bluuebunny/crossref_metadata_2025 huggingface dataset:
Intravital Longitudinal Three-Photon Imaging of Lymphocyte Migration in the Mouse Spleen
Authors: Choe Kibaek , Xu Chris
Published: August 2023

For the last 20 years, intravital two-photon (2P) imaging has been a powerful tool to visualize lymphocyte dynamics in mouse lymphoid organs such as bone marrows and lymph nodes. Although spleen is a key lymphoid organ for adaptive immunity against blood-derived antigens, the lymphocyte dynamics in the spleen are poorly understood because intravital 2P imaging of spleen is challenging. Visualizing the white pulp in the spleen where most lymphocytes are located requires the excitation light to propagate through the red pulp which contains a lot of blood, causing high light-scattering. In addition, intravital spleen imaging suffers from motion artifact due to the mouse breathing motion. To overcome these challenges, we applied three-photon (3P) microscopy for spleen imaging, which can reduce light scattering and improve image contrast in deeper regions compared to 2P microscopy. We also designed a new implantable, chronic imaging window for reducing spleen motion in our up-light microscope. We could identify white pulp regions by in vivo labeling of the marginal zone of white pulp. Using 3P microscopy at 1300 nm wavelength, we could visualize EGFP+ lymphocytes in white pulps at approximately 400–500 μm depth below the surface while 2P microscopy at 920 nm could only reach imaging depths of 160–250 μm in the same region. We then determined the temperature of the imaging window that is required for maintaining normal lymphocyte motility during imaging by monitoring the lymphocyte motility at different temperatures. This intravital longitudinal 3P imaging technique has the potential to examine lymphocyte dynamics in blood-derived antigen-induced inflammatory process in the spleen.
A novel method of digital ELISA using single molecule amplification enables sub-attomolar detection limits for blood-based proteomics and biomarker measurements
Authors: Lambert Jeremy
Published: August 2023

The application of measuring low-abundance protein biomarkers in blood (serum or plasma) can often present challenges due to limits of sensitivity or precision. Digital proteomics technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional immunoassay method. However, current digital ELISA technologies can lack sufficient sensitivity to quantitatively measure many low abundance proteins in blood samples.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule amplification with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (5000 vs. 500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency). Using this approach, we characterize limit of detection (LOD) of of 10–100X more sensitive than the standard digital ELISA. A digital ELISA with improved sensitivity was used to measure cytokines in serum and plasma samples with values consistently above the assay lower limits of quantitation (LLoQ). The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity. The novel approach to multiplex single molecule proteomics with attomolar sensitivity provides the potential to accelerate insights in proteomics biomarkers research, clinical applications and diagnostics.
Sex and age differences in B cell frequencies and antibody responses following influenza vaccination in highly vaccinated adult populations
Authors: Park Han-Sol , Yin Anna , Kuo Helen , Shapiro Janna , Barranta Caelan , Lee John S , Dhakal Santosh , Shea Patrick , Fenstermacher Katherine , Li Huifen , Ochsner Susan Park , Morgan Rosemary , Gearhart Patricia J , Rothman Richard , Leng Sean X , Pekosz Andrew , Klein Sabra L
Published: August 2023

Annual influenza vaccination is required for healthcare workers (HCWs, 18–49 years of age) at Johns Hopkins and is highly recommended to older adults (75+ years of age) to limit severe outcomes from annual influenza. As a result, vaccination rates and pre-vaccination immunity are high in both populations. We previously reported that sex differences in vaccine-induced antibody responses are not observed in these highly vaccinated populations, but that age-associated reductions in antiviral immunity following vaccination are more pronounced in males than females against both H1N1 and H3N2 viruses. Here, we conducted a clinical study of immune responses to the quadrivalent influenza vaccine to evaluate sex and age differences in influenza A vaccine (IAV) B cell including CD19+ B, plasmablasts (PB, CD3− CD19+ CD38++ CD71+ CD20− IgD− H1+ H3+), memory B cells (MB, CD3− CD19+ CD71− IgD− H1+ H3+), and age-associated B cells (CD3− CD19+ CD21− CD11c− T-bet+ H1+ H3+) following receipt of the seasonal influenza vaccine in 2019–2020 and 2021–2022 seasons among male and female young adult HCWs and older adults. Both young and older females consistently have more total and IAV CD19+ B cells than males, with the greatest percentage being H1. Older adults, however, consistently have greater frequencies of IAV MB cells than young adults. Even with these cellular differences, males and females have equivalent post-vaccination neutralizing antibody titers against H1 and H3 influenza viruses. Whether the transcriptional profiles of B cells differ based on sex and age is currently under investigation. Taken together, these studies may provide mechanistic insights into variability in vaccine outcomes and protection based on the sex and age of the individual.

U54AG062333
Immuno-profiling of Individuals with Opioid Use Disorder : Insights for antibody development and immunological efficacy
Authors: Zhang Yue , Efstathiou Paraskevi Maria Vlachou , Triller Gianna , Luba Rachel , Verdi Joseph , Papavisiliou Nina , Comer Sandra D , Pravetoni Marco
Published: August 2023

Opioid use disorders (OUD) and overdose are a public health emergency in the United States and worldwide. Immunotherapeutics such as vaccines and monoclonal antibodies (mAbs) offer potential solutions in treatment and prevention of OUD and overdose. To accelerate their translation, our work focuses on immunoprofiling of opioid-specific antibodies and B cell repertoires in drug-naive individuals and those with OUD. Using antigen-based enrichment paired with flow cytometry, we found that individuals with OUD display increased frequency of opioid-specific B cells. When the individuals with OUD are compared to the drug-naive individuals, the increased opioid-induced B cell population was not paired with increased opioid-specific IgG polyclonal antibodies, changes in the B cell repertoires were associated with cytokine patterns. Sequencing of the B cell receptor (BCR) revealed that opioids affect the V(D)J rearrangement in B cells, as shown by isolation of a human mid-affinity monoclonal antibody against oxycodone and morphine. Results indicate that immunoprofiling could corroborate diagnosis of OUD, and could provide biomarkers to support testing of novel immunotherapeutics in individuals with OUD.

Supported by grants from NIH U01 DA051658 Antibody-based countermeasures against fentanyl and its analogs and UG3 DA 0477117 Phase 1a/1b clinical trials of multivalent opioid vaccine components.
In silico optimization of prenatal vaccine regimens to promote early-life immunity
Authors: Erdogan Remziye Rachel , Mauldin Ileana S , Dudley Donald J , Dolatshahi Sepideh
Published: August 2023

Newborn children are vulnerable to infection due to their dampened humoral immune response. Maternal immunoglobulin G (IgG) transferred across the placenta provides passive immunity to newborns, and prenatal vaccination to boost pathogen-specific IgG transfer successfully reduced the incidence of neonatal tetanus, diphtheria, and acellular pertussis (Tdap) infections. Despite this, higher rates of infection among premature neonates signifies a deficiency in current immunization programs. To identify regulators of IgG transfer and refine prenatal vaccine regimens, we used a novel in silico approach. To disentangle the role of placental Fc gamma receptors (FcγRs) in IgG transfer, we conducted multiplexed immunofluorescence (mIF) to measure FcγR expression in paraffin-embedded placental tissue. A mechanistic model of placental IgG transfer informed by single cell RNA sequencing and the mIF data revealed FcγRIIb as a key driver of dynamic subclass-specific IgG transcytosis in placental endothelial cells (ECs). In model simulations and experiments in vitro with human umbilical vein ECs, we found the maternal IgG profile fine-tunes antigen- and subclass-specific IgG transfer dynamics. To identify immunization windows of opportunity for maximum IgG transfer, we simulated maternal vaccination by integrating a model of B cell activation and antibody production into the mechanistic model. This revealed first trimester vaccination as a potential strategy to maximize Tdap-induced IgG transport to premature newborns. We conclude that optimal prenatal vaccine regimens depend on gestational length and that novel prenatal vaccines can leverage these determinants of IgG transport to maximize neonatal immune protection.

Supported by grants from NIH (T32 GM145443) the Jeffress Trust Awards Program.
Baseline HMGB-1 Concentrations Are Predictive of Neutralization Efficacy after SARS-CoV-2 mRNA Vaccination
Authors: Umeda Lesley K , Kunihiro Braden P , Rubas Noelle C , Wells Riley K , Phankitnirundorn Krit , Peres-David Rafael Aniu , Maunakea Alika K
Published: August 2023

Determining vaccine efficacy in individuals is a concern when managing the spread of a virus. In this study pre-vaccination levels of a panel of inflammation factors were measured in order to predict the efficacy of the SARS-CoV-2 mRNA vaccines by measuring neutralization percentages, SARS-CoV-2 antibody concentrations, and inflammation factors over the course of ten weeks after the initial dose of the mRNA vaccine. Blood and stool samples were collected at a time point before administration of the vaccine (pre-vaccination) and in 2-week intervals after the administration of the first dose (post-vaccination). A cohort of 12 adults (n=12) were separated into two groups, high neutralization ability (n=6) and low neutralization ability (n=6), based on the neutralization percentages measured from samples provided ten weeks post-vaccination. Bivariate correlation analysis was performed to measure correlation between the panel of inflammation factors measured from pre-vaccination samples and neutralization ability at ten weeks post-vaccination. Correlation analysis indicates a significant (P=0.0321) inverse relationship (R =−0.5269) between HMGB-1 concentrations and neutralization ability at time points ten weeks post-vaccination. Unpaired t-test between the high and low neutralization groups and HMGB-1 concentrations taken pre-vaccination are also significantly different (P<0.001). Preliminary results indicate that HMGB-1 is a promising biomarker in predicting SARS-CoV-2 mRNA vaccine efficacy.

This study was supported in part by funding provided by Hawaii Community Foundation under award 20HCF-101573. The comments expressed in this report are the sole responsibility of the authors and do not represent the official view of the Hawaii Community Foundation.
Intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate confers potent mucosal and systemic immunity and protection
Authors: Adam Awadalkareem , Kalveram Birte K , Zou Jing , Rodriguez Leslie , Singh Ankita , Chen John Yun-Chung , Xie Xuping , Shi Pei-Yong , Wang Tian
Published: August 2023

Development of effective SARS-CoV-2 vaccines to induce potent and long-lasting immunity and to provide cross-reactive protection against emerging variants remains a high priority. We recently reported a live-attenuated SARS-CoV-2 vaccine candidate with re-engineered viral transcription regulator sequences and deleted open-reading-frames (ORF) 3, 6, 7, and 8 (Δ3678) is highly attenuated in mice and hamsters and vaccination with Δ3678 SARS-CoV-2 protect hamsters from wild-type virus challenge and transmission. Here we report that a single dose intranasal vaccination with the Δ3678 SARS-CoV-2 live attenuated vaccine candidate induced strong SARS-CoV-2-specific systemic and mucosal humoral and T cell-mediated immune responses at one-month post-vaccination and day 4 post wild-type virus challenge, which are at similar levels to those infected with wild-type virus. High titer of SARS-CoV-2-specific IgG and IgA were detected in sera and BAL of vaccinated mice respectively. Furthermore, mice vaccinated with the Δ3678 vaccine showed markedly diminished viral loads and tissue inflammation in the lung following wild-type SARS-CoV-2 challenge. In summary, our results suggest that the Δ3678 SARS-CoV-2 live attenuated vaccine protects mice from wild-type SARS-CoV-2 infection by induction of protective mucosal and systemic cell-mediated and humoral responses.
Molecular fate-mapping of serum antibodies reveals the effects of antigenic imprinting on repeated immunization
Authors: Schiepers Arien , Victora Gabriel
Published: August 2023

The ability of serum antibody to protect against pathogens arises from the interplay of antigen-specific B cell clones of different affinities and fine specificities. These cellular dynamics are ultimately responsible for serum-level phenomena such as antibody imprinting or “Original Antigenic Sin” (OAS), a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells that responded to a stimulus upon exposure to related antigens. Imprinting/OAS is thought to pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2. Precise measurement of the extent to which imprinting/OAS inhibits the recruitment of new B cell clones by boosting is challenging because cellular and temporal origins cannot readily be assigned to antibodies in circulation. Thus, the extent to which imprinting/OAS impacts the induction of new responses in various settings remains unclear. To address this, we developed a “molecular fate-mapping” approach in which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that, upon sequential homologous boosting, the serum antibody response strongly favors reuse of the first cohort of B cell clones over the recruitment of new, naÏve-derived B cells. This “primary addiction” decreases as a function of antigenic distance, allowing secondary immunization with divergent influenza virus or SARS-CoV-2 glycoproteins to overcome imprinting/OAS by targeting novel epitopes absent from the priming variant. Our findings have implications for the understanding of imprinting/OAS, and for the design and testing of vaccines aimed at eliciting antibodies to evolving antigens.

This study was funded by NIH/NIAID grants R01AI119006 and R01AI139117 to Gabriel Victora. Work in the Victora laboratory is additionally supported by NIH grant DP1AI144248 (Pioneer award) and the Robertson Foundation. Ariën Schiepers was supported by a Boehringer-Ingelheim Fonds PhD fellowship.
Regulatory maturation of lung cDC1 is required for resolution of Influenza infection
Authors: Wang Xinran
Published: August 2023

Pulmonary infection with Influenza A virus (IAV) is responsible for 20,000–30,000 deaths in the US yearly. During the acute stage of infection, pattern recognition receptors (PRRs)-dependent recognition of the virus leads to DC maturation and subsequent expansion of virus-specific effector T cells in the lung-draining lymph node. Recruitment of cytotoxic T cells into the lungs is crucial for the control of IAV infection, however unchecked effector T cell activation often leads to lung immunopathology even after the virus has been cleared. Reducing the availability of IL-2 by CD25+ regulatory T cells is thought to be the principal mechanism limiting the expansion and differentiation of effector CD8 T cells. Recently, efferocytotic maturation of cDC1 (mregDC) has been implicated in impairing CD8 T cells responses, however mregDC has not been studied in the context of IAV infection. Here we described expression of maturation markers, CCR7, PD-L1, and CD40 of lung cDC subsets. PD-L1+ CCR7+ CD40+ cDC1 differentiates in the lungs several days after the virus has been cleared (12–16 days post-infection, dpi). The accumulation of mature cDC1 coincides with increased frequency of Caspase 3+ cells and recovery of disease. At 8dpi, mature cDC1 produce IL-12p70 and induce IFNg+ CD8 T cells, in contrast, at 16dpi mature cDC1 only produce IL-12p40 and fail to induce IFNg+ CD8 T cells. In conclusion, our data suggest that mature DC1 play a role in limiting CD8 T cells mediated immunopathology after IAV infection.
Mechanisms of lung resident memory B cell activation during secondary influenza infection
Authors: Canter Hannah , Allie Rameeza , Silva-Sanchez Aaron , Mudunuru Uma , Randall Troy D
Published: August 2023

B cells and the antibodies they produce are essential components of immunity to respiratory viruses, including influenza virus (IAV) and SARS-CoV-2. B cells responding to viral infection can differentiate into antibody secreting cells (ASCs), or resting memory B cells, some of which are tissue-resident (BRM cells). ASCs provide protection by secreting virus-specific antibodies, whereas BRM cells rapidly differentiate into ASCs after secondary infection. We previously published that IAV-specific BRM cells are retained in the lungs and do not recirculate. We now show that many lung-resident BRM cells reside in the airways and can be collected by bronchoalveolar lavage (BAL). BRM cells in the BAL are phenotypically and functionally different than those in the lung tissue and lymph nodes and express markers at various levels that possibly effect function. There is also a distinction in the role of the ASCs between the lungs and the BAL that will secrete certain antibodies at different time points during the IAV challenge. To understand BRM cell homing, differentiation, and function, we are using single cell approaches to define the transcriptional and epigenetic programs of airway-resident BRM cells at resting memory and during the response to IAV challenge. We are also using this technique to identify the changes among the entire immune system during a secondary IAV challenge to gain an understanding of how the entire immune system plays a role in protection. By understanding the programs that regulate BRM placement in the lung and their response to challenge infection, we expect to identify ways to improve vaccines against respiratory viruses and ameliorate B cell-dependent lung pathology in the context of autoimmunity or allergy.

Supported by grants from NIH (R01 AI152476)
Survivors of Ebola virus disease exhibit elevated levels of autoantibodies
Authors: Velazquez Jalene V , Bond Nell G , Schieffelin John S , Gunn Bronwyn M
Published: August 2023

Survivors of Ebola virus disease (EVD) report a wide range of symptoms following recovery from acute viral infection, but underlying mechanisms are unclear. Many of these symptoms, collectively termed Post-Ebola Syndrome (PES), occur in the absence of detectable virus and resemble symptoms associated with autoimmune disease, such as arthralgia and myalgia, suggesting that EVD survivors may be experiencing a virus-induced autoimmune response. Studies in mice have shown that mice develop autoantibodies (autoAb) against HSP-60 and double stranded (ds) DNA that can bind to host tissues following Ebola virus (EBOV) infection. To determine if human EVD survivors also develop autoAb that may be associated with PES development, we profiled the humoral immune response of 354 survivors and 195 household contacts (HHC) from the 2014–2016 outbreak in Sierra Leone. Using Systems Serology, we measured the levels of antibodies against common autoantigens and found that EVD survivors had elevated levels of autoAb against dsDNA, HSP-60, IFNα, and citrullinated histone compared to HHC. As a subset of HHC were seropositive for EBOV glycoprotein (GP)-specific antibodies, separation of HHC into GP-seropositive and seronegative groups showed that the levels of autoAb in GP-seropositive HHC were elevated compared to GP-seronegative HHC, strongly suggesting that exposure/infection to EBOV tracks with presence of autoAb. As antibodies can activate innate immune cells via the antibody Fc domain, current studies are focused on analyzing the innate effector response mediated by these autoAb in EVD survivors and seropositive HHC. Together, our study highlights the potential role of autoAb in long-term sequelae associated with survival from EVD.
Notch2 amplification of B cell division and differentiation fates
Authors: Londregan Jennifer , Gaudette Brian T , Allman David
Published: August 2023

The efficiency with which B cells respond to activation signals varies depending on the subset. Whereas marginal zone (MZ) B cells exhibit rapid activation kinetics following stimulation, follicular (FO) B cells adopt delayed effector programs. The development and maintenance of MZ B cells uniquely requires signaling through transmembrane receptor family member Notch2, but whether this signal underlies distinctive response patterns among B cell subsets is not known. To examine the functional consequences of Notch2 signals on B cells with no Notch requirement, we cultured FO B cells on Notch2-ligand expressing OP9 stromal cells and subsequently stimulated them. Notch2-experienced FO B cells displayed a marked enhancement of several aspects of proliferation, including two-fold more rounds of division. Further, sensitivity to BCR cross-linking was enhanced by this signal, enabling cell cycle entry earlier and in response to lower concentrations of anti-BCR. Additionally, Notch2 signals modified responses to TLR stimuli Poly(I:C) and LPS by amplifying the time to first division and the propensity to become a plasma cell. Intriguingly, TLR3 responsiveness appeared to be an entirely Notch2-dependant mechanism. Employing a published mathematical model for dividing lymphocytes, we found Notch2 amplified the division destiny of both graded and quantal patterns of B cell responses. We next examined the timing and magnitude of Myc induction, a mitotic regulator and known Notch target. Remarkably, Myc levels prior to the first division appeared to be a direct predictor of division destiny. Together these observations highlight a novel role for Notch2 in modifying two distinct response patterns and their respective outcomes for B cells.

Supported by grants from NIH (T32-AI00763,R01 AI139123,T R01 AI154932)
Dietary L-tryptophan determines the number of colonic regulatory T-cells and susceptibility to colitis via GPR15
Authors: Kim Sangwon V , Van Nguyen T , Zhang Karen , Wigmore Rachel M , Kennedy Anne I , DaSilva Carolina R , Huang Jialing , Ambelil Manju , Vilagomez Jose H , O’Connor Gerald J , Platten Michael , Kasenty Gerard , Sigal Luis J , Prendergast George C
Published: August 2023

Environmental factors play a prominent role in the onset of immunological disorders such as ulcerative colitis, but the mechanisms are unclear. Diet is a major environmental factor, mostly known to influence the gut microbiota, which in turn affects the host immune system. Here, we demonstrated the microbiota-independent effect of the ubiquitous dietary component, L-tryptophan (L-Trp) on mucosal immune responses. We discovered that the amount of ingested L-Trp determines the number of colonic CD4+ T-cells through the aryl hydrocarbon receptor (AhR), which triggers the direct transcriptional activation of the colon T-cell homing receptor, GPR15. However, the default pathway connecting L-Trp, AhR, GPR15, and T-cells is microbiota-independent and involves host IDO1 and IDO2 enzymes, selectively increasing GPR15+ Tregs. Consequently, two weeks of L-Trp supplementation nearly doubled the colonic GPR15+ Tregs and significantly decreased the future risk of colitis. Our extensive, in vitro screening of AhR ligands suggests that AhR ligand-specific variations in target gene expression may account for the Treg-selective response to L-Trp. We also identified benzo[a]pyrene, a compound enriched in cigarette smoke and grilled meat, as an AHR agonist that strongly promotes GPR15+FOXP3+ Treg generation. Thus, our findings uncover mechanisms by which routine dietary and behavioral practices have a significant impact on colonic Treg responses, suggesting a potential non- invasive therapeutic approach for colitis.

Supported by grant from NIH (R01AI141787, R21AI142318, T32 AI134646, 5P30CA056036-22) Supported by grant from CCFA (Career Development Award 329388)
Investigating the Interaction of Inflammation-Associated Fibroblasts (IAFs) with Colon Epithelial Cells in Inflammatory Bowel Disease (IBD)
Authors: Dong Yi , Johnson Blake , Chiu Ian , Larman Tatianna , Li Rong
Published: August 2023

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease that affects the epithelial lining of the gastrointestinal tract. Recent single cell RNA-sequencing (scRNA-seq) studies suggested that inflammation-associated fibroblasts (IAFs) play important roles in the disease pathogenesis and drug resistance. To elucidate IAF’s role in IBD, we isolated fibroblasts from patients with IBD and profiled their responses to a panel of pro-inflammatory cytokines. We found a cocktail of the cytokines can induce IAF marker genes in the patient-derived fibroblasts. These cytokine-stimulated fibroblasts recapitulate many characteristics of IAFs, including increased ECM and chemokine secretion. Furthermore, when co-cultured with colon organoids, the cytokine-stimulated fibroblasts secrete prostaglandin E2 (PGE2) to induce organoid swelling through EP4-cAMP-PKA-CFTR signaling, which mimics diarrhea and crypt distortion of IBD. Furthermore, rapid swelling increases the chance of mitosis error and activates p53 pathway, which increases the risk of tumorigenesis. Thus, we are reporting novel mechanisms of fibroblasts that regulates the pathogenesis of IBD.

This study was funded by Takeda Development Center Americas, Inc.
Depressive-like behavior associated with colitis is altered after treatment with aryl hydrocarbon receptor ligand
Authors: Bogdon Raymond K , Roark Kasie , Saxena Archana , Mitchell Chandani , Hailey Michelle , Staley Shanieka Caroline , Busbee Phillip B
Published: August 2023

Colitis is a chronic inflammatory disease with no cure and an overall reduction in patient quality of life. Due to decreased mucous production and weakening of the colonic epithelial lining, gut -microbiota and their metabolites can invade the intestinal lamina propria and circulate systemically, a term known as “leaky gut”. Recent studies show a relationship exists between peripheral inflammation and depression. The gut-brain axis (GBA) acts as a bidirectional communication pathway between the enteric and central nervous system (CNS), and colitis patients are known to have increased incidence of depression. In the current study, we aimed to investigate the relationship between colitis, and its impact on depressive-like symptoms in mice. Colitis was chemically induced in mice and treated with indole-3-carbinol (I3C), a naturally-occurring aryl hydrocarbon receptor (AhR) our lab previously showed reduced colitis severity. Untargeted metabolomic profiles of over 200 metabolites revealed that during disease induction a potential neurotoxic and kynurenine pathway metabolite, quinolinic acid (QA), was upregulated in colitis models, but reduced after I3C treatment. As high QA levels are linked to depression, we performed tail suspension tests (TST) over the course of disease and treatment to assess depressive-like behavior in experimental mice. Our results show that colitis- induced mice had a higher immobility time as compared to healthy controls, but this was reversed upon treatment with I3C. These results suggest that colonic inflammation can lead to altered behavior and increased depressive-like symptoms through dysregulation of the gut metabolome, but restored after treatment with AhR ligand, I3C.

NIH P20GM103641
Oral CD8+ TRM surveil taste buds where their local reactivation drives papillary inflammation and bitter receptor expression
Authors: Stolley Michael , Masopust David
Published: August 2023

Resident memory T cells (TRM) expedite pathogen detection and clearance at typical portals of pathogen entry. While paradigms in TRM biology were largely established in a few select locations, comparatively little is known about how CD8+ TRM integrate within the oral cavity, which encompasses a functionally organized constellation of absorptive, secretory, and chemosensory tissues. To fill this knowledge gap, we developed a Viral-Prime, Epitope-Pull (VPEP) model for locally augmenting tractable populations of oral TRM to manipulate and study. Leveraging this approach, we document CD8+ TRM within and surrounding taste buds and papillae of the soft palate and tongue. Reactivation of oral T cells induced discernable inflammation within taste structures that was abrogated with prior in vivo CD103+ TRM depletion. Taste buds represent a collection of ~50–100 individual taste receptor cells (TRCs) which sense nutritive and potentially harmful dietary components. We hypothesized that reactivation of taste bud associated TRM would provoke TRC transcriptional changes including genes involved in gustation (taste). Bulk RNAseq was performed on TRM-reactivated circumvallate papillae and soft palate; primary taste-sensing locations in mice and enriched sources of TRCs relative to other oral tissues. Here we found the significant induction of several Tas2r (bitter receptor) genes not observed in control mice or TRM reactivated buccal mucosa. Teleologically, a heightened sensitivity to bitter compounds downstream of oral TRM reactivation may represent an evolved mechanism to protect against the accidental ingestion of noxious chemicals during oral or upper respiratory tract infections when other chemosensory modalities faulter.

T90 DE 022732 K99DE031014
Single-cell RNA sequencing reveals heterogeneous immune landscape of tumor infiltrated immune cell population in B cell deficiency against glioblastoma
Authors: Kim Yumin , Kim Chae Won
Published: August 2023

The distinct immune landscape of glioblastoma (GBM) from other cancer is an obstacle to GBM treatment. To overcome the limited efficacy of GBM therapy, identifying the interaction between the tumor and infiltrated immune cells is needed. The function of B cells against tumors is recently focused on to understand tumor microenvironments. However, there are still few studies on the function of B cells in glioblastoma. Here, we identify tumor infiltrated immune cell population in B cell deficient mouse GBM by landscape analysis against glioblastoma. Using single-cell transcriptome analysis, we find that the frequency of tumor infiltrated immune cells including T cells and monocyte/macrophage is changed by the absence of B cells in mouse GBM. The frequency of effector CD8 and CD4 T cells decreases in B cell deficient mice. Also, expression of the cytotoxic signature protein is downregulated in tumor infiltrated T cells which are known to play an important role in tumor cell killing. In the case of tumor infiltrated macrophage and microglia, expression of M2 signature increased in B cell deficient GBM which can lead immunosuppressive tumor microenvironment. This study reveals changes in the immune cell landscape according to the presence or absence of B cells and shows how B cells contribute to antitumor immunity through immune interaction.
Single-cell RNA sequencing reveals heterogeneous immune landscape of tumor-infiltrating lymphocytes in the glioblastoma tumor microenvironment in the absence of IFNAR
Authors: Lim Juhee , Lee Heung Kyu
Published: August 2023

Glioblastoma (GBM) is known to be the most common malignant brain cancer with a very poor prognosis. Despite standard conventional therapies patient survival rates still remain poor and reasons for this may be due to the extensive tumor cellular heterogeneity of the tumor microenvironment. Type I interferons (IFNs) are a family of cytokines produced by multiple cell types and are known to play a role in anticancer immunity through their signaling via the interferon alpha receptor (IFNAR). We find that absence of IFNAR compromises anti-tumor immunity against GBM. Single-cell RNA analysis of the immune landscape of GBM tumor-bearing IfnαR−/− mice reveals that there is an overall decrease in tumor-infiltrating lymphocytes into the brain along with overall decreased expression of IFNγ, TNFα, and Granzyme A. Analysis of lymphoid cells including T cells and NK cells shows substantial decreased infiltration in the presence of GBM in IFNαR deficient mice. Notably, CD4 and CD8 T cell infiltration is considerably reduced, although how the functions of these cells are affected is yet unknown. Analysis of tumor-infiltrating myeloid cells in the absence of interferon signaling reveals decreased infiltration of monocytes and tumor-associated macrophages as well. Thus, in this study, we are able to see that the absence of type I interferon signaling affects the overall recruitment of lymphoid and myeloid cells during the progression of GBM and likely contributes to the exacerbation of anti-tumor immunity.
Application of Next Generation Sequencing technology to determine breast cancer risk among Bangladeshi women
Authors: Rabbi Mohammad Fazle Alam , Pathan Kawsar Iqbal
Published: August 2023

Recent advancement in sequencing technology had led to the accurate and precise diagnosis of deadly diseases like breast cancer. Breast cancer is the second most common cancer in women, therefore, early diagnosis of this critical disease is important for prevention and therapeutic implications. BRCA1 and BRCA2 genes are significantly associated with breast cancer aetiology. Individuals carrying a germline mutation in BRCA genes increase their chance of developing breast or ovarian cancer in women and pancreatic cancer in both sexes. The purpose of this study was to determine the germline mutations of BRACA among the risk group of Bangladeshi women using Next Generation Sequencing (NGS). One hundred patients were selected randomly from the outdoor breast cancer clinic of DNA Solution Ltd. Using ‘Six-Point Scale’ twenty individuals were selected for BRCA gene mutation study. A clinical database was maintained to record personal and familial cancer history of the selected individuals. Personal identification was preserved according to Helsinki principals. Out of twenty enrolled individuals, seventeen individuals had a family history of cancer. However, upon sequencing data analysis, we observed only six patients carrying the pathogenic mutation of BRCA gene. This study only involved germline mutation and did not consider somatic mutation. Recent reports also suggest various genes other than BRCA are also associated with breast cancer occurrence. To our knowledge, this is the first time to use NGS technology to screen genetic mutations of breast cancer in Bangladesh. These data will help to develop early screening and possible prevention with better disease management in breast cancer.

No Funding Support
High-throughput Multidimensional Characterization of CD8 T cells in MOG Antibody-Associated Disease
Authors: Chizari Shahab , De Jager Philip L , Jiang Jenny
Published: August 2023

Inflammatory demyelinating diseases of the central nervous system (CNS) include clinically and pathologically distinct disorders. Myelin oligodendrocyte glycoprotein-associated disease (MOGAD) is a recently characterized antibody-mediated inflammatory demyelinating disorder of the CNS with clinical presentations which include optic neuritis, transverse myelitis, and acute disseminated encephalomyelitis. The heterogenous clinical course of MOGAD along with unclear prognostic biomarkers makes treatment challenging, as some individuals have monophasic disease while others relapse. Recent work has attempted to identify biomarkers by performing scRNA-seq on the peripheral blood mononuclear cells (PBMC) of MOGAD patients and has highlighted an enrichment for a cytotoxic effector CD8 +T cell subset in MOGAD compared to healthy controls. However, this subset of cells remains under characterized, with questions regarding their antigen specificity, clonality, and overall involvement in MOGAD.

To further characterize this cell subset, we utilize a single cell technology, TetTCR-SeqHD, to evaluate the antigen-specificity, clonality, gene expression, and surface protein expression of CD8 +T cells isolated from the PBMC of MOGAD patients. In this approach, a large library of fluorescently labeled, DNA barcoded peptide-MHC tetramers are generated utilizing peptides derived from MOG or associated proteins such as myelin basic protein and proteolipid protein which are then used to isolate antigen specific CD8+ T cells for single cell analysis. Preliminary results show an enrichment of a distinct CD161 ++TRAV1–2 +MAIT cell population in MOGAD, which has been previously implicated in other chronic inflammatory diseases.

This work is supported by the Chan Zuckerberg Initiative Neurodegeneration Challenge Network Ben Barres Early Career Acceleration Award.
Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis
Authors: Hao Jiaqing , Yu Jianyu , Yorek Matthew , Jabbari Ali , Li Bing
Published: August 2023

Psoriasis is a common chronic inflammatory skin disease. One of the hallmarks of psoriasis is the abundance of neutrophil infiltration in the skin lesions. However, the detailed molecular mechanisms of neutrophil chemotaxis and activation remains unclear. Here we demonstrated an upregulation of epidermal fatty acid binding protein (E-FABP, also known as FABP5) in skin tissue from both human psoriasis and imiquimod (IMQ)-induced psoriatic mouse model. Genetic deletion of FABP5 in global knockout mice and keratinocyte conditional (Krt6a-Cre) knockout mice, but not in myeloid cell conditional (Lysm-Cre) knockout mice, attenuated the skin erythema, thickness, and desquamation. Immunophenotypic analysis showed that FABP5 deficiency reduced the recruitment of Ly6G+ neutrophils in the dermis and ear of IMQ-treated mice. Mechanistically, activated keratinocytes in IMQ-treated mice produced chemokines and cytokines (e.g., CXCL8, IL-36γ) that triggered neutrophil chemotaxis and activation in a FABP5-dependent manner. Proteomic and IP analyses further identified that FABP5 interacted with a valosin-containing protein (VCP), a key player in NFκB signaling pathway in human keratinocytes. Silencing of FABP5 inhibited VCP-mediated IκBα phosphorylation and NFκB signaling activation. Collectively, these data suggest that FABP5 in keratinocytes interacts with VCP to activate NFκb signaling, thus playing an essential role in the neutrophil infiltration in psoriatic skin lesions.

R01AI137324, #LINK#R01CA180986#ENDLINK#
Spatial Transcriptomic Profiling Reveals the Pathophysiology of Early Stage Hidradenitis Suppurativa
Authors: Joh Christine , Jeong Soyoung , Lee Jung Ho , Lee Hee Jung , Kim Gwangil , Lee Cheol , Shin Jung U , Kim Hyun Je , Park Chung-Gyu , Jin Seon-Pil
Published: August 2023

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disorder that ranges from superficial nodules and deep abscesses to draining dermal tunnels in the intertriginous regions. It causes significant pain, scarring, and a poor quality of life, yet there are currently no effective treatment options. The role of tunnels or sinus tracts—a unique feature of moderate to severe HS—was reported that the epidermal tunnels are the source of inflammatory mediators in Hurley stages II and III. However, the pathophysiology of early stage HS needs further investigation. Here, we examined the intradermal epithelial cells surrounding these nodules in early HS in order to explore their features and function. Utilizing spatial transcriptomics, a cutting-edge technique that offers both spatial information of cells and their gene expression profiles, we compared the intradermal epithelial cell of HS patients in the early Hurley stage with the dermal wall of epidermal cysts in epidermal cyst patients. As expected, keratinocytes in the epidermis of individuals with HS were significantly activated, with increased levels of inflammatory cytokines, including IL-36G and CXCL8. In addition, the increase of IL-4R and IL-13RA1 suggests the increase of the sensitivity of Th2 cytokine on the epidermis and in dermal-infiltrating immune cells. Moreover, genes associated with B lymphocytes were highly expressed in the infiltrating immune cells of HS, implicating the importance of “epidermis-derived” inflammatory cytokines and the role of B cell infiltration in the pathomechanism of early HS.
Astrocytic expression of CD137 in the lumbar spinal cord following sciatic nerve crush model of neuropathic pain in mice
Authors: Cao Ling , Withers James , Bean Elizabeth N
Published: August 2023

Previously we have shown that CD137–CD137 ligand (CD137L) mediates pro-nociceptive behaviors in the sciatic nerve crush (SNC) model of neuropathic pain. Global CD137L knockout (KO) mice and wildtype (WT) mice intrathecally treated with neutralizing antibody against either CD137L or CD137 displayed significantly reduced neuropathic pain-like behaviors and faster functional recovery compared to WT mice following SNC. To identify the cellular sources of CD137 and CD137L in the spinal cord, a series of immunohistochemistry analyses was performed. While we could not detect significant expression of CD137L (in part due to both technical and biological issues), we observed CD137 expression exclusively co-localized with astrocytic marker glial fibrillary acidic filament (GFAP). We then conducted a time course study of CD137 expression in the lumbar spinal cord dorsal horn region, the region relevant to nociceptive behaviors, in B6-CD137L KO and B6 WT mice. Significant increases in CD137 expression (represented by integrated density) were observed in WT mice from days 7 to 28 following either SNC or sham surgery. The expression of CD137 in KO mice was slightly (however not statistically significantly) higher than that in WT mice at baseline, and did not change overtime post-either surgery. When RNA expression of CD137 was evaluated, although SNC induced significant upregulation of GFAP RNA expression in WT mice, no changes in CD137 RNA expression were detected in any treatment groups or any genotype of mice. Altogether, we report a novel expression of CD137 on murine spinal cord astrocytes. The underlying mechanism through which astrocytic CD137 mediates SNC-induced nociceptive behavior requires further investigation.

NIH/NINDS R01NS098426 (Cao) and Peter Caradonna from the COBRE Histology and Imaging Core funded by NIH/NIGMS P20GM103643 (Meng).
Elucidating Mycosis Fungoides Gene Expression in Skin of Color Patients from Howard University Dermatology
Authors: Nwade Nina , James Nia , Nagarkar Akanksha , Desse Sachi , Yildiz-Altay Ummugulsum , Okoye Ginette A , Byrd Angel S , Richmond Jillian M
Published: August 2023

Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma.There are notable differences in MF presentation in Skin of Color (SOC) versus White skin, such as asymptomatic hyperpigmented and hypopigmented lesions resulting from malignant T-cell interaction from keratinocytes and melanocytes. Therefore, this work aims at elucidating MF immunopathogenesis and gene expression in SOC patients and determining whether clinical presentation differences are dependent on the cytokine type produced by the tumor. We used both hypopigmented and hyperpigmented MF biopsy samples from SOC patients with MF from Howard University Dermatology and compared their gene expression to that of biopsy samples from SOC healthy patients. Preliminary data showed an upregulation of genes such as PRDX1, HLA-DRA, CTNNB1, CSTB, and S100A4 in MF samples versus their healthy counterparts. The MF samples also exhibited downregulation of CCL3, CCRL2, PDGFB and HOXD4 genes when compared to their healthy counterparts. Lastly, the pathways ‘cell cycle and apoptosis’, ‘antigen presentation’, and ‘interferon signaling’ were increased in MF and decreased in the healthy samples. Overall, these data will elucidate MF gene expression in SOC patients as well as the immunopathogenesis that results in varying presentations. This may facilitate the development of more concise diagnostic criteria and personalized targeted immunotherapies to better health outcomes within a minority population.

Supported by grants from Dermatology Foundation DRSA (SD), the Skin of Color Society Research Grant (JMR), and the American Skin Association Milstein Research Scholar Award for Melanoma/Non-Melanoma Skin Cancer (ASB).
Treatment with doxorubicin and PD-1/CTLA-4 blockade improves T cell activation and anti-tumor efficacy in MCA-205 murine fibrosarcoma
Authors: Therrien Nicholas R , Tellez Cristiam Morreno , Kemp Lindsey E , Powers Kyle N , Levey Daniel , Chand Dhan , Wilkens Margaret , Grossman Joseph , Goodspeed Andrew , Davila Eduardo , Wilky Breelyn A
Published: August 2023

Immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 produce responses in about 20% of adult soft tissue sarcomas (STSs), however, most patients do not respond, potentially from poor T cell activation. Standard chemotherapy for STSs includes the anthracycline doxorubicin (DOX) which has been shown in preclinical cancer models to induce immunogenic cell death (ICD) via tumor production of type I interferons (IFN). We hypothesized that induction of ICD and IFN release by doxorubicin would enhance T cell activation, infiltration, and boost anti-tumor efficacy when combined with dual anti-PD-1/CTLA-4 blockade in the MCA-205 murine fibrosarcoma model. MCA-205 tumors were generated in C57BL/6 mice and were treated with DOX, anti-PD1 (RMP1–14), anti-CTLA4 (9D9 mouse IgG2b) or a mouse surrogate of botensilimab, an anti-CTLA4 (9D9 mouse IgG2b.DLE) antibody with enhanced binding to FcγR, or combination DOX plus anti-PD-1/CTLA-4. We found reduction of tumor growth and improved survival (median survival not reached) with DOX/PD-1/CTLA-4 compared to DOX (median survival 29.5 days). This correlated with a 2-fold increased influx of TILs as measured by flow cytometry and immunohistochemistry, with a marked increase in CD8:CD4 T cell ratio with increased expression of activation markers (PD-1, 4–1BB and TIM3). TCR Vβ clonotyping revealed a shift in clonotype frequency with DOX/PD-1/CTLA-4 relative to other treatment groups. Pathway analysis from bulk RNA sequencing demonstrated upregulation of T cell activation in DOX/PD-1/CTLA-4 tumors compared to control tumors. Further work will correlate these findings with human STS patients receiving DOX plus CTLA-4/PD-1 blockade in an ongoing clinical trial (NCT04028063).

This work was supported by grants from the University of Colorado Cancer Center (P30CA046934) and the University of Colorado School of Medicine.