bluuebunny/crossref_metadata_2025 huggingface dataset:
RGC-32 regulates TGF- β extracellular matrix expression in reactive astrocytes (HUM1P.320)
Authors: Tegla Cosmin , Cudrici Cornelia , Boodhoo Dallas , Martin Alvaro , Danoff Jacob , Sugarman Adam , Rus Violeta , Rus Horea
Published: January 2023

Excessive extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and modify immune reactions. In this study we investigated first the expression of collagen types I-V, decorin and RGC-32 in MS lesions. Collagens I, III, IV and V and decorin were expressed in the perivascular space and parenchyma, and collagens I, IV and V were expressed by astrocytes that also expressed RGC-32. Since RGC-32 is involved in mediating some of TGF-β effects we investigated its role in TGF-β-induced ECM expression. In cultured astrocytes, both fibronectin and collagens I, IV and V were significantly induced after 18 h of stimulation with TGF-β. Silencing RGC-32 expression by transfecting astrocytes with siRGC-32 caused a significant reduction in the TGF-β-induced expression of collagen I (p<0.01), collagen IV (p<0.02), collagen V (p<0.05) and fibronectin (p<0.05) when compared to treatment with control siRNA. We also found that α-smooth muscle actin (α-SMA) was significantly reduced after RGC-32 silencing (p<0.05). The effect on α-SMA suggests that RGC-32 is also required for the transition of astrocytes to a reactive state. These data indicate that RGC-32 plays an important role in TGF-β-mediated induction of ECM expression in astrocytes. RGC-32 may therefore represent a useful new target for therapeutic intervention in MS.
B cells from relapsing remitting multiple sclerosis patients modulate T cell behavior in a neuroantigen-specific manner (HUM1P.322)
Authors: Ireland Sara , Guzman Alyssa , Davis Laurie , Cowell Lindsay , Remington Gina , Greenberg Benjamin , Frohman Elliot , Monson Nancy
Published: January 2023

The role of B cells in the pathoetiology of relapsing remitting multiple sclerosis is not well understood. B cells may contribute to MS by antibody secretion, cytokine production and antigen presentation. Antigen-specific B cells are highly efficient antigen presenting cells in the context of their cognate antigens. Since antibodies from multiple sclerosis patients bind to neuroantigens we hypothesized that neuroantigen specific memory B cells promote disease in multiple sclerosis patients by serving as antigen presenting cells for auto-reactive T cells. We observed that B cells from a subset of multiple sclerosis patients induce naïve and memory T cell proliferation in the presence of recombinant neuroantigens, myelin oligodendrocyte glycoprotein and myelin basic protein, but not MOG 35-55 peptide, in B-T cell co-cultures. Similarly, in the presence of neuroantigen proteins cells from multiple sclerosis patients secreted greater levels of IFN-γ, IL-6, -17A, -17F, -21, -22, -31, -33, TNF-α and soluble CD40L than healthy donors, while levels of IL-4 and IL-25 were similar. These data support a role for human B cells as antigen presenting cells in multiple sclerosis and highlight the role of B cells in the context of autoimmune disease beyond antibody production.
Matrine suppresses glutamate excitotoxicity in experimental autoimmune encephalomyelitis (HUM1P.323)
Authors: Xiao-Jian Zhang , Ju-Kui Song
Published: January 2023

Multiple sclerosis (MS) is T cell-mediated inflammatory diseases characterized by lymphocyte infiltration and demyelination. Matrine (MAT) is a quinolizidine alkaloid derived from the herb Radix Sophorae Flave and has long been used in the treatment of hepatitis B in clinical without obvious side effects. Previous reports have revealed that MAT restrained the central inflammation after experimental autoimmune encephalomyelitis (EAE), the most common animal model of MS due to similar symptoms and mechanisms; however whether MAT is effective to inhibit glutamate excitotoxicity is still unclear. In this study, we provide evidence that MAT attenuated the EAE disease severity, accompanied by down-regulated glutamate level, increased GABA level, as well as promoted the expressions of two dependent glutamate transporters (GLT-1 and GLAST). In addition, MAT treatment significantly decreased the content of the NMDA- and AMPA- glutamate receptor in EAE rats. Together, these data indicate that MAT treatment inhibited glutamate excitotoxicity after EAE, and suggest that MAT may be a novel choice in MS therapy.
Amelioration in generalized anxiety disorder is associated with decreased Treg number and function (HUM1P.327)
Authors: Akimova Tatiana , Negorev Dmitri , Luthra Lalita , Berton Olivier , Hancock Wayne , Lohoff Falk
Published: January 2023

In preclinical models, chronically stressed and highly anxious animals displayed impaired protective immunity and associated increased risks of cancer progression. These observations may reflect stress-linked increases in Treg numbers or function. We therefore assessed the number and function of FOXP3+ T-regulatory (Treg) cells in 9 patients with generalized anxiety disorder (GAD). These patients had Hamilton Anxiety Scale (HAM-A) scores of >20, and provided blood samples for Treg evaluation at baseline and after 8 weeks of treatment with the antidepressant, Desvenlafaxine, or placebo. After treatment, patients with significant improvement of HAM-A scores (i.e. responders) showed decreased Treg suppressive function in comparison with non-responders (23.8±9.8 vs. 83.4±50 units for CD4+ responder cells, p=0.035). Decreased HAM-A scores correlated with decreased suppression of CD4 cell proliferation (Spearman r=0.745, p=0.021) and with decrease in Treg numbers (Spearman r=0.775, p=0.041). Treg ability to suppress CD8 cell proliferation after treatment was also inversely correlated with decreases in HAM-A scores (Spearman r=-0.75, p=0.02). Conclusion: clinical improvement in GAD is accompanied by normalization of Treg numbers and function, which may have long-term impact on patient health and, potentially, risk of cancer progression.
Monocyte aggregates and intermediate (CD14++CD16+) cells that are associated with cardiac events are increased in human psoriatic blood and CD11b +Ly6C high cells and aggregates are increased in the KC-Tie2 psoriasiform mouse model (HUM1P.307)
Authors: Golden Jackelyn , Groft Sarah , Wang Yunmei , Diaconu Doina , Simon Daniel , McCormick Thomas , Ward Nicole , Cooper Kevin
Published: January 2023

Psoriasis confers an increased risk for developing and dying of cardiovascular disease (CVD). We found increased circulating CD14++CD16+ intermediate monocytes (IntMo), a cell type associated with increased cardiovascular risk, in psoriasis patients compared to healthy controls (N=42, 16.5%±2.7 vs. 11.9%±1.4, P=.056). Amnis imaging flow cytometry of the psoriatic IntMo cells revealed increased doublets and aggregates. Overall, CD14++ doublets are increased in psoriasis patients compared to controls (2.72%±0.58 vs. 1.09%±0.20; P=0.006). Murine psoriasiform skin models (KC-Tie2) exhibit increased vascular thrombosis and monocyte doublets, indicating a skin inflammation-driven process. KC-Tie2 mice are pro-thrombotic, develop aortic root vascular lesions, and have increased splenic CD11b +Ly6C high (Ly6Chigh) monocytes (33.8%±3.8, n=7) compared to controls (8.05%±0.55, n=9; P=0.0002), suggesting that chronic skin inflammation generates Ly6Chighcells which mediate vascular inflammation. KC-Tie2 mice also show an increase in Ly6Chighcells in skin-draining lymph nodes compared to controls (8.83%±0.77, n=18, vs. 1.94%±0.8, n=3, P=.002). Crossing IL-6 KO mice with KC-Tie2 decreases these splenic Ly6Chighcells, reverses thrombosis and aortic root vascular lesions, and eliminates skin inflammation. Psoriasis-driven accumulation, increase, and aggregation of monocytes in circulation suggests a novel mechanistic linkage between psoriatic skin disease and its CVD comorbidity.
Do particular antibody genes recognize gray matter cellular targets? (HUM2P.338)
Authors: Monson Nancy , Ligocki Ann , Rounds William , Li Min , Henson Paul , Rivas Jacqueline , Guzman Alyssa , Graves Donna , Greenberg Benjamin , Frohman Elliot , Ward Sally , Stowe Ann
Published: January 2023

My lab’s recent work on the antibody genetics of multiple sclerosis (MS) patients has led to the astonishing discovery that patterns of mutation accumulation into the antibody genes of B cells in the central nervous system (CNS) of MS patients are unique and can be used to predict clinically isolated syndrome (CIS) conversion to MS with 94% accuracy. This Antibody Gene Signature (AGS) is a novel, potentially diagnostic biomarker that is currently in clinical trials and heralds the advent of antibody genetics as a means to categorize patients early in their disease course in order to provide prompt treatment and prevent extensive CNS damage. Our goal for this project was to determine what antibodies expressed by these B cells from MS patients reacted against. We cloned and expressed more than 30 antibodies that contain the AGS-MS from cerebrospinal fluid derived single B cells of MS and CIS patients, and tested their capacity to bind to brain tissue. We found that these AGS-MS enriched antibodies bind to neurons and astrocytes that reside in the brain gray matter tissue with high specificity. Some of these antibodies recognize both neurons and astrocytes in the gray matter, whereas others are specific for a single cell type. We hypothesize that AGS-enriched rhAbs and B cells expressing these antibodies could be participating in gray matter disease pathology in both early CIS and later MS stages.
Loss of AMPKα1 in neuroinflammatory phenotype of X-linked adrenoleukodystrophy (HUM2P.335)
Authors: Singh Jaspreet , Giri Shailendra , Suhail Hamid , Deshpande Mandar
Published: January 2023

X-linked adrenoleukodystrophy (X-ALD) is the most common inherited neuroinflammatory demyelinating leukodystrophy. All X-ALD patients present with mutations in the ABCD1 gene, which encodes a peroxisomal adenosine triphosphate (ATP)-binding cassette transporter protein (ABCD1). X-ALD is a complex disease where the same mutation in the ALD gene (ABCD1) can lead to clinically very distinct phenotypes; a fatal neuroinflammatory childhood cerebral ALD (ALD) or the relatively benign disease of adrenomyeloneuropathy (AMN). Further, no relationship could be established between genotype and severity of the disease as same mutation is known to give different phenotypes, even within a family. We recently documented a role for decreased AMP kinaseα1 (AMPKα1) levels in initiating severe inflammatory and demyelinating phenotype in the CNS of AMPKα1-knockout mice. Abundant data is also available that implicates loss of AMPK in generating spontaneous inflammatory response in several cell types including immune cells. Our data using healthy control, AMN and ALD patient-derived skin fibroblasts and lymphocytes documents, for the first time, loss of a metabolic gene- AMPKα1 in ALD patient-derived fibroblasts and lymphocytes. Consistent with this, the basal expression of inflammatory cytokines (iNOS and IL-6) were increased in ALD patient-derived lymphocytes. Together these observations suggest that AMPKα1 may be involved in the progression of benign AMN to neuroinflammatory ALD phenotype.
Fluvastatin enhances IL-33-mediated cytokine production in mouse mast cells (HYP3P.410)
Authors: Taruselli Marcela , Kolawole Elizabeth , Ryan John
Published: January 2023

The HMG-CoA reductase inhibitor Fluvastatin is a commonly used treatment for hypercholesterolemia. This drug recently has been shown to have immunological suppressive effects. Interleukin 33 (IL-33), an alarmin, has been suggested to contribute to allergies and anaphylaxis by activating mast cells. Due to the suppressive functions of Fluvastatin, we investigated the role of Fluvastatin on interleukin IL-33-stimulated mast cell function. Unlike previous studies, we have found that Fluvastatin increased production of the pro-inflammatory cytokines IL-6 and TNF-α in IL-33 stimulated mast cells. Furthermore the increase in cytokine production appeared to be stem cell factor (SCF)-dependent, since in the absence of SCF, Fluvastatin decreased cytokine production. Investigating the role of Fluvastatin in IL-33-activated mast cells will reveal a better understanding of how innate immunity may be altered by a widely-prescribed class of medications.
Preliminary investigation on the role of micro-RNAs in IL-10 resistance in mast cells (HYP3P.412)
Authors: Abdul Qayum Amina , McLeod Jamie , Ryan John
Published: January 2023

Mast cells are immune cells that play a critical role in allergic diseases both as effector and immunoregulatory cells. A well understood mast cell activation pathway is the binding of IgE to its high affinity IgE receptor (FcεRI). Possible regulators of mast cell homeostasis are regulatory cytokines produced by the immune response. We have shown that IL-10 hinders mast cell cytokine production by selectively targeting the Fyn-Stat5/Akt cascade proteins activated downstream of FcεRI. Interestingly, these effects are altered by genetic background, as cytokine suppression is observed in mast cells from Th1-prone C57BL/6 but not Th2-prone 129/Sv mice. We hypothesize that IL-10 responsiveness is mediated by induction of MicroRNAs (miRNAs), and that a failure to induce these miRNAs results in IL-10 non-responsiveness. miRNAs could potentially be targeting Akt, Stat5B, and Fyn to antagonize cytokine production. We demonstrate that miR-135a-5p expression increases in IL-10-treated BMMC derived from Th1-prone C57BL/6 mice, but not BMMC from Th2-prone 129/Sv mice. Unraveling how mast cell homeostasis is maintained will shed light on the predisposition to numerous inflammatory diseases.
IgE receptor induces Stat5b serine phosphorylation in mast cells (HYP3P.346)
Authors: Kiwanuka Kasalina , Ryan John
Published: January 2023

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate a number of cellular responses including but not limited to cell survival, proliferation and differentiation. This protein family consists of seven members, STAT1-6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid, and erythroid cell development and function. We recently identified Stat5b as the critical regulator of IgE-mediated cytokine production in mast cells. We found that Fyn-mediated Stat5 tyrosine phosphorylation is critical for Stat5-dependent effects. Our recent work demonstrates that Stat5b is also phosphorylated on serine residues during IgE signaling in mast cells. We find that STAT5b serine phosphorylation occurs through a Src-independent pathway requiring PI3-kinase function. Understanding the pathways leading to Stat5b serine phosphorylation and its role in the inflammatory response will yield new targets for mast cell-associated diseases.
Modulation of mast cell function by TIM-3 signaling (HYP3P.351)
Authors: Phong Binh , Kane Lawrence
Published: January 2023

Transmembrane, immunoglobulin and mucin domain-3 (Tim-3) is an inducible negative regulator of Th1 function and anti-tumor immune responses. Recent studies found that Tim-3 is constitutively expressed on mast cells and that an anti-Tim-3 pAb enhanced secretion of pro-inflammatory cytokines by antigen-stimulated bone marrow-derived mast cells (BMMC). However, the mechanism(s) by which Tim-3 modulates mast cell function are unknown. Here we show that Tim-3 enhances FcεRI-derived signals to co-stimulate NF-AT, AP1, NF-κB, and IL-6 promoter transcriptional activation. This activity requires specific tyrosines in the Tim-3 cytoplasmic tail. Furthermore, various antibodies to Tim-3 significantly enhanced IgE+Ag-stimulated tyrosine phosphorylation and up-regulated BMMC cytokine secretion. Conversely, we observed impaired IgE+Ag-dependent activation of mast cells in which Tim-3 expression was knocked down. Using BMMC generated from Nur77-GFP reporter mice, we found that cross-linking Tim-3 synergizes with the antigen receptor FcεRI to enhance Nur77-GFP expression. Our work suggests that Tim-3 acts proximal to the antigen receptor, leading to enhancement of the NF-κB, NF-AT, and AP-1 pathways. Thus, these studies are consistent with Tim-3 being a positive regulator of mast cell function. Mast cells contribute to both innate and adaptive immune responses, so Tim-3 may serve as an important modulator of mast cell effector functions in autoimmune and allergic diseases.
Effect of NADPH oxidase 2 (Nox2) deficiency on the function of Th2 cells (HYP4P.415)
Authors: Yuk Chae Min , Kwon Bo-In , Lee Seung-Hyo
Published: January 2023

Nox2/gp91phox, one of phagocyte NADPH oxidase subunits, is mainly expressed on phagocytes. They transport electrons across the plasma membranes and kill pathogens by producing ROS. Many evidences have shown that Nox2 deficiency causes susceptibility to infections and immunosuppression. Recent studies have identified functional phagocyte-type Nox2 is also expressed in helper T cells through TCR stimulation. So, many papers attempted to define the role of Nox2 on Th1 and Th17 cell differentiation, but the exact function of Nox2 in Th2 differentiation remains unclear. To elucidate this, we adopted experimental allergic asthma that Th2 cells have been considered as a pivotal mediator for asthma pathogenesis. Here, we revealed that Nox2 deficient mice showed enhanced allergic asthma phenotypes including increased airway hyper responsiveness, eosinophilia, glycoprotein secretion and IL-4 production in bronchoalveolar lavage through augmented Th2 differentiation, regardless of challenge routes and allergen types. Based on our results, we conclude that Nox2 could be a molecular target for controlling Th2 cell differentiation and allergic asthma.
Altered neutrophil homeostasis and DARC regulate CXCL5-mediated pulmonary immune responses (INM2P.435)
Authors: Mei Junjie , Dai Ning , Liu Yuhong , Hudock Kristin , Liu Peihui , Deshmukh Hitesh , Lee Janet , Worthen G. Scott
Published: January 2023

CXCL5 is a neutrophil chemokine, predominantly expressed by epithelial cells and platelets. We recently demonstrated the critical role of CXCL5 in chemokine scavenging and innate immunity to severe bacterial pneumonia, and in regulating neutrophil homeostasis through IL-17/G-CSF axis. Here we show that CXCL5-inhibited chemokine scavenging and innate immune response to severe E.coli pneumonia is completely dependent on G-CSF-mediated increases in BM neutrophils and CXCR2 concentrations, but not on DARC. However, DARC functions as a chemokine reservoir rather than a sink in this pneumonia model. Darc-/- mice showed increased neutrophils in the BM, with normal blood neutrophil counts under basal conditions, and defective neutrophil release from BM upon systemic CXCL2 stimulation. Furthermore, DARC deficiency altered neutrophil homeostasis independently of G-CSF and exhibited additive effect with CXCL5 deficiency on increasing BM neutrophil percentages in Cxcl5-/-Darc-/- mice. These data provide the first evidence for a beneficial role of delicate regulation of neutrphil homeostasis in chemokine scavenging and innate immunity to bacterial pneumonia. Moreover, these results substantiate a mechanistic role for DARC deficiency in neutrophil homeostasis, which may provide insight into benign ethnic neutropenia and other inflammatory diseases seen in Duffy null humans.
Vagal modulation of catecholamines in the adrenal gland controls systemic inflammation in experimental sepsis (INM2P.437)
Authors: Ulloa Luis , Inclan Rico Juan Manuel , Morcillo Patrick , Peña Geber , Torres Rosas Rafael
Published: January 2023

Sepsis is the leading cause of mortality in non-coronary Intensive Care Units accounting for 9.3% of overall deaths in the USA. Sepsis and other critical conditions like hemorrhage, resuscitation, shock, and trauma are characterized by overzealous inflammatory responses that cause lethal multiple organ failure. Despite its recent identification, multiple investigators have already reported that the vagus nerve controls systemic inflammation in experimental ischemia and reperfusion, hemorrhage and resuscitation, pancreatitis, colitis, endotoxemia, septic shock and severe sepsis. Vagus nerve stimulation controls systemic inflammation, but its clinical use is precluded by the surgical exposure of the vagus nerve, the use of anesthetics, and its temporal effect(1,2). Transcutaneous activation of the vagus nerve improves survival in experimental sepsis without surgery. Transcutaneous activation of the vagus nerve inhibits the production of multiple inflammatory cytokines including TNFα, IL6 and INFγ. This anti-inflammatory potential has a lasting effect and does not merely delay the pathogenesis of sepsis. Transcutaneous activation of the vagus nerve controls systemic inflammation by modulating catecholamine's production in the adrenal gland. Studies funded by NIH-GM084125 to LU. REFERENCES 1. Wang, H., et al. Nature medicine 10, 1216-1221 (2004). 2. Wang, H., et al. Nature 421, 384-388 (2003).
Galectin-12 in macrophage polarization (INM2P.425)
Authors: Wan Lei , Hsu Yu-An
Published: January 2023

Galectin-12 (Gal12) is a member of a family of animal lectins. Gal12 is expressed in adipocytes and it has been shown to play an important role in adipocyte differentiation. We found that Gal12 is expressed in macrophages and the ablation of Gal12 did not influence bone marrow cells to differentiate into macrophages. The ablation of Gal12 reduced the phagocytic activity against E. coli and lowered the secretion of nitro oxide. Gal12 KO demonstrated the polarization of macrophages to M2 macrophages, as indicated by the increases in the expression levels of FIZZ1 and Ym1 as well as the reduction in the expression levels of TNF-α, IL-6, IL-12, IL-1β, MCP-1, and CXCL1 pro-inflammatory cytokines. We also detected a higher expression level of IL-10, an anti-inflammatory cytokine, in Gal12 KO macrophages. Moreover, the activation of IKKα/β, Akt and ERK was lower in Gal12 KO macrophages, which subsequently reduced the activation of NF-κB and AP1, resulting in lower expression levels of pro-inflammatory cytokines. The activation of STAT1 and STAT6 was lower in Gal12 KO macrophages, indicating that Gal12 may modulate the activation of the IRF3 and subsequently lower the expression of type I interferon by LPS treatment. The activation of STAT3 was much higher in Gal12 KO macrophages activated by LPS, which indicated higher expression levels of IL-10 in Gal12 KO macrophages. Our data indicates Gal12 KO predisposed macrophage to polarize into M2 macrophage.
Nlrc4 inflammasome is dispensable in vitro for IL-1β production and caspase-1 activation upon infection by paracoccidioides brasiliensis. (INM3P.443)
Authors: Carneiro Natália , Rocha Fernanda , Silva Grace , Zamboni Dario , Silva João
Published: January 2023

The fungus Paracoccidioides brasiliensis (Pb), causative agent of chronic systemic mycosis endemic in rural areas of Latin America, is sensed by the TLR-2, 4 and dectin-1 receptors. However, during this infection, the upstream pathways responsible for the cytokines production by macrophages as IL-1β and IL-18 are completely unknown. In Nlrp3-/- macrophages (BMMs), although amounts of IL-1β were reduced, it is still detectable when compared with uninfected macrophages, suggesting that there are additional receptors required for caspase-1 activation and IL-1β production. Then, we evaluate if Nlrc4 could induce IL-1β production and caspase-1 activation in vitro. Strikingly, we observed higher IL-1β release and caspase-1 activation in Nlrc4-/- BMMs than WT cells. To characterize the role of Nlrc4 during experimental infection, WT and Nlrc4-/- mice were infected intravenously with 1x10^6 yeasts forms of Pb18 and fungal burdens in the lung were analyzed at day 15 and 30 post infection. Whereas at 15 dpi WT and Nlrc4-/- mice had similar fungal counts in the lung tissue, at 30 dpi the lung homogenates from Nlrc4-/- mice contained lower numbers of Pb colony-forming units. Moreover, we showed that the reduction in number of viable yeasts was followed by compact and well defined granulomas formation. Taken together, these results suggest that Nlrc4 mediates efficient granulomatous organization and host resistance to P. brasiliensis infection. Financial support: FAPESP, CNPq, CAPES
The nervous system modulates CD8+ T cell function through the beta2-adrenergic receptor (IRC2P.456)
Authors: Estrada Leonardo , Agac Didem , Farrar David
Published: January 2023

There is growing evidence to suggest that various aspects of immune function are controlled by neural networks. Both cholinergic and adrenergic receptors are expressed by various major immune cell types. Specifically, expression of the β2-adrenergic receptor (ADRB2) has been characterized in different cells of the immune system; however, our lab has demonstrated for the first time a differential expression of this receptor in subsets of effector and memory CD8+ T cells. We found that the ADRB2 is upregulated in human effector memory CD8+ T cells compared to central memory/naïve counterparts. Further, human CD8+ T cells that are acutely activated in the presence of the ADRB2 natural ligand, nor-epinephrine (NE), secrete significantly less IFN-γ and TNF-α. Similarly, the cytolytic capacity of these cells is reduced when NE is present. This downregulation by NE was reversed in the presence of a ADRB2-specific antagonist, but not a pan α-adrenergic or β1-adrenergic receptor specific antagonists. Additionally, mouse CD8+ T cells behaved similarly to human cells to these antagonists, suggesting species conservation of this neural-immune network. Moreover, ADRB2-KO CD8+ T cells were completely resistant to the effects of NE. Current experiments using an adoptive transfer model will elucidate how ADRB2 signaling modulates CD8+ T cell function in vivo. These experiments will provide insight into how the nervous system controls CD8+ T cell function during the course of an infection.
TCR strength of signal is upstream of and dominates over cytokine effects in controlling CD4+ T cell polarization in vivo (IRC2P.449)
Authors: van Panhuys Nicholas , Klauschen Frederick , Germain Ronald
Published: January 2023

Both quantitative antigenic signal strength and the qualitative effects of cytokine signaling have been reported to direct the polarization of CD4+ T cells into effectors with distinct patterns of cytokine secretion. Most current models of the polarization process attribute a dominant role to the effects of cytokines, with polarizing adjuvants presumed to work mainly through elicitation of the relevant cytokines. Here we have varied both the adjuvants used for DC activation and the strength of TCR signaling to examine whether quantitative or qualitative aspects of CD4+ T cell activation play the primary role in effector choice in vivo. Using two-photon intravital microscopy we compared DC stimulated with TH1 or TH2 promoting adjuvants and found that it is the duration of interaction and the strength of signal that dominates in controlling CD4+ T cell fate. Adjustments to antigen level that affect interaction duration can reverse the fate outcome irrespective of the adjuvant used to treat the DC, clearly pointing to the quantitative aspects of signaling as dominant over qualitative effects arising from adjuvant-induced cytokine production. Indicating that cytokines operate as a secondary step in a developmental system in which the strength of initial CD4+ T cell signaling regulates the capacity of the cells to respond to cytokine mediators.
Interplay of phosphorylation kinetics and methylation dynamics leads to complex behavior of Foxp3 and PTEN in computational model of T cell differentiation (IRM6P.709)
Authors: Miskov-Zivanov Natasa , Hawse WIlliam , Morel Penelope , Faeder James
Published: January 2023

We have recently published a mathematical model that demonstrates that differential activation of signaling pathways downstream of TCR by high and low concentrations of antigen can be modeled by varying the duration of antigen stimulation, which results in a mixed population of regulatory (Treg), helper (Th) and inactive cells, a prediction we confirmed experimentally. Analysis of the dynamics of this scenario demonstrated an important role for a feedback loop involving Foxp3, PTEN, and Akt-mTOR signaling, which affects the timing of transient Foxp3 expression and PTEN activity, and the ratio of different phenotypes in the mixed population. We hypothesized that the correlation in the dynamics of Foxp3 and PTEN, which we previously modeled as a direct link between Foxp3 and PTEN, stems from the regulation of both Foxp3 and PTEN expression by the transcription factor Foxo1. We updated the model to test this hypothesis, and preliminary results suggest that the interplay of rapid modulation of Foxo1 activity by Akt with the slower kinetics of methylation/demethylation at the Foxp3 promoter mediated by mTOR can produce dramatic changes in the resulting distribution of phenotypes when the timing of these events is varied. To calibrate this timing in the model, we are currently conducting experiments that will help us determine the time scale for Foxo1 inactivation by Akt and for the effect of Foxo1 on Foxp3 and PTEN expression.
IL-17-mediated regulation of C/EBPβ alternative translation: Implications for signaling and response to opportunistic fungal infections (IRM6P.721)
Authors: Simpson-Abelson Michelle , Bishu Shrinivas , Childs Erin , Conti Heather , Gaffen Sarah
Published: January 2023

Th17 cells and IL-17 play essential host-defensive roles in immunity to pathogens, particularly fungi such as Candida albicans. However, the specific signaling pathways used by IL-17 and its receptor are still poorly defined. We have shown that IL-17 activates the CCAAT enhancer binding protein β (C/EBPβ) transcription factor, which is required for induction of many IL-17 target genes. C/EBPβ is translated into 3 distinct protein isoforms whose expression is regulated by the differential use of different translational initiation start sites, known as LAP* (38kDa), LAP (35kDa) and LIP (22kDa). We have shown that IL-17 induces the alternative translation of C/EBPβ, inducing a marked increase in LAP* and LIP. However, the functional significance of differential C/EBPβ isoform expression remains enigmatic. To determine the biological significance of C/EBPβ alternative translation, we used C/EBPβ-/- mice and a knockin that cannot generate the LAP isoform (C/EBPβM20A). We then assessed susceptibility of these mice to oral C. albicans infection, immunity to which is a strongly IL-17-dependent event. We found that C/EBPβ-/- but not C/EBPβM20 mice were highly susceptible to disease, indicating that LAP is dispensable but C/EBPβ is required. In evaluating a panel of downstream genes to elucidate mechanisms of C/EBPβ-dependent immunity, β-defensin 3 (BD3) was strongly downregulated in C/EBPβ-/- mice. These results highlight a key role for C/EBP in antifungal immunity mediated by IL-17.
MicroRNAs, T follicular helper cells, and inflammatory status during aging (IRM6P.714)
Authors: Hu Ruozhen , Kagele Dominique , Huffaker Thomas , Runtsch Marah , Alexander Margaret , Bake Erin , Su Wei , Rao Dinesh , Möller Thomas , Garden Gwenn , Round June , O'Connell Ryan
Published: January 2023

Chronic, low-grade inflammation is associated with many life-shortening human diseases that afflict the elderly. By understanding the molecular mechanisms underlying chronic inflammation effective treatments can be developed to limit such conditions as metabolic disorders, autoimmunity and cancer. Using a mouse model of age-dependent inflammation, we demonstrate that miR-155 is a promoter of disease symptoms in aging mice. Upon analyzing lymphocytes from inflamed versus healthy mice, we observed increased numbers of T follicular helper (Tfh) cells and germinal center (GC) B cells, correlating with increased production of autoantibodies as mice reach middle age. Interestingly, this inappropriate humoral response occurred in a miR-155-dependent manner. Next, we generated T cell-specific miR-155-/- mice and discovered a T-cell intrinsic role for miR-155 in promoting Tfh cell formation, GC B cell development, and production of antigen-specific IgG antibodies. To begin unraveling the mechanistic basis for this phenotype, we employed a pipeline involving RNA-seq, bioinformatics and experimental validation. This approach identified a subset of miR-155 target genes that are relevant to Tfh cell formation. Taken together, our study demonstrates that miR-155 promotes Tfh cells development during age-dependent chronic inflammation, and identifies miR-155 as a potential therapeutic target to diminish age-related inflammatory conditions.
PTEN regulates CD4+ T cell differentiation (IRM6P.716)
Authors: Hawse WIlliam , Miskov-Zivanov Natasa , Faeder James , Morel Penelope
Published: January 2023

Naïve CD4+ T cells differentiate into either helper (Th) or regulatory (Treg) cells following encounter with antigen. T cell fate is in part determined by signaling strength where lower TCR and AKT/mTOR signaling favors Treg differentiation and higher TCR and AKT/mTOR signaling drives Th differentiation. AKT/mTOR signaling is regulated by PTEN, a phosphatase that is highly expressed in Tregs. We recently published a computational model that suggested a critical role for PTEN in T cell differentiation. Our current work focuses on validating the model predictions and explaining how PTEN is regulated during T cell differentiation. We demonstrate that PTEN inhibition increases Akt/mTOR signaling leading to increased Th induction. When the TCR signal strength is high, PTEN is ubiquitinated and phosphorylated and PTEN protein levels are dramatically reduced. Conversely, PTEN levels only decrease transiently when TCR signal strength is low and the resulting Treg express higher PTEN levels. We are exploring the transcriptional regulation of PTEN. One potential regulator is FOXO1, which has been shown by ChIP-Seq to bind the PTEN promoter. High Akt/mTOR signaling results in phosphorylation of FOXO1 and exclusion of FOXO1 from the nucleus. Low Akt/mTOR signaling results in minimal phosphorylation and sustained FOXO1 nuclear localization. Thus, PTEN protein levels are tightly controlled by TCR signal strength and regulation of PTEN plays an important role in determining T cell fate.
Mechanisms of CD4+ T Cell Exhaustion by persisting antigen (LYM3P.737)
Authors: Lober Benedikt , Obst Reinhard
Published: January 2023

When faced with chronic antigen presentation T cells are functionally impaired over time. Since detailed studies of antigen-exhausted CD4+ T cells are scarce, we followed AND TCR transgenic T cells transferred into antigen-transgenic recipients where they received chronic TCR signals of varying strength. In one of the recipient lines, transient antigen expression regulated by doxycycline treatment allowed for the efficient differentiation of memory T cells, with IL7R, Ly6C and swift cytokine production as their hallmarks, while chronic TCR signals exhausted the cells. We found two kinds of exhaustion: Strong stimulation was accompanied by reversible TCR downregulation, signaling impairment and an increase of PD-1 and the anergy markers CD73 and FR4. When stimulated with PMA/Ionomycin, which circumvent TCR-proximal signaling components, these cells produced effector cytokines almost like memory T cells. In contrast, T cells that experienced a weaker TCR stimulus chronically showed no cytokine production following PMA/Ionomycin restimulation. Since in such T cells TCR signaling via the AKT, MAPK and Ca2+ pathways was found only partially impaired, we conclude that strong signals impair TCR-proximal signaling components while weaker signals affect more distal elements. We suggest that the degree of T cell exhaustion correlates with antigen strength, leading either to a fast TCR and signaling downmodulation or to an irreversible functional impairment.
Ndfip1 limits TH17 differentiation and proinflammatory cytokine production. (LYM3P.741)
Authors: Layman Awo A. , Oliver Paula
Published: January 2023

Ubiquitination and subsequent degradation of pathway proteins is an important way of terminating an effector cell response. Three classes of enzymes: E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases, work sequentially to ubiquitinate selected substrates. Ndfip1 (Nedd4-family interacting protein 1) is a protein that activates the catalytic function of several related E3 ubiquitin ligases. Previous work from our lab showed that mice lacking Ndfip1 have increased numbers of TH17 cells and that Ndfip1 is expressed in T cells. Thus, we hypothesized that Ndfip1 has a T cell-intrinsic role in limiting TH17 cell differentiation. Using a mixed chimera model, we show that T cells that lack Ndfip1 are much more likely to produce IL-17A and GM-CSF, two proinflammatory cytokines produced by TH17 cells. Additionally, Ndfip1-deficient T cells produce more IL-17A and GM-CSF when cultured under TH17 polarizing conditions. These data may help to explain why mice that lack Ndfip1 are more susceptible to IL-17-mediated pathology in a DSS colitis model, compared to their Ndfip1-sufficient counterparts. This work reveals a biologically relevant role for Ndfip1 in limiting TH17 differentiation and GM-CSF production, as well as associated inflammatory pathology. We are now working to identify components of Ndfip1-mediated ubiquitin complexes and determine the underlying mechanism by which Ndfip1 limits proinflammatory cytokine production by TH17 cells.
The role of Foxp3 in Treg homeostatic proliferation: Necessary for nTregs, but insufficient for iTregs. (LYM3P.734)
Authors: Lin Yan , Rothstein David
Published: January 2023

NTregs exhibit higher homeostatic proliferation (HP) than Tconv which may provide a competitive advantage during Treg adoptive immunotherapy. Increased Treg HP could be 2° to a self-reactive TCR repertoire and/or Foxp3-mediated increase in survival/fitness. To address this, CFSE-labeled CD4 populations from Foxp3-reporter mice, were compared 10d after transfer into wt mice. We generated Foxp3+ iTregs by activating Tconv and adding TGFβ, or transducing with Foxp3-retrovirus (RV). iTregs did not increase HP over Tconv controls (no TGFβ; RV-vector). Thus, Foxp3 itself is not sufficient to augment HP. To determine the role of Foxp3, we examined GFP+ nTreg from Filig mice (F-nTreg; Foxp3 ~10% wt levels due to unstable mRNA). F-nTreg exhibited reduced HP (10%) vs. wt nTregs (50%). Foxp3-RV transduction increased HP of F-nTregs, while RV-control had no effect (40 vs. 12%). Thus, Foxp3 is necessary for high HP by nTreg. We also examined role of Foxp3 during thymic ontogeny, irradiated Thy1.1 hosts received Filig (Thy1.2) bone marrow that was transduced with Foxp3-RV vs. RV-control. Surprisingly, in this setting, Tconv cells (no endogenous Foxp3) constitutively expressing Foxp3, exhibited increased HP vs. Foxp3- RV-controls (32 vs. 12%). Thus, Foxp3 increases HP in nTreg and when expressed during thymic ontogeny but not in mature Tconv. Our findings provide new insight into the role of Foxp3 expression in Treg homeostasis and highlight additional distinctions between iTreg and nTreg.
Signals through GITR program CD4+ T cells to enhance interleukin-9 production, which mediates antitumor CTL responses (LYM3P.735)
Authors: Kim Il-Kyu , Kim Byung-Seok , Seok Jae-Won , Koh Choong-Hyun , Park Jun-Seok , Shin Kwang-Soo , Lee Ga-Eun , Jeon Hyewon , Chung Yeonseok , Kang Chang-Yuil
Published: January 2023

IL-9-producing T helper cells called Th9 have been a recently identified Th subset diverted from Th2 cells and proved to mediate allergic inflammation and tumor immunity. However, the regulation of Th9 development and functions in diseases remain to be elucidated. Here, we showed that GITR costimulation could induce tumor regression by promoting IL-9 expression on CD4+ T cells. GITR signaling intrinsically enhanced IL-9 expression regardless of cytokine milieu from CD4+ T cells, and furthermore in the presence of TGF-β, could induce IL-9-producing T cells independently of IL-4 in vitro. GITR activation increased the expression of Th9-related transcription factors, Irf4, Gata3 and Batf. In addtion, GITR activated the NF-κB signaling pathway and blockade of NF-κB signaling diminished the increase of IL-9 production from CD4+ T cells by GITR. In vivo GITR ligation augmented cytotoxic T cell responses in mouse tumor models and neutralization of IL-9 led to decrease the cytotoxicity of CD8+ T cells, resultingly impeded tumor regression by anti-GITR Ab. Upregulation of CTL responses was accompanied by the amplication through increasing the maturation and cross-presentaion of infiltrating DCs, regulated by IL-9. Thus, our studies reveal the link between GITR signaling and IL-9, providing a rationale for GITR-induced antitumor immune responses.
BDNF integrates cognitive and physical resilience with immune function in successful aging (LYM4P.753)
Authors: Griffin Patricia , Mueller Robert , Dube John , Huysman Kristy , Michel Joshua , Studenski Stephanie , Vallejo Abbe
Published: January 2023

Successful aging has been defined as functional resilience in late life despite history of chronic disease and/or concurrent medical conditions. Old age therefore may not be synonymous with ill health. We reported that octo-/nonagenarians who are survivors of a long-term cohort of aging have a distinct T cell repertoire expressing diverse NK-related receptors (NKR). Frequency of these unusual T cells is correlated with unimpaired physical/cognitive function. To determine whether broader repertoire remodeling relates to general well being, we recruited a new cohort of community dwelling elders aged >65 years. Results showed physical/cognitive ability is unrelated with systemic cytokines, but with high levels of NKR on both T and NK cells, and brain-derived neurotrophic factor (BDNF) in plasma. Bioassays showed NKR ligation is sufficient to induce T/NK cell activation, and an unexpected production of BDNF. BDNF in culture supernatants and plasma was biologically active as determined by measurable impact on autocrine T/NK cell activation, and on paracrine neurite sprouting and myotube function. Clearly, physical/cognitive resilience is linked to remodeling of the immune cell repertoire. T/NK-derived BDNF indicate unique communication circuit(s) of immune, physical, and cognitive function domains. Unraveling NKR-driven non-TCR centric immunopathway(s) will be key to the development of age-appropriate, group-targeted strategies to promote healthy longevity.
Regulation of c-Myc from costimulatory signals modulates the generation of memory T cells (LYM4P.757)
Authors: Song Jianxun , Haque Mohammad
Published: January 2023

Costimulatory signals are critical for developing high numbers of memory T cells, which can generate optimal secondary immune responses. However, the mechanisms that cosimulatory signals regulate the generation of memory T cells remain to be elucidated. Here we show that the transcriptional factor c-Myc is controlled by costimulatory molecules, including CD28 of the Ig superfamily and OX40 of the TNFR family. Expression of c-Myc in Cd28-/- or Ox40-/- CD8+ T cells partially reversed the defects in the generation of memory T cells to viral infection. c-Myc regulated expression of survivin that promoted the generation of virus-specific CD8+ memory T cells. Moreover, expression of survivin and bcl-xL in Cd28-/- or Ox40-/- CD8+ T cells fully reversed the defects in the generation of memory T cells to viral infection. These data identify c-Myc, together with Nfkb1, as regulators of the generation of memory T cells from costimulatory signals.
miR-155 regulates lymphocyte homeostasis in LAT mutant mice (LYM4P.749)
Authors: Sommers Connie , Rouquette-Jazdanian Alexandre , Li WenMei , Nguyen Phan , Kortum Robert , Samelson Lawrence
Published: January 2023

LAT Y136F mutant mice carry a tyrosine-to-phenylalanine mutation in the LAT adapter gene that leads to a lymphoproliferative disease (LPD) initiated by CD4+ helper T cells. In a screen to detect microRNAs (miRs) specifically regulated in LAT Y136F CD4+ T cells, we found that levels of miR-155 were higher in these T cells than in other proliferating CD4+ T cells from wild type C57BL/6 mice. In order to reveal a potential role of miR-155 in LAT Y136F LPD, we crossed LAT Y136F mice to miR-155-deficient mice yielding double mutant (DM) mice. This resulted in a delay and decreased severity of LPD in DM mice. DM CD4+ T cells showed equal or higher proliferation rates than CD4+ T cells from age-matched LAT Y136F mice, however levels of apoptosis in DM CD4+ T cells were higher. One direct target downregulated by miR-155 is the forkhead transcription factor FOXO3a. In DM T cells, increased expression of FOXO3a led to accumulation of the pro-apoptotic factor Bim and enhanced apoptosis. In addition, elevated Pak1 kinase activity in DM T cells led to activation of Jnk leading to phosphorylation of FOXO3a, FOXO3a nuclear translocation and increased FOXO3a activity. Consequently, direct and indirect effects of miR-155 on FOXO3a led to an increase in Bim-mediated apoptosis and decreased LPD in DM mice. Investigation of downstream targets of miR-155 has led to new information on signaling molecules that control LPD and lymphocyte homeostasis.
Mir-17∼92 promotes terminal differentiation of effector CD8 T cells by lowering TCR stimulation threshold (LYM4P.750)
Authors: Baumann Florian , Yuzefpolskiy Yevgeniy , Sarkar Surojit , Kalia Vandana
Published: January 2023

MicroRNAs are small RNAs (∼22 nucleotides) that regulate gene expression via post-transcriptional mechanisms, and are emerging as major players in immune cell development and function. Using dual strategy of conditional deletion or constitutive overexpression of miR-17∼92, we have recently shown that miR-17∼92 has a critical CD8 T cell-intrinsic role in driving terminal effector differentiation by promoting proliferation (Khan, et al., 2013, Blood). We hypothesized that miR-17∼92 increases the sensitivity of CD8 T cells to TCR stimulation, thus driving proliferation and terminal differentiation. Indeed, when we overexpressed miR-17∼92 in effector CD8 T cells, we observed heightened levels of proximal phosphorylation events downstream of TCR. While PTEN and PD-1 (negative regulators of T cell stimulation) were downregulated upon overexpression of miR-17∼92, dramatic potentiation of TCR signal transduction occurred as evidenced by 50-fold upregulation of ribosomal S6 Kinase activity compared to WT. Converse results were obtained when miR-17∼92 was deleted, and about 10-folds higher levels of peptide-MHC were needed to induce TCR signals to levels similar to WT. Consistent with these observations, under conditions of limiting TCR stimulation, in vivo expansion of virus-specific miR-17∼92-deleted CD8 T cells was also severely compromised. Our data, altogether present a novel role of miR-17∼92 in regulating TCR sensitivity during CD8 T cell activation and effector differentiation.