bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV-1 NEF ligands predominate in the HLA class I of infected CD4+ T cells (APP2P.108)
Authors: Yaciuk Jane , Smith S. , Skaley Matthew , McMurtrey Curtis , Bardet Wilfried , Schafer Fredda , Mojsilovic Danijela , Stewart Christopher , Cate Steven , Jackson Kenneth , Weidanz Jon , Olvera van der Stoep Alex , Brander Christian , West John , Hildebrand William
Published: January 2023

Class I HLA reveal virus-derived ligands to cytotoxic T lymphocytes (CTL) whose function is to eliminate infected cells. The Los Alamos National Laboratory (LANL) database reports numerous HIV-1 CTL epitopes, none of which have elicited protective immunity in vaccine testing. The goal of this study was to determine the number and nature of HIV-1 ligands available for CTL targeting through presentation by the HLA class I of virus-infected cells. Class I presented peptides were recovered from immunoaffinity purified HLA-A*11:01 gathered from HIV-1 (NL4-3)-infected human CD4+ SUP-T1 T cells. These peptides were fractionated by RP-HPLC and mapped by tandem mass spectrometry (MS/MS). Seven HIV-derived peptides were confirmed by MS/MS as unique to infected cells. Twelve LANL HIV epitopes, including the well-studied Gag p24 epitope ACQGVGGPGHK (AK11), were clearly absent from the HLA-A*11:01 of infected cells. Of the 7 HIV-1 ligands found to be presented by A*11:01, 4 were derived from Nef, 2 from Gag, and 1 from Pol. When these 7 ligands were tested for T cell reactivity in a gamma interferon ELISPOT assay using PBMC from HIV-1 infected individuals (including elite controllers), the Nef-derived peptides were found to be highly reactive. These data demonstrate that the class I HLA of HIV-1 infected CD4+ T cells present a handful of viral peptide ligands for recognition by CTL and that reported HIV-1 CTL epitopes might not be presented by the HLA class I of infected cells.
LC3-associated phagocytosis mediates IFN-alpha secretion in response to DNA-immune complexes. (BA3P.207)
Authors: Mehta Payal , Henault Jill , Martinez Jennifer , Riggs Jeffery , Tian Jane , Clarke Lorraine , Sasai Miwa , Latz Eicke , Brinkmann Melanie , Iwasaki Akiko , Coyle Anthony , Kolbeck Roland , Green Douglas , Sanjuan Miguel
Published: January 2023

TLR9 compartmentalization is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the secretion of large amounts of type I IFNs in plasmacytoid dendritic cells. Here, we show that TLR9-mediated production of IFN-α depends on the convergence of the phagocytic and autophagic pathways in response to DNA-containing immune complexes, a process called LC3-associated phagocytosis (LAP). LAP is required for TLR9 trafficking into a specialized IRF7-signaling compartment (ISC) by a mechanism that involves ATG7 but not the conventional autophagic pre-initiation complex. Progression to this compartment did not require AP-3, a protein that mediates TLR9 trafficking and IFN-α secretion in response to CpG oligonucleotides. Our findings unveil a new role for autophagy in inflammatory responses, and provide one of the mechanisms by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict TLR9 activation to pathogenic DNA. Further, we have established phagosomal enrichment assays that will allow us to characterize precise nature ISC as well as the role of LAP in ISC regulation .
The arginine deminase PAD2 is activated by ATP-induced P2X7R signaling in mast cells and modulates the disease course in an inflammatory arthritis model (BA3P.208)
Authors: Mowen Kerri , Guimond David , McKenney Katherine , Arandjelovic Sanja
Published: January 2023

Rheumatoid arthritis is a chronic, systemic inflammatory disease. Citrulline-containing proteins are generated through posttranslational modification of arginine residues in a reaction catalyzed by the Ca2+-dependent peptidyl arginine deiminases or PAD enzymes. Plasma and synovial biopsy specimens from RA patients contain high levels of citrullinated proteins. PAD2 is highly expressed in synovial tissue of RA patients, in close association with citrullinated protein deposits, and its expression correlates with inflammation intensity. The process by which PAD2 is activated and contributes to protein citrullination during RA is unknown. We have identified mast cells as a major source of the PAD2 enzyme. Activation of the P2X7 purinergic receptor by the inflammatory “danger” signal ATP induces PAD2 activity and robust protein citrullination in mast cells. P2X7-mediated activation of PAD2 is sensitive to p38 MAPK and protein kinase C inhibitors, and PAD2 regulates the expression of the TNFR2, Adamts-9, and Rab6b transcripts in mast cells. The PAD2 enzyme and its citrullinated substrate proteins are released from mast cells on activation with ATP. Importantly, mast cells have been shown to play a key role in the development of RA. Our results suggest that P2X7 activation of mast cells may play a role in inflammation by providing PAD2 and PAD2 substrates access to the extracellular space.
Diferuloylmethane suppresses the inflammatory response that mediates type 1 diabetes development (BA3P.133)
Authors: Alard Pascale , Lee Tricia , Rahmouni Faiza , Kosiewicz Michele
Published: January 2023

Because of their various immune defects including the excess production of proinflammatory cytokines by dendritic cells (DC), non-obese diabetic (NOD) mice provide a powerful model to understand type 1 diabetes. Diferuloylmethane (cucurmin), a natural phenol chemical isolated from the plant Curcuma longa (turmeric), is believed to be anti-inflammatory, and has been shown to ameliorate autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease in human and/or animal models. We have, therefore, examined the effects of diferuloylmethane on the inflammatory response leading to type I diabetes. We have found that DC from NOD mice produce decreased pro-inflammatory cytokines, including IL-12, following treatment with diferuloylmethane in vitro. More importantly, diferuloylmethane-treated NOD DC induce fewer IFNγ-producing T cells, shown to be responsible for beta islet destruction. Cell viability was not affected by treatment with diferuloylmethane as shown by 7AAD labeling. Finally, T cells from the spleens of NOD mice treated in vivo with diferuloylmethane for several weeks produced significantly less IFNγ. Overall, our data show that diferuloylmethane suppresses in vitro and in vivo the differentiation of inflammatory cells involved in type 1 diabetes development. Diferuloylmethane may, therefore, have therapeutic potentials for preventing the development of type 1 diabetes.
Central nervous system-specific expression of C-reactive protein exacerbates experimental autoimmune encephalomyelitis. (BA3P.202)
Authors: Wright Tyler , McCrory Mark , Morgan Todd , Bali Namrata , Hou Xiaogang , Finch Caleb , Szalai Alexander
Published: January 2023

Long considered merely a marker of inflammation, new evidence indicates that C-reactive protein (CRP) may also affect the maintenance of health and propagation of disease. Our lab previously showed that human CRP, hepatically synthesized by human CRP transgenic mice (CRPtg), delays onset and reduces severity of Experimental Autoimmune Encephalomyelitis (EAE; a model of Multiple Sclerosis). Though no causal data are available for humans, research suggests that unlike CRPtg, expression/deposition of CRP in the CNS associates with disease, rather than protection. Though human CRP expression in both man and CRPtg mouse is largely hepatic, studies have shown that it is also expressed by multiple cells of the CNS, particularly during neuroinflammation. To test CNS-expressed CRP’s impact on EAE, we made transgenic mice wherein the neuron-specific Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) promoter drives human CRP expression. We confirmed that human CRP expression is restricted to the CNS in these neuronal CRPtg (nCRPtg) mice. In contrast to CRPtg, CNS-specific expression of CRP neither delays EAE onset nor improves its symptoms. As in humans, CNS-expressed CRP associates with more severe disease. In summary, observations made with CRPtg vs. nCRPtg reveal a previously unappreciated site-specificity to the impact of CRP on the CNS. The possibility that CRP mediates beneficial vs. detrimental actions depending on the local cellular environment will be investigated.
Transcription factor Fli-1 directly regulates the expression of IL-6 (BA2P.114)
Authors: Zhang Xian , Sato Shuzo , Lennard Richard Mara
Published: January 2023

Fli-1, a member of the Ets family of transcription factors, is highly expressed in immune cells and endothelial cells and is implicated in the development of systemic lupus erythematosus (SLE) in both human patients and mouse models. Increased expression of IL-6 is also involved in the SLE and other autoimmune disease development. We examined the role of Fli-1 on production of IL-6 in this study. We found expression of IL-6 was significantly reduced in sera from Fli-1 heterozygous knockout MRL/lpr mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal), a murine model of lupus, compared to wild-type littermates. The production of IL-6 were significantly decreased in endothelial cells transfected with specific Fli-1 siRNA compared to cells transfected with negative control siRNA after LPS stimulation. We demonstrated that Fli-1 directly binds to the IL-6 promoter by Chromatin Immunoprecipitation (ChIP) assay and transient transfection assays show that Fli-1 drives transcription from the IL-6 promoter in a dose-dependent manner. Our data indicate that transcription factor Fli-1 binds to the promoter of IL-6 and directly regulates the expression of IL-6.
Autoimmune susceptibility imposed by public TCRβ chains (BA2P.125)
Authors: Geiger Terrence , Zhao Yunqian , Nguyen Phuong , Vogel Peter
Published: January 2023

The MHC is the largest genetic risk factor for autoimmunity. It imposes risk in association with a complementary autoantigen specific TCR repertoire. How this occurs is unresolved. Each individual’s TCR repertoire is largely unique, and should therefore not contribute to population-wide disease risk. However, due to biases in TCR recombination, a subset of α and β sequences are shared by most individuals, or public. If these public α or β chains modulate a TCRαβ heterodimer’s likelihood of productively engaging autoantigen-MHC, they could influence autoimmune susceptibility. We demonstrate that disease-associated regulatory and effector TCRβ repertoires in autoimmune encephalomyelitis preferentially incorporate public TCR. Some disease-associated public TCRβ, when transgenically expressed, could further endow T cells with autoantigen specificity in the absence of priming, and demonstrate a subordinate role of the non-shared, endogenous TCRα chains in autoantigen specificity. Further, 2 of 7 Tg public and 0 of 7 Tg private TCRβ isolated from high frequency CNS-infiltrating T cells, provoked spontaneous, early-onset autoimmunity when associated with endogenous TCRα. These findings support a disproportionate contribution of public TCR to immunopathology and implicate them, in association with risk-associated MHC, in disease susceptibility. They further suggest that subsets of public sequences may serve as disease-specific biomarkers or therapeutic targets in autoimmunity.
The affinity of MOG specific CD4 + T cells does not predict cross-recognition to NFM in demyelinating autoimmune disease (BA2P.127)
Authors: Blanchfield J. Lori , Evavold Brian
Published: January 2023

TCRs can recognize and cross-react to multiple antigens, but little is known of how cross-reactivity might contribute to the etiology of demyelinating autoimmune disease. Here we extend the analysis of monoclonal 2D2 TCR cross-reactivity for MOG38-49 (myelin oligodendrocyte glycoprotein) and NFM18-30 (neurofilament medium protein) to polyclonal T cells. Utilization of the 2D micropipette adhesion frequency assay allows definition of antigen specificity and affinity of individual T cells for peptide:MHC ex vivo. We show that the frequency of CD4+ T cells from the CNS recognizing MOG, NFM, or both fluctuate during the course of MOG35-55 induced experimental autoimmune encephalomyelitis. Higher affinity T cells are maintained as a rare population within the repertoire yet the frequency of higher affinity, MOG specific T cells increase to a greater extent than NFM specific T cells during disease and MOG specific T cells exhibit higher affinities (10-4 to 10-3) than NFM specific T cells (<10-4). Importantly, cross-recognition is not limited to higher or lower affinity clones. We have cloned an encephalitogenic T cell that is high affinity for MOG and low affinity for NFM, which contrasts the 2D2 TCR exhibiting a medium affinity for NFM and a very low MOG affinity. On a population level, TCR recognition of one or more CNS antigens is dynamic within the chronic autoimmune repertoire warranting future research to understand their individual contributions to disease.
Linker for activation of T cells (LAT) in the regulation of T cell activation and autoimmunity (BA2P.121)
Authors: Zhang Weiguo , Sullivan Sarah , Zhu Minghua , Lewis Catherine
Published: January 2023

The transmembrane adaptor protein LAT (Linker for Activation of T cells) is essential in T cell development, activation, survival, and homeostasis. Upon T cell activation, it is tyrosine phosphorylated and interacts with many signaling proteins, such as Grb2, Gads, and PLC-γ1. The mutation of tyrosine 136 on LAT abrogates its interaction with PLC-γ1, causing defective TCR-mediated signaling. Mice harboring this mutation, LATY136F, have significantly impaired thymocyte development; however, they rapidly develop a severe lymphoproliferative disease marked by the uncontrolled expansion of Th2-skewed CD4+ T cells, high levels of IgE and IgG1, and autoantibody production. In this study, we assessed the contribution of multiple signaling pathways in this disease using mice deficient in Gads and RasGRP1 and mice with constitutive activation of the NFAT and NF-κB pathways. We also examined the role of MHC and CD4 in the development of these pathogenic T cells and further investigated how T follicular helper cells and cytokines contribute to LAT-mediated autoimmunity.
Phenotypic and functional characterization of neutrophils from lupus-prone mice (BA4P.223)
Authors: Cowan Catharine , Dai Rujuan , Heid Bettina , Ahmed S.
Published: January 2023

Neutrophils constitute a critical component of cellular immunity that eliminate infectious organisms through oxidative burst, phagocytosis and the formation of neutrophil extracellular traps (NETs). Recently it has been reported that neutrophils contribute to autoimmunity through NET formation and interactions with auto-reactive immune cells. Our goal is to characterize phenotypic and functional properties of neutrophils in mouse models of systemic lupus (SLE) to determine if and how neutrophil dysregulation occurs. Using flow cytometry we have measured neutrophil populations in healthy control and diseased SLE mouse models. We found neutrophils are consistently increased in the spleen of MRL-Faslpr, C57BL/6-Faslpr and NZBWF1 lupus mice compared to controls. Further characterization of neutrophil functions including phagocytosis, reactive oxygen production, NET formation, protease activity and histone citrullination indicate increased DNA release, increased citrullination and alterations in serine protease activity in lupus neutrophils. Differences in the results between the NZBWF1 and MRL-Faslpr models suggest the background genetics play a role in neutrophil dysregulation in these two models. Ultimately we hope to understand the cause of altered neutrophil function in mouse models of SLE, whether they contribute to lupus disease progression, and hopefully develop newer targeted therapies for lupus.
A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)
Authors: Spatz Linda , Novak Brittany , Samuel Sherin , Paredes Jenny
Published: January 2023

One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. Anti-EBNA-1 antibody obtained from mice from all three strains was observed to cross-react with dsDNA. However, the frequency of mice producing cross-reactive antibody was greater and the amount of cross-reactive antibody was higher in BAFF than wild type mice. This suggests that a viral protein such as EBNA-1 may trigger the development of antibodies that cross-react with dsDNA and that genetic susceptibility to lupus may enhance the development of cross-reactive antibodies upon viral exposure.
Lymph nodes and Peyer’s patches harbor resident CD4+ T cells that accumulate after prolonged antigen exposure (CAM1P.230)
Authors: Ugur Milas , Schulz Olga , Pabst Oliver
Published: January 2023

CD4+ T cells can acquire various migratory properties after immune responses to provide enhanced immuno-surveillance together with effective protection. However, the comprehensive analysis of these migratory properties has been difficult due to the highly dynamic nature of T cell circulation. We developed two independent methods to analyze the migration of effector/memory CD4+ T cells by long-term in vivo cell tracking. We identified a resident population of effector/memory CD4+ T cells that stays in lymph nodes and Peyer’s patches (PPs) without circulation or proliferation. Resident CD4+ T cells constitute up to 50% of all effector/memory cells, including, but not limited to, follicular helper T cells. This heterogeneous population of resident cells expresses low levels of egress-promoting sphingosine-1-phosphate receptor 1 (S1pr1) and possesses a distinct TCR repertoire. Furthermore, resident cells constituted a significant portion of all CD4+ T cells in PPs and accumulated in these organs after continuous antigen exposure. Our results define a previously unrecognized population of effector/memory CD4+ T cells in lymphoid tissues which might perform similar functions as non-lymphoid tissue-resident memory T cells.
Effective mentoring of undergraduates in an immunology research laboratory (EDU1P.252)
Authors: Chatterjea Devavani , Tonc Elena
Published: January 2023

Rapid turnover, limited time availability, steep learning curve for bench skills, need for close supervision are challenges in the effective engagement of undergraduate students in active research laboratories. We have met this challenge in our immunology research group with early recruitment of first and second year students, year-round research activity and weekly lab meetings, scaffolded transition to independent bench work, peer-to-peer mentoring, an integrated majors’ research methods course, and a “5th year internship” for a selected student to work full-time for a year after graduation. Early recruitment allows motivated students to spend up to 3-4 years in the lab. Year-round journal clubs and lab meetings support a culture of productivity and accountability. Students collaborate, as appropriate, in the entire arc of research from generating preliminary data for grants to submitting manuscripts. In the past 7 years, 30% of 40 students trained have earned co-authorship on five peer-reviewed, published manuscripts to date. A critical outcome of this approach is an authentic ownership of research problems and motivation to contribute to the broader scientific community that is rare among undergraduates. This experience prepares them to pursue their own research careers after college. Of my 26 research students who have graduated, ten (38%) are currently enrolled in PhD programs in the biomedical sciences and 12 (46%) have worked in biomedical research.
Academic civic engagement in the Immunology classroom: from grant proposals to immunology theater (EDU1P.255)
Authors: Walser-Kuntz Debby
Published: January 2023

There is a growing recognition and sense of urgency that scientists need to be better equipped to communicate scientific ideas to the general public. Improved communication is an opportunity with significant policy, behavior, and funding implications. For the past 5 years, I have integrated civic engagement projects into the upper-level Immunology course I teach at Carleton College - a small liberal arts college located in Northfield, MN - in order to provide an opportunity for students to practice translating complex, jargon-rich information into engaging materials for use in the local community. These projects are by nature reciprocal; they provide an authentic learning opportunity for students and a very real benefit for our community partners. Additional student outcomes include increased awareness of local public health issues, enhanced understanding of the connection between immunology and real world problems, and improved scientific communication skills, including in some cases, grant writing skills. Civic engagement projects range from writing grant proposals or research reports for local nonprofit organizations to performing “Immunology Theater” with at-risk high school students or developing comics and videos to teach about sexually transmitted diseases. Assessment of the effectiveness and impact of the civic engagement projects included alumni surveys (2009-2013 graduates; n=192), evaluation of student reflections, and community partner surveys.
Self-renewing HSCs respond directly to TLR4 ligand and are compromised in obesity, a disease linked to chronic TLR4 stimulation (HEM2P.270)
Authors: Liu Ailing , Kumar Rashmi , Stefanovic-Racic Maja , O'Doherty Robert , Ding Ying , Borghesi Lisa
Published: January 2023

Hematopoietic stem cells (HSCs), the sole source of blood cells throughout life, sense and respond to pathogen via toll like receptors (TLRs). Two TLR4 ligands that are persistently elevated in disease include bacterial lipopolysaccharide (LPS) and obesity-associated dietary lipids. Here, we show that CD150+ HSCs and downstream multipotent progenitors express TLR4 at levels comparable to bone marrow derived macrophages. When stimulated with LPS in culture, purified HSCs exhibit a 3-fold increase in myeloid output relative to vehicle controls. Moreover, LPS sensitivity is completely ablated in TLR4 KO HSCs. Thus, HSCs respond directly to LPS in a TLR4-dependent manner. We recently showed that chronic exposure to low-dose LPS drives HSC exhaustion and myeloid bias in vivo. We tested if diet-induced obesity, a disease marked by chronically elevated levels of the TLR4 agonists LPS and dietary fatty acids, compromised HSCs. Mice fed high fat diet for 16-20 weeks but otherwise unmanipulated had a 15-20% reduction in CD150+ self-renewing HSCs. The magnitude of HSC reduction strongly correlated with elevated blood glucose, a metabolic indicator of insulin resistance, and liver triglycerides, a morphometric risk factor for diabetes. This is the first study showing a link between diet-induced obesity and HSC integrity. Our findings imply that blood glucose levels may predict the severity of the HSC defect.
The essential role of Ligase 4 in hematopoietic stem cell homeostasis (HEM2P.257)
Authors: Park Jihye , Welner Robert , Tenen Daniel , Yan Catherine
Published: January 2023

Increased DNA damage is the principal factor that contributes to the functional decline in hematopoietic stem cell (HSC) renewal. Non-homologous DNA end-joining (NHEJ) is critical for the repair of DSBs in all cell types. In recent studies, NHEJ is implicated in the repair of DNA damage in quiescent long-lived HSCs in ex-vivo cell-based assays. However whether NHEJ plays a physiological role in the repair of DNA damage required for maintaining HSC homeostasis remains unclear. Here we assess whether Lig4 is critical role in HSC homeostasis by assaying HSC functions in a knock-in mouse model of the human recessive hypomorphic R278H mutation in Lig4. Whereas long-lived HSCs are quiescent and are continually replenished, we find that these HSCs in R278H mice have a severe defect in quiescence instigated by increased reactive oxygen species ,oxidative DNA damage, cycling and apoptosis. Our studies implicate the attenuation of Sirtuin 1, a NAD+-dependent histone protein deacetylase , as the key causal mechanism. Our preliminary data show treatment with the Sirt1 activating compound resveratrol rescues the survival of Lig4R/R HSC progenitors in long-term cultures. Our hypothesis is that increased ROS caused by impaired Sirt1 activities and autophagy underlies Lig4R/R HSC defects. These observations we have made implicate a critical role for Lig4 in HSC homeostasis, and in regulations impacting the DNA damage response-signaling cascade and anti-oxidant defense system.
Mechanisms of TLR4-mediated impairment of murine and human HSCs (HEM2P.258)
Authors: Borghesi Lisa , Wang Yujuan , Lu Ailing , Santos Patricia , Ding Ying , Payne Kimberly
Published: January 2023

Chronic exposure to toll-like receptor 4 (TLR4) ligand drives murine hematopoietic stem cell (HSC) exhaustion and loss of lymphoid potential. This finding is significant because TLR4 is activated by plasma lipopolysaccharide (LPS) that is elevated in patients with chronic infection as well in obese individuals. For the first time, we show that human HSCs are severely compromised by TLR4 stimulation. Mice engrafted with human cord blood cells and then exposed to low-dose LPS have variably reduced human CD34+ HSCs with near complete ablation of downstream common lymphoid progenitor (CLP) and B cell precursor subsets in bone marrow. By contrast, myeloid precursors are grossly intact. Peripheral tissue subsets similarly exhibit myeloid>lymphoid skewing. We have shown that LPS-exhausted murine HSCs have reduced levels of the major transcription factor E47. Our newly published findings demonstrate a mechanistic link between E47 and the p21 cell cycle regulator. CD150+ HSCs from mice doubly haploinsufficient for E47 and p21 exhibit hyperproliferation, poor self-renewal, and selective myeloid bias. Finally, we show that murine CD150+ HSC and multipotent progenitors (MPP) express TLR4, and directly respond to LPS in a TLR4-dependent manner. Together, our findings suggest that chronic activation of TLR4 damages the functional integrity of not only murine HSC but also human HSC in vivo, via a mechanism linked to disruption of the E47-p21 pathway.
Rpl22 deficiency predisposes to leukemic transformation by blunting DNA damage responses (HEM2P.263)
Authors: Harris Bryan , Perrigoue Jacqueline , Verma Amit , Sykes Stephen , Fahl Shawn , Wiest David
Published: January 2023

Mutations that impair DNA repair mechanisms lead to accumulation of DNA damage within hematopoietic stem cells (HSC), resulting in their functional exhaustion or development into leukemic stem cells (LSC). Surprising, we have found that one such mutation in the ribosomal protein Rpl22 is able to predispose to leukemic transformation and this appears to involve impaired responses to DNA damage. Indeed, Rpl22-defiicency renders lineage negative Sca-1+, c-kit+ (LSK) hematopoietic progenitors resistant to killing by the DNA-damaging agent 5-fluorouricil (5FU), which is used to treat a variety of malignancies. Interestingly, while 5FU treatment increases γH2AX phosphorylation in Rpl22-deficeint LSK, indicating that cellular DNA has been damaged, these cells fail to induce transcriptionally active p53 because of a defect in their DNA-damage response signaling. This leads to an accumulation of LSK harboring damaged DNA, which we hypothesize predisposes them to transformation. Consistent with this viewpoint, we have found that Rpl22-/- LSK do exhibit an enhanced predisposition to transformation by the oncogenic function of MLL-AF9 in mouse models. This also appears to be true in human cancer patients, since AML patients that express less Rpl22 exhibit reduced survival. Taken together, these data support our hypothesis that Rpl22-deficiency impairs the activation of p53 in response to DNA damage and this predisposes cells to transformation and more aggressive leukemias.
Regulation of γδ T cell effector fate specification by ribosomal protein paralogs (HEM3P.285)
Authors: Fahl Shawn , Lee Sang-Yun , Coffey Francis , Perrigoue Jacqueline , Wiest David
Published: January 2023

γδ T cells are key mediators of inflammation that play a crucial role in preserving epithelial barriers and eradicating cutaneous tumors. The molecular mechanisms that control γδ T cell development and function, however, remain poorly understood. We have previously reported that the ribosomal protein paralogs Rpl22 and Rpl22l1 have antagonistic roles during hematopoietic stem cell emergence. Consequently, we sought to determine if Rpl22 and Rpl22l1 might play roles in more differentiated hematopoietic lineages. We have now demonstrated that γδ T cell development is not arrested in the absence of Rpl22 or Rpl22l1, suggesting these proteins are dispensable γδ T cell development; however, Rpl22 and Rpl22l1 do appear to regulate the specification of γδ effector fate during development in the thymus and have opposing roles in this process. Specifically, Rpl22-deficiency enhances the generation of IL-17-producing γδ T cells. In contrast, Rpl22l1-deficiency leads to an expansion of IFN-γ-producing γδ T cells and, in particular, the Vγ1.1+ innate-like γδ T cell subset. Rpl22 and Rpl22l1 are RNA-binding proteins that exert their control over developmental processes extraribosomally (i.e., away from the ribosome) by binding and regulating the expression of cellular RNA targets. Our efforts to identify the key cellular RNAs through which Rpl22 and Rpl22l1 influence γδ T cell effector fate will be presented.
A committed hemopoietic precursor to innate lymphoid cells (HEM3P.277)
Authors: Constantinides Michael , McDonald Benjamin , Verhoef Philip , Bendelac Albert
Published: January 2023

Innate lymphoid cells specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers. Their growing diversity and close similarities with previously characterized NK and LTi cells have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 based solely on cytokine properties. Elucidating their development pathways is essential to resolve their lineage relationships and dissect their respective functions in disease and homeostasis. Using lineage tracing and transfer studies, we identified and characterized a novel subset of lymphoid precursors in fetal liver and adult bone marrow that transiently expressed high amounts of PLZF, a transcription factor previously associated with NKT cell development. PLZF-high precursors were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 lineage differentiation potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.1+ DX5- ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the frequency and function of several ILC subsets, but did not affect LTi or NK cells. These findings establish novel lineage relationships between ILC, NK and LTi cells and identify the elusive precursor to ILC, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes.
mTOR signaling controls iNKT cell differentiation and effector function (HEM3P.278)
Authors: Zhang Lianjun , Rüegg Markus , Hall Michael , Mach Jean-Pierre , Romero Pedro , Donda Alena
Published: January 2023

Invariant natural killer T (iNKT) cells represent a unique T cell lineage that bears NK cell markers and a semi-invariant TCR recognizing glycolipids in the context of CD1d presentation. iNKT cells rapidly produce a variety of cytokines such as IFNg, IL-4, IL-2, IL-10 upon stimulation. Thus, iNKT cells play critical roles in bridging innate and adaptive immunity and have been implicated in multiple physiological and pathological settings. iNKT cells exhibit high proliferative capacity compared with conventional T cells during early stage development in the thymus. The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that integrates various environmental cues to regulate cell growth, proliferation and metabolism. Given the significance of mTOR signaling in controlling cell proliferation and metabolism, we have explored the role of mTORC1 and mTORC2 in iNKT cell development and function by using mice with T cell-restricted deletion of Raptor or Rictor. Here, we show, on the one hand, that mTORC1 is selectively required for iNKT cell but not for conventional T cells or Treg thymic development. On the other hand, mTORC2 is required to a lesser extent for thymic development as well as effector function of iNKT cells. In this regard, proper iNKT cell development and acquisition of effector functions is under the control of a dedicated mTOR signaling.
Macrophages show distinct profiles and origins between the exocrine and endocrine pancreas (HEM3P.279)
Authors: Calderon Boris , Epelman Slava , Carrero Javier , Unanue Emil
Published: January 2023

Islets of Langerhans contain myeloid-derived cells with important features and properties. Our studies have shown islet myeloid cells to play a central role in retaining diabetogenic T cells during early stages of autoimmune diabetes and the initiation of the inflammatory process. We have studied in-depth the phenotype of the islet myeloid cells in the NOD and non-diabetic mouse strains. The major phagocyte in non-inflamed islet is a macrophage (MΦ) identified by cell markers as well as by gene expression analysis. Under steady state, islet MΦ did not arise from circulating monocytes: parabiosis of CD45.1 with CD45.2 mice showed minimal mixing of islet MΦ. Ccr2-/- mice showed no impairment in the number of islet MΦ. In sum, islet MΦ are self-maintained. Ongoing experiments indicate a slow replication rate. Lineage tracing using a Flt3-cre reporter mouse showed MΦ in the exocrine pancreas and in the islets of the adult mouse to derived from different lineages. Majority of the exocrine pancreatic MΦ were yolk sac/fetal liver-derived, while islet MΦ were bone-marrow derived. Islet MΦ required the expression of CSF-1, while exocrine pancreatic MΦ did not. Absence of islet MΦ in CSF-1 deficient mice resulted in reduced β-cell mass and impaired glucose tolerance, highlighting a symbiosis role between islet MΦ and β-cells for islet homeostasis. Current studies are aimed to replace the islet MΦ, since MΦ are involved in many diseases and represents an attractive therapeutic target.
The role of plasma gelsolin in cardiopulmonary bypass induced acute lung injury in infants and young children: a pilot study (HUM1P.310)
Authors: Shu Qiang , Shi Shanshan , Chen Chi , Zhao Dongyan , Liu Xiwang , Cheng Baoli , Wu Shuijing , Lin Ru , Tan Linhua , Fang XiangMing
Published: January 2023

Background Acute lung injury induced by CPB (CPB-ALI) is a common and serious complication. Infants and children are more prone to CPB-ALI. The purpose of this study was to investigate the perioperative changes of plasma gelsolin (pGSN) in patients ≤3 years of age undergoing cardiac surgery with CPB, and determine whether pGSN are associated with the occurrence and severity of CPB-ALI. Methods 77 patients ≤ 3 years of age with CHD were enrolled and assigned to ALI and non-ALI groups according to the American-European Consensus Criteria. Plasma gelsolin and total protein were measured at 8 perioperative time points. Results 27 patients developed CPB-ALI. The normalized pGSN (pGSNN) of ALI group decreased significantly at 6 hours after CPB (p=0.046), which was much earlier than that of non-ALI group (p=0.009); The pGSNN of ALI group before CPB (p=0.001), 6 hours after CPB (p=0.001) and 48 hours after CPB (p=0.032) were all significantly lower than those of non-ALI group; PGSNN before CPB was the only independent risk factor predicting the occurrence of CPB-ALI (OR, 1.069; 95%CI, 1.001-1.035; p=0.033) with an AUC of 0.753 (95%CI, 0.626-0.880); The optimal cutoff value of pGSNN before CPB was 2641.8. Conclusions Patients developing CPB-ALI had lower plasma gelsolin reservoir and a much more amount and rapid consumption of plasma gelsolin early after operation; PGSNN before CPB was a useful early and sensitive predictor of CPB-ALI in infants and young children.
A pathogenic role for SHP-1 activity in macrophages (HUM1P.321)
Authors: Watson Neva , Massa Paul
Published: January 2023

Microbial infections elicit inflammatory responses that eradicate infection and remove damaged cells. Regulation of these events is critical as excessive inflammation may cause substantial bystander tissue destruction in the process of pathogen clearance while a deficient response may be insufficient to clear infections and damaged cells. Defective clearance of dying cells can trigger devastating chronic inflammatory diseases, and we report an unanticipated role for SHP-1 in facilitating this process. SHP-1 restricts innate immunity to pathogens, and therefore mice lacking SHP-1 suffer macrophage-mediated destruction of myelin in the CNS following infection with Theiler’s murine encephalomyelitis virus (TMEV). To the contrary, we have discovered that although resistant to CNS disease, TMEV-infected wild type animals develop a severe debilitating skeletal muscle disease not seen in SHP-1-/- mice. We show that SHP-1-mediated restriction of macrophage responses in WT mice corresponds with insufficient clearance of damaged tissue and increased muscle dysfunction. As SHP-1 controls macrophage phagocytic activity, our data suggests that increased macrophage activity in SHP-1-/- mice restricts muscle disease through rapid clearance of infected and damaged myofibers. These findings demonstrate a unique role of SHP-1 in exacerbating rather than resolving disease pathology by inhibiting clearance of cellular debris. These data suggest novel therapies for chronic inflammatory diseases.
Genomic polymorphism of CD209 gene is associated with sickle cell disease in African children (HUM2P.328)
Authors: Thomas Bolaji , Navarro Kristen , Guindo Aldiouma , Li Yi , Diallo Dapa
Published: January 2023

Sickle cell disease (SCD) presents with multiple complications and marked variability in severity among individuals and possible underlying differences in ethnic-specific disease pathogenesis. The CD209 gene otherwise called DC-SIGN encodes a transmembrane receptor and is expressed on the surface of dendritic cells with multiple studies showing association between its variants and susceptibility to infections. In SCD, vaso-occlusive crises and infections are common occurrences, and the role of CD209 is currently unknown. We examined the genotypic and allelic diversity of CD209 gene and determined its association with SCD in African children. Genomic DNA from 145 children (HbSS) and 244 controls (HbAA) were analyzed for polymorphic variants of CD209 by PCR-RFLP. All SNPs follow Hardy-Weinberg equilibrium (P>0.05). There was a significant difference in both genotypic and allelic frequencies (P=0.004 and P=0.015 respectively) of a promoter variant in CD209, DC-SIGN1-336 between SCD and controls. Additionally, genotype GG variant is less frequent (10.3% versus 22.1%) and significantly associated with sickle cell disease (OR=0.3868; P=0.0010) and this reduced frequency reveals an impaired or reduced capacity to mount an immune response to infectious pathogens. The implications of this finding for delineating disease co-morbidity or as genetic modifiers of SCD pathophysiology, and its significance in African American children with SCD will be explored
A “reduced function” PTPN22 variant modulates the Toll-like receptor-driven type 1 interferon response in systemic lupus erythematosus (HUM2P.329)
Authors: Wang Yaya , Yamamoto Ami , Gillespie Emily , Fazeli Parastoo , Peterson Erik
Published: January 2023

Most systemic lupus erythematosus (SLE patients) exhibit whole-blood increased expression of type I interferon (IFN)-inducible transcripts, known as the "IFN signature". A variant allele of the PTPN22 gene is associated with increased risk of SLE. We recently demonstrated that PTPN22-encoded lymphoid tyrosine phosphatase (Lyp) promotes Toll-like receptor (TLR)-driven type 1 IFN production and type I IFN-dependent host defense and immunoregulation. Further, the disease-associated variant LypR620W (“LypW”) shows reduced function in promoting TLR-driven type 1 IFN-dependent immunity. Here we report that PBMC from LypW carrier SLE patients exhibit reduced type 1 IFN and type 1 IFN-inducible gene expression upon TLR7 activation. The percentages of plasmacytoid dendritic cells (pDC) producing IFNα, as well as the per-cell production of IFNα, are decreased LypW carrier SLE patients. Notably, we found that similar numbers of LypW carriers and non-carriers in SLE patients exhibit elevated IFN gene signatures in whole blood. Together, these data indicate that LypW SLE patient display reduced mononuclear cell capacity for TLR-induced type 1 IFN, despite exhibiting increased systemic type 1 IFN signaling. The findings raise the possibility that autoimmune phenomena in LypW SLE patients may reflect defective type 1 IFN-dependent host defense. Further, they suggest that therapeutic strategies to boost type 1 IFN-driven processes may benefit the LypW carrier subgroup of SLE patients.
Circulating CD4+ T cells in narcolepsy patients react to hypocretin and cross-react with hemagglutinin from an influenza strain linked to increased narcolepsy incidence (HUM2P.336)
Authors: Mellins Elizabeth , Macaubas Claudia , Jiang Wei , De la Herrán-Arita Alberto , Kornum Birgitte , Mahlios Joshua , Hou Tieying , Mignot Emmanuel
Published: January 2023

Narcolepsy, a disorder characterized by daytime sleepiness, cataplexy, and REM sleep abnormalities, is caused by the loss of ~70,000 hypothalamic neurons that produce the wake-promoting neuropeptide hypocretin (HCRT). The strong association of narcolepsy with HLA DQA1*01:02/DQB1*06:02 (DQ0602) has led to the hypothesis that narcolepsy is an autoimmune disease, but direct evidence has been lacking. We recently reported discovery of circulating CD4+ T cells reactive to two homologous DQ0602-binding HCRT epitopes (HCRT56-68, HCRT87-99) in narcolepsy patients, but not in DQ0602+ healthy control subjects. Based on epidemiologic evidence linking narcolepsy with the 2009 H1N1 influenza A strain (pH1N1) we measured levels of circulating HCRT56-68, HCRT87-99 reactive T cells in narcolepsy patients who had received the seasonal influenza vaccine (containing pH1N1) and found them to be increased. We identified a pH1N1-specific hemagglutinin epitope with homology to the narcolepsy-associated HCRT epitopes; in vitro stimulation of narcolepsy CD4+T cells with this epitope increased the frequency of HCR-reactive T cells. To directly test for molecular mimicry between the HCRT and pH1N1 epitopes, we have used tetramer staining and single cell TCR sequencing. We conclude that narcolepsy is associated with an autoimmune response to HCRT, which can be triggered by cross-reactive T cell responses to pathogen-derived peptides.
Fluvastatin-mediated suppression of IgE-induced cytokine production can be enhanced by TGFβ1 (HYP3P.403)
Authors: Haque Tamara , Abdul-Qayum Amina , Ryan John
Published: January 2023

Fluvastatin, an HMG-CoA reductase inhibitor known for its role in the treatment of hypercholesterolemia and cardiovascular disease, has recently been shown to play a role in the immune response. Given the critical role that mast cells play in allergy and inflammatory diseases such as asthma, we assessed the effect of Fluvastatin on mast cell function. We also examined the impact of TGFβ1, a cytokine we have previously shown capable of limiting mast cell function, in combination with Fluvastatin. We find that Fluvastatin suppresses IgE-mediated cytokine production in mouse mast cells. Prior exposure to TGFβ1 enhanced the effects of Fluvastatin, such that the IC50 for Fluvastatin-mediated suppression was 3-4-fold lower in the presence of TGFβ1 than in its absence. Interestingly, this effect was found for IgE-induced production of IL-6 and TNF, while TGFβ1 did not alter the suppressive effects of Fluvastatin on IL-13 or MCP-1. Current experiments seek to unravel the mechanism behind this selective effect of TGFβ1.
SPE-7 but not Epsilon-26 anti-dinitrophenyl-Immunoglobulin E induces antigen-dependent thermal hyperalgesia accompanying equivalent inflammatory responses during passive cutaneous anaphylaxis reactions in mice (HYP3P.405)
Authors: Chatterjea Devavani , Ashbaugh Alyssa , Tonc Elena , Mack Madison , Martinov Tijana , Sykes Akilah
Published: January 2023

Sensitization of mast cells with SPE-7 anti-DNP-IgE alone initiates inflammatory cytokine production in vitro. In contrast, ε26 anti-DNP IgE does not induce inflammatory cytokines independent of antigen-stimulation. We tested these two anti-DNP IgE clones in our model of murine thermal hyperalgesia. We found pronounced hind paw hyperalgesic responses lasting to at least 6 hours following antigen challenge 24 hours after sensitization with SPE-7 but not ε26. SPE-7/DNP did not induce greater levels of mast cell degranulation in the hind paw. Differential hyperalgesic responses were accompanied by otherwise equivalent inflammatory changes measured including histamine release, neutrophil influx, expression of TNF-α, IL-1β, and IL-6 genes and changes in levels of TNF-α, IL-1, and IL-6 proteins. Inflammatory responses and hyperalgesia were dependent on antigen challenge and were not detected in sham-challenged mice. Hyperalgesia responses or other inflammatory changes were not detected in the hind paw tissue post-sensitization in the 24 hours prior to antigen challenge. We demonstrate a novel behavioral, antigen-dependent difference between reactions mediated by these IgE antibody clones in vivo in mice. We are currently further investigating the inflammatory cytokine milieu and measuring changes in mRNA and protein levels of nociceptive signaling molecules provoked by IgE/Ag-induced cutaneous anaphylaxis to elucidate the mechanisms underlying this differential pain outcome.
TGFβ1 inhibits IL-33-mediated mast cell activation: a role for genetic background (HYP3P.408)
Authors: Ndaw Victor , Kolawole Elizabeth , Ryan John
Published: January 2023

IL-33 is a recently discovered cytokine that functions as an alarmin member of the IL-1 family that plays a critical role in Th2 responses and mast cell activation. Numerous recent studies have demonstrated IL-33-mediated function via the T1/ST2 receptor on mast cells to elicit cytokine production and adhesion, as well as enhancing IgE-mediated degranulation. However, because of reported pro- and anti-inflammatory effects, the role of IL-33 in the pathogenesis of allergic diseases is not completely understood. In this study, we found that TGFβ1 suppresses IL-33-mediated production of TNFα, IL-6 and MCP-1, and reduces T1/ST2 receptor expression on mast cells. These responses were concentration-dependent. Consistent with our previous work, we found that bone marrow-derived mast cells (BMMC) from 129/Sv mice were more resistant to TGFβ1 treatment than C57BL/6 BMMC. We propose that TGFβ1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.