bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel imaging cytometry method for quantitative cell viability assays (65.13)
Authors: Lai Ning , Lyettefi Emily , Pirani Alnoor , Smith Tim , Chan Leo , Lin Bo
Published: January 2023

Accurate measurement of viability is essential for many cell based research. The traditional cell viability analysis instrumentation includes microscopy, flow cytometry and plate reader. However, these systems are often expensive to purchase and maintain. Also, the operational processes are often tedious or complicated. Here we present a novel Cellometer Vision system, which incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. This method utilizes both bright-field and fluorescence imaging of a disposable cell counting chamber to quickly provide concentration and viability measurements of cell populations. In this poster, we demonstrate the application of Cellometer Vision system for cell viability assays that test for different characteristics of cells death. Jurkat cells were treated with cytotoxic compounds and heat treatment. Cell viability was assessed by mitochondria function assay using JC-1 dye, apoptosis assay using Annexin-V, membrane integrity assay using trypan blue and propidium iodide, metabolic assay using calcium AM and alamrBlue, and cell proliferation assay. Differences in cell viabilities were observed from different assays for cells intermediate stages of cell death. Cellometer Vision provides a simple, rapid and cost-effective tool for cell viability measurements. This novel imaging cytometry method is demonstrated here to support viability assays that test different stages of the cell death process.
Quantitative analysis of cellular interactions using imaging flow cytometry (65.15)
Authors: Friend Sherree , Burbach Brandon , Jacobs Evan , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia , George Thaddeus
Published: January 2023

Direct cell to cell contact is critical for an effectively coordinated immune response to pathogens. Cells of the innate immune response are specialized to bind, process, and present antigen in the context of appropriate co-stimuli to direct specific responses from cells of the adaptive immune system. T cell dependent antibody responses require B-T cell interaction. Nibbling can directly transfer proteins from an infected cell to an APC for antigen processing and presentation to a lymphocyte. Interactions between virus-infected cells and antigen presenting cells can induce specific cell-cell fusion. Discrimination amongst these types of cellular interactions requires visualization, but these events are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. The ImageStream automatically and rapidly collects large numbers of high resolution cell images per sample and is thus ideally suited for robust statistical image-based analysis of cellular interaction. Multispectral images of each conjugate provides objective measurement of conjugate numbers and the location information needed to determine the type of interaction on a per cell basis. We will demonstrate three types of conjugate analysis using the ImageStream. 1. The measurement of a protein in the immune synapse between a T cell-APC. 2. The specific nibbling that occurs between an APC and a virally infected cell. 3. Cell fusion between an APC and HIV-infected cell.
A novel multiplexed immunoassay using salivary antibodies to detect human exposure to waterborne pathogens in drinking and recreational water (65.19)
Authors: Simmons Kaneatra , Curioso Clarissa , Eason Tarsha , Griffin Shannon , Fout G. , Wade Timothy , Egorov Andrey , Augustine Swinburne
Published: January 2023

Water is our most important resource and the world’s supply is becoming alarmingly limited with rising concern about the high levels of microbial contamination. We are developing a novel strategy to assess human exposure to microbial contaminants in drinking and recreational water using a multiplexed, high-throughput immunoassay applying the Luminex xMAP platform. In this assay, antigens from waterborne pathogens are coupled to spectrally unique microspheres that are exposed to either pathogen-specific antibodies or human saliva. Our results indicate that multiplexed microsphere sets coated with antigens to Helicobacter pylori, Salmonella typhimurium, Escherichia coli O157:H7, Campylobacter jejuni, Toxoplasma gondii, Cryptosporidium parvum, and Giardia lamblia are able to capture both pathogen-specific antibodies and human salivary antibodies at the lowest-limit of detection of the assay. Additionally, we implemented a Design of Experiment (DoE) approach to evaluate the impact of antigen and antibody concentration on the detection level of H. pylori. From this work, we were able to generate a mathematical model describing the detection level as a function of critical process parameters to determine optimal conditions for protein coupling to Luminex beads. This immunoassay will allow for a sensitive, rapid, and cost-effective analysis of human exposure to waterborne pathogens with the potential to reduce the cost of epidemiological surveillance of waterborne infections.
Ethanol-dispersed polymer nanofiber net for highly-selective capture of lymph node CD4+ T cells (65.21)
Authors: Kim Kwanghee , Jun Seung-Hyun , An Hyo Jin , Kim Byoung Chan , Sonn Chung Hee , Kim Tae-Jin , Lim Seon Ah , Kim Jungbae , Lee Kyung-Mi
Published: January 2023

The hydrophobic polymer NFs could be dispersed in the aqueous solution easily by simple ethanol treatment, which increases the loading of enzyme or antibody effectively. The degree of dispersion of nanofibers was somewhat proportional to the concentration of ethanol, the loading and activity of immobilized enzymes were proportional increased to the degree of dispersion of nanofibers. Next, the feasibility of anti CD4 monoclonal antibodies conjugated on ethanol dispersed nanofibers (anti CD4 mAbs/EtOH-NF) are developed as a useful vehicle for biomedical application such as specific cell capture and separation. The anti CD4 mAbs/EtOH-NF were examined for their ability to bind CD4+ T cells, bound to anti-CD4 mAbs, isolated from the mixture of lymphocytes. The results show that the anti CD4 mAbs/EtOH-NF successfully isolated CD4+ T cells from the mixture of lymphocytes with high specificity. Therefore, biocompatible of alcohol treated nanofibers provide an efficient tool for the cell separation process and further present the potential to be applied to other areas of biomedical application.
An HLA allele frequency-based approach to resolving allelic and genotypic ambiguity in HLA typing data and its implementation in ImmPort (65.22)
Authors: Guidry Paula , Marthandan Nishanth , Smith Thomas , Campbell John , Dunn Patrick , Erlich Henry , Karp David , Single Richard , Thomson Glenys , Wiser Jeffrey , Scheuermann Richard , Mack Steven
Published: January 2023

The classical human leukocyte antigen (HLA) loci are the most polymorphic loci in the human genome, with hundreds of new alleles reported yearly. HLA polymorphisms in exons 2 and 3 of class I loci and in exon 2 of class II loci distinguish the peptide binding regions of HLA proteins. Current HLA typing methodologies focus on identifying variant positions in these exons, and most do not detect allelic variation in other exons or non-coding regions of HLA genes. Because of the limitations in detecting polymorphic variants and the pace of new allele discovery, commonly used HLA typing methodologies cannot unambiguously identify all known HLA alleles or genotypes. Consequently, many HLA genotyping results are ambiguous in that both alleles of each HLA locus for a given subject may have multiple possible identities, and each subject may have multiple possible genotypes. Here, we describe an approach to allelic and genotypic ambiguity resolution that uses published HLA allele frequencies in 10 world regions to determine the most likely diploid alleles and genotype for each subject in an HLA typing study. This method has been implemented in an online tool at www.ImmPort.org, and is freely available for use by the immunology community. This ambiguity reduction tool will allow investigators studying autoimmune disease, adverse drug reactions, transplantation outcome and other HLA associated conditions to more accurately determine the contribution of specific HLA alleles to disease.
A novel assay for imaged-based analysis of leukocyte migration in a stable chemoattractant gradient (65.36)
Authors: Vu Elizabeth , Meyvantsson Ivar , Lamers Casey , Skoien Allyson , Echeverria Victoria , Hayes Steve
Published: January 2023

Current approaches to study chemotaxis are limited by gradient instability, low throughput, cumbersome manual manipulations, high cell number requirements, and inability to acquire high-content data. We present here a new microfluidics-based approach to study leukocyte chemotaxis. The device features strong miniaturization of the use of cells (<12,000 cells per data point). The microfluidic channels in the device are arrayed in 96 well format and can be operated by standard liquid handling robotics or hand pippettes. Cells migrate along a surface in the presence of a stable gradient of chemoattractant that is maintained for at least three hours. We show the ability to obtain consistent dose response curves to chemoattractants as well as chemotactic inhibitors with the device. Because cells are microscopically accessible, the information content is high. Not only can the number of cells migrated be assessed, but also migration distances of individual cells, and even scoring of cell activation and polarization. In addition, we have developed a smaller slide-sized device embedded within an enclosure that prevents evaporation. This feature allows live cell tracking which can deliver highly detailed information on cellular behavior during chemotaxis.
A bioanalytical method for detection of phosphorodiamidate morpholino oligomers in cells. (65.44)
Authors: Crumley Stacy , Mourich Dan , Iversen Patrick
Published: January 2023

Phosphorodiamidate morpholino oligomers (PMOs) are neutrally charged antisense agents that interfere with target gene expression by inhibition of translation or by altering splicing of pre-mRNA. PMOs have been shown to be highly specific and potent therapies after cellular uptake, yet a method to detect PMO inside the cell has not been developed. We offer in this report a novel method for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) which takes advantage of locked nucleic acid (LNA) oligonucleotides as probes. A biotinylated LNA probe complementary to an Influenza A specific PMO (IA-PMO) was used to specifically detect and quantify intracellular IA-PMO after in vitro or in vivo administration with or without Influenza A infection. While IA-PMO was detected in non-hematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, it was detected at higher levels in influenza infected hematopoietic lung cells as compared to uninfected cells. Coincident measurement of target knock down to intracellular IA-PMO concentration allowed for the calculation of an EC50. Furthermore, it was observed that an increase in IA-PMO uptake occurs in infected hematopoietic cells.
4-1BB agonist antibody treatment causes intra-tumoral accumulation of KLRG1+ CD8+ and CD4+ T-cells with a novel highly cytotoxic phenotype driven by the master-regulator Eomesodermin (66.17)
Authors: Curran Michael , Kim Myoung-Joo , Al-Shamkhani Aymen , Allison James
Published: January 2023

Antibodies which block the co-inhibitory receptor CTLA-4 or which activate the co-stimulatory receptor 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma. We find that combining these two antibodies in the context of a Flt3-ligand based B16 melanoma vaccine promoted high CD8+ and CD4+ effector to FoxP3+ Treg ratios in tumors, highly elevated TH1 type cytokine production, increased T-cell proliferation and synergistic levels of tumor rejection. A hallmark of 4-1BB agonist antibody treatment is the upregulation of the lectin KLRG1 on tumor infiltrating CD8+ and CD4+ T-cells. We find that these KLRG1+ T-cells in the tumor express high levels of multiple cytotoxic molecules, kill tumor cells better than their KLRG- counterparts and may be responsible for the increased anti-tumor cytotoxicity attributed to α4-1BB treatment. We found that formation of these cells is driven by high-level expression of the master-regulatory transcription factor Eomesodermin (Eomes) in both the CD8+ and CD4+ T-cell compartments. These Eomes+KLRG1+ cytotoxic CD4+ and CD8+ T-cells represent a novel phenotype distinct from any described in the literature (KLRG1expression is not IL-12/T-bet dependent, they are not exhausted, and the Eomes expression exceeds that of normal TH1). The origins and applications of these novel T-cells have broad implications for tumor immunotherapy as well as basic T-cell immunology.
Effect of BCNU on myeloid-derived suppressor cells in anti-tumor immune response (66.20)
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Nagarkatti Mitzi
Published: January 2023

We attempted to understand if 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) may prevent the generation of myeloid-derived suppressor cells (MDSCs) during tumor growth and modulate anti-tumor immunity. After intraperitoneal injection of EL4 tumor cells into mice, tumor cells grew rapidly in the peritoneal cavity, and metastasized into liver and lung. Mice died around day 14 after tumor challenge. MDSCs appeared at day 7 and massive MDSCs accumulated in spleen, liver and lung at day 11. In vitro assay indicated that MDSCs from tumor-bearing mice significantly inhibited T cell proliferation and anti-tumor immune response. After mice received BCNU injection (20mg/kg body weight) at day 5, however, tumor cell growth was completely inhibited and all of mice survived after BCNU treatment and tumor challenge. FACS analysis showed that MDSC accumulation was completely inhibited in tumor-challenged mice after BCNU injection, suggesting that BCNU may inhibit MDSC development and thus induce tumor rejection in mice. TUNNEL assay demonstrated that BCNU induced MDSC apoptosis. Interestingly, BCNU-cured mice rejected tumor after EL4 tumor cell re-challenge, suggesting that BCNU may not only inhibit MDSC production but also induce anti-tumor immune memory. The results indicate that BCNU may inhibit the production of MDSCs, increase anti-tumor immune response and thus facilitate tumor rejection.
Costimulation through 4-1BB/CD137 improves the expansion and function of CD8+ melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy (66.21)
Authors: Chacon Jessica , Wang Yijun , Wu Richard , Hwu Patrick , Radvanyi Laszlo
Published: January 2023

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a powerful therapy for Stage IV melanoma patients who have failed first and second line therapies. TIL for adoptive transfer are expanded from melanoma surgical specimens with IL-2 followed by a “rapid expansion protocol” (REP), where the cells are activated with anti-CD3 antibody, PBMC feeder cells and IL-2 for two weeks, generating a final infusion product. The REP yields billions of highly-differentiated cytolytic CD8+ T cells that lose costimulatory molecules CD28 and CD27, and are hyporesponsive to further proliferative signals. We found “post-REP” CD8+ TIL express an alternative costimulatory molecule of the TNF-R family, 4-1BB. Our study tests the effects of an agonistic anti-4-1BB antibody added at the time of initiating the REP. We found adding an agonistic anti-4-1BB antibody increased CD8+ T-cell recovery, antigen specificity, BCL-2 gene expression and increased the frequency of CD28+ cells in the recovered CD8+ post-REP TIL. Adding anti-4-1BB improved post-REP TIL survival capacity in the absence of cytokine, with TIL continuing to divide even without added IL-2. Our findings suggest adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.
Tumor-derived mesenchymal stem cells promoted tumor development through CCR2-dependent recruitment of macrophages (66.40)
Authors: Ren Guangwen , Zhao Xin , Zhang Liying , Yuan Zeng-rong , Zheng Betty , Shao Changshun , Shi Yufang
Published: January 2023

Mesenchymal stem cells (MSCs) residing in tumor stroma have been recognized to originate from normal tissues, such as bone marrow. Although they present as a major component of tumor stroma, the exact role they play in tumor progression and how they distinguish from normal tissue MSCs are largely unknown. In this report, we characterized the function of mesenchymal stem cells in promoting tumor development. Compared to BM-MSCs or tissue-MSCs, lymphoma-derived MSCs (L-MSCs) exhibited a strong tumor-promoting effect in a tumor transplantation model. While L-MSCs did not differ from BM-MSCs in their ability to suppress the adaptive immunity, they induced more abundant accumulation of CD11b+Gr-1+ myeloid suppressor cells and F4/80+ macrophages in tumors. Depletion of macrophages, but not myeloid suppressor cells, completely ablated the effect of L-MSCs, indicating that the tumor-promoting effect of L-MSCs was mediated via the recruitment of macrophages. Furthermore, chemokines CCL-2, CCL-7 and CCL-12, all of which utilize CCR2 as a receptor, were found over-expressed in L-MSCs. Correspondingly, the tumor-promoting effect of L-MSCs was largely abolished in CCR2-/- mice. Collectively, these data demonstrated that L-MSCs contribute to tumor development through CCR2-dependent recruitment of macrophages to tumor sites. Our findings thus established a mechanistic link between two major types of tumor stromal cells, providing critical implications for the future cancer therapy.
A novel subset of myeloid suppressor cells in pediatric cancer patients (66.9)
Authors: Zhang Hua , Galli Susana , Mackall Crystal
Published: January 2023

Human myeloid cells contain several phenotypically and functionally distinct subpopulations. Among these are myeloid derived suppressor cells (MDSC), which are identified as CD11b+Gr-1+ in mice, but are more heterogeneous in humans. A clear consensus does not exist regarding the phenotypic definition of human MDSC, but some have described them as CD33+CD11b+CD14- HLA DR- cells, within which granulocytic MDSC express CD15, whereas monocytic MDSC express CD66b. More studies are needed to characterize and delineate MDSC subsets in humans. Here we report a preliminary study of myeloid subsets found within a monocyte fraction elutriated from apheresis samples of pediatric cancer patients vs healthy controls. In 4 of 20 pediatric cancer patients studied, we observed accumulation of CD14-CD123-CD11cint or hi cells, which were not present in healthy donors. Further analysis of these abnormal myeloid populations revealed HLA DR+CD127+CD15+CD66b+CD124+ as well as CD68+CD11b+PDL1+, but CD33lo or -. Histologic examination showed these were immature neutrophils and eosinophils. Functional studies demonstrated diminished T cell proliferation in the presence of suboptimal concentrations of OKT3, compared to cultures with CD14+ monocytes and CD14-CD123+CD11c+ myeloid cells, which enhanced T cell proliferation. Studies are underway to further characterize function of this unique subset and to explore expression of suppressive mediators such as iNOS, arginase and IDO.
Plasmacytoid dendritic cells are potent mediators of mucosal B cell responses to rotavirus (67.1)
Authors: Deal Emily , Lahl Katharina , Narvaez Carlos , Butcher Eugene , Greenberg Harry
Published: January 2023

Humoral immunity to rotavirus, an intestinal pathogen, confers protection against re-infection. We utilize both the human in vitro and murine in vivo models of rotavirus infection to delineate the role of primary plasmacytoid dendritic cells (pDCs) in initiating B cell responses. Here, we demonstrate that primary human pDCs are necessary and sufficient for B cell activation by rotavirus. This requires very few pDCs, indicating the potency of the stimulus. Intact type I interferon (IFN) recognition by B cells is essential for activation. We extend these findings in vivo, demonstrating that murine pDCs and IFNα/β also mediate B cell activation following oral rotavirus infection. Furthermore, rotavirus-specific antibody responses are defective in mice lacking functional pDCs at the time of infection. These data demonstrate, for the first time, that B cell activation and virus-specific antibody secretion in response to mucosal viral infection occur as a direct result of pDC-derived type I IFN. Importantly, we observe increased viral shedding in pDC-deficient mice, indicating that pDC-instructed B cells impact viral clearance. We conclude that pDCs, through rotavirus recognition and subsequent type I IFN production, critically influence the course of rotavirus infection. Thus, mucosal pDCs can display powerful adjuvant properties to initiate humoral immunity. Our findings may extend to other mucosal pathogens, such as HIV or influenza.
Bi-specific MHC heterodimers for the characterization of cross-reactive T cells (67.13)
Authors: Shen Zu , Brehm Michael , Daniels Keith , Sigalov Alexander , Selin Liisa , Welsh Raymond , Stern Lawrence
Published: January 2023

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex withMHCproteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2Kb carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify crossreactive CD8[|#1#|] T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.
Preferential induction of protective T cell responses to Theiler’s virus in resistant (C57BL/6 x SJL)F1 mice (67.14)
Authors: Jin Young-Hee , Kang Hyun Seok , Mohindru Mani , Kim Byung S.
Published: January 2023

Infection of the CNS with Theiler's murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2(s)), but not in strains such as C57BL/6 (H-2(b)). In addition, it has been shown that (SJL/J x C57BL/6)F1 mice, which carry both resistant and susceptible MHC haplotypes (H-2(b/s)), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2(b)-restricted CD8(+) T cell responses vs. the H-2(s)-restricted CD8(+) T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice compared to that in susceptible SJL mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulating viral replication and inducing protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.
TGF-beta 1 as outcome marker in patients with influenza A H1N1 (67.18)
Authors: Martinez-Mejia Corazon , Rendon-Ramirez Erik , Rendon Adrian , Salinas.Carmona Mario , Rosas-Taraco Adrian
Published: January 2023

Cytokines and interferons are important molecules involved in influenza control. However, the relation between those cytokines and the outcome of those patients is not clear. The aim of this work was to analyze interferons, proinflammatory and anti-inflammatory cytokines in patients with influenza A H1N1 and other respiratory diseases. Methodology: Healthy controls (n=8), patients with influenza A H1N1 confirmed by PCR (n=14), and patients with other respiratory diseases (n=22) were enrolled. Blood samples were collected from each individual and sera were obtained for cytokines analysis. A panel of 14 cytokines was analyzed: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-15, IL-17, TNFa, IFNa and IFNg by Luminex; and IL-29 and TGF-b1 by ELISA. Results: IL-6, IL-8, and IL-10 were higher (P<0.05) in sera of patients with influenza A H1N1 and other respiratory diseases compared with control group at admission day. These cytokines were also higher in A H1N1 patients who deceased as compared to those that recovered (P<0.05). TGFb1 was lower in all patient groups compared with control (P<0.05). Interestingly, TGF-b1 increased in A H1N1 patients that recovered. This phenomenon was not observed in other respiratory diseases. No differences were found in the other analyzed cytokines. In conclusion, the anti-inflammatory response, under control of IL-10, is coadjuvated by TGF-b1 which seems important for A H1N1 patient survival.
Caveolin-1 suppresses HIV-1 replication through NF-kappa B. (67.21)
Authors: Simmons Glenn , Hildreth James
Published: January 2023

Caveolin-1 is an integral membrane protein expressed in macrophages. Caveolins are primarily responsible for the formation of intracellular vesicles known as caveolae, which are involved in protein trafficking and cholesterol homeostasis. It has been demonstrated that overexpression of caveolin-1 in cells suppresses HIV-1 protein expression. We have shown that HIV-1 transcription was down-modulated by caveolin-1 expression and the reduction of endogenous caveolin-1 showed an enhancement of HIV-1 replication in cells. Caveolin has been previously demonstrated to blunt inflammatory response via Nf-kappa B. Therefore we tested the role of Nf-kappa B in caveolin-1-mediate HIV-1 suppression. Using HIV-1 reporters with mutations in the Nf-kappa B binding site, we observed a loss of caveolin-mediated suppression on the HIV-1 promoter. From the work presented in this study we concluded that caveolin-1 requires Nf-kappa B in order to suppress HIV-1.
Role of the mannose receptor (CD206) in ricin toxin pathogenesis (99.16)
Authors: Gage Emily , Hernandez Maria , O'Hara Joanne , McCarthy Elizabeth , Mantis Nicholas
Published: January 2023

The entry of ricin toxin into macrophages and other cell types is proposed to occur through two distinct pathways. The first involves the toxin’s B subunit (RTB), an R-type lectin that binds to cell-surface glycolipids and glycoproteins bearing terminal β1,4-linked galactose residues. The second involves recognition of ricin’s mannose side chains by the mannose receptor (MR; CD206), a C-type lectin that functions in both scavenging of serum glycoproteins and uptake of microbial pathogens. While much is known about the role of RTB in ricin uptake, much less is understood about the contribution of the MR in ricin pathogenesis. In this study, we demonstrate using the human monocyte cell line, THP-1 that the MR does indeed mediate toxin uptake and contribute to ricin-induced cell killing in vitro. To determine the role of the MR in ricin toxicity in vivo, MR knock-out mice (MR-/-) or age-matched wild type controls were challenged with 2.5 or 5 lethal dose 50s (LD50s) of ricin, administered by intraperitoneal injection. We found that mice lacking the MR were more susceptible to toxin-induced death than control animals, suggesting that the MR promotes toxin clearance rather than productive internalization, as has been proposed. These findings have implications for the development of biodefense therapeutics aimed at rapidly ridding the toxin from circulation.
Identifcation of new antigen targets for leprosy serodiagnosis (99.24)
Authors: Estrada-Garcia Iris , Talavera-Paulin Moises , Serafin-Lopez Jeanet , Estrada-Parra Sergio
Published: January 2023

Mycobacterium leprae , a non-cultivable bacteria, is the causative agent of leprosy. Despite huge effort from WHO leprosy remains a public health problems in some parts of the world. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. In this work, M. habana a cultivable non-pathogenic mycobacterium and candidate vaccine for leprosy, was used to obtain soluble antigenic extracts (MHSE). By Western blot (WB) analysis MHSE proteins were reacted with sera form lepromatous leprosy patients (LL), active tuberculosis patients (TB) and healthy donors (H) from an endemic leprosy area. LL sera diluted 1:2,000 showed reactivity with a 28-30 KDa doublet antigen in MHSE. No reactivity with these bands was observed at 1:2,000 or lower sera dilutions of TB or H donors. After performing two dimension PAGE and WB analysis with the same sera, the doublet band was shown to have several spots. Further characterization was done using Tandem Mass Spectrometry (LC/ESI-MS/MS) and two proteins were identified: enoyl- coenzyme A hydratase (lipid metabolism) and antigen 85B (Ag85B, mycolyltransferase). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of the infectious form of leprosy.
Induction of a polyreactive IgM response by bacterial infection (99.26)
Authors: Jones Derek , Winslow Gary
Published: January 2023

Infection of mice with the bacterium Ehrlichia muris induces a protective T cell-independent (TI) IgM response. A unique property of the TI IgM produced during early infection is that it is polyreactive; that is, it binds a number of unrelated foreign and self antigens, including influenza virus, Borrelia burgdorferi, ss- and dsDNA, insulin, and thyroglobulin. We hypothesized that polyreactivity results from cross-reactivity of antigen-specific IgM, or that antibodies of unrelated specificity were generated via polyclonal B cell activation. To address these hypotheses, we generated B cell hybridomas from infected spleen cells and determined whether they produced pathogen-specific IgM. Eighty percent of the IgM-producing hybridomas were ehrlichia-specific; 10% of the IgM bound an immunodominant ehrlichial antigen, OMP-19, and the remainder bound yet unidentified ehrlichial antigen(s). Polyreactivity was exclusively associated with OMP-19-specific IgM, however, indicating that the properties of the antigen (e.g., spatial distribution on the bacterium) influence the selection of polyreactive IgM. These data indicate that polyreactive IgM is generated by antigen-specific B cells. Our data support a model whereby polyreactivity among antigen-specific IgM allows for the heteroligation (Mouquet et al., 2010) of OMP-19 and additional ehrlichial antigens, and serves to increase the relative affinity of IgM.
Suppression of lethal toxicity in Mycoplasma arthritidis-infected mice by blocking PAI-1 is mediated by TLR4-dependent iTreg function (99.28)
Authors: Mu Hong , Cole Barry , Jiang Hua , Nourian Maziar
Published: January 2023

Mycoplasma arthritidis causes arthritis and toxic shock syndrome in natural infections of rodents. Here we show that the level of type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), are markedly increased in toxic shock-susceptible mouse strains injected with live M. arthritidis. We also showed that M. arthritidis-infected mice exhibited severe toxic shock syndrome with elevated levels of pro-inflammatory cytokines TNFα, IL-1β, but lower levels of TGFβ and IL-10 production. Repeated injections of animals with anti-PAI-1 blocking antibody effectively prevented mice from lethal toxicity and suppressed production of inflammatory cytokines. Importantly, anti-PAI-1 treatment resulted in increased levels of TGFβ and IL-10 expression and production in vivo. Parallel results demonstrated that the expression of Foxp3, GITR and CTLA-4, the signature markers of inducible regulatory T cells (iTreg) in splenic CD4+ cells, were significantly increased in anti-PAI-1-injected mice with M. arthritidis infection. In addition, we also demonstrated that presence of functional TLR4 is indispensable for the induction and function of iTreg in our model system. Thus these results reveal a novel role of PAI-1 and its association with TLR4 in lethal toxicity caused by M. arthritidis, and suggest it could be a potential therapeutic target for the treatment of inflammatory diseases.
Th17 cells play a central role in experimental Candida albican keratitis in mice (99.32)
Authors: Wang Yiqiang , Zhang Hongbo , Li Hongxia , Zou Yanli , Song Wengang , Jia Changkai , Li Siyuan , Xu Liang
Published: January 2023

Our previous studies showed that experimental Candida albican keratitis (CaK) induced an adaptive immune response. Here we explored the role of T lymphocytes in the progress of CaK. Balb/c mice and nude mice on Balb/c background were used for CaK model. CaK was induced by intrastromal injection of Candida albicans spores into the corneas. Keratitis was measured by scoring the lesions. One day before CaK induction, unsorted splenocytes or purified T cells from either Balb/c, C57BL/6 or OVA-TCR Tg DO11.10 mice were adoptively transferred into nude mice, or various T cell subtypes were depleted in Balb/c mice using specific antibodies. We found that, upon challenge with same amount of fungal spores, Balb/c mice developed typical CaK, but nude mice did not show any sign of CaK. Reconstitution of nude mice with either splenocytes or purified T cells built sensitivity of nude mice to CaK induction in a way dependent of numbers but not of MHC- nor TCR-restriction of transferred cells. Depleting Th17 cells with anti-IL17 or anti-IL23 antibodies completely abrogated the capability of Balb/c mice to develop CaK. But depleting Treg cells with anti-CD25 or γδT cells with anti-γδ antibody did not show any effect. Th17 depleted mice did not set up immunological memory either after primary CaK. We conclude that a minimum of Th17 cells are essential in the initiation of fungal keratitis, and T cells play a dual role in the pathogenesis and prognosis of fungal keratitis.
Parallel expansion of natural Foxp3+ regulatory T cells and effector CD4+ T cells recognizing the same Mycobacterium tuberculosis epitope (99.4)
Authors: Shafiani Shahin , Kaur Sukhraj , Siddiqui Imran , Dinh Crystal , Urdahl Kevin
Published: January 2023

We have previously shown that regulatory T (T reg) cells restrict immunity during early Mycobacterium tuberculosis (Mtb) infection. Our experiments using T cell receptor (TCR) transgenic T reg cells suggest that T reg cells responding during Mtb infection are pathogen-specific. Nevertheless, the epitopes recognized by T reg cells have not been defined in tuberculosis or other infections. Using MHC class II tetramers containing an immunodominant Mtb epitope (specific for ESAT-64-17:I-Ab), we found that a fraction of Foxp3+ T reg cells in the pulmonary lymph node (pLN) of B6 mice during early Mtb infection were specific for ESAT-64-17, the same epitope recognized by effector CD4+ T cells. These T reg cells peaked in number at day 21 when their abundance ranged between 7-20% of all tetramer-binding cells, and disappeared by day 30-40. Interestingly, they were never observed in the lungs. ESAT-64-17-specific T reg cells expressed the transcription factor Helios, whose expression has been linked to thymically-derived natural T reg cells, suggesting that they expand from the population of pre-existing T reg cells and are not derived from conventional ESAT-64-17-specific CD4+ T cells. Moreover, ESAT-64-17-specific T reg cells use a different profile of T cell receptors than effector CD4+ T cells. We hypothesize that the expansion of pathogen-specific T reg cells may not occur in all infections, but may be driven by Mtb to assist in establishing persistent infection.
Group B streptococci Ib monoclonal antibodies protect against invasive aspergillosis. (99.7)
Authors: Wharton Rebekah , Dizon Brian , Kearney John
Published: January 2023

Aspergillus fumigatus (A.f.) the cause of invasive Aspergillosis (IA) has become a leading cause of death in immunosuppressed populations, due to the lack of effective therapies or vaccines. Studies using anti-A.f. monoclonal antibodies indicate that they can protect against IA. However, the lack of detailed information on the antigenic targets of protective anti-A.f. antibodies has hampered the development of an effective anti-A.f. vaccine. It is known that sialylated epitopes found on a variety of pathogens including fungi and Group B Streptococci (GBS) are involved in attachment to host cells, uptake by phagocytes, and complement activation. A.f. sialylated epitopes may therefore provide a potential target for new therapies. Our preliminary data show that a purified, calcium-dependent GBSIb-specific antibody, SMB19 (IgM,κ) also binds to A.f. and provides a degree of protection in a murine model of IA. Likewise, immunization of wild type and SMB19 VH bearing immunoglobulin transgenic mice with a GBSIb vaccine also provides protection. These results support our hypothesis that induction of GBSIb specific antibodies, perhaps supplemented with antifungal drugs, will lead to new vaccines or passive antibody prophylaxis to prevent and treat IA.
The impact of methamphetamine use on host immune response to herpes simplex virus type 2 (105.15)
Authors: Valencia Frances , Milligan Gregg , Cunningham Kathryn , Bourne Nigel
Published: January 2023

Methamphetamine (METH) use is associated with increased risk for sexually transmitted infections (STIs) that is not believed to be due to increased behavioral risk factors alone. However, the impact of METH at the time of exposure to an STI has not been explored. We hypothesized that METH would impact normal host immune responses to an STI pathogen, herpes simplex virus type 2 (HSV-2), resulting in alterations in disease. Female C57BL6 mice were treated with METH (10 mg/kg) or saline daily for 5 days, and inoculated intravaginally with HSV-2 on treatment day 3. METH treated mice experienced significantly earlier onset of disease and enhanced disease progression compared to controls. Unexpectedly, METH treated mice had increased IFNγ levels in the genital tract early after challenge, but this did not correlate with decreased vaginal virus titers. Subsequently, later after virus challenge there were significantly less IFNγ secreting CD4+ T cells in the genital tract of METH treated mice, but an increased number of these cells in the iliac lymph nodes. Our studies confirm our hypothesis that METH alters immune responses to intravaginal HSV-2 challenge with a corresponding impact on disease progression. The alterations in IFNγ production in the genital tract are of particular interest because of the important role of this cytokine in HSV-2 control. These findings have important public health implications and may be more broadly applicable to other important STI’s such as HIV.
The Relationship of Cytomegalovirus (CMV) Infection with circulatory IFN-α levels and IL-7 receptor α expression on CD8+ T cells in Human Aging (105.16)
Authors: Lee Naeun , Lee Won-Woo , Shin Min Sun , Kang Youna , Jeon Sangchun , Kang Insoo
Published: January 2023

The IL-7 receptor alpha (IL-7Rα) is the high affinity receptor for IL-7 which is essential for T cell homeostasis. We recently reported an age-associated expansion of human effector memory (EM) CD8+ T cells expressing IL-7Rα low (IL-7Rαlow), which could be detrimental to hosts by occupying “immunological space”. We investigated the potential mechanisms for this phenomenon, focusing on cytomegalovirus (CMV) infection and IFN-α. In the elderly (age≥65), CMV infection was associated with a decreased frequency of naïve CD8+ T cells as well as with an increased frequency of total EM and IL-7Rαlow EM CD8+ T cells. However, in the young (age≤40), this viral infection was associated only with an increased frequency of IL-7Rαlow EM CD8+ T cells. There was no association found between CMV immune status and plasma levels of IFN-α. In CMV-infected young and elderly people, IFN-α levels had no correlation with the frequency of IL-7Rαlow EM CD8+ T cells although this cytokine levels correlated with the frequency of IL-7Rαlow CD45RA+ EM CD8+ T cells in CMV-uninfected elderly people. Our findings suggest that the effect of CMV infection on CD8+ T cell subsets may begin with IL-7Rαlow EM CD8+ T cells and spread to other subsets with aging. Also, IFN-α could be associated with the expansion of IL-7Rαlow CD45RA+ EM CD8+ T cells in the CMV-uninfected elderly.
Memory CD4 T cells protect against influenza A virus through both an interferon gamma-dependent and multiple interferon gamma-independent mechanisms (105.31)
Authors: McKinstry Kai , Strutt Tara , Swain Susan
Published: January 2023

Memory CD4 T cells provide strong protection against influenza A virus (IAV), but the mechanisms and cellular interactions critical for their antiviral impact are not well described. A more complete understanding of how memory CD4 T cells contribute to viral clearance could improve T cell-based vaccine strategies aimed at protecting against both seasonal and pandemic IAV strains. Here, we show that IAV-specific Th1- and Th17-polarized memory CD4 T cells, as well as memory cells primed by IAV, protect otherwise naïve mice against lethal IAV challenge. We systematically deconstruct this protective response utilizing mice deficient for B cells, T cells, or both, to reveal multiple, distinct mechanisms of protection. First, in the absence of other T cells, memory CD4 T cells synergize with B cells to provide protection that is dependent on neutralizing antibodies but independent of characterized CD4 T cell helper functions. Second, in the absence of B cells, memory CD4 T cells synergize with CD8 T cells to promote equivalent viral clearance. These mechanisms of protection are both fully independent of interferon gamma (IFNγ) production by memory CD4 T cells. Third, in the absence of both B and CD8 T cells, memory CD4 T cells directly control IAV. Strikingly, this direct control is critically dependent on IFNγ. These results support a central role for memory CD4 T cells in orchestrating and directly participating in distinct forms of heterosubtypic immunity against IAV.
Characterization of IL-10 producing CD8 T cells that limit immune surveillance during chronic virus infection. (105.34)
Authors: Hu Zhuting , Molloy Michael , Zhang Weijun , Usherwood Edward
Published: January 2023

Gammaherpesvirus infection in AIDS patients is associated with severe disease. Progressive loss of CD4 T cells results in an erosion of immune surveillance, which may lead to gammaherpesvirus associated tumors. Studies by our group and others have used murine γHV-68 infection in combination with CD4 depletion to model this breakdown in immune surveillance. Mice initially control the infection, but virus reactivates after 6 weeks, indicating a lack of long-term immune surveillance. We previously showed mice had elevated IL-10 and therapeutic IL-10R blockade reduced reactivation of the virus, implicating IL-10 as a key factor suppressing immune surveillance. We identified a population of IL-10 producing CD8 T cells only in the infected mice lacking CD4 T cells but not in intact mice. Here we characterize IL-10+ CD8 T cells and elucidate how they arise in both the acute and chronic phase of the infection. In the acute phase of the infection less IL-10 producing CD8s arose in the absence of CD4 help or CD40-CD40L signaling, while in the chronic phase IL-10+ CD8s only arose in the absence of CD4 help. IL-10 producing CD8s in the chronic phase showed an anergic phenotype in terms of proliferation in vitro. In addition, they suppressed the proliferation of effector CD8 T cell in vitro. Dissection of the mechanisms involved in the generation and the function of these suppressive IL-10 producing CD8 T cells may expose new targets for therapeutic intervention for AIDS patients.
Viral acute lower respiratory infections rapidly impair CD8+ T lymphocyte functions through the PD-1/PD-L1 pathway (105.44)
Authors: Erickson John , Gilchuk Pavlo , Hastings Andrew , Tollefson Sharon , Downing Melissa , Boyd Kelli , Johnson Joyce , Joyce Sebastian , Williams John
Published: January 2023

Viruses are leading causes of severe acute lower respiratory infection (LRI) yet evoke incomplete immunity as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid CD8+ cytotoxic T lymphocyte (CTL) functional impairment in the respiratory tract via programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated pulmonary CTL impairment occurred as early as day 7 during human metapneumovirus or influenza virus infections. Viral antigen alone was sufficient to drive PD-1 upregulation, but PD-1 ligation by infection-induced PD-L1 was required for impairment. Therapeutic inhibition of PD-1 signaling prevented CTL impairment and reduced viral titers without exacerbating lower airway pathology. Additionally, PD-1 and PD-L1 were upregulated in the lungs of pediatric patients with severe H1N1 pandemic influenza virus or respiratory syncytial virus infections. Our results indicate that PD-1 signaling mediates CTL functional impairment rapidly at primary sites of infection due to the presence of local viral antigen and represents a conserved response against different viruses that cause serious LRI in children and adults. Therefore, the PD-1/PD-L1 pathway may represent a novel target for therapeutics and vaccine development against respiratory viruses.
Narrowing of influenza A virus specific T-cell receptor alpha and beta repertoire with increasing age (105.46)
Authors: Gil Anna , Yassai Maryam , Selin Liisa , Naumov Yuri
Published: January 2023

Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age. Here, for the first time we show that the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype. This was the case for all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TCR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity with age.
EXPLORING THE TRANSITION OF CD4 EFFECTOR INTO MEMORY T CELLS IN RESPONSE TO LCMV INFECTION (105.50)
Authors: Spasova Darina , Surh Charles
Published: January 2023

CD4 T cells play an essential part in generating protective memory following an infection. The mechanism through which CD4 effector T cells turn into memory however remains elusive. Previous research has shown that targeting mTOR (the mammalian target of rapamycin), results in an extended contraction period during an infection. This extended contraction period gives a chance to a large number of effector T cells to turn into memory, resulting in a larger memory T cell population. In this study, we utilize the LCMV infection model to try to better characterize the transition of Smarta CD4 T cells from effector into memory.