bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy
Authors: Baracho Gisele Vanessa , Song Hye-Wong , Taylor Ian , Mortimer Stefanie , Widmann Stephanie , Tyznik Aaron J 
Published: January 2023

Abstract
In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.
Comparison of cesium and X-ray irradiation for generation of bone marrow chimeras
Authors: Eng Jason Samuel , Orf Jessica , Perez Kristy , Sawant Deepali , DeVoss Jason J 
Published: January 2023

Abstract
Bone marrow chimeras are mice which have been irradiated with ionizing radiation, thus killing their bone marrow and all bone marrow derived cells, followed by injection of bone marrow from another mouse. Generation of bone marrow chimeras has been a useful tool to interrogate stromal and hematopoietic contributions to immune function. Cesium sourced irradiators have been demonstrated to be effective in generating bone marrow chimeras and have long historic use in academia and industry. However, the increasing cost to maintain a cesium irradiator and greater security measures set in place for using a radioactive source material has led to a need for utilizing an alternative technology – X-ray irradiation. To our knowledge, limited number of publications compare these two modalities.
In our study, we evaluated the X-ray irradiator by analyzing and comparing the reconstitution profile of specific immune cell subsets in the blood and spleens of mice irradiated with a cesium source versus mice irradiated with an X-ray source. Our data suggest that the two modalities provide functionally comparable degrees of chimerism in mice. Thereafter, we transitioned to using the X-ray irradiator and evaluated the effects of irradiation and reconstitution on tumor implantation. Our data shows that there is delayed tumor growth after total body irradiation.
In summary, we have established a protocol for an alternative irradiation modality, confirmed comparable levels of chimerism to traditional generation of bone marrow chimeras, and observed delayed tumor growth following irradiation and reconstitution.
Targeted CRISPR/Cas9-based in vitro and in vivo screening of metabolic pathways in primary T cells
Authors: Sugiura Ayaka , Beier Katherine L. , Rathmell Jeffrey C. 
Published: January 2023

Abstract
Chronic inflammation and immunosuppression are characterized by a dysregulation in the balance between pro-inflammatory effector T (Teff) cells and anti-inflammatory regulatory T (Treg) cells. While many currently available therapies broadly target the immune compartment, selectively targeting the specific T cell subsets that contribute to disease may provide a new avenue for development of improved immunotherapies. We have previously shown that Teff and Treg cells can be distinguished by their reliance on distinct metabolic programs, and that this exposes a way to preferentially target specific T cell subsets. We have developed in vitro and in vivo approaches to CRISPR/Cas9-based genetic screening in primary T cells to interrogate metabolic pathways. In this method, we design small-scale targeted guide RNA libraries, which are transduced into primary T cells. For the in vitro assays, these cells can be cultured under selective pressures, such as with differentiation cytokines, and sorted for populations of interest. For the in vivo assays, the transduced cells are adoptively transferred into host mice of inflammatory or cancer models, such as airway inflammation or melanoma. We have constructed and tested a library targeting the solute carrier (SLC) family of transporters, which import and export essential nutrients required to fuel the metabolic programs. Preliminary results show the glutamine transporter, SLC38a2 (SNAT2), in addition to the previously identified transporter SLC7a5 (LAT1), to be critical for primary T cell proliferative capacity. This approach provides a powerful tool for identifying critical nodes within metabolic pathways of interest in primary T cells that can potentially serve as therapeutic targets.
Detergent wash improves vaccinated lymph node handling ex vivo
Authors: Ball Alexander , Pompano Rebecca R 
Published: January 2023

Abstract
Vaccines target the lymph node (LN) in order to promote an adaptive immune response, but few ex vivo models are available to study this process. We recently developed ex vivo LN slices as a unique tool to study the dynamics of adaptive immunity; however, a pervasive issue occurred between the LN tissue with the embedding biomaterial. Intact lymph nodes from vaccinated animals, which had expanded into the surrounding adipose tissue, consistently failed to adhere to the agarose matrix, preventing downstream analysis. Here we tested the hypothesis that removal of excess lipids with a liquid detergent from the collagen capsule surrounding the LN would improve the interaction with agarose without damaging viability or function. Specifically, we investigated the use of digitonin, to wash LNs before embedding. Harvested LNs were quickly dipped into digitonin solution and washed in saline prior to embedding in agarose and sliced. Fat was visibly removed by this procedure. Slice viability was determined through flow cytometry using Calcein-AM and 7-AAD. Phagocytic capacity across the entire tissue sample was assessed by measuring uptake of fluorescent microparticles through widefield microscopy. Tissue-level responsiveness was tested by incubating the slices with R848 (TLR7 & 8 agonist) for 24 hours and quantified secreted IFNγ by ELISA. None of the above metrics were affected by washing intact LNs with digitonin prior to slicing and we observed significant improvement in adherence to agarose. We anticipate that this novel approach will enable downstream analysis of lymph nodes undergoing an active immune response to vaccination and may improve the slicing other small organs and organ fragments embedded in agarose, such as liver, thymus, and gut.
Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression
Authors: Tyznik Aaron J , Corselli Mirko , Baracho Gisele V , Lomas Chip , Sa Silin , Widmann Stephanie , Saksena Suraj , Ghanekar Smita 
Published: January 2023

Abstract
Cancer treatment has been revolutionized with the development of immunomodulatory therapies. While these therapies have primarily focused on enhancing T-cell responses, there has been interest in harnessing the potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. However, there is a need for better understanding of these recently described cells.
In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs were enriched using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel enabled simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of 3 distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis.
For Research Use Only.
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BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
A humanized mouse platform supporting in vivo maturation of human antibody responses
Authors: Chupp Daniel P , Acosta Ometeotl , Zan Hong , Casali Paolo 
Published: January 2023

Abstract
Mice are widely used models for research and drug discovery, yet provide limited predictive value due to their divergence from humans in immune elements and functions. This underscores the need for more faithful models of the in vivo human immune response. To address this shortcoming, we have generated a HuMice platform by grafting NSG KitW-41J mice with fresh human cord blood CD34+ stem cells via intracardiac injection. This yielded up to 98% human CD45+ cell reconstitution within all major lymphoid compartments, including GALT. Human leukocyte development and long-term persistence in CD34+ cell-engrafted HuMice revealed robust T and B cell maturation, leading to emergence of IgM, IgG, IgA and IgE forming cells. B cells from HuMice underwent in vitro CSR and plasma cell differentiation as efficiently as B cells isolated from healthy humans. Once conditioned with our immune potentiation treatment, HuMice supported T-dependent and T-independent antibody responses involving formation of germinal center-like structures, class-switching, somatic hypermutation, as well as plasma cell and memory B cell differentiation, this entailed further maturation of the B cell compartment resulting in high B2/B1 cell ratio. HuMice were amenable to hydrocarbon-induced autoimmunity leading to lupus-like disease and were susceptible to infection by and respond to human pathogens such as vaccinia virus and EBV. Thus, we have devised a novel in vivo HuMice platform, which allows for generation and maturation of human antibody responses to microbial agents and lends itself to in vivo modeling of autoimmunity, thereby offering opportunities for identification of therapeutic targets and vaccine development.
Evaluating the WOLF® Cell Sorter impact on neutrophil viability and activation
Authors: Krasny Aly , Morachis Jose 
Published: January 2023

Abstract
Neutrophils have been associated with antimicrobial functions in acute infections and also play critical roles in chronic inflammation. The half-life of circulating neutrophils is estimated at between 13–19 hours and, if activated, they may be cleared even faster. The shear forces and pressure of traditional FACS can be harsh on such sensitive cells and as a result, the use of magnetic beads and centrifugation gradients to isolate neutrophils is sometimes preferred. However, these methods lack the ability to select for viability or multiple markers needed for specific subset gating. NanoCellect’s WOLF Cell Sorter allows for multi-parameter detection and sorting like traditional FACS. However, the WOLF’s microfluidic sorting mechanism uses far lower pressure (&lt;2 psi vs 10–70 psi) that results in gentler sorting and a higher quality of cells post-sort. Here, the ability of this novel instrument to sort neutrophils with minimal cell death or activation was investigated. Live CD16+ neutrophils were sorted from whole blood and evaluated for purity, viability, and expression levels of CD11b, and CD62L. Results demonstrate that the WOLF Cell Sorter can enrich for neutrophils with high purity, viability, and a limited effect on activation, making it an effective method for neutrophil separation. This indicates that this novel microfluidic cell sorting technology can be used to study emerging neutrophil functions, mechanisms of action, and novel cell-based therapeutic approaches.
Analysis of BCR-IgH from genomic DNA with UMI incorporation
Authors: Berngruber Chelsea , Pan Wenjing , Byrne-Steele Miranda , Li Song , Han Jian 
Published: January 2023

Abstract
NGS of T and B cell immune receptors has become routine in immunological studies. While using RNA as a template has its benefits, gDNA is often preferred by those collecting the sample as DNA is often easier to obtain, especially from difficult samples or samples that have been in long-term storage. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. Here, we describe the development of a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted uniquely since the relationship of one V(D)J per cell will be maintained. Thus, both the identity and the frequency of the B-cell can be captured directly, while allowing for the elimination of PCR and sequencing error. The newly developed panel was tested on a BCR-IgH Synthetic Library. The results demonstrate a high degree of correlation with the known BCR-IgH Synthetic Library (R20.956) including coverage of all known V and J gene targets. Currently, gDNA extracted from B-cell enriched PBMC samples are being tested. This method has important implications for monitoring aberrant clonotypes in blood cancers since the frequency of a clonotype can be directly quantified, even under conditions when one clonotype is expanded.
Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases
Authors: Liu Jeffrey M , Chen Pauline , Cieniewicz Brandon , Cepika Alma-Martina , Bacchetta Rosa , Roncarolo Maria Grazia 
Published: January 2023

Abstract
Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.
Deubiquitinase BRCC3 ameliorates acute graft-versus-host disease and promotes graft-versus leukemia effects via promoting type I-IFN secretion
Authors: Liu Yin , Cheng Qiao , Zhang Wenli , Wang Dong , Xu Yang , Wu Depei 
Published: January 2023

Abstract
Acute graft-versus-host disease (aGVHD) remains the leading cause of transplant related deaths after allogeneic hematopoietic stem cell transplantation (allo-HSCT). aGVHD is mediated by the activation of donor lymphocytes in response to the host antigens, and involves a variety of immune cells, inflammatory cytokines and multiple other cytokines. Previous studies have shown that type I-IFN plays an important role in balancing aGVHD and graft-versus leukemia (GVL) responses. Ubiquitination plays essential roles in the production of type I-IFN, but deubiqutinases (DUBs) were reported rarely. Here, we showed that BRCC3 overexpression enhanced IFNβ-luciferase activity, and knockdown of BRCC3 decreased the expression of IFNβ. Interestingly, BRCC3 was down-regulated in allo-HSCT mice compared to syngeneic HSCT (syn-HSCT) mice. We further found that overexpression of BRCC3 ameliorated aGVHD, while promoting GVL effects. Relevant mechanistic studies exhibited that BRCC3 promoted IFNβ production in aGVHD mice, and reduced the absolute numbers of CD4+ T cells and CD8+ T cells in aGVHD target organs. Furthermore, we found that BRCC3 physically interacts with the E3 ligase Trim25, which is involved in RIG-I signaling pathway. In addition, BRCC3 de-ubiquitinates and up-regulates Trim25. These findings suggest that BRCC3-mediated type I-IFN production may be a strategy to control aGVHD and promotes GVL responses.
Supplementation of dietary zinc promotes T cell reconstitution after hematopoietic stem cell transplant
Authors: Iovino Lorenzo , Kinsella Sinéad , Cooper Kirsten , Jain Reema , deRoos Paul , Gagnon Alex , Ugrai Tamas M , Galimberti Sara , Petrini Mario , Dudakov Jarrod A 
Published: January 2023

Abstract
Hematopoietic stem cell transplant (HSCT) is a well-established therapy with curative potential for a variety of malignant and non-malignant diseases. However, delayed T cell reconstitution is an important contributor to transplant-related morbidity and mortality due to infections and malignant relapse. Optimal T cell development requires a functional thymus and many patients who could benefit from enhancing immune regeneration after HSCT have poor thymic function due to their age or their exposure to chemo- and radiation therapy. Here, we show that zinc, which is the second most abundant trace metal in the body, is crucial for thymic function and can be used to promote T cell reconstitution after HSCT. Mice that had been fed a zinc deficient diet for 3 weeks showed reduced thymic cellularity as zinc was required for the expansion and transition from DN to DP thymocytes. Conversely, supplementation of zinc in drinking water could significantly enhance T cell reconstitution after HSCT. Mechanistically, we found a switch in the bioavailability of zinc following damage, likely due to the death of thymocytes; and its release from thymocytes was able to directly stimulate the zinc-gated receptor GPR39 on endothelial cells to directly induce the production of BMP4; which we have previously identified as a key mediator of endogenous thymic regeneration. Consistent with this hypothesis, mice that had been given a pharmacologic inhibitor of BMP4 signaling did not demonstrate any improved thymic regeneration after zinc supplementation. In conclusion, we have demonstrated that zinc is important for steady-state T cell development and zinc supplementation offers a simple but innovative therapeutic strategy to improve T cell reconstitution.
Susceptibility of genetically diverse outbred mice to acute graft vs. host disease (aGVHD)
Authors: Enriquez Josue , McDaniel Brianyell T , Furr Kathryn , Grisham Matthew B 
Published: January 2023

Abstract
Mice are the primary animal of choice for modeling aGVHD. One aspect of these mouse models that may limit translation of promising therapeutic strategies to patient treatment is the use inbred mice as surrogates for genetically diverse human populations.

Objective
Quantify and compare tissue inflammation and injury in inbred vs. genetically diverse outbred mice using a well-characterized model of aGVHD-mediated bone marrow (BM) failure and spleen hypoplasia.

Methods
C57Bl6 (Bl6) CD4+ T cells (20,000 T cells/g b.w.) were injected (i.p.) into sub-lethally irradiated Bl6-H2-Ab1bm12(BM12) recipients (Bl6→BM12) or into Collaborative Cross (CC) or CD1 outbred recipients.

Results
Adoptive transfer of allogeneic Bl6 T cells into BM12 recipients induced marked weight loss (25%) at 20 days post T cell transfer that was associated with dramatic reductions in hematocrit as well as large and significant decreases in circulating granulocytes, platelets and erythrocytes when compared to their syngeneic controls (BM12→BM12). Total cell numbers in BM and spleen were also found to be dramatically reduced in these mice. Histopathological inspection confirmed severe hypocellularity in BM and spleen whereas no aGVHD was present in the lungs, liver, skin and colon. In contrast, adoptive transfer of Bl6 T cells into sub-lethally irradiated CC or CD1 or CC mice induced little or no weight loss at 25–30 days post T cell transfer with no significant reductions in hematocrit, circulating leukocytes, erythrocytes or platelets. BM- and spleen-associated cell numbers were not reduced when compared their CC controls.

Conclusions
Genetically diverse outbred mice are resistant to aGVHD-mediated BM failure and spleen hypoplasia.