bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of lysine dimethylation in Bcl-2 function and T cell activation (LYM4P.744)
Authors: Li Wenqing , Xiao Zhen , Ravichandran Sarangan , Luke Brian , Collins Jack , Durum Scott
Published: January 2023

Bcl-2 is an anti-apoptotic protein that can protect developing and mature T cells from apoptosis. Interaction between anti-apoptotic and pro-apoptotic members of Bcl-2 family proteins is a key event in the regulation of apoptosis. Using mass spectroscopy to analyze a murine T cell line, we identified dimethylation of two lysine residues, K17 and K22, in the BH4 domain of Bcl-2, a domain critical for its anti-apoptotic function. Mutation of K22 (but not K17) to alanine abrogated Bcl-2 binding to pro-apoptotic proteins and greatly reduced Bcl-2 anti-apoptotic function in cytokine withdrawal. This assigns functional importance to K22, which is conserved across species and other anti-apoptotic members, and suggests that dimethylation could affect its function. Structure model of Bcl-2 was generated using computer simulation. The Bcl-2 model showed that the K22 appears to be involved in stabilizing peptide secondary structure, rather than directly interacting with pro-apoptotic proteins. With our newly developed monoclonal antibody to detect the K22 dimethylation of Bcl-2, dimethylated Bcl-2 was found in murine T and B cells, but not in thymocytes. There is no evidence that the dimethylation of Bcl-2 is regulated by IL-7, a growth factor required for T cell development and homeostasis. Anti-CD3 stimulation abolished Bcl-2 dimethylation in CD4 or CD8 cells. This suggests that Bcl-2 dimethylation may regulate activated T cell apoptosis but not be involved in thymic development.
Differential restimulation-induced cell death sensitivity between CD8+ memory T cell subsets (LYM4P.754)
Authors: Larsen Sasha , Katz Gil , Snow Andrew
Published: January 2023

CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in longevity and protective capacity after infectious challenge. However, their respective sensitivity to cell death has been overlooked, despite its importance in maintaining immune homeostasis. Here we show that effector T cells derived from sorted human CD8+ CM and EM subsets demonstrate differential sensitivity to T cell receptor (TCR) restimulation induced cell death (RICD), a critical self-regulatory apoptosis pathway for constraining excessive T cell expansion. By quantitating differences in AnnexinV binding, mitochondrial membrane potential and caspase activation, we found that EM-derived effector T cells are significantly more sensitive to RICD than CM-derived effectors, whereas responses to other extrinsic or intrinsic apoptotic stimuli were equivalent. Our data suggests that EM-derived effectors demonstrate a stronger, more robust TCR signal, via enhanced LCK activity, for potent RICD induction. These data point to “imprinted” TCR signaling differences preserved between CD8+ CM and EM progeny that govern their relative sensitivity to RICD. Our findings offer new insight into why CM-derived T cells display superior effector expansion and persistent memory responses in vivo, beyond simple proliferation capacity. Moreover, our use of primary human T cell subsets is a novel comparative approach for defining molecular mechanisms that control RICD sensitivity.
A role for Noxa in human primary T cell activation and metabolic reprogramming (LYM4P.756)
Authors: Benson Jenna , Lowman Xazmin , Hanse Eric , Kelekar Ameeta
Published: January 2023

Upon activation, T cells undergo changes to meet the metabolic demands of proliferation, including the upregulation of aerobic glycolysis. The expression of the Bcl-2 protein Mcl-1 and paradoxically that of its pro-apoptotic binding partner Noxa is also upregulated. Studies showing Noxa is post-translationally modified in both primary and leukemic human T cells, such that its death function is masked, suggest that Noxa plays a direct role in T cell proliferation and survival. Our data show that Noxa also regulates metabolic flux in Jurkat T cells. Here we evaluate Noxa’s role in early proliferation and metabolic reprogramming of human primary T cells. Surprisingly glutamine, but not glucose, was essential for Noxa induction following stimulation. T cells stimulated in the absence of glucose or glutamine exhibit an activated phenotype comparable to controls although cell viability was low in the absence of glucose. The high oxygen consumption and low extracellular acidification rates (OCR and ECAR) of glucose-deprived cells pointed to an early switch to glutamine for mitochondrial respiration during activation. Conversely, glutamine-deprived cells decreased OCR but increased glycolysis to maintain an elevated ECAR, reminiscent of the Warburg effect. We also traced the path of [13C] glucose and glutamine during this metabolic switch. Our data demonstrate for the first time how human primary T cells reprogram metabolism upon activation and how Noxa contributes to these pathways.
Oral infection drives a distinct population of resident memory CD8 T cells with enhanced protective function (LYM4P.751)
Authors: Sheridan Brian , Pham Quynh-Mai , Cauley Linda , Puddington Lynn , Lefrancois Leo
Published: January 2023

The intestinal mucosa is designed for promoting specialized T cell responses which are distinct from responses generated systemically or even from other mucosal surfaces. Oral infection with Listeria monocytogenes induced a robust CD8 T cell response. Unlike splenic short-lived effector cells (SLEC) early after infection, intestinal SLEC rapidly underwent apoptosis resulting in a predominant memory precursor (MPEC) population between 9 and 12 dpi. Similarly, the resulting IEL population was functionally superior to their splenic counterparts and provided rapid protection upon secondary oral challenge. These early memory cells, but not other effector cell subsets, rapidly upregulated CD103 identifying them as mucosal resident memory precursors. Surprisingly, CD103 expression promoted T cell accumulation in the epithelium but did not regulate long term retention within the epithelial layer or CD8 T cell fate. Indeed, intranasal or intravenous Listeria monocytogenes infection generated memory CD8 T cell populations within the intestinal epithelial compartment which were heterogeneous for CD103 expression and displayed distinct effector cell subsets. Collectively, this data demonstrates the importance of priming location for generating intestinal resident and protective T cell memory and demonstrates that priming location and tissue of residence impacts resident memory T cell formation and maintenance.
A foil to itself: TLR2 signaling protects and damages host tissue during Mycobacterium tuberculosis infection (MPF1P.773)
Authors: Konowich Jill , Rafi Wasiulla , Gopalakrishnan Archana , Dietzold Jillian , Salgame Padmini
Published: January 2023

TLR2 polymorphisms have been associated with increased susceptibility to tuberculosis (TB). We have demonstrated that TLR2 is critical for host resistance against Mtb infection and limits immunopathology in the lung. Recent publications illustrating a role for epithelial cells in host immunity against Mtb led us to investigate TLR2’s role in hematopoietic (H) cells and nonhematopoietic (NH) cells in host defense against Mtb. We generated radiation bone marrow chimeric mice, which lack TLR2 either in the H, NH, or both compartments, and infected these mice with Erdman Mtb. Our results show that TLR2 signaling on the H component is crucial for control of chronic murine Mtb infection. Loss of TLR2 in the H component resulted in increased bacterial burden, decreased accumulation of regulatory T cells, and disruption of granuloma architecture. In contrast, loss of TLR2 on the NH compartment resulted in decreased inflammation and dissemination of Mtb to extrapulmonary sites. These data illustrate a novel role for TLR2 and a paradox within TLR2 signaling: TLR2 signaling on H cells is protective to the host both by controlling infection and regulating inflammation while on NH cells it promotes inflammation induced immunopathology. This balanced response is essential to maintaining host control of TB and could lead to the development of cell-specific TLR2 inhibitors.
Adoptive transfer of Treg cells to B6 mice during Toxoplasma gondii infection induces down-modulation of exacerbated inflammatory immune response but does not generate protection (MPF1P.777)
Authors: Olguín Jonadab , Fernández Jacquelina , Castellanos Carlos , Campuzano Jaime , Terrazas Luis I. , Saavedra Rafael
Published: January 2023

Toxoplasmosis is an infection disease affecting one third of the world population and is caused by the protozoan parasite Toxoplasma gondii. TH1 immune response is necessary for protection, IFN-γ being the major mediator of resistance. An exacerbated TH1 immune response however, is detrimental. Regulatory T (Treg) are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, thereby contributing to the maintenance of immune homeostasis. It has been reported that during acute toxoplasmosis in B6 mice, Treg cell number decreases. In order to analyze if Treg cells are involved in protection during T. gondii infection, we carried out adoptive transfer of Treg cells to infected mice. We observed a decreased mortality when Treg cells were adoptively transferred but an increased number of cysts in brain was detected. A decreased pathology in the small intestine, a decreased production of IFN-γ and TNF-α in serum, a decreased activation in CD4+ cells, and a decreased production of IFN-γ by CD4+ cells was also observed in the same mice. All these results suggest that adoptive transfer of Treg cells during T. gondii infection down-modulate the exacerbated immune response, but it does not generate protection in B6 mice. Thus Treg cells are not involved in protection during T. gondii infection.
Microbiota-derived GABA exacerbates Clostridium difficult-associated intestinal inflammation and suppresses development of protective immunity (MPF1P.768)
Authors: Dann Sara , Eaves-Pyles Tonyia , Peniche Alex , Choudhury Barun , Feng Hanping , Savidge Tor
Published: January 2023

Background: Clostridium difficile infection (CDI) is a major cause of nosocomial antibiotic-associated diarrhea and colitis. Previously we performed metagenomic and metabolomic analysis on stool samples collected from patients with and without CDI, and found a significant correlation between disease recurrence and intestinal lumen levels of the neurotransmitter GABA. Study objectives: To determine the role of GABA in CDI pathogenesis and the effects on host immune defenses. Methods & Results: C57BL/6 mice were treated with an antibiotic mixture +/- GABA in their drinking water and infected orally with C. difficile. Intestinal tissue was collected and examined for histopathology and gene expression. Administration of GABA exacerbated CDI- induced mucosal inflammation and damage, whereas inhibition of GABA with picrotoxin reduced disease severity. In addition, mice given daily doses of probiotic bacteria that secrete GABA had greater disease severity compared to controls. In vitro, GABA modulated epithelial cell cytokine responses to C. difficile toxins, LPS and flagellin. In vivo, GABA altered the cellular response to infection, and promoted the recruitment of CD11b+Gr1+ cells to the site of infection. The early immune changes were associated with decreased protective antibody production at week 2 post-infection. Conclusion: Microbial-derived GABA promotes CDI disease pathogenesis by modulating innate immune responses which leads to suppression of protective immunity.
Association of miRNA expression profile with Chlamydial-induced genital tract pathology (MPF2P.803)
Authors: Yeruva Venkat , Maurelli Anthony , Spencer Nicole , Praskievicz Nancy , McChesney Grant , Myers Garry , Rank Roger
Published: January 2023

Currently, with respect to chlamydial infections, one cannot predict the probability of an infected patient developing pelvic inflammatory disease. To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (M4, M9) that showed differences in the development of upper tract pathology. M4 has a slower growth rate in vitro, induces pathology in 6 of 30 C57Bl/6 oviducts versus 25 of 30 oviducts in M9-infected mice. To determine if chemokine cytokine production within 24 h of infection will define the outcome of pathology, 15 chemokines and cytokines were measured. M4 infection induced significantly lower levels of CXCL 1, CXCL 2, TNF-α and CCL 2 in comparison to M9 infection with similar ribosomal RNA (rs16) levels for chlamydiae. To understand if chemokine cytokine responses would also be reflected in miRNA expression profile within 24 h of infection, 134 inflammation-related miRNAs were measured. Interestingly, 12 miRNAs (miR-135a-5p, miR155-5p, miR325-3p, miR338-5p, miR105, miR132-3p, miR142-3p, miR142-5p, miR147-3p, miR223-3p, miR298-5p and miR299a-3p) were expressed significantly higher during M4 infection than M9 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge this is the first report demonstrating that early biomarkers elicited in the host could differentiate between two pathologic variants of chlamydiae and be predictive of upper tract disease.
Inhibitory role of β-arrestin2 in T cells protects against colitis. (MUC2P.823)
Authors: Sharma Deepika , Malik Ankit , Parameswaran Narayanan
Published: January 2023

β-arrestins have shown to be important regulators of inflammation. The aim of this study was to determine the role of β-arrestin2 in intestinal inflammation. In response to DSS-induced colitis, β-arrestin2 knockout (KO) mice exhibit greater body weight loss, higher disease activity index, shortened colon and loss of thymic weight as compared to wild type (WT) mice. Additionally, number of CD4+T cells and proportion of IL-17A+ (and RORγT+) and IFNγ+ CD4+ T cells were markedly higher in DSS-treated KO colonic lamina propria (cLP). Surprisingly, control KO mice too had higher number of CD4+ T cells and il17a mRNA expression in colon tissue compared to control WT mice. Furthermore, T-cell receptor (TCR) stimulation of MLN and splenic cells led to significantly higher production of IFNγ and IL-17 from KO compared to WT cells. Additionally, naïve CD4+ T cells from KO mice produced higher IFNγ and IL-17 and differentiated into Th1 and Th17 cells to a greater extent following TCR stimulation. These results suggest that the KO colon is likely predisposed to exacerbated mucosal inflammation by virtue of enhanced CD4+ T cell numbers and function. This overt response however, was independent of microbiota since, KO animals co-housed with WT mice also exhibited higher weight loss and colon shortening following DSS. Together, our results demonstrate that β-arrestin2 negatively regulates T cell activation and thereby modulates mucosal response under both basal and inflammatory conditions.
Development of non-overlapping multiplex antibody arrays for the quantitative measurement of 400 human and 200 mouse proteins in parallel (TECH1P.849)
Authors: Mao Yingqing , Yen Haw-han , Sun Yanni , Lv Zhiqiang , Huang Ruopan
Published: January 2023

Accurate detection of multiple cytokines in complex biological samples is essential to progress in immunological research. However, development of high-density multiplex sandwich-based ELISA panels is hampered by nonspecific cross-reactivity between and among target-specific reagents (i.e., capture antibodies, detection antibodies, and/or antigens); this is particularly challenging for bead-based assays, in which all three are free to interact in solution. To overcome this bottleneck, we tested for nonspecific cross-reactivity among reagents used on our quantitative antibody arrays (in which capture antibodies are affixed to a glass slide). Combinations of reagents exhibiting nonspecific interactions were separated on discrete arrays. Using this divide-and-conquer strategy, we developed and validated 10 human and 5 mouse non-overlapping 40-plex arrays for parallel, quantitative detection of 400 human and 200 mouse proteins respectively. These markers cover the most analyzed secretory proteins such as cytokines, chemokines, growth factors, soluble receptors, and other regulatory factors. To our best knowledge, this is the highest density multiplex antibody arrays developed to date. Such a system hold a great promise in determining key factors and mechanisms in immune cell physiology and pathology, in identifying novel drug targets, and in screening for putative biomarkers from patients' samples.
Real-time monitoring of MMP-9 secretion from monocytes using a nanoplasmonic-microfluidic platform (TECH1P.834)
Authors: Cheng Xuanhong , Wu Shu-han , Wei Pei-kun , Chiou Arthur
Published: January 2023

Leukocyte secretion is fundamental to the understanding of many immunological phenomena and is widely used in clinical diagnoses. Conventional methods, such as ELISA, ELISPOT and flow cytometry reveal significant information of cell secretion at fixed time points, but miss the secretion dynamics. To address this issue, we constructed an optofluidic platform for real-time monitoring of secretory activities of monocytes. The sensing element is a nanoslit array that uses the surface plasmon resonance mechanism to detect refractive index changes at the sensor/liquid interface. When functionalized with an antibody against matrix metalloproteinase 9 (MMP-9) and integrated with a cell-trapping microfluidic device, the sensor is able to monitor dynamic MMP-9 secretion from human acute monocytic leukemia cells in situ. Upon continuous lipopolysaccharide (LPS) stimulation, MMP-9 secretion is detected within two hours. This sensitivity is a result of ultralow surface detection limit, small culture compartments and close proximity of the sensor to the target cells. The average secretion measured using our system well matches previous reports while it requires orders of magnitude less cells. Furthermore, the cell secretion dynamics reveal an intermittent profile and the cause is still under investigation. Current effort is focused on imaging secretion from individual cells and multiplexing the sensors to study the secretion interplay of multiple molecules.
Auto-antibody profiling with over 9,500 human expressed proteins (TECH1P.853)
Authors: Lazar James , Amariei Flavia , Qin F. Xiao-Feng
Published: January 2023

Autoantibody profiling is a powerful method to probe the immune system for early signs of disease. However, progress has been limited by the availability of suitable antigens and high-throughput tools for screening large numbers of samples. Most antigens available have been produced in bacterial or insect cells and thus may not be optimal for autoantibody profiling. Although protein microarrays have been widely used to identify common autoantibodies in many diseases, creating smaller panels of antigens for larger screening studies has been difficult and costly. We have developed rapid methods for coupling proteins to Luminex beads allowing us to create and validate focused beads arrays in just a few days using native human proteins that have been expressed and purified in HEK293 cells. We have also developed a variable-stringency assay system that allows the user to optimize the detection specificity according to their particular application. Internal and external controls validate the presence of each protein and the IgG from the patient sample. Bead arrays for classic autoimmune antigens, as well as human breast, prostate, ovarian, pancreatic and lung cancer antigens have been prepared and custom arrays can be prepared from any of OriGene’s over 9500 purified proteins. Preliminary results with serum samples from traditional autoimmune patients, breast cancer, and Type II Diabetes support the utility of the approach.
A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents (TECH1P.859)
Authors: Reed Brendan , Chopp Laura , Girtman Megan , Van Keulen Virginia , Johnson Aaron , Schrum Adam , Pease Larry
Published: January 2023

MHC multimers including tetramers and pentamers provide a powerful way to visualize antigen specific T cell responses in both experimental and clinical immune assays. The multimers can be acquired commercially, from the NIH, or assembled in the laboratory for measuring immune responses to commonly-studied or novel-hypothetical antigens. The multimer reagents can be expensive and time consuming to acquire and in many cases are uncontrolled reagents as robust positive control T cells used to assess the reagents functionally may not be available. This is particularly problematic when using MHC multimers to investigate less well-defined T cell responses, such as those characterized by low T cell frequencies or by specificity to hypothetical antigens relevant to cancer, infection, or autoimmune disease. These problems are magnified when studying irreplaceable clinical trial samples where having confidence that reagents are biochemically intact is critical. To address this problem, we have devised a versatile, flow cytometry-based, capture assay to probe the structural integrity of MHC multimer reagents. In principle, the idea is to immobilize on a bead antibody specific for conformational determinants expressed on a properly folded MHC molecule. The particle becomes labeled and can be visualized and quantified by flow cytometry when a fluorescently tagged tetramer is adsorbed to the particulate upon binding to the antibody.
Big data approach towards the characterization of normal peripheral immune cells with data from ImmPort (TECH1P.860)
Authors: Andorf Sandra , Bollyky Jennifer , Bhattacharya Sanchita , Shankar Ravi , Dunn Patrick , Thomson Elizabeth , Wiser Jeffrey , Butte Atul
Published: January 2023

To date, our understanding of a normal immune system is far behind that of other healthy organ systems. One reason for this is the lack of a widespread adoption of standardization in the lab techniques, especially flow cytometry. Here we demonstrate the use of the Immunology Database and Analysis Portal (immport.niaid.nih.gov) as a source of publicly available flow cytometry files from hundreds of participants in several clinical trials, to study immune cells from the blood of healthy individuals. To characterize well-defined cells in a normal immune system, we used an unbiased, gate-free method to compare data from different cytometers and antibody staining panels, based on the distribution of fluorescence intensity of the markers. In order to perform the meta-analyses, the marker information from each file was obtained in an automated fashion and their nomenclature was made consistent. Various transformation and normalization strategies were applied and evaluated to make the flow cytometry data comparable across studies. Initial promising results were observed for the cell-surface markers used to define T and B cells in general, and some specific subsets including regulatory T cells as well as plasmablasts. We found high concordance in normal individuals from two studies performed at different times measured within the same facility. The extension of this approach to studies of healthy individuals across facilities promises to give insights towards the normal immune system.
Proteomic profiling of activated immune cells using quantitative multiplex bead-based assays to identify secretome and cell signaling protein signatures (TECH1P.869)
Authors: Lie Wen-Rong , Mistry Jehangir
Published: January 2023

Multiplex bead-based assays using the Luminex® xMAP® technology allows the simultaneous quantitation of soluble mediators involved in adaptive immune responses. Here we report on the quantitative profiling of the immune secretome and cell signaling target proteins to uncover the characteristics of a CD40L/IFN-γ-activated mature dendritic cell (DC)-based vaccine and the characteristics of T-cell responses generated upon vaccination. Over 200 secreted proteins were evaluated using the Human MILLIPLEX® MAP magnetic bead panels (Cytokine/Chemokine Panels 1-3, High Sensitivity T Cell Panel, CD8 Panel, Th17 Panel, Soluble Cytokine Receptor Panel, Angiogenesis/Growth Factor Panel , MMP & TIMP Panels, IGFBP Panel, Sepsis Panels, Cancer Panels 1-4, CVD Panels 1-3, and others). Additionally, the activation status of a DC-vaccine was profiled using MILLIPLEX® MAP intracellular MAPK/SAPK Pathway multiplex panel. Representative heat maps indicated differences in secreted protein concentrations, biomarker signatures and phosphorylation status in GM-CSF/IL-4-derived immature DCs and CD40L/IFN-γ-activated mature DCs. Furthermore, vaccine-induced antigen-specific CD8+ T cells presented 2 distinct phenotypes based on secretome profiling, and these profiles correlated with clinical outcomes. Consequently, these data demonstrate the utility of MILLIPLEX® panels using the Luminex® xMAP® technology for the characterization of adaptive immune responses in vaccine/immunotherapy setting.
Combinatorial approach to enumerate immunophenotypes in flow cytometry panels (TECH1P.872)
Authors: Bhattacharya Sanchita , Pouliot Yannick , Dunn Patrick , Thomson Elizabeth , Wiser Jeffrey , Butte Atul
Published: January 2023

The identification of cell populations from flow cytometry has rarely been mapped in a multi-dimensional space. Instead, the focus is typically limited to a small number of pre-specified cell types already described in the literature. We propose to re-conceptualize the flow cytometry panel as a discovery tool to disentangle complex flow data into the simplest, most clinically relevant cell types. We present a combinatorial method to enumerate all possible immunophenotypes within a flow panel from a clinical trial study. We applied this approach to 11,000 raw flow cytometry files from a multi-panel flow experiment in a ragweed allergy clinical trial (ImmPort:SDY1), available from the publicly accessible, NIAID-funded Immunology Database and Analysis portal (ImmPort; immport.niaid.nih.gov). Files were processed through an automated pipeline that applied ImmPort’s FLOCK autogating tool to segregate cell populations based on the expression of individual surface markers. This yielded roughly 59,000 unique cell populations. Unsupervised hierarchical clustering revealed cell populations that were highly correlated with treatment conditions, based on the Pearson similarity measure. From these results we identified a subset of monocytes with positive expression for CD23 that were correlated with clinical outcome. We believe this approach can help identify the minimal set of markers needed to characterize functionally distinct cell populations associated with clinical outcome.
Optimized antibody labeling efficiency through continuous centrifugal diafiltration (TECH1P.838)
Authors: Cappione Amedeo , Mabuchi Masaharu , Briggs David , Nadler Timothy
Published: January 2023

Immuno-detection encompasses a range of analytical methodologies. These assays, which quantify and/or localize expression of protein(s) in complex samples, are reliant upon tagged target-specific antibodies. While a range of labeling kits is available, the workflow is hampered by multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. Also, due to differences between labeling targets (and dyes), prior determination of labeling reaction parameters must occur. We present an alternative method for small-scale protein labeling based upon continuous flow diafiltration. By exploiting the rapid kinetics of labeling reactions, continuous diafiltration minimizes reaction time and exposure to excess dye, guaranteeing maximal target labeling and thus eliminating the need for up-front optimization of reaction conditions. All phases of the conjugation workflow can be performed in a single centrifugal device. When compared to other buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting, recovery, integrity). Originally designed for resin-based affinity purification, the device also provides a platform for initial antibody purification or albumin carrier removal. Overall, the method offers a simplified workflow with reduced processing time and fewer hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.
Impact of mammalian target of rapamycin inhibition by everolimus on peripheral blood αβ and γδ T lymphocytes in renal transplant recipients (TRAN1P.875)
Authors: Gonzales Josephine , Dutt Shelley , Steinberg Steve , McKay Dianne , Jameson Julie
Published: January 2023

The kidney is an essential organ necessary for the human body to discharge harmful toxins and is imperative for filtering blood. According to the Centers for Disease Control and Prevention, kidney disease is the eighth leading cause of death in the United States. Diabetes and hypertension are two major factors that influence kidney function, which can consequently lead to end-stage renal disease. The primary method of treatment for end-stage renal disease is kidney transplantation, which requires a regimen of immunosuppressant drugs to prevent allograft rejection. Previously, our lab has shown that administration of the immunosuppressant drug, sirolimus a mammalian target of rapamycin inhibitor, also known as mTOR, impairs skin homeostasis and wound repair. To further understand the mechanism behind this adverse effect of mTOR suppression, we examined renal transplant recipients who were administered the sirolimus derivative everolimus. The number and activation of αβ and γδ T cells in the blood of patients administered everolimus were compared to a control group of patients not administered everolimus. Several of these T cell populations, including Vγ9Vδ2 T cells, have been previously displayed to home to sites of tissue damage to improve wound repair. This data explores which T lymphocyte populations require mTOR to maximize function in transplant recipients with implications in tissue repair and graft rejection.
A mutation in Transportin3 (Tnpo3) leads to generation of an individually distinct tumor-specific Kd-restricted epitope in the Meth A fibrosarcoma (TUM2P.899)
Authors: Duan Fei , Duitama Jorge , Al Seesi Sahar , Blanchard Tatiana , McMahon David , Sidney John , Sette Alessandro , Mandoiu Ion , Srivastava Pramod
Published: January 2023

The mutational repertoire of cancers creates the epitopes that make cancers immunogenic. Here, through a comprehensive genomic, bioinformatic and immunological analyses, we uncover over a hundred new neo-epitopes in the Meth A fibrosarcoma of BALB/c mice. Interestingly, the predicted affinities of neo-epitopes for MHC I have no bearing on protective anti-tumor immunogenicity; instead, the numerical difference of such affinities between the mutated and un-mutated sequences, named the Differential Agretopic Index (DAI), is a significant although imperfect predictor. The mutated Transportin 3 (Tnpo3)-derived epitope, the highest ranking (by DAI) epitope of Meth A is naturally presented by Meth A cells. Immunization with whole Meth A cells induces mutated Tnpo3 epitope-specific CD8+ T-cell responses. Immunization with mutated Tnpo3 peptide elicits CD8+ T-cell responses that recognize Meth A cells ex vivo, as well as significant protection from a tumor challenge. This tumor immunity is further enhanced by combination of immunization with mutant Tnpo3 with a Toll-like receptor 9 (TLR9) ligand or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody.
Targeting gammadelta regulatory T cell recruitment for breast cancer immunotherapy (TUM2P.904)
Authors: Peng Guangyong , Ye Jian , Ma Chunling , Hsueh Eddy , Toth Karoly , Varvares Mark , Hoft Daniel
Published: January 2023

Immunosuppressive microenvironments induced by regulatory T cells (Treg) present a major barrier for successful anti-tumor immunotherapy. Understanding the mechanisms for the accumulation of different subtypes of Treg cells in the immunosuppressive tumor microenvironment is essential to improving cancer treatment. Enriched gammadelta1 T cell populations in tumor-infiltrating lymphocytes (TILs) suppress T cell responses and dendritic cell maturation in breast cancer, where their presence is correlated negatively with clinical outcomes. However, mechanism(s) that explain the increase in this class of gammadelta Treg cells in breast cancer patients have yet to be elucidated. In this study, we showed that IP-10 secreted by breast cancer cells attracted gammadelta Treg cells. Using neutralizing antibodies against chemokines secreted by breast cancer cells, we found that IP-10 was the only functional chemokine that causes gammadelta Treg cells to migrate toward breast cancer cells. In a humanized NSG mouse model, human breast cancer cells attracted gammadelta Treg cells as revealed by a live cell imaging system. IP-10 neutralization in vivo inhibited migration and trafficking of gammadelta Treg cells into breast tumor sites, enhancing tumor immunity mediated by tumor-specific T cells. Together, our studies show how gammadelta Treg cells accumulate in breast tumors, providing a rationale for their immunological targeting to relieve immunosuppression in the tumor microenvironment.
Investigating the mechanism underlying Ly-6A, a highly expressed protein on activated and transformed lymphocytes, induced growth inhibition and cell death in transformed T-cell lines (TUM2P.879)
Authors: Lang Melissa , Jenkins Sultan , Balzano Philip , Bamezai Anil
Published: January 2023

The Ly-6 family of glycosylphosphatidylinisotol proteins is excellent differentiation markers of immune cells during their development and they regulate signaling responses. Specifically, Ly-6A is up-regulated on a variety of transformed cells therefore making it a potential biomarker for cancer detection and therapy. Previous work in our laboratory revealed that a transformed CD4+ T lymphocyte cell line shows growth inhibition and apoptotic cell death when Ly-6A proteins are engaged. We have investigated whether this mechanism involves p53 protein, which is known to regulate the cell cycle, trigger growth inhibition and apoptotic cell death in several normal cell types. In cancer cells, p53 is often mutated, resulting in uncontrollable cell growth. The cell cycle is regulated by several proteins known as cyclin dependent kinases that bind cyclin proteins and usher the cell past checkpoints in the cell cycle. Therefore, we have examined the expression of relevant cell cycle and apoptotic proteins. We find that while growth inhibition and cell death in the T cell line was p53 independent, the cell cycle inhibitor gene p27 becomes significantly up-regulated. Furthermore, we extended this study beyond the analysis of one Ly-6A antibody and one T-cell line. We find that the responses were tied primarily to the extent of cross-linking with anti-Ly-6A antibody. Other transformed T-cell lines also exhibited growth inhibition and cellular death when the Ly-6A protein was engaged.
TIGIT mediated T cell exhaustion in cancer is dependent on TIGIT/CD226 interaction (TUM2P.907)
Authors: Comps-Agrar Laetitia , Johnston Robert , Hackney Jason , Yu Xin , Yang Yagai , Huseni Mahrukh , Eaton Dan , Grogan Jane
Published: January 2023

The generation of blocking antibodies against co-inhibitory receptors involved in T cell exhaustion is a recent key strategy for cancer immunotherapy. We have shown that co-blockade of TIGIT and PD-L1 results in tumor rejection by restoring the function of exhausted tumor-infiltrating CD8+ T cells. We were then interesting in deciphering the molecular mechanism of action of this anti-TIGIT blocking antibody. We hypothesized that TIGIT might inhibit a complementary co-activating receptor. Interestingly, TIGIT expression on tumor infiltrating CD8+ T cell was correlated with CD226, which binds a common receptor PVR. To better understand the relationship between these co-receptors, we utilized Time-resolved Fluorescence Resonance Energy (TR-FRET), a technology that allows for monitoring of cell surface protein interactions in real time. We showed that CD226 forms homodimers, and that co-expression of CD226 with TIGIT results in 1) the attenuation of CD226 self-association and 2) the formation of a TIGIT/CD226 complex in cis. Strikingly, anti-TIGIT antibody causes a significant decrease of the TIGIT/CD226 interaction. These observations indicate that the interaction of CD226 and TIGIT may play a central role in the antitumor effects of anti-TIGIT. Indeed, anti-CD226 antibodies blocked the ability of anti-PD-L1/anti-TIGIT to inhibit tumor growth in mice. Altogether, these results depict a close relationship between TIGIT and CD226 in CD8+ mediated T cell exhaustion.
Control of in situ pathogenic effector T cell activities by PD-1-PDL1 interaction in the lung during respiratory syncytial virus infection (VIR2P.1019)
Authors: Yao Shuyu , Jiang Li , Du Jianguang , Zhou Baohua , Braciale Thomas , Sun Jie
Published: January 2023

RSV infection is the most common cause of lower respiratory diseases in infants and young children. It is believed that over-exuberant inflammatory responses are mainly responsible for the disease development. Here, we seek to define host factors that can dampen over-reactive pulmonary inflammation and injury during RSV infection. We have identified that the co-inhibitory receptor PD-1 is up-regulated on pulmonary effector T cells in RSV infected mice and infants. Importantly, acute blockade of PD-1 ligand (PD-L1) function in vivo resulted in the development of excessive pulmonary inflammation and severe host diseases. Using the in vivo and in vitro cytokine staining assays, we found that PD-L1 blockade specifically enhanced the in situ inflammatory activities (i.e. release of inflammatory cytokines) of effector T cells in the lung. Interestingly, we showed that PD-L1 expression is mainly restricted to the monocyte derived dendritic cell (Mo-DC) population in the lung, and that an axis, involving with type I and II interferons (IFN), STAT1 and IRF1, is critical for the induction of PD-L1 expression on Mo-DCs. Collectively, these data suggest that IFN-dependent PD-L1 expression on respiratory DCs is critical to inhibit the pathogenic activities of tissue effector T cells, thereby suppressing the development of excessive pulmonary inflammation and lung injury during RSV infection. Our study also opens the door for novel strategies to treat sever RSV infection.
Impaired CD8+ T cell responses to influenza infection in neonatal mice are due to the immature T cell receptor repertoire (VIR2P.1020)
Authors: Carey Alison , Gracias Donald , Thayer Jillian , Hope Jennifer , Mueller Yvonne , Katsikis Peter
Published: January 2023

To understand the role of virus antigen-specific CD8+ T cell responses in neonates during respiratory viral infection, we developed a model where 3-day old C57Bl/6 mice were infected intranasally with influenza type A virus strain A PR/8/34 (H1N1). When these neonatal mice are infected with influenza virus they mount a greatly reduced and delayed lung NP(366-374)-specific primary CD8+ T cell response. To investigate what was responsible for the reduced response, we performed adoptive transfers of TCR-transgenic OVA(257-264)-specific (OT-I) CD8+ T cells into hosts infected with recombinant influenza virus WSN-OVA, which expresses the OVA(257-264) peptide. These studies revealed that naïve adult OT-I cells expand equally well in neonatal and adult hosts, which excludes the neonatal environment as an inhibitor of the CD8+ T cell response. When naïve neonatal OT-I cells were transferred into neonatal and adult hosts, these neonatal OT-I cells expanded equally as well as adult OT-I cells, excluding an intrinsic defect in the neonatal CD8+ T cells. These studies demonstrate that the TCR repertoire of neonates is responsible for reduced CD8+ T cell immunity in neonates, not a suppressive environment or intrinsic signaling defects. Finally, despite impaired primary response, animals primed as neonates exhibited normal secondary responses to influenza virus. These findings raise important issues that need to be taken into consideration for vaccination of neonates.
T cell promiscuity between susceptible MHC alleles potentiates autoimmune diabetes (HUM3P.259)
Authors: Chow I-Ting , Gates Theresa , McGinty John , Mai Duy , Papadopoulos George , Torres-Chinn Nadia , James Eddie , Greenbaum Carla , Nepom Gerald , Kwok William
Published: January 2023

Major histocompatibility complex (MHC) genes, also known as human leukocyte antigen (HLA) genes in humans, are the prevailing risk factors in multiple autoimmune diseases, including multiple sclerosis (MS), autoimmune diabetes (type 1 diabetes or T1D), and rheumatoid arthritis (RA), yet MHC-linked susceptibility is not fully understood. In T1D, HLA-DQ8trans (DQ8trans), a trans-complementary molecule encoded by DQA1*0501 (HLA-DQ2α) and DQB1*0302 (HLA-DQ8β), confers exceptionally high risk. In contrast to the notion that DQ8trans expands a unique pathogenic T cell repertoire due to its trans-complementary nature, here we report a high degree of epitope overlap between DQ8trans and another high-risk allele, HLA-DQ8 (DQ8; DQA1*0301/DQB1*0302).T cells specific for shared islet-epitopes are cross-reactive to both molecules. Cross-reactivity is driven mainly by the shared β-chain and is observed ex vivo in polyclonal T cell populations in T1D patients. Stronger disease-associated responses to cross-reactive epitopes are elicited by DQ8trans, as the HLA-DQ2 α-chain enhances peptide-MHC-TCR interaction.These results suggest that a functional epistatic interaction between DQ8 and DQ8trans promotes the pathogenesis of T1D and provide an important general mechanism whereby disease risk may be altered primarily byT cell promiscuity among susceptible HLA haplotypes.
Intracisternally localized lupus serum IgG induces meningitis (HUM3P.245)
Authors: Deng Guo-Min , Sun Lingyun , Liao Xiang , Xie Chang-hao
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high autoantibody levels and multiorgan tissue damage, including brain. Headache in SLE patients is frequent clinical manifestations and its etiology and pathogenesis remains unclear. We hypothesized that lupus serum containing high levels of IgG induces meningitis. Information from the analyzed 150 lupus patients demonstrates higher levels of IgG in CSF from lupus patients with headache than that without headache. There is meningitis in brain of MRL/lpr mice. We have intracisternally injected mice and rats with serum from patients with SLE, health individuals, lupus prone mice and normal mice , and found meningitis induced only by serum from lupus patients and lupus prone mice. Meningitis induced by lupus serum appeared 12 h and peaked 3 days after injection. Deleted the monocyte/ macrophage abrogated meningitis but lack of lymphocyte did not affect the development of meningitis induced by lupus serum. Specific interactions with vascular selectin expression and depletion of complement system led to a significant amelioration of meningitis. Depletion of IgG from lupus serum reduced meningitis suggests that IgG is important contributor in lupus serum-induced meningitis. Intracisternally lupus serum may trigger development of hippocampus neuron apoptosis. This study proves that lupus serum IgG induces meningitis, which is important mechanism of headache of lupus patients.
Biomimetic lipid nanoparticles as treatment for bacterial sepsis (HUM4P.275)
Authors: Foit Linda , Thaxton C.
Published: January 2023

Sepsis describes a spectrum of potentially fatal medical conditions caused by the inflammatory response of the human body to an infectious insult. Due to significant morbidity and high mortality, new treatments are urgently needed specifically targeting the cause of sepsis. Gram-negative sepsis is elicited by lipopolysaccharides (LPS). Recognition of these bacterial cell wall components by TLR4 and accessory proteins leads to activation of complement and coagulation systems and the release of pro-inflammatory agents. Human high-density lipoproteins (hHDL) can bind LPS and reduce its levels in the blood. However, remodeling and depletion of HDL during sepsis impairs HDLs’ immune-protective capacity. Thus, we sought to design nanoparticles (NP) that mimic hHDL in shape and surface chemistry, yet exceeded its LPS-binding properties and boosted LPS detoxification. Convincingly, our data show that HDL-like NPs effectively reduce the LPS-induced production of cytokines in human monocytic cell lines and in whole human blood. Our NPs, but not hHDL, exert their immune-protective function at low nM concentrations when stimulated with LPS concentrations above those levels reported in sepsis patients. Further, our NPs are non-immunogenic and non-toxic to target cells. As such, our NPs have the great potential for an LPS antidote that rapidly detoxifies LPS in the blood, drastically inhibits the release of inflammatory mediators, and constitutes a highly effective treatment for sepsis.
Dexamethasone suppresses of IL-33-mediated mast cell activation in vitro and in vivo (HYP4P.314)
Authors: Paranjape Anuya , Chernushevich Oksana , Ryan John
Published: January 2023

Dexamethasone, a synthetic member of the glucocorticoid class of steroid drugs is known for its immunosuppressant and anti-inflammatory effects. Mast cells, critical effectors of allergic disease, can be activated by IL-33, a pro-inflammatory member of the IL-1 family. We found that Dexamethasone potently suppresses IL-33-stimulated cytokine secretion by mouse mast cells. We similarly found that Dexamethasone treatment in vivo reduced IL-33-mediated cytokine production and neutrophil infiltration in the murine peritoneum. We compared the activation status of signaling proteins during IL-33 stimulation, and found that Dexamethasone pretreatment significantly reduced Erk phosphorylation. However, we also observed that Dexamethasone-mediated suppression occurred when it was added simultaneously with IL-33, suggesting that inhibition occurrs even prior to Erk blockade. The effect of Dexamethasone on IL-33 stimulated NFkB-mediated gene transcription is currently under investigation. Our data show that Dexamethasone, a well-established therapy for inflammatory disease, can suppress IL-33-mediated mast cell activation, and may therefore be effective for treating diseases now being attributed to IL-33 effects.
CXCR2 is a critical mediator of Brucella-induced articular inflammation (INC4P.335)
Authors: Skyberg Jerod , Lacey Carolyn , Keleher Lauren , Brown Charles
Published: January 2023

Osteoarticular complications are the most common focal manifestation of brucellosis in humans. Little is known about the pathology of osteoarticular brucellosis due to the lack of relevant models. However, we recently reported that ablation of IFN-γ in mice results in articular inflammation during infection with Brucella melitensis. Here, we show that adaptive immune cells are dispensable for Brucella-induced articular inflammation. Wild-type, µMT-/-, and Rag1-/- mice all developed articular inflammation at a similar rate and severity when infected with B. melitensis and depleted of IFN-γ. In addition, minimal levels of cytokines associated with T cell responses were detected in arthritic joints. However, we found increased numbers of neutrophils and macrophages in joints from mice with Brucella-induced arthritis. In addition, the concentration of CCR2 and CXCR2 ligands, was strikingly enhanced in Brucella-infected joints. Interestingly, CXCR2-/-, but not CCR2-/- mice, displayed markedly lower joint inflammation relative to wild-type mice when infected with B. melitensis and depleted of IFN-γ. Collectively, these results show that chemokines, such as CXCR2 ligands, are important mediators of Brucella-induced articular inflammation while adaptive immune cells are not required. Therefore, the targeting of CXCR2 and/or other chemokine receptors may have potential as a complementary antibiotic therapy for the resolution of Brucella-induced arthritis.