bluuebunny/crossref_metadata_2025_split huggingface dataset:
Islet-derived CD105+ stromal cells release autoantigens via exosomes secretion: activation of autoreactive T and B cells in NOD mice (P4131)
Authors: Rahman Muhammad , Regn Danielle , Bashratyan Roman , Dai Yang 
Published: January 2023

Abstract
Exosomes (EXO) are secreted, nano-size membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXO carry islet antigens and activate autoreactive T- cells in non-obese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes (T1D). To investigate whether primary islet cells in NOD can produce EXO, we isolated cells from the islet of Langerhans and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, self-replicating cells that express mesenchymal stem cell makers such as CD105, Sca-1 and CD90 molecules. These islet stromal cells (iSC) release EXO that are highly immunostimulatory to activate lymphocytes and induce inflammatory cytokines and chemokines. The EXO-induced IFN-gamma production is clearly associated with the autoreactivity of the lymphocytes and the progression of T1D in the mice. These results support that iSC may release unique autoantigens via EXO secretion that may trigger islet-specific autoimmunity. Consistent with these observations, immune histological analysis of pancreata showed that CD105+ stromal cells restrict to the peri-islet area in normal islets but penetrate into beta cell area as lymphocyte infiltration occurs. In addition, serum EXO level in NOD mice is dramatically increased at prediabetic stage, possibly following the islet inflammation. Thus, EXO are autoantigen carriers with unique adjuvant activities and may activate autoreactive cells.
Dysregulated CD8+ T cells promote antibody-mediated autoimmune disease (P4105)
Authors: Hoyer Katrina , Gravano David , Brunello Jacob , Jiang Shuwei 
Published: January 2023

Abstract
IFNγ-producing CD4+ and CD8+ T cells accumulate during systemic autoimmune disease in IL-2-deficient mice. CD4+ T cells are necessary for disease progression, and we have previously shown that IL-2-KO CD4+ T cells can transfer autoimmunity to recipient mice. However, the importance of CD8+ T cells to initiation and propagation of disease is unclear. Several groups have demonstrated a protective role for CD8+ T cells in inflammation and autoimmunity, while others have suggested a pathogenic role. To define the function of CD8+ T cells during spontaneous autoimmune disease, we depleted CD8+ T cells in IL-2-KO mice and evaluated the pathogenesis of autoimmunity. Elimination of CD8+ T cells for only two weeks resulted in a significant augmentation of survival, reduction in the number and activation state of the CD4+ T cells and B cells, and a delay in the development of erythrocyte (RBC)-specific antibodies. In untreated IL-2-KO mice, both CD4+ and CD8+ T cells produced IFNγ in response to RBC antigens. This upregulation indicates that autoreactive CD8+ T cells specific for RBC-antigens are present during disease. Our results suggest that autoreactive CD8+ T cells contribute to the initiation or propagation of lymphoproliferation and pathogenesis of autoimmunity. We are currently addressing the importance of these autoreactive CD8+ T cells relative to self-reactive CD4+ T cells. We are also evaluating the mechanisms by which CD8+ T cells promote the autoimmune disease process.
Temporal fate-mapping study reveals essential roles of Aire in the maturation program of Aire-expressing cell lineage (P4107)
Authors: Matsumoto Mitsuru , Nishikawa Yumiko , Mouri Yasuhiro , Nishijima Hitoshi 
Published: January 2023

Abstract
We are proposing that one important function of Aire might be the promotion of mTEC differentiation program. In this case, reduced expression of self-Ag genes from Aire-deficient mTECs could be the result of the failure in full maturation program where promiscuous gene expression is a unique property of terminally differentiated mTECs of Aire-expressing cell lineage (maturation model). To test this hypothesis, we took advantage of Cre/loxP based fate-mapping approach. We created timing-controlled fate mapping using tamoxifen-inducible CreER/loxP system to generate fluorescence-labeled Aire-expressing cell lineage(s). Sequential treatment of fate-mapping mice with tamoxifen for 6 days generated 3 to10% of GFP+ mTECs, and we observed percentages of GFP+ mTECs were decreased in a time-dependent manner. During the observation of 10 days following tamoxifen administration, CD80 and MHC-II expression levels were decreased. By 3 weeks after tamoxifen treatment, almost all GFP+ mTECs (but already lacking Aire expression) had disappeared. Interestingly, absence of Aire did not prolong the lifetime of Aire+ mTECs, which does not obviously support the possible proapoptotic activity of Aire. We then combined this timing-controlled fate mapping with Aire/GFP-knockin system to investigate the Aire-dependent mTEC differentiation program more in detail.
VCAM-1/α4β1 integrin interaction is important for T cell recruitment to prevent reactivation of chronic infection with Toxoplasma gondii in the brain (P3326)
Authors: Sa Qila , Michie Sara , Xu Baohui , Payne Laura , Wang Xisheng , Suzuki Yasuhiro 
Published: January 2023

Abstract
T. gondii establishes a chronic infection by forming cysts preferentially in the brain, and IFN-γ production by T cells is required to maintain the latency of infection. We examined the adhesion molecules important for T cell recruitment to prevent reactivation of the infection. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of these animals as well as those of infected wild-type animals. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Immune T cells from wild-type mice were treated with anti-α4 integrin or control mAbs and transferred into infected SCID or nude mice, and animals received the same mAb every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Cerebral expression of mRNA for CD3, CD4, CD8, IFN-γ, and NOS2 (an effector molecule to inhibit T. gondii growth) was 5-34 times less in mice treated with anti-α4 integrin mAb than those treated with control mAb at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA were 17 times greater in the former than the latter at 6 days after sulfadiazine discontinuation. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for recruiting immune T cells into the brain to prevent reactivation of chronic T. gondii infection.
Characterization of the CD8+ T cell response in rickettsial infections (P3330)
Authors: Caro Gomez Erika , Cespedes Maria , Texeira Bruno , Gazi Michal , Valbuena Gustavo 
Published: January 2023

Abstract
Natural and experimental rickettsial infections provide strong and cross-protective immunity if the individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8+ T cells, the characterization of the cellular immune response has not been systematically addressed. Here, we report the systematic, kinetic and ex vivo analysis of CD8+ T cells from spleen, liver, lungs and lymph nodes by flow cytometry with specific panels to address the activation, effector, and memory phases of the anti-rickettsial response in C3H/HeN mice infected with R. typhi. Our findings suggest that the instauration of the effector phase occurs around 7dpi which coincides with the rickettsial clearance. This phase was characterized by an increased frequency of CD8+ T cells with a phenotype compatible with effector cells:CD44+CD43+CD27-/+ and CD44+CD43-/+CD127-; along with an increased production of IFN-γ and GZM B. A more robust production of IFN-γ and GZM-B was detected in the target organs, highlighting the compartmentalization of the immune response. Furthermore, at 7dpi, differences in the production of IFN-γ and GZM-B between controls and mice immunized with a vaccine candidate were also observed. These findings are relevant for vaccine development, providing insights about meaningful time points and specific correlates of protection that can be used as a paradigm for anti-rickettsial vaccine testing.
Increased apoptosis of intrahepatic central memory CD8 T cells in the absence of IL-15 is attributed to the loss of long-lasting protection against malaria infection (P3334)
Authors: Zarling Stasya , Krzych Urszula 
Published: January 2023

Abstract
Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity amongst residents of malaria endemic areas indicate that memory responses to Plasmodia antigens are not adequately developed or maintained. In contrast, multiple exposures to radiation-attenuated Plasmodia sporozoites (γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge. In the P. berghei (Pb) γ-spz mouse model, lasting protection is associated with the presence of intrahepatic CD8 T cells that comprise two phenotypically and functionally discrete sets: effector/effector memory (TE/EM) cells that produce IFN-γ, and central memory (TCM) cells which express elevated levels of IL-15Rβ, suggesting that CD8 TCM cells depend upon IL-15 for maintenance and/or function. We asked whether immunization with Pb γ-spz could protect IL-15 KO mice against sporozoite challenge. The results demonstrate that although protective immunity is inducible in IL-15 KO mice, protection is short-lived, owing to reduced expression of Bcl-2 and increased apoptosis of proliferating intrahepatic CD8 TCM in the absence of IL-15. Therefore, we hypothesize that the maintenance of long-lasting protection induced by Pb γ-spz depends on a process whereby intrahepatic CD8 TCM cells, maintained by IL-15-mediated survival and basal proliferation, are conscripted into CD8 TE/EM cell pool during subsequent infections.
Tissue-specific antigen presentation to CD4 T cells during persistent Salmonella Typhimurium infection (P3300)
Authors: Frederick Daniel , McLachlan James 
Published: January 2023

Abstract
Antigen presentation is essential for the initiation of a CD4 T cell immune response and is commonly associated with dendritic cells (DCs) presenting foreign antigen to CD4 T cells in the context of MHCII in lymphoid tissue. It is currently unclear as to which antigen presenting cells (APCs) are displaying antigen, and what the impact this has on CD4 T cells throughout a persistent bacterial infection. In this study, we use a murine model of typhoid fever by orally infecting resistant mice with a virulent strain of Salmonella typhimurium tagged with an immunogenic epitope called Eα52-68 peptide (pEα). This allows for the visualization of antigen presentation directly ex vivo at various times after infection through the use of the Y-Ae antibody that specifically recognizes the pEα:MHCII complex. We show that while DCs are the most prominent APC in the infected spleen and mesenteric lymph nodes early, macrophages seem to predominate in the liver. Surprisingly, we could still detect antigen presentation in the spleens of mice infected with pEα-tagged S. typhimurium as late as 120 days post infection. Additionally, interferon-γ responses from effector T cells could be detected directly ex vivo at this late time. These results demonstrate that that cells presenting antigen may differ greatly between persistently infected organs and paves the way to understanding what role this might have in regulating CD4 T cell responses during this type of infection.
The role of Notch signaling in host defenses to Cryptococcus neoformans (P3310)
Authors: Olszewski Michal , Qiu Yafeng , Sandy Ashley , Hadd Zachary , Davis Michael , Chapman Rebekah , Carolan Jacob , Maillard Ivan 
Published: January 2023

Abstract
Cryptococcus neoformans (C. neoformans) is a major opportunistic fungal pathogen in individuals with impaired T cell-mediated immunity. Notch pathway is an important signaling component of immunological synapse during APC/T-cell engagement. Little is known about the role of Notch signaling in fungal infections. We sought to determine the role of Notch signaling in C. neoformans infection. Wild-type C57BL/6 (WT) and CD4-Cre+×ROSA DNMAML (DNMAML) mice were infected intratracheally infected with C. neoformans. The fungal burden, leukocyte recruitment, and cytokine profile were assessed at 3 and 6 weeks post-infection (wpi). A 50-fold greater fungal burden was detected in both lungs and brains of DNMAML mice compared to those in the WT mice at 6 wpi. Although, equivalent fungal burdens were found at 3 wpi in WT and DNMAML groups, the production of IFN-γ, IL-4, and IL-13 was significantly decreased in lung leukocytes of DNMAML mice. In contrast, an equivalent induction of IL-17 was observed in both WT and DNMAML mice at this time. Notch signaling plays a crucial role in the host defense against C. neoformans. Notch signaling contributes to control of pulmonary fungal growth and inhibition of CNS dissemination by promoting cytokine induction. This is the first demonstration of the importance of Notch signaling in cryptococcal infection. The mechanisms by which Notch signaling regulate the immune response against cryptococcal infection are under investigation.
Intracellular Staphylococcus aureus fails to induce DC apoptosis. (P3312)
Authors: Johnson Anne , Lehtimäki Mari , Neal Stephanie , Wark Wendy , Kanevsky-Mullarky Isis 
Published: January 2023

Abstract
Staphylococcus aureus causes chronic mastitis in the bovine and is a major health concern in humans. The goal of this study is to elucidate the mechanism by which S. aureus infects DC and evades immune responses and antibiotic treatments. Inhibiting DC apoptosis would provide S. aureus with a replicative niche, an escape from treatments, and a reprieve from the immune system. DC were treated with either live S. aureus, live Streptococcus agalactiae , γ-irradiated S. aureus, or UV. Apoptosis was measured via annexin V/FITC and PI staining by flow cytometry at 48 hours post-infection. Treatment with either S. agalactiae or UV induced a significant increase in apoptotic DC (annexin V+/PI-) percentages as compared to untreated DC. However, there was no significant increase in percentage of apoptotic DC following treatment with live or irradiated S. aureus as compared to no treatment. We concluded that S. aureus does not induce, but may inhibit, DC apoptosis. These data supply knowledge about the ability of S. aureus to manipulate the host immune system and will provide information from which to design efficacious vaccine strategies.
FlowCAP: comparison of automated and manual gating of standardized lyoplate flow cytometry data (P3374)
Authors: Scheuermann Richard , Finak Greg , Ramey John , Taghiyar Jafar , Stanton Rick , Brandes Aaron , De Jager Philip , Qiu Peng , McCoy J. , Hafler David , Maecker Holden , Mosmann Tim , Brinkman Ryan , Gottardo Raphael 
Published: January 2023

Abstract
Standardization of immunological assays, including flow cytometry, in terms of reagents, sample handling, instrument setup, and data analysis, is essential for successful cross-study and cross-center analysis in order to mitigate the effects of technical variability in assay results. The Human Immunology Project and the Federation of Clinical Immunology Societies (FOCIS) have partnered to develop five standardized, lyophilized, eight-color staining reagent panels (termed lyoplates) for this purpose. In collaboration with the FlowCAP consortium, standardized samples (Cytotrol control cells) were distributed to nine participating centers and analyzed by flow cytometry using the lyoplate reagents and SOP's to minimize experimental variability. Data from two of these panels (T-cell and B-cell) were entered into the FlowCAP-III challenge, where participants analyzed the data using automated gating methods for comparison against cell population statistics for major T and B-cell subsets as defined by a consensus manual gating scheme. This evaluation showed that several automated gating algorithms could successfully recapitulate centralized manual gating statistics for T-cell and B-cell subsets with little statistical bias, and with within-center and between-center variability as low or lower than centralized manual gating. These results demonstrate that automated gating algorithms are ready for use in performing reproducible analyses and comparisons of immunological data.
A rapid method to isolate and test the γ2a switched variants from a γ2b antibody producing B cell hybridoma (P3382)
Authors: Liu Haolin , Zhang Gongyi , Marrack Philippa , Kappler John 
Published: January 2023

Abstract
The heavy chain isotype switch is reported to occur at a rate of 10-6 to 10-5 in B cell hybridomas. Because of the rare occurance, usually two or three rounds of cell sorting and subcloning are needed to isolate these variants, which is labor and time consuming. We find that the switch rate of γ2a variants in a γ2b antibody producing B cell hybridoma, S212, is around 5*10-6 by ELISPOT assay. Using the Alexa Fluor 647 conjugated goat anti-mouse IgG2a heavy chain specific antibody, we first enriched γ2a switched variants in S212 with the use of anti-AF647 microbeads. We then used the FACS to sort the single AF647 positive cell from this enriched population into a single well to ensure the single cell cloning. 18 cells were sorted into 18 wells, and there were hybridoma cells growing in 12 wells after 10 days of culture. We tested the isotype of antibody produced in the supernatant by passing it through the four channels of a CM5 chip which were respectively coated with goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 heavy chain specific antibody in a Biacore instrument. The result showed that all of the 12 antibodies were IgG2a and none of them secreted IgG2b antibody any more. This method will be useful in isolating downstream Ig class switch variants of monoclonal antibodies to obtain antibodies with different Fc functions.
Novel method for next generation sequencing of KIR by Roche 454 (P3383)
Authors: Brayton Bonnie , Byron Mary Margaret , Chew Glen , Tamate Trent , Crawford Timothy Quinn , Barbour Jason 
Published: January 2023

Abstract
Natural Killer cells are regulated by the polymorphic Killer Ig-like Receptor family. KIR genotype and expression has been associated with HIV-1 disease progression. We developed a high-throughput method to characterize both DNA genotype and RNA expression of KIR using a novel 454 sequencing method (Roche), for study of HIV and innate immunity. This method uses a 299bp and 395bp amplicon to distinguish and assign long - and short-tailed KIR. These amplicons, from motifs common to 3’ UTR of known KIR genes, amplify both KIR DNA and mRNA. We developed a custom bioinformatics pipeline to analyze 454 sequence reads and it compares each sequence (average 3000 reads per specimen) to a reference alignment of KIR genes via the BLAT algorithm. Using this method, we characterized KIR in a cohort of 700 treatment-naïve adults in early HIV-1 disease. Initial results indicate that our KIR DNA 454 sequencing data agree with KIR DNA genotyping results from mass spectrometry-based typing as well as sequence-specific oligonucleotide typing. KIR3DL1 expression on NK cells by flow cytometry shows 80% agreement with KIR mRNA expression from PBMCs by 454. The use of mRNA- and DNA-based next generation sequencing for human immune receptor genes, such as KIR, will accelerate genetic association and functional genetic studies of diseases of the immune system.
Luminescence-based protease biosensor as a novel method to measure cytotoxic T lymphocyte function (P3386)
Authors: Li Jinzhu , Ferrell Julie , Figueira Sarah , Binkowski Brock , Risma Kimberly 
Published: January 2023

Abstract
Cytotoxic lymphocytes (CTL) induce target cell apoptosis through delivery of granzymes and subsequent activation of caspases. CTL function is usually assessed by either traditional Cr51 release assay or flow-based assay. A 96 well, non-radioactive assay is desirable to meet the need of the fast growth of the research and clinical application of CTL. We aimed to develop a new luminescence-based CTL assay, using protease biosensor technology. For this purpose we utilized a circularly-permuted, firefly luciferase to contain a caspase 3/7 proteolytic activation site, allowing luminescent activity upon cleavage during apoptosis. When we expressed the caspase biosensor in EL4 tumor cells and co-incubated with antigen-specific CD8 effector cells, the biosensor was triggered in minutes and allowed for evaluation of the kinetics of caspase induction in the target cells in real time. The biosensor signal correlates with the Cr51 release assay and was dependent upon perforin and granzyme B signal. The caspase-activated, luciferase biosensor has substantial advantage over the traditional methods to measure lymphocyte cytotoxicity, and allow, for the first time, measuring cytotoxic function in real time.
Isolation of functional murine naïve and memory T cell subsets in as little as 15 minutes (P3387)
Authors: Kokaji Andy , Posarac Vesna , Tonelli Luciana , Sun Jeong Hun , Molavi Armon , Fairhurst Maureen , McQueen Karina , Thomas Terry 
Published: January 2023

Abstract
Effective cellular immunity requires naïve CD4+ and CD8+ T cells to undergo activation, proliferation and differentiation into various effector cell populations following antigen exposure. CD4+ and CD8+ T cell activation induces the production of cytokines and cytolytic effector proteins which function to coordinate immune responses or directly eliminate infected cells. While most effector T cells become senescent and apoptotic, some effector T cells further differentiate into memory T cells which can provide long lasting immunity. Although the signals that regulate these processes have been widely investigated, continued efforts will eventually construct a definitive model for the role of T cells in adaptive immunity. In many cases, highly purified cells are required for these studies and current methods for their isolation from mice require either lengthy protocols or flow based cell sorting. We have recently developed three new column-free, immunomagnetic cell isolation kits to isolate untouched naïve and memory CD4+ T cells, and naïve CD8+ T cells in as little as 15 minutes. Purities of up to 98% can be achieved as assessed by CD44 and CD62L expression using the manual EasySep™ pour-off method or the fully automated RoboSep™ cell separator. The purified naïve and memory T cells can be immediately used for downstream assays and are fully functional as assessed by in vitro proliferation and cytokine production assays.