bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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

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

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

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

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

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.
Minimizing PCR bias in high-throughput adaptive immune repertoire sequencing assays (P3389)
Authors: Carlson Chris , Emerson Ryan , Sherwood Anna , Desmarais Cindy , Chung Moon , Parsons Joe , LaMadrid-Herrmannfeldt Marissa , Livingston Robert , Rieder Mark , Robins Harlan
Published: January 2023

The human adaptive immune system is a primary defense against the vast world of potential pathogens. To counter this, B and T cells rearrange a diverse repertoire of surface receptors. This system functions in part by clonal expansion, so measuring changes in the abundance of both unique and total clones is necessary to understand the immune response. We developed immune profiling assays to amplify and sequence rearranged receptors. To efficiently capture receptor diversity we use a multiplex PCR approach. However, small variations in annealing kinetics can have large impacts on amplification efficiency causing PCR amplification bias (PAB). To address this issue, we developed a method to quantify and correct PAB introduced by multiplex PCR. We demonstrate this method using our T cell receptor γ (TCRG) assay. Our method uses a quantitated, synthetic immune repertoire standard consisting of every possible Variable(V) and Joining(J) gene combination. Following multiplex TCRG PCR amplification, we assessed the difference between the synthetic VJ composition before and after amplification and quantified PAB. We adjusted primer concentrations and redesigned primers to generate an unbiased multiplex PCR assay. Using this method we reduced PAB for the TCRG assay from a 340-fold to a 4.5-fold dynamic range. Now the abundance of receptor sequences is representative of the frequency of each receptor chain in the input sample, allowing for accurate quantification of the TCRG repertoire.
Live cell RNA detection aids in the understanding of cellular responses (P3362)
Authors: Weldon Don , Ko Alex , Williams Yuko , Koong Victor
Published: January 2023

Understanding the immune response can be aided by analyzing the genes that immune cells express in response to stimuli. Since all cells from the same orgainism have the same genetic components it is the selection of which genes are expressed that gives each cell its unique characteristics. Once a cell senses cues from its environment it begins to respond by transcribing its DNA into RNA which is later translated into protein. In order to monitor responses at the earliest possible point the RNA levels of a given gene can be assessed giving the researcher insight to how the cell has chosen to react before the protein is translated. Detecting gene expression has traditionally been limited to technologies that examine RNA in lysed or fixed cell populations. The ability to detect gene expression in live cells would allow for more physiologically relevant information. We have developed a novel RNA detection technology capable of detecting specific mRNA and miRNA in live, intact cells. It allows for carrier-free cellular uptake of the reagent, followed by detection of target RNA, with the ability to perform downstream analysis in the same sample.
Cell type-specific role of autophagy against Citrobacter rodentium infectious colitis (P3268)
Authors: Inoue Jun , Hoshi Namiko , Nishiumi Shin , Fujishima Yoshimi , Masuda Atsuhiro , Shiomi Hideyuki , Yoshida Masaru , Azuma Takeshi
Published: January 2023

Recently, genome-wide association studies linked some autophagy-related genes to increased inflammatory bowel disease susceptibility, which suggests that autophagy has an important role in the control of gut immunity. To examine the function of autophagy in different cell compartments in the intestine, we generated the Atg7, an essential gene for autophagy induction, conditional knockout mice in the intestinal epithelium (Atg7FL/FL;Villin-Cre mice). We injected mice with LPS and tested the inflammatory response in the intestine. Atg7FL/FL;Villin-Cre mice exhibited stronger activation of NF-κB and higher levels of TNF-α and IL-1β than WT mice. We also tested the autophagy function in fibroblasts NIH3T3 cells overexpressing mutant Atg4B, which have defect in autophagosome closure, and found that those cells showed enhanced NF-κB activation to LPS stimulation as well. These results suggest that autophagy deficiency in non-hematopoietic compartment can lead to excess inflammatory response against intestinal microorganisms. To confirm this point, we infected mice with C. rodentium and found that Atg7FL/FL;Villin-Cre mice showed aggravated histological intestinal injuries and higher TNF-α, IL-1β and IFN-γ levels in the colon than WT mice. Our findings suggest that autophagy in non-hematopoietic cells play an important role in the regulation of inflammation and host defense against C. rodentium. We are currently testing the function of Atg7 in the hematopoietic cell compartment.
Mice deficient in IL-25 are less susceptible to dextran sulfate sodium-induced colitis (P3270)
Authors: Li Yanfei , Wang Anjiang , Grinchuk Viktoriya , Smith Allen , Ramalingam Thirumalai , Wynn Thomas , Urban Joseph , Shea-Donohue Terez , Zhao Aiping
Published: January 2023

IL-25 is a member of IL-17 cytokine family and is highly expressed in gastrointestinal epithelial cells. The biological activities of IL-25 have been associated with promotion of Th2 but inhibition of Th1/Th17 immunity. Whether IL-25 plays a role in the development of colonic inflammation is unresolved. In this study, we investigated the contribution of IL-25 to DSS-induced colitis in mice. C57BL/6 WT and IL-25-/- mice received 3% DSS in the drinking water ad libitum and were euthanized eight days later. When compared to WT mice, IL-25-/- mice lost significantly less body weight and had delayed appearance of diarrhea and bloody stool, as well as less shortened colon length. Histological evaluation of H&E-stained tissue sections of colon showed that IL-25-/- mice had only mild inflammation, while severe inflammation developed in WT mice based on the extent of focal crypt lesions, loss of goblet cells, and infiltration of inflammatory cells. IL-33 purportedly contributes to intestinal inflammation and qPCR analysis of colonic cytokine expression showed an up-regulation of IL-33 in DSS-treated WT but not IL-25-/- mice. In addition, there were significantly fewer transcripts of the pro-inflammatory cytokines (TNFα, IL-6, IL-17A) in the colon of DSS-treated IL-25-/- than WT mice. Overall, these data suggested that a deficiency in IL-25 partially protected mice from DSS-induced colitis, revealing a previously unidentified pro-inflammatory role for IL-25 in colonic inflammation.
Bone marrow precursors require β7 integrins to give rise to intestinal mononuclear phagocytes with tolerogenic potential (P3290)
Authors: Mora J. Rodrigo
Published: January 2023

Eduardo J. Villablanca 1, Jaime De Calisto 1, Patricia Torregrosa Paredes 1, 2, Barbara Cassani 1, Susanne Gabrielsson 2 & J. Rodrigo Mora 1 1 Gastrointestinal Unit, Massachusetts General Hospital & Harvard Medical School, Boston, MA; 2 Translational Immunology Unit, Karolinska Institutet, Stockholm, Sweden Intestinal mononuclear phagocytes, including dendritic cells (DC) and macrophages (MF), develop from at least two different bone marrow (BM) precursors. CD103- MF derive from lineage negative (Lin-) Ly6Chigh monocytes, whereas CD103+ DC, which metabolize vitamin A into all-trans retinoic acid (RA), derive from Lin-Ly6Clow BM precursors. However, how mononuclear phagocytes precursors are recruited to the intestinal mucosa remains unknown. Here, we show that BM Lin-Ly6Clow cells require β7 integrins to reconstitute intestinal mononuclear phagocytes and to give rise to RA-producing mesenteric lymph node DC. Interestingly, the BM contains a distinct population of α4β7+ Lin-Ly6Clow cells, which was markedly reduced in vitamin A-depleted mice. Importantly, mice lacking β7 integrins in the CD11c+ compartment showed decreased generation of antigen-specific regulatory T cells and were impaired in developing oral tolerance. Thus, BM progenitors require α4β7 to give rise to intestinal mononuclear phagocytes with tolerogenic potential.
A short-term model of COPD identifies a role for mast cell tryptase (P3242)
Authors: Hansbro Philip , Beckett Emma , Stevens Richard , Jarnicki Andrew , Kim Richard , Hanish Irwan , Hansbro Nicole , Deane Andrew , Keely Simon , Horvat Jay , Yang Ming , Oliver Brian , van Rooijen Nico , Inman Mark , Adachi Roberto , Soberman Roy , Hamadi Sahar , Wark Peter , Foster Paul
Published: January 2023

Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short time-frame.We have created a mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short amount of time. Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathological features of COPD were assessed. The roles of macrophages and mast cell (MC) tryptase in pathogenesis were evaluated using depletion and in vitro studies and MC protease-6 deficient mice. After 8 weeks of smoke exposure, wild-type mice developed chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid-resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart, and increased susceptibility to respiratory infections also were observed. We demonstrate here that macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced pro-inflammatory responses from cultured macrophages. This model can be used to better understand multiple aspects of COPD pathogenesis.
TWEAK/Fn14 pathway promotes chronic colitis and fibrosis mediated by IL-13-TSLP axis (P3244)
Authors: Dohi Taeko , Kawamura Yuki , Kawashima Rei , Son Aoi , Oshio Tomoyuki , Wu Ping , Burkly Linda
Published: January 2023

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF ligand superfamily that is constitutively expressed by many immune cell types including monocytes/macrophages, DC, neutrophils, natural killer cells, and also T cells. The only known signaling receptor for TWEAK, FGF-inducible molecule 14 (Fn14), is a highly inducible molecule in epithelial cells (EC), endothelial cells, and other mesenchymal cell types wherein TWEAK signals through Fn14 to promote the canonical and noncanonical NF-κB, MAPK and potentially other pathways. We previously reported that TWEAK promoted colitis in mice by enhancing production of inflammatory mediators by EC. TWEAK also induced EC damage in the presence of IL-13, which is known as a pathogenic profibrotic T helper 2-type cytokine. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2-type immunity were significantly reduced in Fn14 knockout (KO) mice when compared with wild type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon EC than in WT EC. TWEAK/Fn14 was required for the induction by IL-13 of TSLP in colon explants. TWEAK and IL-13 synergistically promote embryonic fibroblast proliferation. Thus we propose an IL-13-TWEAK/ Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.
IL-10 modulates DSS-induced colitis through a macrophage -ROS-NO axis (P3254)
Authors: Li Bofeng , Alli Rajshekhar , Vogel Peter , Geiger Terrence
Published: January 2023

IL-10 is a critical regulator of colitis development. Yet, its cellular targets and mechanisms of action are not fully resolved. To identify lineages and mechanisms responsible for IL-10's protective effects in colitis, we generated mice with cell type-restricted deficiencies in IL-10 responsiveness by using DSS colitis model with mice bearing deletions of the IL-10Rα in T cells, B cells, DCs, or macrophages. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced colitis that did not significantly differ from disease in IL- 10-/- or IL-10Rα-/- mice; no impact of IL-10Rα-deficiency in other lineages was observed. IL-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Quantitation and phenotypic assessment of lamina propria macrophages did not identify differences from controls. IL-10Rα-deficient macrophages also did not display a competitive advantage over wild type cells in mixed chimeras. Pro-inflammatory cytokine production, and particularly TNF-α, was increased, though TNF-α neutralization failed to reveal a defining role for the cytokine in the aggravated disease. IL-10RαMdel lamina propria macrophages produced substantially greater levels of NO and ROS than control macrophages. Inhibition of these more dramatically reduced colitis severity in IL-10RαMdel mice, and largely eliminated the differential effect of DSS in IL-10RαMdel and control mice.
1,25-dihydroxyvitamin-D3 reversibly prevents EAE by modulating Th cells trafficking without affecting priming or effector functions (P5123)
Authors: Grishkan Inna , Calabresi Peter , Gocke Anne
Published: January 2023

Pharmacologic targeting of chemokine receptors alters Th cell trafficking and thus poses an attractive opportunity for amelioration of autoimmune diseases such as multiple sclerosis (MS). MS risk is associated with vitamin D deficiency, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to prevent experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. However, the mechanism of its in vivo action is poorly understood. In the present study, we utilized FACS analysis and in vivo tracking of bioluminescent Th cells to investigate the effect of 1,25(OH)2D3 on Th cells in MOG EAE. We demonstrate that MOG-specific Th cells are successfully generated in the presence of 1,25(OH)2D3, are able to enter peripheral circulation, but are not present in the CNS. 1,25(OH)2D3 treatment does not affect the ability of Th cells to produce IFNγ and IL-17, but reversibly reduces their expression of CXCR3, a chemokine receptor essential for migration to the CNS. Remarkably, mice rapidly develop EAE upon treatment cessation, demonstrating that pathogenic Th cells are present but unable to cause disease while exposed to 1,25(OH)2D3. Herein, we provide novel evidence that 1,25(OH)2D3 prevents EAE by modulating encephalitogenic Th cells migration without affecting their priming or effector functions. The disease rebound further emphasizes that 1,25(OH)2D3 is not an immunosuppressive but rather a reversible immunomodulatory agent that specifically targets pathogenic Th cells in EAE.
Rexinoids as modulators of central nervous system inflammation (P5125)
Authors: Nowak Elizabeth , Chandraratna Roshantha , Dmitrovsky Ethan , Noelle Randolph
Published: January 2023

Transcription factors that interact with retinoid x receptors as heterodimers, such as retinoic acid receptors, liver x receptors, and peroxisome proliferator-activated receptors, demonstrate profound immunoregulatory activities. Therefore small molecule agonists and antagonists to these receptors are attractive drug candidates to control immunity. Studies in our laboratory show that in vivo treatment of mice developing experimental autoimmune encephalomyelitis, a model of central nervous system (CNS) inflammation, with the rexinoid IRX4204 delays the onset and reduces the severity of disease. In accordance with reduced disease severity, we find significantly decreased CNS infiltration of myeloid dendritic cells and CD4+ T cells. Studies to determine the mechanism of action of this drug are in progress.
Vitamin D reduces development and progression of virus-induced demyelinating disease (P5131)
Authors: Olson Julie , Hall Samuel
Published: January 2023

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that is associated with an inflammatory immune response. Microglia are the CNS resident immune cells which have been shown to be activated during MS. Virus infections have been suggested as a causative agent for MS, and vitamin D insufficiency has been correlated with disease development. Thus, we wanted to determine whether higher vitamin D levels at the time of virus infection could alter development of demyelinating disease. Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is an animal model of MS. TMEV infection of susceptible mice leads to development of a demyelinating disease associated with an inflammatory immune response in the CNS. Our studies showed that increased levels of vitamin D3 at the time of virus infection reduced development and progression of demyelinating disease. Vitamin D reduced the number of CD4+ T cells expressing IFNγ and increased the number of CD25+CD4+ T cells. Most interestingly, vitamin D reduced the expression of pro-inflammatory cytokines and increased the expression of IL-10 and type I interferons by microglia. The reduction in the inflammatory immune response by microglia was dependent on IL-10 induction of SOCS3. Overall, these results show that vitamin D reduced the inflammatory immune response in the CNS and reduced the development and progression of demyelinating disease following virus infection.
The peroxisome proliferator-activated receptor-γ agonist rosiglitazone in the prevention and treatment of murine systemic lupus erythematosus (P5135)
Authors: Aprahamian Tamar , Bonegio Ramon , Weitzner Zachary , Rifkin Ian
Published: January 2023

Peroxisome proliferator-activated receptor-γ agonists are known to have many immunomodulatory effects. We have previously shown that the peroxisome proliferator-activated receptor-γ agonist rosiglitazone is beneficial when used early in prevention of lupus and lupus-related atherosclerosis. In this study, we attempt to define the ability of peroxisome proliferator-activated receptor-γ agonists to ameliorate established autoimmune disease using two mouse models: the MRL.lpr mouse which is an established lupus-like model, and the gld.apoE-/- mouse, which is a model of accelerated atherosclerosis and lupus. We demonstrate that, in contrast to the marked amelioration of disease seen when rosiglitazone was started before disease onset, treatment with rosiglitazone after disease onset in MRL.lpr or gld.apoE-/- mice had minimal beneficial effect on the development of autoimmune phenotype; however, rosiglitazone treatment remained highly effective at reducing lupus-associated atherosclerosis in gld.apoE-/- mice on both normal diet or high cholesterol Western diet. These results suggest that beneficial effects of peroxisome proliferator-activated receptor-γ agonists on the development of autoimmunity might be limited to the early stages of disease but that atherosclerosis, a major cause of death in SLE patients, may be ameliorated even in established or severe disease.
Priming of regulatory DNA elements activates inflammatory cytokine genes synergistically with TLR signaling to increase transcription (P1230)
Authors: Qiao Yu , Giannopoulou Eugenia , Elemento Olivier , Ivashkiv Lionel
Published: January 2023

Synergistic activation of inflammatory cytokine genes by IFN-γ and Toll-like receptor signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of Toll-like receptor signaling events, a previously proposed mechanism, is insufficient to explain massive activation of cytokine production in human macrophages. We found that IFN-γ induced sustained occupancy of STAT1 and IRF1 at proximal and distal regulatory regions of the TNF, IL6 and IL12B gene loci in human macrophages, which was associated with increased histone acetylation, an indicator of an active histone state. This opening of chromatin greatly facilitated further recruitment of other transcription factors and the assembly of the RNA polymerase II complex upon Toll-like receptor activation, which resulted in increased and prolonged transcription of these inflammatory cytokines. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Furthermore, IFN-γ-mediated priming conferred sensitivity of cytokine gene transcription to inhibitors of Jak kinases or chromatin regulators of the bromo and extra terminal protein family. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment Toll-like receptor-induced gene transcription, and suggest therapeutic approaches that selectively target priming mechanisms.
The pro-inflammatory and anti-inflammatory TLR2 responses to Staphylococcus aureus can be uncoupled (P1242)
Authors: Peres Adam , Stegen Camille , Cousineau Benoit , Desrosiers Martin , Madrenas Joaquín
Published: January 2023

Microbial detection by pattern recognition receptors (PRRs) is typically associated with a pro-inflammatory cytokine response that primes adaptive immunity. However, some PRRs may also elicit an anti-inflammatory response. Most notably, LPS engagement to Toll-like receptor 4 (TLR4) leads to a PI3K-dependent internalization of TLR4, and the production of Type I interferons and, subsequently, of the anti-inflammatory cytokine IL-10. We have previously reported that peptidoglycan-embedded molecules in the gram-positive bacterium Staphylococcus aureus cell wall, acting on TLR2 on monocytes and macrophages, trigger a PI3K/Akt-dependent IL-10 response that can suppress adaptive T cell immunity. We now report that, unlike the anti-inflammatory TLR4 response, the TLR2-mediated anti-inflammatory IL-10 response is independent of actin-mediated receptor internalization and Type I interferon production. More importantly, the anti-inflammatory response triggered by S. aureus isolates through TLR2 can be uncoupled from the pro-inflammatory response, as they have different kinetics and TLR2 internalization requirements. Our data reveal a novel mechanism of TLR-mediated anti-inflammatory response that may be triggered by selective ligands on the staphylococcal cell wall. Identification of these anti-inflammatory TLR2 ligands may be a first step toward designing novel therapies to treat staphylococcal infections.
Dual-specificity phosphatase 3 knockout female mice are resistant to LPS and to polymicrobial induced septic shock in TNF dependent manner. (P1222)
Authors: Rahmouni Souad , Singh Pratibha , Dejager Lien , Maneesh Singh , Amand Mathieu , Musumeci Lucia , Moutschen Michel , Mustelin Tomas , Libert Claude
Published: January 2023

We report the generation of dual-specificity phosphatase 3 (DUSP3) deficient mice. These mice develop normally and do not exhibit any spontaneous phenotype. However, VHR-/- females, but not males, are resistant to LPS- and to polymicrobial infection-induced septic shock. After LPS injection, while VHR-/- males and VHR+/+ mice of both genders, displayed an increased serum levels of TNF-α and IFNγ, the levels of these cytokines remained significantly low in the VHR-/- females. In vitro experiments using peritoneal macrophages showed the same results suggesting that the systemic cytokines profiles observed are macrophages-dependent. Adoptive transfer of VHR-/- females bone marrow to irradiated VHR+/+ female mice, but not to VHR-/- or VHR+/+ males, protected them from death after administration of LPS. Interestingly, VHR-/- females were sensitive to TNF-α- induced lethality. We also report that the decrease of TNF-α production observed in VHR-/- female’s macrophages after LPS activation was associated with a decreased ERK1/2, but not MEK1/2, activation. Interestingly, pervanadate (PTP pan inhibitor) treatment prior to LPS activation restored ERK1/2 activation in the VHR-deficient macrophages, suggesting that VHR is targeting one of the ERK1/2 PTPs or DUSPs. These results, together with our observation that DUSP3 is the most highly expressed phosphatase in macrophages, suggest a key non-redundant role of VHR as positive regulator of TNF-α in innate immune response in females.
Regulation of naturally-occurring CD4+CD25+ regulatory T cell development by E3 ubiquitin ligase Cbl-b (P1041)
Authors: Zhang Jian , Guo Hui , Qiao Guilin
Published: January 2023

Naturally-occurring CD4+CD25+Foxp3+ regulatory T cells ("nTregs") are involved in shutting down immune responses after they have successfully eliminated invading organisms, and also in preventing autoimmunity. CD28 costimulation is essential for the development of nTregs in thymus. E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. Here, we report that the loss of Cbl-b partially but significantly rescues the defective development of nTregs in CD28-/- mice. This partial rescue is likely to be independent of IL-2 because treating CD28-/- mice with IL-2/anti-IL-2 complex, which has been shown to selectively stimulate nTregs, fails to rescue defective development of CD4+CD8-CD25+Foxp3+ nTregs in thymuses of CD28-/- mice. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation, and targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in CD28-/- T cells, the defective development of nTregs in CD28-/- mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Cbl-b. Therefore, we provide a previously-unappreciated function for Cbl-b in regulating Foxp3 expression via an ubiquitination-dependent mechanism.
Scope and mechanisms of peripherally generated regulatory T cell mediated suppression in inflammatory contexts (P1031)
Authors: Niec Rachel , Rudensky Alexander
Published: January 2023

The peripheral Treg population, which prevents fatal autoimmunity in mice and man, consists of Tregs that develop in the thymus (tTregs) and Tregs derived from naïve T cells in the periphery (pTregs). Mice with a specific deficiency in pTregs do not develop the spontaneous multi-organ autoimmunity observed in Treg deficient mice. Instead, specific deficiency in pTregs results in spontaneous Th2 inflammation at mucosal surfaces. While loss of pTreg cells under homeostatic conditions favors Th2 inflammation, we hypothesized that pTregs also participate in control of other types of inflammation, such as induced Th17 inflammation. In a mouse model that is prone to development of spontaneous intestinal Th17 disease, we observed that the absence of pTregs significantly exacerbated Th17 inflammation in intestinal tissues without affecting Th2 inflammation. Next we investigated the potential mechanisms of pTreg mediated T-helper type-specific immune regulation. Unlike tTregs, which enter the periphery as a dedicated suppressor T cell subset, pTregs arise from a precursor population that likely has broad T-helper lineage potential. Here we explore the alternative differentiation of would-be pTregs unable to induce Foxp3 to assess the role of pTreg differentiation as a mechanism of “T-helper immune detraction”. Thus, we explore if Foxp3 induction in the periphery is essential to control a range of inappropriate inflammatory responses and the mechanisms by which this is achieved.
Dasatinib is an effective adjuvant to therapeutic vaccination against melanoma (P4269)
Authors: Lowe Devin , Taylor Jennifer , Tawbi Hussein , Kirkwood John , Storkus Walter
Published: January 2023

Dasatinib (DAS) is an FDA-approved inhibitor of receptor tyrosine kinases (RTKs) (i.e., EphA2 and PDGFRβ) involved in vasculature development/maintenance of solid cancers such as melanoma. Administration of DAS transiently normalizes tumor blood vessels and reduces lesional interstitial pressure, hypoxia, and acidosis (which typically impede the efficacy of standard of care therapies). We hypothesized that DAS-induced vascular changes would lead to improved recruitment of therapeutic effector T cells, particularly after specific vaccination. As a model system, C57BL/6 mice bearing established day 7 subcutaneous B16 melanomas were vaccinated (weekly x 2; VAC) with a tumor-specific DC-based vaccine +/- DAS (i.p. daily for days 7-13). We confirmed that DAS reconditioned tumor blood vessels based on a disruption of hypoxia (reduced expression of HIF-1α/HIF-2α and cancer stem cell markers) and pericyte/endothelial cell architecture. Interestingly, DAS also abrogated systemic frequencies of the MDSC/Treg cell immunosuppressor cell populations. Combination VAC + DAS therapy provided superior anti-tumor protection versus either monotherapy, which correlated with the enhanced trafficking of antigen-specific CD8+ T effectors into tumor lesions. Overall, these results support the clinical translation of this combination immunotherapy for the treatment of melanoma.