bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-idiotypic small molecules neutralize pathogenic anti-ADAMTS13 autoantibodies in autoimmune Thrombotic Thrombocytopenic Purpura in vitro - new treatment tools in vivo? (P5173)
Authors: Schaller Monica , Hiltbrunner Sabine , Sulzer Irmela , Vogel Monique , Kentouche Karim , Laemmle Bernhard , Kremer Hovinga Johanna
Published: January 2023

A therapy aiming at neutralization of inhibitory autoantibodies (Abs) causing autoimmune thrombotic thrombocytopenic purpura (aTTP) would be highly desirable. We screened a large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins, Molecular Partners AG, Switzerland; library size 1015-23) for anti-idiotypic binders to an equimolar pool of three spleen-derived inhibitory anti-ADAMTS13 monoclonal Abs of two aTTP patients containing 1 of 4 shared CDR3 motifs. Four unique anti-idiotypic DARPins were purified and their neutralization potential tested against 8 spleen-derived anti-ADAMTS13 Abs (200 nM), belonging to the same, shared CDR3 motif used for selection. Anti-idiotypic DARPins restored ADAMTS13 activity up to 100% (2000 nM). By ELISA we found Abs in plasma of 27 of 37 randomly selected aTTP patients that bound specifically to the anti-idiotypic DARPins. Pre-incubation with anti-idiotypic DARPins (960 nM) prevented binding of plasma anti-ADAMTS13 Ab to recombinant ADAMTS13 in a competition ELISA (n=5). Our findings of anti-idiotypic DARPins able to neutralize several, although similar (belonging to the same CDR3 motif) pathogenic anti-ADAMTS13 Abs and the fact that the DARPins are also specifically recognized by plasma-derived anti-ADAMTS13 Abs of different aTTP patients are promising and suggest that possibly only a limited number of the selected DARPins are required to develop a general treatment for aTTP.
Specific targeting of PI 3-kinase p110δ for the treatment of rheumatoid arthritis (P5177)
Authors: Reif Karin , Shen Fang , Wang Yugang , Huang Tao , Lin Zhonghua , Zhang Juan , Suto Eric , Kaplan Charles , Liao Cheng , King Beverly , Nguyen Allen , Yeh Sherry , Delarosa Donnie , Liimatta Marya , Chiu Henry , Sweeney Zachary , Safina Brian , Johnson Adam , DeMartino Julie , DeForge Laura , Salphati Laurent , Lee Wyne , Balazs Mercedesz , Sutherlin Dan
Published: January 2023

The p110δ isoform of PI3K is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which PI3K mediates inflammation are poorly understood. Here we describe a novel highly selective p110δ small molecule inhibitor, G-286, that allowed dissecting the contribution specifically of p110δ to cellular pathways that are implicated in arthritis disease pathogenesis in human and mice. Using G-286, we demonstrate that p110δ regulates B cell- but not myeloid-cell dependent inflammatory arthritis in mice. G-286 blocks B cell receptor dependent proliferation and reduces autoantibody levels in collagen-induced arthritis abrogating disease. However, G-286 does not significantly inhibit FcγR-mediated inflammatory cytokine production in murine macrophages. Accordingly, selective p110δ inhibition did not significantly affect disease progression in FcγR- and myeloid cell-dependent autoantibody-induced arthritis. Importantly, we provide evidence that human and mice diverge in their relative dependency on p110δ to regulate FcγR responses. In addition to B cells and myeloid cells, T cells and osteoclasts contribute to rheumatoid arthritis pathogenesis. Utilizing G-286, we dissect the relative impact of p110δ inhibition on human and mouse T cell and osteoclast function. These results provide new insight in the role of p110δ in arthritis disease processes and offer a compelling rationale for targeting p110δ for the treatment of human rheumatoid arthritis.
Anti-diabetic drug, metformin protects oligodendrocytes under CNS pathological conditions: implication for multiple sclerosis (P5205)
Authors: Paintlia Ajaib , Paintlia Manjeet , Mohan Sarumathi , Singh Avtar , Singh Inderjit
Published: January 2023

We earlier documented that AMPK activators attenuate inflammation in activated mixed glial cultures and autoimmunity in EAE. Recent studies established that AMPK signaling is crucial to protect neurons under CNS pathological conditions however its influence on oligodendrocytes (OLs) loss is unclear. We here demonstrated that the observed loss of OLs as evident from the reduction of cell numbers and decrease in the expression of OL signatory genes in mixed glial cultures stimulated with LPS/IFN-γ was attenuated by AMPK activator (metformin). This effect of metformin was ascribed to the attenuation of inflammatory response and induction of myelinogenic milieu in similarly treated mixed glial cultures via AMPK dependent mechanism. In addition, metformin attenuated oxidative stress and mitochondrial membranal depolarization in immature OLs against cytokine toxicity and enhanced their differentiation into myelin-forming OLs. These in vitro findings were consistent with the attenuation of clinical symptoms, cellular infiltration and myelin loss in EAE animals treated with metformin. In addition, levels of OL-lineage cells signatory genes, neurotrophic factors and MnSOD mRNA transcripts including AMPK phosphorylation were elevated with corresponding reduction of cytokines and chemokines expressions in the CNS similarly treated EAE animals. Together, these data provide evidence that AMPK signaling partcipate in the protection of OLs under CNS pathological conditions.
Transcriptional control of tolerance associated gene TCAIM is CREB, NFATc1 and C/EBPβ dependent resulting in generation of tolerogenic dendritic cells (P2152)
Authors: Panov Ivo , Schlickeiser Stephan , Vogel Simone , Schumann Julia , Schliesser Ulrike , Gossen Manfred , Berberich-Siebelt Friederike , Sawitzki Birgit
Published: January 2023

Cell therapy by tolerogenic dendritic cells has become an attractive therapeutic option in transplantation. We could recently show that high expression of TCAIM, formerly known as TOAG-1, in dendritic cells (DCs) is associated with stable tolerance following transplantation. Here, we investigated transcriptional control of TCAIM in murine bone marrow derived DCs (BMDCs) and human MUTZ-3 derived DCs (M3DCs). We have cloned the human TCAIM promoter to identify regulatory elements by site directed mutagenesis. In addition, we performed luciferase reporter as well as ChIP assays. Furthermore, BMDCs or M3DCs were pre-incubated with cAMP elevating drugs prior to LPS-mediated maturation. Phenotype and tolerogenic potential of DCs were investigated by qPCR, flow cytometry, CBA and MLR. Site-directed mutagenesis, reporter assays and ChIP defined TCAIM core promoter containing three CREB responsive elements. Co-transfections revealed positive regulation of promoter activity by NFATc1 and C/EBPβ. Furthermore, pre-incubation of M3DCs or BMDCs with cAMP elevating drugs leading to CREB activation resulted in high TCAIM expression, inhibited DC maturation, diminished pro-inflammatory cytokine release and induced high frequency of CD4+CD25+FoxP3+ Tregs in DC-T cell co-cultures. These results indicate that transcriptional activation of TCAIM is cAMP/CREB dependent and further amplified by NFATc1 and C/EBPβ, resulting in induction of tolerogenic potential in human M3DCs and murine BMDCs.
Sodium cromoglycate treatment of CD200R1KO.CD200tg mice enhances graft survival (P2154)
Authors: Gorczynski Reg , Chen Zhiqi , Yu Kai
Published: January 2023

Allograft survival is not increased in CD200R1KO mice, lacking the primary inhibitor receptor for CD200, unlike in CD200tg mice. Rejection in both control, and CD200R1KO mice is associated with graft infiltration by activated mast cells, with evidence for degranulation (>80%). There is evidence that alternate CD200Rs in mice can contribute to immunosuppression through altered dendritic cell differentiation, and enhanced induction of Foxp3+Tregs. We asked whether, in CD200R1KO.CD200tg mice, attenuation of mast cell degranulation by sodium cromoglycate could foster enhanced graft survival following induction of Tregs. BALB/c skin grafts were transplanted to 10/group control, CD200tg or CD200R1KO.CD200tg mice, with/without treatment with cromoglycate (150mg/Kg, alternate days). All animals received low-dose rapamycin (0.5mg/Kg), previously shown to prolong survival only in CD200tg mice. Graft survival was monitored daily from d10, and splenic MLC responses measured in vitro on d14/21. Immunohistology was performed on all grafted tissue at d14. Graft survival was increased only in CD200tg mice, and in CD200R1KO.CD200tg mice receiving cromoglycate (p<0.05). This was accompanied by decreased degranulation of graft-infiltrating mast cells, and increased Foxp3+Treg infiltration. Survival was abolished by ongoing infusion of neutralizing anti-CD200 mAb, or infusion of anti-CD200R1/2mAb (in CD200tg or CD200R1KO.CD200tg mice respectively).
Prostaglandin E2-induced changes in alveolar macrophage scavenger receptor profiles alter phagocytosis of bacteria post-bone marrow transplantation (P2156)
Authors: Domingo-Gonzalez Racquel , Katz Samuel , Serezani C. Henrique , Moore Thomas , LeVine Ann Marie , Moore Bethany
Published: January 2023

The effectiveness of hematopoietic stem cell transplantation as a therapy is complicated by pulmonary infections. Using our syngeneic bone marrow transplant (BMT) mouse model, BMT mice displayed increased susceptibility to Pseudomonas aeruginosa and Staphylococcus aureus. BMT alveolar macrophages (AMs) exhibited a defect in P. aeruginosa phagocytosis while S. aureus uptake was enhanced. This difference was due to an altered scavenger receptor (SR) profile. Interestingly, MARCO expression was decreased while SR-AI/II was increased. Phagocytosis assays with SR-AI/II transfected-CHO cells revealed SR-AI/II was important for S. aureus uptake but not P. aeruginosa. Conversely, AMs treated in vitro with soluble MARCO exhibited similar defects in P. aeruginosa internalization as did BMT AMs. miR-155 targets the 3’UTR of SR-AI, however, its expression is decreased post-BMT. Anti-miR-155-transfected AMs exhibited an increase in SR-AI/II expression and S. aureus phagocytosis. In vitro treatment of AMs with prostaglandin E2 (PGE2) (elevated post-BMT) increased SR-AI/II, and decreased MARCO and miR-155. Despite a difference in phagocytosis, BMT AMs harbor a killing defect to both P. aeruginosa and S. aureus. Thus, our data suggest that PGE2-driven alterations in SR and miR-155 expression account for the differential phagocytosis of P. aeruginosa and S. aureus but impaired killing ultimately confers increased susceptibility to pulmonary infection.
Surfactant protein A regulation of host defense post-bone marrow transplantation (P2158)
Authors: Ashley Shanna , Domingo-Gonzalez Racquel , Wilke Carol , Agarwal Manisha , Levine Ann Marie , Moore Bethany
Published: January 2023

Patients who undergo stem cell transplant are at increased risk of developing bacterial lung infections. We have developed a murine model of syngeneic bone marrow transplant to try to understand the mechanisms which may be responsible for impaired innate immunity post-transplant. Our results demonstrate that syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa post-transplant. This is mediated in part by reduced expression of the MARCO scavenger receptor. We now report that the BMT mice are also deficient in the production of surfactant protein A post-BMT. Levels of SpA in the bronchoalveolar lavage fluid are diminished post-BMT. Phagocytosis assays demonstrate that phagocytosis of Pseudomonas aeruginosa can be enhanced in the presence of exogenous SpA. We have evidence to suggest that exogenous SpA allows Pseudomonas aeruginosa to be phagocytosed by binding to the scavenger receptor A (SRA) I/II. Current studies are underway to determine why SpA is reduced post-BMT and to determine the influence of SpA on bacterial killing post-BMT.
Tonsil-derived stromal cells reduces CCl4-induced liver fibrosis in mice via autophagy activation (P2180)
Authors: Woo So-Youn , Park Minhwa , Lee Hye Jin , Kim Ji-Yon , Ryu Kyung-Ha
Published: January 2023

Liver transplantation is the treatment of choice for chronic liver failure due to fibrosis, however, it faces several difficulties, including donor shortage, surgery-related complications, immunological rejection, and high medical cost. Cell transplantation therapy would be the minimally invasive and alternative methods, potentially with fewer complications. As cell source, we isolated mesenchymal stromal cells from human palatine tonsils (T-MSCs) and injected into CCl4-induced liver injury model of the mice. We found that T-MSCs were differentiated into hepatocyte-like cells under 25 ng/ml insulin-like growth factor, 25 ng/ml hepatocyte growth factor, 10-7 M dexamethasone, and 10 ng/ml oncostatin M for three weeks in vitro. After injecting into CCl4-liver damaged mice, T-MSCs were differentiated into albumin positive cells but were not detected in the group of mice with normal liver. During this process, T-MSCs showed accumulation of autophagosome-bound LC3B, upregulation of proautophagic beclin-1, and ubiquitin positive in vivo. Our data imply that T-MSCs contributed to reduction of CCl4-induced liver fibrosis through autophagy activation pathway.
Nonmyeloablative conditioning employing busulfan and sirolimus permits donor-dominant stable chimerism in a murine model of haploidentical allogeneic stem cell transplantation (P2192)
Authors: Weitzel R. , Fitzhugh Courtney , Phang Oswald , Hsieh Matthew , Tisdale John
Published: January 2023

Allogeneic stem cell transplant (SCT) is a potentially curative option for many hematologic diseases, yet donor availability remains a challenge, and haploidentical SCT is associated with a higher risk of rejection. We successfully translated a model based on total-body irradiation (TBI) with to humans; however, TBI involves toxicities and risk of malignant side-effects, and we hypothesize that busulfan may be a superior alternative in some settings. G-CSF-mobilized F1/J (C57BL/6-BALB/c hybrid) splenocytes were injected into C57BL/6 hosts who had received 10-30mg/kg IP busulfan the previous day. All recipients received 31 days of 3mg/kg IP sirolimus beginning the day before transplant. No GvHD was observed and counts recovered by week 8 in all groups. Mice conditioned with 30mg/kg busulfan displayed remarkably high donor chimerism (>90%), which was significantly higher than 300cGy TBI-conditioned control mice (64-86%) across all timepoints. Mice receiving 30mg/kg or 20mg/kg busulfan maintained stable chimerism for over 18 months. Mice conditioned with 10mg/kg busulfan showed only transient chimerism through 4 weeks. Proliferation of CD4+ cells in response to donor increases with decreasing busulfan, and mice receiving 30mg/kg responded more strongly than mice receiving TBI. A memory effect was observed in mice that received 10mg/kg busulfan and lost their grafts. Notably, a slightly higher incidence of host-derived Tregs in mice was also observed after graft rejection.
Development of engineered heart tissue from MHC-I downregulated non-cardiac fibroblasts and stem cell derived cardiomyocytes. (P2224)
Authors: Karabekian Zaruhi , Ding Hao , Jamshidi Aria , Sarvazyan Narine
Published: January 2023

Our goal was to test a hypothesis that downregulation of MHC class I molecules via inhibition of beta2-microglobulin gene expression can minimize immunogenicity of non-cardiac fibroblasts and stem cell derived cardiomyocytes without impairing ability of these cells to form functional cardiac tissue. When co-cultured with mouse stem cell cardiomyocytes, two different types of non-cardiac mouse embryonic fibroblasts (NIH3T3 and WT19) supported the development of three-dimensional cardiac fibers. Fibroblasts had to be irradiated to ensure optimal growth, so that they did not overgrow non-replicating cardiomyocytes and a proper ratio of fibroblasts to cardiomyocytes was needed to create optimal fibers. The fibers also formed when both cell types underwent downregulation of MHC-I molecules. The latter was achieved by inhibition of beta2-microglobulin gene expression using shRNA technology and was confirmed by qPCR and FACS analyses. The downregulation of MHC-I molecules made fibroblasts and cardiomyocytes less susceptible to T-cell recognition in both in vitro and in vivo experiments. We conclude that readily obtainable non-cardiac fibroblasts can be used to engineer functional cardiac tissue and that downregulation of MHC-I can make such tissue less prone to immune rejection.
Inhibition of CD1d-mediated antigen presentation by the TGF-β/Smad signaling pathway (APP3P.113)
Authors: Iyer Abhirami Kannan , Bailey Jennifer C. , Gourapura Renukaradhya , Lin Yinling , Nguyen Hoa B. , Brutkiewicz Randy
Published: January 2023

CD1d is a MHC class I-like molecule that presents lipid Ags to a subpopulation of innate lymphocytes called natural killer T cells. In a prior study, we showed that the mitogen-activated protein kinase p38 is a negative regulator of Ag presentation by CD1d. It is unknown how p38 is activated to induce this regulation; however, several studies have implicated a role for the immunoregulatory cytokine, TGF-β, in the activation of p38. Therefore, we hypothesized that TGF-β negatively regulates the CD1d-mediated Ag presentation pathway. Indeed, a dose-dependent decrease in CD1d-mediated Ag presentation and impairment of lipid Ag processing was observed in TGF-β-treated antigen presenting cells. However, this inhibition was not through p38 activation. Instead, by employing a lentiviral shRNA-based approach, we found that the canonical TGF-β signaling elements, Smads 2, 3 and 4, contributed to the TGF-β-mediated inhibition of Ag presentation by CD1d. Furthermore, a side-by-side comparative analysis of the effect of TGF-β on CD1d and MHC class II-mediated Ag presentation revealed that in contrast to that observed with CD1d, TGF-β actually enhances MHC class II-mediated Ag presentation. Overall, these results suggest that the canonical TGF-β/Smad pathway has distinctly different effects on the regulation of CD1d- and MHC class II-mediated antigen presentation pathways.
HLA-DM and HLA-DO: bi-directional regulation of function, tuned by pH (APP3P.103)
Authors: Jiang Wei , Strohman Michael , Wang Nan , Hou Tieying , Mellins Elizabeth
Published: January 2023

Peptide processing and loading onto MHC-II in endosomes occurs across a wide pH range. We studied pH effects on DM/DO regulation of peptide loading in vitro, using soluble, recombinant molecules, including a stabilized mutant DO dimer (sDOAP11A) that inhibits DM normally. We tested different DO:DM ratios at pH 4.6-7.2 during loading of 4 HLA alleles. DO alone had no effect. DM inhibition was diminished at lower DO:DM ratios, implying stable DM association is required for DO effect. Inhibition occurred across the pH range, but was reduced at pH 4.6. Using a narrower pH gradient (4.56-5.30), across which DM function is equivalent, we identified DO as the primary target of pH effects. DO activity was reduced after pre-incubation at pH 5.4, and longer exposure, lower pH and higher temperature enhanced this effect. FRET analysis of DM/DO at low pH was consistent with DO release rather than conformational change of intact complexes. SPR, bio-layer inferometry and ELISA assays showed reduced DM/DO association at pH≤4.85, suggesting released DO binds back inefficiently. With pre-incubation at pH 4.6 or 5.4, DM-DOwt complexes acquired DM-like activity; analysis of active SEC fractions indicated presence of free DM. Pre-incubation of sDM alone at these pHs caused aggregation. Complexes of full-length DM and DOwt showed similar behavior to soluble molecules. We conclude that low pH generates free DM by releasing DO, which both inhibits and chaperones DM.
Potentiation of antigen-specific CTL generation by caTLR4 gene transduction into leukemic plasmacytoid dendritic cell line (PMDC11) (APP3P.108)
Authors: Takahashi Masuhiro , Iwabuchi Minami , Satoh Naoya , Uchiyama Takayoshi , Iwaya Shunpei , Oiwa Rie , Nishizawa Yoshinori , Hashimoto Shigeo , Bonehill Aude , Kasahara Noriyuki , Narita Miwako
Published: January 2023

Leukemic plasmacytoid dendritic cell (pDC) line with HLA-A02/24, PMDC05, which was established in our laboratory, acquired a higher antigen presenting ability by being transduced with lentiviruses encoding the CD80 gene. CD80 gene-transduced PMDC05 was named PMDC11. In order to establish a more potent antigen presenting cells for cellular immunotherapy in tumors, PMDC11 cells were transduced with a constitutively active (ca) TLR4 gene by using the Tet-On system (caTLR4-PMDC11). CD8+ T cells from healthy donors with HLA-A24 were co-cultured with WT1 peptide-pulsed caTLR4-PMDC11 cells. Priming with WT1 peptide-pulsed caTLR4-PMDC11 cells was performed every week. Immediately before the priming with caTLR4-PMDC11 cells, cultured cells were analyzed for the percentage and number of CD8+/WT1 tetramer+ T cells. CD8+/WT1 tetramer+ T cells were generated after 2-3 weeks of culture and much increased in caTLR4-PMDC11-primed CD8+ T cell culture compared with parental PMDC11-primed CD8+ T cell culture. These CD8+ T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to be cytotoxic to WT1-expressing HLA-A24+ target cells. These data revealed that the antigen-specific cytotoxic T lymphocyte (CTL) inducing ability of PMDC11 was enhanced by being transduced with caTLR4 gene, and that caTLR4-PMDC11 cells could be applied as potent antigen presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infection.
Human peripheral invariant natural killer T are adaptive lymphocytes, whose TCR CDR3β regulates their antigen specificity and reactivity to avoid harmful autoimmune responses (BA8P.124)
Authors: Chamoto Kenji , Imataki Osamu , Guo Tingxi , Saito Toshiki , Saito Akiko , Elia Andrew , Chiu Priscilla , Butler Marcus , Hirano Naoto
Published: January 2023

Human invariant natural killer T (iNKT) cells carry a sole variable element, complementarity determining region 3β (CDR3β) in their T cell receptors (TCRs). It has been widely accepted that iNKT cells recognize foreign and self-ligands via CD1d in a pattern recognition-like innate-type manner. Here, we have created a thymically unselected human iNKT Vβ11 TCR repertoire and isolated a series of iNKT TCRs with ultra-high to low autoreactivity. We have found three distinctive CDR3β sequence motifs that dictate high iNKT TCR autoreactivity. In vitro and in vivo studies demonstrated that highly autoreactive iNKT cells carry innate-type TCRs, recognize CD1d molecules in a ligand-independent manner and evoke severe systemic autoimmune responses within a few hours upon adoptive transfer. In contrast, iNKT cells with intermediate to low autoreactivity express adaptive-type TCRs, which selectively recognize self-ligands and induce tissue-specific moderate autoimmune responses in vivo. High throughput CDR3β sequencing revealed that the frequency of highly autoreactive human iNKT cells positive for all 3 motifs was significantly reduced among iNKT cells in the periphery compared to the thymus. We conclude that intermediately and lowly autoreactive iNKT cells which prevail in the human periphery are adaptive lymphocytes and that the sole adaptive CDR3β sequences regulate the antigen specificity and reactivity of iNKT cells to avoid harmful autoimmune responses.
IL-33/ST2 promotes tissue mastocytosis under conditions of inflammation (CCR3P.219)
Authors: Wang Jun-Xia , Kaieda Shinjiro , Nigrovic Peter
Published: January 2023

Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues, where they have been implicated in multiple allergic and inflammatory diseases. MC express the IL-33 receptor ST2, and this new member of the IL-1 cytokine family is recognized as an important pathway both for direct MC activation and for priming MC to respond to other pro-inflammatory signals. However, IL-33 has no known role in tissue mastocytosis. Using primary MC cultured from human skin, we found that IL-33 did not alter MC proliferation but instead protected MC from apoptosis, principally through upregulation of the anti-apoptotic molecule BCLXL. Murine bone marrow-derived MC demonstrated a similar response to IL-33, dependent entirely upon ST2, allowing us to test the role of this biology in vivo. Mice lacking ST2 exhibited fewer peritoneal MC as well as reduced accumulation of MC in inflamed arthritic joints and helminth-infected intestine. Upon engraftment into the peritoneum, WT MC exhibited a clear, IL-33-mediated survival advantage over ST2-deficient MC, in particular after thioglycollate-induced peritonitis. Together, these data reveal a novel, cell-intrinsic role for the IL-33/ST2 axis in tissue mastocytosis, especially under conditions of local inflammation.
Significance of CXCR6-CXCL16 in prostate cancer progression and therapeutic efficacy (CCR3P.222)
Authors: Gupta Pranav , Sharma Praveen , Grizzle William , Lillard James , Singh Shailesh
Published: January 2023

Despite recent advances prostate cancer (PCa) is still a leading cause of cancer related deaths among men, primarily because etiopathogenesis of PCa is not well defined. Chemokines and their corresponding receptors have been shown to play major role in PCa progression and poor therapeutic outcomes. The focus of this study is to determine the potential role of CXCR6 and its natural ligand CXCL16 in PCa pathogenesis and impact of this axis on docetaxel (DTX) efficacy. We investigated CXCR6/CXCL16 expression in clinical samples and prostate cell lines. CXCR6/CXCL16 expression was significantly higher in PCa samples compared to their respective controls, which showed positive correlation with tumor stage and grades. Similar to patient’s samples, PCa cell lines showed higher expression of CXCR6 and CXCL16 compared to normal prostatic epithelial cells. Furthermore, molecules involved in cell survival, matrix digestion and epithelial-mesenchymal transition were differentially modulated by CXCL16 treatment. Interestingly, expression of CXCR6/CXCL16 was elevated in PCa cells following DTX treatment compared to untreated cells. Taken together, better efficacy of DTX was observed when given with anti-CXCR6 is presumably due to inhibition of CXCR6-mediated survival signals. These findings suggest that CXCR6-CXCL16 axis play a crucial role in PCa pathogenesis and inhibition of this axis may have significant impact on disease progression and therapeutics outcomes.
HMGB1 is a key mediator of anemia in a murine model of sepsis survivors (CCR3P.224)
Authors: Valdes-Ferrer Sergio , Blanc Lionel , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Anemia is omnipresent in patients with severe sepsis, and correlated to long term outcome. Its mechanism is incompletely understood. High mobility group box 1 (HMGB1) is persistently elevated in murine sepsis survivors. To study the role of HMGB1 in anemia of sepsis, we used a murine model of severe sepsis induced by cecal ligation and puncture (CLP). Anemia developed 3 days after onset of sepsis and persisted for no less than 28 days. The compensatory regenerative response was blunted, as demonstrated by reduced and erratic reticulocytosis. Splenomegaly was present in CLP survivors for as long as the follow up period. Microscopy revealed splenic architecture disruption, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation using flow cytometry demonstrated an increase in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast, while mature erythroid cell populations were significantly reduced. An anti-HMGB1 (2G7 clone; 50ug/day x3 days I.P.) administered 9 days after after CLP significantly protected septic mice from anemia. In contrast, exogenous administration of HMGB1 (500ug/mouse/day x7d I.P.) to healthy mice was sufficient to induce a significant decrease in hemoglobin (p>0.05) and splenomeglay. These results indicate HMGB1 is necessary and sufficient for developing anemia in murine sepsis, and suggest HMGB1 may be a novel therapeutic target of anemia in sepsis survivors.
BAFF induces IL-6 production by monocytes through a BAFF receptor and promotes IgG production by B cells in patients with primary Sjögren's syndrome. (CCR3P.210)
Authors: Yoshimoto Keiko , Ishioka Eriko , Nishikawa Ayumi , Suzuki Katsuya , Kameda Hideto , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

Backgrounds and Objectives: We have found that the elevated expression of BAFF receptor (BR3) on monocytes is involved in activation of monocytes to promote IL-6 production in patients with primary Sjögren's syndrome (pSS). In addition, it is well known that pSS is often accompanied by hypergammaglobulinemia (HγG). In our present study, we investigated the possible involvement of monocytes in the development of HγG. Methods: Peripheral monocytes were cultured in vitro with or without peripheral B cells and stimulated with soluble BAFF (sBAFF). The amounts of IL-6 and IgG in the culture supernatants were measured by ELISA. FACS analysis of whole blood samples was employed to analyze the expression of BR3. Results: The serum level of IgG and the proportion of BR3-positive monocytes (BR3+/CD14+) were elevated in pSS patients as compared to those in normal individuals. Remarkably, the BR3+/CD14+ ratio was positively and significantly correlated with the serum IgG level. In addition, the ratio was positively and significantly correlated with the amount of IL-6 produced by pSS monocytes stimulated with sBAFF. Stimulation of co-culture of B cells and monocytes with sBAFF drastically enhanced IgG production by B cells. Conclusions: Our data collectively suggest that the abnormal expression of BR3 on monocytes is responsible for the overproduction of IgG by B cells in pSS patients.
Metabolic maturation, migration, and liver retention of CD4 single positive thymocytes and recent thymic emigrants (HEM4P.238)
Authors: Ge Qing , Xu Xi , Zhang Shusong
Published: January 2023

CD4 or CD8 single positive (SP) thymocytes, recent thymic emigrants (RTEs), and peripheral naïve T cells represent three consecutive cell populations along T cell maturation program. Except for negative selection in SP thymocytes, the importance and the regulation of this maturation is not well understood. In particular, little is known about the transition from mature SP to RTE and key events of this transition during and after thymic egress. We identified the phenotype of CD4+ pre-RTEs, a population of SPs that has acquired the thymic egress capability. A pro-oxidant shift and an increase of mitochondria content was found in pre-RTEs when compared to SPs with less functional maturity. The elevation of intracellular ROS was involved in promoting the functional maturation of these young T cells. After egress, autoreactive CD4+ pre-RTEs were retained and some were activated in the liver. Education from secondary lymphoid organs were not required for hepatic retention but TCR-MHC interaction in the liver was important for the activation of CD4+ liver RTEs. When transferred into RAG-/- mice, liver but not lymph node RTEs caused T cell infiltration and severe pulmonary inflammation, suggesting that the liver may be the first checkpoint in the periphery to filter and retain autoreactive CD4+ RTEs that escape from negative selection. Together, our results emphasize the complexity of SP-RTE maturation and the impact of various lymphoid tissue microenvironments on this maturation.
Cell types important for fostering normal thymic medullary epithelial cell development (HEM4P.242)
Authors: Jeon Hyein , Williams Joy , Hodes Richard
Published: January 2023

Tolerance in developing T cells is dependent on medullary thymic epithelial cells (mTEC) and mTEC development in turns requires signals from mature SP thymocytes. Several TNF family receptor-ligand pairs have been shown to be important players in the thymic crosstalk required to foster mTEC development including RANK-RANKL, LTαβ-LTβR and CD40L-CD40. In addition to confirming the important roles of TNF family members in thymic crosstalk, we have recently identified CD28-CD80/86 interactions as an additional pathway important for normal thymic medullary compartment development. CD80 and CD86, similar to LTβR, CD40 and RANK, are expressed by mTEC as well as by bone marrow (BM) derived cells in the thymus. To address the question of which cell type(s) need to express CD80/86, LTβR, CD40 and RANK in order to support mTEC development, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. For LTβR and RANK, we find that expression on radiation-resistant non-BM-derived cells, is required. Interestingly, however, we find that for CD80/86 and CD40, expression on either non-BM-derived or BM-derived cells is sufficient to promote mTEC development. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other bone marrow derived cell types as well.
Exploring the thymic niche requirements for regulatory T cell development (HEM4P.230)
Authors: Weist Brian , Robey Ellen
Published: January 2023

Development of regulatory T cells in the thymus is crucial for maintenance of tolerance to self. Here we demonstrate a new approach to systematically study the niche requirements for development of regulatory T cells in thymus. Using a thymic slice model, delivery of a model antigen leads to synchronized Treg development over the course of 72 hours. Antigen delivered by an exogenous antigen loaded dendritic cell population, or expressed as a transgene in medullary thymic epithelial cell populations, or a mixture of the two results in similar Treg numbers developing, and in all cases the number of developing regulatory T cells does not exceed 20% of antigen specific thymocytes. Addition of IL2 to thymic slices enhanced development, providing evidence that IL2 availability is a limiting factor for Treg development. We observed diminished Treg development when IL2-/- dendritic cells are used to present antigen, providing evidence that the APC population can be an important source of IL2 for developing Tregs. Overall, the thymic slice model provides an excellent system to dissect the niche requirements for the development of Tregs. Here we show that both antigen availability and IL2 exposure from a novel source can influence Treg development, but additional factors remain to be uncovered.
Evidence of T cell lymphopenia in the colonic mucosa in idiopathic CD4 lymphocytopenia patients (HEM4P.250)
Authors: Kovacs Stephen , Sheikh Virginia , Rupert Adam , Thompson William , Estes Jacob , Sandler Netanya , Roby Gregg , Freeman Alexandra , Sereti Irini
Published: January 2023

Idiopathic CD4 lymphocytopenia (ICL) is a syndrome of unknown etiology characterized by low peripheral CD4 T cell counts and susceptibility to opportunistic infections. One hypothesis for its etiology is trapping of T cells in effector sites. 11 ICL patients and 16 healthy controls (HC) underwent colonic biopsies for CD3, CD4, and CD8 T cell enumeration by flow cytometry (FC) and immunohistochemistry (IHC) in lamia propria (LP). Mucosal IL17+ CD4 T cells were assessed after PMA/ionomycin stimulation. Plasma levels of LPS (for microbial translocation), I-FABP (for gut epithelial integrity), sCD14 (for monocyte activation), D-dimer (for coagulation) and CRP, IL-6 and TNFalpha (for inflammation) were tested in 33 ICL patients and 44 HC. ICL patients had fewer CD3 (2.84-fold, P=0.0080), CD4 (3.04-fold, P=0.013) and CD8 (2.28-fold, P=0.043) T cells in the colonic mucosa compared to HC by FC and fewer CD3 (2.31-fold, P=0.036) and CD4 (3.81-fold, P=0.0003) T cells in LP by IHC. The proportion of IL-17+ CD4 T cells was higher in ICL compared to HC (6.04 vs 3.35%, P=0.013). Levels of sCD14 levels were slightly higher in ICL than in HC (1.42 vs 1.21 µg/ml, P=0.003). Levels of LPS, I-FABP, IL-6, TNFalpha, D-Dimer, and CRP levels were not significantly different between ICL and HC (all P>0.05). These data suggest that colonic mucosal lymphopenia accompanies peripheral T cell lymphopenia in ICL, albeit with preserved gut epithelial integrity and a lack of systemic inflammation.
An obesity-associated cytokine profile for immune cell differentiation and activation (HEM4P.253)
Authors: Nikolajczyk Barbara , Zhu Min , Xiao Qiang , Kuchibhatla Ramya , Apovian Caroline
Published: January 2023

Immune cells are dominant sources of the obesity-associated inflammation that predisposes type 2 diabetes (T2D), but triggers of immune cell hyper-responsiveness in obesity/T2D are poorly understood. T cells are key sources of cytokines that promote immune cell differentiation/activation, although T cells generally require contact with other immune cells to produce obesity-associated amounts of cytokines. To test the possibility that obesity associates with cytokine changes that impact immune cell differentiation/activation, we stimulated T cells from lean, obese non-diabetic (ND), or obese pre-diabetic (PD; HbA1c 5.6-6.5) subjects in the context of PBMCs and quantitated cytokines with the Milliplex Human High Sensitivity T Cell Panel. Samples from ND and PD subjects produced more T cell/Th1-supportive IL-2, IL-7 and IL-12p70, myeloid-supportive GM-CSF, and B cell-supportive IL-21 compared to samples from leans. In contrast, anti-inflammatory IL-10 was highest in samples from leans. Based on the anti-inflammatory actions of the T2D drug metformin, we also measured cytokine production by samples from PD subjects that were taking metformin. Metformin partially restored IL-10 production in samples from PD subjects, but had no impact on immune cell-activating cytokines. We conclude that obesity associates with inflammation due to over-expression of cytokines that support immune cell differentiation/activation, and that anti-inflammatory action of in vivo metformin is limited.
Towards understanding the expansion of plasmablasts in dengue virus infections (HEM4P.254)
Authors: Dobbs Andrea , Nascimento Eduardo , Garcia-Bates Tatiana , Smith Amanda , Marques Ernesto , Barratt-Boyes Simon
Published: January 2023

An increased prevalence of dengue has resulted in its emergence as an important public health threat. Pre-immunity to any of the 4 dengue virus (DENV) serotypes is the main risk factor for developing severe disease during heterologous DENV infections. We have recently published that during secondary DENV infections there is a major expansion of DENV-specific antibody secreting plasmablasts (CD19+CD27+CD38+) that correlates with the development of severe disease. The mechanisms driving this response remain unknown and may be important in understanding the pathogenesis of DENV infections. We are using in vitro approaches to define the possible mechanisms responsible for driving expansion of plasmablasts during heterologous DENV exposures. PBMCs from a cohort of healthy DENV seropositive and seronegative donors were collected for this study. We used flow cytometry to assess the differentiation of B-cells into plasmablasts 7 days after stimulating PBMCs with each DENV serotype. Preliminary in vitro studies suggest that B-cells from DENV immune donors are more apt to differentiate into plasmablasts when stimulated with DENV with possible variances between the different serotypes. Our results with isolated B-cells also suggest that the presence of other cell populations, potentially monocytes, is required for the expansion of plasmablasts. Ongoing experiments will reveal further cellular requirements for the in vitro expansion of plasmablasts after exposure to heterologous DENV.
Vitamin D augments house dust mite-mediated Th2 responses in human pulmonary dendritic cells (HYP6P.267)
Authors: Hrusch Cara , Sullivan Meghan , Tjota Melissa , Williams Jesse , Blaine Kelly , Sperling Anne
Published: January 2023

Low serum levels of vitamin D (VD3) have been implicated in multiple immune disorders including atopic asthma. Other studies have shown that VD3 promotes monocyte-derived dendritic cell (MDDC) differentiation into tolerogenic DCs, which in turn induce regulatory T cells. However, we have found that VD3 actually augments allergen-induced Th2 cytokines. Specifically, cells isolated from human lung draining lymph nodes produced more IL-13 when treated with a combination of VD3 and the common allergen house dust mite (HDM) than either treatment alone. Similarly, when peripheral blood-derived MDDCs were co-treated with VD3 and HDM, these cells were able to induce less IFNγ, but more IL-4, from allogeneic CD4 T cells in a mixed lymphocyte reaction. In both MDDCs and pulmonary DCs, co-treatment with VitD and HDM led to a phenotype distinct from either treatment alone, as the DCs expressed both the stimulatory molecules HLA-DR and CD86 as well as the VD3-induced molecule thrombomodulin (CD141). Additionally, while the inhibitory receptors ILT3 and ILT4 are induced in MDDCs by VD3, we found that HDM was able to block this induction. Interestingly, the ILT4 ligand HLA-G was increased in this population. Future studies will explore the role of DC-expressed HLA-G and its receptors on DC function. Together, these data indicate that while VD3 may induce markers of tolerance, common allergens abrogate this response in DCs to augment the induction of a Th2 response.
Exploring the role of miRNA 210 and regulatory T cells during TDI sensitization (HYP6P.270)
Authors: Long Carrie , Marshall Nikki , Siegel Paul , Meade Barbera , Lukomska Ewa , Anderson Katie , Beezhold Donald , Anderson Stacey
Published: January 2023

TDI (Toluene 2, 4-diisocyanate) is one of the leading causes of chemical-induced occupational asthma. MicroRNAs are small, noncoding RNAs that exhibit profound functional significance through the inhibition of gene expression of mRNA targets. Recently, the robust regulatory potential of miRNAs has become apparent, including their functional role in the regulation of allergic disease. Our laboratory has previously showed that miRNA 210 expression is significantly augmented in the draining lymph nodes of mice dermally dosed with TDI at various time points post exposure, however, the specific role of miRNA 210 in chemical sensitization is unknown. In this work, to elucidate the functional role of miRNA 210 during TDI sensitization, BALB/c mice were dermally exposed to TDI (4% v/v) or vehicle and endpoints were evaluated via RT-PCR, magnetic cell separation, and flow cytometry. Draining lymph node CD4+ cells showed significantly increased miRNA 210 expression following a single exposure to TDI. Confirmed (foxp3) and predicted (runx1t1, runx3, smad4, and stat6) miRNA 210 transcription factor target expression was evaluated and augmentations in foxp3 cellular protein expression and decreases in runx1 and foxp3 mRNA were observed. This data indicates that miRNA 210 has a functional role in TDI sensitization which may be related to regulatory T cell differentiation and function.
Aspirin induces IL-4 mRNA expression via activation of MAPKs pathway (HYP6P.272)
Authors: Kong Su-Kang , Chung Il Yup
Published: January 2023

Aspirin-exacerbated respiratory disease is characterized by asthma, nasal polyps, and intolerance to aspirin. IL-4 is mainly produced by allergic cells, including Th2 cells, mast cells, and eosinophils, and plays a central role in pathogenesis of allergic diseases such as asthma. In the present study, we demonstrated that aspirin induced IL-4 expression. Aspirin dose-dependently induced IL-4 mRNA and IL-4 promoter reporter activity in these three cell types. Treatment with pharmacologic inhibitors showed that the aspirin-induced IL-4 expression occurred through the MAPK ERK1/2 pathway, which was consistent with the observation that aspirin induced ERK phosphorylation. Analysis of serially deleted IL-4 promoter reporter showed that aspirin elevated the promoter activities regardless of the deleted constructs. When nuclear extract from these cells treated with aspirin were reacted with the probes harboring P0, P1, P2, P3, P4, P5 elements, and a region between P1 and P2 elements, DNA-protein complexes were remarkably augmented in the probes. Some of the DNA-protein complexes were supershifted by anti-NFAT. Collectively, these data indicate that aspirin induces IL-4 expression via ERK1/2 activation and availability of transcription factors that bind critical cis-acting elements in IL-4 promoter, thereby contributing to pathogenesis of aspirin-intolerant asthma.
Inhibition of LPS-induced acute lung inflammation in mice by adenovirus mediated Foxp3 expression (HYP6P.275)
Authors: Chung Hwan-Suck , Park Soojin , Baek Hyunjung , Bae Hyunsu
Published: January 2023

Forkhead transcription factor 3 (Foxp3) is critical transcription factor in CD4(+)CD25(+) regulatory T cells. There are increasing evidences that the functions of Foxp3 proteins are not restricted to regulatory T cells. The aim of this study is to evaluate the functions of Foxp3 proteins in LPS induced lung inflammation. In this study, LPS was administrated to Balb/c mice to induce a disease that resembles COPD. Two days prior to LPS administration, mice were infected once with the Foxp3-GFP or control GFP expression adenovirus to intra-trachea. The total cells and neutrophil numbers in bronchoalveolar lavage (BAL) fluid were counted and pro-inflammatory cytokine levels in BAL fluid were measured after 3 days later from LPS administration. Lung tissues were stained with hematoxylin and eosin (H&E) for histologic evaluation. Total cells, including eosinophil, macrophage and lymphocytes was significantly reduced in the foxp3 expression adenovirus infected mouse BAL fluid compared with the control adenovirus infected mouse BAL fluid. Foxp3-GFP or GFP was only detected in the lung epithelial cells by confocal microscopy. Using a mouse lung epithelial cell line, LA-4, Foxp3 adenovirus infected cells reduced the expression of chemokines such as CCL3, CXCL2 and CXCL5 by LPS stimulation. These results suggest that Foxp3 expression in lung epithelial cells suppresses inflammation via inhibition of chemokines secretion in lung inflammation.
House dust mite increases IL-33 and thymic stromal lymphopoietin (TSLP) gene expression in response to human rhinovirus 1B in murine bone marrow-derived macrophages. (HYP6P.276)
Authors: Akbarshahi Hamid , Menzel Mandy , Uller Lena
Published: January 2023

Acute viral infections are the most frequent cause of asthma exacerbations, and are therefore responsible for the majority of healthcare costs attributed to asthma. Human rhinovirus is the most common identified one in children and adults. It has been shown that IL-33- driven mechanism initiates TH2 responses to the clinically relevant allergens house dust mite (HDM). We have shown that TSLP is increased in asthma exacerbation. Alveolar macrophages have higher expression of IL-33 and TSLP in asthma. To determine whether HDM can have an effect on IL-33 and TSLP gene expression in response to human rhinovirus (RV), we treated bone marrow-derived macrophages (BMM) with HDM in the presence of M-CSF and infected them with RV1B. RV1B induced expression of IL-33 (2-fold, P-value<0.001) and TSLP (2-fold P-value<0.05) in HDM- treated macrophages after 24 hours compared to HDM non-treated BMM. RV1B infection caused an increase in IL-33 in IL-33 (P-value<0.01) and TSLP ( P-value<0.01) in HDM treated BMM compared to UV-treated RV1B group. There was no increase in IL-33 and TSLP gene expression in response to RV1B in the HDM-treated BMM at eight hours compared to UV-treated RV1B group, while HDM non-treated BMM showed increase in IL-33 (P-value<0.01) and TSLP (P-value=0.059 ) in response to RV1B compared to UV-treated RV1B. These results suggest that HDM increases IL-33 and TSLP gene expression in macrophages in response to RV1B which can play a role in asthma exacerbations.