bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective T cell receptor dependencies provoke a revised model of innate-like T cell development (P1431)
Authors: Wencker Melanie , Cope Andrew , Hayday Adrian 
Published: January 2023

Abstract
γδ cells include prototypic “innate-like” T cells that cast new light on lymphocyte biology. For example, many have their functions developmentally pre-programmed in the thymus so that they can respond more rapidly to challenge than can conventional T cells. Likewise, rather than deleting them or converting them to T-regs, agonist engagement in the thymus reportedly skews γδ cells toward IFN-γ-producing effectors. Conversely, those cells that do not engage agonist default to IL-17 production. Paradoxically however, thymic progenitors of CD27(-) IL-17-producing γδ cells are TCRhiCD127hiCD44hi which is typical of pre-activated cells, whereas the phenotype of most CD27(+) IFN-γ producers is typical of naïve T cells. To explore this paradox, we examined the SKG mouse which carries a hypomorphic ZAP70 mutation, thus attenuating TCR signaling but retaining TCR expression so that γδ T cell subsets can be identified. Unexpectedly, reducing TCR-signaling largely prevented the development of IL-17-producing γδ cells, whereas IFN-γ-producing γδ cells appeared normal. Nonetheless, deeper analysis showed that IFN-γ-producing γδ cells with the phenotype of agonist selection were depleted. These data permit a revised model in which there are two pathways of γδ T cell development. One gives rise to innate-like T cells that require a developmental TCR signal; from the other, cells biased toward IFN-γ emerge as adaptive cells, without significant dependence on developmental TCR signaling.
Natural killer cell education results in prolonged survival through a cytokine receptor balance mediated mechanism (P1435)
Authors: Felices Martin , Lenvik Todd , Ankarlo Dave , Curtsinger Julie , Cooley Sarah , Tolar Jakub , Blazar Bruce , Anderson Stephen , Miller Jeffrey 
Published: January 2023

Abstract
Although NK cell differentiation leads to acquisition of function, little is known about how cell survival affects the NK cell repertoire. We found that Killer-cell immunoglobulin-like receptor positive (KIR+) NK cells, which exhibit enhanced cytotoxicity and cytokine production, survived better than KIR- NK cells under serum starving conditions. Accordingly, KIR+ NK cells have more anti-apoptotic proteins, while they express less Fas and FasL. As NK cell survival and homeostasis are mediated by cytokine signaling, we studied expression of IL-15 and IL-2 signaling components: the shared IL-2Rβ and the common γ chain, and the individual “private” components, IL-2Rα for IL-2 and IL-15Rα for IL-15. IL-2Rα was expressed at lower levels on KIR+ NK cells while the common γ chain was higher in these cells. Interestingly, IL-15 has been shown to induce survival signals better than IL-2. We hypothesized that IL-15Rα and IL-2Rα compete for common components to influence survival and tested this by overexpressing or knocking down IL-2Rα. IL-2Rα expression negatively influenced IL-15 mediated survival confirming our hypothesis. Since no crosslinking or IL-2 signals were present in this experiment, this role differs from that of IL-2 in AICD, supporting our competition mechanism. Finally, we found a direct correlation between NK cell education/licensing and enhanced survival. In the clinic these findings could lead to better NK cell persistence required for clinical efficacy.
Luminal microbial sensing regulates colonic goblet cell associated antigen passages through the activation of p42/p44 MAPK and the EGFR (P3154)
Authors: Knoop Kathryn , McDonald Keely , Miller Mark , Newberry Rodney 
Published: January 2023

Abstract
The intestinal tract is full of proteins ranging in antigenicity from inert food antigens to potential pathogens and bacterial toxins. A major focus of mucosal immunology is to better understand how these antigens are acquired and introduced to the immune system. Using intravital two-photon imaging we recently showed that in conventionally housed mice, small intestinal goblet cells, but not colonic goblet cells, act as a route of antigen delivery to intestinal dendritic cells by forming goblet cell-associated antigen passages (GAPs), in a mechanism linked to goblet cell secretion. In contrast to conventionally housed mice, we observed that GAPs were present in the colon of germfree mice, mice given antibiotics, and, surprisingly, neonatal mice. We found that the activation of p44/p42 MAPK and EGFR in colonic goblet cells correlated with the presence of luminal microbiota and the ability to sense the flora via TLRs. Inhibition of either p42/p44 MAPK or EGFR allowed GAP formation to occur in the colon of SPF housed mice, and colonic GAPs in germfree mice were blocked by activation of the EGFR. These observations indicate that sensing the enteric flora inhibits colonic GAP formation. This regulation of antigen delivery would allow sampling of luminal antigens when the environment is permissive, such as in the homeostatic small intestine, but would prevent sampling luminal antigens in a hostile environment, such as the high microbial load in the colon or during infection.
Microbiota down-regulates intestinal DC expression of miR-107 which targets IL-23p19 and IL-10RB (P3158)
Authors: Yao Suxia , Xue Xiaochang , Cao Anthony , Evans Heather , Elson Charles , Cong Yingzi 
Published: January 2023

Abstract
Commensal flora plays important roles in development of mucosal immune system and maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we observed that lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice compared to that in normal control mice. miR-107 was preferably expressed by intestinal epithelial cells and dendritic cells (DC). Consistent with high levels of proinflammatory cytokines and more commensal bacteria translocation across the intestinal epithelium in colitic mice, we demonstrated that IL-6, IFNγ and TNFα down-regulated, whereas TGFβ promoted miR-107 expression. In addition, microbiota down-regulated miR-107 expression in both mouse and human DC in MyD88- and NF-κB-dependent manners. We identified IL-23p19 and IL-10RB, the key molecules in innate immune responses to commensal bacteria, as direct targets of miR-107, in that ectopic expression of miR-107 specifically repressed the expression of IL-23p19 and IL-10RB. We concluded that microbiota regulation of miR-107 could serve as an important pathway for maintaining the intestinal homeostasis to microbiota.
Characterization of allergen-specific T-cell responses in a humanized mouse model relying on a human mugwort-specific T-cell receptor and HLA-DR1 (P6208)
Authors: Neunkirchner Alina , Mager Lukas , Wojta-Stremayr Daniela , Schmetterer Klaus , Leb-Reichl Victoria , Rosloniec Edward , Ronald Naumann , Jahn-Schmid Beatrice , Bohle Barbara , Pickl Winfried 
Published: January 2023

Abstract
While mice expressing an I-Ad restricted murine T-cell receptor (TCR) specific for chicken ovalbumin (Ova) are widely used to mimic allergic diseases induced by aeroallergens in vivo, implications for human allergic diseases remain questionable, since Ova is not a human-relevant aeroallergen. We here created double tg mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1. In tg mice ninety percent of peripheral blood CD4+ T lymphocytes expressed the Art v 1-specific TCR, while CD14+ monocytes and B220+ B lymphocytes revealed HLA-DR1 expression. Splenocytes of tg mice specifically proliferated upon incubation with the human-relevant immuno-dominant Art v 125-36 peptide or whole Art v 1 protein when compared to control mice. In vivo, after allergen-specific sensitization, only tg mice showed enhanced airway-hyperreactivity (AHR) as assessed by methacholine or allergen-specific challenge. Three consecutive exposures to nebulized allergen were found to be sufficient for induction of AHR. The effects on bronchoalveolar lavage fluids, lung histology and specific antibody titers will be described and discussed. Humanized allergy models are amenable to the evaluation of human-relevant allergen formulations or cellular intervention protocols and will thus contribute to the further understanding of allergic diseases and their cure.
Granulysin requires heparin sulphate proteoglycan for the uptake by target cells and moves to mitochondria matrix in CHO cells. (P1320)
Authors: Okada Satoshi , Natsuya Ryosuke , Nishida Jiro 
Published: January 2023

Abstract
9 kD granulysin (GLN) is one of cytotoxic molecules released by cytotoxic T cells and NK cells. GLN-derived peptide G8 which covers a part of second and third helixes (Q23-R51) is much more potent than GLN in killing target cells. It was previously shown that GLN and G8 enter Jurkat cells and cause mitochondria-mediated cell death. In Jurkat cells, GLN and G8 are diffusely localized inside cells. In the present study, we evaluated the uptake of G8 by CHO, PgsA-745 and PgsD-677 cells. PgsA-745 and PgsD-677 are mutants of CHO and incapable of producing proteoglycan (PG) and heparin sulphate PG (HSPG), respectively. The uptake of G8 was about ten times less efficient in PgsD-677 and PgsA-745, strongly suggesting that HSPG is pivotal in the uptake of GLN and G8 by target cells. Surprisingly, both GLN and G8 eventually became localized in mitochondria in 45 minutes after the uptake by CHO cells although they did not enter the nucleus. A study using isolated mitochondria in sucrose buffer and proteinase K showed that GLN and G8 enter mitochondria matrix. However, replacement of one the cysteine residue of GLN and G8 with serine inhibited the transfer to inter-membrane space and matrix. Our study may help further understanding the mechanism by which internalized protein is sorted to organelles. Furthermore, GLN-derived peptide may provide a clue in developing a therapeutic tool to transfer nucleotides or peptides into mitochondria.
DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells (P1323)
Authors: Mulrooney Tiernan , Hurley Carolyn 
Published: January 2023

Abstract
Killer Ig-like receptors (KIR) aid in the regulation of natural killer (NK) cell activity. In this study, the effect of the interaction between the two domain stimulatory KIR (KIR2DS) and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when co-transfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12 specific siRNA. Treatment of the KIR2DS and DAP12 transfected cells with either cycloheximide or Brefeldin A repressed KIR2DS surface expression revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS indicating the interaction likely initiates within the endoplasmic reticulum. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.
Identification of gene targets of PLZF in NKT cells (P1289)
Authors: Newsome Courtni , Sant'Angelo Derek 
Published: January 2023

Abstract
The transcription factor PLZF (promyelocytic leukemia zinc finger) is required for the development of NKT cell effector functions. Studies described here investigate the gene targets of PLZF in NKT cells. The aims are: 1) to determine the gene expression in wild type and PLZF deficient NKT cells, and 2) to define direct genetic targets of PLZF. A hurdle to carrying out gene expression analysis has been the need to use CD1d tetramers to identify invariant NKT cells. Crosslinking of the TCR, as a result of binding the tetramer, can cause changes in signaling and gene expression. Therefore, for aim 1, we used a BAC eGFP/PLZF reporter mouse system. In this mouse, PLZF expressing cells are labeled with eGFP, which obviates the need for CD1d tetramer. Importantly, GFP expression is retained in NKT cells that develop in PLZF deficient mice. Therefore, “untouched” NKT cells can be collected from PLZF expressing and deficient mice. RNA was isolated from these cells and subjected to microarray analysis. Microarray analysis indicated many gene changes, of which only some will be direct targets of PLZF. Aim 2 will be to carry out Chromatin Immunoprecipitation assays followed by high-throughput sequencing. We will utilize an NKT cell hybridoma cell line that expresses PLZF and has retained the capacity to produce IL-4 and IFN-γ. By combining the data, we expect to define genes that are regulated by PLZF and are necessary for the development of NKT cells.
Molecular characterization of an alternative model of inflammation (P1347)
Authors: Myers Michael , Deaver Christine , Peters Sharla , Swaim Heidi , Yancy Haile 
Published: January 2023

Abstract
Non-steroidal anti-inflammatory drugs (NSAIDs) for food-producing animals such as swine need to control systemic and local inflammatory responses. However, there are no NSAIDs approved to control inflammation in swine due to an absence of qualified measures of efficacy needed to substantiate those claims. The absence of systems and approaches to substantiate a claim to control inflammation has led to the total absence of approved products for swine. The goal of this study was to develop an in vitro carrageenan model of local inflammation for use in studies of novel non-steroidal anti-inflammatory drug efficacy, and to determine if there are common pathways/biomarkers for local and systemic inflammatory responses. We evaluated carrageenan induced stimulation and its capacity to affect previously identified systemic inflammatory biomarkers in swine, using swine whole blood cultures. We found that lambda carrageenan increases the production of IL-8 and MCP-1 via ELISA. Conversely, TNF-α and SAA demonstrated a no change in production. Additionally, Phosphokinase-3, Monocyte Chemotactic Protein-1, Alveolar Macrophage Chemotactic -II showed increased gene expression. These results suggest lambda carrageenan impacts ubiquitination on immune cells. These results also demonstrate that carrageenan initiates an inflammatory response that differs from that induced by endotoxin in terms of the pathways and gene products altered as a consequence of carrageenan exposure.
Rapamycin-resistant mTORC1 restrains dendritic cell B7-H1 expression that requires IL-1β to enhance regulatory T cell induction (P1349)
Authors: Rosborough Brian , Raïch-Regué Dàlia , Matta Benjamin , Lee Keunwook , Boothby Mark , Turnquist Heth , Thomson Angus 
Published: January 2023

Abstract
Introduction: The mammalian Target of Rapamycin (mTOR) is a central regulator of dendritic cell (DC) function that performs the catalytic activity of mTOR complex (mTORC)1 and 2. mTORC2 functions independently from mTORC1 and is resistant to inhibition by rapamycin (RAPA); however, mTORC1 has both RAPA-sensitive and -resistant outputs. Our goal was to ascertain the role of RAPA-resistant mTOR in DC. Methods: WT C57BL/6 or B7-H1-/- bone marrow-derived DC were generated with the addition of RAPA or ATP-competitive mTOR inhibitor, which blocks all mTOR signaling. DC lacking rictor, an mTORC2-specific subunit, were generated from conditional rictor KO mice. DC induction of regulatory T cells (Treg) was determined in MLR, using BALB/c CD4+CD25- T cell responders. Results and Conclusion: RAPA and mTORC2 deletion reduced DC B7-H1 expression, but ATP-competitive mTOR inhibitors enhanced B7-H1 expression. Augmented B7-H1 expression was blocked by STAT3 inhibition and correlated with reduced expression of the STAT3 negative regulator, SOCS3. DC exposed to ATP-competitive mTOR inhibitors increased Treg induction, which was dependent on DC B7-H1. IL-1β neutralization additionally reduced Treg induction by B7-H1-/- ATP-competitive mTOR inhibitor-exposed DC, suggesting that IL-1β and B7-H1 act additively to promote Treg induction by these DC. These findings establish a RAPA-resistant mTORC1 pathway that acts through SOCS3 and STAT3 to regulate DC B7-H1 expression and Treg induction.
Transcription factor Ets2 negatively regulates IL-6 production in innate immunity (P1351)
Authors: An Huazhang , Wen Mingyue 
Published: January 2023

Abstract
Transcription factor Ets2 was reported to increase production of proinflammatory cytokine TNF-α. We investigated the regulatory role of Ets2 in LPS-induced inflammatory cytokine production. Upon LPS stimulation, Ets2 was translocated into nucleus. ERK1/2 and p38 inhibitors inhibited LPS-induced Ets2 nuclear translocation, suggesting that Ets2 was activated by ERK1/2 and p38 pathways. Although Ets2 knockdown decreased LPS-induced TNF-α production, it increased LPS-induced IL-6 production in mouse peritoneal macrophages. Similarly, Ets2 knockdown also increased VSV infection-induced IL-6 production in mouse peritoneal macrophages. Upon LPS stimulation, Ets2 was induced to bind the promoter of IL-6 gene, suggesting that Ets2 functions as a negative transcription factor in IL-6 gene transcription in innate immunity.
Sex differences in IRF5 levels and implications for pDC IFNα response (P1365)
Authors: Griesbeck Morgane , Tomezsko Phillip , Doyle Erin , Sirignano Michael , Diefenbach Thomas , Altfeld Marcus , Chang J. Judy 
Published: January 2023

Abstract
Background: Differential sex-dependent immune activation is a common feature in both HIV-1 and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) derived from females have been shown to produce significantly more IFNα in response to HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary T cell activation. Given their crucial role in the regulation of IFNα production by pDCs, we investigated the impact of the interferon regulatory factors (IRF) on the observed differences. Methods: Fresh PBMCs were isolated from healthy donors, and the quantity of IRF7 and IRF5 were measured by flow cytometry directly ex vivo and after TLR7 stimulation. IRF5 nuclear translocation following TLR7 stimulation was confirmed by confocal microscopy and quantified using TissueFAXS. Results: Baseline levels of IRF5 in pDCs were found to be significantly higher in females than in males (p&lt;0.05). No differences between males and females in baseline levels of IRF7 in pDCs were observed. Importantly, levels of IRF5 were significantly higher in pDCs secreting IFNα compared to the non-secreting pDCs after TLR7 stimulation (p&lt;0.05). Conclusion: These data indicate that sex differences in IRF5 levels might account for the described higher IFNα production upon TLR7 stimulation in females, providing new insights into the mechanisms underlying higher inflammation observed in females for both HIV-1 infection as well as autoimmune diseases.
Methionine sulfoxide reductase A attenuates heme oxygenase-1 induction through inhibition of Nrf2 activation (P1377)
Authors: Kim Hwa-Young , Kim Jung-Yeon , Choi Seung Hee , Lee Eujin , Kang Young Jin 
Published: January 2023

Abstract
Methionine sulfoxide reductase A (MsrA) functions as a protein repair enzyme by catalyzing the stereospecific reduction of methionine-S-sulfoxide to methionine. We previously identified that MsrA deficiency inhibits normal cell growth via activation of the p53-p21 pathway. In this study, we report a critical role of MsrA in expression of heme oxygenase-1 (HO-1), a highly inducible enzyme that has an anti-proliferative effect mediated by up-regulation of p21. Down-regulation of MsrA induced HO-1 expression in mammalian cells with increased p21 levels, but MsrA overexpression did not affect HO-1 expression. MsrA depletion activated Nrf2 by increasing its expression and nuclear translocation. Nrf2 activation was associated with increased reactive oxygen species production. MsrA overexpression in MsrA-depleted cells led to the reduction of increased HO-1 expression, and suppressed nuclear accumulation of Nrf2. Taken together, the data suggest that MsrA attenuates HO-1 induction by inhibiting Nrf2 activation.
MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal regulatory protein α. (P1303)
Authors: Zen Ke , Zhu Dihan , Li Limin 
Published: January 2023

Abstract
Myeloid cell-specific signal regulatory protein α (SIRPα) is an essential signaling molecule that modulates leukocyte inflammatory responses. The regulation of SIRPα expression and its dynamic changes in leukocytes, however, remains incompletely understood. Here we study the role of microRNAs in regulating SIRPα expression at posttranscriptional level and macrophage inflammatory responses. We identified SIRPα as a common target gene of miR-17, miR-20a and miR-106a. During SIRPα induction in promyelocytic cells by retinoic acid or phorbol-12-myristate-13-acetate, the levels of these three miRNAs were all reduced and their downreulation occurred through suppression of the c-Myc signaling pathway. All miR-17, miR-20a and miR-106a specifically bound to the same seed sequence within the SIRPα 3’-untranslated region and correlate inversely with SIRPα protein level in various cells. In macrophages, up-regulation of miR-17, miR-20a and miR-106a by lipopolysaccharide (LPS) served as the mechanism underlying LPS-induced SIRPα reduction and macrophage activation. Both in vitro and in vivo assays demonstrate that miR-17, miR-20a and miR-106a regulate macrophage infiltration, phagocytosis and proinflammatory cytokine secretion through targeting SIRPα. In summary, these findings demonstrate for the first time that miR-17, miR-20a and miR-106a regulate SIRPα production and SIRPα-mediated macrophage inflammatory responses in a redundant fashion.
The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of CD74 (P1108)
Authors: Beisner Daniel , Langerak Petra , Parker Albert , Dahlberg Carol , Otero Francella , Poirot Laurent , Barnes Whitney , Young Michael , Niessen Sherry , Wiltshire Tim , Bodendorf Ursula , Martoglio Bruno , Cooke Michael 
Published: January 2023

Abstract
B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. Here we report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis (RIP) of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.