bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intestinal commensal bacteria promote gut barrier repair through non-canonical inflammasome pathway activation
Authors: Wu Wan-Jung , Kim Myunghoo , Tsai Ming-Ting , Morales Fatima Beatriz Saldana , Ruiz Daniel Fernando Zegarra , Hill Andrea , Song Hyo Won , Norwood Kendra , Diehl Gretchen 
Published: January 2023

Abstract
The intestinal epithelial barrier critically separates exogenous microbes from our internal tissues. Signals from commensal microbes are known to support intestinal barrier integrity and repair. However, the role of specific microbes in maintaining the intestinal barrier is not well-defined. We hypothesize that signals from distinct commensal microbes play different roles in regulating the barrier. We find that mice develop severe colitis with decreased ability to repair the intestinal barrier after antibiotic depletion of intestinal microbes. We further isolated a mouse commensal E. coli that protects from increased colitis after antibiotic treatment. This protection depends on the presence of intestinal antigen presenting cells (APCs) that express the chemokine receptor CX3CR1. We screened a panel of human mucosal-associated E. coli and identified a subset that also protect from colitis in a CX3CR1+ APC dependent manner. All protective E. coli induce IL-1β secretion by activation of the non-canonical inflammasome pathway in CX3CR1+ APCs. In vivo protection is lost if IL-1β signaling is blocked with anti-IL-1β antibody. This E. coli induced IL-1β production activates type 3 innate lymphoid cells (ILC3) to produce IL-22 which drives epithelial proliferation and barrier repair. Collectively, we identified a novel mechanism by which a subset of mucosal-associated intestinal E. coli activate IL-1β production by APCs to protect the intestinal barrier from damage.
CD8αα intestinal intraepithelial lymphocytes derived from two thymic precursors seed the intestine in early life
Authors: Ruscher Roland , Lee Seungeun Thera , Barrero Oscar Salgado , Breed Elise , Isakson Sara , Jameson Stephen , Hogquist Kristin Ann 
Published: January 2023

Abstract
TCRαβ+CD4−CD8αα+ intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ+ thymocytes: a nascent PD-1+ population and a T-bet+ population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1+ cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed “time-stamp” experiments: We crossed Cd4CreERT2 with Rosa26TdT (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4+CD8+ stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells.
Potentiating lung-localized immune responses to conserved influenza virus antigen through a novel vaccine platform
Authors: Nelson Sean Alexander , Fischer Nicholas O , Rasley Amy , Sant Andrea J 
Published: January 2023

Abstract
Current influenza vaccines are primarily delivered at peripheral sites and provide immunity characterized by neutralizing antibody responses. However, this strategy often fails to provide broadly-protective immune capacity. Numerous studies have implicated lung localized T cells as key mediators of broadly protective immunity to influenza infection. The goal of our current research is to expand virus-specific CD4 T cells via the respiratory route in order to understand how this mode of priming poises the host for future protective immunity to infection. We have explored a novel nanolipoprotein (NLP) vaccine conjugated to influenza nucleocapsid protein (NP) to boost lung localized immunity. ELISA assays revealed that NLP immunization boosted anti-NP antibody titers, which have been shown to contribute to heterosubtypic immunity. Importantly, immunization with NLP elicited NP-specific CD4 and CD8 T cell responses in the lung, draining lymph node, and spleen. Intravascular labeling and flow cytometry were performed to assess the localization and phenotype of CD4 T cells in the lung. Analyses revealed that intranasal immunization with NLP elicited an increased abundance of lung parenchyma-localized CD4 T cells expressing surface markers associated with tissue residency and cytolytic potential. Using a novel pMHC class II tetramer, we observed that the majority of antigen-specific CD4 T cells were localized to the lung tissue and can rapidly produce antiviral cytokines upon restimulation. These studies will improve our understanding of vaccine strategies deigned to elicit effector T cells at mucousal sites and demonstrate the potential of tissue resident cells to mediate broadly protective immune responses to influenza.
The C-X-C motif chemokine 17 plays a minimal role during lung infection with hypervirulent Mycobacterium tuberculosis
Authors: Choreno-Parra Jose Alberto , Dunlap Micah , Swanson Rosemary , Jiménez-Álvarez Luis A. , Zúñiga Joaquín , Khader Shabaana A 
Published: January 2023

Abstract
The C-X-C motif chemokine 17 (CXCL17) is a novel mucosal chemotactic factor for myeloid cells that displays bactericidal activity against respiratory pathogens in vitro. Although CXCL17 is constitutively expressed along the respiratory tract, its function during lung infections has not been tested in vivo. We previously showed that the CXCL17 gene is overexpressed in innate lymphoid cells (ILCs) isolated from the lung of patients with active pulmonary tuberculosis (TB). Thus, in the current study we further explored the role of CXCL17 in the defense against Mycobacterium tuberculosis (Mtb). In a mouse model of aerosol infection with the prototype W-Beijing lineage Mtb strain, hypervirulent HN878, we found that CXCL17 is actively produced at the lung during the acute and chronic stages of disease, with levels peaking at 30 days post-infection. Using in vitro infection assays, we observed that lung epithelial cells, bone marrow-derived macrophages (BMDMs), and bone marrow-derived dendritic cells (BMDCs) can become cellular sources of CXCL17 in response to Mtb. In humans, the serum levels of CXCL17 are significatively higher among individuals with latent TB infection (LTBI) than in active TB patients, suggesting a protective role. However, low serum levels of CXCL17 do not correlate with increased lung damage among active TB subjects. Furthermore, CXCL17−/− mice do not exhibit increased susceptibility to Mtb HN878 infection as compared to wild type controls. Finally, administration of exogenous recombinant CXCL17 into the airways of Mtb-infected animals does not impact their lung bacterial burden despite increased recruitment of myeloid cells to the pulmonary tissue. Our data suggest that CXCL17 plays a redundant role during TB.
Altered expression of Surfactant and CD74 in the alveolar epithelial cells during Influenza infection disease
Authors: Periolo Natalia , Ibañez Lorena Itati , Peralta Liliana Martinez 
Published: January 2023

Abstract

Background
Influenza A virus (IAV) causes substantial morbidity and mortality in the human population. In the lower lung, the primary targets for IAV replication are type II alveolar epithelial cells (AECII), which are increasingly recognized for their immunological potential. Surfactant components are synthesized primarily by AECII, which play an important role in host defense against infection. On the other side, AECII express high levels of CD74 and contributes to repair the damaged alveolus during infection.

Aim
To analyze the expression of surfactant (proSP-C), CD74 and activation markets (HLA-DR, CD80, CD86) expressed by AECII cells from infected mice and A549 cell line.

Methods
IAV used include the mouse adapted influenza strains. Pandemic H1N1 (pH1N1) and A/Victoria/3/75 (H3N2) were used to infect BALB/c mice. AECII cells were obtained from mice lungs, and cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/CD74/proSP-C) followed by FACS analysis. The A549 cell line was used as a human in vitro model and to compare the results.

Results
Analysis of AECII phenotype showed a significant decrease of proSP-C+CD31−CD45−and CD74+CD31−CD45− expression in infected mice comparing to controls, and also in the group infected with H1N1 vs H3N2 (MFI p &lt;0.01; p&lt;0.05, respectively). Expression of HLA-DR was very low for AECII and A549 cell line in infection condition. However, CD80 and CD86 they did not show changes in their expression.

Conclusions
Our results indicate that morphological changes in AECII phenotype suggest very active participation of alveolar epithelial cells in inflammatory viral processes in the lung, and higher effects of H1N1 comparing to H3N2 strain.
Expansion of natural killer cells with optimal killing activity for cancer immunotherapy
Authors: Flynn Kevin , Goetz Christine A. , Van Etten Jamie , Hammerbeck Christopher , Peng Li , Lee Neil , Liang Joyce , O-Reilly Daniel , Herr Greg , Tong Thuy , Finkel Dave , Kubik Mandy , Bonnevier Jody L 
Published: January 2023

Abstract
Natural killer (NK) cells play a key role in the elimination of microbial pathogens and cancer cells. NK cells rapidly respond to stressed cells that are infected or bear tumorigenic mutations that reduce “self” markers of MHC class 1 and/or display increased presentation of activating ligands. These signals prompt NK cells to form an immunological synapse with an infected or tumor cell and discharge lytic granules containing cytotoxic proteins such as IFNγ, perforin and granzyme B leading to target cell destruction. Because of their natural ability to recognize and kill cancer cells, a plethora of preclinical studies and clinical trials have shown that NK cell therapies offer a potentially powerful weapon to fight both advanced-stage blood cancers and solid tumors. Given the great potential of NK cell therapies, we developed clinically relevant serum- and xeno-free methods for human NK cell expansion. Using optimized media and cytokines, either with anti-NKp46 coated tissue culture dishes or Cloudz™ CD2/NKp46 microspheres we have greatly expanded highly purified NK cells from human peripheral blood mononuclear cells (PBMCs) that are capable of potent cytolytic activity, as indicated with a novel flow cytometry-based NK cytotoxicity assay. Using this NK expansion methodology, we performed high-throughput screening methods to identify novel agents that enhance NK cell killing activity as assessed by IFNγ and Granzyme B quantikine ELISA assays which were then confirmed with NK cytotoxicity assays. The methods established here provide a novel workflow for generating highly purified NK cells that are applicable to clinical translation. Furthermore, the agents identified in this work provide insight for augmenting NK killing potential.
Use of real-time live-cell analysis with enhanced data analytics to quantify immune cell tumor killing
Authors: Bevan Nicola , Lovell Gillian , Kelsey Lauren , Szybut Clare , McBain Kirsty , Perez-Garrido Sandra , Oliver Miniver , Patel Kalpana , Trezise Del , Dale Tim 
Published: January 2023

Abstract
To develop new therapeutics, researchers are exploring the role of the immune system in defending the body against tumors. Modelling induced malignant cell death in vitro is of paramount importance.
Tumor and immune cell co-cultures were created in 96 well plates and using live-cell analysis, various parameters of tumor killing were quantified in real-time. Red nuclear labeled target cells and various densities of pre-activated PBMCs (α-CD3/IL2, 4 d) were seeded in combination with IncuCyte Annexin V green apoptosis detection reagent. Images were acquired every 2 h for 3 d using IncuCyte. Analysis of the fluorescence images provides measurement of target cell number and apoptosis. Enhancement of the phase contrast image analytics enabled single cell segmentation, permitting determination of effector cell parameters; cell number, shape and, using fluorescently labeled surface marker antibodies, protein expression levels. In addition, studies into spatial interactions of target and effector cells were conducted. To exemplify how these new analytical features can be used to investigate the biology of tumor cell killing, studies of a α-hCD3xCD19 bi-specific T-cell engager antibody induced cytotoxicity were performed.
Further characterization of effects on cell cycle during target cell death and use of more advanced 3D models of immune cell killing were also assessed, demonstrating the flexibility of live-cell analysis as a powerful tool for analyzing immune cell killing.
Advances in data analytics has enabled the multiplexing of target cell quantification alongside the interrogation of effector cell properties in live cells. The added insight gained from these approaches will hopefully lead to improved immuno-therapeutics.
Development of a cells and bead-based, advanced flow cytometry assay for exhausted T cell quantitation
Authors: Carter Mark , Chauhan Sarita , O’Rourke John , Senutovitch Nina 
Published: January 2023

Abstract
T cells can become exhausted due to persistent antigen exposure during chronic infections and cancer. Exhausted T cells exhibit decreased effector functions and express inhibitory receptors (IRs). The exhausted phenotype may be protective in the context of chronic infection, however, it allows tumors to avoid immune surveillance and immune cell mediated killing. Recent therapies block the IRs to reinvigorate exhausted T cells.
A miniaturized, multiplexed flow cytometry assay was developed to measure expression of IRs on T cells while simultaneously measuring viability, proliferation, and cytokine secretion in microtiter plates. Data was acquired on the Intellicyt® iQue platform and analyzed using Forecyt®. Templates were prepared for ease of data analysis, to auto-generate gating, standard curves and sample quantitation and rapid, actionable results.
For proof of concept studies, human PBMCs, purified CD3+, or in vitro exhausted purified CD3+ cells were treated with anti-CD3/CD28 and cells were analyzed at 1, 2, and 3 days post-stimulation. Non-exhausted cells showed a concentration and time dependent increase in IR expression. Persistent, high levels of the IR markers PD-1, LAG-3 and TIM-3 were measured in exhausted cells. Stimulated exhausted T cells secreted stable and moderate levels of IFN-γ (&lt;1650 pg/ml) and TNF (&lt;750 pg/ml) over time whereas non-exhausted cells showed time and concentration dependent increases in IFN-γ (13,319 pg/ml) and TNF (3,711 pg/ml) upon stimulation.
The ability of this assay to examine T cell response allows screening for molecules that reverse T cell exhaustion, screening of modified cells such as CAR-Ts or selection of clones that exhibit low levels of IRs and show robust response to stimulation.
New microfluidic system provides automated alternative to Ficoll® for leukocyte isolation
Authors: Scully Melanie M , Mickool Sarah M , Rivelli Nicholas D , Jonca Aleksander J , Arnal Gustavo , Culligan Laura G , Kou Peng Meng , Smith Kyle C 
Published: January 2023

Abstract
Despite its limitations, Ficoll® has remained the primary method of leukocyte isolation for decades due to a lack of viable alternatives. To address this, MicroMedicine has developed SorterraTM, an automated microfluidic system that enables highly consistent label-free white blood cell separation and concentration.
In a comparative study, Sorterra recovered 25% more viable lymphocytes than Ficoll, 88 ± 7% and 69 ± 13%, respectively (n=48). Sorterra retained &lt;0.01% red blood cells (RBCs) and &lt;0.1% platelets, resulting in a highly pure leukocyte isolate, while Ficoll retained &lt;0.01% RBCs and 8% platelets. Sorterra also demonstrated more consistent performance across operators than Ficoll.
Sorterra and Ficoll isolates were assessed for B cell and T cell functionality. To assess B cells, the isolates were stimulated with R848 and IL-2 (n=9), and total IgG and IgM secretion were evaluated with ELISpot. Secretion from Sorterra and Ficoll isolates was shown to be equivalent using distribution free resampling analysis. To assess T cell functionality, negatively selected T cells from Sorterra and Ficoll isolates were activated with CD3/28 beads and expanded with IL-2 (n=12). The isolates displayed similar T cell activation (89±4% vs. 88±5% CD25+/CD69+), proliferation, and T cell phenotypes (Tn TCM, TEM, TEFF) at 7 and 10 days post activation.
Compared to Ficoll, Sorterra enables rapid, automated isolation of leukocytes, consistently yielding more lymphocytes with negligible RBCs and platelet contamination while maintaining T cell and B cell function. By minimizing operator variability, Sorterra has the potential to transform cell separation by automating an outdated, variable, and labor-intensive process.
Intravital three-photon microscopy of entire mouse lymph node
Authors: Choe Kibaek , Hontani Yusaku , Wang Tianyu , Ouzounov Dimitre , Lai Kristine , Signh Ankur , Béguelin Wendy , Melnick Ari M. , Xu Chris 
Published: January 2023

Abstract
For the last 20 years, intravital confocal and two-photon microscopy have been powerful tools to explore dynamic immune cell behavior in mouse lymph node. However, they can only image ~100 and ~300 μm depth respectively while a peripheral lymph node of adult mouse has 600 – 1000 μm thickness. Here, we visualized whole thickness of adult mouse popliteal lymph node with intravital three-photon microscopy. Three-photon excitation significantly improved a signal-to-background ratio compared to two-photon excitation even at the same excitation wavelength. The capability to image the full depth enabled to achieve the 3D volume of entire lymph node vasculature in vivo by tiling multiple z-stack images. We observed DsRed-expressing lymphocyte migration in LYVE1+ lymphatic sinus at 600 μm depth of lymph node where conventional two-photon microscopy was normally inaccessible. In addition, we demonstrated the capability multi-color imaging by using Cγ1-Confetti mice of which each germinal center B cells stochastically expresses one of 4 different fluorescent proteins (CFP, GFP, YFP and RFP). Intravital three-photon microscopy has the potential to shed light on unknown immune cell behavior in deeper regions of the lymph node in vivo just as two-photon microscopy did during the last 20 years at the shallower depth.
Development of a Semi-automated Monocyte Monolayer Assay (MMA) to Determine the Clinical Significance of Red Cell Alloantibodies
Authors: Dean Melinda M , Tran Thu V , Ji Yu , Powley Tanya , Gu Yuan Tong , Sauret Emilie , Flower Robert L 
Published: January 2023

Abstract

BACKGROUND
One risk of allogeneic blood transfusion is the development of alloantibodies against red blood cell (RBC) antigens not found on the recipient’s RBC. The monocyte monolayer assay (MMA) is an in vitro model which has been used to determine the clinical significance of alloantibodies and predict post-transfusion RBC survival. We aimed to develop a semi-automated procedure to reduce assay time and facilitate improved patient management.

METHODS
PBMCs isolated using Sepmate tubes (STEMCELL Technologies) were added to Lab-Tek 8-well chamber slides (37°C, 1 hr, CO2). Antigen matched RBC were sensitised with plasma with known anti-RBC antibodies (anti-D n=3, anti-Yta n=4) (37°C, 1 hr, CO2). A modified Wright-Giemsa staining protocol was performed (Aerospray Hematology Pro 2 stainer, ELITechGroup) and the proportion of bound and/or phagocytosed RBC was determined via microscopy (&gt;5% considered clinically significant).

RESULTS
The introduction of PBMC isolation using sepmate tubes and automated cell staining reduced technical requirements, improved assay reproducibility and reduced assay time by &gt;2 hours. For the alloantibodies tested, 67% of the anti-D and none of the anti-Yta were clinically significant.

CONCLUSIONS
We developed a MMA with improved reproducibility and reduced turnaround time. This assay will improve blood transfusion safety and facilitate better patient management by reducing the risk of incompatible blood transfusion.
STING Negatively Regulates Allogeneic T Cell Responses by Constraining Function of Antigen Presenting Cells
Authors: Wu Yongxia , Mealer Corey , Sofi Mohammed , Tian Linlu , Bastian David , Schutt Steven , Choi Hee-Jin , Tang Chih-Hang Anthony , Hu Chih-Chi Andrew , Yu Xue-Zhong 
Published: January 2023

Abstract
Stimulator of interferon genes (STING) plays an important role in eliciting innate immune responses by sensing tumor and microbial DNA in anti-tumor and anti-infection responses, respectively. How the STING signal affects allogeneic response is not clear. To address this question, we utilized murine models of allogeneic hematopoietic stem cell transplantation (allo-HCT). By transferring donor bone marrow (BM) and T cells into allogeneic recipients, we found that significantly more severe graft-versus-host disease (GVHD) was induced in STING−/− recipients as compared to WT controls. By generating BM-chimeric mice in which STING was deficient in hematopoietic or non-hematopoietic antigen-presenting cells (APCs), we confirmed that STING on hematopoietic cells was primarily responsible for constraining host APC function. We further demonstrated that STING on host CD11c+ APCs played a predominant role in the regulation of allogenic T-cell responses. Mechanistically, we found that host CD11c+IAb+ cells deficient for STING could survive better and be activated more strongly after allo-HCT. As a consequence, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues, resulting accelerated/exacerbated GVHD after allo-HCT. Using pharmacologic approaches, we further demonstrated that systemic administration of STING agonist (c-diGMP) on recipient mice before irradiation significantly reduced GVHD mortality. In conclusion, we reveal a novel role of STING in APC activity that dictates T-cell allogenic responses, and validate STING as a potential therapeutic target for controlling GVHD after allo-HCT.