bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a novel, murine, peritoneal leukocyte recruitment model to overcome the lack of C5a receptor cross reactivity between human and mice (60.5)
Authors: MacLeod Heather , Serpa Megan , Zhang Qin , McAllister Kathryn 
Published: January 2023

Abstract
The ability to test target-specific small molecules in vivo can be hampered by a lack of species cross reactivity. In the absence of knock-in animals, novel methods to evaluate in vivo efficacy by a target-specific mechanism can be developed. In vivo models of peritoneal recruitment of leukocytes are often used to evaluate cell migration and inflammation by different pro-inflammatory stimuli. An example of a potent chemoattractant capable of inducing migration of inflammatory cells into the peritoneum is the anaphylatoxin C5a which binds to the C5a receptor (C5aR) on many cell types. The aim of this study was to develop a novel mouse peritoneal leukocyte recruitment model using C5a as the pro-inflammatory stimulus and a cell line expressing the human C5aR. Mouse B lymphocytes (L1.2 cells) were stably transfected to express human C5aR and were fluorescently labeled with Qdot® nanocrystals. Labeled cells were injected into mice i.v. followed by an i.p. injection of human C5a. At multiple time points following C5a administration, the peritoneum was lavaged and the recovered cells subjected to flow cytometry to quantify the number of the labeled cells that migrated to the peritoneum. Our data show that cells expressing the human C5aR are recruited to the peritoneum 24 hours following human C5a administration. This assay demonstrates a novel mouse model that can detect human receptor-driven chemotaxis in mice.
PD-1:PD-1 ligand interactions suppress B-1b cell responses to T cell-independent type 2 antigens (61.7)
Authors: Haas Karen 
Published: January 2023

Abstract
B-1b cells play a key role in producing antibodies (Ab) in response to T cell-independent type 2 (TI-2) antigens (Ags). However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, the role of the PD-1 immunoinhibitory receptor in regulating B-1b cell responses to TI-2 Ags was investigated in normal mice. Following TNP-Ficoll immunization, Ag-specific B-1b cells transiently expressed PD-1, which suppresses BCR-induced B-1b cell proliferation when ligated in vitro. In vivo administration of an Ab that blocks PD-1 from interacting with its ligands during the first 3 days following TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and differentiation relative to mice that had received control Ab. Serum levels of Ag-specific IgG3, the major IgG isotype produced in response to TI-2 Ags, remained significantly elevated in PD-1 mAb-treated mice for at least 6 weeks post-immunization. Remarkably, PD-1 mAb administration during the first few days following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3, but not IgM, secreting cells at both early (day 5) and late (6 week) timepoints. Thus, PD-1:PDL interactions occurring shortly after initial TI-2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG3-producing bone marrow and spleen cells.
Structural and functional studies on PD-1: from crystals to mice (63.17)
Authors: Lazar-Molnar Eszter , Ramagopal Udupi , Almo Steven , Nathenson Stanley 
Published: January 2023

Abstract
Programmed death-1 (PD-1) inhibits T and B cell responses upon binding to its ligands, PD-L1 and PD-L2, and has crucial role in maintaining peripheral tolerance. We have recently reported the crystal structure of the complex between PD-1 and PD-L2. Performing structure-based mutagenesis studies, high-affinity, and PD-L2-specific PD-1 mutants were identified. High-resolution crystal structures of the high-affinity PD-1 mutant with PD-L1 and PD-L2, respectively, provide the atomic details of the tighter receptor-ligand binding. The crystal structure of the complex between the PD-L2-specific PD-1 mutant and PD-L2 provides the atomic details of the selective interaction of this mutant with PD-L2. Soluble Ig fusion proteins of the two PD-1 mutants were designed to modulate the PD-1 pathway for immunotherapy. Finally, these mutations were introduced into mice by generating “knock-in” mouse strains, which provide novel animal models to study the role of PD-1 in immunity. Current work is focused on analyzing the functional effects of these mutations in PD-1 mediated immune responses. In summary, our data demonstrate the value of structural studies in developing novel immunotherapy and animal models for in vivo studies.
Stat3 activation plays a role in mTEC development (64.20)
Authors: Jain Manju , Lomada Dakshayani , Reddy Madhava , Kang Rhea , DiGiovanni John , Richie Ellen 
Published: January 2023

Abstract
The thymic medulla is central to the establishment of central tolerance. Development of medullary thymic epitthelial cells (mTECs) in the fetal thymus requires lymphoid tissue inducer cells, whereas in the adult, signals from positively selected thymocytes engage different components of NF-κB signaling pathway to regulate the formation of an optimal medullary stromal niche. Thus, mTEC development is impaired in RAG-1-/- and RAG-2-/- mice due to the early block in thymocyte differentiation. We find that introduction of a constitutively active Stat3 transgene regulated by a keratin 5 promoter (K5.Stat3C) in Rag 2-/- mice rescues mTEC development bypassing the requirement for positively selected thymocytes. Although K5.Stat3C mice exhibit thymus hypoplasia, the medullary compartment is expanded. Immunohistochemical analysis demonstrates K5+K14+ and UEA+ mTECs. However, FACS analysis reveals an increase in immature mTECs based on low expression of MHC class II and CD80 markers, as well as a higher mTEC to cTEC ratio. The results suggest a partial block in the terminal differentiation of the expanded immature mTEC population. Conditional deletion of Stat3 in mTECs, achieved by crossing Stat3fl/fl mice with K5.Cre mice, leads to a decrease in mTEC cellularity and a reduced mTEC to cTEC ratio. These findings implicate a novel role for Stat3 signaling in molecular regulation of the thymic mTEC compartment.
FlowCAP: critical assessment of flow cytometry population identification methods (65.2)
Authors: Scheuermann Richard , Aghaeepour Nima , Brinkman Ryan , Gottardo Raphael , Mosmann Tim , Qian Yu , Schoenfeld Jill 
Published: January 2023

Abstract
Traditional methods for flow cytometry (FCM) data processing have relied on manual gating of cell events to define cell populations for statistical analysis. However, this approach has become increasingly problematic with the advances in instrumentation and reagents that allow for evaluation of larger numbers of cell properties. Recently several groups have developed computational methods for automatically identifying cell populations in multidimensional FCM data obviating the need for manual gating. In order to compare the performance of these methods, the Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) competition was established to make available a common set of FCM data together with manual gating results for comparative analysis. The first FlowCAP competition included 5 different data sets with data from 12-30 samples containing 5000-100,000 cell events stained with 3-10 fluorochrome markers. We received 36 analysis result submissions from 14 research groups. Both model fitting and density-based clustering methods were found to perform well in comparison with manual gating by domain experts as the gold standard, using statistical tests to measure and rank algorithm performance. In addition, combining results using a computational “ensemble” method was found to outperform all individual methods. These results suggest that, in the near future, automated computational methods may become an integral part of routine FCM data analysis.
Optimiser™ microplate and OptiMax™ reagent systems enable femtogram/ml level sensitivity employing standard ELISA equipment and protocols (65.32)
Authors: Kai Junhai , Santiago Nelson , Puntambekar Aniruddha , Lee Se Hwan , Sehy David , Durnell Randy , Schultheis Ron , Han Jungyoup , Ahn Chong 
Published: January 2023

Abstract
Optimiser™ Microfluidic ELISA Plates dramatically enhance ELISA sensitivity through the incorporation of microfluidic channels into a standard ELISA plate configuration. Validation studies with Optimiser™ microfluidic ELISA plates demonstrate the Optimiser™’s ability to utilize available sandwich ELISA reagents, equipment, and protocols to attain unparalleled levels of sensitivity. Limits of Detection and Limits of Quantification are demonstrated for a variety of assays in the range of 30-100 fg/ml, representing a 20-1000 fold improvement over the same assays run in conventional ELISA plates. OptiMax™ ELISA Reagent protocols on Optimiser™ Microfluidic ELISA Plates employ the similar reagents, but abbreviated assay steps because, unlike conventional sandwich ELISAs, no traditional plate washing is required. Optimiser™ Microfluidic ELISA Plates are compatible with existing pipetting systems (manual or automated) and ELISA plate readers. Furthermore, the surface area to volume ratio of each microfluidic reaction chamber represents a 50-fold increase when compared to the well of a conventional 96-well immunoassay plate. The improvement in surface to volume ratio increases binding kinetics dramatically, enabling assays to be run in less than half the time of traditional ELISA assays and providing researchers with the flexibility to use dramatically smaller sample and reagent volumes (e.g., 5 ul).
The application and demonstration of a user-friendly, antibody-based biosensor for environmental monitoring (65.33)
Authors: Spier Candace , Vadas George , Unger Michael , Kaattari Stephen 
Published: January 2023

Abstract
Monoclonal antibodies (mAbs) are a valuable resource in both clinical and environmental health. In clinical applications, mAbs have been extensively used as a diagnostic tool for the rapid evaluation of specific diseases and in targeted anti-cancer therapies, among others. Recently, mAbs have been employed in environmental monitoring applications for the detection of oil, pesticides, and many other pollutants. MAb-based biosensors have become an excellent means to achieve rapid, sensitive monitoring of a specified target. Here I describe the application of a mAb employed in a portable, user-friendly, sensor format for the detection and quantification of polycyclic aromatic hydrocarbons (PAHs) in environmental samples. PAHs are carcinogenic and found widespread in the environment. One application of this biosensor included the monitoring of estuarine water during the dredging of PAH-contaminated sediments, revealing a plume of PAHs emanating from the dredge site. The second application was the near real-time monitoring of PAHs in stormwater run-off. The ease of technology transfer was demonstrated by the operation of the biosensor by high school students in two science programs: VIMS/NASA Governor’s School Summer Internship and NSF’s GK-12 Program. Overall, these opportunities demonstrated the utility of this biosensor to rapidly and precisely quantify PAHs in environmental samples. Moreover, this biosensor technology was successfully operated by non-professionals.
Novel methods for simultaneous assessment of CD4 and CD8 T cell mitochondrial potential changes (65.39)
Authors: Clor Julie , Tyagarajan Kamala 
Published: January 2023

Abstract
The study of mitochondrial membrane potential (ΔΨm) changes in immune cells is important in understanding immune cell apoptosis, mechanism of diseases, and evaluating the action of compounds and drugs that cause mitochondrial stress. Simultaneous identification of T cells along with ΔΨm changes is challenging due to the broad fluorescence of traditional mitochondrial dyes, such as JC-1, in multiple channels. Here we demonstrate results from a novel approach for the simultaneous evaluation of ΔΨm in CD4 & CD8 T cells using simplified cocktails and a mitochondrial responsive dye followed by microcapillary flow cytometry. PBMC’s were treated with a range of compounds including staurosporine and gambogic acid and evaluated for mitochondrial membrane potential changes and annexin V binding on T cells. Our results demonstrate that with most treatments, a greater % of cells exhibit earlier and more sensitive changes in ΔΨm, than Annexin V binding. Further, some donors treated with staurosporine, a significantly greater % of CD8 T cells demonstrated changes in ΔΨm vs. CD4 T cells. Gambogic acid demonstrated a dramatic reduction of ΔΨm and showed increased apoptosis on both CD4 and CD8 T cells simultaneously. The ability to measure mitochondrial membrane potential changes in CD4 and CD8 T cell simultaneously can allow more sensitive identification of immune cell perturbation and provide deeper insights into the mechanism of immune cell apoptosis in disease and drug discovery.
SMAD3 deficiency causes immune dysregulation, colitis and colon tumorigenesis (66.13)
Authors: Bommireddy Ramireddy , Ball Corbie , Cherukuri Durga , Nasr Michael , Doetschman Tom 
Published: January 2023

Abstract
Introduction: TGFβ plays important role in T cell development, differentiation, tolerance and autoimmune diseases. However, recent studies have shown an important role for TGF-β1 and β3 in pathogenic helper T-cell (Th17) differentiation in the presence of IL-1β, IL-6 and IL-23. SMAD3 is mediates TGFβ signaling in many cell types including lymphocytes. Smad3 /- mice develop inflammation-induced colon cancer upon Helicobacter hepaticus infection suggesting that TGFβ1 signaling through SMAD3 is essential to prevent inflammation and progression to colon cancer. In this study we investigated the role of SMAD3 in inflammation using mice deficient in Smad3. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines are determined by ELISA, CBA and MSD methods. Cells are stained with antibodies for CD4, CD25, CD62L and intracellular FOXP3 and Helios. Results: Smad3-/- mice exhibit inflammatory response upon infection with commensal bacterium H. hepaticus. We find that while SMAD3 signaling is important for inducible Treg cell generation and for their maintenance, deficiency of SMAD3 increases IL-6 and IL-17 levels suggesting a regulatory role for SMAD3 in Th17 cell differentiation. Conclusions: While SMAD3 signaling is important for iTreg cell generation and maintenance, it is not required for Th17 cell differentiation. In addition, we find that SMAD3 signaling negatively regulates Th17 differentiation through induction of FOXP3.
Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice (66.33)
Authors: Liu Jinlin , Wang Honglin 
Published: January 2023

Abstract
Background: Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. Regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown. Methodology/Principal Findings: CRC was either induced by MNU and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. Recruitment of FoxP3GFP+ Treg-cells was assessed using the IVIS Imaging System. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Macrophages produced a large amount of CCL20, the sole ligand of CCR6 when co-cultured with CRC cells. Injection of recombinant CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice. Conclusions/Significance: TAMs recruit CCR6+ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.
CD73: an emerging target for cancer immunotherapy (66.7)
Authors: Wang Long , Fan Jie , Thompson Linda , Shin Tahiro , Curiel Tyler , Zhang Bin 
Published: January 2023

Abstract
CD73 is over-expressed in many types of human and mouse cancers, and is implicated in the control of tumor progression. However, the exact contribution of tumor and host CD73 expression to tumor growth remains unknown to date. We here show that host CD73 promotes tumor growth in a T cell-dependent manner, and that the optimal anti-tumor effect of CD73 blockade required inhibiting both tumor and host CD73. Notably, CD73 on bone marrow (BM)-derived cells (CD4+CD25+ T regulatory cells), but not on nonhematopoietic cells, inhibited systemic anti-tumor T cell expansion and effector function. On the other hand, CD73 on non BM-derived cells (presumably endothelial cells) limited anti-tumor T cell homing to local tumors. Thus, CD73 has distinct roles in hematopoietic and nonhematopoietic cells in regulating anti-tumor T cell responses. In addition, pharmacological blockade of CD73 using its selective inhibitor α,β-methylene adenosine 5′-diphosphate or an anti-CD73 monoclonal antibody, inhibited tumor growth and completely restored efficacy of adoptive T cell therapy. These findings suggest that both tumor and host CD73 cooperatively protects tumors from incoming anti-tumor T cells, and point to a novel cancer immunotherapy strategy by targeting CD73.
Characterization of the mechanism by which L-asparaginase II of Salmonella enterica serovar Typhimurium induces T cell inhibition (99.12)
Authors: Torres AnnMarie , Kullas Amy , van der Velden Adrianus 
Published: January 2023

Abstract
T cells play a key role in controlling and clearing infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). However, T cell-mediated immunity against S. Typhimurium takes months to develop and has been described as slow and inefficient. Previously, we have shown that S. Typhimurium have a direct inhibitory effect on T cells, down-modulating expression of the beta chain of the T cell receptor (TCR-beta) and inhibiting T cell proliferation. Furthermore, we have shown that a soluble, proteinaceous factor produced or induced by S. Typhimurium is responsible for this inhibition. Most recently, we have found that STM3106 is required for S. Typhimurium to inhibit T cells and that L-asparaginase II, which is encoded by STM3106, is present in the supernatants of T cells cultured in the presence of S. Typhimurium. In addition, we have found that L-asparaginase II produced by S. Typhimurium is both necessary and sufficient for the inhibition of T cells. With further characterization, this research should provide new insight into the mechanism by which S. Typhimurium inhibit T cells, avoiding immune clearance.
TRAIL-dependent suppression of naïve and memory CD8 T cell responses to secondary bacterial infection after sepsis (99.14)
Authors: Griffith Thomas , Gurung Prajwal , Rai Deepa , Badovinac Vladimir 
Published: January 2023

Abstract
Sepsis is the leading cause of death in most ICUs, and the death of septic patients usually does not result from the initial septic event but rather from subsequent nosocomial infections. Patients who survive severe sepsis often display severely compromised immune function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and the induction of sepsis-induced immune suppression. Using a clinically-relevant “two-hit” sepsis model that better reflects the delayed mortality seen during sepsis due to secondary infection, we have extended this work to investigate sepsis-induced immune suppression of naïve and memory Ag-specific CD8 T cell responses to an experimental heterologous bacterial (LM-OVA) infection. CLP-treated WT mice had a reduced ability to clear LM-OVA vs. sham-treated WT mice, which was paralleled by suppressed T cell responses in the CLP-treated mice. In contrast, bacterial clearance and T cell responses were similar in CLP- and sham-treated TRAIL-/- and DR5-/- mice. Administration of a blocking anti-TRAIL mAb to CLP-treated WT mice led to bacterial clearance and T cell responses like those seen in sham-treated mice. These data further suggest the importance of TRAIL in the induction of sepsis-induced immune suppression and that TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.
HIV triggers interleukin 21-mediated induction of granzyme B-secreting B cells with antiviral properties (105.18)
Authors: Kaltenmeier Christof , Dahlke Karen , Gawanbacht Ali , Hofmann Stefanie , Beyer Thamara , Härter Georg , Grüner Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd 
Published: January 2023

Abstract
Certain lymphocyte subsets including plasmacytoid dendritic cells and regulatory T cells can secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB in response to interleukin (IL) 21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we demonstrate for the first time, that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21. We further demonstrate that such T cells induce GrB in co-cultured B cells in an IL-21-dependent fashion. In support of these data, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of B cells (36.2 ± 12.9%) from patients infected with HIV, but not normal B cells, express GrB. Importantly, co-culture of HIV-infected CD4+ T cells with GrB+ B cells resulted in GrB transfer, and strongly suppressed both, proliferation of T cells and virus replication as indicated by significantly reduced p24 levels. The observed effects were enhanced by IL-21, and reduced by GrB inhibition. In summary, we demonstrate that HIV induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV.