bluuebunny/crossref_metadata_2025 huggingface dataset:
Human liver CD69+CD103−CD8+ memory T cells are regulated by HIF-2α
Authors: Kim Jong Hoon , Han Ji Won , Choi Young Joon , Rha Min-Seok , Koh June-Young , Park Jun Yong , Park Su-Hyung , Joo Dong Jin , Shin Eui-Cheol
Published: January 2023

Human liver CD69+CD8+ T cells are ~95% CD103− and ~5% CD103+. Although CD69+CD103+CD8+ T cells show tissue residency and robustly respond to antigens, CD69+CD103−CD8+ T cells are not yet well understood. Here, we report that human liver CD69+CD103−CD8+ T cells exhibit a phenotype of tissue residency and terminal differentiation with hypoxia-inducible factor (HIF)-2α upregulation. CD103− T cells included both hepatotropic and non-hepatotropic viruses-specific cells whereas CD103+ T cells did only hepatotropic virus-specific cells. Although CD103− cells were weaker effectors on a per cell basis than CD103+ cells following T-cell receptor or interleukin-15 stimulation, they remained the major CD69+CD8+ effector population in the liver, surviving with less cell death. HIF-2α inhibitor or knockdown suppressed the effector functions and survival of CD69+CD103−CD8+ T cells. In addition, HIF-2α expression in liver CD69+CD103−CD8+ T cells was significantly increased in patients with acute hepatitis A or liver cirrhosis (LC). In patients with LC, the activation and increased function of liver CD69+CD103− CD8+ T cells were associated with their HIF-2α expression, which could be controlled by HIF-2α inhibition. In summary, we show that human liver CD69+CD103−CD8+ T cells showed terminally differentiated tissue resident phenotype regulated by HIF-2α.
Specific Subset of Dendritic Cells Control the Priming of Mucosal Th17 Cells
Authors: Ngoi Soo , Yang Yi , Iwanowycz Stephen , Liu Bei
Published: January 2023

Phagocytic mononuclear cells play an essential role in regulating gut immune homeostasis by sensing, internalizing microbial parts, and subsequently driving adaptive response in an antigen- and cytokine-dependent context. In a healthy host, physiological Th17 cells are predominantly generated in the gut, induced by local commensal microbiota. However, the precise subpopulation of antigen-presenting cells responsible for imprinting Th17 program in naïve CD4 T cells have not yet been definitively identified.

Objective

Mapping the functional subset of Th17-inducing dendritic cells (DCs) will uncover new targets for mucosal vaccine design.

Methods & Results

Using transgenic CD4 T cells specifically recognizing a gut-colonizing commensal bacterial antigen, we found that migratory DCs (CD11chiMHCIIhiZbtb46+) in the mesenteric lymph nodes are indispensable for the initial Th17 cell priming. Whereas the absence of CD103+ or CX3CR1+ DCs did not impact Th17 cell differentiation. Single cell RNA sequencing revealed that the migratory DCs are highly heterogeneous and consist of 8 clusters of transcriptionally specialized subsets. Interleukin-6, the Th17-polarizing cytokine, is expressed by a minor population of DCs with a type I interferon signature. Our results indicate that during homeostasis, Th17 cells activation in the mesenteric lymph nodes and later persistence in the small intestine may be controlled and sustained by different groups of antigen-presenting cells. The complexity and heterogeneity of mucosal DCs imply that other effector and regulatory T cell responses may be regulated by different DC subsets at this unique tissue environment.
Immune phenotypic analysis using pre-designed BD® AbSeq pan immune-response panel
Authors: Song Hye-Won , Narayan Punya , Tyznik Aaron J , Martin Jody , Lazaruk Katherine , Mortimer Stefanie
Published: January 2023

High-throughput single cell RNA sequencing (scRNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional scRNA-seq captures information at transcript level, recent technology using DNA-barcoded antibodies enable multiomic analysis, examining protein alongside mRNA. Although this approach allows high parameter antibody panels, the sequencing cost can escalate quickly for high plexy panel with high antigen density markers. Therefore, thought should be put on panel design in order to gain meaningful results efficiently. Here, we have designed a panel for analyzing multiple immune responses, BD® AbSeq pan immune-response panel. Our immune response panel excludes abundant cell lineage markers which take up a huge amount of reads but provide only cell lineage information that can be easily resolved by RNA profiles only. This panel is focused on analyzing the various immune responses in multiple cell lineages. Therefore, it has a potential to be used to uncover phenotypic changes upon disease status with various immune stimuli. To test the capability of this AbSeq panel in discovery of phenotypic changes in multiple immune response, we have tested it in the various scenarios. We propose that the BD® AbSeq pan immune-response panel can be used to evaluate unpredictable disease phenotypes to identify the cell types affected from the disease as well as the responsible molecular pathways of the disease at a single cell level.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
A Novel IL2-based Immunotherapeutic Protein Prevents the Development of Atherosclerosis in ApoE−/− mice and LDLR−/− mice
Authors: li Qiongzhen , Zhu Xiaoyun , Spanoudis Catherine , Ravelo Kristine , Dee Mike , Liu Bai , Prendes Caitlin , You Lijing , Wong Hing
Published: January 2023

Atherosclerosis is the leading cause of coronary heart disease and stroke. Sterile Inflammation, resulting from activation of the inflammasome NLRP3, plays a key role in the pathophysiology of atherosclerosis. Studies suggested that low-dose rIL-2 may be effective in treating autoimmune and inflammatory diseases by inducing Treg cells. Since rIL-2 has unfavorable pharmacokinetic profile in humans, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain, to extend the half-live of IL-2 for Treg expansion. In vitro treatment of human PBMCs with HCW9302 significantly expanded CD4+/CD25+/FoxP3+ Treg cells without activating CD4+ and CD8+ T cells over a broader concentration range than rIL2. Subcutaneous injection of HCW9302 (3 mg/kg every 2 – 3 weeks) into atherosclerosis-prone mice (ApoE−/− mice and LDLR−/− mice fed the Western Diet (WD) for 6 weeks) significantly increased the percentage of Treg cells, NK cells and CD8+ T cells in blood compared to the PBS-treated control group (p<0.001). Cytokines associated with atherosclerosis, including IL-1β and MCP-1, were also significantly decreased in the HCW9302-treated group, suggesting HCW9302 can moderate inflammasome NLRP3 activities. Remarkably, HCW9302 lowered the fasting blood glucose and insulin resistance. In aortas evaluated by the en face method at week 13 of WD, the ratio of plaque size to aortic surface was also significantly lower in HCW9302-treated mice compared to controls. HCW9302 treatment was well tolerated in both models. In summary, HCW9302 can effectively prevent the development of atherosclerosis in ApoE−/− mice and LDLR−/− mice, potentially through expansion of Treg cells.
Infinity Flow: comprehensive single-cell protein profiling via massively parallel flow cytometry and machine learning
Authors: Becht Etienne , Headley Mark , Newell Evan , Gottardo Raphael
Published: January 2023

Modern flow cytometers can quantify around 20 proteins at the single-cell level. Higher-dimensional alternatives exist, such as mass cytometry, or oligonucleotides-tagged antibodies - but they feature lower cellular throughput, higher cost and are not as widely available. To overcome these limitations, we propose a pipeline which allows the quantification of hundreds of proteins across millions of single cells.

Experimentally, cells are stained with a customizable 10–20 “backbone” antibody panel which defines the cellular population structure of the sample. This is followed by aliquoting into hundreds (200–300) of individual wells, each containing a unique “exploratory” antibody. Data acquisition is performed using conventional flow cytometry.

Computationally, multivariate non-linear regression models are trained, one for each well. These models learn how to impute an exploratory antibody signal from backbone measurements. The models are each applied to the backbone data to obtain hundreds of imputed measurements across millions of single cells.

We applied this workflow cell suspensions from the lungs of C57/B6 mice, resulting in 2,660,000 events with imputed expression levels for 266 proteins. Imputed expression and co-expression patterns are accurate and consensual across regression models, allowing near-exhaustive annotation of both immune and non-immune cells, and finer granularity than dimensionality reduction or clustering.

By enabling broad characterization of cellular networks at the protein level, this pipeline is well-suited for systems immunology approaches. Given the wealth of data generated by this pipeline and its accessibility, we anticipate it will be widely adopted.
Cyto-feature engineering: a pipeline for flow cytometry analysis to uncover immune populations and association with disease
Authors: Fox Amy , Dutt Taru S. , Karger Burton , López Mauricio Rojas , Obregón-Henao Andrés , Anderson G. Brooke , Henao-Tamayo Marcela I
Published: January 2023

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data, from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline’s modular nature allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.
Detection and Quantification of Extracellular Vesicles with Cargo Protein and RNA Using the Amnis® CellStream® Flow Cytometer
Authors: Kong Raymond K , Pugsley Haley Renee
Published: January 2023

Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. These EVs—released under normal physiological conditions as well as in the pathogenesis of neurological, vascular, hematological, and autoimmune diseases—have been shown to transfer biological molecules such as protein and RNA between cells, potentially transmitting signals. To understand more about these signaling mechanisms, there is a need for detecting and quantifying EVs with cargo protein and RNA in a reproducible and reliable manner. However, this has been challenging due to the small size of EVs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. Here, we showcase the Amnis® CellStream® Flow Cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. We demonstrate that a charge-coupled device (CCD)-based, time-delay-integration image capturing system can be used to detect and quantify EVs and their cargo labeled with ExoGlow™-Protein or ExoGlow™-RNA.
A cloud-based bioinformatic and analytic infrastructure for the Expanded Program on Immunization Consortium
Authors: Vignolo Sofia , Diray-Arce Joann , McEnaney Kerry , Rao Shun , Consortium EPIC-HIPC , Ozonoff Al
Published: January 2023

The overarching goal of the Expanded Program for Immunization Consortium - Human Immunology Project Consortium (EPIC-HIPC) is to identify and characterize vaccine-induced neonatal responses and define biomarkers that may predict immunogenicity. Key to this effort is the establishment of the Data Management Core (DMC) to provide reliable clinical data and bioinformatic infrastructure for centralized curation, storage, and analysis of multiple deidentified ‘omics datasets. The DMC established a cloud-based platform to track, store, and share data according to set standards using Amazon Web Services (AWS). In our clinical core sites, biosamples collected and shipped across sites are tracked using ItemTracker via AWS Elastic Compute Cloud while their associated clinical data are captured using Research Electronic Data Capture software. Multi-omic datasets are stored in access-regulated Amazon Simple Storage Service (S3) for file version control. All data must complete quality control (QC) processes by the site generating said data, which is then exported to the DMC for quality assurance (QA). Data integration is performed using RStudio Server Pro which directly imports the data files from Amazon S3 via a controlled computing environment. The DMC deposits finalized datasets onto public repositories to be shared openly upon publication. Completion of our goal will provide the resources, planning, and scientific expertise to make this discovery platform possible. Robust DMC operations will allow rapid sharing of integrative results across the entire team. Maintenance of standards and public deposition of high quality ‘omics data will further advance scientific progress for the benefit of vaccine development and public health.
A single-marker method of renal cDC2 isolation
Authors: Bates Briana L , Mikulin Joseph A , Wilson Timothy J
Published: January 2023

Because of their ability to present antigens, respond to inflammatory signals, and produce critical acute-phase cytokines, renal dendritic cells are of particular interest to those studying various disease states of the kidney. Markers such as CD11c, MHC-II, CX3CR1, and CD11b have historically been used in the murine system to isolate renal dendritic cells from the various parenchymal cells among which they’re embedded. In this study, we have determined that signaling lymphocyte activation molecule family member 9 (SLAMF9) is sufficient to mark the population of CD11b+ MHCIIhi CX3CR1+ CD11c+ F4/80+ cells in the murine kidney under homeostatic conditions. Using SLAMF9, we have devised a single-marker method of renal cDC2 isolation via fluorescence-activated cell sorting (FACS). This method will allow isolation of mouse CD11b+ dendritic cells with minimal surface receptor interference.
Regulation of body temperature in Canadian beef cattle of various immune response phenotypes when the temperature humidity index is above normal
Authors: Husseini Nasrin , Altvater-Hughes Tess , Beard Shannon , Hodgins Douglas , Mallard Bonnie A
Published: January 2023

Climate change with increases in ambient temperature and humidity, affect animal life dramatically decreasing their production and reproduction potential, as well as making them more susceptible to different diseases. This leads to an increase in their core body temperature and respiration rate which in turn decreases their production, reproduction, and immune function. The High Immune Response (HIR™) technology which has been developed at the University of Guelph ranks animals using two arms of adaptive immune system to high (H), average (A), and low (L) immune responders. (H) immune responders have been reported to have fewer incidents of disease and robust immune response. (H) immune responders have been also reported to have better colostrum and milk quality making them an ideal model to examine the effects of climate change on health traits. The objective of this study was to check if (H) immune responder beef cattle are able to regulate their rectal temperature better. In this study, rectal temperatures of 36 beef cows were recorded during normal Temperature-Humidity Index (THI<74) and above normal THI (THI ≥ 74) once in the morning and once in the afternoon. Results indicated that 62.5% of cows showed increases in rectal temperature in THI ≥ 74. However, mean rectal temperatures of (H) antibody-mediated immune responders did not differ significantly during THI < 74 and THI ≥ 74; indicating that these high antibody responder beef cows were better able to regulate their body temperatures.

This is the first time that genetic regulation of an immune response trait has been shown to influence the response to in vivo heat stress indicating that it may be possible to select cattle with both improved health and heat tolerance.
Development of a competition ELISA (cELISA) for specific detection of Zika virus antibodies in human sera
Authors: Katz David , He Dan , Patrusheva Irina , Maria Clara Echevery G , Ortiz Carolina , Hughes Holly R , Moreno Alberto , Walia Vishakh , Hilliard Julia
Published: January 2023

Infections by Zika virus (ZIKV), a member of the Flavivirus genus, can be diagnosed by virus isolation, detection of virus RNA or by serological methods for detecting ZIKV immunoglobulin M antibodies (IgM), a marker of recent infections, and immunoglobulin G antibodies (IgG), a marker of past infections. In some cases, where IgM antibodies may last for longer periods, accurate diagnosis of recent infections is not always possible. Because commercial and non-commercial serological tests are often confounded by cross-reactivity with other Flaviviruses there is a need for simple, ZIKV-specific serological tests. Here, we present the development of a ZIKV-specific ELISA in which cross-reacting antibodies in serum samples are competed and blocked by Dengue virus antigens (DENV). The competed sera are then tested by ELISA on ZIKV and DENV coated wells to enable a calculation of a ZIKV/DENV (Z/D) cutoff ratio. For the development and validation of the ZIKV specific cELISA we have used three groups of sera from Colombia. 49 sera were obtained in 2018 from individuals that have been infected with ZIKV during a 2016 outbreak. The outbreak was in a DENV endemic area. Another group of 19 sera were obtained from a known DENV endemic area with no evidence of any ZIKV infections. The third group of 12 sera were obtained from patients from an area with no known Flavivirus infections and therefore considered as negative for both ZIKV and DENV. A Z/D ratio of 1.5 was determined as the cutoff value for the specific determination of ZIKV positive or negative sera. Our analysis indicated a 85% agreement with the BOB assay, and a 78% agreement with the commercial IgG ELISA test. The estimated specificity and sensitivity for the cELISA were 70% and 82%, respectively.
The EPIC data analytics platform for clinical mass cytometry
Authors: Wasser Martin , Yeo Joo Guan , Kumar Pavanish , Chew Valerie , Lim Chun Jye , Arkachaisri Thaschawee , Poh Su Li , Leong Jing Yao , Yeo Kee Thai , Albani Salvatore
Published: January 2023

Single cell technologies, such as high-dimensional cytometry, promise to enable the discovery of immune cell populations that will serve as clinical biomarkers. Recently, we reported the creation of a reference database of the healthy immune system from birth to old age and the development of the EPIC (Extended Poly-dimensional Immunome characterisation) data mining platform (Nature Biotech, accepted). Here we extend the analytics pipeline to facilitate detection of clinically stratifying cell populations in mass cytometry (CyTOF or cytometry by time-of-flight) data. Data structures called immune maps are used to integrate single cell protein expression data (over 40) of multiple samples with clinical metadata and phenotypic information inferred from automated clustering and assisted cell type annotation. Clustering is combined with batch effect correction to reduce technical while preserving biological variations. Subsequent single cell exploratory data analysis and statistical tests help to identify cell populations whose frequencies differ significantly between groups of patients. To gain further insights, users can compare their cytometry data with the healthy reference immunome by performing two types of analysis; (1) mapping to existing clusters helps obtain abundance estimates of immune cell types, (2) reclustering of uploaded data merged with reference immunomes uncovers differences to healthy immune profiles of different ages. We implemented the EPIC pipeline using R Shiny to provide interactive visualisation and an intuitive user interface. We will show examples on how the EPIC platform can characterise cellular diversity in the context of disease and the morphogenesis of the healthy immune system.
Alpha-synuclein specific regulatory T cells expanded with bee venom phospholipase A2 improves the neuroprotective effects in MPTP-induced mouse model of Parkinson’s disease
Authors: Park Seon-Young , Min Daeun
Published: January 2023

Parkinson’s disease (PD) is a long-term degenerative autoimmune disease that mainly affects the motor system. Cell therapy using regulatory T cells (Tregs) has been shown to be effective on PD progression. To improve the efficiency of Treg therapy on PD, alpha-synuclein (a-syn) was used as a self-antigen for enrichment of a-syn specific Tregs during ex vivo expansion. In addition, to solve the problem of changing the phenotype of Tregs during expansion, bee venom phospholipase A2 (bvPLA2) was treated. The results showed that treatment of bvPLA2 remains Tregs to naïve phenotype that possessed optimal suppressive activities. And a-syn presenting changed the TCR repertoire in Tregs. As a result, these changes in Tregs by bvPLA2 and a-syn improve the neuroprotective effects on MPTP-induced mouse model of PD. Motor function deficits induced MPTP intoxication was palliated by adoptive transfer of a-syn specific and bvPLA2 treated Treg. Adoptive transfer of a-syn specific and bvPLA2 treated Treg also reduced activated microglia, especially pro-inflammatory microglia in MPTP-induced PD model. Dopaminergic neurons in substantia nigra (SN) was dramatically protected from MPTP intoxication by the Tregs that has treated with a-syn and bvPLA2. Thus, adoptive transfer of a-syn specific and bvPLA2 treated Tregs shows substantial neuroprotective effects on MPTP-induced PD mice.
B Cell Maturation Antigen deficiency increases inflammatory B-cell functions in multiple sclerosis
Authors: Kumar Gaurav , Ko Rose M. , Axtell Robert C.
Published: January 2023

B cells have emerged as a therapeutic target for multiple sclerosis (MS). Depleting B cells with anti-CD20 antibodies are effective in treating MS. Yet, TACI-Ig treatment, which blocks BAFF and APRIL (two cytokines important for B cell development and function), paradoxically increased disease activity in MS patients. The stark differences in clinical outcomes with these therapies demonstrate that B cells have both inflammatory and regulatory effects in MS. B cell Maturation Antigen (BCMA), a receptor that binds BAFF and APRIL, plays a prominent role in B cell function. The role BCMA plays in MS remains enigmatic and therefore we explored the impact BCMA deficiency has on the development and progression of the experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Here, we identified that the expression of BCMA is dysregulated in B cells from MS patients and is important in inhibiting inflammatory B cell responses in EAE. We discovered that BCMA expression is decreased on transitional B cells in MS. In mice we found that EAE induced with MOG35–55 was severe in BCMA−/− mice compared to BCMA+/+. The increased disease was associated with increased CNS inflammation, significantly reduced transitional B cells and a skewing of B-cells toward a mature/inflammatory phenotype. Bone marrow chimeric and in vitro experiments demonstrated that BCMA directly inhibits memory B cell expansion and anti-inflammatory cytokine production by B cells. Strikingly, BCMA impacts the efficacy of B cell depleting therapies, as anti-CD20 antibody treatment attenuated EAE in BCMA-deficient mice but not BCMA-sufficient mice. These data provide novel insights into B-cell dysregulation in MS.
Immunomodulatory networks in multiple sclerosis
Authors: Ntranos Achillefs , Lee Brian , Xie Hui , Rockoff Jonathan , Brandstadter Rachel , Kim-Schulze Seunghee , Lublin Fred
Published: January 2023

Background

Dimethylfumarate (DMF) and ocrelizumab are two effective immunomodulators for multiple sclerosis but their mechanism of action involves different immune pathways. In this study we constructed immunomodulatory networks from cytokine and immunoprofiling data to evaluate the similarities and differences between these two disease modifying therapies in multiple sclerosis (MS).

Methods

Plasma and PBMCs from 16 and 13 MS patients were collected at baseline, 3 months and 6 months after treatment with DMF and ocrelizumab respectively. Cytokine analysis from plasma was performed with Olink assays and immunophenotyping was done by Cytof. Data were integrated with weighted correlation network analysis and highly interconnected modules were identified. Associations between modules and therapy was done with linear mixed effect models.

Results

DMF treatment was associated with a significant reduction of a module responsible for chemokine signaling pathways and cytokine-cytokine receptor interactions, whereas ocrelizumab treatment led to the increase of a module of soluble factors that are involved in tryptophan metabolism and foxp3 activation. DMF also reduced both T and B cell memory populations and ocrelizumab exerted its immunomodulatory effect by mostly reducing the frequency of memory B cells.

Conclusions

Our results suggest that DMF and ocrelizumab affect different immune networks to exert their effect in multiple sclerosis patients.
Recipient expression of C-type lectin receptor Mincle promotes rapid T cell homeostatic recovery following antibody mediated lymphoablation
Authors: Hasgur Suheyla , Fan Ran , Valujskikh Anna
Published: January 2023

Antibody-mediated lymphocyte depletion is widely used in transplantation. We recently showed that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) depends on B cells activated via CD40 and is mediated by B cell-derived IL-1β and IL-6. Mincle (Clec-4e) is a FcRγ-coupled C-type lectin receptor that binds ligands released by pathogens or damaged cells and signals through the Card-9 pathway. While Mincle functions are well characterized in myeloid cells, its significance in B cells remains to be determined. Our goal was to investigate the role of Mincle in initiating B cell cytokine production in lymphopenic heart allograft recipients. The NanoString and RT-PCR analyses of B cells showed that mATG depletion resulted in B cell up-regulation of Mincle and Card-9 gene expression. Flow cytometry confirmed cell surface Mincle expression in B cells from mATG-treated recipients but not from naïve mice. Treatment with anti-CD154 mAb in addition to mATG decreased the B cell expression of Mincle, IL-1β, IL-6 and TNF-α. Moreover, B cell IL-1β and TNF-α expression were reduced in mATG treated Mincle KO allograft recipients compared to WT controls. Most importantly, Mincle KO recipients treated with mATG had severely impaired T cell recovery and significantly prolonged allograft survival compared to WT recipients. We propose that B cells activation via CD40 results in up-regulation of cell surface Mincle. Endogenous ligands released from necrotic graft cells bind to Mincle thus inducing B cells to secrete proinflammatory cytokines and facilitate CD8+ T cell proliferation. These data identify Mincle as a novel therapeutic target to improve the efficacy of lymphoablation in allograft recipients.
Tertiary lymphoid organs in renal chronic allograft rejection
Authors: Daya Khodor Abou , Zhao Daqiang , Biery Kyle , Oberbarnscheidt Martin H.
Published: January 2023

Chronic allograft rejection remains a major obstacle to long-term allograft survival. The immunologic role of tertiary lymphoid organs (TLO) in allograft rejection is unclear. Here, we employed a chronic renal allograft rejection model in mice and intravital 2-photon microscopy to investigate the function of TLO in transplant rejection.

CB6F1 (F1) RIP-LTα (preformed TLO) or F1 (no TLO) kidney grafts were transplanted to WT B6 recipients and survival monitored. To investigate immunologic function of TLO, we adoptively transferred B6-RIPLTα CD11c-YFP mice with 10m naïve dsRed OT-I T cells or 10m CTR-labeled NP-specific B cells + 10m CFP+ OT-II cells and immunized with NP-OVA + alum. Intravital 2P imaging of renal TLO was performed at time points 0, 3, 6, 24 or 72 hours after immunization. 4D image analysis was performed and mean speed, displacement, arrest coefficient (AC) and contact times (CT) with DC were calculated for OT-I, OT-II and NP-B cells.

F1 RIP-LTα grafts rejected significantly faster (MST= 54) than F1 grafts (MST= 225), demonstrating that TLO contribute to allograft rejection. F1 RIP-LTα grafts contained similar numbers of, but larger TLO than F1 grafts as demonstrated by histology. Mean speed and displacement of OT-I and OT-II cells significantly decreased over time after immunization while AC and mean CT significantly increased. B cell mean speed, displacement and AC increased after immunization. These data are consistent with B cell activation and productive T cell-DC interactions and mirror previously reported data in secondary lymphoid organs.

We provide first evidence that TLO provide a local structure for T and B cell activation that propagates anti-graft immune responses in the setting of chronic rejection.
Circulating exosomes from human lung transplant recipients with respiratory viral infections contain nucleic acids potential to activate innate immune signaling (cGAS/STING and RIG-1 Pathways)
Authors: Bansal Sandhya , Limaye Ajit , Omar Ashraf , Smith Michael , Bremner Ross , Mohanakumar Thalachallour
Published: January 2023

Exosomes are 50–200nm size vesicles released by cells into the body fluids. We reported the presence of circulating exosomes with lung self-antigens (SAgs) (Collagen-V,K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Respiratory viral infections (RVI) is a known risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant. We postulated immune mechanism by which RVI increases risk for CLAD is by induction of exosomes with SAgs containing viral DNA/RNA capable of activating innate immune signaling via DNA/RNA sensing. Exosomes were isolated using ultracentrifugation. Exosomal DNA and RNA were used to generate libraries using Kapa Biosystem’s kit. Illumina 2×150bp pair-end reads were checked on FastQC, aligned to human/viral genome from CHIP seeker Database. Role of exosomes inducing cell signaling in human airway epithelial cells (KCC266) and Hep2 were analyzed by incubating exosomes from LTxRs with RVI or stable. Viral nucleic acid sequences were higher in exosomes from LTxRs with RVI while comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in exosomes from RVI. We observed upregulation of proteins associated with cGAS/STING and RIG1 in KCC266 and Hep2 cells (STING; 1.5, cGAS; 1.9, pIRF3; 2.5, pTBK; 3.1, MAVS; 1.7 and IFNβ; 3.8 fold respectively) incubated with exosomes from LTxRs with RVI. We conclude that LTxRs with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling. This can lead to activation of innate immune signaling resulting in adaptive immune responses to viral and donor antigens resulting in CLAD.
The effect of SLA expression on genetically-engineered pig renal cells on the human T cell xenoresponse
Authors: Wang Zheng-Yu , Ladowski Joseph M. , Li Juan , Martens Gregory R. , Reyes Luz M. , Estrada Jose L. , Tector A. Joseph
Published: January 2023

Background

Xenotransplantation using genetically-engineered (GE) pig organs offers an unlimited supply of organs for patients with end-stage organ failure. Disrupting the porcine GGTA1/CMAH/β4GalNT2 genes (TKO) markedly reduces binding of human natural anti-pig antibodies in human to carbohydrate antigens expressed on the pig organ. However, more evident have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. The object of this study is at an organ-specific level to evaluate human T cell-mediated response against GE pig renal microvascular endothelial cells (RMEC) with or without swine leukocyte antigen (SLA) class I or II.

Methods

Primary RMEC were isolated from the GGTA1/CMAH knockout (DKO) pigs, and immortalized using Lenti-SV40 T. The gene editing was performed to create specific TKO RMEC cell lines expressing SLA class II. Stimulation of human T cell activation or proliferation by RMEC was performed flow cytometry-based mixed lymphocyte reaction (MLR).

Results

The specific GE cells lacking carbohydrates or expressing SLA class I or II were confirmed. MLR data shows that human CD4+ T cell proliferation by CD31+ RMEC was higher than that by fibroblast-like renal cells (RC), and was significantly inhibited by immunosuppressive drugs. Furthermore, both of CD69+ and CD25+ T cells were significantly enhanced by RMEC with expression of SLA class I and II compared with SLA class I alone.

Conclusion

Porcine RMEC express SLA class I/II and known costimulatory molecules and have the ability to simulate human T cell proliferation. RMEC will be a useful reagent for the further study of xenotransplantation.
Developing a novel biologic for the treatment of high-risk B cell acute lymphoblastic leukemia
Authors: Ruiz-Jiménez Caleb , Coats Jacqueline , Meng Xianmei , Stoian Cornelia , Baez Ineavely , AlKashgari Hossam , Paz-Aldana Andy , Doublet Sarah , Choi Hannah , Williams Roland , Farzin-Gohar Shadi , Dovat Sinisa , Payne Kimberly J
Published: January 2023

The subtype of B cell acute lymphoblastic leukemia (B-ALL) at highest risk for relapse overexpresses surface CRLF2 (CRLF2 B-ALL) and disproportionately affects Hispanic children and adults. CRLF2 partners with the IL-7Ra to form a receptor complex activated by the cytokine, Thymic Stromal Lymphopoietin (TSLP). TSLP activates the JAK/STAT and PI3/AKT/mTOR pathways which are known to promote cell survival and proliferation. Our in vitro studies of CRLF2 B-ALL cells showed that supra-physiological levels of TSLP (1 ng/ml) induced a complete shutdown of TSLP receptor signaling, following the initial activation event. This effect was dose dependent and did not occur at physiological levels (20 pg/ml) of TSLP. This signal shutdown was correlated with the loss of surface IL-7Ra. Using a novel patient-derived xenograft (PDX) model that expresses human TSLP (hTSLP, mouse TSLP does not activate the human TSLP receptor) created in our lab, we showed that high dose hTSLP virtually eliminated human CRLF2 B-ALL cells in vivo. The goal of work presented here was to test whether treatment of CRLF2 B-ALL PDX with high doses of recombinant hTSLP (rhTSLP) could re-capitulate anti-tumor effects of hTSLP observed in our engineered mice and to evaluate its effects on prolonged survival. After 24 days of treatment, rhTSLP reduced the leukemia cell burden by 90% in treated PDX as compared to control PDX that received physiological levels of rhTSLP. Similarly, survival was prolonged in PDX mice receiving high-dose rhTSLP as compared to controls: all 11 control mice died before any of the 11 treated mice died. Taken together, these data suggest that TSLP has promise as a biologic for the treatment of CRLF2 B-ALL.
Overexpression of LLT1 (CLEC2D) in childhood Acute Lymphoblastic Leukemia (ALL)
Authors: Mathew Stephen O , Powers Sheila , Daniel Steffi , Jose Roslin , Aryal Subhash , Bowman W. Paul , Mathew Porunelloor
Published: January 2023

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer in the developed world with peak incidence between the ages of 2 and 5 years. Despite current treatment protocols achieving significant improvement in survival of ALL patients, serious acute and late complications are frequent and resistance to chemotherapy often develops. Although NK cells have long been known to mediate antigen independent tumor killing, the therapeutic potential of NK cell-based immunotherapy has yet to be realized. NK cells have the capacity to kill virus-infected or tumor-transformed cells and they express several surface molecules that regulate NK cell function both positively and negatively. We have previously identified 2B4 (CD244), CS1 (CRACC) and LLT1 (CLEC2D) to play a major role in NK cell activation. In this study, we evaluated the cell surface and mRNA expression of NK cell receptors in 42 ALL subjects and 20 healthy subjects both male and female in the age range of 2 – 20 yrs from different racial/ethnic groups. The peripheral blood samples were collected and analyzed by flow cytometry and real-time PCR for expression. A significant increase in the expression of LLT1 in the T and NK cell populations was observed in ALL subjects as compared to healthy subjects. NK cells from 3 ALL subjects also showed impaired cytotoxic function as they demonstrated lower killing of K562 cells compared to a healthy subject. These results indicate that LLT1 may play a role in NK-mediated surveillance of childhood ALL.
Circulating Low Density Neutrophils In Advanced Lung Cancer Patients Exhibit Unique Immune Signatures and affect prognosis
Authors: Krieg Carsten , Shaul Merav , Guglietta Silvia , Weber Lukas , Robinson Mark , Fridlender Zvi
Published: January 2023

The role of innate immunity, in especially neutrophils, in supporting anti-tumor immunity in lung cancer is understudied. Neutrophil "flavors", such as low-density neutrophils (LDN) and high-density neutrophils (HDNs), have been described in cancer patients and associated with tumor-supportive properties. Nevertheless, HDN and LDN phenotypic properties and their cancer-driven modulations are still largely unknown. Here, we aimed to evaluate the clinical relevance and significance of circulating LDN in lung cancer patients, and by using data-driven artificial intelligence supported analysis tools evaluated phenotypic states of circulating neutrophils subsets during lung cancer progression. COPD patients and healthy volunteers served as controls. Using mass cytometry (CyTOF), we aimed to identify major sub-populations within circulating LDN and HDN subsets and determine a possible change in phenotype due to cancer. LDN were highly enriched in advanced lung cancer patients (12.9±2.4%), but not in early stage patients (1.2±0.2%), healthy individuals (1.3±1.1%), or stable COPD patients (2.2±0.6%). Elevated LDN (>10% of LDF) showed a remarkable correlation with poorer prognosis in advanced patients. Our CyTOF analysis revealed the presence of three main neutrophil subsets with a great variability in the presence of CD10+ vs. CD10− LDNs between patients. We further demonstrated that both HDN and LDN retain a degree of inherent spontaneous plasticity. Deep characterization of cancer-related circulating neutrophils, as well as their modulation along tumor progression, will assist in suggesting new biomarkers and novel targets to improve response to current conventional therapies and immunotherapies.
Dynamic regulation of TRIM37 expression in macrophages facilitates pancreatic cancer development
Authors: Chen Hui-Chen , Chang Hung-Chih , Chang Yi-Hao , Wu Chia-Wei , Wu Guo-Wei
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer as the overall five-year survival rate was less than 5%. More than 90% of PDAC patients harbor Kras mutation, but only mutant Kras is insufficient to give rise to PDAC. Inflammation is essential to cooperate with mutant Kras for the initiation of pancreatic precancerous lesion, and attracted M1-polarized macrophages-induced inflammation are known to contribute to the process. Among TRIM family proteins, TRIM37 is the only protein containing MATH domain that endows it being able to interact with proteins containing TRAF domain, indicating its possible role in regulating pattern recognition receptor-, TNFR- and cytokine-mediated signaling pathways. We have observed that, when TRIM37 was knocked-down, macrophage cytokines production, such as IL-1β, IFN-β, IL-12, TNF-a, IL-6 and CCL-2 in response to LPS was reduced, and the regulation was both epigenetically and non-epigenetically. Interestingly, pro-inflammatory cytokines also upregulated TRIM37 expression in macrophages. Thus our data implicated that upregulated TRIM37 that positively regulate macrophage cytokine production might contribute to the initiation of PDAC. However, when macrophages were co-cultured with pancreatic cancer cells, the expression level of TRIM37 in macrophages were downregulated, this led to the loss of its ability in maintaining macrophages in a stable M1 phenotype, and instead, skewing the macrophages into a M2 phenotype, which promotes cancer progression. In conclusion, our data demonstrate that during pancreatic cancer development, the expression of TRIM37 in macrophages are differential regulated to favor cancer development and progression.
STAT3 induces the expression of fatty acid transport proteins (FATP2 and FATP4) to promote myeloid-derived suppressor cells function in tumor
Authors: Adeshakin Adeleye Oluwatosin , Yan Dehong , Zhang Mengqi , Afolabi Lukman Olalekan , Adeshakin Funmilayo Oladunni , Wan Xiaochun
Published: January 2023

Myeloid-derived suppressor cells are critical to regulation of immune responses; their accumulation in cancer patients inhibit antitumor immunity and suppress T-cells activity thereby promoting cancer progression and metastases. Metabolic reprogramming has been identified as a crucial factor in modulating MDSCs function; however, how lipid metabolism promotes MDSCs activity during tumor remains unknown. Here we report the upregulation of fatty acid transport proteins (FATP2 and FATP4) enhanced the exogenous uptake of fatty acids resulting in hyperlipidemic MDSCs during tumor. Accumulation of unsaturated fatty acids in GM-CSF-induced MDSCs in-vitro released IL-6, ARG1 and iNOS thereby inhibiting T-cells function. Increased fatty acid uptake in MDSCs induces the expression of FATP2 and FATP4 via activation of STAT3. Interestingly, therapeutic blockade of STAT3 decreased lipid accumulation and repressed FATP2 and FATP4 expression in MDSCs. Also, FATP2 specific inhibitor (Lipofermata) abrogated tumor progression in immunocompetent B16F10-tumor bearing mice in MDSCs dependent manner. Therefore, our data suggests therapeutic manipulation of lipid metabolic signaling pathways in MDSCs could be a promising approach to effective cancer immunotherapy.
CD226hiCD8+ T cells are a prerequisite for anti-TIGIT immunotherapy
Authors: PARK YOON , Jin Hyung-seung
Published: January 2023

Multiple clinical trials are evaluating the efficacy of anti-TIGIT antibodies for use as single-agent therapy or in combination with PD-1/PD-L1 blockade. However, TIGIT’s mechanism of action and whether a TIGIT blockade will synergize with current immunotherapies is not fully understood. Here we show that CD226loCD8+ T cells are accumulated at the tumor site and have an exhausted phenotype with an impaired functionality. In contrast, CD226hiCD8+ tumor-infiltrating T cells possess a higher self-renewal capacity and potent responsiveness. Anti-TIGIT antibody treatment selectively affects CD226hiCD8+ T cells by promoting CD226 phosphorylation at tyrosine 322. CD226 agonist antibody-mediated activation of CD226 augments the effect of TIGIT blockade on CD8+ T cell responses. Finally, mFOLFIRINOX treatment, which increases CD226hiCD8+ T cells in patients with pancreatic ductal adenocarcinoma, potentiates the effects of TIGIT or PD-1 blockade. Our results implicate CD226 as a predictive biomarker for cancer immunotherapy and suggest that increasing CD226hiCD8+ T cells may improve anti-TIGIT therapy.
Identification of novel ligands that alter the function of type II NKT cells
Authors: Nishio Kumiko , Camara Kaddy , Pasquet Lise , Bloom Anja C , Richardson Stewart K , Cardell Susanna L , Howell Amy R , Terabe Masaki , Olkhanud Purevdorj , Berzofsky Jay A
Published: January 2023

Natural killer T (NKT) cells are distinct innate lymphocytes which recognize lipid antigens in the context of the major histocompatibility complex-like molecule CD1d. When activated, these cells can rapidly produce a variety of cytokines and modulate innate and adaptive immune responses. There are two main subsets of NKT cells, termed type I and type II NKT cells. Type I NKT cells are characterized primarily on the basis of their invariant TCRα expression and reactivity to the glycolipid α-galactosyl ceramide (αGalCer), whereas type II NKT cells express a different and more diverse TCR repertoire than type I NKT cells. Currently, the most widely studied antigen for type II NKT cells is sulfatide. Sulfatide-reactive type II NKT cells are reported to have the immunosuppressive function in autoimmune diseases and cancer. We previously showed a role for sulfatide-reactive type II NKT cells in suppressing tumor immunosurveillance in a murine model of lung metastasis. The activation of type II NKT cells in this model by the injection of sulfatide increased the development of lung metastasis. We synthesized diverse sulfatide-analogues varying in the number of double bonds and hydroxyl groups in the ceramide. In vitro, analogues having a phytosphingosine sphingoid base activated both type II and type I NKT cell hybridomas. On the other hand, the analogue C24:2 with a sphingosine base activated only the type II NKT cell hybridoma. In vivo, the C24:2 analogue significantly reduced the development of lung metastasis, showing distinct and opposite effects from sulfatide. The identification of new ligands inducing altered functional activity of type II NKT cells could be of great interest for developing new anti-tumor immunotherapies.