bluuebunny/crossref_metadata_2025 huggingface dataset:
Hippuric acid inhibits survival of germinal center B cells via GPR109A in kidney disease
Authors: Peroumal Doureradjou , Jawale Chetan , Antos Dani , Li De-Dong , Choi Wonseok , Wang Shuxia , Alcorn John , Biswas Partha
Published: November 2024

The formation of germinal center (GC) response is crucial for antibody mediated immunity against infection. However, compromised humoral immunity to infection was seen in patients having pre-existing comorbid conditions including kidney disease. The mechanisms by which kidney disease account for poor antibody response is unknown. Here we show that kidney disease negatively impacted GC formation, antibody affinity maturation, isotype switching and titer. GC B cells exhibit increased apoptosis in kidney disease. Uremic toxin hippuric acid drives loss of mitochondrial membrane potential leading to increased apoptosis of GC B cells in G-protein–coupled receptor109A (Gpr109A) dependent manner. GC B cells express Gpr109A and mice without kidney disease treated with Gpr109A agonist exhibit similar defect in GC formation, affinity maturation and antibody titer. Conversely, immunized Gpr109A global knock-out or mice in which B cells are lacking Gpr109a show no defect in GC formation, affinity maturation and antibody titer following kidney disease. These results shed lights on hippuric acid/Gpr109A axis in causing GC B cells apoptosis and provide a mechanistic understanding of how renal dysfunction suppresses humoral immunity in patients with kidney disease.
Investigating Enzymatic Control of Inflammation Oxo-eicosanoid Synthesis in Live Zebrafish
Authors: Ma Yanan , Hui King Lam , Niethammer Philipp , Gelashvili Zaza , Lengyel Miklos
Published: November 2024

Eicosanoids, derived from arachidonic acid (AA), are bioactive lipids with chemokine-like function. While prostaglandin and leukotriene have been extensively studied, the genetics of oxoeicosanoid metabolism remain relatively unexplored. The proto-type oxoeicosanoid 5-KETE is generated from AA by 5-lipoxygenase and the long-elusive redox-sensitive enzyme 5-HEDH. Pure 5-KETE attracts leukocytes in vitro and in vivo via the G-protein coupled receptor OXER1. 5-KETE is induced in response to Ischemic, allergic challenges, and colitis. OXER1 inhibition suppresses neutrophilic inflammation in zebrafish and primate asthma models and frameshift mutations of OXER1 in mucosal tumors suggest a yet undescribed cancer role. However, the lack of OXER1 orthologs in rodents and uncharacterized 5-HEDH enzyme have severely stalled oxoeicosanoid research for the past 30 years. Through a combination of lipidomics, enzymatic activity screening and genetic perturbation in human cell culture, coupled with intravital imaging of acute inflammation in zebrafish, we here identify the first-in-class 5-HEDH member. Its genetic deletion severely compromises 5-KETE synthesis in human cells and abrogates rapid neutrophil and macrophage recruitment to zebrafish tail fin wounds in vivo, indicating its role in neutrophil and macrophage interplay. Our work will open new therapeutic avenues for treating acute and chronic mucosal inflammation.
Aryl hydrocarbon receptor control of α4β7 and chemokine receptor 9 expression on B cells is partially determined by sex
Authors: Bauerle Logan , DeKrey Gregory
Published: November 2024

The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor that regulates immune function differentially by sex. In this study, we compared the impact of AhR activation on the expression of chemokine receptor (CCR) 9 and α4β7 integrin on male and female B cells in vitro. Splenic B cells were isolated from C57Bl/6J mice and then stimulated in culture with 300 ng/mL anti IgD-dextran, 2 ng/mL human TGF-β, and 25 μg/mL LPS. Cells were also exposed to either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, agonist, 50 nM) or CH223191 (100 μM, antagonist). After 96 hours, cells were analyzed by flow cytometry. Under antagonist conditions, the percentage of viable cells expressing CCR9 was 3.8% (male) and 5.2% (female), and α4β7 expression was 1.5% (male) and 1.6% (female) (N=6). AhR activation significantly increased (P < 0.05) the percentage of viable cells expressing either CCR9 or α4β7 by approximately 3-fold for male cells and >4-fold for female cells. Relative to males, a significant increase in the proportion of female cells expressing CCR9, but not α4β7, occurred following AhR agonism. The level of CCR9 expressed on individual cells (mean fluorescence intensity) was also significantly increased by AhR activation (approximately 50%, log scale) in both males and females, but the level of α4β7 expression was significantly increased only in female mice. These results suggest that AhR activation may enhance B cell migration into the gut preferentially in females.
Aryl hydrocarbon receptor activation ameliorates colitis via transcriptional regulation of -defensin1
Authors: Palrasu Manikandan , Kakar Khadija , FNU Hamida , Marudamuthu Amarnath Satheesh , Carter Tayler , Wilson Kiesha , Saxena Archana , Yang Xiaoming , Singh Narendra , Busbee Brandon , Nagarkatti Prakash , Nagarkatti Mitzi
Published: November 2024

Ulcerative colitis (UC), a chronic inflammatory disease of the colon and rectum accounts for 1.5 million individuals, with an annual incidence of 15 per 100,000 individuals. The etiology of colitis includes host, genetic, environmental, and microbiological factors. The intestinal epithelium can produce antimicrobial molecules for epithelial protection from commensal microorganisms. Aryl hydrocarbon receptor (AhR) plays an important role as an environmental sensor and a regulator of the immune response. Indeed, studies have shown that the expression of AhR was correlated with the expression of a-defensins in inflammatory bowel disease patients. Therefore, the present study was designed to investigate how AhR-ligand activation regulates a-defensin1 in a colitis model. Here, we discovered that in intestinal epithelial cells from colitis mice, dietary Indole-3-carbinol (I3C), a ligand for the AhR, induced AhR and α-defensin1. We also found that AhR-null animals, which are more likely to develop colitis, did not exhibit an increase in α-defensin1 following I3C treatment, indicating that AhR regulates α-defensin1. Similar findings were observed in MC38 cells. Furthermore, the DRE region in the a-defensin1 promoter was involved in the upregulation of a-defensin1 by AhR upon I3C treatment. Collectively, these results provide evidence that AhR activation followed by a-defensin1 production may be a vital prophylactic and therapeutic measure for colitis.
Perturbation of azurophilic granule integrity drives NLRP3-independent IL-1b processing and release in neutrophils
Authors: Miller Brandon , Dubyak George
Published: November 2024

IL-1β is an inflammatory cytokine secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from monocytes downstream of activated NLRP3 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD and N-GSDMD plasma membrane (PM) pore formation to permit release of IL-1β and induction of pyroptosis. NLRP3 is a cytosolic sensor of cellular homeostasis including [K+]. In monocytes, K+ efflux mediated NLRP3 activation is triggered by agents that disrupt lysosomal integrity. Neutrophils assemble competent NLRP3 inflammasomes and release IL-1β via GSDMD-dependent mechanisms, but they resist N-GSDMD PM pore formation and pyroptosis. We tested whether lysosome-disrupting stimuli in neutrophils would phenocopy the monocyte mechanism of NLRP3-dependent IL-1β release. Surprisingly, our data indicates that lysosome disruption induces release of mature IL-1β and lytic cell death in an NLRP3-independent manner. Kinetic analysis demonstrates an alternate mechanism of release such that early phase IL-1β release is NLRP3-dependent, but prolonged stimulation leads to NLRP3-independent release. Collectively, our data supports a signaling mechanism by which azurophilic granule disruption and cytosolic accumulation of neutrophil serine proteases disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β as part of a novel neutrophil-specific signaling mechanism of IL-1β processing and export.
Single-cell proteomic analysis of peripheral blood mononuclear cells in patients with pulmonary long COVID
Authors: Guinto Elizabeth , Kuo Grace , Messing Melina , Cheung Chung , Yip Will , Gerayeli Firoozeh , Eddy Rachel , McNagny Kelly , Sin Don
Published: November 2024

An estimated 15% of patients are expected to continue to exhibit respiratory symptoms after their SARS-CoV-2 infection. The mechanisms behind these symptoms remain unknown. We recruited twenty-three patients (65% female) who were on average 406 ± 251 days post-COVID-19. The cohort consists of primarily mild (non-hospitalized) patients (87%), with an average age of 43 ± 15 years. Nine of these patients had self-reported dyspnea, which we defined as pulmonary long COVID (PLC). The remaining 14 patients recovered without persistent dyspnea. We performed a 38-panel cytometry by time-of-flight on the peripheral blood mononuclear cells of this cohort. Quality control was performed using FloJo. Further analysis, was done using the CyCombine, CATALYST, and FlowSom packages in R. Our final annotation uncovered 17 different cell clusters. In PLC patients, double-positive T-cells (DPTs) were significantly increased (Wilcoxon p<0.05), indicating that T-cell development or differentiation may be altered in these patients. Though DPTs are known to be increased in females, there was no significant difference in the proportion of DPTs between males and females in our cohort. PLC stem cells had increased expression of CD304 and CD200R (Wilcoxon p<0.05). CD304 is as a co-receptor for SARS-CoV-2, while CD200R is a limiter of pro-inflammatory activity. These findings indicate that the immune landscape in PLC patients is altered, compared to those who recover without residual pulmonary symptoms.
Gpx4 regulates iNKT cell homeostasis and function by preventing ferroptosis and lipid peroxidation
Authors: Sok Sophia P M , Pipkin Kaitlyn , Van Remmen Holly , Fan Zhichao , Zhao Meng
Published: November 2024

Invariant natural killer T (iNKT) cells are a group of innate-like T cells recognizing lipid Ags and plays important roles in immune responses. We found iNKT cells, compared to CD4T cells, have significantly higher level of lipid peroxidation, lower labile iron pool, and higher level of Gluthathione peroxidase 4 (GPX4), a major antioxidant enzyme protecting cells from ferroptosis. T cell-specific KO of Gpx4 reduced iNKT cells, especially NKT1 cells, the IFNg producing subset. RNAseq revealed Th1 pathway, cell cycle, and mitochondrial function are perturbed by Gpx4 deletion in iNKT cells. Indeed, we detected impaired cytokine production, elevated cell proliferation and cell death, accumulation of lipid peroxides and mitochondrial ROS in Gpx4 KO iNKT cells. Ferroptosis inhibitor, iron chelator, vitamins E, and mitochondrial antioxidant (MitoQ) prevented the ferroptotic cell death induced by Gpx4 inhibition in iNKT. Moreover, the function of the Gpx4 KO iNKT cells can be restored by antioxidants or iron chelator. Notably, vitamin E rescued iNKT cells at homeostasis in Gpx4KO mice. Furthermore, vitamin K2 prevented the cell death and restored the cytokine production in Gpx4-cKO iNKT cells in vitro, and it also recovered the percentage of iNKT1 in Gpx4 cKO mice when administrated in vivo, consistent with a protective role of vitamin K cycle during ferroptosis.
Complement C4 and Autoimmune Diseases
Authors: Zhou Danlei , Coss Samantha , Abdul Aziz Rabheh , Drew Joanne , Miller Katherine , Driest Kyla , Sivaraman Vidya , Spencer Charles , Barbar-Smiley Fatima , Ardoin Stacy P , Yu Chack-Yung
Published: November 2024

Complement activation on autologous cells causes tissue injuries but the process is not well studied. We seek to elucidate how C4 variants, their activation products and isotype deficiencies contribute to increased risks of children systemic lupus erythematous (cSLE) and juvenile dermatomyositis (JDM). We performed kinetic studies of C4A and C4B reactivities by measuring hemolysis of sensitized sheep erythrocytes (EA). EDTA-plasma diluted to 1% showed that C4B lysed EA immediately, as revealed by released hemoglobin at OD415 nm. Those of C4A exhibited hysteresis property. C4A probably mitigates damaging effects of activated C4B on host cells. We performed flow cytometry to evaluate cell-bound complement split products on blood cells using specific monoclonals for samples with cSLE (n=119), JDM (n=159), and controls (HC, n=109). Non-parametric analyses for median fluorescent intensities were compared with median and IQR presented. The three sets of data among cSLE, JDM and HC were 795 (523, 1161), 350 (181, 779) and 252 (163, 252), respectively, for E-C4d (cSLE/HC, p=3.8x10-22; JDM/HC, p=8.8x10-8); 201 (95, 328), 33 (4, 101), and 26 (6, 70), respectively, for E-C4Ad (p=5.3x10-10, cSLE/HC; p=0.02, JDM/HC); and 68 (30, 154), 11.9 (1.9, 23.8), and 13.5 (8, 19.2) for E-C3d (p=1.8x10-6, cSLE/HC; p=0.16, JDM/HC). In conclusion, complement activation on host cells is retarded after activation of C4. Moreover, activation of C4 is substantially higher in cSLE and JDM than HC.
Proton therapy inhibits the production of immune suppressive exosomes in head and neck squamous cell carcinoma
Authors: Chimote Ameet , Lehn Maria , Bhati Jay , Mascia Anthony , Sertorio Mathieu , Lamba Michael , Ionascu Dan , Tang Alice , Langevin Scott , Khodoun Marat , Wise-Draper Trisha , Conforti Laura
Published: November 2024

Proton therapy (PT) is a promising new alternative to conventional X-ray-based photon therapy (XRT) for patients with advanced head and neck squamous cell carcinomas (HNSCC) owing to its precise dosimetric characteristics and reduced toxicity. While tumor-derived exosomes contribute to immune suppression in HNSCC, the effects of PT on tumor-derived exosomes and anti-tumor immune responses are poorly understood. In this study, we generated primary HNSCC cell cultures from resected tumors and irradiated them with 5 Gy PT or XRT, and isolated exosomes from cell culture supernatants. We observed that compared to XRT, PT-exposed HNSCC cells produced 75% fewer exosomes. To assess the immunomodulatory effects of PT, we exposed peripheral blood mononuclear cells (PBMCs) to exosomes from PT- and XRT-irradiated HNSCC cells and conducted preliminary single-cell studies by transcriptome analysis (NanoString), secretome profiling (Isoplexis) and immune function (IFN-g release). Transcriptomic studies revealed that exosomes from PT- and XRT-irradiated cells comparably reduced cytotoxic and Th1 cell abundance, and inhibited immune responses associated with cytotoxicity, NK, and T cell functions. Exosomes from PT- and XRT-irradiated cells equally suppressed IFN-g release and reduced the polyfunctionality (the ability to secrete multiple cytokines from a single cell) of CD8+ T cells. Our results suggest that PT limits the immune suppressive effect of exosomes by reducing their production.
Postprandial changes to systemic metabolism imprint durable changes on T cell immune responses
Authors: Kumar Alok , Rivadienera Dayana , Delgoffe Greg , Poholek Amanda , Kohan Alison
Published: November 2024

Here we show short periods of fasting and refeeding can have long-lasting effects on T cell immunity. Mouse or human T cells from fed hosts have higher mitochondrial quality and respiratory capacity than those from fasted hosts ex vivo. These metabolic phenotypes persist after activation and expansion, both in vitro culture and in vivo in response to vaccination. Fed T cells outcompete fasted T cells in a co-transfer model, such that T cells from fasted hosts form far fewer memory T cells. Serum profiling revealed triglycerides as a driver of postprandial metabolic reprogramming: chylomicrons enriched from the lymphatics of fed mice were sufficient to enhance mitochondria of fasted T cells. LDLR-deficient T cells had the metabolic phenotype of fasted T cells and were insensitive to fed serum, confirming the role of triglyceride uptake in postprandial T cell metabolic programming. Transcriptomic analysis revealed higher expression of LDLR, OXPHOS proteins, and fatty acid metabolism enzymes in fed T cells over fasted T cells. Finally, human CAR T cells manufactured from the same donor after a meal show a therapeutic advantage over T cells collected in the fasted state. In summary, postprandial metabolism imparts durable metabolic and functional advantage on T cells. Our study highlights the need to consider diet content and timing as key factors in immune cell analysis, vaccination strategies, and the generation of cellular therapies for cancer.
Electrical stimulation of the arterial splenic nerve has durable therapeutic efficacy on murine collagen-induced arthritis.
Authors: BONDUE Alexandre
Published: November 2024

Vagus nerve (VN) stimulation has anti-arthritic effects in animal models and in patients with rheumatoid arthritis (RA). However, VN stimulation may affect several physiological functions. The splenic nerve (SpN) has been implicated in VN-mediated immunomodulation. We tested the therapeutic efficacy of arterial SpN stimulation (SNS) on collagen-induced arthritis (CIA) in mice as a single treatment or in combination with etanercept or Jak inhibitor. SNS was evaluated in mice with CIA before or after disease onset and applied one or six times daily. Alternatively, mice were treated with etanercept or propranolol three times a week. SNS treatment of CIA mice before the onset of clinical symptoms prevented disease in a β1/β2-adrenoceptor-dependent manner. After SNS treatment, splenic inflammatory monocytes, B cells and autoantibody-secreting cells were reduced, as were serum levels of TNF and anti-collagen antibodies. When SNS was tested as a therapeutic modality in already diseased mice, clinical scores were significantly reduced compared to sham-treated animals. Importantly, the therapeutic efficacy of SNS was enhanced when combined with etanercept treatment. The results of this study suggest that SNS may have potent therapeutic anti-arthritogenic effects.
Efficacy of a novel oral, potent, and highly selective PKCt inhibitor in the SCID T-cell transfer colitis model
Authors: Harden Jamie , Whitener Robert , Kim Andrea , Hofland Hans , Patel Jeegar
Published: November 2024

Protein kinase C family theta isoform (PKCt) is a key kinase downstream of T-cell receptor (TCR) and CD28 co-signaling in T-cells, leading to T-cell activation and production of effector cytokines. As T-cells are key pathogenic cells in a multitude of chronic inflammatory diseases, inhibition of PKCt with an oral small molecule may provide a novel mechanism to treat chronic inflammatory diseases, such as colitis. In this study, we evaluated the efficacy of two orally bioavailable, potent, and highly selective PKCt inhibitors in the severe-combined-immunodeficient (SCID) T-cell transfer colitis model; this model is considered the most relevant to human inflammatory bowel disease (IBD), and displays characteristics of both Crohn’s disease (CD) and ulcerative colitis (UC). PKCt inhibition resulted in a dose-dependent prevention of colitis symptoms, including maintenance of body weight and stool score, with efficacy similar to an anti-IL-12 monoclonal antibody. Histological assessment and tissue cytokine and chemokine protein quantification of the colon were also performed. These data suggest that an oral PKCt inhibitor may have utility in the treatment of IBD, and other chronic inflammatory diseases driven by pathogenic T-cell activation.
Efficacy of lymphatic pump treatment in conjunction with methotrexate on rats with adjuvant-induced arthritis
Authors: Volin Michael , Patel Saahil , Zanotti Brian , Monroy Del Toro Rosalinda , Incrocci Ryan , Swanson-Mungerson Michelle
Published: November 2024

Rheumatoid Arthritis (RA) is an autoimmune disease targeting synovial joints in which self-reactive T cells release cytokines and B cells produce self-reactive antibodies resulting in an inflammatory response. Lymphatic pump treatment (LPT) promotes lymphatic flow resulting in a flushing out of the immune-complexes and cytokines. A rat mode on RA (AIA) was used to determine whether LPT in conjunction with the anti-rheumatic drug, methotrexate (MTX), enhanced its therapeutic impact on RA. A preventative study was performed in which MTX was given along with LPT (3 times a day for 7 days) . Ankles were assessed daily for signs of inflammation. Flow cytometry was performed on spleen and popliteal lymph nodes (PLN) to determine changes in leukocyte populations. Cytokine levels were determined on ankle lysates. The study showed that MTX treatment delayed the onset of arthritis. MTX reduced the spleen weights (p<0.05). LPT also reduced spleen weights but did not enhance the MTX reduction. LPT reduced PLN weights compared to MTX. MTX resulted in higher numbers of CD4+ T cells and lower numbers of CD8+ T cells and B cells in spleens. PLNs treated with MTX had reduced lymphocyte numbers. MTX treatment reduced CINC-1 and TNFa levels (p<0.05). LPT also reduced CINC-1 levels. In conclusion, MTX treatments delayed arthritis formation in AIA rats while LPT affected both spleen and PLN weights. Thus, the addition of LPT to RA treatment showed potential benefits without negative effects.
Joint single-cell transcriptomic and epigenomic analysis reveals key regulators of CAR T cell stemness and antitumor immunity
Authors: Wu Tuoqi , Zhu Ziang , Wizzard Safuwra , Yao Chen
Published: November 2024

The differentiation of antitumor T cells is tightly controlled by transcriptional and epigenetic pathways. Understanding the programs governing the differentiation of therapeutic T cells such as chimeric antigen receptor (CAR) T cells is crucial for improving their efficacy. Using a single-cell multi-omics approach, we simultaneously profiled chromatin accessibility and transcription within the same single CAR T cell in mice with B-cell leukemia or melanoma. One of the CAR T-cell subsets displayed stem cell-like properties and greater proliferative potential upon tumor challenge. In addition, we uncovered primed chromatin states foreshadowing transcriptional changes and lineage-defining genes heavily regulated at the epigenetic level. By constructing gene regulatory networks in CAR T cells based on the single-cell multi-omics data, we identified hub transcription factors of stem-like CAR T cells including BACH2. By knocking out BACH2 in CAR T cells, we demonstrated an essential role of BACH2 in the development of the stem-like subset and antitumor immunity of CAR T cells. Thus, our study elucidated the transcriptional and epigenetic mechanisms underlying CAR T-cell differentiation and revealed key transcriptional regulators controlling the differentiation and antitumor immunity of CAR T cells.
CyTOF enables unprecedented resolution of intracellular readouts to reveal novel functional diversity of human T Cells
Authors: Mcconney Christina , Smith-Mahoney Erika , Cohen Michael , Belkina Anna , Selvanantham Thirumahal , King David , Loh Christina , Cappione Amedeo , Snyder-Cappione Jennifer
Published: November 2024

Measuring functional signatures at the single-cell level in a cross-lineage manner provides key insights into several facets of immunology, including the plasticity of innate T cells, T/B memory cell diversity following infection/vaccination, and the panoply of activities associated with cell therapy success/failure. To better understand the dynamics of the immune system, flow cytometry phenotyping panels often include intracellular markers of functional potential, such as cytokines, phosphorylation events, and transcription factors. Many of these readouts, including cytokines of the Th2/Treg lineages, have been historically difficult to detect in human cells using flow cytometry. We compared the signal resolution of three 13-parameter panels on a Cytek™ Aurora spectral cytometer and the CyTOF™ XT mass cytometer. Each panel measured surface and intracellular analytes; fluorescent spectral panels were designed with little/no spectral overlap. PBMC-derived data was analyzed using PhenoGraph clustering and visualized by opt-SNE. Data collected on CyTOF XT™ demonstrated superior signal resolution for a range of intracellular readouts, including cytokines IL-5, IL-10, and IL-13. Also, when better signal/noise enabled more accurate clustering, a higher number of immune cell subsets possessing unique signatures resulted from CyTOF datasets. Overall, cytometry by CyTOF provided clearer understanding of the immuno-diversity present in a sample in the context of unbiased analysis.
Chromatin remodeler ACTL6A in CD8+ T cells promotes antitumor immunity
Authors: Wu Bogang , Thant Win , Bitman Elena , Liu Ting , Ellisen Leif
Published: November 2024

Chromatin remodeling plays a pivotal role in dictating epigenetic and transcriptomic regulation governing cytotoxic T lymphocyte differentiation and effector function. However, the regulatory mechanisms underlying chromatin remodeler assembly and actions remains unclear. Actin-like gene family members have been implicated in the organization of chromatin landscapes, however their role in T cell functional regulation remains unknown. We identified Actin-like protein 6A/Actl6a as the most upregulated gene within actin-like family members upon CD8+ T cell activation across multiple species. Additionally, ACTL6A is highly expressed in human tumor-infiltrating CD8+ mitotic tissue-resident memory T cells, suggesting a functional importance of ACTL6A in this subset. Deletion of mouse Actl6a in mature T lymphocytes impairs immune responses to transplanted syngeneic mouse tumors. T cell-specific Actl6a deletion mice exhibit a reduced effector memory population and attenuated cytotoxicity in their tumor infiltrating CD8+ T cells. T cell adoptive transfer further corroborates a CD8+ T cell-intrinsic role of Actl6a on antitumor adaptive immunity. Transcriptomic/epigenetic investigations provide mechanistic insights into the action of ACTL6 in the CD8+ T cells regulation. Furthermore, our study suggests that pharmacological or genetic approaches to increase Actl6a expression in CD8+ T cells may have the potential to boost immune based anticancer therapies including CAR-T therapies.
introduction of expanded tools for spatial biology applications
Authors: Serafin Jen , Montoya Leticia , Harvey Austin , Voeun Mae , Clark Scott
Published: November 2024

Fluorescence labelling is a highly versatile approach to immunohistochemistry (IHC), offering the ability to detect numerous targets simultaneously with excellent sensitivity and specificity. Currently, the fluorescent labelling method utilized for IHC is the use of secondary antibodies. This multi-step process is restricted by targets that share the same species isotype, limiting the number of targets possible in one sample. Another method used for multiplex staining is cyclic labeling, the process of staining and stripping a tissue sample with different antibodies to detect multiple targets. This process is time-consuming and increases the risk of tissue damage or loss of antigenicity. Our new reagents will enable researchers to stain in fewer steps with preserved antigenicity, ensuring accurate and reliable detection of target antigens in tissue samples. One of the key benefits of these products is the ability to achieve higher-plex labeling, allowing researchers to multiplex with 8-10 biological targets on a single sample in one staining cocktail. This reduces the need for cyclic labeling, streamlines the workflow, and reduces overall time required for analysis. We provide a process that enables successful detection of multiple protein markers across diverse tissue organs while simultaneously detecting transcriptomic targets, providing comprehensive insights into complex biological processes and disease mechanisms. Intended for research use only.
Using single cell CITE-seq and high parameter flow cytometry to characterize human regulatory T cells
Authors: D'Cruz Louise , Shi Xiaoshan , Rezvan Ali , Sen Moen , Sakofsky Cynthia , Genero Gracia , Widmann Syephanie , Tyznik Aaron
Published: November 2024

Regulatory T cells (Tregs) are an essential population of T cells that are critical for maintenance of peripheral tolerance. Imbalanced Treg frequency or dysregulation of Treg function can lead to the development of autoimmune diseases. Thus, the modulation of Treg frequency or function has become an important therapeutical approach to treat autoimmune disorders. Despite increased study, whether distinct populations of Tregs with unique functions exist is unclear. Here, we used a combination of single-cell CITE-Seq (scCITE-Seq) and high parameter flow cytometry to resolve the heterogeneity of Tregs in humans. Using magnetic cell enrichment in conjunction with fluorescence-activated cell sorting (FACS), we isolated Tregs and performed downstream scCITE-Seq. By using more than a hundred antibody-oligo conjugates, we identified distinct populations of Tregs, uncovered the proteomics signature associated with these populations, and revealed donor-to-donor variability in the peripheral Treg compartment. The protein expression pattern and antigen density on Tregs revealed by scCITE-Seq correlated to that of high dimensional flow cytometry. Trajectory analysis segregated Treg subsets into distinct differentiation pathways with unique proteomic signatures ranging from naïve, activated to memory Tregs. These findings improve our understanding of the heterogeneity of Tregs and provide a single-cell proteomic atlas for dissecting the complex roles of Tregs in health and disease.
Ce3D-IBEX: Achieving Multiplex 3-dimensional Imaging for Deep Phenotyping of Cells in Tissues
Authors: Arroyo Armando , Ichise Hiroshi , Hor Jyh Liang , Chu Colin , Croteau Joshua , Radtke Andrea , Germain Ronald
Published: November 2024

2D multiplexed antibody-based imaging provides a framework to study cell-cell interactions in thin tissue sections but lacks the ability to interrogate spatial relationships in larger 3D micro-anatomical structures. Several methods for 3D imaging of optically cleared tissue exist, but current approaches do not provide a deep phenotyping capability as afforded by high content 2D imaging. Here,we developed a method to overcome this constraint by combining a fast hydrophilic tissue clearing technique, clearing-enhanced 3D (Ce3D), and the recently developed Iterative Bleaching Extends Multiplexity (IBEX) technique together with a robust image registration algorithm suite to create a highly multiplex 3D imaging method: Ce3D-IBEX. It has been used to analyze murine lung, lymph nodes, small intestine, retina, tumor, and cornea as well as human retina, kidney, liver, tonsil, and jejunum, yielding seamless views of large-scale tissue structures while enabling visualization of high dimensional phenotypic markers at single-cell resolution, permitting the identification of discrete cell subsets, quantification of protein expression level and their mutual spatial association within tissue structures. To date, 25+ markers have been visualized in a single 3D sample. This represents a major advance in the emerging field of highly multiplexed 3D imaging. With aims to open new avenues for characterization of spatial dynamics of immune cells in healthy and diseased tissue.
A comprehensive analytical validation framework for immunofluorescence assay design using CellScape precise spatial multiplexing
Authors: Braubach Oliver , Jackson Charle , Christians Arne , Boog Jannik , Ingalls Matthew , Meshik Xenia , Northcutt Adam , Cashion Kayla , Schwarz John
Published: November 2024

Multiplex immunofluorescence (mIF) is a powerful immunology research tool that enables deep phenotyping of cells within the tissue microenvironment. Careful optimization of multiplex antibody panels for specificity, sensitivity, and reproducibility, is crucial to achieving robust data and is a requirement for assay repeatability and standardization. Here, we describe our validation procedure for multiplex assay panels.

Experiments were performed using the CellScape™ platform for precise spatial multiplexing. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility.

Pairing the CellScape platform with comprehensively validated antibody panels streamlined deep immune profiling. The 15-plex, ready-to-use VistaPlex™ immune profiling panel for human FFPE tissues was validated on human tonsil, lung, breast, and colon tissues demonstrating the ability to positively identify epithelial cells and 22 cellular immune phenotypes most critical to understanding tumor immune responses.
High-throughput and high-dimensional single-cell analysis of antigen-specific CD4+ T cells in COVID-19 and influenza
Authors: Guo Yuwan , Huang Chao , Jiang Ning
Published: November 2024

This study aims to elucidate the tissue-specific dynamics of antigen-specific CD4+ T cells in response to SARS-CoV-2 and influenza. We have developed TetTCRII-SeqHD, a novel high-throughput, high-dimensional single-cell analysis platform that utilizes barcoded tetramers for antigen-specific TCR sequencing. It enables simultaneous profiling of CD4+ T cells' antigen specificities, TCR sequences, gene and surface-protein expression in human tissues. Using this technology, we have profiled >50,000 SARS-CoV-2-specific CD4+ T cells from lung, lung-associated lymph node, spleen, and blood.

Our results show distinct TCR repertoires and responses of CD4+ T cells in COVID-19 and influenza. Tissue-isolated CD4+ T cells display overlapping core signatures, yet distinct tissue cues influence their phenotype, pointing to site-specific functions. In COVID-19, unique transcriptional profiles of CD4+ T cells is characterized by a pronounced cytotoxic signature and a reduction in Treg, in contrast to flu-specific cells. We also observed that tissue context influences the Treg TCR repertoire, with clonal diversity tending to be more restricted in lung tissues compared to the spleen.

This study provides a comprehensive understanding of CD4+ T cell heterogeneity and function in tissues during viral infections. The novel TetTCRII-SeqHD platform presents an advancement in analyzing tissue-localized immune responses, offering potential implications for future immune research and vaccine strategies.
Streamlined 96-well plate method for simultaneous single-cell TCR immune receptor repertoire analysis and immunophenotyping
Authors: Chenchik Alex , Liu Tianbing , Makhanov Mikhail , Hu Dongfang , Kobzik Lester , Ghias Khadija , Diehl Paul
Published: November 2024

This study describes an efficient immune profiling method by FACS-sorting single cells in a 96-well plate to assess clonotype repertoire diversity, paired-chain information, and cell phenotypes in a single, multiplex RT-PCR-based assay without the use of any specialized equipment. It is a cost-effective alternative to conventional single-cell technologies such as microwell arrays or droplet microfluidics. The 96-well plate contains primers for either T-cell receptor (TCR) / or TCR / chains, together with primers for 30 key T-cell markers. T cells from PBMCs are sorted in a single-cell-per-well format. We conduct RT-PCR library prep using the DriverMapTM Adaptive Immune Receptor (AIR) Repertoire Profiling Assay and sequence the CDR3 region. The results show clonotype frequencies, chain pairing details for / and / chains, and T-cell subtype classifications based on gene expression profiling. This enables in-depth analysis of TCR gene rearrangements at the single-cell level, enhancing understanding of T cell development, proliferation, and clonality, which are important in studying cancer, immunodeficiencies, and autoimmune disorders. Additionally, we can effectively identify and characterize γδ T cells by integrating single-cell TCR sequencing with RNA sequencing data, potentially useful in the development of γδ cancer immunotherapies. Overall, this method provides an efficient way to simultaneously analyze clonotypes and immunophenotypes in a single assay.
Lactobacillus gasseri KBL697 ameliorates psoriatic inflammation through the modulation of gut-skin axis
Authors: Kim Jun-Hyeong , Lee Kiuk , Jang Sung Jae , Jo Eun Jung , Kim Daehwan , Cho Bo-Ram , Lim Sang Kyun , Ko GwangPyo
Published: November 2024

Psoriasis is a chronic inflammatory disease arising from aberrant interactions between various immune cells and cytokines. The current standard therapy includes topical steroids, methotrexate or anti-IL-17A/IL-23 antibodies. However, due to the limited efficacy and associated side effects, there is still an unmet medical need for the development of novel therapeutic agents for this disease.

KBL697 is a strain of Lactobacillus gasseri, isolated from the vaginal samples of healthy donors. We previously reported that KBL697 ameliorated the symptoms of an inflammatory bowel disease mouse model. At the molecular level, KBL697 decreased pro-inflammatory cytokines including IFN-gamma, IL-6, and IL-17A, while an anti-inflammatory cytokine IL-10 was increased.

In the current study, we tested KBL697 in pre-clinical models for psoriasis. Oral administration of KBL697 showed a significant beneficial effect in imiquimod-induced mouse models, judged by the skin inflammation, PASI score and immune profile change. Further studies revealed that IL-10 is induced by lipoteichoic acid (LTA) of KBL697, indicating the critical role of LTA/IL-10 in the anti-inflammatory function of KBL697. In the skin analysis, key cytokines in psoriasis, such as IL-17A, IL-23, and IL-36, are downregulated while genes for skin regeneration are upregulated, supporting the model for KBL697 working in the gut-skin axis. Therefore, KBL697 has great potential as a novel therapeutic agent for the treatment of psoriasis.
Siglec-E is a novel co-inhibitory receptor in the modulation of innate activation after transplantation
Authors: Rodrigues Lima Karina , Gassen Rodrigo , Liu Kaifeng , Ganchiku Yoshikazu , T. Ribas Guilherme , J. Borges Thiago , Riella Leonardo
Published: November 2024

Early after transplantation, inflammation and tissue injury are associated with significant activation of the innate immune cells, such as DCs. This activation drives adaptive immune responses, triggering acute rejection while hindering the development of transplant tolerance. Current immunosuppressive drugs primarily target T cells, while innate immunity is not efficiently inhibited in transplantation. The sialic acid–binding immunoglobulin-like lectin E (Siglec-E, or SigE) is an innate receptor that binds to ligands carrying sialic acids and inhibits TLRs-triggered inflammatory responses. Here, we demonstrate the role of SigE in experimental solid organ transplantation mouse models. Upon heart transplantation in mice, SigE is upregulated in graft-infiltrating APCs, including DCs. In humans, Siglec-7 and -9, the human homologs of SigE, are also upregulated in human heart biopsies following transplantation. Genetic ablation of recipient SigE leads to accelerated rejection of heart, kidney, and skin allografts. Mechanistically, SigE-deficient DCs are more susceptible to activation by DAMPS with an enhanced NF-kB activation and production of TNF-a. Moreover, DCs lacking SigE have a higher ability to induce allo-T cell responses in vitro and in vivo. In contrast, overexpression of SigE on DCs decreases their activation by DAMPs and reduces their T cell allostimulatory capacity. Thus, Siglec-E is a major inhibitory receptor in transplantation by controlling APC activation.
Respiratory mucosal immunity against SARS-CoV-2 after vaccination and infection
Authors: Tang Jinyi , Chaudhuri Arka , Qu Panke , Wu Yue , Wavell Kristin , Spano Marthajoy , Taylor Justin , Liu Shan-lu , Teague William , Sun Jie
Published: November 2024

The current COVID-19 mRNA vaccine is effective in systemic immune memory but lacks in generating mucosal anti-SARS-CoV-2 immunity. While prior SARS-CoV-2 infection establishes respiratory immunity, its strain-specific nature and limited duration pose challenges for sustained protection. Recent research has shown that hybrid immunity, a combination of vaccination and infection, results in a more robust, durable, and broadly reactive immune response in circulation compared to vaccination or infection alone. However, the characteristics and protective mechanisms of hybrid immunity in the respiratory mucosa are not fully understood. Moreover, it remains to be determined whether mucosal booster vaccines could potentially induce responses that are even stronger and more broadly reactive than hybrid immunity. Here, we collect nasal wash, bronchoalveolar lavage, and blood from controls, vaccinee, convalescents, or hybrid individuals. We found that hybrid individuals displayed higher levels of specific Ab, nAb, and B cells than other groups in the respiratory mucosa. In our mouse models, although hybrid immunity showed significant mucosal humoral and cellular immune responses, an adenovirus-based mucosal vaccine booster induced more robust protective respiratory immunity against SARS-CoV-2 variants. Our findings reveal the mechanisms of protection of hybrid immunity and underscore the importance of developing mucosal vaccine boosters that can emulate heightened mucosal immunity.
Persistence of Bordetella pertussis as aggregates within nasal tissues of aPV immunized mice prevents recruitment of Siglec-F+ neutrophils and bacterial clearance
Authors: Hall Jesse , Gutiérrez-Ferman Jessica , Shamseldin Mohamed , Guo Myra , Gupta Yash , Deora Rajendar , Dubey Purnima
Published: November 2024

Despite global vaccination, pertussis caused by Bordetella pertussis (Bp) is resurging. One explanation for the increased incidence of infections in immunized individuals is the inability of acellular pertussis vaccines (aPV) to clear Bp from the nose. To understand the host and bacterial mechanisms that contribute to Bp persistence, we evaluated bacterial localization and the immune response in the nasal associated tissues (NT) of naïve and immunized mice following Bp challenge. Bp resided in the NT of naïve and aPV-immunized mice as aggregates, while aggregate formation was not observed in mice immunized with a whole cell pertussis vaccine (wPV). CXCL1 was detected in the NT of wPV immunized mice. Siglec-F+ neutrophils, critical for eliminating Bp from the nose, were elicited at higher levels in wPV immunized mice compared to aPV immunized mice. Together, our data suggest that the TH2 polarized immune response generated by aPV vaccination permitted Bp to persist in the NT by establishing a niche in epithelial tissues that formed a protective barrier to infiltration of immune effectors. In contrast, immune responses generated by wPV, including the neutrophil chemoattractant CXCL1, recruited neutrophils that rapidly eliminated the bacterial burden and prevented establishment of a nasal reservoir. Overall, development of a nasal Bp reservoir and subsequent transmission may be prevented by a modified aPV that elicits a TH1/TH17 phenotype generated by wPV.
Dal Magistero del Santo Padre
Authors: ] [anonimo
Published: November 2024

Not available
From the heart of the Church
Authors: Marchionni Paolo
Published: November 2024

In recent weeks, the Apostolic Constitution of John Paul II on Catholic Universities, entitled Ex corde ecclesiae, was published. This document, which bears the date 15 August, the Solemnity of the Assumption of the Blessed Virgin Mary, is intended to be a kind of reference text, a sort of ‘magna charta’ (n. 8) to which all Catholic Universities on the various continents must refer to in order to continue their mission to seek and serve the truth.
Clinical profile, risk factors, disease severity, and outcome for COVID-19 disease in patients with tuberculosis on treatment under the National Tuberculosis Eradication Program: a cohort of 1400 patients
Authors: Singla Neeta , Gupta Amitesh , Khalid U.K. , Dewan Ravindra Kumar , Singla Rupak
Published: November 2024

COVID-19 affected millions of people worldwide, and tuberculosis (TB) continues to affect millions of people each year. The combined pandemic of COVID-19 and TB had a catastrophic effect on healthcare policies and healthcare setups around the globe. The clinical profile and factors affecting the outcome of COVID-19 disease in TB patients on treatment in field conditions have not been studied in detail. The present study attempted to study the occurrence of COVID-19 among patients on TB treatment in terms of severity of COVID-19 disease and outcome of both COVID-19 and TB in patients at National Tuberculosis Eradication Program treatment centers over a period of one year during peak COVID-19 times. Out of 1400 TB patients enrolled, 65 (5%) suffered from COVID-19 disease. Of the 65 TB patients with COVID-19 disease, 37 (57%) were male and under 45 years old, 33 (51%) had a TB diagnosis after first receiving a COVID-19 diagnosis, 29 (45%) had a TB diagnosis first, and received anti-TB treatment before receiving a COVID-19 diagnosis, and only 3 patients (5%) had a COVID-19 and TB diagnosis concurrently. The majority of 59 (91%) patients had mild COVID-19 disease. The outcome of TB treatment was available in 25 patients out of these 65 COVID-19-positive patients, with 21 (84%) patients having a favorable outcome. Out of the 65 COVID-19-positive patients, 4/25 (16%) had unfavorable outcomes, with one patient (4%) failing TB treatment and two patients (8%) dying. This is the first study from India that studied the occurrence and course of COVID-19 among a large number of TB patients taking anti-TB treatment under programmatic conditions. Due to the similarity in symptoms of TB and certain viral respiratory illnesses, a protocol should be established for health care to check patients for both illnesses.
Rehabilitation, the disciplining of the body, and disability identity: Reflections from psychotherapy with disabled people
Authors: Watermeyer Brian P.
Published: November 2024

Background: Recently, attention has been paid to how rehabilitation not only provides medical treatment and instrumental skills but also impacts psychological well-being and identity. We all have psychological structures that discipline the self, enforcing norms internalised during early life and exacting judgments when we fail to ‘make the grade’. In cases of congenital disabilities, rehabilitation interventions may span many years, involving strict programmes of therapy, exercise and self-discipline. These regimes may align with internalised rules in harmful ways, as striving for functional improvements takes on a moral dimension, affecting psychological health and empowered disability identities.Objectives: This study explores rehabilitation by examining the experiences of adults with congenital disabilities, who have undergone childhood medical and rehabilitative interventions.Method: This study was based on the experience of a psychoanalytic psychotherapist working with adults with disability, and presents composite case material to illustrate how interactions with medical authority figures, such as rehabilitation professionals, can have a formative influence on self-identity and entitlement to inclusion.Results: The findings vividly reflected how ‘medical socialisation’ created meanings of disability that were enacted and repeated well into adulthood.Conclusion: The discipline of rehabilitation still has much to do in examining its value-laden assumptions and practices, and how these may shape the internal and relational worlds of people with disability.Contribution: This article contributes to the debate in critical rehabilitation studies, focussing on the issue of constructions of disability which may be communicated to people with disability, with implications for self-advocacy and the growth of the disability movement.
Acknowledgement to reviewers
Authors: Office Editorial
Published: November 2024

No Abstract required.