bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative analysis of T Cell Homeostatic Proliferation Reveals Genetic Influence (89.4)
Authors: Li Cheng-Rui (Michael) , Lo David D 
Published: January 2023

Abstract
Conditions of lymphopenia can drive proliferation of residual T cells, a process known as T cell homeostatic proliferation. This situation can be reproduced experimentally by transferring T cells into lymphopenic recipient animals; transferred T cells undergo several rounds of proliferation without antigen stimulation. However, detailed mechanisms have not been fully elucidated. In this study, we developed a mathematical model of the proliferating T cells, considering factors such as apoptosis and proliferation. The modeling results show differential behaviors of T cells between CD4+ and CD8+ subsets. This model predicts that, at least for CD8+ T cells, both the proportion of cells entering cell cycle and the proportion of cells undergoing apoptosis increase with each successive division. These predictions can be confirmed by short-term BrdU labeling and AnnexinV staining. Using same modeling method, we analyzed T cell homeostatic proliferation from different mouse strains, including C57BL/6, BALB/c, CB6F1 and NOD. The results show that homeostatic proliferation is also subject to genetic influence. As homeostatic T cell proliferation has been implicated in precipitating autoimmune diseases, better understanding of the genetic influences over the parameters in this modeling system may yield insights into the behavior of T cells not only in lymphopenic conditions, but also in the context of antigen responses and autoimmune disease susceptibility.
Role of the SH2 domain-containing Phosphatase-1, SHP-1 in Dendritic Cell Activation (89.6)
Authors: Ramachandran Indu Rekha , Levitt Jonathan Michael 
Published: January 2023

Abstract
SHP-1 is an SH2 domain containing phosphatase activated upon recruitment to inhibitory receptors through their immunotyrosine inhibitory motif domains. Once activated, SHP-1 can dephosphorylate a wide range of receptors (e.g. antigen, chemokine and cytokine receptors), as well as their downstream signaling molecules, dampening cellular signals. Although SHP-1 is highly expressed in dendritic cells (DCs), its role in DC signaling and function is not well defined. By blocking SHP-1 activity with shRNA or a phosphatase dead dominant negative mutant (dn), our studies in the murine DC line, D2SC/1 show that SHP-1 inhibited AP-1 activity, upon LPS stimulation but did not affect NFκB activity. This is in contrast to SHP-1 function in RAW264.7 macrophages where it inhibits both AP-1 and NFκB activity after LPS and/or IFNγ treatment. Further, phenotypic characterization of primary bone-marrow derived DCs from SHP-1 deficient motheaten viable (MeV) mice shows, surprisingly, that the MeV DCs have drastically lower and uninducible levels of MHC class II surface expression compared to wild-type DCs. We are currently investigating the mechanisms by which SHP-1 affects class II surface expression in DCs. Taken together, these studies elucidate differences between SHP-1 signaling in DCs and macrophages which may have bearing on their differential ability to modulate antigen presentation and subsequent T cell activation.
Supported by DOD New Investigator Award PC061027 to JL.
High concentrations of CpG-B ODNs induce a novel IFNαβ-specific inhibitory pathway in dendritic cells (89.9)
Authors: Gray Reginald Courtney , Kuchtey John , Harding Clifford V 
Published: January 2023

Abstract
CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) and other immunomodulatory responses (i.e., TNF, IL-12p40, and MHC-I, etc.) by antigen presenting cells. CpG ODNs are currently in preclinical and clinical studies as vaccine adjuvants and therapies for allergies and cancers. Caveats to CpG ODN therapies are the possible provocation of autoimmune diseases such as systemic lupus erythematosis, which may be exacerbated by the induction of CpG-induced IFNαβ. Thus, further studies are needed to examine CpG induction of IFNαβ and to develop agents to inhibit the induction of IFNαβ by CpG ODNs. These studies address a mechanism for inhibiting induction of IFNαβ that could potentially offer an approach for therapeutic inhibition of IFNαβ expression in vivo. Our data suggest that high concentrations of CpG-B ODNs (H/CpG-B) induce an IFNαβ specific inhibitory mechanism as early as 3 h and sustained inhibition as long as 48 h, without similarly affecting TNF and IL-12p40 levels. Additionally, H/CpG-B inhibits MyD88-dependent (CpG-A ODNs) and -independent (Sendai virus) induction of IFNαβ. However, H/CpG-B fails to inhibit IFNαβ expression following induction with recombinant IFNαA protein, suggesting that H/CpG-B IFNαβ inhibition is upstream of the IFNαβ receptor and possibly due to specific obstruction of early type-I IFN induction (IFNα4/β). These data demonstrate a novel IFNαβ specific inhibitory mechanism induced by stimulation of DCs in vitro with H/CpG-B ODNs.
The DNA degradation product uric acid directly promotes T cell autoreactivity (90.1)
Authors: Webb Ryan , Jeffries Matlock , Sawalha Amr H 
Published: January 2023

Abstract
Purpose: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune response. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation.
Methods: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150 and 200 μg/ml. This range of uric acid concentrations covers the normal physiologic serum uric acid concentration as well as a spectrum of hyperuricemic values in human. The expression of T cell activation markers CD25 and CD70 was assessed by flow cytometry. Purified human T cell proliferation response to the various uric acid concentrations was quantified using a colorimetric proliferation assay.
Results: Uric acid treatment resulted in antigen-independent T cell activation as determined by increased expression of CD25 in both CD4+ as well as CD8+ T cells. CD4+ T cells also overexpressed CD70, a co-stimulation molecule important in T cell-dependent B cell activation. In addition, we demonstrate a dose response relationship between T cell proliferation and uric acid concentration in vitro.
Conclusion: The finding that uric acid directly promotes T cell proliferation and activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gout and other uric acid associated diseases.
This work is supported by NIH Grant Number P20-RR015577 and the University of Oklahoma Health Sciences Center.
Temporal development of B and T cell areas in the neonatal rabbit appendix. (90.14)
Authors: Lanning Dennis Keith , Hanson Nicholas B. 
Published: January 2023

Abstract
The rabbit appendix is organized structurally into multiple B cell follicles separated, at their sub-apical regions, by well-defined T cell areas. We found, by RNA in situ hybridization, that the B cell chemoattractant CXCL13 (B-lymphocyte chemoattractant, or BLC) is highly expressed in the B cell follicles. In addition, we found that the T cell chemoattractant CCL21 (secondary lymphoid tissue chemokine, or SLC) is highly expressed in the T cell areas. The localized expression of these chemoattractants likely organizes the B and T cell areas of the appendix. Surprisingly, however, we found that the localized expression of CXCL13 and CCL21 differs dramatically in early neonatal appendix. At two days after birth, CXCL13 expression was confined to the appendix domes and CCL21 expression was localized in a continuous layer significantly beneath the domes. Over subsequent days, as B cells accumulated in the domes and began proliferating, developing follicles gradually distended downward through the layer of CCL21 expression, ultimately generating the characteristic organization of B and T cell areas seen in the adult appendix. This developmental process might be important in establishing an environment supportive of B cell proliferation and antibody repertoire diversification, which occur in the rabbit appendix between 3 and 6 weeks of age.
IgG1, IgG2a, complement and FcR in immune complex disease (90.16)
Authors: Strait Richard , Meyer Cathy , Lambris John , Magotti Paola , Finkelman Fred 
Published: January 2023

Abstract
Immune complexes (IC) contribute to tissue damage in some diseases but prevent it in others. We hypothesize that this contrast occurs because different immunoglobulin (Ig) isotypes involved in the IC interact differently with both stimulatory and inhibitory FcγR and complement (C). To study this we compared IgG1 (which poorly activates C and stimulatory FcγR) to other Ig isotype-mediated IC processing using a model in which wild-type (WT) mice immunized with goat anti-mouse IgD antiserum (GaMD), produce a large, predominantly IgG1 Ab response, produce large amounts of IC, and eliminate them without developing renal disease. In GaMD-immunized IgG1-deficient mice, IgG2a (which activates C and interacts with stimulatory FcγR) predominates and mice develop severe, lethal IC-mediated renal disease that can be prevented by immunization with antigen-specific IgG1. Renal disease also develops in C3-deficient, FcγRIIb-deficient and anti-FcγRII/III mAb treated WT but not in FcRγ-deficient mice immunized with GaMD. Surprisingly, GaMD-immunized IgG1-deficient mice that are also lacking both FcRγ and C3 or lacking FcRγ and treated with a C5aR antagonist still develop renal disease. Together these findings suggest that IgG1 may protect against IC disease through a C- and FcγRIIb-dependent mechanism, while non-IgG1, presumably IgG2a, may mediate IC injury through a FcRγ- and either C3 or C5aR-independent mechanism.
Testosterone attenuates TLR4 expression on murine macrophages (90.5)
Authors: Rettew Jennifer , Marriott Ian 
Published: January 2023

Abstract
Gender has long been known to be a contributory factor in immune responses following infection, with females generally exhibiting greater immune responses than males. These differences have generally been assumed to be a consequence of the actions of reproductive hormones and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study we have investigated the acute effects of testosterone on the level of expression of a key microbial pattern recognition receptor, Toll-like receptor 4 (TLR4), on isolated murine macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in cell surface TLR4 expression within 24 hours. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression. We report that macrophages isolated from orchidectomized animals have significantly less (~30%) TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, this effect was not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data represent a novel means by which reproductive hormones can modulate immune responsiveness to infectious organisms.
Toll-like Receptor 4-Dependent activation of Dendritic cells by heme binding protein from Ascaris suum (90.9)
Authors: MADALA SATISH KUMAR , Bundoc Virgilio Garcia , Hodges Marcus Gomez , Trivedi Shweta , Urban Joseph F. , Keane-Myers Andrea 
Published: January 2023

Abstract
Toll-like receptors (TLRs) recognize specific molecular patterns on invading pathogens leading to the development of host immunity. A number of pathogens including helminths have used pattern recognition by TLRs to modulate host immunity and inflammation to establish a chronic infection. To understand the molecular basis for the TLR-induced tolerance induction observed in individuals infected with helminth parasites, we have established a mouse model combining allergic inflammation and Ascaris suum infection. These in vivo studies demonstrated that concomitant infection with chronic A. suum can inhibit allergic inflammation in response to a non-parasite allergen, ragweed (RW). In the current study, we have purified heme-binding protein (HBP) from pseudocoelomic fluid of A.suum and studied its antigenic and immunomodulatory properties using bone marrow derived dendritic cells. Recognition of HBP by antiserum from pigs infected with Ascaris larvae indicate that HBP is one of the antigenic proteins present in PCF. Furthermore, HBP exposure increases activation marker surface expression on DC including CD40, CD86 and MHC II. This increase in activation marker expression is abrogated in TLR4, but not TLR2, knockout mice implicating that HBP is a TLR4 ligand. Pretreatment of dendritic cells with HBP prior to LPS stimulation results in increased production of IL-10 but not IL-12 compared with DC exposed to LPS alone. These results provide the first evidence for a novel TLR4-mediated response to Ascaris suum HBP resulting in altered APC activation and cytokine production.
The role of LacCer inhibitor in modulation of established Pneumocystis pneumonia (91.1)
Authors: Vuk-Pavlovic Zvezdana , Standing Joseph E , Limper Andrew H 
Published: January 2023

Abstract
Pneumocystis pneumonia is caused, at least partly, by inflammatory action of β-glucan, the major component of Pneumocystis cell wall. Glucan induces release of pro-inflammatory cytokines in alveolar macrophages and lung epithelial cells. While macrophages recognize glucan mainly by dectin receptors, epithelial cells utilize membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer) as glucan-binding molecule. The use of GSL inhibitors in vitro causes reduced NF-κB activation and inhibited IL-8 release in epithelial cells. To assess the impact of LacCer in lung inflammation, we treated Pneumocystis-infected mice with GSL inhibitor PDMP. The animals were immunosuppressed by GK 1.5 antibody, infected with Pneumocystis by intratracheal injection and, starting at 8 weeks post infection, treated with either 150 mg PDMP/kg of body weight - dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). The analysis of sacrificed animals showed that PDMP-treated mice had fewer leukocytes in the bronchoalveolar lavage fluid (BALF) than saline-treated controls with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP-2 and TNF-α (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q-PCR, showed a trend of decreased Pneumocystis-specific DNA copies in PDMP-treated animals. These results suggest that PDMP might have an impact on inflammatory response to Pneumocystis. Other GSL inhibitors are yet to be tested in similar in vivo experiments. This might lead to new options in treatment of Pneumocystis pneumonia.
Supported by NIH RO1 HL62150 and Mayo
Target APC polarization at immunological synapses (IS) in vivo: infected astrocytes polarize towards CTL cells during the brain antiviral immune response. (91.13)
Authors: Lowenstein Pedro R , Barcia Carlos , Liu Chunyan , Wawrowsky Kolja , Kroeger Kurt M , Castro Maria G 
Published: January 2023

Abstract
IS mediate intercellular communication between T cells and APCs. T cells’ structure and membrane proteins are polarized at IS. T cell polarization at the IS consists of a peripheral supramolecular activation cluster (pSMAC), which is high in LFA-1, and low in TCR, and a central SMAC, high in TCR, and low in LFA-1. The MTOC, Golgi and secretory vesicles are also polarized towards the IS. Here we demonstrate that during an anti-viral immune response in the brain, APCs are also polarized. Brain astrocytes were infected with an adenoviral (Ad) vector expressing Thymidine Kinase (TK), a marker of infected cells. 30 days later we immunized systemically against Ads, and 14 days later animals were sacrificed and brains prepared for microscopical analysis. Brain sections were immunostained for TCR, LFA-1, TK, EAAT2, γ-Tubulin, α-Tubulin and GM130. Confocal analysis revealed that target infected astrocytes, normally a non-polarized cell, became completely repolarized. Target astrocytes retract all processes and form a single large protrusion towards the T cell. The MTOC and Golgi are then targeted towards and into the protrusion, and membrane proteins become polarized. These results demonstrate that CTL signaling, potentially mediated through IS, induces profound morphological changes in target APCs.
This work was funded by grants from NINDS/NIH.
Antigen receptor-dependent and -independent regulation of NKT patrolling and activation in liver sinusoids (91.7)
Authors: Velazquez Peter , Cameron Thomas O , Kinjo Yuki , Nagarajan Niranjana , Kronenberg Mitchell , Dustin Michael L 
Published: January 2023

Abstract
Natural killer T (NKT) cells are most abundant in liver and spleen and, recognize lipid antigen presented by CD1d. Utilizing intravital fluorescence microscopy, we previously demonstrated NKT patrolling in the liver sinusoids, meaning cell migration both with and against the blood flow in surveillance of antigen. Here, we report the relationship between patrolling and activation. First, both a Sphingomonas bacteria derived weak agonist, GalA-GSL at 2 μg/g, and a synthetic strong agonist, aGalCer at 10 ng/g, induced serum IL-4, CD69 upregualtion and acute NKT arrest. Surprisingly, a low dose of aGalCer (2 ng/g) did not significantly affect NKT patrolling, although a comparable biological response was elicited. Second, IL-12 and IL-18 synergized to activate NKT cells in the absence of exogenous antigen. These cytokines activated liver NKT cells and induced serum IFN- ? but not IL-4. NKT patrolling was not significantly affected. Therefore, glycolipid antigens can induce NKT cell activation and arrest, although the requirement for arrest may be restricted to weak agonists. Importantly, IL-12 and IL-18 activate NKT cells without affecting NKT patrolling thereby allowing these primed cells to continue to survey tissue for antigen. Current studies are aimed at understanding the hierarchy of antigen receptor versus cytokine driven activation and further defining their relationship to NKT cell patrolling.
CD45RB expression refines delineation of memory CD4 T cells and aids in understanding their development (91.8)
Authors: Caucheteux Stephan M , Younes Souheil A , Paul William E 
Published: January 2023

Abstract
Upon immunization, CD4 T cell stimulation leads to the development of effectors cells and of 2 major subsets of memory cells: central and effector memory cells, the latter often defined by their differential expression of CD62L and CD44. It is still unclear whether central memory cells derive from effectors or are directly induced. Our results have shown that CD44hi cells can be divided into 2 different subtypes depending on their expression level of CD45RB, providing a precise distinction among central and effector memory CD4 T cells. Using these markers, we show that while central memory CD4 T cells are absent in young mice, they are enriched in old and thymectomized animals To understand the properties of the subsets defined by CD45RB and the establishment of the central and effector memory pools, we followed the fate of transferred naïve 5C.C7 T cell receptor transgenic cells, specific for pigeon cytochrome. During the first days following immunization, virtually all the transferred cells adopt the phenotype associated with effector or effector memory cells. After a month, essentially all 5C.C7 cells in lymphoid organs express phenotypes associated with central memory cells, while in the lung effector T cells are still found. Analysis of the mean “life” of these memory cells and their cycling within the lymphoid and the non-lymphoid organs is under investigation.
Taken together, these observations suggest that CD45RB, together with CD44 and CD62L, provides a powerful tool to identify and quantitate naïve, effector and memory cells in lymphoid and non-lymphoid organs.
This work was supported by the Intramural Research Program of the NIAID.
Identification of human B cells with immunoregulatory properties (92.12)
Authors: Tretter Theresa , Venigalla Ram KC , Eckstein Volker , Lorenz Hanns-Martin 
Published: January 2023

Abstract
Regulation of immune responses and tolerance is mediated by a variety of mechanisms and cells. So far the role of B cells in this process is not well known, yet.
Methods: Highly purified CD19+B and CD4+T cells were separated from human PBMC by MACS. B cells were prestimulated with SAC, αIgM/IgG or αCD40, washed and set up in cocultures with freshly isolated autologous CD4+T cells under addition of αCD3+IL-2 or αCD28. CD4+ T cell proliferation was determined after 3–6d by 3H Thymidine incorporation and PKH-26; apoptosis by AnnexinV.
Results: under optimal stimulatory conditions T cell proliferation was inhibited more than 50% in presence of SAC-activated B cells and to a lesser extent by αIgM/IgG stimulated B cells. Enrichment of SAC-activated large CD25+ B cells by FACSsorting enhanced suppression further up to 68%, while the small CD25− population had no significant effect. Separation of B and T cells by cell culture inserts abolished inhibition, suggesting a requirement for direct cell-contact. In addition to growth inhibition, T cell specific cytokine production (IFNγ, IL-10) was downregulated, and a significant proportion of T cells (37% vs 17%) went into apoptosis.
Conclusions: B cells are able to inhibit T cell mediated responses depending on their mode of activation. Further experiments are dealing with the mechanisms of B cell mediated suppression and their pathophysiological impact also in autoimmune diseases.
The Proinflammatory Cytokine Interleukin-32 is expressed in Keratinocytes and Dendritic Cells Obtained from Patients with Chronic Plaque Psoriasis (CPPs) (92.15)
Authors: Hu Ling-Jia , Li Li , Fitzpatrick James E. , Francis Shayla O. , Fujita Mayumi , Takashi Miyatake K. , Shroyer Kenneth R. , Azam Tania K. , Kim Soo-Hyun K. , Dinarello Charles A. , Norris David A. , Edwards Carl K 
Published: January 2023

Abstract
CPPs is characterized by hyperproliferation of keratinocytes (KC) and T-cell (Tc)/DCs activation. IL-32, which induces TNF and activates NFkB and p38 MAPK, is involved in early inflammation. We investigated the role of IL-32 in the pathogenesis of CPPs by IHC, ECL and qRT-PCR analysis. Biopsy tissues were obtained from CPPs patients with active disease (N=20) and healthy human controls (N=15). A mouse Mab against human IL-32 and mouse IgG1 isotype antibody were used. IHC staining of IL-32 was performed on deparaffinized sections of formalin-fixed tissues by an indirect imunoperoxidase method (EnVision, Dako). IL-32 was expressed in basal KC with cytoplasmic granular staining. IL-32 was also expressed in DCs with strong nuclear and cytoplasmic staining in dermis. IL-32 was weakly expressed in the nucleus of KC in healthy human skin. Induction of IL-32 was detected in human KC lysates after stimulation with TNF and IL-1 or cell contact with activated Tc, but not in KC supernatants, indicating IL-32 is not secreted from KC in vitro. ECL/qRT-PCR results showed IL-32 and other CPPs-relevant cytokines (IL-18, IL-20, 22 and 23) were significantly (P&lt;0.05) increased. Our data support the hypothesis that IL-32 is a novel proinflammatory cytokine which is involved in the pathogenesis of CPPs, especially in epidermal regions of the skin where innate immune mechanisms are central to host defense.