bluuebunny/crossref_metadata_2025_split huggingface dataset:
B lymphocytes secrete FasL+ exosomes capable of inducing apoptosis in CD4+ T cells (61.6)
Authors: Klinker Matthew , Reed Tamra , Shaw Campbell , Fox David , Holoshitz Joseph , Lundy Steven 
Published: January 2023

Abstract
B lymphocytes possess immunosuppressive properties such as secretion of the anti-inflammatory cytokine interleukin-10 and the expression of apoptosis-inducing molecules. We have previously identified a murine splenic B cell population which expresses the death-inducing molecule Fas Ligand (FasL) and showed that these cells can directly kill antigen-specific T cells. Upon screening a panel of human B cell-derived lymphoblastoid cell lines (LCLs) for expression of FasL, we found that a high frequency of LCLs constitutively produce intracellular FasL as determined by immunoblot and intracellular staining. Secretory exosomes (&lt;100nm diameter microvesicles) were isolated from LCL culture supernatants and it was found that all lines tested secreted exosomes containing MHCII and FasL. The presence of MHCII+FasL+ exosomes was confirmed by incubating exosomes with anti-MHCII-coated beads and showing the presence of FasL on MHCII+ exosome-coated beads by flow cytometry. Exosomes derived from LCLs were capable of inducing significant levels of apoptosis in autologous CD4+ T cells in the presence of superantigen. Finally, preliminary data suggest that splenic murine exosomes bound to anti-IgM-coated beads stain positive for the presence of FasL. These data indicate a novel role for B cells as producers of immunosuppressive exosomes and suggest that LCL-derived MHCII+FasL+ exosomes may represent a novel therapy for T cell-mediated autoimmunity.
The V(D)J recombination machinery is organized as large complexes anchored on the nuclear matrix (62.1)
Authors: Xie Wanqin , Lange Miles D , Hong Sang Yong , Huang Lin , Xue Weihua , Ding Shi-jian , Jones Dallas , Yu Zhihong , He Ti , Yu Yangsheng , Burrows Peter D. , Lu Runqing , Swanson Patrick C. , Su Kaihong , Zhang Zhixin 
Published: January 2023

Abstract
V(D)J recombination provides the foundation for adaptive immunity. This complicated process involves the cleavage of specific recombination signal sequences (RSS) by the recombination activating gene products, RAG1 and RAG2, followed by repair of the DNA double-stranded breaks (DSBs) by the non-homologous end joining (NHEJ) factors, including DNA dependent protein kinase (DNA-PK), Artemis, Ku86, Ku70, DNA ligase IV, XRCC4, XLF, and HMGB1. Currently, it is not clear how these factors are organized inside of the nuclei for recombination. Accumulating studies indicate that the multiple-component DNA replication and RNA transcription machineries are organized as large complexes on the nuclear matrix (NM). Here we show that RAG1, RAG2, and many NHEJ factors are associated with the NM, where they appear to form large complexes and are distributed closely at discrete foci. Ongoing RSS cleavage mainly occurs on the NM in developing B, T lineage cells and HEK293 cells with an artificial recombination system; Purified NMs containing RAG and NHEJ proteins possess the entire enzymatically active recombination system to cleave RSS substrates and generate coding joints in vitro. These results support a novel model that the V(D)J recombination machinery is organized as large complexes anchored on the NM to conduct recombination.
Profiling the pattern of human immunoglobulin SHM and CSR in B memory cells via high-throughput sequencing (62.11)
Authors: Han Jian , Sanders Catherine , Yang Qunying , Byrne-Steele Miranda , Pan Wenjing , Wang Chunlin 
Published: January 2023

Abstract
One of the defining features of the adaptive immune system is that it adapts its response during an infection to improve its recognition of the pathogen. The adaptive feature is best embodied in the genetic alteration mechanism of somatic hypermutation (SHM) and class-switch recombination (CSR) as they are only activated to increase recognition to a previously encountered pathogen. Due to the limited number of sequence data, the hypermutation pattern such as mutation rate, and the distribution of mutation spots remains poorly understood. Using the combination of arm-PCR and high-throughput sequencing, we have determined about one million cDNA sequences of human immunoglobulin genes in B memory cells isolated from the peripheral blood from two subjects at various time points after administration of the 2009-2010 flu vaccine. We identified the location of the hypermutations, the distribution of mutation rate, mutation patterns of different classes of immunoglobulin, and the impact on primer design for immunoglobulin genes.
Modulation of biallelic TCRα expression in influenza-specific CD8 T cells (62.6)
Authors: Dash Pradyot , McClaren Jennifer , Oguin III Thomas , Morris Melissa , Doherty Peter , Thomas Paul 
Published: January 2023

Abstract
Our understanding of complementary T cell receptor (TCR)α and β chain utilization is very limited for pathogen-specific immunity. This problem has been solved with the development of a novel, multiplex nested RT-PCR method for the simultaneous amplification of TCRα and TCRβ chain transcripts from single cells. Using this technique, we found surprisingly, 25% of influenza-epitope-specific CD8+ cytotoxic T lymphocytes recovered directly from the pneumonic lung expressed a dominant, non-productively rearranged TCRα transcript, an effect that is never seen for TCRβ. T cells with these out-of-frame TCRα mRNAs also express an alternate, in-frame TCRα, while approximately10% have two productive TCRα transcripts. The proportion of cells with bi-allelic transcription increases over the course of a response; stayed high in the memory compartment but decreased in the recall response. However these bi-allelic cells appear to expand with low efficiency. Further, cells with out-of-frame expression appear to be specifically excluded from the recall response, a finding that has implications for immune memory and for autoimmunity.
AID-dependent gene expression in activated B cells (62.9)
Authors: Unniraman Shyam , Arya Rahul , Gregory Briana , Agrawal Anurodh , Carico Zachary , Upton Dana 
Published: January 2023

Abstract
Activation induced deaminase (AID) initiates both somatic hypermutation (SHM) and class switch recombination (CSR) by deaminating cytosines to uracils in the immunoglobulin genes. AID is also mistargeted to the transcribed portion of several other genes in activated B cells. In contrast, during early stages of development, AID appears to play a very different role in some non-B cells. Specifically, it targets methylated cytosines in CpG islands leading to demethlylation and induction of silent genes. Notably, both antibody diversification (SHM and CSR) as well as demethylation rely on the same deamination activity of AID but the choice of co-factors determines the genes and portions within the genes that are deaminated. Therefore, we wanted to test whether AID-mediated demethylation could occur in activated B cells as well. Towards this end, we compared gene expression profiles between wild-type and AID-deficient B cells, and identified several genes that are induced in an AID-dependent manner. Here, we present the role played by some of these genes in regulating antibody diversification and mature B cell development. These results reveal a non-canonical function of AID by activating silent genes in B cells and have implications both in normal B cell function as well as tumorigenesis.
Modulation of T-dependent recall humoral immunity by Staphylococcal enterotoxin B (63.12)
Authors: Lee William , Janik David 
Published: January 2023

Abstract
Some microbial toxins are superantigens (SAg) that target large numbers of CD4 T cells. We previously showed that the SAg SEB can lead to memory CD4 T cell inactivation (anergy). Given that SAgs target TCRs independently of their peptide-binding properties, memory T cell help for unrelated antigens might be impaired. Here we show that SEB prevented T-dependent recall humoral immunity. In a mouse model, ovalbumin (OVA)-specific, Vβ8-expressing memory CD4 T cells provided help to hapten-specific B cells after challenge with the T-dependent antigen, TNP-OVA. Only when specific memory T cells were present did the mice make TNP-specific secondary (IgG) humoral responses; otherwise a primary (IgM) humoral response was observed. Exposure of the immune mice to SEB prior to challenge with TNP-OVA led to a loss of IgG production, indicating impaired memory helper function. Aberrant recall immunity was long-lived, as even 3 weeks after exposure to SEB, immunization with TNP-OVA resulted in IgM, but not IgG, anti-TNP Ab. Comparable numbers of carrier-specific T cells were present in SEB-treated and control mice, suggesting that anergic memory cells were maintained in vivo. Memory cell anergy occurred due to Fyn kinase-dependent negative signaling, and Fyn-deficient memory cells promoted IgG production even after SEB exposure. Hence, bacterial toxins can target memory helper cells to eliminate recall responses to unrelated antigens, suggesting a potential need for re-vaccination.
PD-L1 costimulation of the CD8 T cell response to Listeria monocytogenes infection (63.16)
Authors: Xu Daqi , Fu Han-Hsuan , Obar Joshua , Park Jang-June , Tamada Koji , Lefrancois Leo 
Published: January 2023

Abstract
It is well known that programmed death ligand-1 (PD-L1) transduces a suppressive signal, increasing the activation threshold of T cells via its counterreceptors PD1 and CD80 (B7.1). On the other hand, other studies have suggested a possible role of PD-L1 as costimulator. In this study, we analyzed the role of PD-L1 in the primary CD8 T cell response to Listeria monocytogenes infection. PD-L1 blockade using the monoclonal antibody (mAb) 10F.9G2 impaired antigen-specific CD8 effector T cell expansion, particularly limiting short-lived effector cell differentiation. Simultaneous CD4 T cell depletion and anti-PD-L1 blockade exacerbated the effect of either treatment alone demonstrating that PD-L1 provides important costimulation even in the absence of CD4 T cells. Most importantly, specific blockade of PD-L1 binding to CD80 (43H12 mAb) or PD-1 (RPM1-14 mAb) during Listeria monocytogenes infection did not recapitulate blockade with 10F.9G2. These data suggest a 3rd unknown binding partner for PD-L1 that is responsible for positive costimulation of antigen-specific CD8 effector T cells. We conclude that PD-L1 plays an important role in antigen-specific CD8 effector T cells during Listeria monocytogenes infection through a novel costimulatory pathway.
Cre-mediated excision of exon 8 of the Smad4 gene specifically in T cells is not associated with enhanced CD4+ T cell activation and differentiation (63.19)
Authors: Lv Ming , Zhang Jiyan 
Published: January 2023

Abstract
Numerous studies have revealed the critical importance of TGF-beta signaling on T cells. As a central mediator of TGF-beta signaling, Smad4 (homolog 4 of mothers against decapentaplegic, Drosophila) has been demonstrated to play essential roles during vertebrate development, tumorigenesis and the maintenance of gastrointestinal tract homeostasis. Recently, it has been reported that Cre-mediated excision of exon 1 of the Smad4 gene specifically in T cells has little role in the in vivo production of IFN-gamma, IL-10, IL-17, IL-17F, and IL-22 after KLH immunization of 4-week-old mice. Here, we report that Cre-mediated excision of exon 8 of the Smad4 gene specifically in T cells showed no significant effect on T cell proliferation and T cell development. In the peripheral tissues, the number of Treg cells, CD4+ T cell activation and differentiation were similar between aged wild-type and Smad4-deficient mice. Thus, our data are consistent with the previous report and suggest that Smad4 is indispensable for the maintenance of CD4+ T cell homeostasis.
Targeting the PD-1/PD-L1 complex with drug-like small molecules to induce T-cell tolerance (63.26)
Authors: Fuhrman Christopher , Gubernick David , Brusko Todd , Ostrov David 
Published: January 2023

Abstract
The interactions between T-cell costimulatory molecules and cognate receptors expressed on antigen presenting cells result in important signaling events that control T cell homeostasis and tolerance. The costimulatory ligand Protein Cell Death Ligand 1 (PD-L1) binds T-cell expressed Protein Cell Death-1 (PD-1) and promotes tolerogenic signaling and regulates T-cell responsiveness. Blocking PD-1/PD-L1 interactions inhibits tolerogenic signaling thus accelerating the onset of autoimmunity in several in vivo model systems. In contrast, PD-1 agonists are expected to promote T-cell tolerance in autoimmune settings. We developed a novel strategy for the development of tolerogenic PD-1 agonists. A large library of drug-like small molecules were targeted to the PD-1/PD-L1 interface via virtual high throughput screening. We coupled the grid-based energy scoring function with the AMBER molecular mechanics scoring function of Dock 6.4 to prioritize compounds for screening. The highest-ranking compounds are under investigation to characterize their effects on the association and dissociation kinetics of the PD-1/PD-L1 complex. The discovery of small molecules that increase the duration of PD1/PD-L1 contact (dwell time) and promote T-cell tolerance, may offer an invaluable tool in exploring T-cell homeostasis and regulating autoimmunity in vivo.
The generation of a B7 conditional knockout mouse (63.27)
Authors: Watanabe Masashi , Bhatia Sumeena , Hodes Richard 
Published: January 2023

Abstract
B7.1/B7.2 molecules are ligands for CD28 and CTLA-4, major T cell co-stimulatory and co-inhibitory molecules, respectively. B7.1/B7.2 molecules are essential not only for eliciting normal immune responses through CD28 mediated T cell activation, but also for maintaining immune homeostasis via CTLA-4 mediated negative regulation of T cell function. B7/CD28 interaction is critical for development and maintenance of specific immune cell populations, including iNKT cells and Foxp3+ regulatory T cells. Although many cell types (i.e., TEC, DC, MΦ, B cell and T cell) express B7 molecules in vivo, the precise role of B7 on each cell type for immune cell development and immune response is not well defined. To address these questions, we here report the generation of a B7 conditional KO mouse. Since B7.1 and B7.2 have largely redundant roles in vivo, we generated B7.1flox BAC Tg mouse on B7.1/B7.2 DKO background (designated as B7flox). The pattern of B7.1 expression in the B7flox mouse is comparable to that of WT mouse regarding both expression levels and tissue distribution. Cre-dependent deletion of B7.1 expression appears to be efficient in the B7flox mouse, and studies are in progress to evaluate tissue specific roles of B7 in immune system development, homeostasis, and response by tissue-specific Cre-mediated deletion.
Preformed CD40L is stored in an intracellular, secretory compartment in Th1, Th2, Th17, and T follicular helper cells as well as CD4 single positive thymocytes and invariant NKT cells, but not in Treg cells (63.5)
Authors: Parker David , Buenafe Abigail , Thauland Timothy , Gardell Jennifer , Bivins-Smith Elizabeth , Jacoby David , Slifka Mark , Koguchi Yoshinobu 
Published: January 2023

Abstract
CD40L is essential for development of adaptive immune responses. It is generally thought that CD40L is made from new mRNA following antigen recognition like other cytokines. However, we and others have shown that some CD4+ effector and memory cells also store preformed CD40L (pCD40L) in an intracellular secretory compartment and mobilize it onto the plasma membrane immediately upon antigenic stimulation, suggesting that CD4+ T cells may use pCD40L to activate APC during brief encounters in vivo that are too short to allow de novo CD40L synthesis. In this study, to understand relevance of pCD40L in different immune responses, we investigated the presence of pCD40L among different effector CD4+ T cells and Treg cells. We show that pCD40L is present in Th1 and follicular helper T cells that develop during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from CNS of experimental autoimmune encephalitis (EAE) animals. pCD40L is absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, but is turned off in Treg cells to avoid compromising their suppressive activity.
PPARγ promotes age-related thymic involution (64.12)
Authors: Youm Yun-Hee , Leff Todd , Dixit Vishwa 
Published: January 2023

Abstract
Aging of the thymus precedes aging of other organs in healthy adults. The decline in naïve T cell production from thymus is related with ectopic adipocyte development within the lympho-stromal thymic zones. The regulators responsible for age-related increase in thymic adiposity and reduced thymopoiesis are poorly understood. Our prior data suggests that age-related elevation of PPARγ expression in aP2 expressing thymic stromal cells in correlated with thymic adiposity. To directly investigate whether PPARγ activation induces thymic involution, we created transgenic mice with constitutive active PPARγ (CA-PPARγ) fusion protein driven by an aP2 promoter. The CA-PPARγ transgene was expressed in thymus and CA-PPARγ fusion protein mimicked the liganded PPARγ receptor. The CA-PPARγ mice had increased thymic adipogenesis, thymic involution, reduced thymopoeisis and restricted T cell receptor (TCR) repertoire diversity. In addition, the ligand activation of PPARγ by insulin sensitizing class of thiazolidinediones drug, rosiglitazone accelerated the PPARγ driven thymic adipogenic cascade and promoted thymic involution andreduced naïve T cells. Our data suggest that activation of PPARγ induces thymic adiposity and accelerates age-related thymic involution.
Activation of the non-classical NF-kB pathway in thymic epithelial cells drives mTEC development in the absence of TCR+ thymocytes (64.14)
Authors: Williams Joy , Jenkinson Rhiannon , Zhang Jingjing , Xie Ping , Bishop Gail , Hollaender Georg , Hodes Richard 
Published: January 2023

Abstract
Activation of the non-classical NF-kB signaling pathway is required for the formation of a normal medullary thymic epithelial cell (mTEC) compartment, critical for tolerizing developing thymocytes. Mice deficient for key signaling molecules in this pathway fail to support normal mTEC expansion and differentiation. Known activators of this signaling pathway include tumor necrosis receptor family members; of these, Rank, CD40 and LTbR are all expressed on mTEC and have been shown to be important at various stages of mTEC organization, expansion and differentiation. The ligands for these receptors: RankL, CD40L and LT are provided by TCR-expressing thymocytes; in the absence of TCR+ thymocytes medullary formation is substantially impaired. To test directly whether activation of the non-classical pathway would be sufficient to support mTEC development independently of receptor signaling we made use of a mouse model in which Traf3, a negative regulator of the non-classical NK-kB pathway, is conditionally deleted only in epithelial cells. In B cells where Traf3 has been deleted, constitutive activation of the non-classical NF-kB pathway has been observed in the absence of receptor signaling.We find that epithelial-cell specific Traf3 null mice develop a robust mTEC compartment even in the absence of TCR+ thymocytes, suggesting that engagement of the non-classical NF-kB pathway in mTEC progenitor cells is the critical signal provided by TCR+ thymocytes to promote mTEC formation.
Use of surface enhanced resonance Raman scattering for 96 well plate based immunodetection of biomolecules (65.24)
Authors: Park Jun , Brewster David , Utzat Christopher , Moskow John , Chernokalskaya Elena 
Published: January 2023

Abstract
We report here a novel detection method in which the intrinsic affinity of proteins toward silver is used to induce Surface Enhanced Resonance Raman scattering (SERRS) of Raman active R6G dye conjugated to detection antibodies. We found a simple method of passive adsorption of silver nanoparticles onto PVDF membrane produces readily useful substrate for inducing the enhancement of Raman signal which could then be used to discern the presence of specific target molecules in quantitative manner. The effect is specific for PVDF membrane, other synthetic membranes such as nitrocellulose fail to produce the same effect. The method can be used for an accurate quantitative measurement of proteins labelled with Raman-active dyes. For example, it can be useful for quantification of detection antibodies eluted off after immunodetection of a target protein and subsequently allowed to adsorb onto silver substrate which can induce enhancement of Raman signal. Using mouse IgG molecules as a model antigen, adsorbed mIgG molecules were detected with limit of detection (3x S/N ratio over blank) down to 0.4ng (3 femtomoles) per well with greater than 1 log linear dynamic range. We also tested this method under sandwich immunodetection targeting c-peptide and show detection limit of 20 picogram (7 femtomoles).
Brighter is better: the introduction of Brilliant Violet™ conjugated antibodies for flow cytometry (65.26)
Authors: Nguyen Dzung , Koh Carolyn , Lundsten Kelly , Jiang Nan , Wormsley Susan , Lay Gene 
Published: January 2023

Abstract
There is always a need for improved and more sensitive fluorophores in research applications. Brilliant Violet 421™ and Brilliant Violet 570™ are novel polymeric, highly sensitive fluorophores excited by the violet laser, for use in flow cytometry. These are ideal for producing antibody conjugates that provide significantly improved signal-to-noise ratios, as much as 12-fold, compared to comparable fluorochromes excited by the violet laser, Pacific Blue™ and Pacific Orange™. These antibodies were fully compatible for use in multi-color flow cytometry applications, maintaining expected phenotypic frequencies, and were also useful for intracellular staining of cytokines. Furthermore, they work well with common commercial cell staining, fixation, and permeabilization solutions, proving their versatility and ease of use. Thus, antibodies conjugated to these novel Brilliant Violet™ fluorophores could prove to be an enabling technology allowing researchers to resolve lowly expressed antigens with greater resolution and accuracy, providing new and expanded capabilities in flow cytometry using the violet laser.