied for years however the question of their MHC restriction has not been elucidated. Therefore, we decided to clone TCR isolated from naturally arising CD8aa T-IEL and retrovirally express these TCRs in BM chimera. First, we cloned four TCRab that were expressed retrovirally in RagKO BM chimera. Analysis of the chimera showed that all TCR clones gave rise to T cells. The T cell that developed were CD4-CD8b- but CD8a+ and phenotypically identical to CD8aa T-IEL isolated from wild type mice. In addition, they were preferentially found in the gut. In order to define the MHC restriction of these particular TCRs, BM chimeras in various MHC-deficient backgrounds were generated. Subsequent analysis of the chimera for the presence of CD8aaTCRab IEL demonstrated that they are dependent either on Kb or Db MHC I or b2m-dependent MHC I aside from KbDb. In conclusion, our results indicate that CD8aa T-IEL develops from precursors expressing particular TCRs that allow their engagement in this unique T cell lineage. In addition, CD8aa T-IEL can harbor clones selected on different MHC molecules.
Regulation of NKT cell development by SLAP2 (P1470)
Authors: Tuttle Kathryn , Peterson Lisa , Bedel Romain , Dragone Leonard , Gapin Laurent
Published: January 2023

NKT cell function in animal models is highly variable. This variation is likely due to functionally distinct subpopulations of NKT cells that are present to different degrees in each mouse strain. NKT precursors proceed through several developmental stages marked by the sequential expression of activation markers and NK cell receptors. At each step of development, NKT precursors receive signals that promote progression to the next stage. The nature of these signals is not completely understood, however T cell receptor (TCR), SLAM receptor and cytokine signaling are important. Src-Like adaptor protein 2 (SLAP2) is an adaptor protein that has been implicated in regulation of TCR and cytokine signaling in cell lines. While TCR signaling and development of conventional αβ T cells are normal in SLAP2-/- thymocytes, NKT cell development is altered. SLAP2-deficient thymocytes are enriched for immature NKT cells, a population known to be biased towards IL-4 secretion. Consequently, a large increase in the proportion of IL-4-dependent "innate CD8 lymphocytes" is detected in the thymus of SLAP2-/- mice. We hypothesize that SLAP2 modulates currently undefined signaling pathways that are important for NKT cell development.
Innate lymphoid cells are critical regulators of host-microbiota interactions and intestinal homeostasis (P3152)
Authors: Sonnenberg Gregory , Hepworth Matthew , Fung Thomas , Monticelli Laurel , Artis David
Published: January 2023

Mammals are colonized with trillions of commensal bacteria that are essential for normal intestinal physiology and immune cell development. However, dysregulated host-microbiota interactions have been causally linked with the pathogenesis and progression of numerous chronic infectious, inflammatory and metabolic diseases, such as HIV, cancer, obesity, viral hepatitis and inflammatory bowel disease (IBD). Innate lymphoid cells (ILCs) are an emerging family of immune cells that have recently been identified to play critical roles in regulation of immunity, inflammation and homeostasis in the intestine of mice and furthermore alterations in ILC populations have been observed in several chronic human diseases. However, whether ILCs are involved in the regulation of host-microbiota interactions is poorly understood. Here we show that ILCs are critical regulators of host immune responses to the microbiota through several distinct pathways. Surprisingly, abrogation of ILCs or their effector pathways resulted in pathologic host adaptive immune cell responses to resident intestinal microbiota and induction of spontaneous inflammation in the intestine and peripheral tissues. Collectively, these results identify an essential role for ILCs in regulating interactions between the mammalian adaptive immune system and commensal microbiota that dictate the state of intestinal health, and may represent a novel therapeutic target in chronic human disease.
Effect of the green tea catechin, epigallocatechin gallate, on innate immune responses in HIV-1 infection as a microbicide (P3170)
Authors: Nance Christina , Mata Melinda , Shearer Willliam
Published: January 2023

Our preclinical data showed binding of green tea catechin, epigallocatechin gallate(EGCG), to CD4 with inhibition of HIV-1-gp120 binding to human CD4+Tcells throughout a broad spectrum of virus strains lead to implementation of our Phase I/IIa clinical trial in HIV-1-infected indivduals. As mucosal dendritic cells(DC), Langerhans cells(LC) and monoctye-derived dendritic cells(MDDC), with CD4 receptor binding to HIV-1-gp120, act as first line defense in mucosa against invading pathogens via innate immune responses,EGCG in the context of an HIV-1 microbicide was studied. Human CD34+ hematopoietic stem cells and CD14+ cells differentiatied to LC and MDDC,respectively were assessed for HIV-1-gp120 binding, Annexin V-binding apopotosis and receptor profiling by flow cytometry, cytokine profiling by Luminex, toll-like receptor(TLR) profiling by RT-PCR, antigen uptake/presentation(APC) by RIA, cytotoxicity by MTT and statistical significance by Student’s t test. EGCG(1-50uM) downregulated TLR-6,-8 mRNA expresion (25%;p<0.05;43%;p<0.01, respectively),not TLR-2 or-3. TLR9 ligation downregulated CD86 expression and was reversed by EGCG (50uM) (62%;p<0.05).EGCG (1-50uM) did not significantly increase DC cytotoxicity, apoptosis, APC nor promote proinflammatory ctyokine secretiion of TNFa,IL1a,IL1B,orIL6. R5 chemokines, MIP1a,MIP1BandRANTES were upregulated by EGCG (25uM) (29%,33%,37%,respectively;p<0.01). EGCG has potential as an HIV-1 microbicide.
Inducible bronchus-associated lymphoid tissue reduces Th2-driven pathology by redistributing leukocytes within the lung (P6214)
Authors: Hwang Ji Young , Rangel-Moreno Javier , Garcia-Hernandez Maria de la Luz , Randall Troy
Published: January 2023

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid tissue formed in the lung after pulmonary infection or inflammation. This local lymphoid tissue is structurally similar to conventional secondary lymphoid organs, with separated B and T cell areas, specialized stromal cells and lymphoid dendritic cells (DCs). Here, we tested whether the presence of iBALT affects pulmonary immune responses to allergens. We induced iBALT formation in neonatal mice by pulmonary administration of LPS, and when the mice were adults, challenged them intranasally with OVA. We found that although mice with iBALT had more lymphocytes in their lungs, they had fewer eosinophils, less goblet cells and had reduced Th2 cytokine production in bronchoalveolar (BAL) fluid and lung tissue after OVA challenge. Surprisingly, using IL-4 reporter and by transferring OVA-specific OT-II cells, we found that the presence of iBALT did not reduce the number of Th2 cells that accumulated in the lung. Despite similar numbers of Th2 effectors in the lungs of mice with and without iBALT, we found that Th2 cells were differentially distributed in these two groups of mice. Those in the lungs of mice without iBALT were more widely dispersed, whereas the cells in the lungs of mice with iBALT were predominantly concentrated in the lymphoid tissue. These results suggest that the reduction in Th2-driven pathology with iBALT is due to the redistribution of leukocytes within the lung after iBALT formation.
Molecular mechanisms of TRAF6 regulation and intranuclear localization (P1305)
Authors: Su An-Jey , Adamik Juraj , Galson Deborah , Auron Philip
Published: January 2023

Tumor-necrosis factor receptor associated factors (TRAFs) are widely regarded as cytoplasmic adaptor proteins, that are important for the assembly of signaling complexes necessary for cell-receptor mediated immunological response. Among the TRAFs, TRAF6 is uniquely able to couple TLR/IL-1 and TNFR activation of NF-κB. Insomuch, the regulation of TRAF6 is critical for normal cellular physiology and responses to activation events. Accumulated evidence suggests that TRAF6 is regulated by both 1) interactions with other molecules as well as 2) an intramolecular interaction with itself. We have previously reported autoinhibition by the intramolecular interaction of TRAF6’s N-terminal Ring-zinc (RZ) and C-terminal MATH domains in trans. We now provide evidence that IRAK1, which contains a TRAF-Interaction Motif (TIM), can counteract TRAF6 autoinhibition. Further, we newly identify that the transcription factor Sox4 interacts with TRAF6 and that overexpression of either Sox4 or Syntenin, a dual PDZ domain containing protein, alters TRAF6 activation of NF-κB and translocates the normally cytoplasmic TRAF6 into the nucleus. While many studies have focused on cytoplasmic TRAF6 and its activation, there are few reports on the inhibition of TRAF6 and none have provided a mechanism by which it enters the nucleus.
Platelet CXCL7 and CXCL4 inhibit chemokine scavenging and improve innate immunity to bacterial infection (P1317)
Authors: Mei Junjie , Kowalska M. Anna , Dai Ning , Liu Yuhong , Hudock Kristin , Jeyaseelan Samthamby , Lee Janet , Guttentag Susan , Poncz Mortimer , Worthen G. Scott
Published: January 2023

We recently demonstrated the critical role of CXCL5 in chemokine scavenging, neutrophil homeostasis and host defense to bacterial pneumonia. Here we generated Cxcl7-/- mice and found that Cxcl7 deletion also disrupted expression of CXCL4 and CXCL5, but did not affect expression of two other neutrophil chemokines, CXCL1 and CXCL2. Cxcl4, 7, 5 form a conserved genomic locus (14.5 kb in length) in both human and mice, suggesting their importance during evolution. Heparin treatment of blood led to over 10-fold more plasma CXCL4, but comparable plasma CXCL7 as compared to PBS treatment. In addition, the inactive PPBP form of murine CXCL7 showed considerable binding affinity with red cell DARC. We further delineated the role of CXCL7 and CXCL4 by comparing the phenotypes of Cxcl7-/- and Cxcl5-/- mice. CXCL7 and CXCL4 inhibit chemokine scavenging not only in blood, but also in tissues, and platelet-derived CXCL7 is present in alveolar space and peritoneum. In CXCL1- and nebulized LPS-induced acute lung inflammation models, K. pneumoniae pneumonia and bacteremia models, as compared to Cxcl5-/- mice, Cxcl7-/- mice showed attenuated neutrophil chemokine levels in tissues and blood, decreased neutrophil influx to the lung, and impaired innate immunity. This study is the first to reveal the central role of homeostatic CXCL7 and CXCL4 in increasing chemokine levels in blood and tissues, and potentiating neutrophil transmigration during inflammation and infection.
Mechanisms and intervention strategies for chronic low-grade inflammation. (P1361)
Authors: Morris Matt , Li Liwu
Published: January 2023

Low-grade inflammation contributes to diseases such as atherosclerosis, arthritis, and diabetes. Contributors to low-grade inflammation include circulating microbial products such as lipopolysaccharide (LPS). High plasma levels of LPS cause acute shock, but low levels of LPS predispose individuals to mild inflammatory conditions. This predisposition results from mild expression of pro-inflammatory cytokines without compensating anti-inflammatory modulators, leading to persistent, unresolved inflammation. Despite the clinical importance of chronic inflammation, its underlying mechanisms are poorly understood. Glycogen synthase kinase 3 (GSK3) is implicated in the pro-inflammatory conditioning of innate immunity. We tested whether inhibition of GSK3 and upstream kinases can prevent inflammatory skewing by low dose LPS. We measured the expression of pro-inflammatory IL-6 and anti-inflammatory IL-33 in pre-monocytic THP-1 cells treated with low dose LPS (50 pg/mL) and a GSK3 inhibitor. We observed that while low dose LPS alone induced mild expression of IL-6, GSK3 inhibition skews THP-1 cells into an anti-inflammatory state by augmenting the induction of IL-33 by low dose LPS while diminishing the induction of IL-6. We found that broad inhibition of tyrosine kinases also diminishes IL-6 induction. These observations suggest a complex intracellular circuit that dynamically modulates the expression of pro- and anti-inflammatory mediators upon treatment with low dose LPS.
MiR-155 regulates NK cell activation (P1366)
Authors: Sullivan Ryan , Leong Jeffrey , Schneider Stephanie , Keppel Catherine , Romee Rizwan , Fehniger Todd
Published: January 2023

MiR-155 is a well-documented small RNA regulator of a variety of immune cells, including adaptive T and B lymphocytes. However, its role in regulating the innate NK cell response is unclear. We evaluated the expression of miR-155 using real-time qPCR and found that miR-155 is expressed at modest levels in resting mouse and human NK cells, but is upregulated upon stimulation, especially with IL-12+15+18 (30-fold). Therefore, we hypothesized that miR-155 regulates NK cell function following activation. We utilized miR-155-/- mice (155KO), NK-specific miR-155 overexpression mice (155OE), and miR-155 lentiviral transduction to examine the role of miR-155 in NK cell functional responses. miR-155KO, miR-155OE, and miR-155-transduced NK cells were all found to secrete more IFN-γ (by ELISA) upon ligation-mediated or cytokine stimulation, with no apparent defects in maturation or development. When further dissected by flow cytometry, we found that 155OE and transduced NK cells had increased IFN-γ MFI, indicating more “per-cell” production. In contrast, the 155KO NK cells had a higher percentage of IFN-γ+ NK cells, with similar MFI, indicating a potential activation threshold defect. Though both 155KO and 155OE NK cells produce more IFN-γ, the cellular basis for each, i.e. miR-155 target, likely differs. Therefore, we used unbiased RISC-IP to identify relevant miR-155 targets, which included previously validated miR-155 targets (e.g., SHIP-1) as well as novel miR-155 NK cell targets.
DDX41 recognizes bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response (P1375)
Authors: Parvatiyar Kislay , Zhang Zhiqiang , Teles Rosane , Ouyang Songying , Jiang Yan , Liu Zhi-Jie , Iyer Shankar , Zaver Shivam , Schenk Mirjam , Zeng Shang , Zhong Wenwan , Modlin Robert , Liu Yongjun , Cheng Genhong
Published: January 2023

Cytosolic detection of bacterially derived secondary messengers cyclic-di-GMP (c-di-GMP) or cyclic -di-AMP (c-di-AMP) by the host immune system activates an innate immune response characterized by the induction of type I interferons (IFNs). Induction of IFN by c-di-GMP or c-di-AMP has been shown to be dependent on a stimulator of IFN genes-TANK binding kinase 1-IFN regulatory factor 3 (STING-TBK1-IRF3) signaling axis. Although STING has been shown to interact with c-di-GMP, an upstream sensor of these cyclic dinucleotides is unknown. Here we identify the helicase, DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41) as the pattern recognition receptor (PRR) that senses both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacts with c-di-GMP. Knockdown of DDX41 via shRNA in murine or human immune cells inhibits the induction of innate immune genes and results in defective STING, TBK1 and IRF3 activation in response to c-di-GMP or c-di-AMP. Our findings suggest a mechanism whereby c-di-GMP and c-di-AMP molecules are detected by the DDX41 PRR, which complexes with the STING adaptor to signal to TBK1-IRF3 and activate the IFN response.
Multiple pools of dynein and their roles in T cell effector functions (P1028)
Authors: Nath Shubhankar , Christian Laura , Lee Siu , Artz Jonathan , Yuan Denley , Poenie Martin
Published: January 2023

Dramatic cytoskeletal activities and protein movements are seen when T cells engage cognate targets or antigen presenting cells. These include formation of a specialized contact site known as the immunological synapse (IS), clustering of vesicles around the microtubule organizing center (MTOC) and translocation of the MTOC up to the IS. Ultimately, this leads to focused secretion towards the cognate target. In an effort to understand how MTOC translocation takes place, we have investigated the roles of dynein, p150Glued, Lis1,NDE1 and NDEL1 using Jurkat cells as a model system. Using expressed molecular traps aimed at dynein intermediate chain (DIC) or LC8 (a dynein light chain), and Jurkat cells expressing fluorescent CTLA-4 constructs targeting secretory vesicles, we have shown that dynein is required for both vesicle clustering and MTOC translocation. When p150Glued expression was reduced, vesicles became dispersed but MTOC translocation was unaffected suggesting that dynactin was needed for vesicle movements but not MTOC translocation. MTOC translocation was potently blocked when Lis1 expression was reduced. We also find that NDE1 accumulates at the IS and pulls down with DIC and Lis1. Overexpression of GFP-NDE1 blocks MTOC polarization. The combination of localization and functional data suggests that there are two separate dynein complexes, a dynein-Lis1-NDE1/NDEL complex that mediates MTOC translocation and a dynein-dynactin complex that mediates vesicle clustering.
Th2 cytokines IL-4 and IL-13 negatively regulate lymphangiogenesis (P1038)
Authors: Sin Ki-Hyuk , Kataru Raghu , Lee Seung-Hyo
Published: January 2023

The vascular system consists of two major components, blood vessels (BVs) and lymphatic vessels (LVs). Both vascular systems are essential for homeostasis in healthy organism and related to the pathogenesis of many diseases. Other than fluid homeostasis LVs are also important for immune surveillance. In the present study, we show novel anti-lymphangiogenic factors IL-4 and IL-13 which is major effector cytokine produced by type 2 helper T cells (Th2 cells). By using murine lymphatic endothelial cells (mLECs) and human LECs (hLECs), we demonstrate that IL-4 and IL-13 down-regulate Prox-1 which is essential transcription factor of LECs. These cytokines inhibited tube formation of LECs also. Surprisingly, co-culture LECs with Th2 cells inhibited tube formation, but this effect was fully restored by blocking IL-4 and IL-13. Furthermore, in vivo blockade of IL-4 and IL-13 in a murine asthma model increase not only the density of LVs in the lung but also the function of LVs. Taken together our findings demonstrate a novel anti-lymphangiogenic function for Th2/IL-4 and IL-13, suggest that these effects could be involved in pathogenesis of asthma.
Systematic identification of immunodominant CD8+ T cell responses to influenza A virus (P6170)
Authors: Chen Weisan , Wu Chao , Grant Emma , Zanker Damien , Valkenburg Sophie , Chan Kok-Fei , Eckle Sidonia , Bharadwaj Mandvi , Tan Bee , Kedzierska Katherine , Zou Quan Ming , Doherty Peter
Published: January 2023

Immunodominant T cell responses are important for virus clearance. However, the identification of immunodominant T cell peptide+HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for new, immunodominant CD8+ T cell epitopes. The analysis targeted healthy HLA-A2+ and HLA-A2- donors. While M158A2 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three out of eight while, for the other five, prominent CD8+ T cell responses tended to focus on various peptides from the influenza nucleoprotein (NP) that were not presented by HLA-A2. In all the tested 7 A2- individuals, immunodominant responses all focused on NP. Importantly, most novel, immunodominant T cell epitopes identified by our study would not have been predicted by the current prediction programs as they are either too long or lacking typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8+ T cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity.
Co-immunization induces tolerogenic response against asthma via generation of antigen specific iTreg (P5233)
Authors: Wang Bin , Geng Shuang
Published: January 2023

Asthma is a chronic inflammatory disorder caused by T cell-mediated inflammation within airways. No antigen specific treatment has been available. Using an Der p1 induced murine asthma model, we find that co-immunization of an Der p1 protein with a DNA vaccine encoding the same antigen is able to prevent this experimental asthma as evidenced in the marked reduction of infiltrations of eosinophils and lymphocytes into the site of the allergen challenge. We demonstrate that the prevention of experimental asthma was directly related to the induction of a population of Der p1 specific T regulatory (iTreg) cells exhibiting a CD4+CD25-FoxP3+ phenotype and expressing IL-10, and TGF-β following the co-immunization. Blockade of IL-10 and TGF-β of the iTreg cells by anti-IL-10 and TGF-β antibodies is partially able to reverse the suppression in vitro and in vivo, which caused the recurrence of the inflammation. Furthermore, adoptive transferred Der p1 induced iTreg cells is able to suppress the Der p1 induced asthma, but not to OVA induced asthma, and vice versa, suggesting that the iTreg exerts an inhibition in an antigen specific manner. To our knowledge, the co-immunization of protein with its cognate DNA vaccine protect/treat experimental asthma via the induced antigen specific iTreg cells may lead to a novel immunotherapy against asthma in humans.
Pegylated adenosine deaminase prevents immune cell migration and infiltration into the brain in experimental autoimmune encephalitis (P5171)
Authors: Kim Do-Geun , Krenz Antje , Bynoe Margaret
Published: January 2023

Multiple sclerosis (MS) is a debilitating neuroinflammatory disease that is characterized by infiltration of inflammatory immune cells into the central nervous system (CNS). Currently, there is no cure for MS and the few available treatments only lessen symptoms in some patients, and can have debilitating side effects. Our lab has pioneered studies demonstrating that blockade of broad spectrum or A2A specific adenosine receptor signaling inhibits lymphocyte infiltration into the CNS and protects mice from a multiple sclerosis animal model called experimental autoimmune encephalomyelitis (EAE). Pegylated adenosine deaminase (PEG-ADA) is an FDA-approved drug used to treat patients with severe combined immune deficiency (SCID) due to a mutation in their adenosine deaminase (ADA) gene which converts adenosine into inosine. In this study, we used PEG-ADA to assess its therapeutic potential to protect progression and alleviate the severity of EAE. We hypothesized that similar to adenosine receptor blockade, depletion of extracellular adenosine by PEG-ADA may lessen the influx of inflammatory immune cells into the brain and protect mice from EAE. Our results showed that indeed mice treated with PEG-ADA developed significantly less severe EAE and had less immune cell infiltration in their brain. Because a common characteristic of MS in general is the invasion of pathogenic immune cells into the CNS, this study has the promising potential to benefit MS patients.
In vitro and in vivo anti-arthritic study of a novel histone deacetylase inhibitor YH508 (P5187)
Authors: Yang Chia-Ron , Hsieh I-Ni
Published: January 2023

The pathology of rheumatoid arthritis (RA) includes synoviocytes proliferation, expression of inflammatory mediators, and persistent recruitment of immune cell; these might result from dysregulation of epigenetic control by histone acetylase (HDAC). The aim of this study is to examine the anti-arthritic effect of YH508, a histone deacetylse (HDAC) inhibitor, by in vitro and in vivo models. The IC50 values of YH508 against enzymatic activity of HDAC1, 2, 3, 6, and 8 significantly lower than an approved HDAC inhibitor SAHA (Vorinostat), indicates that YH508 is more potent than SAHA. Treatment with YH508 showed dose-dependent inhibition of cytokines (IL-6, nitric oxide, PGE2) secretion in RAW264.7 macrophages and RA synovial fibroblasts. In addition, YH508 dramatically suppressed M-CSF/RANKL induced osteoclastogenesis from macrophages at low concentration. Furthermore, these inhibitory effects were reversed by overexpression class I/II HDAC. In vivo anti-arthritic effects of YH508 were evaluated in a M. butyricum-induced arthritis model in rats. Oral administration of YH508 significantly inhibited paw swelling, bone destruction and reduced serum cytokine levels. Our results demonstrate that HDAC inhibitor YH508 inhibits the development of arthritis, suggest that it provides a new therapeutic approach for the treatment of inflammatory arthritis.
FTY720 ameliorates murine Scl-cGVHD by promoting expansion of splenic regulatory cells and inhibiting immune cell infiltration into the skin (P5153)
Authors: Matsushita Takashi , Le Huu Doanh , Takehara Kazuhiko , Fujimoto Manabu
Published: January 2023

Objective: Murine sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) is a model for human Scl-cGVHD and systemic sclerosis (SSc). Sphingosine-1-phosphate (S1P) exerts a variety of activities in immune response, inflammation, and vascular systems. It was reported that regulation of S1P on fibrotic diseases as well as systemic sclerosis. FTY720, an oral S1P receptor modulator, has been shown to be a useful agent for the prevention of transplant rejection and the treatment of autoimmune diseases. In this study, the effects of FTY720 were investigated in Scl-cGVHD. Methods: FTY720 was orally administered to allogeneic recipients from day 0 to day 20 (short-term, early-treatment group), day 0 to day 42 (full-term, early-treatment group), or day 22 to day 42 (delayed-treatment group) after bone marrow transplantation (BMT). Results: Delayed-treatment of FTY720 attenuated and early-treatment inhibited Scl-cGVHD severity and fibrosis. In early-treatment, FTY720 induced expansion of splenic myeloid-derived suppressor cells, regulatory T cells and regulatory B cells. Vascular damages in Scl-cGVHD were inhibited by FTY720 through downregulating serum levels of S1P and sE-Selectin. Furthermore, FTY720 inhibited the infiltration of immune cells into the skin and diminished the expression of mRNA for proinflammatory cytokines in the skin. Conclusion: These results suggest that FTY720 could be a promising strategy for treating Scl-cGVHD and SSc patients.
Natura-alpha, a novel STAT3-Y705 inhibitor in treating systemic lupus erythematosus in NZB/W female mice (P5157)
Authors: Wang Longgui , Han Liying , Fallon John , Tsao Allen , Chiao Jen-Wei
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Increases in Th1/Th17 cells, and decreases in Treg cells, have been observed in patients with SLE. Natura-α, a synthetic small molecule inhibits STAT3-Y705 phosphorylation and regulates Th1/Th17 and Treg. The potential of Natura-α as a SLE therapeutics was explored. The SLE model NZB/W female mice which develops lupus nephritis were used after the disease was confirmed at week 19. One group (n=10) was given orally vehicle, and the other groups given Natura-α at 25 or 75 mg/kg, once/day, 5 days/week for 29 weeks. Mice were monitored until 52 weeks of age. Proteinuria in control mice was elevated at week 31, followed with sharp increase reaching greater than 1000 mg/dL at week 44. The levels in the two Natura-α-treated groups essentially maintained at stable lower level (150+100mg/dL at 25mg/kg, and 115+127mg/dL at 75mg/kg). Control mice began to die at week 38. At week 48, 60% of mice died in the vehicle group. In sharp contrast, all mice in the Natura-α-treated groups were survived (P<0.001). Glomerular lesion in the control mice reached maximal at week 38, with over 85% exhibited severe lesions. The glomerular lesions in the Natura-α-treated groups were around the baseline level, i.e. approximately 20%. The treatments with the Natura-α significantly reduced proteinuria, prevented and reversed glomerular lesions, and improved survival, suggesting this novel STAT3-Y705 inhibitor is a potential SLE inhibitor.
Immune modulation and prevention of alloimmunization following transfusion with pathogen reduced platelets (P2144)
Authors: Jackman Rachael , Muench Marcus , Heitman John , Inglis Heather , Law Jacqueline , Marschner Susanne , Goodrich Raymond , Norris Philip
Published: January 2023

Blood transfusion is the most commonly performed allogeneic transplant, and results in alloimmunization of a number of recipients, potentially complicating subsequent transfusions as well as solid organ transplants. The use of a pathogen reduction method using riboflavin and UV light has been shown to induce changes in WBCs that result in a failure to stimulate allogeneic PBMCs in vitro. We assessed the degree of alloimmunization in vivo using mice given pathogen reduced versus untreated allogeneic platelets. WBC-enriched platelet rich plasma (PRP) was prepared from C57Bl/6 and Balb/cJ donor mice and either left untreated or pathogen reduced before transfusion via tail vein into Balb/cJ mice. Two weeks after transfusion circulating alloantibodies were measured, as were splenocyte cytokine responses to challenge with donor type cells ex vivo. Pathogen reduction treatment of allogeneic PRP prior to transfusion blocked alloimmunization. Mice that were transfused with untreated PRP following previous transfusion with pathogen reduced PRP had a normal alloantibody response, but their splenic lymphocytes had a reduced cytokine response to allogeneic challenge ex vivo. This immune modulation required the presence of WBCs in the transfused PRP and was donor specific. These results imply that UV treatment of blood products has the potential to modulate subsequent exposure to alloantigens, with implications for tolerance and organ transplantation.
Innate recognition of allogeneic non-self by monocytes initiates the rejection of transplanted organs (P2128)
Authors: Oberbarnscheidt Martin , Zeng Qiang , Williams Amanda , Rothstein David , Lakkis Fadi
Published: January 2023

It is generally assumed that inflammation or ‘danger’ at the time of transplantation triggers rejection. Here we tested the alternate hypothesis that the innate immune system distinguishes between self and allogeneic non-self, causing DC maturation and initiation of adaptive immunity. B6 Rag-/-γc-/- CX3CR1-GFP+/- reporter mice, which lack T, B, and NK cells, received syngeneic (B6) or allogeneic (Balb/c) vascularized heart grafts. >90% of DCs present in the grafts by d.1 after Tx were derived from host monocytes. Mono-DC (GFP+lineage-Ly-6G-CD45+CD11b+CD11c+F4/80lo) were observed in greater numbers in allo than syn grafts at early time points and persisted at later time points (>21d) only in allografts. Mono-DC in allografts were mature (MHCII+CD80+), expressed IL-12p40, and presented endotoxin-free OVA to CD4+ (OT-II) and CD8+ (OT-I) cells, causing proliferation and IFNγ production. Mono-DC from syn grafts were less mature, did not express IL-12p40, and failed to induce IFNγ production. In vivo exposure of monocytes to allogeneic non-self precipitated T cell-mediated rejection of single minor Ag-mismatched grafts that are otherwise accepted by the host. Transient depletion of host CD11b+ cells in WT mice abrogated T cell accumulation and IFNγ production in heart allografts and prevented acute rejection. Therefore, sensing allogeneic non-self by monocytes is a key, and previously unrecognized, innate pathway by which alloimmune responses are initiated.
Epitopes involved in indirect recognition of pocine MHC molecules induce potent T cell responses in HLA transgenic mice (P2184)
Authors: Lee Chong-Kil , Park Chan-su , Kim Ki-Hyang , Im Sun-A , Kim Narae , Song Sukgil
Published: January 2023

Indirect xenorecognition has been implicated as the major mechanism of chronic rejection, but precise definition of the epitopes involved has been limited. We identified swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. The SLA class I-derived peptides that bind to HLA-DRB1*0401, a representative of the DR4 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and their binding capacities to HLA-DRB1*0401 were compared in a competitive ELISA using biotinylated hemagglutinin reporter peptides [HA307-319]. Peptide-11 (LRSWTAADTAAQISK) was determined to exhibit the most potent binding capacity to HLA-DRB1*0401 in vitro and thus selected for in vivo immunization. Immunization of HLADRB1*0401-transgenic mice with peptide-11 elicited potent CD4+ Th1 responses. Peptide-11 shares homology to α2 domains of three SLA-1 alleles, six SLA-2 alleles, and 14 SLA-3 alleles. Additionally, we predicted HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes of SLA, and then performed peptide/HLA-A*0201 affinity and complex stability assays. Of candidate peptides, we selected two peptides that have high affinity and stability. Collectively, the present study identifies indirect xenorecognition epitopes shared by diverse SLA alleles.
Human iPS cell-derived CD34+ hematopoietic progenitor cells induce T cell anergy in alloreactive CD8+ T cells (P2188)
Authors: Kim Eun-Mi , Manzar Gohar , Zavazava Nicholas
Published: January 2023

Human induced pluripotent stem (iPS) cells have emerged as an alternative source of pluripotent stem cells that can be used in regenerative medicine. However, knowledge about the immunological characteristics of their derivatives remains enigmatic. Here, human iPS cells were differentiated into CD34+ hematopoietic progenitor cells (HPCs).These iPS cell-derived HPCs poorly express MHC class-I antigens, and are MHC class-II negative. They also lack expression of costimulatory molecules CD80 and CD86, but show relatively high expression of PD-L1. To investigate whether these HPCs stimulate CTLs, we generated CD8+ T cells against HLA-A2. Subsequently, HPCs were used as target cells for the CTLs. Interestingly HPCs were not susceptible to alloreactive CTL killing in a cytotoxicity assay. HPCs also failed to trigger release of IL-2 by CTLs as a sign of their activation. This lack of CTL activation was partially due to low MHC class I expression by the HPCs because HPC cell lysis was partially restored after MHC class I upregulation by treatment with IFN-γ. Interestingly, the CTLs became anergic when incubated with HPCs. As expected, T cell anergy was abrogated by treatment with rIL-2. Collectively, anergy was a result of low expression of CD80 and CD86 and high expression of PD-L1. These data indicate for the first time that human iPS cell-derived CD34+ HPCs induce T cell anergy, a property that could be exploited for cellular allo-transplantation of iPS cell-derived tissues.
Donor CD19-targeted T cells exert potent graft versus lymphoma activity without GVHD (P2134)
Authors: Ghosh Arnab , Davila Marco , Young Lauren , Jenq Robert , Sadelain Michel , van den Brink Marcel
Published: January 2023

We evaluated the relative effects of donor CD19-targeting chimeric antigen receptors (CAR)+T cells on the elimination of CD19+B cells and endogenous TCR-mediated GVHD in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: CD19-delta, a tail-less CAR lacking the CD3ζ signaling and CD19-CAR signaling through CD28 and CD3ζ. CD19-CAR+T cells mediated specific lysis of CD19+tumors in vitro, while CD19-delta+T cells did not. We next transferred the transduced donor T cells into lethally irradiated recipients of T cell-depleted allografts and CD19+lymphoma A20-TGL (B6→BALB/c+A20-TGL). CD19-CAR+T cells mediated clearance of A20 tumor leading to significantly improved tumor free survival (p<0.01), while mediating significantly less acute GVHD, resulting in improved survival (p<0.001). Donor CD19-delta+T cells caused lethal GVHD, indicating persistent endogenous TCR mediated alloreactivity in the absence of CAR signaling. Compared to controls, fewer alloactivated CD19-CAR+T cells were found in the lymphoid organs of allo-HSCT recipients. This was accompanied by a lower expression of IFN-γ and lower frequencies of phenotypically activated T cells (p<0.05). LPAM, associated with increased gut GVHD, was also significantly lower on CD19-CAR+T cells. CAR stimulation did not inhibit TCR-mediated killing. These results provide the first preclinical evidence suggesting that CAR+ donor T cells may be safely applied in an allogeneic context.
Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells (APP3P.114)
Authors: Subrahmanyam Priyanka , Webb Tonya
Published: January 2023

Natural killer T (NKT) cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. Once activated, NKT cells can mount strong anti-tumor responses. However, little is known about the regulation of CD1d-mediated antigen processing and presentation to NKT cells, particularly in the context of B cell lymphoma. Pro-survival factors of the Bcl-2 family, such as Bcl-xL are often upregulated in B cell lymphomas, and are associated with changes in the endocytic pathway, which is paramount for CD1d-mediated antigen presentation. We hypothesized that Bcl-xL regulates this process, and found that over-expression or induction of Bcl-xL led to increased antigen presentation to NKT cells. Conversely, inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Knockdown of Bcl-xL also resulted in the loss of CD1d trafficking to LAMP1+ compartments. Furthermore, Rab7, a late endosomal marker was upregulated following Bcl-xL knockdown, and CD1d molecules accumulated in the Rab7+ compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering the intracellular localization of CD1d. Thus, we have identified a new pathway involved in antigen presentation that can impact current therapies targeting the Bcl-2 family, as well as emerging NKT cell based cancer immunotherapeutic strategies.
Increased expression of IL-15 and IL-15Ra in the islets of patients with T1D (CCR3P.217)
Authors: Chen Jing , Feigenbaum Lionel , Awasthi Parirokh , Butcher Donna , Anver Miriam , Golubeva Yelena , Bamford Richard , Zhang XiaoJie , St. Claire Mark , Thomas Craig , Discepolo Valentina , Jabri Bana , Waldmann Thomas
Published: January 2023

We previously showed that transgenic mice with islet expression of IL-15 and IL-15Ra developed diabetes that mimics early human T1D. Inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122) or with the JAK 2/3 inhibitor tofacitinib reversed the diabetes. This suggested a causative role of IL-15 and IL-15Ra in the induction of insulin dependent diabetes in mice. To further investigate whether IL-15 and IL-15Ra play a role in the pathogenesis of human type 1 diabetes, we investigated the expression of IL-15 and IL-15Ra in the islets of patients with type 1 diabetes, anti-insulin autoantibodies and normal donors. Immunohistochemistry demonstrated IL-15 expression in the islets in all T1D patients examined whereas IL-15 was detected only in the pancreatic islets in one out of four normal donors and two out of five autoantibody positive donors. Interestingly, in two cases where IL-15 was positive, we also detected islet expression of MxA, a GTPase induced by type 1 IFN (α/β) that has been reported to interfere with virus multiplication and spread. Furthermore, IL-15 and IL-15Rα mRNA levels were also higher in the islets from T1D patients compared to that from autoantibody positive donors or normal donors. Moreover, the serum sIL-15Rα levels were significantly higher in patients with T1D compared with those of normal controls. Taken together, our data suggest that disordered IL-15 and IL-15Rα may be involved in the pathogenesis of human T1D.
Histone H2A deubiquitination by 2A-DUB/Mysm1 regulates critical checkpoints in early T cell development (HEM4P.226)
Authors: Gatzka Martina , Hainzl Adelheid , Tasdogan Alpaslan , Maity Pallab , Singh Kamayani , Wilms Christina , Wlaschek Meinhard , Scharffetter-Kochanek Karin
Published: January 2023

Developmental processes rely on collaboration of epigenetic modifications and sequence-specific transcription factors for timely activation and silencing of lineage-specific genes. Because histone H2A deubiquitination by 2A-DUB/Mysm1 was recently shown to affect hematopoiesis, in this investigation, we used 2A-DUB-deficient mice to systematically characterize the function of this epigenetic regulator in early T cell development. Thymi of 2A-DUB-/- mice were severely hypoplastic with >10-fold decrease in total cell numbers and increased fraction of apoptotic thymocytes compared with wild-type littermates. Diminished cell numbers resulted from a reduction of early thymocyte progenitors (ETPs) in context with defective HSCs, a partial block at the double-negative (DN)1-DN2 transition and increased apoptosis of double-positive (DP) thymocytes. In qPCR and IF analyses, we identified p19ARF and other genes involved in early T-cell development and protection from apoptosis as potential 2A-DUB target gene. ChIP analyses were conducted to explore binding of 2A-DUB to the Ink4a/Arf promoter in thymocytes. In addition, genetic approaches are used to validate the interaction of 2A-DUB/Mysm1 with known regulators of apoptosis and T cell development. We conclude that 2A-DUB/Mysm1 is critical for early T cell development via regulation of lineage-specific genes and that insufficient histone H2A deubiquitination induces apoptotic programs in HSCs, thymocytes and potentially other cell types.
FOXG1 is required in thymic epithelial cells for normal development and expression of interleukin 7 (HEM4P.245)
Authors: Jarvis Barton , Wei Qiaozhi , Condie Brian
Published: January 2023

Thymic epithelial cells (TECs) are required in the vertebrate thymus microenvironment for normal differentiation of T-cells. Cortical (cTEC) and medullary TEC (mTEC) are the two subtypes of TECs and each component performs different functions in the production of T-cells. The regulatory pathways that control the specification and differentiation of TEC during development are not completely known. Foxg1 has previously been shown to be expressed in TEC before the onset of differentiation. Analysis in mutants showed that specification and differentiation is initially normal but that differentiation of TEC becomes abnormal at E13.5-E14.5. Further analysis of Foxg1 mutants has shown that the expression of the essential cytokine IL-7 is altered. Postnatal examination of Foxg1 conditional mutants has shown that many of the fetal phenotypes persist into adulthood which suggests a potential role for Foxg1 in TEC maintenance. Our results suggest that Foxg1 is an upstream regulator of IL-7 expression in addition to its role in the development and survival of TEC.
Successful β-selection depends on TCF-1 (HEM4P.228)
Authors: Harly Christelle , Yang Qi , Bhandoola Avinash
Published: January 2023

The transcription factor TCF-1 is expressed very early during thymic development and is important for early T cell specification. TCF-1 expression further increases after b-selection and is maintained at high levels in differentiated T cells, but its function at these later stages has not been examined. We used mice with conditional alleles of TCF-1 (TCF-1f/f) to delete TCF-1 at two time points of T cell development. Whereas deletion of TCF-1 in double-positive thymocytes (CD4-cre TCF-1f/f) had little impact on later T cell populations, the effect of the deletion at double-negative 3 (DN3) stage (CD2-cre TCF-1f/f) was dramatic. Thymic cellularity of CD2-cre TCF-1f/f mice was decreased more than 10-fold compared to littermates control, and competitive BM chimeras clearly revealed a sharp defect at the b-selection checkpoint in these mice. Analysis of TCF-1-deleted DN3 thymocytes did not reveal any defects in T cell gene expression and pre-TCR expression and signaling. However, post b-selection DN3 and DN4 thymocytes failed to display the increase in cell size and proliferation characteristic of successful b-selection. Because these two phenomena have been previously attributed to c-Myc, we examined its expression and found that c-Myc protein is reduced in TCF-1-deleted post b-selection DN3. We are currently examining the mechanism by which TCF-1 controls c-Myc. Our work to date implicates TCF-1 downstream of the pre-TCR during the development of αβ T cells.
Aging of thymic epithelial progenitor pool is determined by the p63-FoxN1 regulatory axis (HEM4P.233)
Authors: Burnley Preston , Ruan Linhui , Coder Brandon , Su Dong-Ming
Published: January 2023

The postnatal thymic epithelial progenitor (TEP) pool is proposed to be regulated by the p63 and FoxN1 genes through proliferation and differentiation, respectively. However, the combined role of these two genes in the aging TEP is still a mystery. Evidence from murine models has elucidated contrasting roles of the p63 isoforms during the aging process. We found that TAp63+, but not ΔNp63+, thymic epithelial cells (TECs) were increased with age, accompanied with increased senescence associated β-gal clusters and p21+ TECs. Senescent clusters also developed after intrathymic infusion of exogenous TAp63 cDNA into young wild-type mice. Using our conditional FoxN1 gene knockout mouse model to disrupt TEP differentiation accelerated this senescent phenotype to early middle age. However, upon infusion of exogenous FoxN1 cDNA into aged wild-type mice resulted in only an increase in ΔNp63+ TECs, but no change in TAp63+ TECs in the partially rejuvenated aged thymus. Interestingly, using a novel FoxN1 transgenic mouse model to enhance TEP differentiation, ΔNp63+ TECs were decreased in young thymus. Additionally, the TAp63+ population contained a high percentage of phosphorylated-p53 and apoptotic TECs, but showed no changes in BrdU-labeled proliferation. As a result, FoxN1 controlled TEC differentiation as a bottleneck to determine TEP pool via affecting TAp63 and ΔNp63 levels. Thus, TEC homeostasis during aging has been determined through the p63-FoxN1 regulatory axis.