bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Capacity Immunomagnetic Isolation of Apheresis Samples in an Automated Closed System
Authors: Hsu Faye , Sa Silin , Yu Liping 
Published: February 2023

Abstract
Cell therapy development, including Chimeric antigen receptor (CAR)-T cells, involves many steps and requires processing on average 3 billion white blood cells or more. Automated large scale immunomagnetic cell isolation with high efficiency and throughput in a closed system is desired to enrich T cells from apheresis products.
Current automated, high capacity methods are time consuming, high cost, low yield and can potentially influence the physiology of the target cells. We have developed an automated closed magnetic separation platform, MARS® Bar to address these problems. This platform uses three uniquely designed magnetic separation modules and each magnetic module can perform positive or negative selection of target cells in a fluidic chamber at high flow rate (1mL/min per module). On this platform we have developed cell isolation reagents and program with minimal cell preparation. In the pilot study, we isolated 116 million CD3+ T cells from 267million CD45+ cells in a single LSR chamber on a single MARS® module. The entire automated process took less than 15 minutes to complete and resulted in &gt; 95% purity and &gt; 96% recovery of CD3+ T cells. The isolated cells were then activated and expanded, with results showing high proliferation and the dominant population being central memory T cells. The unique MARS® Bar matrix-free design has unlimited capacity because of the automated release of positively captured cells and very low carry-over. This was validated by processing greater than 3 billion leukocytes at 20 million cells per mL within the three modules in parallel and intermittent release of captured cells for collection. The MARS® Bar can be easily integrated into standard T cell production workflow.
High-Throughput and High-Dimensional Single Cell Analysis of Antigen-Specific CD8+ T cells
Authors: Jiang Ning , Ma Ke-yue , Schonnesen Alexandra A. , He Chenfeng , Xia Amanda , Sun Eric , Chen Eunise , Sebastian Katherine , Balderas Robert , Kulkarni-Date Mrinalini 
Published: February 2023

Abstract
Although critical to T cell function, antigen specificity is often omitted in high-throughput multi-omics based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell receptor sequencing (TetTCR-SeqHD) method to simultaneously profile TCR sequences, cognate antigen specificities, targeted gene-expression, and surface-protein expression from tens of thousands of single cells. Using polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrated over 98% precision for detecting the correct antigen specificity. We also evaluated gene-expression and phenotypic differences among antigen-specific CD8+ T cells and characterized phenotype signatures of influenza- and EBV-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we applied TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in type 1 diabetes patients compared to healthy controls, and discovered a TCR that cross reacts between diabetic and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses.
An Accessible Automatic Cell Counting Methodology Using Trainable Weka Segmentation in ImageJ Validated on Murine Microglia
Authors: Kataras Theo J , Koury Jeffrey , Singh Hina , Kaul Marcus 
Published: February 2023

Abstract
Counting cells is a cornerstone of tracking disease progression in neuroscience. A common approach for this process is having trained researchers individually select and count cells within an image, which is not only difficult to standardize but also very time-consuming. We present an automatic cell counting methodology which leverages the Trainable WEKA Segmentation (TWS) plugin available in the FIJI distribution of ImageJ (Schindelin 2012, Schneider 2012). TWS provides access to machine learning tools for object segmentation with an accessible graphical user interface. However, to analyze whole datasets requires further automation through the use of macros which we constructed within ImageJ maintain this high level of accessibility.
We compared our automatic counts to a published, hand counted dataset of Immunofluorescence stained mouse cortex microglia (Singh et al. 2020). For this analysis we used a wild-type (WT) control and a transgenic mouse model for HIV-induced brain injury (HIVgp120). HIVgp120 mice showed behavioral deficits, and increase in microglia numbers presumably caused the gp120 protein. 10X images were collected in the sagittal plane from brain slices of three 11–14 month-old male mice for each genotype.
The auto counting strategy with TWS replicated the increase in mean microglial density in images from the HIVgp120 mice (Manual p =0.0001; Auto p=0.0002). The auto count also correlates significantly with the manual counts in both genotypes (WT p= 2.297 e−07, adj. R2= 0.65; HIVgp120 p=2.591 e−04 adj. R2=0.423). The auto counting strategy took less than a fifth of the time for the expert manual count, allowing for the efficient exploration of large sets of image data.
Lumit&[trade] Immunoassays for Simple and Rapid Determination of Cytokine Release from Cell Culture Models
Authors: Lazar Dan F , Hsiao Kevin , Sondgeroth Casey , Gilden Julia , Kupcho Kevin , Thompson David , O’Brien Martha , Cali James 
Published: February 2023

Abstract
Rapid (&lt; 70 min), add-and-read assays for cytokine determination were developed with Lumit™ immunoassay technology. Separate antibodies labeled with complementary SmBiT or LgBiT subunits bind to their shared cytokine target, resulting in proximity-driven reconstitution of NanoBiT luciferase and luminescence signal. Lumit immunoassays for human IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α exhibit LLODs &lt; 10 pg/ml and linearity &gt; 3 logs of analyte in 96-well format. Maximal S/B ratios are greater than 1000, and an assay signal T1/2 of 2 h accommodates batch plate processing. 384-well, assay performance (Z′ &gt; 0.7) on treated human peripheral blood mononuclear cells (hPBMC) indicates the assay format is amenable to screening applications.
Assay reagents were added directly to cell wells for multiple culture models. For hPBMC, LPS, R848, or a combination of PMA and ionomycin (CSC) produced dose-and time-dependent release of cytokines with a wide range of responses, all measurable without sample dilution. In a mix of purified CD8+ T cells and target Raji B cells treated with the bispecific T-cell engager Blincyto®, IL-2, IFN-γ, and TNF-α release was observed with an EC50 of ~0.2 ng/ml and max S/B ratios of 82-, 168-, and 31-fold, respectively. For Th2 cells, significant release of IL-4 was observed in response to CSC treatment. An alternative to the no-transfer assay method, split-sample analysis, can be performed to easily determine the levels of multiple cytokines released in the same cell well.
The implementation of this novel detection chemistry will enable simple and rapid assay of cytokine release from cells for both low-and high-throughput applications, including quantitative assessments of T cell activation and differentiation.
StarBright Dyes: New Tools for Flow Cytometry
Authors: Green Angela M , Cichewiz Magdalena , Li Lei 
Published: February 2023

Abstract
StarBright Dyes are fluorescent nanoparticles, with superior brightness over conventional flow cytometry dyes. They have narrow excitation and emission peaks, hence low spillover, spreading and compensation, providing greater flexibility when building multiplex panels in flow cytometry. StarBright Dyes have been conjugated to numerous antibodies against surface markers found on human and mouse cells. High stability and batch consistency have been confirmed using the 5-laser and 27-detector ZE5 Cell Analyzer. The antibody conjugates also showed high reagent compatibilities, without a need for special cell staining buffers even in multiplex panels with additional conjugates and can be pre-mixed and used at a later date. Furthermore, they are able to reliably identify positive cells regardless of the staining buffer and conditions used, providing flexibility and helping researchers avoid false negatives. StarBright Dyes are new valuable tools to fulfill unmet needs in immunology-based flow cytometry.
Flt3 ligand expressed on the surface of T-cells contributes to effector function
Authors: Molina Megan Stanley , Stokes Jessica , Hoffman Emely , Simpson Richard J , Katsanis Emmanuel 
Published: February 2023

Abstract
Flt3 ligand (FL) is a growth factor that contributes to the homeostasis of the dendritic cell (DC) compartment. FL provides signals for differentiation, development, and survival by binding to the Flt3 receptor tyrosine kinase. Flt3 expression is maintained on DCs through their terminal differentiation. Endogenously, FL is produced by stromal cells and select immune cells, including T-cells and some DC subsets. FL may be expressed as a membrane-bound form (mFL), with no intracellular signaling domain, and may undergo cell-autonomous shedding to generate soluble FL. This is believed to contribute to a localized source of FL for those DC subsets, however T-cells do not express Flt3 and have no FL requirement. As such, it remains unclear why T-cells express mFL on their surface. Interestingly, FL-driven DCs are more steady state-like, secreting less pro-inflammatory cytokines, and can suppress graft-versus-host disease (GvHD). We hypothesized that mFL on T-cells may regulate effector responses through binding to Flt3 on DCs. To test this, we compared GvHD in a murine bone marrow transplant model using WT compared to FL−/− donor cells. Engraftment, T-cell reconstitution, and activation were monitored weekly. We also performed mixed leukocyte reactions (MLR) to measure T-cell proliferation in WT versus FL−/− T-cells. Infusion of FL−/− donor cells resulted in significantly worse GvHD and higher expression of T-cell activation markers CD25 and CD69 in vivo. The absence of mFL also resulted in significantly enhanced alloreactive T-cell proliferation in vitro. These studies indicate that mFL expressed on the surface of T-cells can alter their effector function, which may be a result of the engagement of mFL with Flt3 receptor expressed on DCs.
Vγ2+ gamma-delta (γδ) T cells promotes the survival of skin allografts in the presence of anti-CD40L induced transplantation tolerance
Authors: Lal Girdhari , Giri Shilpi 
Published: February 2023

Abstract
Donor-specific transfusion (DST) and anti-CD40L treatment is one of the best tolerogenic strategies for promoting the survival of allografts. The effect of DST and anti-CD40L treatment on the phenotype and function of most of the innate and adaptive immune cells are known. Gamma delta (γδ) T cells uniquely possess features of both innate and adaptive immune cells and perform crucial functions during cancer and autoimmune diseases. Recent reports have suggested that γδ T cells promote skin allograft rejection but their contribution during costimulatory blockade induced skin allograft survival is not known. In the present study, we show that depletion of γδ T cells or specifically Vδ2+ γδ T cells led to acute rejection of skin allografts even with DST and anti-CD40L mAb treatment. Tolerogen treatment increased the expression of regulatory receptor CD39 on Vδ2+ γδ T cells. Tolerogen treatment reduced IFN-γ expression in Vγ2+ γδ T cells which in-turn suppressed the Th1 differentiation of naive CD4+T cells. Depletion of Vγ2+ γδ T cells induced hyper IFN-γ response in tolerogen-treated allograft recipients. Tolerogen-treated and Vγ2+ γδ T cells-depleted recipients showed enhanced infiltration of activated macrophages in the skin allografts. Adoptive transfer of Vγ2+ γδ T cells purified from tolerized mice in naive mice promoted the survival of skin allografts. Taken together, our results showed that Vγ2+ γδ subset of T cells promote costimulatory blockade-induced prolonged survival of allografts and suggest its importance in adoptive cellular therapy to control the allograft rejection.
The effects of UW Solutions in preventing ischemia induced apoptosis of retinal ganglion cells after whole eye transplantation
Authors: Khatter Neil J. , Matlock Tyler , Owens Charles R. , Li Bing , Wong Yong , Huang Christene A. , Nickells Robert W. , Su An-Jey A. , Washington Kia M. 
Published: February 2023

Abstract

Background
Retinal ganglion cell (RGC) death from ischemia reperfusion injury and optic nerve transection are challenges to successful restoration of vision by whole eye transplantation (WET). We examined whether University of Wisconsin Solution (UW) or a modified version which includes valproic acid and BaCl2 (mUW) can preserve RGCs in a WET model.

Methods
24 hemifacial rat flaps prepared for WET were flushed with heparinized saline, UW, or mUW. After 85 minutes of ischemia, donor eyes were injected with the same solutions and prepared for immunohistochemistry (IHC) analysis before (n=6/group) or POD7 after (n=3/group) syngeneic WET. Donor and untreated non-ischemic naïve (n=4) retinas were stained for Brn3a and p-cJun (PNJ) to assess RGC viability and apoptosis respectively.

Results
After 85 minutes of ischemia, all donor eyes across treatments (saline, UW, or mUW), had equal levels of Brn3a+ and PNJ+ expression (P&gt;0.05). Naïve eyes averaged ~100k Brn3a+ and ~18k PNJ+ RGCs. At POD7, saline and UW donor eyes each had ~20% loss in Brn3a+ RGCs as compared to control naïve eyes (P&lt;0.05). At POD7, apoptotic signaling increased in both UW and saline donor eye, doubling in PNJ+ signal compared to non-ischemic naïve eyes (P&lt;0.05).

Conclusions
Using a novel system to asses RGC degradation and apoptosis in an experimental rodent WET model, we found that UW and mUW solutions as formulated did not preserve RGCs after WET, despite reports of their anti-ischemic properties in other studies.
Durvalumab and Lenvatinib in Participants With Locally Advanced and Metastatic Hepatocellular Carcinoma (Dulect2020-1)
Authors: Feng Hao , Tong Huan , Xia Qiang 
Published: February 2023

Abstract

Background:
This study will evaluate the safety and efficacy of durvalumab in combination with lenvatinib in participants with locally advanced hepatocellular carcinoma before liver transplant and metastatic unresectable HCC. The primary hypothesis of this study is that patients with locally advanced HCC could benefit from durvalumab plus lenvatinib before liver transplant; patients with metastatic unresectable HCC could also benefit from durvalumab plus lenvatinib concerning: 1) Progression-Free Survival (PFS); or recurrence-free survival (RFS) if patients with locally advanced HCC underwent liver transplant; 2) Objective Response Rate (ORR), and 3) Overall survival (OS).

Methods:
The investigators design a clinical study to explore whether the combination above as a treatment in patients with advanced and recurrent endometrial carcinoma could prolong PFS and analyze potential immune biomarkers of therapeutic response. This trial is designed as a prospective, open-label study for patients with locally advanced hepatocellular carcinoma before liver transplant and metastatic unresectable HCC (NCT04443322).

Results:
20 patients with pathologically or cytologically proved locally advanced or metastatic hepatocellular carcinoma were recruited. The combination therapy of Lenvatinib 80–120mg daily orally and durvalumab 1500mg by IV infusion every 4 weeks were performed. PFS and RFS were evaluated as the primary endpoint. ORR, OS, and Percentage of Participants who Experience an Adverse Event (AE) were assessed as the secondary endpoints.

Conclusion:
Durvalumab, combined with Lenvatinib in participants with locally advanced and metastatic hepatocellular carcinoma, is safe and effective.
Distinct phenotype of antibody-suppressor CXCR5+CD8+ T cells
Authors: Zimmerer Jason M , Han Jing , Hart Madison , Chaudhari Sachi , Peterson Chelsea M , Bumgardner Ginny L 
Published: February 2023

Abstract
The novel CXCR5+CD8+ T cell subset has been shown to inhibit alloantibody production (termed CD8+ TAb-supp cells) but do not reject allografts. In contrast, CXCR3+CD8+ T cells reject allogeneic transplants (but do not inhibit antibody). Recent literature indicates there are many CXCR5+CD8+ T cell subsets with different effector functions (anti-viral, anti-tumor, anti-autoimmune, or antibody enhancement). By investigating CD8+ TAb-supp cell by RNA-seq and flow cytometry, we aimed to determine a profile that distinguishes this novel subset. To investigate this, C57BL/6 (H-2b) were transplanted with FVB/N (H-2q) hepatocytes and CD8+ T cell subsets were analyzed on day 7. Alloprimed CXCR5+CD8+ T cells were compared to naïve CD8+ T cells and alloprimed CXCR3+CD8+ T cells. Fluorescence minus one controls were used to determine background staining (flow cytometry). RNA-seq analysis show that 1670 transcripts were upregulated or downregulated when comparing between flow-sorted alloprimed (CD62L−CD44+) CXCR5+CD8+ T cells or naïve (CD62L+CD44−) CD8+ T cells. Transcripts of note include Bcl-6 (3.3-fold upregulated), CXCR3 (8.4-fold downregulated), and S1PR3 (140-fold upregulated). Flow cytometry analysis suggests that CD8+ TAb-supp cells are short-lived effectors cells based on the expression of CD44, KLRG1, and CD122 (not CD62L or CD127). An expansive flow analysis panel comparing differences in transcript and protein expression between alloprimed CXCR5+CD8+ TAb-supp cells and other reported CXCR5+CD8+ T cell subsets indicates that the absence of PD-1, IL-10, and FoxP3 expression distinguishes CD8+ TAb-supp cells from all reported CXCR5+CD8+ T cell subsets (including CD8+ T follicular regulatory cells).
MHC II expression by donor bone marrow-derived macrophages underpins neuroinflammation and cognitive deficits during murine chronic GVHD
Authors: Adams Rachael C , Shaik Samreen N , Llanes Genesis T , Carter-Cusack Dylan , Blazar Bruce B , Vukovic Jana , MacDonald Kelli PA 
Published: February 2023

Abstract
Chronic graft-versus-host disease (cGVHD) remains the leading cause of non-relapse mortality after allogeneic stem cell transplantation (SCT) for haematological malignancies. Manifestations of GVHD in the central nervous system (CNS) present as neurocognitive dysfunction in a significant proportion of patients. Neuroinflammation in the cortex of mice and humans in acute GVHD is dependent on host resident microglia-derived TNF. However, the mechanisms driving cGVHD in the CNS are yet to be elucidated. Here, our studies of murine cGVHD revealed a hippocampal-dependent spatial learning and memory deficit associated with inflammation and persistent IFN-γ upregulation. Hippocampal RNA sequencing identified perturbations to structural and functional synapse-related gene expression, in the context of upregulated genes associated with antigen presentation and IFN-γ responses. Brain T cell infiltration was initially dominated by CD8 T cells with an eventual expansion in CD4 T cells, in parallel with a marked infiltration of MHC Class II-expressing bone marrow-derived macrophages (BMDM). After day 70, BMDM constituted 50% of the brain macrophage pool and exhibited a transcriptional signature distinct from host resident microglia. Transfer of MHC class II–deficient BM grafts resulted in attenuated neuroinflammation, implicating donor BMDM as critical mediators of CNS cGVHD. Our identification of the hippocampus as a cGVHD target, and disease mediators distinct from those in the acute phase, highlights the necessity to pursue alternative therapeutic targets for late-stage neurological manifestations post-SCT.
Intranasal adjuvant immunotherapy increases survival in murine metastatic Lymphangioleiomyomatosis (LAM)
Authors: Amosu Mayowa , Maisel Katharina 
Published: February 2023

Abstract
Pulmonary lymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age. LAM is characterized by the abnormal growth of smooth muscle-like cells leading to cystic destruction of the lungs. Rapamycin, the only FDA approved treatment for LAM, slows disease progression but patient responses are incomplete. Therefore there is a critical need for new LAM treatments. Research has shown that LAM has several characteristics of cancer including suspected immune suppression. We and others recently found that LAM may be treated using immune checkpoint inhibitors (ICI), a common cancer immunotherapy. Here, we investigate immune-activating adjuvants as local anti-LAM immunotherapies.
Using an established murine model of metastatic LAM, we determined survival after twice weekly intranasal treatments of CpG, PolyI:C, or MPLA. We used flow cytometry and immunofluorescence to determine how adjuvants alter the immune response in LAM.
We found that intranasal treatment with CpG and PolyI:C, but not MPLA, increased survival in mice with LAM. In more progressive LAM disease, we observed an increase in activated CD8 T cells, and a decrease in B cell, macrophage, and eosinophil infiltration in lungs of CpG treated vs untreated LAM mice. We also observed increased expression of MHC-II on alveolar macrophages but decreased numbers of migratory dendritic cells in CpG treated LAM lungs.
These results suggest that CpG immunotherapy in LAM lungs activates cytotoxic lymphocytes while tempering other inflammatory cells like eosinophils. Our findings also suggest that LAM lungs may respond favorably to adjuvant immunotherapy, and that combination ICI-adjuvant therapy could be synergistic.
Humanized mice challenged with TNBC tumor develop graft versus host disease and anemia limiting vaccine immunotherapy studies
Authors: Bommireddy Ramireddy , Munoz Luis E , Pack Chrstopher , Ramachandiran Sampath , Reddy Shaker , Selvaraj Periasamy 
Published: February 2023

Abstract
Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).