bluuebunny/crossref_metadata_2025_split huggingface dataset:
The human CD8β isoforms differentially affect signal transduction pathways and monoubiquitination of the M4 isoform regulates signaling through the mTORC1/2/pathway (IRM4P.497)
Authors: Kavathas Paula , Coman Maria 
Published: January 2023

Abstract
The CD8αβ coreceptor contributes to T cell signaling by bringing the tyrosine kinase p56lck associated with CD8α to theTCR-CD3 complex. Four isoforms (M1 to M4) of the CD8β chain exist in the great apes and humans that differ in the amino acid sequence of their cytoplasmic tails and their expression pattern. We previously found that the M1 isoform, equivalent to murine CD8β, is the predominant isoform in naïve CD8+ T cells, while the M4 isoform is predominant in effector memory T cells. We expressed each isoform in either T cell tumor lines or peripheral blood T cells, stimulated the cells by crosslinking with anti-CD8 or anti-CD3 antibodies and determined effects on signal transduction pathways using phosphoflow. Differences were observed with anti-CD8 crosslinking. For instance, the M1 isoform showed greater amounts of phospho-Erk (2-3 fold) and the M4 isoform showed greater amounts of phospho-Akt (2-3 fold). In addition, because the M4 isoform uniquely binds the adaptor protein AP-2 and is mono-ubiquitinated on lysines, we analyzed mutant forms of the M4 isoform in signal transduction as well. Mono-ubiquitination appears to be a regulator of the mTORC1/2/pathway important in metabolism as the lysine mutant demonstrated a large increase in phosphorylation of the ribosomal protein S6, in the mTORC1 pathway. This supports the hypothesis that the different CD8β isoforms evolved to make unique contributions to CD8 coreceptor function.
TGF-β1 enhances T-cell PD-1 expression through a Smad3-dependant increase in transcription. (IRM4P.499)
Authors: Park Benjamin , Chattergoon Michael , Pan Fan , Pardoll Drew , Cox Andrea 
Published: January 2023

Abstract
Programmed cell death-1 (PD-1) is an inhibitory receptor highly expressed on T cells specific for viral and tumor antigens. TGF-β1 affects the tumor microenvironment and high serum levels are associated with both malignancy and pathogenesis in some chronic viral infections. However, the effect of TGF-β1 on PD-1 expression is unknown. PD-1 expression requires nuclear factor of activated T cells (NFATc1) activation through T cell receptor (TCR) signaling. We found that TGF-β1 signaling increases NFATc1-induced pdcd-1 mRNA and PD-1 expression on human peripheral CD4+ and CD8+ T cells. Smad2 and Smad3 are phosphorylated following TGF-β Receptor I binding and ultimately mediate gene transcription. We found that inhibition of TGFβRI kinase activity or Smad3 phosphorylation blocked TGF-β1-dependent PD-1 enhancement on T cells. Similarly, when compared to TCR stimulation alone, TGF-β1 increased PD-1 expression on murine wild-type (WT) but not on Smad3 knock-out (KO) CD4+ T cells. In contrast, Smad2 KO CD4+ T cells maintain increased PD-1 expression in the presence of TGF-β1, demonstrating a specific role for Smad3. We show that Smad3 increases pdcd-1 transcription by directly binding to a proximal region of the human PD-1 promoter and stabilizing NFATc1 binding. In addition to TGF-β1’s previously known effects on T cell function, our findings suggest that TGF-β1 mediates suppression via PD-1 upregulation. Smad3 may represent an additional target in therapeutic modulation of PD-1.
Vitamin D regulates CD8 T cell expansion and immune memory to infections (IRM4P.502)
Authors: Yuzefpolskiy Yevgeniy , Baumann Florian , Kalia Vandana , Sarkar Surojit 
Published: January 2023

Abstract
Epidemiological studies implicate a potential role of vitamin D in modulating immune responses to infections and vaccines. In this study we analyzed the contribution of vitamin D signals to the development and maintenance of antigen-specific memory CD8 T cell responses during a viral infection. Using the vitamin D receptor (VDR)-deficient murine model, we found that lack of vitamin D signals resulted in a dysregulated CD8 T cell expansion program, which was largely attributable to cell-extrinsic effects. Investigation of events proximal to TCR activation demonstrated a delayed yet sustained priming of antigen-specific CD8 T cells in the absence of vitamin D signals. In spite of a larger ensuing effector CD8 T cell pool generated in the absence of vitamin D signals, the final memory pool was smaller. Interestingly, within both effector and memory CD8 T cell pools, we observed a bias towards CD8 T cells specific for immunodominant determinants, resulting in a restricted repertoire of antigen-specific CD8 T cells in the absence of vitamin D signals. Collectively, these studies delineate a critical role of vitamin D in regulating the quantity and quality of adaptive T cell immunity. Importantly, our studies have direct impact on the efficacy of vaccines, owing to disparate nutritional states of vitamin D that are currently seen cross-sectionally around the world.
Investigating the mechanism of TLR signaling crosstalk in mouse macrophages (IRM5P.702)
Authors: Lin Bin , Fraser Iain 
Published: January 2023

Abstract
It is well established that the TRIF- and MyD88-dependent signaling branches in the TLR4 pathway cooperate to induce a strong innate immune response. It is also known that certain toll-like receptor (TLR) ligand combinations can induce synergistic production of some cytokines. However, the molecular mechanisms underlying this signaling pathway cooperation remain elusive. We conducted a microarray analysis of mouse bone marrow-derived macrophages stimulated with all pairwise combinations of the TLR ligands LPS, Pam3CSK4, R848 and poly I:C to characterize synergy in transcript induction. We find that the crosstalk between the different TLR pathways is most prevalent in combinations which induce both the TRIF and the MyD88 pathways, which is essentially a mimic of the TLR4 pathway. Genes synergistically induced under dual TLR stimulation are mainly cytokines and immune effectors, including IL-6, IL-10, IL-1α, IL-1β, haptoglobin, lipocalin 2, and SOCS3. Based on these data, over 200 synergy factor candidates were picked and targeted using RNAi screening, with IL-6 and IL-12p40 secretion as read outs. A total of 32 hits were identified in the primary screening, including well-known regulators of secondary response gene transcription such as Nfkbiz, Irak2, Tnfaip3 and Tnip1. Secondary screening is ongoing to verify what role the 32 identified genes play in the MyD88/TRIF-dependent synergistic induction of immune regulators.
Protein kinase C delta (PKCδ)−extracellular-signal regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition−mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)−induced endotoxemia (IRM5P.704)
Authors: Noh Kyung Tae 
Published: January 2023

Abstract
Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)−induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular-signal regulated kinase 1/2 (ERK1/2) activation under LPS−stressed conditions via protein kinase C delta (PKCδ) activation. Furthermore, PKCδ−induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS−treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition restored in ERK1/2−dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition−induced ERK1/2 activation via PKCδ relieved LPS−mediated endotoxemia. This finding suggests that IL-10 hyper-expression resulted from GSK-3 inhibition−induced ERK activation could be a new therapeutic pathway for endotoxemia.
Critical role for extracellular-regulated kinase signaling in endotoxin priming of human monocyte NLRP3 inflammasome (IRM5P.705)
Authors: Ghonime Mohammed , Shamaa Obada , Eldomany Ramadan , Srabani Das , Gavrilin Mikhail , Wewers Mark 
Published: January 2023

Abstract
Inflammasomes play key roles in the innate immune response to pathogens and tissue injury. Inflammasome assembly involves two distinct steps; priming and activation, which together trigger caspase-1 to process and release IL-1β, IL-18 and in some settings to induce pyroptosis. Current understanding suggests that priming is the de novo synthesis of proIL-1β and the up-regulation of NLRP3. However, since proIL-18 is constitutively present in monocytes, we studied the mechanisms and timing of endotoxin induced priming events that precede new protein synthesis. Within 15 min of endotoxin priming, exogenous ATP induces caspase-1-dependent mature IL-18 release. This priming is not impaired by inhibition of gene transcription or protein translation. Knockdown of NLRP3 supports a role for constitutive NLRP3 in this rapid IL-18 processing. To dissect the early signaling events we investigated upstream signaling pathways. Interestingly, ERK phosphorylation was found to be essential for priming as proven by chemical inhibition and knockdown approaches. Scavenging of reactive oxygen species blocked both priming and ERK phosphorylation. These findings suggest that priming involves ERK-mediated posttranslational modifications which license the NLRP3 inflammasome to respond to a second, activation signal. Thus, understanding what these modifications are may provide new opportunities to modulate inflammasome activation in disease.
Transcription repressor Hes1 is a selective regulator of TLR-induced CXCL1 expression and neutrophil responses (IRM5P.708)
Authors: Shang Yingli , Au Karmen , Hu Xiaoyu 
Published: January 2023

Abstract
Chemokine-mediated neutrophil recruitment to sites of inflammation is a critical initial step in host defense and inflammatory responses. However, neutrophil recruitment must be tightly regulated to avoid inappropriate immune activation which may lead to autoimmune and inflammatory diseases. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses TLR-induced expression of CXCL1, a chemokine crucial for recruiting neutrophils. Interestingly, Hes1 deficiency does not affect expression of other pro-inflammatory cytokines such as Tnf and Il1b. Hes1 negatively regulates Cxcl1 expression and neutrophil recruitment in vivo in LPS-induced peritonitis. In addition, deficiency of Hes1 exacerbated severity of K/BxN serum-induced arthritis in which neutrophils play a key pathogenic role. Mechanistically, regulation of Cxcl1 expression by Hes1 is at the transcriptional level but not via modification of TLR-induced canonical signaling events. Instead, Hes1 represses Cxcl1 productive transcription by attenuating occupancy of a positive-transcription elongation complex P-TEFb at the Cxcl1 gene locus and thus preventing subsequent recruitment of serine 2-phosphorylated RNA polymerase II. In summary, our results identify Hes1 as a novel homeostatic regulator of CXCL1 expression and neutrophil responses and suggest that targeting Hes1-mediated repressive pathway may represent a new therapeutic approach to selectively curbing neutrophil-mediated inflammation.
Selective ROCK2 inhibitor down-regulates pro-inflammatory T cell responses via shifting Th17/Treg balance (IRM6P.711)
Authors: Zanin-Zhorov Alexandra , Mo Rigen , Scher Jose , Nyuydzefe Melanie , Weiss Jonathan , Schueller Olivier , Weiss Sara , Poyurovsky Masha , Dustin Michael , Abramson Steven , Waksal Samuel 
Published: January 2023

Abstract
Targeting pro-inflammatory cytokines is a promising approach to the treatment of certain autoimmune diseases. However, the signaling mechanisms involved in the initiation and effector phases of auto-aggressive pathways are still an enigma. Rho-associated kinase 2 (ROCK2) regulates the secretion of inflammatory cytokines interleukin (IL)-21 and IL-17 and the development of autoimmunity in mice. Our data from a phase 1 clinical trial demonstrates that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects significantly down-regulates the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g in response to TCR stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORgt protein levels in T cells derived from healthy subjects or rheumatoid arthritis (RA) patients. Simultaneously, ROCK2 inhibition induces phosphorylation of STAT5 and SMAD2/3, and subsequently increases the percentage of Foxp3+ T cells. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between pro-inflammatory and regulatory T cell subsets in man. Targeted inhibition of ROCK2 may therefore have a role in the treatment of autoimmune disease with disturbed immune homeostasis.
Identification of molecular mechanisms that regulate T helper 1 cell plasticity (IRM6P.715)
Authors: Oestreich Kenneth , Read Kaitlin , McDonald Paul 
Published: January 2023

Abstract
In order to mount an effective adaptive immune response, the body relies on a number of specialized CD4+ T cells to perform a variety of functions. These specialized cell types arise from a common naïve CD4+ T cell progenitor and include Th1, Th2, Th17, Treg, Th9, Th22, and Tfh populations. Recent work, including our studies, has demonstrated that a considerable amount of flexibility between T helper cell subsets exists to allow the cells to appropriately respond to changing environmental conditions. Initially, we demonstrated that in response to low IL-2 signaling, Th1 cells upregulate Bcl-6 expression, shifting the gene expression program away from a Th1 profile in favor of a Tfh-like program. In this setting, the interaction between T-bet and Bcl-6 acts as a regulatory switch that senses changing environmental conditions to fine-tune the immune response. We have now extended these findings by using RNA-seq to examine the gene expression signatures of both Th1 and Tfh-like cells. This approach has led to the identification of novel transcriptional regulators that may play roles in promoting Th1-to-Tfh cell flexibility. Furthermore, we have demonstrated that in addition to IL-2, IL-15 signaling is a regulator of Th1 cell flexibility. These findings are critical first steps in elucidating the mechanistic players in Th1-to-Tfh plasticity and the environmental signals they respond to, so that we may be able to therapeutically manipulate T helper immune cell potential.
A novel population of interleukin 21-producing pre-follicular T helper cells develop spontaneously in young naïve mice (LYM3P.743)
Authors: Adkins Elisabeth , Wang Xulong , Sproule Thomas , Christianson Gregory , Park Giljun , Carter Gregory , Morse Herbert , Roopenian Derry 
Published: January 2023

Abstract
Interleukin 21 is a cytokine vital for driving the proliferation of antigen-stimulated B cells within the germinal center and their subsequent differentiation into antibody-secreting plasmablasts and plasma cells. IL21 is predominately produced by a subset of activated CD4+ T cells known as T follicular helper cells. These cells develop at the T cell-B cell border and express ICOS, PD1, CD44, CXCR5 and BCL6. While this cell type has been identified, the processes that ultimately result in TFH differentiation are poorly understood. Here we use a novel IL21-venus fluorescent protein reporter mouse to investigate the signals and pathways involved in the earliest stages of development of TFH cells and production of IL21. We identify and characterize a naturally occurring population of ICOShi CD44hi CD4+ T cells that produce IL21 and are present within 4 weeks of age in naïve healthy C57BL/6J mice. These T cells show characteristics of TFH cells, including the upregulation of Bcl6 and Sostdc1, but lack surface staining for CXCR5 and PD1 and, surprisingly, develop in the absence of B cells. Therefore, these spontaneously arising cells actively producing IL21 appear to identify the earliest precursor to the mature TFH state.
The TNF family member TL1A promotes Th9 differentiation and Th9-mediated immunopathology (LYM3P.733)
Authors: Richard Arianne , Tan Cuiyan , Hawley Eric , Gomez-Rodriguez Julio , Goswami Ritobrata , Yang Xiangping , Cruz Anthony , Penumetcha Pallavi , Hayes Erika , Pelletier Martin , Gabay Odile , Walsh Matthew , Ferdinand John , Keane-Myers Andrea , Choi Yongwon , O'Shea John , Al-Shamkhani Aymen , Kaplan Mark , Gery Igal , Siegel Richard , Meylan Francoise 
Published: January 2023

Abstract
The TNF family cytokine TL1A (Tnfsf15) costimulates T cells through its receptor DR3 (Tnfrsf25). Currently known effects of TL1A on T cell differentiation or systemic immune responses cannot account for the resistance of Tnfrsf25-/- mice to diverse animal models of autoimmune and allergic inflammation. IL-9 is a cytokine that promotes inflammatory and allergic immune responses and is expressed by a distinct subset of T cells early after T cell activation or restimulation. We have found that TL1A potently promotes generation of T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. Unlike the TNF family member OX40 ligand, TL1A increases Th9 differentiation independently of STAT6 and the TNF-signaling adaptor TRAF6. Instead, TL1A affects Th9 differentiation through enhancing IL-2-dependent STAT5 activation and allowing increased binding to the IL-9 promoter. In two models of allergic asthma, we demonstrate that Tnfrsf25-/- mice have reduced disease severity accompanied by fewer IL-9 producing T cells in the lung. In addition to promoting Th9 differentiation, we find that TL1A enhances pathogenicity in Th9 transfer models of both uveitis and allergic asthma, where endogenous TL1A signaling is required for maximal pathology and IL-9 production at the site of inflammation. Taken together, these data identify TL1A as a novel cytokine that promotes Th9 differentiation and pathogenicity and as a possible therapeutic target in diseases dependent on IL-9.
The forkhead transcription factor, Foxo1, may coordinate naïve CD8+ T cell homeostasis with energy availability in mice. (LYM4P.758)
Authors: Gubbels Bupp Melanie , Litvin Seth , Edwards R. , Mihalcoe Brittany , Byrne Frances , Hoehn Kyle , Anoff Joshua 
Published: January 2023

Abstract
The naïve CD8+ T cell population must be large enough so that sufficient diversity exists to respond to potential pathogens, yet not so large as to unnecessarily drain an organism’s resources. Thus, we propose that a molecular network links the size of the naïve CD8+ T cell pool with energy availability. We report that short-term calorie restriction in C57BL/6 mice resulted in reduced numbers of naïve CD8+ T cells which efficiently engaged survival signals and minimized energy usage. For example, calorie restricted naïve CD8+ T expressed higher levels of CD8 and CD127 than ad libitum cells, both of which are responsible for receiving homeostatic survival signals in vivo. Naïve CD8+ T cells isolated from calorie restricted mice also experienced less death-by-neglect and engaged in less glycolysis than cells isolated from mice fed ad libitum. Finally, neither the size of the naïve CD8+ T cell pool nor the sensitivity of those cells to death-by-neglect were affected by calorie restriction in the absence of the forkhead transcription factor, Foxo1, indicating that Foxo1 may be involved in synchronizing the size of the naïve CD8+ T cell pool with energy availability.
NKT cells require IL-7, not IL-15, for survival and homeostasis (LYM4P.761)
Authors: Park Joo-Young , Keller Hilary , Sato Noriko , Park Jung-Hyun 
Published: January 2023

Abstract
NKT cells are thymus-generated T lineage cells with shared phenotypic and functional characteristics of innate NK cells. Both T and NK cells require γc cytokines for their survival and homeostasis with T cells being dependent on IL-7 while NK cells being dependent on IL-15. As NKT cells carry features of both T and NK cells, it has been a long standing question which γc cytokine would drive the survival and homeostasis of NKT cells. In contrast to the current view, here we report that NKT cells survival is not dependent on IL-15 but requires IL-7. Specifically, we found that IL-15-deficient mice have intact numbers of peripheral NKT cells and that overexpression of IL-15 failed to expand NKT cell numbers even as it significantly increased CD8 T cell numbers. On the other hand, using a new in vivo model of IL-7-deficiency selectively in peripheral lymphoid tissues (K7 mice), we demonstrated that IL-7 is a non-redundant survival factor for NKT cells. Furthermore, transgenic IL-7 expression dramatically increased peripheral NKT cell numbers, suggesting that IL-7 availability determines size of the NKT cell pool. Mechanistically, we identified STAT5 as the transcriptional effector molecule downstream of IL-7 signaling to promote survival and maintain NKT cell homeostasis. Collectively, these data provide a new perspective on γc cytokine requirements for NKT cell survival, proposing a new role for IL-7 in NKT cell biology.
ITK signals tune CD8+ T cell homeostatic proliferation and anti-tumor immunity (LYM4P.747)
Authors: Huang Weishan , Luo Junjie , Huang Lu , Huang Fei , August Avery 
Published: January 2023

Abstract
IL-2 inducible T cell kinase (ITK) is a Tec family non-receptor tyrosine kinase. Loss of ITK perturbs CD8+ T cell homeostasis and leads to spontaneous acquisition of innate memory/effector phenotype. However, Itk-/- naïve CD8+ T cells develop on an OTI Rag-/- background, and we use these cells to show here that ITK tunes lymphopenia-induced proliferation (LIP) and antigen sensitivity during CD8+ T cell homeostatic expansion. Experimental analysis and computational simulations revealed that in a lymphopenic environment, naïve Itk-/- CD8+ T cells exhibit massive and immediate homeostatic expansion, accompanied by significant death and followed by population collapse. The enhanced immediate expansion of Itk-/- cells is CD8+ T cell-intrinsic and is independent of recipient MHCI, but dependent on donor CD8+ T cell-T cell interaction. The lack of ITK resulted in enhanced antigen sensitivity and effector program in homeostatically expanded (HP) CD8+ T cells. These Itk-/- HP CD8+ T cells exhibit robust anti-tumor immunity in an antigen specific manner, independent of CD4+ T cell help. These data suggest that ITK intrinsically regulates TcR tuning of homeostatic cytokines during homeostasis of CD8+ T cells and that targeting ITK may have clinical benefit in cancer therapy by generating superior anti-tumor responses.