bluuebunny/crossref_metadata_2025_split huggingface dataset:
STAT5 is a negative regulator of Bcl6-mediated TFH differentiation (63.2)
Authors: Johnston Robert , Diamond Jeffrey , Crotty Shane 
Published: January 2023

Abstract
Follicular helper T cells (TFH) constitute the CD4+ T cell effector subset that is specialized to provide germinal center B cell help. TFH differentiation is driven by the transcription factor Bcl6, and recent studies have identified cytokine and cell-cell signals that are required for optimal expression of Bcl6. However, the mechanisms underlying the negative regulation of TFH differentiation have been less clear. Here, we show that STAT5 was a key inhibitor of TFH differentiation and function. Constitutive STAT5 signaling in activated CD4+ T cells selectively blocked TFH differentiation, resulting in a collapse of the germinal center reaction. Conversely, STAT5-deficient CD4+ T cells (mature STAT5flox/flox CD4+ T cells transduced with a Cre-expressing retrovirus) rapidly upregulated Bcl6 expression and preferentially differentiated into TFH cells. STAT5 signaling failed to inhibit TFH differentiation in the absence of the transcription factor Blimp-1. We have previously shown that Blimp-1 is a direct repressor of Bcl6-mediated TFH differentiation (Science, 2009). These results demonstrate that STAT5 and Blimp-1 collaborate to negatively regulate TFH differentiation.
Two different pathways contribute to cytotoxicity of mouse cytotoxic T lymphocytes (63.25)
Authors: Dudenhöffer-Pfeifer Monika , Krause Elmar , Hoth Markus , Rettig Jens 
Published: January 2023

Abstract
Cytotoxic T lymphocytes (CTLs) are an important part of the immune system and kill cells which are infected by pathogens like viruses or bacteria (named antigen-presenting-cell or APC). Two different killing mechanisms are being used by CTLs - (1) exocytosis of lytic granules which contain cytotoxic substances and (2) the so called Fas/FasL pathway which relies on agonist/receptor interaction and induces apoptosis in the APC. So far the quantitative contribution of either pathway to the total killing capacity of mouse CTL was not known. To address this question we used a knock out mouse strain (Jinx) which lacks the SNARE associated protein Munc13-4. As a result CTLs from these mice display a severe defect in the secretion of cytotoxic substances, as we could demonstrate by a Granzyme B release assay (reduction of 85% ± 4%) and a FACS based degranulation assay (reduction of 70% ± 9%). Interestingly we found that despite the lytic granule release failure CTLs from Munc13-4 -/- have a remaining killing capacity of about 43%, arguing for a significant contribution of the Fas/FasL pathway. To strengthen this finding we established a single cell assay in which the increase of intracellular Ca2+ in the APC was used as a measure of apoptosis and found that about 50% of killing was induced by the Fas/FasL pathway alone. We conclude from these data that both killing mechanisms are equally important and function synergistically to maintain the cytoxic activity of a mouse CTL.
Embryonic stem cell-derived factors inhibit T effector activation and induce T regulatory cells by modulating PKC-θ activation. (63.29)
Authors: Mohib Kanishka , Al-Khamees Bodour , Allan David , Wang Lisheng 
Published: January 2023

Abstract
Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. However, the mechanisms by which ESC inhibit immune activation remain mostly unknown. We have previously shown that ESC derived factors block dendritic cell maturation and thereby affect T cell activation indirectly. Our ongoing studies have elucidated that ESC derived factors also have a direct effect on T cell activation. In mixed lymphocyte reaction assays, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating IL-10 and TGF-β expression. Also, ESC derived factors suppressed the expression of Th1 transcription factor Tbet, while enhancing the expression of Treg transcription factor Foxp3. Furthermore, PBMC/purified T cells activated with CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Incubation of ESC derived factors with recombinant GST-PKC-θ resulted in the pull-down of four proteins. The purified proteins are currently being analyzed by mass spectrometry. In conclusion, ESC’s are able to directly impact T cell activation and polarization, likely by negatively regulating PKC-θ through potentially novel factors.
Activation of the protein kinase A pathway down regulates spleen tyrosine kinase in human T cells (63.30)
Authors: Ghosh Debjani , Kis-Toth Katalin , Juang Yuang-Taung , Tsokos George 
Published: January 2023

Abstract
Cyclic adenosine monophosphate (cAMP)-mediated activation of the protein kinase A (PKA) pathway has an overall negative impact on T cell proliferation and down regulates or neutralizes signaling pathways of many genes. Spleen tyrosine kinase (SYK) is an important gene expressed differentially during T cell development and functions by phosphorylating downstream effector molecules in both T and B lymphocytes. We report here that PKA inhibitors promoted, whereas cAMP analogues suppressed SYK expression. Overexpression of a catalytic subunit of PKA significantly abrogated SYK protein and mRNA expression as well as the levels of phosphorylated SYK. A complete cAMP response element (CRE) site on SYK promoter was found functional and capable of binding CREMα and CREB as detected by EMSA and chromatin immunoprecipitation. CREMα overexpression reduced, while disruption of the CRE site enhanced the basal promoter activity of SYK. Collectively, the cAMP-mediated PKA pathway functions as the negative regulator of SYK expression by promoting direct binding of a repressor element to the SYK promoter.
Tracking subcellular distribution of fluorescent fusion proteins during thymocyte migration and development (64.2)
Authors: Melichar Heather , Dzhagalov Ivan , Ross Jenny , Herzmark Paul , Robey Ellen 
Published: January 2023

Abstract
T cell receptors (TCRs) on developing thymocytes are screened for appropriate recognition of MHC molecules and self-reactivity before differentiating into CD4, CD8, or other non-conventional T cell subsets prior to exiting the thymus. However, how thymocyte interactions and T cell receptor signaling relate to developmental outcomes during these selection processes is unclear. Dynamic re-localization of proteins within cells can provide information about cell polarity and cell signaling. Here we use fluorescent fusion proteins to identify the subcellular localization of cell polarity proteins (PKCζ) and markers of TCR signaling (LAT) in conjunction with time-lapse two-photon microscopy of thymic lobes. In our current study, we have generated chimeras with bone marrow engineered to express fluorescent fusion proteins, and observe the re-localization of these proteins during thymocyte migration and intercellular interactions. We present a semi-automated approach to quantify fluorescent fusion protein distribution in cells of thymus tissue and show asymmetric distribution of the PKCζ-GFP fusion protein during migration as well as differential accumulation of the LAT-GFP fusion protein during developmental checkpoints. These observations may lead us to an understanding of how the quality of TCR interactions in the thymus affects developmental outcome.
mTOR complex 2 mediates Notch signaling in thymocyte and T-cell leukemia development (64.8)
Authors: Lee Keunwook , Gudapati Prathyusha , Volanakis Emmanuel , Cho Sunghoon , Boothby Mark 
Published: January 2023

Abstract
Mammalian target of rapamycin (mTOR) is an important component of the PI3-kinase (PI3K) pathway that regulates cell growth and proliferation. PI3K signaling cascades can be initiated by activation of Notch, a critical regulator for T lineage commitment and leukemia development. Here we show that mTOR complex 2 (mTORC2) is functionally vital as a downstream target of Notch in thymocytes and T-cell acute lymphoblastic leukemia (T-ALL). Conditional deletion of rictor, an essential component of mTORC2, results in impaired proliferation and differentiation of pre-T cells driven by Notch ligation. Moreover, mTORC2 is a primary kinase for Akt S473 and enhances Akt activity in thymocytes. Furthermore, mTORC2 regulates NF-κB activity, such that constitutively active Akt can restore nuclear NF-κB levels in the rictor-depleted pre-T cells, as well as normalizing Notch-initiated proliferation and differentiation. Strikingly, interruption of mTORC2 significantly delays T-ALL induced by Notch1 activating mutation and suppresses infiltration of leukemic cells into the non-lymphoid organs. Our study provides evidence of a key role of mTORC2 in relaying Notch signaling in thymocyte development, and reveals that mTORC2 promotes Notch-driven leukemia progression.
Monoclonal mouse antibodies against camel immunoglobulins (65.11)
Authors: Holzlöhner Pamela , Schliebs Erik , Maier Natalia , Pfister Friederike , Füner Jonas , Micheel Burkhard , Heilmann Katja 
Published: January 2023

Abstract
Camelid antibodies are of special interest because some of them belong to immunoglobulin subclasses (IgG2 and IgG3) which are build up only by heavy chains. To use such antibodies for practical application we need specific identification tools. Immunoglobulins llama (Lama glama) were isolated by Protein G (IgG3 and IgG1) and Protein A (IgG2) sepharose. Fab Fragments were generated from llama IgG1 by papain digestion. Balb/c mice were immunized against the three different IgG subtypes and the Fab fragments and monoclonal antibodies were produce by hybridoma technology. We were hitherto able to obtain the following purified monoclonal antibodies (moAb): - moAb reacting to all subtypes with identical binding strength, - moAb that preferentially bind the conventional IgG1, - moAb that bind only IgG1. All generated monoclonal antibodies react against immunoglobulins from new world (llama and alpaca) and old world camelids (bactrian camel and dromedary) but show no cross reactivity with immunoglobulins from other species (rat, mouse, sheep, rabbit, goat, chicken and human). The monoclonal antibodies are suitable to detect camelid antibodies by enzyme immunoassay (ELISA) but not in western blots, indicating that the binding epitops are discontinuous. The production of monoclonal mouse antibodies binding to camelid heavy chain only antibodies is subject for future experiments.
Immune repertoire high-throughput sequence analysis (IRAS) web service (65.20)
Authors: Wang Chunlin , Sanders Catherine , Yang Qunying , Han Jian 
Published: January 2023

Abstract
An immune repertoire is the sum of functionally diverse B and T cells, which is shaped by genetic polymorphism, antigen exposure history and regulation and modulation of the immune system. High-throughput sequencing techniques allow us to take a snapshot of the diversity of the immune repertoire, which is a fundamental determinant of the competence of the immune system. However, many researchers are overwhelmed by the task of managing and analyzing the flooding amount of sequence data generated by next-generation DNA sequencers. To make the immune repertoire high-throughput sequencing technology accessible to greater numbers of investigators, we have developed a web based immune repertoire sequence data analysis and management system (IRAS). IRAS is backed by a Linux cluster, where V, D, J germline sequences can be mapped onto reads using the Smith-Waterman algorithm in a high-throughput manner. Mapping results are loaded into a MySQL database and reads are annotated with CDR1, 2, 3 regions. We have developed a web portal to allow easy access to those data, where 2D-plot, 3D-plot, various summary tables, statistical results and mapping alignments can be generated on the fly. The IRAS is open to public and welcome to use. Users can submit their sequence data and relevant metadata through the web portal.
Multiplex immunoglobulin isotyping assays for human, mouse or rat serum samples (65.25)
Authors: Wiese Rick , Gessling Aliya , Hayes David 
Published: January 2023

Abstract
The determination of serum immunoglobulin isotype concentrations is an important indicator of immunological health. We have constructed several multiplex isotyping assays on the Luminex® assay platform. These assays allow for the analysis of human, mouse and rat serum, tissue culture or body fluid samples. The human isotyping assay enables the quantification of immunoglobulins A, M, G1, G2, G3 and G4 in a single assay well. While the mouse isotyping assay cover immunoglobulins A, M, G1, G2A, G2B, and G3, and the rat isotyping immunoglobulins A, E, M, G1, G2A, G2B, G2C. Since the concentrations of IgE in human and mouse samples are typically much lower than the other immunoglobulin isotypes we have also designed separate assays for mouse and human IgE analysis. This collection of assays allows for the rapid, multiplex analysis of major immunoglobulin isotypes in the human , as well as the two predominant research maodels, rat and mouse.
Skin-based soluble microneedle array mediated anti-malarial CSP vaccination (65.28)
Authors: Speaker Tycho , Chang Stella , Fairman Jeff , Kaspar Roger 
Published: January 2023

Abstract
A safe, effective, easily administered, and storage-stable anti-malarial vaccine delivery platform would improve vaccination efforts in populations at risk for malaria. Painless, self-blunting soluble microneedle arrays seem ideal for skin-based inoculation. To test the ability of adjuvanted microneedle arrays to induce an immune response, anti-malarial circumsporozoite surface protein (CSP) antigen was delivered directly to skin by water-soluble polyvinyl alcohol microneedles, inducing a robust and specific immune response in BALB/c mouse cohorts. Inclusion of JVRS-100 cationic lipid DNA complex adjuvant boosted immune response and dried arrays induced strong response even after exposure to accelerated thermal storage conditions. To assess storage stability of the needle array dosage form, loaded microneedle arrays and parallel antigen solutions were subjected to one-week storage at 20°, 37°, and 50°C, and subsequently used to inoculate mouse cohorts. Serum anti-CSP IgG titers indicated that while antigen solutions showed profound (10-fold) loss of activity with increasing storage temperature, the dry arrays exhibited no such degradation.
Magnetic bead-based 28-plex immunoassay for the quantification of rhesus and cynomolgus monkey cytokines (65.30)
Authors: Chitnis Vivek , Strange Christina , Sekella Phillip , Spencer James , Huuskonen Jarkko , Blankfard Martin 
Published: January 2023

Abstract
Non-human primates, such as rhesus and cynomolgus monkey share a similar biochemistry and metabolism to humans. These animals are therefore crucial model systems in a number of areas of biomedical research. To simplify cytokine profiling we have developed a 28-plex magnetic luminex bead-based cytokine immunoassay for the analysis of rhesus and cynomolgus monkey samples. Validated sample types include serum, plasma, and conditioned tissue culture medium. Analytes include: EGF, eotaxin, FGF basic, G-CSF, GM-CSF, HGF, IFN-γ, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-15, IL-17, I-TAC, MDC, MIF, MIG, MIP-1α, MIP-1β, MCP-1, RANTES, TNF-α, and VEGF. The utility of the assay is demonstrated by determining cytokine profiles obtained with LPS-stimulated or PMA plus A23187-stimulated peripheral blood mononuclear cells (PBMCs) from rhesus monkeys and cynomolgus monkeys. Linearity of dilution experiments with tissue culture medium and serum produce R2 values of 0.98 and greater, while sample recovery routinely falls between 80 and 120%. Intra-assay and inter-assay precision is ≤10% CV. This multiplex monkey cytokine assay provides a very powerful tool for addressing cytokine profiling in non-human primates. 28 cytokines can be simultaneously analyzed from a single sample greatly simplifying and cutting cost of monkey cytokine analysis.
Magnetic bead-based immunoassays for the quantification of human and mouse cytokines (65.31)
Authors: Chitnis Vivek , Strange Christina , Gu Jun , Curtis Daniel , Spencer James , Huuskonen Jarkko , Blankfard Martin 
Published: January 2023

Abstract
Multiplexing immunoassays have become a very powerful and economic way to measure multiple analytes from a single sample. This is especially important when sample volumes are limited, such as for rodent samples. Life Technologies has recently launched a set of luminex bead-based immunoassays that can conveniently quantify between 10 and 30 cytokines from human or mouse tissue culture, serum and plasma samples. These assays use magnetic beads which offer a convenient way to automate these assays using commercially available plate washers, such as BioTek ELx405 or Tecan HydroSpeed. The mouse analytes in these kits are: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p40/p70, TNF-α (10-plex), supplemented with FGF basic, IL-1α, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α and VEGF (mouse 20-plex). Human analytes are GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-α (10-plex), supplemented with IL-1RA, IL-2R, IL-7, IL-12 p40/p70, IL-13, IL-15, IL-17, IFN-α, MIP-1α, MIP-1β, IP-10, MIG, eotaxin, RANTES, MCP-1, VEGF, G-CSF, FGF basic and HGF (human 30-plex). All the kits have been extensively validated to include sample recoveries routinely between 80 and 120%, intra-assay and inter-assay precision of &lt;10%, and linearity of dilution with tissue culture medium and serum with R2 values of 0.98 and greater. Benchmarking of the kits has been performed against our ELISA kits to ensure equal results between the two immunoassay platforms.
The role of PD-1 in human CD8 prostate infiltrating lymphocytes. (66.12)
Authors: Bruno Tullia , Durham Nicholas , Nirschl Christopher , Ceccato Christina , Getnet Derese , Pardoll Drew , Drake Charles 
Published: January 2023

Abstract
CD8 T cell tolerance to tumors is multi faceted and includes both extrinsic and intrinsic factors. Extrinsically, the tumor microenvironment seems to play a large role in the functional inertness of CD8 T cells; major contributors are persistent antigen exposure in the microenvironment as well as T regulatory cells. Intrinsically, lack of costimulation seems to play a role (anergy), and often, the presence of inhibitory checkpoints such as PD-1, CTLA-4, LAG-3, and 41BB can also facilitate the down regulation of CD8 T cell function. In CD8 prostate infiltrating lymphocytes (PILS) from cancer patients, we observe refractoriness to TCR stimulation accompanied by reduced cytokine production (IFN-γ and TNF-α) that cannot be reversed by addition of IL-2. Thus, in trying to decipher the exact intrinsic factor that could play a direct role on the CD8 PILS, we measured inhibitory checkpoints on the cell surface. PD-1 was a major checkpoint, and seemed to correlate with reduced Granzyme B production. Interestingly, the total CD8 PIL population was not deficient in Granzyme B production, just the PD-1+ CD8 PILS. Additionally, blockade with anti PD-1 restored proliferation and Granzyme B production in the CD8 PILS, which indicates a potential clinical implication of anti PD-1 immunotherapy.
Critical function of CCL1 in the maintenance of T regulatory cell’s suppressive role. (66.14)
Authors: Hoelzinger Dominique , Dominguez Ana , Lustgarten Joseph 
Published: January 2023

Abstract
T-regulatory cells (Tregs) create an immunosuppressive environment that reduces the efficacy of anti-tumor immunotherapy. Depletion of Tregs enhances anti-tumor immune responses, but also affects T-effector cells. Previously we showed that blockade of chemokine ligand 1 (CCL1) inhibits Treg conversion, their suppressive function, and in combination with intra-tumoral CpG-ODN results in complete rejection of tumors in BALB-NeuT tolerant mice. Most importantly, this therapeutic combination does not affect T-effector function and increases tumor-lytic CD8+NKG2D+ cells within the tumor. Here we expand on the role of blocking CCL1 in enhancing anti-tumor immunotherapy. Using CCL1 knockout (CCL1ko) mice, we found no impairment in TGFβ dependent de novo Treg conversion, however CCL1ko Tregs are less suppressive in vitro, than their wild type counterparts. CCL1ko mice treated with intratumoral CpG showed a marked reduction in tumor growth and a significant increase in tumor rejection as compared to similarly treated C57b control mice. Examination of lymphocyte populations within B16 and MucI tumors showed elevated levels of CD8+ cells. Finally the evaluation of levels of intra-tumoral cytokines and chemokines revealed elevated levels of IFNγ, CXCL9, CCL3 and CXCL10 in tumors grown in CCL1ko mice, suggesting a pro-inflammatory environment. Taken together these data support the role of CCL1as a critical factor for the maintenance of the suppressive function of Tregs.
STAT1 and IFNAR1 regulate Treg frequencies and IL-10 expression the tumor microenvironment (66.15)
Authors: Stewart Charles , Metheny Hannah , Dzutsev Amiran , Karp Christopher , Trinchieri Giorgio 
Published: January 2023

Abstract
The efficacy of cancer immunotherapy is limited by immunosuppression in the tumor microenvironment. Regulatory T cells (Treg) are found at high frequency in tumors, and have greater suppressive activity than Treg from spleen or lymph node tissues. In addition, IL-10 limits the activation of antigen presenting cells and production of proinflammatory cytokines at the tumor site. We examined the cellular sources of IL-10 in the tumor. Using IL-10GFP (VERTX) reporter mice and real-time PCR assays, IL-10 expression was detected in Ly6C+ myeloid cells, macrophages and approximately 50% of tumor Tregs. A similar portion of tumor Treg produced IL-10 following stimulation with PMA/ionomycin. Microarray analyses on IL-10 positive and IL-10 negative tumor Treg and total spleen Treg revealed an activation signature associated with IL-10 expression. This signature correlated strongly with expression of the transcription factor Maf and with induction a type I IFN/STAT1-signature. Using knockout mice we observed that both the type I IFN receptor, IFNAR1 and STAT1 were required for normal tumor Treg frequencies and IL-10 production. This indicates that the STAT1/IFNAR1 axis is required to drive immunosuppressive Treg and their production of IL-10 in the tumor microenvironment. Therefore the immunostimulatory effects of type I IFN are paralleled with an increase in Treg function and IFNAR1/STAT1 may constitute an important therapeutic target for tumor Treg.