bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient, cost-effective isolation of functional antigen specific antibodies using Nanovial technology
Authors: Kershaw Kathleen , Bosler Lucienne , Kuo Wei-Ying , Wu Alex , Di Carlo Dino , Liang Jesse , de Rutte Joseph
Published: November 2024

The therapeutic application of monoclonal antibodies has been invaluable in improving the quality of life for countless patients across numerous indications. Despite advances in streamlining the process, enhanced screening methods still necessitate substantial investments in equipment, reagents, and training. To address this gap, we have developed a workflow utilizing Nanovials, hydrogel nanoliter containers, to enable the discovery of functional antibodies from single cells with a simple workflow without any new equipment or protracted hands-on assay steps.

We demonstrate this workflow using a hybridoma producing anti-Hen egg lysozyme (HEL) antibodies. As a proxy for rare “on target” cells, HEL hybridoma was spiked into a non-antigen specific hybridoma at known percentages and functional, HEL specific clones were enriched and isolated with a Nanovial assay. Nanovials specifically enrich the target antigen specific secreting cells at concentrations as low as 1% and isolate desired clones by sorting. Further improving the functional screening capabilities without lengthening the assay, multi-cell Nanovial assays were performed combining a target antibody secreting cell and reporter cell to specifically sort cells based on antibody binding or pathway modulation. By providing increased functional assay capabilities while reducing the time and material needed to isolate functional, antigen specific antibodies, Nanovials enable the rapid discovery of potent antibody therapeutics.
New automation-friendly 96-well plate ready-to-use 8-color dry antibody panel for basic phenotyping of human whole blood and peripheral blood mononuclear cells using flow cytometry
Authors: Bowers Rita , Dupas Nathalie , Kapinsky Michael , Lacaille Vashti , Mazumder Saumyabrata , Edicheri Alinkeezhil Gopikrishnan , Saif Samee
Published: November 2024

Automation-friendly 96-well plate flow cytometry solutions are ideal for pharmaceutical, academic, and CRO laboratories that perform testing on large numbers of samples and/or large number of test conditions. Our ready-to-use pre-formulated dry antibody panel in 96-well plate format provide researchers all the reagents within the wells of the plate where the test is conducted in a stable dry format, resulting in dependable reagent performance and reduction of manual pipetting steps with the added benefit of time and material (reagent and sample) savings. In addition, the 96-well plate format is compatible with automated liquid handlers for walk-away flexibility and higher-throughput sample processing. Here we show flow cytometry data for both whole blood and PBMC samples stained with the first ready-to-use DURAClone IM Phenotyping BASIC plate (RUO) in a shallow 96-well plate containing a dry reagent antibody panel to identify major lymphocyte and monocyte subpopulations for the following markers: CD45, CD16, CD56, CD19, CD14, CD3, CD4 and CD8. Processing of whole blood samples using the shallow 96-well plate format was achieved using FLEXLyse, a lysing solution with low volume ratio of lysis/whole blood, applicable for no-wash and wash protocols. Our experiments were performed using automation on the Biomek i7 liquid handling automated workstation integrated with a CytoFLEX LX flow cytometer and microplate centrifuge demonstrating a fully automated flow cytometry workflow.
Quantitative immunology protein panel built on the SomaScan assay platform
Authors: Katilius Evaldas , Keeney Tracy , Tabacman Eduardo , Liu Rong , Yearwood Graham , Schafer Peter
Published: November 2024

The SomaScan™ assay is a highly multiplexed proteomic assay that uses SOMAmer™ reagents to detect proteins in various biological samples. The latest version of the SomaScan assay allows researchers to measure over 11,000 proteins in human blood. The SomaScan assay is designed to provide protein epitope abundance measurements by reporting relative SOMAmer reagent abundance quantified using DNA microarrays. This is common for highly multiplexed proteomic assays but different from clinical protein assays, which report protein concentrations. We set out to develop a Quantitative Immunology Protein Panel (QuIPP) built on the SomaScan 7K assay, focused on a set of 58 immunologically-relevant cytokines, chemokines, and growth factors. Panel qualification was performed by developing calibration curves and demonstrating reproducibility for the biomarker measurements in serum samples with defined LLOQ and ULOQ acceptance criteria. Intra-run and inter-run CVs were calculated for high, medium, and low QC samples. 54 analytes met the standard curve, QC sample reproducibility, and plate edge effect acceptance criteria, with 34 analytes meeting strict parallelism acceptance criteria.

We have demonstrated the process for developing the QuIPP using SomaScan assay as a core technology. The ability to generate quantitative results will allow SomaScan users to compare protein concentrations across datasets and further validate biomarker discoveries on the SomaScan platform.
Purine catabolism regulates the production of IL-1beta in macrophages
Authors: Archambault Anne-Sophie , de Brito Monteiro Lauar , Starchuck Lucas , Oh Juhee , Perraso Emilia , Patterson Annette , Verchere C Bruce , Klein Geltink Ramon
Published: November 2024

Activation of pro-inflammatory (M1-like) macrophages leads to the production of pro-inflammatory cytokines, such as IL-1b, TNFa and IL-6. Those cytokines are important in the control of infections, but overproduction can lead to chronic inflammation. M1-like macrophages undergo metabolic remodeling, including the upregulation of purine metabolism. Using inhibitors of purine breakdown or purine synthesis, we aimed to identify the role of purine metabolism in M1-like macrophage function.

Blocking purine synthesis or purine breakdown did not affect the frequency of CD80+CD86+iNOS+macrophages, nor the accumulation of the M1-like macrophages hallmark metabolites itaconate and succinate, critical for immune function. However, inhibition of purine synthesis significantly reduced IL-1bsecretion. Mature IL-1b release is dependent on both transcriptional activation of the pro-IL1 gene and NLRP3-inflammasome. Blockade of purine synthesis sustained pro-IL1 transcription, an accumulation of pro-IL1b and significantly reduced caspase-1 activity. When purine degradation was inhibited, we observed no change in IL-1b production, but a reduced TNFa and IL-6.

In summary, purine metabolism is dispensable for the immunophenotype of M1-like macrophages, but it regulates macrophage cytokine production. Targeting specific nodes in purine metabolism in macrophages provides opportunities for immunomodulation in inflammatory diseases affected by pro-inflammatory macrophage-linked cytokines.
Tertiary Lymphoid Organs promote allograft rejection
Authors: Feizi Neda , Feizi Neda , Zhang Gang , Halesha Latha , Abou Daya Khodor , H. Oberbarnscheidt Martin
Published: November 2024

Tertiary lymphoid organs (TLOs) are ectopic lymphoid structures that arise in non-lymphoid tissues and are frequently observed in tissue affected by non-resolving chronic inflammation. If TLOs are beneficial or detrimental in transplantation is controversial. We investigate the role of TLO and LTbR in transplantation by manipulating the LTa-LTbR pathway.

Using a mouse allo kidney transplantation model, we found that preformed intragraft TLO are sufficient to precipitate rejection in recipients lacking all secondary lymphoid organs (SLO)(LTbRko). In WT recipients with a complete set of SLO, intragraft TLO accelerated rejection (MST=63 vs. 225 d). Donor grafts that cannot form TLO (LTbRko) prolonged allograft survival (MST 24 vs 11 d) in an acute kidney rejection model. Intravital imaging confirmed that T and B cell activation takes place in renal TLO upon migration of naïve T and B cells and prolonged interactions with dendritic cells. T cells produced IFNg and Confetti+/+ B cells clonally expanded within TLO.

In summary, intragraft TLO are sufficient for and accelerate allograft rejection. Local immune responses are initiated and maintained in intragraft TLO, while disruption of TLO formation in the allograft leads to prolonged allograft survival. TLO support T and B cell activation as well as local DSA formation. These findings highlight the importance of TLO in local immune responses with implications for cancer, autoimmunity and transplantation.
The role of virtual memory CD8+ T cells in pulmonary viral and bacterial infection
Authors: Li Dong-Lin , Lin Jr-Shiuan , Tai Chi , Xie Jia-Xun , Wang Ching-Chun , Lai Meng-Chih
Published: November 2024

Virtual memory CD8+ T (TVM) cells, also known as antigen-inexperienced T cells with memory phenotypes, have been reported to mediate bystander protection against primary infection. It is known that the expansion of TVM cells depends largely on IL-4; thus, we used α-galactosylceramide (α-GalCer), which activates invariant natural killer T (iNKT) cells to trigger a strong IL-4 response to expand TVM cells in mice. Whether expanded TVM cells could grant protection against influenza A virus (IAV) and Streptococcus pneumoniae (SPN) infections and the functions of TVM cells in the lungs in these settings remain unknown. Here, we revealed that TVM cells significantly increased in the respiratory tract after α-GalCer treatment and maintained for at least 14 days. In contrast, the percentage of iNKT cells has returned to basal levels at this time. Furthermore, α-GalCer treatment generated more effector functional CD8+ cells and the proportion of TVM cells in the cytokine-producing CD8+ cell population significantly increased in the lungs and mediastinal lymph nodes. Notably, α-GalCer treatment decreased the viral titer after mice were challenged with a high dose of IAV as well as significantly improved the survival of SPN-infected mice. These results suggested that the protection may be attributed to TVM cells. Therefore, we further established a reliable intranasal transfer model to investigate the direct role of TVM cells in pulmonary viral and bacterial infections.
Loss of TET2 increases cell number and reduces immunoglobulin CDR3 diversity in B-1 cells
Authors: Dennis Emily , Murach Maria , Blackburn Cassidy , Marshall Melissa , Root Katherine , Pattarabanjird Tanyaporn , Erickson Loren , Binder Christoph , Bekiranov Stefan , McNamara Coleen
Published: November 2024

Ten-Eleven Translocation-2 (TET2) is an epigenetic modifier and potent regulator of immune cells, yet its role in B-1 cell biology is largely unknown. B-1 cells protect against infection and clear apoptotic cells via the production of natural IgM. Our study analyzed B-1 cell subsets from mice with global knockout of TET2 compared to WT controls using flow cytometry to determine the frequency and number of B cell subsets, RNAseq to determine differences in global gene expression as well as BCR repertoire analysis, and ELISA to evaluate plasma IgM levels. Results revealed that mice with TET2-KO had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow and spleen. Consistent with this finding, circulating IgM was elevated in TET2-KO mice compared to WT. We observed reduced expression in both heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, IgM was by far the most abundant isotype expressed by genes in B-1 cells. Yet, only in B-1a cells was there a significant increase in the proportion of IgM isotypes in TET2-KO mice compared to WT. Analysis of the CDR3 revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. The results from our study suggest that TET2 may regulate the pool of antigen-specific IgM-producing cells.
Modulation of neuromuscular function through extracellular traps in septic shock models
Authors: ADAM Lucille , Bourcier Camille , Emam Laila , Vinit Stéphane , Mansart Arnaud
Published: November 2024

Sepsis via innate immune system activation causes organ dysfunction. Among these, the central nervous system (CNS) is particularly affected by sepsis-associated encephalopathies. These symptoms are linked with the activation of microglia and leukocyte infiltration. These immune cells have been discovered to have the ability to produce extracellular traps (ETs). While these components capture and destroy pathogens, deleterious effects occur such as reduced neuronal excitability with excessive production. In this study, the objectives were to determine whether (1) immune cells form ETs in the CNS by using two models of sepsis: lipopolysaccharide (LPS) and cecal ligation and puncture (CLP); (2) ETs produce neuromuscular disorders, and (3) a threshold is necessary to produce these disorders. Our results demonstrate a systemic inflammation for both models. As expected, the LPS administration increases leukocyte infiltration into the CNS, activates immune cells and produces ETs, which directly impair the gastrocnemius motoneuron excitability. Sivelastat, an inhibitor of ET formation significantly decreases the production of ETs in immune cells and induces a preservation of the neuromuscular function without decrease of the inflammatory response. This infiltration of leukocytes and production of ETs is also observed using the CLP model, at a lower stage, without neuromuscular dysfunctions, suggesting that the CNS has an ET threshold in order to display these deleterious effects.
Allogeneic CD4+ iNKT cellular immunotherapy promotes anti-tumoral T cell responses in a human B-lymphoma xenograft model
Authors: Baiu Dana , Sharma Akshat , Schehr Jennifer , Basu Jayati , Smith Kelsey , Kenney Shannon , Gumperz Jenny
Published: November 2024

iNKT cells are innate-like CD1d-restricted T cells that mediate potent anti-tumor responses in murine models and have shown therapeutic potential in clinical trials. In humans, expression of CD4 delineates two functionally distinct iNKT cell subsets, with CD4- iNKT cells mainly producing TH1 cytokines and showing cytolytic activity, while CD4+ iNKT cells display a polyfunctional phenotype and are linked to regulatory pathways. Here, we used an EBV-driven B-lymphoma model to investigate the anti-tumor effects of these two iNKT subsets. In a live cell imaging system, CD4- iNKT cells carried out cell-mediated cytotoxicity and limited tumor spheroid growth in vitro, while CD4+ iNKT cells showed little anti-tumor activity. However, in a xenograft model, the effects of the two subsets were reversed. CD4- iNKT cells had no impact on tumor mass, which was likely due to down-regulation of CD1d by lymphoma cells in vivo. In contrast, administering CD4+ iNKT cells to tumor-bearing mice led to rapid reduction in tumor mass. CD4+ iNKT cells activated the expression of multiple co-stimulatory ligands by DCs, and human T cells from mice given CD4+ iNKT cell immunotherapy had enhanced responses against EBV. These results show that allogeneic CD4+ iNKT cell immunotherapy leads to potent anti-tumor activity in vivo that does not require tumor cell CD1d expression, and instead is associated with adjuvant-like activity that enhances anti-tumor responses by endogenous T cells.
Impaired NK cell-mediated antiviral responses to HIV following BCG vaccination: implications for neonatal vaccination
Authors: Gunasena Manuja , Alles Mario , Mulhern Will , Robinson Richard , Demberg Thorsten , Liyanage Namal
Published: November 2024

Bacillus Calmette-Guérin (BCG) vaccine is the only approved vaccine against tuberculosis (TB). BCG has been shown to elicit nonspecific protection as well as detrimental effects during diseases unrelated to TB. We investigate the impact of BCG on modulating NK cell responses and its effect on altering susceptibility to HIV acquisition. The C57BL/6 mouse model was used to compare splenic NK cell responses of control (n=4) and BCG vaccinated mice (n=4) following subsequent exposure to Mycobacterium bovis (Mtb) and HIV Gag peptide antigens. Statistical comparisons were made using Wilcoxon ranked sum test. The data acquired though flowcytometry suggested that mature, BCG trained memory-like (KLRG1+ Ly49H+) NK cells showed increased proinflammatory cytokine responses and polyfunctionality upon re-exposure to Mtb antigens. However, when these trained cells were exposed to HIV Gag antigen, we observed a significant reduction in cytokine production and polyfunctionality. Specifically, these BCG trained NK cells showed marked decrease in IFNγ, TNFα, IL1β and Granzyme B production alone or in combination (p<0.05). Furthermore, negative correlations between NK polyfunctionality and mature BCG trained NK cells were observed, following HIV Gag peptide exposure. This suggests that NK cells are impaired in their antiviral effects during HIV exposure, soon after BCG vaccination. This may suggest higher risk of HIV acquisition in BCG vaccinated neonates that are frequently exposed to HIV.
Function of tissue-resident helper T cells in chronic lung sequelae following viral pneumonia
Authors: CHEON In Su , SON Youngmin , Narasimhan Harish , Im Jun-Sub , Vassallo Robert , Sun Jie
Published: November 2024

Respiratory viral infections such as influenza and SARS-CoV-2 can lead to the development of chronic lung disease and impaired lung function long-term after the recovery from acute morbidity. However, the mechanisms underlying the development of chronic diseases post-acute respiratory viral infection are largely elusive.

Using a model of age-related development of chronic lung disease after acute influenza infection, we found that a population of lung resident PD1HiCD4+ T helper cells (TRH), which exhibit characteristics of both follicular helper T cells and resident memory cells, was greatly increased after primary influenza pneumonia. Furthermore, TRH cells were located near the dysplastic/inflammatory lung parenchyma after the resolution of primary viral pneumonia. Strikingly, genetic ablation of TRH or the blockade of TRH-derived IL-21 signaling diminished chronic lung pathology and improved lung function following viral pneumonia in aged animals. Moreover, TRH cell presence was associated with worse lung function and increased lung pathology in COVID-19 convalescents. Our data suggest that exuberant TRH responses contribute to the development of chronic lung sequelae following viral pneumonia. Additionally, our findings may pave the way to develop novel preventive and/or therapeutic options treating chronic lung diseases following acute viral pneumonia including long COVID.
Intestinal ILC3 Activation by Commensal Bacteroides thetaiotaomicron: Implication for Liver Protection in Alcohol-Induced Liver Disease
Authors: Sriwastva Mukesh Kumar , Lei Chao , Xu Zhishan , Tan Yi , Deng ZhongBin
Published: November 2024

In recent years, there has been a growing recognition of the intricate interplay between the commensal bacteria Bacteroides thetaiotaomicron (Bt) and immune system. Among the various immune cell subsets, innate lymphoid cells (ILCs), group 3 ILCs (ILC3s) have particularly piqued the interest of researchers, as they appear to wield a significant influence in maintaining gut homeostasis. In order to understand if Bt mediates the crosstalk between intestinal epithelial cells (IECs) and ILC3 that prevents gut dysbiosis and liver injury in alcoholic liver disease (ALD), a disorder that causes lipid accumulation, liver damage, gut bacterial overgrowth and gastrointestinal mucosa dysfunctions, this study narrowed to the exploration of intricate relationship between Bt and ILC3s activation in the modulation of ALD. Bt supplementation leads to lower liver triglyceride content, prevent liver from steatosis, inflammatory lesions, relieves liver fat lesions and enhance intestinal. Bt enhances the Ahr activity and IL23 expression in IEC. The beneficial effect of Bt was abrogated in mice supplemented with Bt lacking capsular polysaccharides (CPS) and diminish AhR dependent IECs IL23 production and activation of type 3 innate lymphoid cells (ILC3). IL22-producing ILC3 cells improved gut homeostasis and this cascade ultimately results in enhance Mucin secretion, reduced systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury through the gut-liver axis.
LRRK2 Kinase Function Regulates NLRC4 Inflammasome Activity and Thereby Modulates Intestinal Inflammation
Authors: Deloer Sharmina , Fuss Ivan , Strober Warren , Suebsuwong Chalada , Kumar Kunal , Peter Inga , Devita Robert J
Published: November 2024

The LRRK2 protein associated with increased risk of IBD. Recently described, this protein influences NLRC4 inflammasome phosphorylation. This LRRK2 function, however, has uncertain significance since it is not clear how NLRC4 phosphorylation affects NLRC4 inflammasome function.This study utilizes commercially available LRRK2 inhibitors, to clarify this knowledge gap. In vitro studies showed that inhibition of LRRK2 kinase activity inhibits both its binding to NLRC4 and phosphorylation upon activation with needle protein, thus establishing that LRRK2 has an indispensable role for NLRC4 phosphorylation. later using THP-1 cells with ASC deletion or human PBMC-derived dendritic cells with shRNA ASC- KD showed that ASC deficiency retards NLRC4 phosphorylation. Finally, we found that inhibition of NLRC4 phosphorylation by LRRK2 inhibitors has a major negative effect on NLRC4 mediated IL-1b cleavage but, surprisingly, it has minor effect on IL-18 cleavage. This indicated that IL-1b expression requires ASC-caspase binding whereas IL-18 expression does not. Next, systemic administration of NLRC4 activator (flagellin protein) caused increased intestinal permeability and that co-administration of inhibitor reversed the increased permeability. These findings concluded that inhibition of IL-1b production with the preservation of IL-18 via LRRK2 kinase inhibition blocks the potentially harmful effects of NLRC4 activity on intestinal permeability during intestinal inflammation.
Highly multiplex volumetric imaging Ce3D-IBEX reveals commensal-induced immune clustering along sensory nerves in the lung
Authors: Ichise Hiroshi , Hor Jyh Liang , Arroyo-Mejías Armando , Jin Hao , Belkaid Yasmine , Ronald Germain
Published: November 2024

The airway mucosa is a primary site of the immune response to inhaled antigens. However, how immune homeostasis is maintained in this organ during continuous exposure to airborne particles and microbial antigens is not known. Previous studies have shown that the spatial organization of immune components in relationship to each other and to non-hematopoietic components in tissues is critical to their proper function. To interrogate such relationships in the lung, 3D information is required. However, current methods for fluorescence 3D imaging do not provide the marker depth afforded by high-content 2D imaging.

To overcome this constraint, we created Ce3D-IBEX, which enables the visualization of 30+ markers in an optically cleared 3D sample. Using Ce3D-IBEX, we have identified a previously uncharacterized immune cluster in the lung of naïve SPF mice that is enriched with T cells and dendritic cells (DCs) and is localized in close proximity to vagal sensory nerves. Agonistic denervation of the sensory nerves reduced the number of clusters, and that cluster formation was disrupted in germ-free mice as well as OT-I and OT-II TCR transgenic mice. These findings indicate that the clustering requires sensory nerve-derived signals and TCR-mediated recognition of bacterial antigens. Functional studies in virus-infected animals suggest that the T cells in these clusters behave like tissue resident memory T cells, providing acute antigen-independent effector activity.
Pre-clinical Pharmacology and Tolerability Characterization of a Novel KIRxICOS Targeting Bispecific CD8 Treg Modulator
Authors: Pham Thi Minh Nguyet , Bogatzki Lisa , Boster Dan , Bowser Justin , Childs Monica , Crane Courtney , Gardell Jennifer , Hermansky Gleda , Julien Susan , Meengs Brent , Quach Phoenicia , Tan Cathy , Therriault Jon , McMahan Catherine J , Swiderek Kristine M
Published: November 2024

Here we present an Fc-based bispecific CD8 Treg modulator that binds in cis to inhibitory KIR and ICOS receptors co-expressed by CD8 Treg designed to restore CD8 Treg functions. CD8 Treg dysfunctions include impaired cytotoxicity and insufficient longevity in autoimmune disease affected tissues. The KIRxICOS targeting bispecific molecule selectively enhances CD8 Treg killing, promoting their recruitment to and survival within disease affected tissues.

Through in vitro, ex vivo, and in vivo evaluations in humanized mouse models, we assessed binding, specificity, and function of the KIRxICOS CD8 Treg Modulator, which boosted ICOS signaling in KIR-expressing CD8 Treg, without activating immune cells expressing only ICOS. It extended survival and reduced disease score in a humanized acute GvHD model, marked by a reduction of activated CD4 T cells and an increase of CD8 Treg. In antigen stimulated tissues, the KIRxICOS CD8 Treg modulator reduced CD4 derived proinflammatory cytokines and epithelial cell death. In vivo, the KIRxICOS molecule demonstrated antibody-like PK parameters. Repeat dose studies in huNSG(IL15Tg) mice indicated good tolerability without terminal toxicity or induction of proinflammatory serum cytokines. The evaluation of binding, impact on immune cell phenotypes, and pharmacology revealed immune cell binding without activation.

Our preclinical data supports development of the KIRxICOS CD8 Treg modulator as a therapeutic in autoimmune disease.
Advancing CD19 CAR T Cell Therapy for Treatment of Primary Biliary Cholangitis
Authors: Sokke Umeshappa Channakeshava , Babu Kolla Harish , Hebbandi Nanjundappa Roopa
Published: November 2024

CD19 Chimeric Antigen Receptor (CAR) T cell therapy is emerging as a revolutionary approach in autoimmune disease management, offering a promising alternative to conventional treatments like anti-CD20 antibody therapy. Unlike challenges associated with existing therapies, CD19 CAR T cells, engineered to recognize CD19 molecules on B cells, exhibit remarkable efficacy in achieving targeted B cell depletion. Upon target recognition, they become activated, efficiently eliminating the harmful B cells. Furthermore, the CAR T cells exhibit a propensity for proliferation upon target recognition, thereby ensuring their prolonged presence within the body. Our findings unequivocally establish the effectiveness of CD19 CAR T cells in specifically targeting and depleting B cells, both in vitro and in vivo. In a well-established PBC model involving NOD.c3 mice, the application of CD19 CAR T cell therapy led to a remarkable reduction in liver pathology scores, indicating a substantial decrease in tissue damage and inflammation compared to control groups. Beyond liver autoimmunity, our research holds promise for treating other autoimmune conditions where B cells play a detrimental role. These insights pave the way for innovative therapies that have the potential to revolutionize autoimmune disease management, offering a hope for affected individuals and improving their quality of life.
Potent and specific killing of SLE donor B Cells with AlloNK (AB-101), an allogeneic cord blood-derived NK cell therapy, in combination with anti-CD19 or anti-CD20 monoclonal antibodies
Authors: Rogers Paul , guerrettaz lisa , Teng Kun-Yu , Palomino La Torre Claudia , Conerty Amanda , Flynn Peter , Raymon Heather
Published: November 2024

B cells play a crucial role in the pathogenesis of SLE by producing autoantibodies that target self-antigens, leading to tissue damage and inflammation. Here we demonstrate proof of concept for the use of a NK cell therapy to enhance the depletion of SLE donor B cells in the presence of anti-CD19 or anti-CD20 antibodies by an antibody dependent cellular cytotoxicity (ADCC) mechanism. AlloNK™ is a non-engineered, allogeneic, off-the-shelf, cryopreserved NK cell product, currently being evaluated in a clinical trial in combination with rituximab in subjects with lupus nephritis. AlloNK, which has been optimized for ADCC, was tested in combination with anti-CD20 (rituximab, obinutuzumab) or anti-CD19 (tafasitamab) mAbs in a short-term ADCC assay to show specific killing of SLE donor B cells. PBMC were isolated from SLE donors and co-cultured with AlloNK at various effector to target (E:T) ratios and mAbs concentrations. After 4h, the percentage of caspase 3/7+ B and T cells was determined by flow cytometry. At a 1:1 E:T ratio, AlloNK mediated killing of B cells in the presence of obinutuzumab (range 79-95%), rituximab (range 19-62%), and tafasitamab (range 24-77%) in a dose-dependent manner. Killing was specific as no off-target apoptosis of T cells was observed. Taken together, these data suggest that AlloNK has the potential to be effective in combination with mAbs to induce deeper B cell depletion and improved efficacy, over the mAbs alone, in SLE.
Notch2 signaling instructs TLR-responsive programs in B cells
Authors: Londregan Jennifer , Gaudette Brian , Galli Francesca , Tursi Nicholas , Allman David
Published: November 2024

The efficiency with which B cells respond to activation signals varies depending on the subset. Whereas marginal zone (MZ) B cells exhibit rapid activation kinetics following stimulation, follicular (FO) B cells adopt delayed effector programs. Further, MZ B cells uniquely exhibit readiness for the plasma cell (PC) fate, differentiating in mere hours independent of mitosis or T cell help. Some of the many ways MZ B cells achieve poised responsiveness is through sustained Myc levels and mTORC1 activity. MZ B cells uniquely require constitutive signaling through the transmembrane receptor Notch2, but whether this signal underlies the functionally poised nature of these cells is not understood. To examine the consequences of forced Notch2 on B cells with no Notch requirement, we co-cultured stimulated FO B cells on Notch2 ligand-expressing OP9 stromal cells. Notch2 signals modified the response to TLR 3 and 4 stimuli by amplifying the time to first division and the propensity toward PC differentiation altogether. Intriguingly, TLR3 responsiveness in B cells appeared to be entirely Notch2-driven and dependent on the signaling adapter Bruton’s Tyrosine Kinase. Employing a published mathematical model for dividing lymphocytes, we found Notch2 signals significantly augmented the division rate and division destiny of activated B cells. Together, these observations demonstrate a novel role for Notch2 in instructing B cell response programs to TLR signals and their effector outcomes.
Mapping the role of ITK (interleukin 2 inducible T cell kinase) and TCR (T cell receptor) signal strength in determining the early fate of CD8 T cells using Nr4a3 “timed” fluorescent reporter
Authors: Hunkins Josh , Ono Masahiro , Perrenoud Loni , Schoenbach Mary , Berg Leslie
Published: November 2024

ITK plays a key role in the rate and magnitude of TCR-dependent transcription factor activity and early genetic programming that determines the extent of CD8 T cell effector and memory function. To study the extent of this role we crossed an OT1 transgenic ITK germline KO to the Nr4a3-Tocky timer model which produces a time dependent fluorescent reporter. Nr4a3 is highly expressed following TCR activation and thus is a good metric for TCR signal strength and duration. The Tocky protein fluoresces blue, at λ 466 and then, after ~7hrs, degrades to a red fluorescence at λ 583. OT-1 Rag1KO ITK (WT, +/-, and -/-) Nr4a3-Tocky bulk splenocytes were stimulated with the altered peptide ligand T4 Ova peptide at varying concentrations for 12/24 hours and sorted on Tocky+ Blue/Red and negative events. ITK -/- CD8s showed elevated CD69 and CD25 but were variable for Tocky expression. Both Tocky Negative and Tocky Blue sorted ITK -/- CD69+ CD8s expressed higher IRF8, cFos, cMyc, Bcl2, and TCF1/7 as well as lower IRF4 versus WT at 12 hours post-stimulation. Analyses of Tocky Blue/Red or persistently active CD8s showed WT having increased expression of TCF1 and similar expression of IRF8, cFos, Bcl2, Eomes and Egr2 compared to ITK -/-. Bcl2 and PDL1 are also highly expressed through all stages of TCR signaling in ITK -/- CD8s. These data indicate that differences in TCR signal strength with and without ITK can affect the time at which transcriptional networks arise after TCR stimulation.
Assessment of molecular mechanisms involved in T-cell receptor (TCR)-dependent metabolic switch into aerobic glycolysis
Authors: Baracho Gisele , Reznan Ali , Shi Sam , Aruna Ayer , Widmann Stephanie , Tyznik Aaron
Published: November 2024

Upon activation, T cells undergo metabolic adaptations to fulfill the energy and biosynthetic requirements for their rapid growth and proliferation. TCR engagement and CD28 co-stimulation trigger the initial metabolic shifts that enable T-cell activation including increased glucose uptake. PI3K signaling plays a pivotal role in regulating Glut-1, the primary glucose transporter in T cells. Using high-dimensional spectral flow cytometry, we found correlated expression between Glut-1 and granzyme B (GZMB) in subsets of in vitro-activated human T cells. Notably, the PI3K inhibitor Ly294002 abrogated TCR-dependent upregulation of Glut-1 abolishing Glut-1highGZMB+ T cells in the cultures. A comprehensive single-cell targeted mRNA profiling of over 56, 000 immune cells captured with the BD Rhapsody™ HT Xpress System further showed a TCR-dependent upregulation of GZMB and various metabolic regulators such as GAPDH, HMGB2 and TK1. The results provide additional details on the involvement of the glycolytic pathway in T cell activation and offer a deeper insight into the molecular mechanisms associated with TCR-dependent cell activation.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademark of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-2938
Characteristics of tumor-rejecting neoantigens identified in a physiologically relevant cutaneous squamous cell carcinoma model
Authors: Hastings Karen , Adams Anngela , Macy Anne , Borden Elizabeth , Herrmann Lauren , Brambley Chad , Ma Tao , Li Xing , Hughes Alysia , Roe Denise , Mangold Aaron , Buetow Kenneth , Wilson Melissa , Baker Brian
Published: November 2024

Challenges in identifying tumor-rejecting neoantigens limit the use of neoantigen-based immunotherapies to treat high mutational burden tumors, such as cutaneous squamous cell carcinoma (cSCC). Most mutations in human cSCC are unique to an individual tumor. Only a small portion of human tumors share neoantigens predicted to bind MHC, supporting the need for personalized strategies. We generated transplantable cell lines, from solar UV-induced cSCC tumors, which recapitulate the UV mutational signature and driver mutations found in human disease. Depletion of CD8 and CD4 T cells revealed a dominant role for CD8 T cells and a supporting role for CD4 T cells in constraining cSCC. Vaccination with irradiated cSCC cells completely protects from tumor challenge and is dependent on CD8 T cells. Neoantigens were prioritized based on predicted binding affinity, MHC presentation and expression. IFN-γ-secreting CD8 T cells recognizing two neoantigens were present in cSCC tumors and tumor-draining lymph nodes. Prophylactic vaccination with these neoantigens constrained cSCC growth. Structural modeling of tumor-rejecting neoantigens in cSCC and other cancer models identified an increased solvent accessible surface area of the mutated residue in tumor-rejecting neoantigens compared to non-immunogenic neoantigens, which is anticipated to improve TCR recognition. This work provides a conceptual basis for consideration of TCR recognition in the prioritization of tumor-rejecting neoantigens.
Characterizing immune-mediated neuronal microtubule destabilization and axonal transport defects in a mouse model of optic neuritis
Authors: Sommers Kelli , Dittel Bonnie
Published: November 2024

Multiple sclerosis (MS) is a central nervous system autoimmune disease that often presents with inflammation of the optic nerve, optic neuritis (ON). The mechanisms of neuronal damage in MS and ON are unknown. In vitro studies modeling MS neurodegeneration demonstrated that cytolytic lymphocytes drive microtubule destabilization in human induced pluripotent stem cell-derived spinal motor neurons. Microtubules are essential for axonal transport, a key function for neuron health. We hypothesize that immune-mediated neuronal microtubule destabilization drives development of ON through disruption of axonal transport. MOG35-55 T cell receptor transgenic mice were used as a model of ON. The non-invasive ophthalmologic technique optical coherence tomography (OCT) allows for confirmation of symptomatic ON. Using OCT, increasing severity of progressive ON was observed over 5 months. To examine transport efficiency, cholera toxin subunit B (CTB), an axonal transport tracer, was injected into the eyes of mice with ON. Optic nerves were collected and stained for β3-tubulin, a marker of microtubules, and imaged by immunofluorescence. Preliminary data found decreased β3-tubulin staining and markedly reduced CTB transport in the presence of immune cell infiltration in mice with ON. These data confirm that OCT can be used to monitor ON progression and provide evidence that immune-mediated neuronal microtubule destabilization causes axonal transport deficits that underly ON development.
Bacterial peptidoglycan drives synovial inflammation in osteoarthritis
Authors: Dickman Jacob , Holub Meaghan , Rouse Joseph , Danner Rebecca , Wahhab Amanda , Jutras Brandon , Edelstein Adam , Lochhead Robert
Published: November 2024

Peptidoglycan (PG) is a potent inflammatory mediator. We have previously demonstrated that PG is present in the synovial tissue of osteoarthritis (OA) patients but its role in OA inflammation remains unknown. We hypothesized that PG in synovial tissue drives inflammation in OA. Intraoperative synovial tissue and synovial fluid samples were obtained from 56 consecutive patients with OA, with no prior history of infection. PG in synovial tissue was detected by immunohistochemical (IHC) staining of serial sections of 10 mm2 of tissue with anti-PG antiserum. PG was found in 33/56 (59%) of the tissue samples using IHC, with a median of eight PG occurrences per 10 mm2 in PG-positive samples. Tissue inflammation and fibrosis were assessed by histopathology. Cellular localization was further characterized in three PG-positive tissue samples by immunofluorescent microscopy (IFM). PG was localized to cells demonstrating mononuclear and fibroblastic morphology primarily in and around areas of vascularization and inflammation. CD68+ macrophages and to a lesser degree CD90+ synovial sublining fibroblasts. In multiplex assay analysis of synovial fluid cytokine levels, IL-6 and PG abundance were positively correlated. Fibroblasts and macrophages stimulated with PG in vitro secreted high levels of innate cytokines, paricularly IL-6 when measured by supernatant cytokine quantification. Together these data support the hypothesis that PG in synovial tissue drives joint inflammation in OA.
Peripheral mucosal-associated invariant T (MAIT) cells are severely depleted in HIV-positive heavy alcohol users
Authors: Li Wei , Khalili Mandana , Huang Laurence , Sanyal Arun , Shah Vijay , Chalasani Naga , Gupta Samir , Yu Qigui
Published: November 2024

MAIT cells are innate-like lymphocytes enriched in mucosal tissues, liver, and peripheral blood, contributing to protection against pathogens. Alcohol abuse and HIV infection independently reduce circulating MAIT cells. To assess their combined impact, we studied peripheral blood MAIT cells in heavy drinkers without overt liver disease (ALC), people with HIV (PWH) on ART, PWH on ART with heavy drinking (HIV-ALC), and healthy donors (HD). MAIT cell frequencies were significantly reduced in ALC (median 0.56, IQR 0.22-1.44; p=0.001), HIV (0.43, 0.20-0.97; p<0.001), and HIV-ALC (0.30, 0.14-0.74; p<0.001) vs HD (1.52, 0.74-2.71), accompanied by increased apoptotic MAIT cells. MAIT cells in HIV and HIV-ALC expressed elevated levels of activation markers (CD38, HLA-DR), with HIV-ALC also having higher CD69 levels than HIV and HD. Activated MAIT cells in HIV-ALC positively correlated with disease severity (prolonged prothrombin time, total bilirubin) and trended inversely with CD4% and CD4/CD8 ratio. However, no correlate was observed with cytokines linked to MAIT cell activation/function (IFN-α, IL-7, IL-12, IL-15, IL-17, IL-18, IL-23, IFN-γ, TNF-α). They also did not correlate with levels of blood biomarkers of gut injury/microbial translocation (TFF3, REG3α, LBP), monocyte/macrophage activation (sCD14, sCD163), and systemic inflammation (CRP). Conclusion: Peripheral blood MAIT cells in HIV-ALC were severely depleted, yet highly activated, and associated with disease severity.
Mechanistic insights into immune amnesia and immunity induced by measles infection and vaccination via comprehensive antiviral antibodies profiling
Authors: Chan Kwan Pui , Liu Liting , Bartlett Maggie , Griffin Diane
Published: November 2024

Measles is a disease known to induce acute immune amnesia and immunosuppression with increased susceptibility to other infections, while paradoxically conferring patients with life-long immunity to measles. However, it is not clear if the live-attenuated measles vaccine (LAMV) induces immunosuppression comparable to the wild-type measles (WT MeV) infection, nor how mechanistically LAMV and WT MeV affect antibodies specific to different pathogens.

Therefore, we utilized VirScan, a phage-display immunoprecipitation and sequencing (PhIP-seq) technology for virome-wide detection of antibodies against viral epitopes, to compare longitudinal antibody diversity in plasma samples from WT MeV and LAMV-infected rhesus macaques (RMs) (n=10 and 6 respectively, with 5 longitudinal timepoints at D0, D14/15, D28, D84/89, D112/168/176).

Preliminary analysis showed that WT MeV led to more long-term depletion of pre-existing antibodies to various viral peptides, while mounting a MeV-specific antibody response with greater intensity and breadth than LAMV-infected RM. Furthermore, in our sex difference analysis, male RMs elicited a stronger MeV response than female, particularly to N protein; also a more polyclonal MeV humoral response

In summary, our work provided crucial mechanistic insights into immune amnesia and immunity induced by natural infection versus vaccination, highlighting the public health value of the measles vaccine in averting MeV-induced immune amnesia.
Perforin production by T cells and Natural Killer cells in CMV-reactivated allogeneic cell transplant patients is associated with early post-transplant death
Authors: Sheikh Daanish , Nesher Lior , Khawaja Fareed , Shields Christina , Grimbergen Andrea , Nahmod Karen , Carrero Longlax Santiago , Ladki Malik , Nishiguchi Tomoki , Chemaly Roy , Makedonas George , DiNardo Andrew
Published: November 2024

Introduction:

Cytomegalovirus (CMV) is a prevalent herpesvirus that poses a serious risk to immunocompromised patients, particularly hematopoietic stem cell transplant (HSCT) recipients. CMV reactivation increases the risk of mortality due to secondary infections. CMV infection has been shown to epigenetically induce immune exhaustion and decrease non-specific immunity.

Methods:

We reviewed flow cytometry data from CMV seropositive HSCT recipients to determine if CMV reactivation predicted mortality via modulation of lymphocyte function. Peripheral blood mononuclear cells (PBMCs) were collected from recipients 30 days following transplant and stimulated with CMV-specific peptide pools of pp65 and staphylococcal enterotoxin B (SEB) to assess CMV-specific and non-specific immune function.

Results:

Non-specific, SEB-induced, polyfunctionality of CD4 and CD8 T cells was diminished in deceased CMV seropositive HSCT recipients. In deceased individuals, T-bet and perforin expression were elevated, with decreased IFNγ, TNFα, or IL2 SEB-induced production.

Discussion:

Mortality in CMV reactivated HSCT recipients was associated with non-specific immune exhaustion phenotypes, but it remains unclear whether this was a result of a higher pathogenic burden, recurrent CMV reactivation, or chemotherapy and myeloablation.
Microglial Differential Transcriptional Profiles in the Late-Stage Chronic Conditions of alpha synucleinopathy
Authors: Raihan Md Obayed , Fazal Nadeem , McGregor Brett A , Hur Junguk , Porter James E
Published: November 2024

Microglial immune function is compromised under pathological perturbation and a dysregulated immune response has been linked to neurodegenerative disorders and their progressive nature, including synucleinopathies. Using a transgenic mouse model of synucleinopathy (mThy1-hα-Synuclein), we have previously reported significant differences in microglial transcriptomic profiles at early and late stages of disease. We are interested in changes to microglia gene expression and regulation which contribute to disease progression or symptoms. Mice were characterized via behavior testing for motor deficit prior to microglia isolation and RNA extracted for RNA-sequencing. A total of 1,591 genes were differentially expressed (DEGs) in transgenic mice compared to age-matched controls. The DEGs were assessed for enriched functional terms via the Gene Ontology (GO) database and were clustered based on gene overlap between terms identifying 89 total clusters. Terms related to cytokine signaling, immune system, and synaptic transmission were enriched, which may suggest a link between declining motor function and αSyn associated pathology. Overall, our present study detailed the profile of αSyn reactive microglial under late-stage chronic conditions. Future studies in microglia regulation in neurodegenerative diseases will be critical to determining their contribution to disease progression as well as for developing novel therapeutics aimed at targeting neuroinflammation.
Natural History study to characterize CC011Unc/J spontaneous Mouse Model of Colitis
Authors: Gonzalo Gil Elena , Bowden Mia , Kramer Joshua , Davis Katherine , Copeland Katherine , Gutierrez Alan , Gott Rachel , Kasper Rachel , Case Laure
Published: November 2024

Objective: Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a complex disorder characterized as an autoimmune response to the host’s intestinal microflora. We evaluate the progression of the colitis using CC01UncJ mice, a recombinant inbred mouse with similar characteristics to human IBD (Rogala AR et al., Mamm Genome, 2014. 25:95-108).

Methods: CC011Unc/J mice (JAX stock# 018854) were monitored weekly. Mice were euthanized at 8, 25, 30, 35 or 40 weeks of age, and colons were fixed and stained with hematoxylin and eosin. Slides were analyzed for leukocyte density, glandular hyperplasia, goblet cell loss, vasculitis, crypt abscesses, mucinous lakes, ulceration, and extent of lesion on a scale from 0-4.

Results: CC011Unc/J mice showed mild to moderate inflammation characterized by multifocal lymphocytes and plasma cell aggregates. A statistically significant scoring increase was observed between 8 weeks and 30 weeks (p<0.01), with a plateau reached at 35-40 weeks of age. Significant differences between males and females were not observed for any of the scored parameters.

Conclusion: In contrast to the publications, CC011Unc/J mice showed only a moderate inflammation, suggesting an important role of environmental conditions to the development of the disease. Additional studies will be performed at JAX preclinical services to fully characterize these mice to offer this model as a platform for performing pre-clinical testing.