bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of genes with bivalent chromatin in memory CD8 T cells (IRM4P.496)
Authors: Kim Jiewan , Subedi Kalpana , Wersto Robert , Zhao Keji , Peng Weiqun , Weng Nan-ping 
Published: January 2023

Abstract
Genes containing both histone methylation H3K4me3 and H3K27me3 markers at the promoter are so called bivalent genes that have been implicated in stem cell differentiation and function. Our previous study identified several dozen bivalent genes in memory CD8 T cells (central memory, TCM and effector memory, TEM). But the role of bivalent genes in CD8 T cells is not understood. Our current focus is to determine the characteristics and functions of these bivalent genes during CD8 memory T cell activation. We used freshly isolated TCM and TEM from human blood that were stimulated by anti-CD3 plus anti-CD28 in vitro, and analyzed histone methylation and mRNA levels of these bivalent genes in resting and activated CD8 memory T cells. Our preliminary results showed that the bivalent status of most genes was stable but corresponding mRNA change was more detectable. We also identified a few bivalent genes whose status changed to become “monovalent”; either H3K4me3 or H3K27me3 after activation. We plan to determine the functional role of these bivalent genes that are altered after activation, and whether the bivalent histone methylation is present on a single gene allele or on two different alleles by sequential ChIP. Our study will shed new insight into the details of histone methylation on gene loci and the function of these bivalent genes in memory CD8 T cells. “This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging “
FoxO1 directly regulates the expression of PD-1 and sustains viral specific CD8+ T cell responses during chronic infection (IRM4P.501)
Authors: Staron Matthew , Gray Simon , Kaech Susan 
Published: January 2023

Abstract
Understanding the signaling and transcriptional pathways that regulate the function, survival and homeostasis of CD8+ cytotoxic T lymphocytes (CTLs) during chronic infection is fundamental to the development of therapies to treat chronic infections such as HIV, HSV, and HCV. Of these, the PI3K/AKT/mTOR signaling pathway is of significant interest because it controls T cell differentiation, proliferation, metabolism and survival. We show that during murine chronic lymphocytic choriomeningitis virus (LCMV) infection, chronic antigen stimulation greatly impairs the ability of virus-specific CD8 T cells to activate PI3K/AKT/mTOR signaling in response to TCR and cytokine stimulation, resulting in enhanced nuclear retention of the transcription factor, FoxO1. Therapeutic blockade of the inhibitory PD-1 receptor, which restores antiviral CTL responses, was dependent on increased PI3K/AKT/mTOR activity. FoxO1 expression was critical to sustain virus-specific CTL responses and control viral infection. Lastly, we demonstrate that FoxO1 was both necessary and sufficient to induce the expression of the inhibitory receptor PD-1 on virus-specific CD8 T cells, and therefore represents a critical regulatory factor in balancing effector and exhausted CTL states during chronic infection. These findings highlight a novel role for AKT-FoxO1 pathway in the regulation of exhaustion CTL responses during chronic infection.
Divergent signaling pathways downstream of TLR7 regulate TNFα production by bone marrow polymorphonuclear leukocytes and monocytes in experimental lupus (IRM5P.700)
Authors: Han Shuhong , Zhuang Haoyang , Xu Yuan , Li Yi , Yang Lijun , Reeves Westley 
Published: January 2023

Abstract
Pristane-treated mice develop TLR7/IFNα dependent lupus autoantibodies and nephritis. In contrast, anemia results from TLR7-driven TNFα production by bone marrow (BM) polymorphonuclear leukocytes (PMN). Little TNFα is produced by BM monocytes. We examined the regulation of TNFα in BM of mice with pristane-lupus, emphasizing the role of IL-10, whose over-productionis implicated in SLE pathogenesis. Intracellular TNFα and IL-10, surface IL-10Rα, and p38MAPK activation were assessed by flow cytometry. In response to TLR7 ligand R848, BM PMN and monocytes both produced TNFα and IL-10. Inhibiting p38MAPK suppressed both PMN and monocyte TNFα production. In contrast, blocking NFκB had opposite effects on TNFα production in PMN (inhibition) vs. monocytes (enhancement). The differential effects were a consequence of NFκB mediated IL-10 production and the higher IL-10 responsiveness of monocytes vs. PMN. IL-10 suppressed TNFα in monocytes to a far greater degree than in PMN, consistent with levels of IL-10Rα expression by the different cell types.In contrast, PMN were more sensitive than monocytes to dexamethasone (which acts in part via NFκB) suppression of TLR7-driven TNFα production. Cell-type-specific regulation of NFκB and p38MAPK downstream of TLR7 leads to production of a complex array of cytokines (IFNα, TNFα, IL-10).Patients with disease mediated by IFNα and monocytes may respond differently to certain therapies than those with PMN-mediated clinical manifestations.
Regulation of TLR4-promoted inflammatory signaling by β-arrestin 2 (IRM5P.706)
Authors: Yin Deling , Denney James , Hu Dan , Yan Hui , Caudle Yi , Li Hui 
Published: January 2023

Abstract
The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that β-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 MAPK-dependent mechanism. β-arrestin 2 forms complexes with p38 mitogen-activated protein kinase (MAPK) and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of β-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking β-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which β-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.
ATF4 is induced by ROS and regulates Th1 and Th17 responses (IRM6P.718)
Authors: Yang Xi , Gao Xin , Yang Qianting , Zhu Yibei , Li Gang , Zhang Xueguang , Xiao Guozhi , Cao Huiling , Chen Xinchun , Lu Binfeng 
Published: January 2023

Abstract
Reactive oxygen species (ROS) play a major role in the pathogenesis of chronic inflammatory and autoimmune diseases. Little is known about underlying molecular mechanisms regulating T cell immune responses to ROS. Here, we demonstrated that ROS induced ATF4 in activated T cells. We found, in the high ROS condition, ATF4-/- T cells failed to proliferate and produced much less IFN-γ when cultured in the Th1 polarization condition. Inhibition of ROS by addition of antioxidants significantly recovered proliferation and IFN-γ production by ATF4-/- CD4 T cells in the Th1 condition, suggesting a crucial role of ATF4 in preventing oxidative stress during T cell activation and Th1 differentiation. In addition, we demonstrated that ATF4-/- mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), which is associated with elevated levels of IL17 but reduced IFN-gamma production. The in-depth molecular mechanisms are further studied and the result will be presented. Our study establishes that the ROS/ATF4 axis promotes Th1 and suppresses Th17 immune responses and likely plays an important role in autoimmunity and cancer immunology.
Identification of candidate genes for type 1 diabetes by microarray expression analysis of islet-specific CD4+ T cells (IRM6P.726)
Authors: Waldner Hanspeter , Berry Gregory , Frielle Christine , Salzberg Anna 
Published: January 2023

Abstract
Type 1 diabetes (T1D) is a complex genetic disease, resulting from the interplay of multiple insulin dependent diabetes loci (Idd) in both humans and nonobese diabetic (NOD) mice. NOD.B10 Idd9 congenic mice contain the Idd9 from T1D-resistant C57BL/10 mice, mediating significant T1D protection compared to NOD mice. However, the genes and the pathways by which Idd9 controls T1D development remain to be fully elucidated. We have developed novel NOD.B10 Idd9 congenic mice that harbor predominantly islet-specific CD4+ T cells expressing the BDC2.5 T cell receptor (BDC-Idd9.905 mice). To establish functional links between Idd9 genotype and its phenotype, we have performed microarray gene expression analyses of purified CD4+ T cells from these mice and BDC2.5 NOD control mice. We found that among the limited number of differentially expressed genes, those located within the Idd9 region were greatly enriched. Bioinformatics analyses identified Eno1, Rbbp4 and Mtor as differentially expressed Idd9 genes that were part of significant gene networks involved in T cell growth and development. Accordingly, antigen-specific proliferation and Th1 and Th17 responses between BDC-Idd9.905 and BDC2.5 splenocytes differed significantly and were impaired in BDC-Idd9.905 splenocytes. Our findings suggest that these Idd9 genes differentially control proliferation and differentiation of islet-specific CD4+ T cells in the two strains, which may contribute to their differential T1D development.
The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation (IRM6P.710)
Authors: Lappas Courtney 
Published: January 2023

Abstract
Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A2A receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A2AR playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A2AR-dependent mechanism. Specifically, zeatin riboside inhibits the production by CD3+CD4+ T cells of IFN-γ, IL-2, TNF-α, IL-4 and IL-13, and the production by CD3+CD8+ T cells of IFN-γ, IL-2, and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A2AR antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.
The antigen sensitivity of effector but not memory CD4 T cells is regulated by the pathogen-induced inflammatory milieu. (IRM6P.712)
Authors: Richer Martin , Kim Marie , Segura Stephanie , Harty John 
Published: January 2023

Abstract
CD4 T cells play an important role in orchestrating immune responses following recognition of antigens through their T cell receptor (TCR). As such, their sensitivity for cognate antigen is likely to be tightly regulated. Previously, we demonstrated that the antigen sensitivity of CD8 T cells was tuned by inflammatory cytokines. Whether inflammatory cytokines also tune the antigen sensitivity of CD4 T cells remains unknown. Herein, we demonstrate that the antigen sensitivity of effector CD4 T cells is tuned by the inflammatory milieu. Mechanistically, we demonstrate that, in contrast to CD8 T cells, increased antigen sensitivity is not linked to enhanced TCR signal transduction capacity. In addition, tuning of the antigen sensitivity of CD4 T cells by inflammatory cytokines is independent of their role in the polarization of these cells towards a Th1 phenotype. Further, we demonstrate that, unlike CD8 T cells, inflammatory cytokines do not regulate the antigen sensitivity of memory CD4 T cells. Taken together, these data demonstrate that while inflammatory cytokines tune the antigen sensitivity of both CD4 and CD8 T cells, they do so through distinct mechanisms. Understanding the regulation of the antigen sensitivity of T cells can yield important insight leading to potential therapeutic avenues aimed at manipulating the antigen sensitivity of T cells. These approaches may be useful to prevent chronic viral infections, allow for clearance of tumors and prevent autoimmunity.
T-bet and Runx transcription factors are required for the ontogeny of pathogenic IFNγ-producing Th17 cells (LYM3P.729)
Authors: Lazarevic Vanja , Wang Yan , Godec Jernej , Ben-Aissa Khadija , Cui Kairong , Zhao Keji , Pucsek Alexandra , Weaver Casey , Yagi Ryoji 
Published: January 2023

Abstract
Functional plasticity of CD4+ Th cells can be advantageous for the host as it creates diversity that is required to establish a productive immune response against a variety of pathogenic microbes. However, the acquisition of a gene expression profile associated with the opposing CD4+ Th lineages can lead to increased susceptibility to autoimmune diseases. For example, Th17 cells can give rise to IL-17A/IFNγ-double producing cells, which have been implicated in the development of autoimmune diseases. However, our understanding of the molecular mechanisms that govern the generation of IFNγ-producing Th17 cells during an inflammatory response remains incomplete. Here we report that, contrary to current concepts, co-expression of Th1- and Th17-cell master transcription factors, T-bet and RORγt, respectively, did not generate Th cells with strong dual Th1 and Th17 features. Instead, the development of Th1-like Th17 cells required T-bet and Runx1 or Runx3. Indeed, interleukin (IL)-12 stimulated Th17 cells upregulated Runx1, which bound to the Ifng locus in a T-bet dependent manner. Conversely, T-bet or Runx1 deficiency, or inhibition of Runx transcriptional activity, impaired the development of Th1-like Th17 cells during experimental autoimmune encephalomyelitis in vivo. Thus, our studies identify a critical role for T-bet and Runx transcription factors in the generation of pathogenic IFNγ-producing Th17 cells.
Analysis of the formation of antigen-specific memory CD4+ T cell populations derived from single naïve precursors (LYM3P.731)
Authors: Tubo Noah , Fife Brian , Jenkins Marc 
Published: January 2023

Abstract
Following infection, a naive antigen-specific CD4+ T cell population proliferates and differentiates into to effector cell progeny that are either Th1 cells, which activate the microbicidal functions of macrophages, or T follicular helper cells, which help B cells make antibodies. After the resolution of infection, most antigen-specific cells die, while a small number of cells survive to form long-lived memory cells. The origins of these memory cells remain unclear. In this study, we tracked the differentiation clonal populations of CD4+ T cells, from single naïve cells into effector cells, and subsequently memory cells. We find that not all naïve CD4+ T cells that produce effector cell populations give rise to memory populations. However, when memory populations are formed, they are phenotypically similar to the effector populations in terms of Th1 and T follicular helper cell differentiation, suggesting that effector cell phenotype is maintained into memory, and that memory cells are derived from effector cells. Ongoing work is focused on determining the contributions of TCR signal strength and effector population burst size has in memory cell differentiation.
Role of lysine dimethylation in Bcl-2 function and T cell activation (LYM4P.744)
Authors: Li Wenqing , Xiao Zhen , Ravichandran Sarangan , Luke Brian , Collins Jack , Durum Scott 
Published: January 2023

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

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

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

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

Abstract
TLR2 polymorphisms have been associated with increased susceptibility to tuberculosis (TB). We have demonstrated that TLR2 is critical for host resistance against Mtb infection and limits immunopathology in the lung. Recent publications illustrating a role for epithelial cells in host immunity against Mtb led us to investigate TLR2’s role in hematopoietic (H) cells and nonhematopoietic (NH) cells in host defense against Mtb. We generated radiation bone marrow chimeric mice, which lack TLR2 either in the H, NH, or both compartments, and infected these mice with Erdman Mtb. Our results show that TLR2 signaling on the H component is crucial for control of chronic murine Mtb infection. Loss of TLR2 in the H component resulted in increased bacterial burden, decreased accumulation of regulatory T cells, and disruption of granuloma architecture. In contrast, loss of TLR2 on the NH compartment resulted in decreased inflammation and dissemination of Mtb to extrapulmonary sites. These data illustrate a novel role for TLR2 and a paradox within TLR2 signaling: TLR2 signaling on H cells is protective to the host both by controlling infection and regulating inflammation while on NH cells it promotes inflammation induced immunopathology. This balanced response is essential to maintaining host control of TB and could lead to the development of cell-specific TLR2 inhibitors.