bluuebunny/crossref_metadata_2025 huggingface dataset:
Obesity-driven IL-18 inhibits the suppressive function of FOXP3+ T-regulatory (Treg) cells
Authors: Akimova Tatiana , Zhang Tianyi , Beier Ulf H. , Jiao Jing , Diamond Joshua M. , Christie Jason D. , Simmons Rebecca A. , Hancock Wayne W.
Published: January 2023

We investigated ways by which obesity affects the immune system and promotes inflammation, given that obesity has a high economic and medical impact, worldwide. IL-18 was the only inflammatory marker differing between our colonies of “obese but otherwise healthy” (hHFD) and “obese with low-grade inflammation” (iHFD) mice. All mice gained weight equally, had signs of low-grade inflammation (increased TNF-a, IL-1b and IL-6 mRNA) in adipose tissues, and high plasma leptin levels. The livers of hHFD mice had no inflammatory phenotype despite evidence of steatosis, whereas livers of iHFD mice had increased TNF-a, IL-1b, IL-6 and IL-7 mRNA. Levels of IL-18 were increased in livers and fat of iHFD, but not hHFD mice, and were associated with significant impairment of Treg suppressive function. Addition of IL-18 had significant inhibitory effects on the suppressive function of murine and human Tregs, and Tregs isolated from obese/overweight patients also had impaired suppressive activity. Human Tregs express high levels of IL-18Ra, and downregulate FOXP3 protein but not mRNA, when stimulated in the presence of IL-18. FOXP3 downregulation compromises the FOXP3-regulated gene network, leading to re-activation of pro-inflammatory genes such as IFN-g, IL-2, IL-6 and IL-17, and downregulation of CTLA-4 protein, which is critical for suppressive function. Our preliminary data indicate that IL-18 activates the MyD88 pathway in Tregs and impairs FOXP3 protein stability, possibly by ubiquitination and proteasomal degradation. This may be a physiologically essential tool to dampen Treg function during acute infection and increase the efficiency of immune responses, but has a detrimental effect in obesity-driven low-grade inflammation.
Antioxidant treatment ameliorates cigarette smoke and respiratory syncytial virus-induced inflammatory response by blocking HMGB1 release
Authors: Hosakote Yashoda Madaiah , Rayavara Kempaiah , Hallberg Lance M. , Ameredes Bill T. , Kurosky Alexander
Published: January 2023

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants, and causes severe respiratory morbidity and mortality in the elderly. Second hand tobacco smoke is the major environmental risk factor for the development and severity of lower respiratory tract infections, however, the molecular mechanism of tobacco smoke-induced RSV severity is not fully understood. High mobility group box 1 protein (HMGB1) has been shown to functions in the regulation of cellular transcription and the activation of proinflammatory responses in many pathogenic conditions, but little is known about its molecular mechanism of action during its release in respiratory viral infections and tobacco smoke-induced lung inflammation. In this study we investigated the mechanism of HMGB1 release and associated cellular signaling in RSV and cigarette smoke (CS)-induced airway inflammation. A549 cells, a human lung carcinoma cell line and normal small alveolar epithelial (SAE) cells were pretreated with antioxidants and exposed to CS followed by RSV infection. CS exposure in combination with RSV infection synergistically enhanced HMGB1 secretion with concomitant decrease in the total cell lysates when compared to uninfected as well as each stimulus alone. NFκB and MAPK were significantly activated in CS exposed and RSV-infected cells. Treatment with antioxidant significantly inhibited RSV and CS-induced HMGB1 secretion, cytokine release, and NFκB and MAPK activation. The results of these studies will help to design new therapeutic strategies not only for RSV infection, but also for other respiratory diseases that have been shown to be caused or exacerbated by environmental toxicants such as tobacco smoke.
Alcohol abstinence partially restores peripheral mucosal-associated invariant T cells in patients with alcoholic hepatitis
Authors: Li Wei , Lin Edward , Liangpunsakul Suthat , Rane Susimita , Lan Jie , Chalasani Sai , Puri Puneet , Kamath Patrick , Sanyal Arun , Shah Vijay , Katz Barry , Radaeva Svetlana , Crabb David , Chalasani Naga , Yu Qigui
Published: January 2023

Alcoholic hepatitis (AH) develops in a small percentage of heavy drinkers. The adaptive immune system of AH patients is hyper-activated, yet dysregulated and ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are found in liver, intestinal mucosa, and peripheral blood and contribute to protection against infectious diseases. To determine whether MAIT cells were dysregulated in heavy drinkers and the effect of alcohol abstinence on MAIT cell recovery, we compared peripheral MAIT cells between AH patients and heavy drinkers without liver disease (HDC) at baseline and 6- and 12-month followup. At baseline, AH patients had severely reduced frequency of MAIT cells than HDC and healthy donor controls (HC), whereas MAIT cell frequency was not significantly different between HDC and HC. In addition, MAIT cells in AH patients expressed higher levels of the activation markers (CD69, CD38, and HLA-DR) as well as the immune checkpoint inhibitor PD-1. Multiplex immunoassays showed that plasma levels of several cytokines related to MAIT cell activation were elevated in AH patients (IFN-s, IL-7, IL-15, IL-17, IL-18, and IL-23, and TNF-a). In AH patients, the frequency MAIT cells and CD38-expressing MAIT cells correlated with levels of several cytokines and AST. At followup, AH patients had increased MAIT cell frequency and decreased MAIT cell activation with cessation of alcohol. However, abstinent AH patients still had higher levels of CD38-expressing MAIT cells than HDC at 6-month followup.

Conclusion

Blood MAIT cells were severely reduced and were dysregulated in AH patients and alcohol abstinence partially reversed abnormalities in MAIT cell frequency and phenotype.
BCAS2 is a neuron protector to inhibit microgliosis
Authors: CHEN SHOW LI.
Published: January 2023

Breast carcinoma amplified sequence 2(BCAS2) is involved in Prp19/CDC5 Lspliceosome complex for regulation of RNA splicing. Exon array analysis indicated that β-catenin was one of BCAS2-targeted gene. Disruption of Wnt/β-catenin signaling is involved in several neurodegenerative diseases such as Alzheimer’s disease (AD). Current report of other group demonstrated that the RNA expression of BCAS2 is reduced in the microarray analysis of AD patients. Recently, we used the Cre/loxP system driven by CaMKIIα to generate BCAS2 conditional knock out (cKO) in postnatal forebrain, which causes microcephaly-like, dendritic malformation, poor learning and memory phenotypes partly reasons from down-regulation of β-catenin. In this study, the reactive gliosis in cKO mice was extensively measured and compared with WT mice. The selective microgliosis was found in the cornu ammonis1 stratumradiatum (CA1 Rad) and dentate gyrus molecular layer (DG Mo) but did not exhibit astrogliosis in cKO compared with WT mice, which confirmed depletion of BCAS2 in the forebrain caused neural defect. Lithium, an inhibitor of glycogen synthase kinase-3β (GSK-3β). Lithium treatment to cKO mice could improve their spatial learning and memory capability using passive avoidance and Morris water maze; and reduced selective microgliosis in the molecular layer (DG Mo)compared with untreated cKO mice. Taken together, BCAS2 may be a neuron protective factor to inhibit microgliosis.
Evaluation of the pig-tailed macaque (Macaca nemestrina) as a model of human Staphylococcus aureus nasal carriage
Authors: Cole Amy Liese , Cosgrove-Sweeney Yvonne , Lasseter Amanda G. , Gray Justin M. , Beavis Ashley C. , Chong Christine , Hajheidari Safarali V. , Beyene Alex , Patton Dorothy L. , Cole Alexander M.
Published: January 2023

Staphylococcus aureus (SA) nasal carriage is a common condition effecting both healthy and immunocompromised populations, and provides a reservoir for dissemination of potentially infectious strains by casual contact. Factors regulating the onset and duration of nasal SA colonization are mostly unknown, and a human-relevant animal model is needed. Here, we screened 17 pig-tailed macaques (Macaca nemestrina) for SA carriage, and 14 of 17 animals tested positive in the nose at one or both screening sessions (8 weeks apart), while the other three animals were negative in the nose but positive in the pharynx at least once. Similar to humans, SA colonization was densest in the nose, and treatment of the nostrils with mupirocin ointment effectively cleared the nostrils and 6 extra-nasal body sites. Experimental nasal SA colonization was established with 104 CFU/nostril, and both autologous and non-autologous strains survived over 40 days without any apparent adverse effects. A human nasal SA isolate (D579/ST398) was carried in 4 of 6 animals for over three weeks. Nostrils that did eradicate experimentally applied SA exhibited neutrophilic innate immunity marked by elevated nasal IL-1β, IL-8, and MCP-1, and a 10-fold decreased IL-1RA:IL-1β ratio within 7 days post-inoculation, analogous to the human condition. Taken together, pig-tailed macaques represent a physiological model of human SA nasal carriage with utility for testing natural colonization and decolonization mechanisms, and novel classes of anti-SA therapeutics.
Sirt1-mediated metabolic modulation of Aicda, class-switch DNA recombination and somatic hypermutation
Authors: Gan Huoqun , shen Tian , Sanchez Helia , Xu Zhenming , Zan Hong , Casali Paolo
Published: January 2023

Maturation of the antibody response entails Ig somatic hypermutation (SHM) and class switch DNA recombination (CSR). SHM and CSR perfect the antigen-binding activity and diversify the effector functions of antibodies. By showing that histone deacetylase (HDAC) inhibitors selective for class I/II HDACs dampened SHM/CSR by decreasing activation induced cytidine deaminase (AID) expression through upregulation of miRNAs that target Aicda, we have outlined an important role of Zn-dependent HDACs in the modulation of the antibody response. Here we have shown that the NAD+-dependent Class III HDAC Sirt1 also plays an important role in the regulation of SHM and CSR. B cells undergoing CSR/SHM profoundly downregulated Sirt1. Sirt1 deletion in AID-expressing B cells of AicdacreSirt1fl/fl mice, led to increased CSR and SHM in response to NP-CGG, as a result of upregulated AID expression, without altering Prdm1 expression. Sirt1-deficient B cells reduced DNA methylation in the Aicda promoter and hyperacetylated p65, thus activating the canonical NF-kB pathway, which mediates Aicda activation, in absence of any change in DNA methylation of the Prdm1 promoter or histone acetylation of the Aicda or Prdm1 promoter. Increased glucose concentration, which reduced NAD+ Sirt1 co-factor, led to hyperacetylated p65 and Aicda upregulation. Conversely, the NAD+ Sirt1 co-factor or SRT1720 small molecule, a specific Sirt1 activator, dampened Aicda expression and CSR once added to B cells. Thus, our findings outline an important B cell-intrinsic role of Sirt1 in the modulation of the antibody response. Such a role is independent from and may be in addition to the histone deacetylation activity of this Class III HDAC in immune cells other than B lymphocytes.
IL-5 signaling protects mice against mortality from acute lung injury
Authors: Hrusch Cara L. , Mills Kathleen A.M. , Krishack Paulette A. , Decker Donna M. , Blaine Kelly M. , Sperling Anne I.
Published: January 2023

Acute respiratory distress syndrome (ARDS) affects almost 200,000 people in the US each year with a mortality rate of 20–40%. ARDS occurs as a result of acute lung injury (ALI)-induced damage to pulmonary epithelium and/or endothelium. In a bleomycin model of ALI, we previously have shown that ICOS−/− mice, which have defective type-2 immune responses, are more susceptible to death. However, exogenous treatment of these mice with IL-5 reduces lung edema and mortality. We hypothesized that IL-5 signaling is necessary for survival from ALI and examined how mice deficient in IL-5Rα respond during ALI. IL-5Rα−/− mice had significantly increased lung edema at day 5 post-bleomycin challenge compared to WT mice (P=0.014), and had 100% mortality by day 15 (P=0.0002). IL-5Rα−/− mice had severely reduced numbers of lung eosinophils before and after challenge compared to WT mice, suggesting that eosinophils may provide protection from ALI. However, we found no difference in survival between eosinophil-deficient PHIL mice and WT mice (P=0.36). In WT mice, IL-5Rα was constitutively expressed by lung eosinophils and neutrophils. However, bleomycin challenge upregulated the receptor on myeloid cells and epithelial cells, indicating that IL-5 may regulate multiple cell populations during inflammation. Our study demonstrates that IL-5 is a critical mediator of protection against lung injury by maintaining barrier function in the lung. Surprisingly, eosinophils may not be required for the protective effect, and our data suggests that many lung cell types are capable of responding to IL-5 in the context of ALI. Thus, IL-5 may be a novel therapeutic strategy to protect against lung injury following pulmonary infection, sepsis, or trauma.
Protection from lethal Flu infections: lessons from a systemic lupus mouse model
Authors: Abboud Georges , Choi Seung-Chul , Titov Anton , Li Wei , Morel Laurence
Published: January 2023

Patients with systemic lupus erythematosus (SLE) are more susceptible to infections than healthy subjects but it is unknown whether this is due to disease or treatment. Using mice expressing three SLE susceptibility loci (triple congenics hereafter called TC mice) we unexpectedly found that these mice are resistant to an otherwise lethal respiratory Flu PR8 (H1N1) infection. At the naïve state, TC mice had not only more activated plasmacytoid dendritic cells, inflammatory monocytes and natural killer cells but also more activated CD8+ and CD4+ T cells with higher TNF and IFNg-producing capacity than C57BL/6J controls. At the primary effector phase following intranasal Flu PR8 infection, TC mice mounted poor antibody responses but their Flu-specific CD8+ T cell responses were greater effectors at the membrane level (higher frequencies of KLRG1+IL7R− and KLRG1+IL7R+) and at the transcriptional level (higher T-bet expression) than CD8+ T cells from C57BL/6J counterparts. PR8-immune TC mice also developed more functional Flu-specific CD8+ T cell responses than C57BL/6J mice following to a heterosubtypic Flu X-31 (H3N2) challenge. While immunosuppressive treatments of SLE in patients might be responsible of mounting poor anti-microbial immune responses, lessons from this SLE mouse model could be taken to prevent and protect against severe Flu infections.
Toll-like receptor-4-interacting surfactant protein-A-derived peptide reduces inflammation in a mouse model of intratracheal lipopolysaccharide challenge.
Authors: Awasthi Shanjana , Kumar Gaurav , Xie Jun , Beierle Jacob , Awasthi Vibhudutta , Ramani Vijay , Singh Bhupinder , Breshears Melanie , Kosanke Stanley
Published: January 2023

Acute lung injury is characterized by inflammation and respiratory failure. In this work, we evaluated the effect of Toll-like receptor 4 (TLR4)-interacting SPA4 peptide on inflammatory and physiological parameters in cells and in mice against lipopolysaccharide (LPS). Cellular toxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Binding of SPA4 peptide was studied in cells transiently transfected with a plasmid DNA encoding TLR4. We assessed the changes in the levels of cytokines and chemokines in supernatants of cells treated with SPA4 peptide simultaneously or post-LPS challenge. The extent of lung edema, levels of cytokines and chemokines, and changes in lung histopathology, blood gas and electrolytes, and serum lactate levels, were then determined after SPA4 peptide treatment in a mouse model of acute lung inflammation and injury induced by intratracheal LPS challenge. Our results demonstrate that the SPA4 peptide is not toxic, and binds to TLR4 at the cellular level. A significant reduction was observed in secreted levels of LPS-induced cytokines and chemokines in cell-free supernatants. Correspondingly, the levels of TNF-α, IL-1β, IL-6, MIP-2, KC, and MCP-1 were reduced in bronchoalveolar lavage fluids of SPA4 peptide-treated mice. Moreover, SPA4 peptide treatment attenuated the LPS-induced pulmonary inflammation, lung edema, serum lactate, and blood gas and electrolyte parameters. Together, our results suggest that the SPA4 peptide treatment can help control lung inflammation and injury.
Versatile system for single cell acquisition and analysis
Authors: Ma Zhongcai , Kudo Lili C. , Karsten Stanislav L.
Published: January 2023

Single cell analysis (SCA) has become an essential part of basic and clinical research. A prerequisite to SCA studies is the isolation of individual cells from cell cultures. There are commercially available technologies including laser based systems and cell sorting instruments. However, they are typically complex and expensive making integration within standard lab/clinical sample processing workflows difficult. We have developed a universal platform for rapid acquisition of single cells from cultures and deposition into the single wells for further molecular analysis or clonal expansion. The instrument may also be used for tissue microdissection and measurements of individual cell adhesion. The main working principle is based on the capillary based vacuum pulse assisted technology (CTAS; UP8797644). Developed instrument collects individual cells from any adherent cultures grown in standard cell culture dishes in as small as 15 nl volume. It may be used with a wide range of inverted microscopes, so that the cells of interest can be identified based on morphology, location or fluorescent label. Automatic extraction and deposition algorithms were developed for acquisition of targeted cells and tissue regions of interest. Here, individual cells were collected from human neuroblastoma SH-SY5Y, CHO, and 3T3 cell lines. Collected cells were dispensed immediately into individual wells for clonal expansion. Clonal expansion and trypan blue assay revealed minimal effect on cellular viability (up to 99% when compared to controls). The benefits of the proposed technology include cost-efficiency, simple operation, complete workflow, and compatibility with a wide range of inverted microscopes and standard culture dishes.
Rapid cell fractionation of whole immune organs using a novel microfluidic device
Authors: Pradhan Isha , Cohen Henry , Wong-Noonan Stephanie , Wells Derrick , Lakomy Robert , Tajima Asako , Bai Shi , Rudert William , Boehm Bernhard O. , Trucco Massimo , Hou Han Wei , Fan Yong
Published: January 2023

Separation and isolation of specific cell populations from solid immune organs are essential steps in immunological research. While antibody-labeling based cell sorting techniques, such as FACS, offer the possibility of isolating rare cells from a large background population, the cost and lengthy preparation time associated with these approaches remain burdensome for many researchers. Here, we report a rapid partitioning method that allows the separation of immune cells based on their size and density using a novel, spiral designed, microfluidic device (MFD) (Hou et al., 2016)

Representing less than 0.5% of the total thymic cells, thymic epithelial cells (TECs) are critical for the generation of diverse T cell populations, essential for acquired immunity. Due to their rarity, separating TECs from the large pool of thymocytes is challenging. Using a MFD, we achieved substantial enrichment of TECs (up to 20X), high cell recovery (>2.5X of the traditional density gradient method) and excellent cell viability (>90%) within 7 minutes/thymus. High-purity TEC populations were obtained through further depletion of CD45+ thymic stromal cells using MACS. Thymus organoids reconstructed with TECs can support the development of both CD4+ and CD8+ T cells in vitro, proving the function of the isolated TECs.

In addition, MFD could also be utilized to separate immune cell subtypes from spleen and lymph nodes. Notably, significant enrichment of CDllc+ MHCII+ dendritic cells (~15X) was achieved without RBC lysis and antibody labeling. In conclusion, our data suggest that the MFD partitioning method is efficient and cost-effective for rapid size-based separation of immune cells from primary and secondary immune organs for downstream applications.
A novel high-throughput method for quantification of microbiota distribution at the single cell level using high dynamic range DNA barcoding
Authors: Shiroguchi Katsuyuki , Jin Jianshi , Takeuchi Tadashi , Miyauchi Eiji , Ohno Hiroshi
Published: January 2023

In the bacteria-host interaction, the dynamics of microbiota contributes to the development of many diseases, e.g., infectious diseases, gastrointestinal cancers, autoimmune diseases, etc. The mechanisms behind the dynamics of microbiota remain unknown, and one of them is cell-cell interaction among the bacteria within microbiota. In order to investigate the mechanisms, a method to characterize the microbiota, including identification of species and the cell-number distribution of all species, is required. However, the current techniques measure the distribution of 16S rRNA copies, which is different from the cell-number distribution, since different bacterium has different 16S rRNA copies on its genome. Here we are developing a novel high-throughput bacterial counting/identification method by 16S rRNA sequencing and our developed cell barcoding. To validate this method, we measured a known ten bacterial community, and successfully identified their correct 16S rRNA sequences. This method identifies sequences independently of database and distinguishes single-base difference, so that it can identify unregistered 16S rRNA sequences as well. Based on these identified 16S rRNA sequences, we quantified the cell numbers of each bacterium. The results were consistent with the cell numbers measured under a microscope. Furthermore, we applied this method for studying the murine gut bacterial microbiota, and we found significant changes between the large intestine and the cecum. We also identified unregistered 16S rRNA sequences in both samples. This method offers a high-throughput experimental way that directly counts each bacterium in a bacterial community including unregistered bacteria at the cell level.
Standardized oligo barcode antibody conjugates, and Veri-Cells™ PBMC for multiplex immunophenotyping and CITE-Seq
Authors: Li Cheng-Rui , Yeung Bertrand , Zhao Xinfang , Divekar Anagha , Monell Craig , Yang Xifeng
Published: January 2023

The CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) platform is a recent advance in single cell analysis, which is based on high-throughput single cell sequencing (scSeq) and combines measurements of cellular proteins and transcriptomes. This platform will potentially transform how complex cell populations (lineage differentiation or tumor infiltrating lymphocytes) are studied. Published data indicated that CITE-Seq analysis on cell surface marker expression are comparable to multi-color flow cytometry, but provides superior capacity in multiplexing to the latter. Currently, individual investigators use their choice of oligo bar-codes for different protein markers and there is no available control. This will make comparison of data from different studies difficult in the future. The availability of standardized oligonucleotide bar-code labeled antibodies as well as control cells will enable reliable comparison data from longitudinal studies or among different studies. FlexSeq™ is our new product line, in which monoclonal antibodies are conjugated with unique oligonucleotide bar-codes. We provide standardized bar-coding system and ready to use oligo-antibody conjugates to support CITE-Seq based multiplex immunophenotyping. Veri-Cells™ is our proprietary lyophilized cell product that can be used as a control for detection of cell surface and intracellular flow cytometry. In this study, we validate the application of FlexSeq™ products in CITE-Seq platform, and compare Veri-Cells™ PBMC with freshly isolated PBMC in the assays.
Enhanced cell growth with physiologically relevant media supplements.
Authors: Naser Lisa , Master Alyssa M.
Published: January 2023

Recent advances in genetic engineering have resulted in exponential growth in cell therapy technologies. This has led to a need for corresponding advances in the cell culture media that is used to recover, sustain and expand these important cells. Cell therapy researchers and manufacturers alike, have spent valuable time and money to optimize media formulations to enhance cell proliferation while maintaining functional capabilities of stem cells and immune cells. Proliferation rates and cell robustness often suffer in serum free media (SFM) or chemically defined formulations due to the lack of physiologically relevant protein sources and concentrations. Physiologix™ XF Human Growth Factor Concentrate (hGFC) is a cell culture media supplement that can be used in place of serum supplements with traditional basal media such as RPMI 1640 or DMEM/F12. In this study, hGFC was compared to various serum free media. CD4+ T cells, bone marrow derived mesenchymal stem cells (BM-MSC) and induced pluripotent stem cells (iPSC) were cultured in the presence of hGFC or SFM. All cell types had preferentially higher cell count when grown in the presence of hGFC than in their corresponding SFM. In addition to this, CD4+ T cell function was examined by looking at cytokine profiles, activation markers CD25 and CD69, and exhaustion marker PD-1. Cells showed higher capability for activation in presence of hGFC. They also exhibited greater ability for exhaustion under constant stimulation, which is indicative of a more physiologically relevant response to constant stress. The implications of this data are particularly important for those in cell therapy manufacturing where cell proliferation rate and viability are critical.
Analytical Validation of Digital Spatial Profiling - a novel approach for multiplexed characterization of protein distribution and abundance in FFPE tissue sections
Authors: Merritt Chris , Barker Kristi , Metz Heather , Dennis Lucas , Webster Philippa , Beechem Joseph
Published: January 2023

Nanostring Technologies’ Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10’s to 100’s of proteins from discrete regions of interest (ROI), providing a morphological context to multiplexed protein analysis. The purpose of this study is to validate an antibody panel designed to characterize key immunology and tumor markers on the DSP platform.

IHC was performed on human FFPE tissues and human cell line pellets to evaluate binding specificity in both unconjugated and oligo-conjugated antibodies. The dynamic range of antibodies was tested using positive and negative FFPE cell pellets at different ratios. Interaction screens were performed to evaluate potential effects of multiplexing antibodies. A tissue microarray (TMA) containing normal and cancer tissues was employed to assess assay robustness.

IHC analysis of antibodies displayed indistinguishable staining patterns for both unconjugated and oligoconjugated antibodies. Mixed-proportion cell pellet assays revealed strong correlations between positive counts and positive cell numbers in an ROI. For example, CD3 displayed a LOD of 4% when assayed using cell pellet mixtures containing CD3+ CCRF-CEM cells and CD3− HEK293T cells. Interaction studies showed similar values for antibodies alone or in combination (R2 > 0.8). TMA hierarchical clustering analysis demonstrated expected patterns for immune and tumor cells across different tissues.

These results demonstrate that indexing oligo conjugation does not interfere with antibody specificity and that these conjugated antibodies are robust reagents for quantification of protein abundance. Continued work on the DSP platform will expand the library of antibodies accessible for profiling.
Biomarker prediction to anti-PD-1 immunotherapy by using high dimensional single cell analysis
Authors: Krieg Carsten , Nowicka Malgorzata , Guglietta Silvia , Schindler Sabrina , Hartmann Felix J. , Weber Lukas M. , Dummer Reinhard , Robinson Mark D. , Levesque Mitchell P. , Becher Burkhard
Published: January 2023

Blockade of checkpoint inhibitors such as programmed cell death protein 1 (PD-1) set the pace for the immunotherapy of cancer. Following the initial success in metastatic melanoma, by showing a dramatic increase in progression-free survival, PD-1 immunotherapy was expanded to other cancers. As PD-1 monotherapy advances into first line treatment and combination of checkpoint inhibitors and other therapies start to show even increased response rates, still a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of clinical response for stratification of patients most likely to respond to immunotherapy are urgently needed. In this study, we employed high-dimensional single cell mass cytometry and a generalized machine-learning bioinformatics workflow for the in-depth characterization of all immune cells in the peripheral blood of stage IV melanoma patients before and at 12 weeks of anti-PD-1 immunotherapy. In agreement with published literature a clear response in the T cell compartment of patients under immunotherapy was observed. To our surprise, prior to commencing PD-1 immunotherapy, a strong predictor of progression free and overall survival in responders to anti-PD-1 was a higher frequency of CD14+CD16−HLA-DRhi monocytes. We confirmed this finding by conventional flow cytometry in an independent, blinded validation cohort. Altogether, in this study, we identified for the first time a novel predictive biomarker for therapy decision in the treatment of metastatic melanoma and propose to extend this approach to select for positive responders in other cancers prior to immune checkpoint therapy.
Chemical screening based machine learning platform to identify MDSC-specific gene programs and immunomodulators to target solid tumor microenvironments
Authors: Chopra Gaurav , Kischuk Erin , Majumder Joydeb , Fine Jonathan , Lantz Travis Christopher , Ratliff Timothy L.
Published: January 2023

Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages can reprogram the pro-tumor microenvironment towards anti-tumor immunity. We hypothesized that MDSC suppressive function can be altered by targeting differentially expressed up-regulated genes of activated monocytic MDSCs in the tumor microenvironment compared to spleen. Using our proteome-scale target/antitarget network based interaction platform we identified and synthesized cell specific non-toxic chemical libraries with exclusive functions for anticancer potency and immunomodulation. MDSC were isolated from the tumor tissue of mice with bladder cancer, incubated with tumor explant supernantant under hypoxic condition to partially recreate the tumor microenvironment. The impact of synthetic compounds was assessed via changes in nitric oxide production and expression of function-associated genes, such as iNos, Arg1, CD274 (PD-L1). We used machine learning to iteratively predict activity that was verified experimentally. Feature selection based on proteome-compounds interactions simultaneously identified cell-specific gene programs and synthetic compound library targeting modular MDSC cell function ex vivo. We selected a compound that show no sensitivity to bladder cancer cells in vitro to assess if our cell-specific predictions translated in vivo. Significant reduction in tumor mass, lower frequency of MDSC expressing suppressive functional markers (iNOS, PD-L1) and increased IFNγ+T-cells was observed following treatment by oral gavage. We conclude that our platform can be used generally for desired immune cells to identify gene-program specific compounds to perturb the tumor microenvironment for combating solid tumors.
Stem cell, low-energy shock wave and phosphodiesterase type 5 inhibitor therapy to promote erectile function recovery in a rat pelvic neurovascular trauma model
Authors: Hertz Alexandria M. , George Evalyn I. , Reed-Maldonado Amanda B. , Brand Timothy C. , Salgar Shashikumar K.
Published: January 2023

Introduction

Battlefield pelvic injuries can have long-lasting debilitating effects, including erectile dysfunction (ED) in young soldiers. We investigated whether phosphodiesterase-5 inhibitor (PDE5i), low-energy shock wave (LESWT) and/or mesenchymal stem cell (MSC) therapy could improve erectile function (EF) after pelvic injury.

Methods

Lewis rats 10–12 weeks old were used. We had nine experimental groups (n=6/group): Sham, Injury alone, PDE5i, LESWT, MSC, LESWT+PDE5i, LESWT+MSC, MSC+PDE5i, and LESWT+PDE5i+MSC. Pelvic injury was created by performing bilateral cavernous nerve crush injury and internal pudendal bundle ligation. Bone marrow-derived MSCs (CD29+, CD90+, CD34−, CD31−) were injected (2×106 cells) into the corpora cavernosum. Rats received LESWT (1000 pulses at 0.06 mJ/mm2 and 3Hz) three times per week or sildenafil (PDE5i) in drinking water at ~10 mg/kg/day for four weeks. At 6thweek intracavernous pressure (ICP; mmHg) was recorded as EF outcome measure.

Results

Erectile function was significantly (P<0.001) improved with PDE5i+LESWT therapy (max ICP 131±16, average ICP 116±11, change ICP 63±13) compared to untreated injury control (max ICP 88±24, average ICP 81±13, change ICP 16±23); ICPs became nearly normal, similar to uninjured sham group. LESWT alone improved (P<0.05) EF (max ICP 105±14; change in ICP 39±15) compared to untreated injury control. Also, MSC alone improved (p<0.05) EF compared to injury control. In both LESWT and MSC alone therapies EF recovery was partial. The other combination therapies and PDE5i alone did not show significant (P>0.05) EF recovery.

Conclusion

LESWT, MSC and/or PDE5i combination therapy appears to be highly promising in EF recovery.
Fluorescent hypoxia probe for flow cytometry
Authors: Low Quentin , Calderon Veronica , Chen Aimei , Hu Yizhen , Mandavilli Bhaskar , Wang Xin , O’Grady Mike
Published: January 2023

Hypoxia is a condition where low levels of oxygen levels are present (1% to 2% O2). Hypoxia play a wide role in physiological and pathological conditions, from developmental angiogenesis to tumor progression and evasion. Hypoxia also modulates a number of immune functions from promoting inflammation to suppressing adaptive immunity. The current method for detecting hypoxic cells relies on an immunochemical approach. Pimonidazole react with peptide thiols in hypoxic cells and the resulting adduct is detected with an anti-pimonidazole antibody. To increase the availability of hypoxia detection reagents, we have developed a rhodamine-based hypoxia reagent (HR) for live cells. Using Jurkat leukemia cells incubated in hypoxic or normoxic conditions and stained with HR, we were able to clearly distinguish hypoxic cells from normoxic cells on a flow cytometer. We were also able to resolve cells incubated at differing levels of O2 (10%, 5% and 2.5% O2). To demonstrate that HR is compatible with other reagents, hypoxic cells were co-stained with a viability dye (SYTOX Red), a dye retained in intact mitochondria (TMRM) and with staining for a marker of apoptosis (annexin V). The results show that hypoxic cells excluded the dead cells dye while retaining TMRM, and were negative for annexin V staining at early time points. As expected, prolong incubation at hypoxia resulted in dead and apoptotic cells as evident in an increase in dead cells staining, a loss of mitochondrial integrity and an increase in annexin V+ cells. In summary, we present a sensitive reagent for detecting hypoxic cells without the need for cellular fixation and permeablization, while retaining it usage with other live cell flow cytometry detection reagents.
Identification of paired heavy and light chains from single B-cells from immunized Malian adults with rapid functional confirmation using iPair-BCR™, NGS, and iScreen™
Authors: Hou Xiaohong , Byrne-Steele Miranda L. , Pan Wenjing , Brown Brittany , Sanders Mollye , Eisenhower Mary , Coelho Camila , Hurtado Patricia Gonzales , Doritchamou Yai , Highsmith Kendrick , Taylor Justin J. , Schwartz Allison , Morrison Bob , Muratova Olga , Sagara Issaka , Doumbo Ogobara , Anderson Charles , Fried Michael , Duffy Patrick E. , Han Jian
Published: January 2023

Information about the cognate pairing of TCR alpha-beta chains and BCR IgH and IgKL chains encoded by individual T and B cells is key to understanding adaptive immune responses and developing therapeutic applications. We have previously reported the development of a sensitive technology that allows the amplification and identification of the paired human TCR alpha and beta chains from single T cells, termed iPair-TCR. Here, we report the extension of this technology to identify paired human BCR IgH and IgKL chains from antigen-specific single B cells. In this proof of concept study, we identified paired-VDJ-receptors from antigen-specific B-cells from nine Pfs230-EPA immunized Malian adults using the iPair-BCR method. Next, we developed a method to rapidly generate Fab fragments and demonstrate the binding of several of these single cell targets to the original Pfs230 antigen. Single cells of interest were identified based upon their repeated frequency on the plate, which indicates a clonal selection. The corresponding heavy and light chains were PCR amplified from selected wells. Using overlap extension PCR, all necessary elements for in vitro transcription and translation and either the CH1 or C-kappa-domain were added to both the 5′ and 3′ ends of the single cell VDJ. After in vitro transcription and translation, four out of five tested Fab fragments demonstrated binding through a colorimetric ELISA assay. The overall process after VDJ identification can be performed in under a week indicating the utility of our technology for rapid identification of antigen-specific BCRs and functional binding characteristics.
The ProQuantum immunoassay platform offers high performance in a simplified workflow for low-level measurement of cytokines in human and mouse sera/plasma
Authors: Bourdon David M. , Alohali Madhawi , Meng Lihao , Le Lucas , Freeman Alisha , Hu Jay
Published: January 2023

Immunoassays are not only a cornerstone of immunology but also biomedical research as a whole. A significant challenge that basic research scientists as well as translational investigators face is limitation of sample volumes obtained from mouse models and/or precious human samples. Researchers may need to compromise, make tradeoffs, and/or spend more time and resources to get enough samples for their experimental studies.

Thermo Fisher Scientific, the world leader in serving science, recently launched the ProQuantum High-Sensitivity Immunoassay platform (www.thermofisher.com/proquantum). These ready-to-use immunoassay kits combine the analyte specificity of high affinity antibody-antigen binding with the signal detection and amplification of real-time PCR.

The ProQuantum immunoassay platform features greater sensitivity and dynamic range than traditional methods using only 2 μL of sample. Assays are compatible with any real-time PCR instrument at 96- or 384-well configurations. Site-specific labeling chemistry preserves variable domain integrity of analyte-specific antibodies. The homogeneous (no wash) assay workflow provides sample-to-answer in 2 hours. An innovative web-browser based software application streamlines assay setup and provides robust data analytics.

Data presented here highlight human and mouse cytokine assay results for analytes important in the study of inflammation/autoimmunity. These performance data demonstrate the advantages of using ProQuantum immunoassays, especially when sample limitation is a concern.
Profiling of soluble immune checkpoint proteins as potential non-invasive biomarkers in colorectal cancer and sepsis
Authors: Pepin D , Godeny Michael , Russell Donna , Mehta Pramod , Lie Wen-Rong
Published: January 2023

In recent years, targeting immune checkpoint proteins has been shown to be an innovation for treatment of cancer and immune-related adverse events. Many checkpoint molecules have been identified on immune cells and/or tumor cells as activating receptors or inhibitory receptors. Many immune checkpoint proteins are also expressed as soluble forms in circulation, easily accessible as potential biomarker candidates. Here we report the development of a bead-based Luminex® multiplex assay for the quantitative profiling of a panel of co-inhibitory and co-stimulating immune checkpoint proteins: BTLA, CD27, CD28, CD40, CD80/B7-1, CD86/B7-2, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, TIM-3, and TLR-2. The aim of this study was to investigate a potential role for serum circulating immune-checkpoint proteins as putative biomarkers in colorectal cancer (CRC) and sepsis patients. We have used this novel 16-plex multiplex assay to measure multiple checkpoint protein expression levels in serum samples from CRC patients (n=20), sepsis patients (n=20), and a corresponding set of normal serum samples (n=20). Analysis of the soluble checkpoint protein signatures generated from this multiplex approach revealed differential expression of 9 soluble form of proteins, sBTLA, sCD27, sTIM-3, sHVEM, sCD40, sPD-1, sPD-L1, sCD86, and sTLR-2, in the CRC or sepsis serum samples, compared to the healthy serum controls. In summary, our results demonstrate that the multiplex immunoassay we developed is a useful research tool for the simultaneous quantitation of circulating immune checkpoint proteins, as well as its potential application in biomarker discovery and translational research.
Phosphoproteomic Analysis Reveals New SLP76 and PLCg1 Feedback Pathways That Regulate Lck in T Cells
Authors: Salomon Arthur R. , Belmont Judson , Gu Tao , Ji Qinqin
Published: January 2023

T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. The exact mechanisms that regulate the spatial equilibrium of Lck between the TCR and downstream regulators are not well defined. While some of the important regulatory proteins involved in Lck feedback loops in T cells are also beginning to be defined, the pathways leading to the activation and recruitment of these inhibitory molecules are much more obscure. Modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways. Our recent data reveal a previously uncharacterized regulatory network where N-terminal tyrosine residues on SLP76 are involved in both positive and negative feedback loops, which regulate the phosphorylation of the activation loop on Lck. We also collected phosphoproteomic data that suggests that Lck targeting but not activity is regulated by PLCγ1. We examined the phosphoproteome of PLCγ1 deficient and reconstituted T cells through a timecourse of TCR stimulation. These data indicate that Lck activity was shifted from TCR to the SLP76 complex in the absence of PLCγ1. Altogether, wide-scale modern methods in quantitative mass spectrometry drive the molecular characterization of T cell signaling circuits and facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states.
Immune cells avoid death from immunoproteasome inhibitors by switching to conventional proteasome
Authors: Ladi Ena , Everett Christine , Daniels Blake , Huestis Malcolm , Dere Ed , Gause Haley , Dirk Matthew , Rajapaksa Kathila , Staben Steve , Purkey Hans , Eidenschenk Celine , Siu Mike , Pappu Rajita
Published: January 2023

The pan-proteasome inhibitor bortezomib has demonstrated sustained clinical efficacy in off label trials in SLE patients by depleting pathogenic immune cells including highly secretory plasmablasts and plasmacytoid dendritic cells. But bortezomib also effects non-immune cells including neurons which raises significant safety concerns thus limiting the feasibility of pan-proteasome inhibitors as a treatment for autoimmune disease. As an alternative, targeting the immune cell specific immunoproteasome has been proposed as a promising therapy to deplete pathogenic immune cells while avoiding toxicity issues. Using highly specific immunoproteasome inhibitors, we show that this strategy does not lead to immune cell death as anticipated. We generated a large panel of small molecule inhibitors with varying specificity to the β5 subunit of the conventional and immuno- proteasome, and found that effects on viability of plasmablasts and pDCs correlate with the inhibition of the conventional proteasome, not the immunoproteasome. Our data indicates that the immune cells upregulate the conventional proteasomal subunits and prevent the accumulation of ubiquitinated protein. Widely used immunoproteasome inhibitors also block the conventional proteasome which, our data would indicate, is key to their ability to deplete immune cells.
The influence of berberine on co-stimulatory molecule expression and T cell activation
Authors: Vita Alexandra Adorno , Pullen Nicholas A.
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and deterioration within the joints, as well as extra-articular and systemic manifestations depending on severity. While there are many immunological factors that contribute to the pathology of RA, its progression is largely due to the activation of CD4+ T cells by antigen presenting cells (APCs), which then subsequently enhances the immune response of other cells within the area. Berberine (BBR), an isoquinoline alkaloid, has demonstrated the ability to attenuate mechanisms involved in RA in vitro and in vivo. Recent studies provide some evidence that BBR may interfere with the co-stimulatory mechanisms involved in a RA inflammatory response, however research specifically concerning BBR’s effect on co-stimulatory molecule expression and IL-12 production remains equivocal. In this study, we used flow cytometry to examine BBR’s effect on the cell surface expression of co-stimulatory molecules on bone marrow-derived dendritic cells (BMDCs) following stimulation with LPS, and on isolated splenic CD4+ T cells following activation with anti-CD3/anti-CD28. On BMDCs, the expression of CD40, CD80/86, and MHC class II was quantified, whereas CD40L, CD28, and IL-2R expression was examined on splenic CD4+ T cells. The amount of IL-12 secreted in BMDC supernatant was measured with an ELISA. The data collected on BBR’s effects will allow further insight into its potential as an alternative therapy for RA.