bluuebunny/crossref_metadata_2025_split huggingface dataset:
Upregulation of Bcl6 expression in Follicular Helper T cells (TFH) requires both dendritic and antigen-specific B cell help. (63.10)
Authors: Weinstein Jason , Poholek Amanda , Hernandez Sairy , Nowyhead Heba , Bertino Sarah , Craft Joseph 
Published: January 2023

Abstract
The transcription factor B cell CLL/lymphoma 6 (Bcl6) was recently found to be both necessary and sufficient for TFH cell development. However, the factor(s) that promotes the upregulation of Bcl6 expression in TFH cells remains incompletely known. Here we explored the roles of B and dendritic cells (DC) in the upregulation of Bcl6 in TFH cells. Antigen-specific, Thy1-mismatched, TCR transgenic CD4 T cells were co-transferred with Ag-specific or polyclonal B cells into CD19-/- or wild type hosts. Upon immunization of CD19-/- mice with NP-OVA, Bcl6 expression increased in donor T cells compared to unimmunized controls, despite the absence of functional B cells. Although Bcl6 expression was not equivalent to T cells transferred to CD19-intact mice post immunization, upon transfer into CD19-/- animals expression of T cell surface markers was consistent with emigration towards the T-B border, albeit without expression of TFH cell markers and follicular entry. The transfer of Ag-specific B cells, but not their polyclonal counterparts, to CD19-/- hosts restored Bcl6 expression to levels approximating that observed from immunized wild type controls, and restored the TFH cell phenotype with follicular entry. Bcl6 was not upregulated in T cells transferred to mice depleted of DCs, followed by immunization. These data demonstrate that CD4 T cells need signals from both DCs and from Ag-specific B cells to fully upregulate Bcl6 and drive TFH cell development and function.
Priming with very low affinity peptide ligands gives rise to CD8+ T cell effectors with enhanced function but with greater susceptibility to TGFβ-mediated suppression (63.15)
Authors: Guevara-Patino Jose-Alejandro , O’Sullivan Jeremy , Zloza Andrew , Kohlhapp Frederick , Moore Tamson , Lacek Andrew , Dulin Nickolai 
Published: January 2023

Abstract
While the effects of TCR affinity and TGFβ on CD8+ T cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that, while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8+ T cells primed with lower affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a noncanonical negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8+ T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed.
Induction of autoimmune pathology linked to asymmetric T cell division (63.21)
Authors: King Carolyn , Koehli Sabrina , Hausmann Barbara , Schmaler Mathias , Palmer Ed 
Published: January 2023

Abstract
Immunological tolerance, defined as the ability to distinguish between self and nonself, begins in the thymus where T cells undergo development. The affinity between T cell receptor (TCR) and pMHC has long been established as a major determinant guiding thymocyte selection and recent data revealed a universal threshold affinity used by all thymocytes with class I MHC restricted TCRs to initiate self tolerance. Nevertheless, the prevalence of autoimmune disease indicates that self reactive T cells can be activated in the periphery. Whether the affinity threshold established during thymic selection plays a role in the maintenance of peripheral self tolerance has not been established. Here we show that only high affinity ligands (above the threshold for negative selection) are able to induce disease in a mouse model of autoimmune diabetes. Immunization with high affinity peptides results in more efficient T cell-APC conjugate formation, T cell proliferation, VLA-4 upregulation and ultimately pancreatic infiltration. Importantly, higher affinity peptides are more efficient at inducing asymmetric cell division resulting in a greater number of fully differentiated effector T cells. In contrast, stimulation with low affinity peptides results in fewer asymmetric divisions and fewer effector T cells capable of promoting disease. These findings underscore the critical importance of TCR in directing T cell fate and maintaining peripheral T cell tolerance.
Inter-follicular germinal center B cell and T follicular helper cell development precedes follicular Tfh maintenance (63.23)
Authors: Kerfoot Steven , Yaari Gur , Patel Jaymin , Johnson Kody , Gonzalez David , Kleinstein Steven , Haberman Ann 
Published: January 2023

Abstract
We identify the interfollicular (IF) zone of the lymph node as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, where they participated in long-lived interactions and upregulated Bcl-6, which controls the differentiation of both cell types. During this time, T cells also acquired the Tfh markers CXCR5, PD-1 and GL7. While T cell immigration into the follicle interior occurred three days post immunization, responding B cells remained largely restricted to the IF zone and the area adjacent to the sub capsular sinus at this timepoint. B cell entry into the follicle interior, and formation of nascent germinal centers, did not occur until a day later. Notably, in the absence of cognate B cells, PD-1hi Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 expression was not maintained on T cells that nevertheless persisted within the follicle. Thus, cognate B cells are not required for Tfh cell differentiation in the IF zone, but are instead required for the long-term maintenance of the activated Tfh phenotype.
Type I interferon promotes PD-1 transcription and limits the duration of T cell-mediated immunity (63.24)
Authors: Chikuma Shunsuke , Terawaki Seigo , Shibayama Shiro , Hayashi Tamon , Yoshida Takao , Okazaki Taku , Honjo Tasuku 
Published: January 2023

Abstract
PD-1 is a co-receptor for T lymphocytes that provides feedback inhibition of T-cell activation. Although PD-1’s expression on T cells is known to be activation dependent, the factors that determine the timing, intensity, and duration of PD-1 expression in immune reactions are not fully understood. To address this question, we analyzed a conserved 5’-flanking region of the PD-1 gene, and identified a putative interferon-stimulation response element (ISRE), which was responsible for PD-1 transcription in the 2B4.11 T-cell line. Consistent with this finding, activation by interferon (IFN) alpha enhanced both the induction and maintenance of PD-1 expression on TCR-engaged primary mouse T cells through an association of interferon-responsive factor-9 (IRF9) to the ISRE. Indeed, PD-1 expression on antigen-specific CD8+ T cells was augmented by IFN alpha in vivo. We also demonstrated that IFN alpha administration in combination with PD-1 blockade in tumor-bearing mice effectively augmented the anti-tumor immunity. Thus we propose that strong innate inflammatory responses promote primary T-cell activation and their differentiation into effector cells, but also cause an attenuated T-cell response in sustained immune reactions, at least partially through type I interferon-mediated PD-1 transcription.
T follicular helper cells require cognate B cell interactions for acquisition of full effector function (63.4)
Authors: Barnett Lisa , Goenka Radhika , Silver Jonathan , O'Neill Patrick , Hunter Christopher , Cancro Michael , Laufer Terri 
Published: January 2023

Abstract
Follicular helper T cells (TFH) are a unique subset of CD4+ helper T cells that are critical effectors of thymus-dependent humoral immune responses, yet much about their development in vivo remains unclear and controversial. The requirements for different antigen presenting cell subsets in TFH formation are not yet fully understood. By transiently ablating or restricting MHCII antigen presentation to CD11chi dendritic cells (DCs), we show that cognate, MHCII dependent interactions with DCs are both necessary and sufficient for the formation of CXCR5+, ICOS+ TFH cells that express the key transcription factor Bcl6, which governs their differentiation. However, TFH cells that do not undergo TCR-MHCII-dependent interactions with B cells lack a small subset of GL-7+ PD1hi cells found in the germinal center (GC) and GCs fail to form. These TFH exhibit a significant reduction in their ability to produce IL-21 when compared to TFH cells generated in the presence of cognate B cell interactions. Consistent with these data, in vitro priming of naïve T cells by B cells, but not DCs, induces optimal IL-21 production, which, in turn, induces a GC B cell transcriptional profile. Thus, this study supports a multi-step model for priming effector TFH in which DCs activate CD4+ T cell towards a poised intermediate which can then interact with responding B cells to sustain the GC status of both cell types.
Modulation of mucosal effector and regulatory T cell responses by Helicobacter pylori (63.6)
Authors: Carbo-Barrios Adria , Climent Montse , Muite Kevin , Viladomiu Monica , Pedragosa Mireia , da Fonseca Isabel , Humedas Merce , Lu Pinyi , Horne William , Hontecillas Raquel , Bassaganya-Riera Josep 
Published: January 2023

Abstract
Helicobacter pylori is a gram-negative, microaerophilic bacterium that colonizes the gastric mucosa and a leading cause of gastritis, peptic ulcer and gastric cancer. While dysregulated immune responses contribute to H. pylori-mediated pathogenesis, its immunoregulatory mechanisms are incompletely understood. To characterize the mechanisms of immune modulation by H. pylori we have performed dose-titration and time-course studies in mice infected with H. pylori strain 26695. Our dose-titration results indicate an increase in the percentages of INFγ+Tbet+ Th1 cells, lymphocytic infiltration into the gastric lamina propria, and disease activity with increasing concentrations of the H. pylori infectious dose (i.e., 106, 108 and 1010 cfu/ml). The profiling of T cell responses in the gastric lymph nodes (GLN) over time illustrates an initial increase in CD4+INFγ+Tbet+ (Th1 cells) on day 7 post-infection followed by a predominant peak of CD4+IL10+ T cells and smaller but significant peak of CD4+IL17+ T cells on day 14 post-infection. In conclusion, gastric H. pylori infection in mice induced mucosal effector Th1 and Th17 responses that were paralleled by strong regulatory responses, thereby lending support to importance of characterizing effector and regulatory networks to better comprehend microbial pathogenesis at the site of infection.
Foxg1 is necessary for thymic epithelial cell differentiation. (64.13)
Authors: Condie Brian , Wei Qiaozhi 
Published: January 2023

Abstract
Thymic epithelial cells (TECs) are an essential component of the thymic microenvironment and are necessary for the production of normal T cells. Cortical (cTEC) and medullary TEC (mTEC) subtypes have different functions and distinct gene expression profiles. However, the transcription regulatory network controlling the specification and differentiation of TECs is poorly understood. We have initiated a screen for additional transcription factors that are likely to be required for TEC development. This screen identified Foxg1 as a candidate regulator of thymic epithelial cell differentiation. We have found that Foxg1 is expressed in TECs before the onset of TEC differentiation and continues to be expressed in them throughout fetal development. Analysis of Foxg1 mutant mouse embryos revealed that TEC differentiation initiates normally but then becomes abnormal at E13.5-E14.5. Foxn1, which is required for the initiation of TEC differentiation, is expressed throughout fetal development in Foxg1 mutant TECs suggesting that Foxg1 activity is not necessary for Foxn1 expression. Overall, our results suggest that Foxg1 may regulate TEC differentiation independently of or in collaboration with Foxn1.
A genetically selective inhibitor reveals ZAP-70 kinase-dependent checkpoints in T cell development (64.15)
Authors: Au-Yeung Byron , Cheng Debra , Zhang Chao , Shokat Kevan , Weiss Arthur 
Published: January 2023

Abstract
The tyrosine kinase ZAP-70 is critical for the activation of signal pathways downstream of the TCR. We previously generated a transgenic mouse expressing a ZAP-70 mutant (ZAP-70AS) that is selectively inhibited by a PP1 analog, called 3-methyl benzyl PP1 (3-MB-PP1). This system enables the rapid and specific inhibition of ZAP-70 catalytic activity. We used a combination of ZAP-70 inhibitor treatment with fetal thymic organ culture (FTOC) to determine when ZAP-70 kinase activity is required for proper T cell development. Our results show that treatment of ZAP-70AS FTOC with 3-MB-PP1 impairs development of the CD4 CD8 double-negative 4 (DN4) subset, indicating that ZAP-70 has an important role in pre-TCR signaling and TCR β-chain selection. This block is not complete, allowing generation of double-positive (DP) thymocytes, possibly due to expression of the related kinase Syk. However, ZAP-70 kinase activity is fully required for positive selection, as characterized by DP cell upregulation of surface levels of TCR, CD5 and CD69. Here we show that ZAP-70 kinase activity has both partial and full requisite roles in T cell development.
β-catenin acts upstream of RORγt in double positive thymocyte survival regulation (64.17)
Authors: Wang Ruiqing , Xie Huimin , Sun Zuoming 
Published: January 2023

Abstract
CD4+CD8+ double positive (DP) thymocytes are subject to positive and negative selection, and thus the lifespan of DP cells determines T cell repertoire. Previously, we have identified RORγt as a critical player in the regulation of DP thymocyte survival. However, the molecular mechanisms responsible for RORγt-mediated survival remain unknown. Here we demonstrated that β-catenin/TCF acts upstream of RORγt to enhance DP thymocyte survival via up-regulation of anti-apoptotic factor Bcl-xL. Activation of TCF-1 pathway by transgenic expression of a constitutively active β-catenin leads to increase of RORγt level and enhanced DP thymocyte survival via upregulation of Bcl-xL, whereas DP thymocytes from Tcf-1-/- mice undergo accelerated apoptosis and have significantly reduced RORγt and Bcl-xL. More importantly, forced expression of RORγt in Tcf-1-/- thymocytes leads to rescue of massive apoptosis and restoration of Bcl-xL level. We further demonstrated that β-catenin/TCF-1 activates RORγt promoter. Our data thus suggest that β-catenin/TCF-1 acts upstream of RORγt in the regulation of Bcl-xL expression and DP thymocyte survival.
FLVCR, a heme export protein, is required for T cell development beyond the CD4+CD8+ double-positive stage (64.18)
Authors: Philip Mary , Zaballa Alexandra , Phelps Susan , Abkowitz Janis 
Published: January 2023

Abstract
Heme is essential for every mammalian cell; however, free heme can induce free radical formation and cellular damage and cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Deletion of Flvcr caused progressive anemia in neonatal and adult mice (Science 319:825-8, 2008). We developed a transplant model in which developing lymphocytes lacked FLVCR while erythroid cells expressed FLVCR, preventing anemia, and found that CD4 and CD8 peripheral T cells were severely decreased while B cell numbers were only mildly affected. We analyzed thymic development and found that although there were similar numbers of CD4-CD8- double-negative thymocytes (DN) and CD4+CD8+ double-positive thymocytes (DP), there was a dramatic decrease in the number of CD4+ and CD8+ single positive thymocytes (SP) in FLVCR KO-transplanted mice. Because sensitive quantification of intracellular free heme is challenging, we measured the relative expression of a heme-inducible gene, heme oxygenase-1 (hmox-1) and found higher hmox-1 expression in FLVCR KO thymocytes compared to controls, suggesting that Flvcr deletion leads to higher intracellular free heme in developing T cells. While heme is required for erythroid function, there is no known specific role for heme in T cell development. Studies are underway to determine whether direct heme toxicity arrests T cell development, or whether excess heme alters T cell developmental gene expression.
IL-18 and IL-12 promote development of early thymic progenitors and can substitute for IL-7 in short-term OP9-DL4 co-cultures (64.7)
Authors: Tan Chibing , Marino Julie , Taylor Ashlee , Wren Jonathan , Teague T. Kent , Van De Wiele C. Justin 
Published: January 2023

Abstract
Determining the most effective parameters for in vitro propagation of T cell precursors is critical for realizing the promise of many envisioned T cell-based immunotherapies. To this end, the consequences of culturing ETP and DN2 thymocytes with IL-18 and IL-1β in the context of the OP9-DL4 co-culture model were evaluated after inference of a hypothesized role for these molecules in thymopoiesis based on results obtained via a novel microarray/literature mining algorithm. Sort-purified mouse ETPs and DN2 thymocytes were cultured on OP9-DL4 stromal layers ±IL-18, IL-1β, IL-12 and/or IL-7 for one week. IL-18 and IL-12 alone promoted proliferation and differentiation of ETPs to the DN3a stage and these cytokines could compensate for absence of IL-7. When IL-18 was used in conjunction with IL-7, synergistic proliferation was observed. IL-1β proved to be deleterious to development and was not overcome by inclusion of IL-7. To evaluate the developmental potential of the derived pre-T cells, culture-generated DN3a-like cells were sort-purified then cultured for an additional 8 days. In these assays, the pre-T cells demonstrated differentiation to the DP stage. Overall, these results indicate that IL-18 and IL-12 can positively impact T cell development and should prove useful in strategies to augment in vitro T cell development in the OP9-DL4 system.
Immunotherapeutic proteins expressed in transgenic soybean seeds (65.1)
Authors: Bost Kenneth , Hudson Laura , Piller Kenneth 
Published: January 2023

Abstract
Soybean seeds expressing transgenic proteins represent a novel, sustainable platform technology which overcomes some of the current limitations for producing recombinant proteins useful as immunotherapeutics. Advantages include low cost of glycoslyated protein production, greenhouse containment, highest protein/biomass ratio, marketable formulations which require no purification from soy, safety, accurate dosing, low cost of protein purification, low-tech sustainability of product lines, reduced risk of contamination, ease of scalability, minimal waste produced, as well as being a green technology. We have successfully expressed a variety of subunit protein antigens, and potential immunomodulatory proteins in transgenic soybean seeds, and are evaluating their usefulness as vaccines and therapeutics. The flexibility of long-term storage of transgenic seeds or powder in the absence of a cold chain and without degradation allows protein expression to occur years in advance of its therapeutic use. Purification of a transgenic protein is simplified by the limited number of soy proteins present in seeds. Furthermore, transgenic protein expression levels of approximately 13 grams per liter of soy powder and 22 kilograms per greenhouse acre represent the highest protein/biomass ratio for the industry, and contribute to low production costs. As protein expression technologies expand and evolve, transgenic soybean seeds represent an emerging technology with significant advantages.
Development of an alternative method for the identification and production of antigen-specific monoclonal antibodies (65.16)
Authors: Byrne-Steele Miranda , Sanders Catherine , Yang Qunying , Pan Wenjing , Wang Chunlin , Han Jian 
Published: January 2023

Abstract
A critical step in the production of monoclonal antibodies (Abs) is the initial identification of the antigen-specific Abs, which is usually performed by multiple rounds of panning in both hybridoma and phage display. We have developed an alternative method that allows for the rapid and direct identification of antigen-specific Abs from peripheral blood via high-throughput immune repertoire sequencing and LC MS/MS peptide matching. Our B-cell repertoire technology combines novel amplicon rescued multiplex PCR (arm-PCR, patent pending) with high-throughput gene sequencing to access the sequence of a broad spectrum of heavy and light chain V-regions. As a proof of concept, we have sequenced the immune repertoire of 2 healthy individuals at various time points after administration of the 2009-2010 seasonal influenza vaccine. Antigen-specific Abs were purified directly from immune peripheral blood serum and identified using LC MS/MS peptide sequencing, exploiting the B-cell repertoire gene sequencing results as a database for identification. During our study, several unique peptides were successfully matched for each individual’s response to both Flu A strains in the vaccine. Future work includes the development of a method to rapidly clone and express these identified Abs in a human in vitro glycoexpression system. The recombinant Abs will then be tested for their ability to bind flu hemagglutinin, demonstrating the utility of our technology towards the production of mAbs.
Acoustic flow cytometry for multicolor immunophenotyping analysis (65.23)
Authors: Zhang Yu-Zhong , Anderson April , Bradford Jolene 
Published: January 2023

Abstract
The Attune® Acoustic Focusing Cytometer utilizes resonant device that focuses cells or particles into a single tight line. This technology enables both longer cell interrogation times and higher throughput, which simultaneously permits better cell interrogation as well as the analysis of a larger numbers of cells. As a result, we were able to accomplish a high throughput of samples with relatively low sample concentration while maintaining a high degree of separation. Human whole blood stained with several IVD and ASR grade pre-mixed cocktails followed by lyse no-wash procedures were analyzed on the Attune® Acoustic Cytometer. The immunophenotyping data indicated that the analyses obtained are both reliable and reproducible. In addition, the 405 nm violet laser in Attune® Acoustic Cytometer allows researchers using Qdot® nanocrystals to add more parameters to their flow cytometry experiments by filling in the spectral landscape around common organic fluorochrome conjugates. The availability of Qdot® nanocrystals in multiple emission wavelengths and ever more Qdot® conjugates, allows the creation of multicolor panel consisting of Qdot® conjugates. We show the effectiveness of using Qdot® -conjugated antibodies in a typical 6-color T-cell phenotyping panel. Experiments were carried out using standard staining protocols with human peripheral blood leucocytes.