bluuebunny/crossref_metadata_2025_split huggingface dataset:
The public T cell repertoire is continuously generated over the lifespan of the individual. (64.11)
Authors: Kondo Kayo , Maverakis Emanual 
Published: January 2023

Abstract
It has been demonstrated that syngeneic mice can respond to antigenic challenge using the same “public” T cell repertoire. This is at odds with the apparent low probability of the same TCR being generated in multiple individuals. To investigate this, two public T cell responses were studied. One response was generated from a germline VDJ recombination lacking N additions and was specific to a foreign antigen, hen egg white lysozyme p106-116, in the context of I-Ed. The second was an autoreactive public response specific to the N-terminus of myelin basic protein, MBP:Ac1-9, presented in the context of I-Au. These two model systems yielded the same results. Both young and very old animals responded to antigenic challenge using the same public T cell response. To determine if this public response could be generated over time, naïve mice underwent CD4+ T cell depletion with antibody and then were allowed to reconstitute their CD4+ T cells. Following antigen priming these mice responded using the characteristic public T cell repertoire. In another experiment, naïve animals underwent total immune ablation via lethal irradiation followed by immune reconstitution via syngeneic bone marrow transplantation. Again, identical public T cell expansions were noted following antigen challenge. Together, these results demonstrate that naive mice, regardless of age, respond to antigen using similar T cells and that these can be continuously regenerated.
Regulation of gene expression during thymocyte differentiation by Protein Phosphatase 1 (64.16)
Authors: Dovat Sinisa , Li Zhanjun , Payne Kimberly , Song Chunhua 
Published: January 2023

Abstract
The goal of our research is to define signal transduction pathways that regulate thymocyte differentiation. We have previously reported that Protein Phosphatase 1 (PP1) directly dephosphorylates and regulates the activity of the Ikaros protein - a master regulator of lymphocyte differentiation (Popescu et al. J Biol Chem 2009 284:13869). We extended our studies to determine whether PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of the terminal deoxynucleotidyl transferase (TdT) gene during thymocyte differentiation. An Ikaros mutant that is unable to interact with PP1 (IK 465/7A), and that is subsequently hyperphosphorylated, was compared to the wild type Ikaros for DNA-binding affinity toward the TdT promoter and for ability to repress transcription of the TdT gene using a luciferase reporter assay. Chromatin immunoprecipitation assays showed that the loss of PP1-mediated dephosphorylation of Ikaros leads to decreased Ikaros DNA-binding affinity for the D’ regulatory element of the TdT promoter in vivo. The IK 465/7A mutant also failed to repress transcription of TdT in the luciferase reporter assay, suggesting that Ikaros’ interaction with PP1 and subsequent dephosphorylation is essential for its function as repressor of TdT transcription. Results suggest that the PP1 signal transduction pathway regulates thymocyte differentiation by controlling the function of Ikaros and transcriptional repression of the TdT gene.
Visualizing acute negative selection in the thymus (64.3)
Authors: Dzhagalov Ivan , Melichar Heather , Robey Ellen 
Published: January 2023

Abstract
Negative selection in the thymus ensures that only the functional and not the useless or harmful thymocytes will be exported into the circulation. The behavior of cells undergoing negative selection has been visualized only recently. However, the steady-state model used did not allow the precise reconstruction of the timeline of the events. To circumvent this drawback, we developed a model for visualizing synchronous negative selection following peptide injection, while avoiding the induction of non-specific cell death due to peripheral T cell activation. We observed that transgenic thymocytes slowed down significantly after the injection of their cognate peptide. This occurred very early, possibly within minutes of the injection. In addition, we established the time-course of cell activation and apoptosis induction in the system. Our data suggest that while stopping of the thymocytes is an event immediately downstream of the T cell receptor signaling, committing to death takes much longer time and most likely requires integration of multiple stimuli.
Identifying molecules that “fine-tune” T cell antigen receptor signaling during thymocyte selection. (64.5)
Authors: Pinkhasov Julia , Lee Jan , El-Khoury Dalal , Lee LiQi , Hwang SuJin , Lesourne Renaud , Zhang Yongqing , Weng Nan-Ping , Love Paul 
Published: January 2023

Abstract
The prevailing view of thymocyte selection is that the fate of developing cells is dictated exclusively by the affinity/avidity of the expressed TCR for self-MHC/self-ligand. We hypothesize that there is significant plasticity in the signaling processes that control T cell development and that the ability to ‘fine-tune’ the TCR signaling response, through the regulated expression of molecules that positively or negatively impact the TCR signaling response operates to maximize the TCR repertoire. One example of a ‘fine-tuning’ molecule that was previously characterized is the cell surface protein CD5. We found that CD5 is an inhibitory tuning receptor, and its surface expression is directly and proportionately regulated by the affinity of the TCR for selecting ligand. In order to identify additional candidates for ‘TCR-tuning’, we employed a TCR transgenic experimental system where TCR expression was fixed on all developing thymocytes and where the affinity of the positively selecting TCR/self-MHC/self-ligand interaction could be altered by changing the MHC haplotype. Thymocytes that successfully underwent selection under conditions of relatively low or relatively high avidity interactions were analyzed by microarray. These experiments identified CD5 as well as several additional potential tuning molecules not previously thought to play a role in ‘TCR-tuning’. Our results provide important new insights into the genetic and molecular processes that control thymocyte selection.
Diethylstilbestrol and the methoxychlor metabolite, HPTE, alter viability and differentiation of embryonic C57BL/6 thymocytes (64.9)
Authors: Mourad Faraj , Escalante Priscilla , Sortillon Don , Leung Liu Lucie , Pierce Katy , Broussard Christine 
Published: January 2023

Abstract
Endocrine disrupting chemicals (EDCs) with estrogenic activity are ubiquitous in the environment. Studies of select EDCs have suggested that human exposure alters the immune system leading to increases in infection and autoimmune disease frequency. How these alterations occur is unclear. Exogenous and pregnancy-related increases in estrogen have been shown to induce atrophy and alterations in thymocyte development, suggesting a potential mechanism for EDC alteration of the immune system. Diethylstilbestrol (DES), a synthetic estrogen once prescribed to pregnant women, and HPTE, the metabolite of the once widely applied pesticide Methoxychlor (a replacement for DDT) are EDCs of interest. Using an in vitro assay, our research probed whether HPTE and DES alter thymocyte development in embryonic C57Bl/6 mice and at what concentrations. We also examined the mechanism by which DES and HPTE affect thymocyte differentiation. Our preliminary data show a dose-dependent decrease in thymocyte viability and alterations in thymocyte population distribution. Over the range of doses tested, increasing DES and HPTE concentration led to significant decreases in CD4+ intermediate and DP T-cells. Furthermore, we designed a time course experiment to probe the cell death mechanism induced by DES and HPTE using Annexin V and PI staining. Our work suggests that one mechanism of action of DES and HPTE on embryonic thymocytes is induction of cell death through apoptosis.
Quantifying immunoreactivity of a dual-labeled imaging agent by near-infrared flow cytometry (65.17)
Authors: Hall Mary , Aldrich Melissa , Azhdarinia Ali , Robinson Holly , Haviland David , Sevick-Muraca Eva 
Published: January 2023

Abstract
Criteria for agent batch release used in clinical diagnostics include validation of biological activity. The Lindmo assay (LA) has been used conventionally to quantify immunoreactivity of radiolabeled antibodies (Ab), however, this method does not provide consistently accurate results. We developed and validated a near-infrared (NIR) flow cytometry (FC) assay for quantifying immunoreactivity of a dual-labeled Ab for use in human and small animal nuclear and NIR fluorescence imaging. Ab specific for epithelial cell adhesion molecule (EpCAM) was conjugated to DOTA-NHS-ester (DOTA), labeled with IRDye 800CW, and further labeled with 64Copper (Cu) or nonradioactive Cu prior to reacting with human prostate cancer (PC3) cells for testing by LA or FC, respectively. Immunoreactivity was 76% and 73%, respectively, by LA and FC. Ab without Cu yielded 79% immunoreactivity by FC. No significant differences were found among these levels of activity (P &gt; 0.05), indicating that NIR FC is an equivalent method for measuring immunoreactivity and that Cu-labeling does not significantly affect the Ab’s ability to bind to its target. Immunoreactivity was significantly reduced when increasing dye-to-protein (D/P) ratios were tested by NIR FC. A similar trend was observed in the LA. Nonspecific, control Ab did not bind PC3 cells. In summary, NIR FC is a validated method with specificity and sensitivity equivalent to the LA for quantifying immunoreactivity of NIR-labeled agents.
Development of an high efficiency peripheral B lymphocyte immortalization tool. (65.18)
Authors: Razanajaona-Doll Diane , Zhu Ying , Clear Olivier , Laurant Stéphanie , Pin Jean-Jacques 
Published: January 2023

Abstract
The low proportion of peripheral B cells (5-10% of PBMC) and their poor capacity to enter cell cycle limited the study of the overall B repertoire. A prerequisite is an in vitro amplification and immortalization of B cells. To date, B cell immortalization was mainly relying on the CD40 system and/or EBV transformation; however, these methods targeted only 1% and 1/106 cells of the starting B cells, respectively. Dendritics company has both optimized previous methods and developed new tools aiming at increasing immortalization efficiency: the irradiated fibroblasts of the CD40 system were replaced by inert reagents, the EBV transformation capacity was significantly increased, a novel monoclonal antibody inducing B cell proliferation was generated, a specific B cell growth booster reagent was developed and a 2 stimulation/transformation combined culture steps, starting with PBMC, was set up. With the Dendritics process, up to 20% of the starting B cell population was transformed within less than 2 months. As proofs of concept, cultures were prolonged, allowing the isolation of very rare specific antibody secreting cells such as anti-HPA5b or anti-Chikungunya virus B cells. Genomic, transcriptomic and proteomic studies of the overall B cell repertoire will be facilitated by this innovative tool. This new Dendritics process was packaged as a transferable, ready to use, and easy to handle product labeled Human Blood B Booster kit which marketing is currently under study.
In situ peptide-MHC-II tetramer staining of antigen-specific CD4+ T cells in tissues (65.37)
Authors: Dileepan Thamotharampillai , Kim Hyeon , Moon James , Linehan Jonathan , Jenkins Marc , Cleary Patrick , Skinner Pamela 
Published: January 2023

Abstract
The invention of peptide-MHC-tetramer (pMHC) technology to label antigen-specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. Although in situ staining of antigen specific CD8+ T cells with pMHCI tetramers has been well characterized and permits visualization and characterization of antigen-specific CD8+ T cells relative to other cell types in stained undisrupted tissue sections, similar staining of antigen specific CD4+ T cells in situ with pMHCII tetramers has been elusive. Thus far, detection of antigen specific CD4+ T cells with pMHCII tetramers has been limited to cells isolated from blood or disrupted tissues that are analyzed by flow cytometry. In this report, we describe our development of an in situ pMHCII tetramer staining method to directly visualize antigen-specific CD4+ T cells in tissues. We used an infectious model system, where Group A streptococcus (GAS) expressing a surrogate peptide (2W) fused to the M1 protein was used to inoculate C57BL/6 mice. NALT from these mice were used in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. Parallel flow cytometric analysis was performed to validate the findings of in situ staining. The results showed pMHCII tetramer stained cells in GAS-2W but not GAS infected mice. Thus, we have developed a pMHCII in situ staining method, and this novel methodology can be used to study the localization, abundance and phenotype of antigen specific CD4+ T cells in tissues.
A novel Quantum Dot/peptide-MHC probe to study recognition of self by T cells (65.38)
Authors: Anikeeva Nadia , Gakamsky Dimitry , Sykulev Yuri 
Published: January 2023

Abstract
We have assembled soluble pMHC proteins on fluorescent nanoparticles, Quantum Dots (QD), to probe membrane associated events and downstream signaling followed by TCR engagement on live T cells. A unique architecture of QD/pMHC probe let us to demonstrate CD8-dependent strong binding of non-cognate QD/pMHC that did not induce any detectable Ca2+ flux in T cells unless at least one pMHC complex per dot was cognate [1]. In contrast, cognate and non-cognate pMHC assembled on Streptavidin scaffold into the tetramer did not show detectable binding to CTL and Ca2+ mobilization. Consistent with these data, we have found very different distribution of the separating distances between pMHC ligands in the two different oligomers as established by analysis of Foster Resonance Energy Transfer (FRET) between MHC bound peptides labeled with either donor or acceptor fluorophores. We have also shown that close proximity between pMHC on QD but not the valency of the probe endows QD/pMHC with unique properties. We propose to utilize this novel tool to study responses of autoimmune T cells recognizing self pMHC as well as the nature of tonic signal required for maintenance of quiescent T cells. [1]. Anikeeva et al. (2006) PNAS, 103:16846
Profiling antigen-specific T cell phenotypes with heavy metal labeled peptide-Major Histocompatibility Complex tetramers and single cell mass-spectroscopy (CyTOF) (65.40)
Authors: Newell Evan , Sigal Natalia , Bendall Sean , Nolan Garry , Davis Mark 
Published: January 2023

Abstract
The direct detection of antigen-specific T cells using fluorescently tagged pMHC-tetramers is widely used in basic and clinical immunology, allowing the unperturbed assessment of T cell phenotypes by concurrent staining with surface and/or intracellular markers. However, the number of T cell specificities and phenotypic markers that can be assessed in a single sample is limited by fluorescence spectral overlap. We recently devised a way to label pMHC tetramers with heavy metals that allows for detection using single-cell mass spectroscopy (Cytometry via Time of Flight - CyTOF). The ability to use 30+ different isotopic labels in this system greatly extends the number of T cell specificities, phenotypic markers and intracellular cytokines that can be simultaneously evaluated. Using this approach, we find that CD8+ T cells from human blood display a much broader diversity of phenotypes and cytokine production capabilities than previously appreciated. Although the phenotypes for different pathogen specific T cells are diverse, cells specific for a given antigen display a distinct subset of phenotypes, which differs depending on the antigen specificity and the nature of the antigen. High-content single-cell phenotypic profiling of antigen-specific T cells should allow for more accurate predictions of T cell response outcomes. Application of CyTOF-based combinatorial tetramer staining, as we previously described for fluorescent labels, will extend the utility of this approach.
Performance of Luminex and MSD high-sensitivity multiplex cytokine assays across multiple lots and laboratories (65.6)
Authors: Breen Elizabeth , Reynolds Sandra , Cox Christopher , Jacobson Lisa , Magpantay Larry , Mulder Candice , Dibben Oliver , Margolick Joseph , Bream Jay , Sambrano Elise , Martínez-Maza Otoniel , Sinclair Elizabeth , Borrow Persephone , Landay Alan , Rinaldo Charles , Norris Phillip 
Published: January 2023

Abstract
Circulating cytokine levels can provide valuable information about immune status, but often require high-sensitivity assays. Multiplex assays hold great promise, especially when limited sample volumes are available. Four high-sensitivity cytokine multiplex assays (BioRad, BioSource/Invitrogen, Linco/Millipore, Meso Scale Discovery [MSD]) were evaluated for ability to detect circulating levels of cytokines, and for lot and laboratory variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women’s Interagency HIV Study (WIHS), and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples IL6, IL8, IL10, and TNFα were detectable with at least three kits, while IL1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (p&lt;0.001) between laboratories and/or lots with all kits. The kits generally detected similar cytokine patterns over the onset of HIV viremia. This multi-site comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma levels of cytokines, and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories, but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.
CD28: a novel therapeutic target for the treatment of multiple myeloma (66.10)
Authors: Murray Megan , Nair Jayakumar , Lee Kelvin 
Published: January 2023

Abstract
Multiple myeloma (MM) is a plasma cell neoplasm which resides in the bone marrow (BM) and is critically dependent upon the BM microenvironment (BMM) for its survival. It has been observed that CD28 is expressed on MM cells and that expression levels correlate with worsening progression and worse prognosis. We have also observed that ligation of CD28 protects MM from chemotherapy. Taken together, these data imply that CD28 is a critical pro-survival molecule for the MM. The ligands for CD28, CD80 and CD86, are expressed on antigen-presenting cells, particularly dendritic cells (DC). We have observed that DCs infiltrate myelomatous portions of patient BM biopsies. We therefore hypothesize that it is the DC in the BMM which is providing the pro-survival signal to the MM via a CD28:CD80/86 interaction. Co-culture of murine or human DC with MM increased MM survival in melphalan. We then treated MM/DC co-cultures with CTLA4-Ig (which blocks CD80/86) and cultured them with or without melphalan. In the presence of melphalan, DC alone increased the survival of MM cells from 30% to 75%; however, addition of CTLA4-Ig completely abrogated this protection. We observed similar results using a CD28 blocking antibody rather than CTLA4-Ig. Mechanistically, such pro-survival signals often involve regulation of apoptosis. We have preliminary data which suggest that CD28 ligation decreases the levels of the pro-apoptotic molecule Bim, suggesting a mechanism for CD28-mediated survival.
Human tumor-infiltrating Th17 cells have the capacity to differentiate into IFN-gamma+ and FOXP3+ T cells with potent suppressive function (66.27)
Authors: Peng Guangyong , Ye Jian , Su Xinming , Hsueh Eddy , Zhang Yanping , Koenig Joyce , Hoft Daniel 
Published: January 2023

Abstract
Accumulating evidence suggests that T-helper 17 (Th17) cells and regulatory T (Treg) cells may exhibit development plasticity, and that CD4+ Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs). We showed that Th17 clones generated from TILs can differentiate into IFN-gamma-producing and FOXP3+ cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells (PBMCs). We further demonstrated that T-cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage-specific transcriptional factor and cytokine genes. In addition to expressing IFN-gamma and FOXP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Tregs. We further demonstrated that the Th17-derived Tregs, unlike naturally occurring CD4+CD25+ Tregs, did not reconvert back into Th17 cells even under Th17-biasing cytokine conditions. These results provide the critical evidence that human tumor-infiltrating Th17 cells can differentiate into Tregs and indicate a substantial developmental plasticity of Th17 cells.
Tumor cell Programmed Death Ligand-1-mediated T cell suppression is overcome by co-expression of CD80 (66.29)
Authors: Haile Samuel , Bosch Jacobus , Zeender Annette , Srivastava Minu , Rodel Sabine , Wolf Julie , Ksander Bruce , Ostrand-Rosenberg Suzanne 
Published: January 2023

Abstract
Programmed death ligand 1 (PDL1 or B7-H1) is expressed constitutively or is induced by IFNγ on the cell surface of most human cancer cells and acts as a “molecular shield” by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma) we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1- mediated immune suppression by tumor cells and restores the ability of tumor cells to activate tumor-specific T lymphocytes. Mechanistically, CD80 mediates its effects through its extracellular domain which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating anti-tumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.