bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)
Authors: McKarns Susan , Miller Patrick , Bonn Michael
Published: January 2023

Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.
Identifying the cytokines provided by antigen-presenting cells critical for generating an encephalitogenic T cell (BA11P.126)
Authors: Lee Priscilla , Smith Alan , Yang Yuhong , Racke Michael , Lovett-Racke Amy
Published: January 2023

Multiple sclerosis (MS) is a CD4 T cell-mediated demyelinating disease; however, what determines the encephalitogenicity of T cells remains unclear. Recognizing myelin peptides is not sufficient, as the frequencies of myelin-reactive T cells are similar between MS patients and healthy individuals. In experimental autoimmune encephalomyelitis (EAE), anti-CD3/CD28 antibodies-generated myelin-specific T cells do not transfer disease, but antigen-presenting cells (APC)-generated ones do. This suggests that the cytokines secreted by APC provide critical signals beyond T cell receptor activation and co-stimulation, contributing to an encephalitogenic phenotype. To identify the critical cytokines, myelin-specific T cells were activated with anti-CD3/CD28 in the presence of various cytokines, and the cells were adoptively transferred into naïve recipients. T cells generated with the combination of IL-6/IL-23 or IL-12/IL-23 induced severe EAE. We showed that IL-6 and IL-12 induced the initial expression of IL-23R on naïve T cells, and IL-23 further enhanced the expression of its own receptor. Neutralizing IL-6 or IL-12 in the APC-T cell culture significantly reduced the encephalitogenicity of T cells. Furthermore, IL-23R signaling induced STAT3 and STAT4 phosphorylation in both IL-6/IL-23 and IL-12/IL-23-generated T cells. Taken together, we have identified the minimal signals required to generate an encephalitogenic T cell, which provides potential targets for novel MS therapy.
Mathematical modeling of lymphocyte kinetics reveals true residence times in and preferential migration of lymphocytes via ovine lymph nodes (CAM4P.157)
Authors: Ganusov Vitaly , McDaniel Margaret
Published: January 2023

There are many published values of lymphocyte residence times in lymph nodes of various mammals such as mice, rats, and sheep. Yet, most of these estimates have been obtained semiquantitatively, without the use of mathematical modeling, which raises a question about assumptions underlying such estimates. We developed a novel mathematical model for migration of lymphocytes in the whole body which takes into account the known biological details of how lymphocyte enter and exit major lymphoid tissues. We applied this novel mathematical model to experimental data on cannulation of different ovine lymph nodes (LNs). In particular, we predicted that in sheep, recirculatory lymphocytes spend on average 3 hours in the spleen and 20 hours in LNs with a distribution of residence times in LNs following a skewed gamma (lognormal-like) distribution. This is in contrast which a recent suggestion that the distribution of residence times of naïve T cells in murine LNs is exponential. We also found that lymphocytes isolated from the prescapular LN have a two-fold increased entrance rate into the prescapular LN as opposed to the intestinal LN. Importantly, the data could not be explained by preferential retention of cells in a specific LN. Taking together, our work illustrates the power of mathematical modeling in predicting the kinetics of lymphocyte migration in sheep and provides quantitative estimates of lymphocyte residence times in major ovine lymphoid tissues.
IL-33/ST2 correlates with severity of haemorrhagic fever with renal syndrome and regulates the inflammatory response in Hantaan virus-infected endothelial cells (CCR5P.205)
Authors: Zhang Yusi , Zhang Chunmei , Zhuang Ran , Ma Ying , Zhang Yun , Yi Jing , Jin Boquan
Published: January 2023

Hantaan virus (HTNV) causes a severe lethal haemorrhagic fever with renal syndrome (HFRS) in humans. Despite a limited understanding of the pathogenesis of HFRS, the importance of the abundant production of pro-inflammatory cytokines has been widely recognized. IL-33 has been demonstrated to play an important role in physiological and pathological immune responses. After binding to its receptor ST2L, IL-33 stimulates the Th2-type immune response and promotes cytokine production. Depending on the disease model, IL-33 either protects against infection or exacerbates inflammatory disease, but it is unknown how the IL-33/ST2 axis regulates the immune response during HTNV infection. We found that the levels of IL-33 and sST2 were significantly higher in HFRS patients’plasma than in healthy donors and the elevated IL-33 and sST2 levels were positively correlated with the disease severity. Furthermore, we found that IL-33 enhanced the production of pro-inflammatory cytokines in HTNV-infected endothelial cells through NF-κB pathway and that this process was inhibited by the recombinant sST2. Our results indicate that the IL-33 acts as an initiator of the "cytokine storm" during HTNV infection, while sST2 can inhibit this process. They may exert their pro- and anti- functions in inflammatory response during HTNV infection, respectively. Our findings indicated that the IL-33/ST2 axis may be exploited as a therapeutic target for Hantavirus infections.
Thymocyte requirements for normal thymic medullary epithelial cell development (HEM7P.227)
Authors: Williams Joy , Jeon Hyein , Hodes Richard
Published: January 2023

The thymic medullary compartment has a central role in tolerizing developing TCRαβ+ CD4 and CD8 single positive (SP) thymocytes. In turn, SP thymocytes are required for normal maturation of medullary thymic epithelial cells (mTEC). In the absence of TCRαβ+ SP thymocytes, the thymic medullary compartment is much smaller and numbers of mTEC are dramatically reduced. We recently demonstrated that the requirement for TCRαβ+ SP thymocytes in mTEC development can be overcome when Traf3, an inhibitor of NIK-dependent NF-κB signaling, is absent in TEC. In contrast to TCRα KO mice, TCRα KO/TEC-specific Traf3 KO mice have mTEC numbers comparable to that of wildtype mice. Traf3 inactivation also overcomes the requirements for LTβR and CD40 in mTEC development. Interestingly, however, we find that mTEC cell numbers are not rescued in TCRβ KO/TEC-specific Traf3 KO mice. TCRβ KO mice, like TCRα KO mice, lack SP thymocytes but also lack post-β-checkpoint TCRβ+ DN and DP thymocytes. This result suggests that a cell type(s) other than a TCRαβ+ SP thymocyte is required for normal mTEC development even in the absence of Traf3 and that this requirement is mediated by molecular interactions other than those involving LTβR and CD40. Studies to determine the identity of this thymic cell population and the corresponding molecular pathways involved in mTEC development are underway.
The cellularity of thymic regulatory T cells is regulated by CCR7 through a non-cell intrinsic mechanism (HEM7P.228)
Authors: Hu Zicheng , Ehrlich Lauren
Published: January 2023

To ensure central tolerance, developing T cells must enter the thymic medulla to encounter self-antigens that induce autoreactive cells to undergo apoptosis or differentiation into the regulatory T cell (Treg) lineage. Both dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) present antigens to tolerize thymocytes. mTECs express a diverse array of self-antigens, while DCs express auto-antigens acquired both in the periphery and from mTECs. Thymocytes must interact efficiently with these medullary antigen presenting cells (APCs) to encounter the full spectrum of self- antigens. CCR7 guides thymocytes into the medulla, promoting encounters with medullary DCs and mTECs. In the absence of CCR7, negative selection of autoreactive thymocytes is impaired, and autoimmunity ensues. Surprisingly, thymic Treg cellularity is increased in Ccr7-/- mice. Diminished negative selection has previously been associated with a concomitant increase in Treg differentiation. Thus, we anticipated that a cell-intrinsic defect in CCR7-/- thymocytes would account for increased Treg generation. Instead, a series of bone marrow chimeras demonstrate that CCR7-deficient non-T, non-B lineage hematopoietic cells account for increased Treg cellularity. Expression profiling revealed expression of Ccr7 by thymic DCs. We present evidence that CCR7 deficiency in thymic DCs is responsible for increased Treg cellularity, suggesting that CCR7 has pleiotropic effects on thymic central tolerance.
DNA-dependent protein kinase plays a role in inflammation independent of DNA-damage repair function and its inhibition blocks allergic inflammation in mice and modulates human CD4+ T cell function without causing SCID (HYP7P.264)
Authors: Pyakurel Kusma , Ghonim Mohamed , Ju Jihang , Rodriguez Paulo , Lammi Matthew , Davis Christian , Naura Amarjit , Boulares Hamid
Published: January 2023

We reported that DNA- dependent protein kinase (DNA-PK) is critical for the expression of NF-κB-dependent genes in TNF-α -treated glioblastoma cells including VCAM-1. Here we show that DNA-PK protein level and function are critical for VCAM-1 expression in TNF-α-treated human endothelial and mouse lung smooth muscle cells and are required for monocytes adhesion. Interestingly, DNA-PK activation and subsequent VCAM-1 expression in presence of TNF occurred independently of DNA breaks/repair. Administration of the DNA-PK inhibitor, NU7441, and DNA-PK heterozygosity reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA-specific IgE production in asthma model of mice. Such effects correlated with a marked reduction in lung VCAM-1 expression and production of several inflammatory cytokines. Remarkably, such protection occurred without causing SCID. These results were confirmed in a chronic model of asthma using house dust mite. DNA-PK inhibition reduced Th2 cytokines production without affecting Th1 cytokines production and cell proliferation in CD3/CD28-stimulated human CD4+T cells potentially by blocking gata-3 expression. In mouse CD4+T cells, DNA-PK inhibition, in vitro, severely blocked CD3/CD28-induced gata-3 and t-bet expression and prevented differentiation of Th1 and Th2 cells. Thus, our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.
Monocyte and Treg phenotypes in two U.S. farming populations mirror differential asthma and atopy risk: studies in Amish and Hutterite children (HYP7P.254)
Authors: Hrusch Cara , Ober Carole , Stein Michelle , Igartua Catherine , Holbreich Mark , Thorne Peter , Vercelli Donata , von Mutius Erika , Sperling Anne
Published: January 2023

The differences in asthma prevalence between farming and non-farming families in central Europe are recapitulated in two unique U.S. farming populations, the Amish of Indiana and the Hutterites of South Dakota. The prevalence of asthma and allergic sensitization was 5.2% and 7.2%, respectively, in Amish children and >15% and >40%, respectively, in Hutterite children. Compared to Hutterite children, Amish children’s PBLs had lower eosinophils, but higher neutrophils. Amish had higher gene expression of IRF7 and TNFA, which may be the result of the elevated levels of endotoxin found in Amish homes. To determine how the Amish environment alters antigen presentation, we measured the activation status of monocytes. The Amish had increased percentages of inhibitory monocytes that expressed low HLA-DR and high ILT3 and ILT5. We investigated how these changes in innate activation influenced the adaptive immune response. Amish had low ICOS expression on their CD4 effector cells and high ICOS expression on their Tregs. The expression of ILT5 on monocytes correlated to the level of CD45RO+ICOS+ Tregs. Consistent with the idea that microbial exposure could be promoting a regulatory immune phenotype similar to endotoxin-induced tolerance, PBLs from the Amish were hyporesponsive to LPS. These results indicate that environmental exposure has a profound impact on baseline immune function and may prevent the induction of atopic disease in children.
Maternal supplementation of allergic female mice with gamma-tocopherol increases the development of select dendritic cell subsets and allergic lung inflammation in neonates (HYP7P.272)
Authors: Abdala-Valencia Hiam , Soveg Frank , Cook-Mills Joan
Published: January 2023

Offspring from allergic mothers have elevated numbers of CD11b+ lung dendritic cells and develop allergic lung responses to suboptimal allergen challenge. We recently reported that maternal supplementation with alpha-tocopherol (αT) reduced development of these dendritic cell subsets and allergic responses in offspring of allergic female mice. It is not known whether gamma-tocopherol (γT) supplementation regulates development of allergic responses. To address this, allergic female mice were supplemented with γT during pregnancy/lactation. Then, offspring were given a suboptimal allergen challenge. γT supplementation of allergic mothers elevated pup responses to allergen challenge. There were increased numbers of pup lung eosinophils, inflammatory mediators, and CD11b+ but not CD11b- subsets of CD11c+ dendritic cells. There were elevated numbers of IRF4+ CD11b+ CD11c+ dendritic cells, a dendritic cell subset critical for development of allergic responses. There were also fewer pups from γT supplemented allergic mothers. In conclusion, maternal αT supplementation reduced and maternal γT supplementation increased development of CD11b+ CD11c+ dendritic cells and allergic responses in offspring from allergic mothers. These results have implications for supplementation of allergic mothers with tocopherol isoforms and for development of allergies in future generations.
TGFb inhibits IL-33-mediated mast cell activation (INC6P.316)
Authors: Ndaw Victor , Paez Patrick , Taruselli Marcela , Ryan John
Published: January 2023

TGFβ is involved in many pathological considtions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGFβ can suppress IgE-mediated mast cell activation. IL-33 is a recently discovered member of the IL-1 family that plays a critical role in mast cell activation and enhances IgE-mediated activation. In this study, we investigated the effects of TGFβ on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGFβ -1, -2, or -3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13 and MCP-1, in a concentration-dependent manner. Furthermore, TGFβ also reduced expression of the T1/ST2 receptor as well as IL-33-mediated ERK activation. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGFβ on IgE-mediated activation, demonstrate that TGFβ can provide broad and substantial inhibitory signals to activated mast cells.
Generation and validation of a reporter mouse for inflammasome assembly (INM6P.335)
Authors: Tzeng Te-Chen
Published: January 2023

Inflammasomes are key signaling platforms that detect pathogenic microorganisms and sterile stressors and then activate the highly pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 in response. Both IL-1β and IL-18 have numerous functions in inducing the production of more cytokines, such as TNFα. Hence, the inflammasome is a central mediator of inflammation. Although we have several tools to test inflammasome activation in vitro, there is a dearth of in vivo tools to probe the role of inflammasome activation during infection. Thus, we generated a transgenic mouse expressing mouse ASC-citrine fusion protein in the Rosa26 locus. This system contains a knock-in of ASC-citrine gene and a proximal floxed stop site, which allows us to conditionally express ASC-citrine in a lineage specific manner. BMDC from this reporter mouse showed ASC speck formation after inflammasome stimulation, as defined biochemically by others. This suggests that inflammasome activation will be detectable in vivo by looking for specks in tissues. Interestingly, our data suggested that non-hematopoietic cells are the major speck formed cells in the early phase of inflammation when infected mice with Influenza A virus. We also showed that specks are released after inflammasome activation that may enhance and amplify the inflammatory responses. Overall, the expression of ASC-citrine molecules in the mouse system has renders it a useful tool to study and monitor inflammasome activation in vivo.
B cell intrinsic effects of the vitamin D receptor on IgE responses. (IRC10P.417)
Authors: James Jamaal , Cantorna Margherita
Published: January 2023

B cells express the vitamin D receptor (VDR). VDR KO mice had higher levels of circulating total IgE than WT mice by 12 wks of age. Mice were sensitized using two OVA/alum injections. VDR KO mice mounted a significantly higher OVA-specific IgE response compared to WT mice. The data also showed that vitamin D deficient mice had higher OVA-specific IgE responses than vitamin D sufficient mice. IL-4, IL-5, IL-2, IFN-γ and IL-17 were not different in OVA re-stimulated spleen and mesenteric lymph nodes (MLN) in WT and VDR KO mice. However, VDR KO spleen and MLN produced significantly less IL-10 than WT. Furthermore B cells and not T cells were the major source of IL-10 in both the WT and VDR KO cultures. Experiments were done using T cell specific-VDR deletion (T-VDR KO), or B cell specific-VDR deletion (B-VDR KO). The serum IgE levels in T-VDR KO mice were comparable to that of WT mice. Conversely, B-VDR KO mice had higher total serum IgE levels than WT mice and, when immunized, produced significantly more OVA-IgE. In our current model, we propose that B cell-derived IL-10 is regulated by the VDR and that autocrine IL-10 is higher in WT mice, therefore inhibiting IgE production to a greater extent in WT than in VDR KO mice. The major finding of this work is that vitamin D receptor expression selectively regulates IgE levels in the serum via a direct effect of the VDR in B cells.
Probiotic preparation modulate immune system response to food proteins (IRC10P.418)
Authors: Zlotkowska Dagmara , Wasilewska Ewa
Published: January 2023

Probiotics are microorganisms which have been reported to have beneficial effect on human health to the different extent. Presented study uses probiotic preparation (one commercial were used) to modulate mucosal immune system response to food allergens beta-lactoglobulin and ovalbumin. Mice were orally fed with 5x107 CFU DICOFLOR® by 24 days. Then mice were orally immunized with antigen on 10, 17 and 24 day of experiment. At 35 days post-immunization, significant decreases in serum Ag-specific IgG and IgA titers were observed: 2^6 and 2^4 for β-LG immunized mice and 2^7 and 2^2 for OVA-immunized mice, respectively . Specific IgA titer in fecal extracts was slightly higher than that in groups not supplemented with DICOFLOR®. On 48th day of the experiment, DTH test was performed. No response was observed 24 and 48 hours after DTH challenge by groups supplemented with probiotics preparation. Lymphocytes from spleen, head and neck lymph nodes, Peyer’s patches and mesenteric lymph nodes were phenotyped for CD8+, CD4+and CD4+CD8+ T cells. In Peyer’s patches, mice immunized β-LG and supplemented with probiotics showed elevated numbers of CD4+CD8+ and CD8+ T cell subsets compared to β-LG group not supplemented with probiotic. Similar results were found for OVA group. Results show that probiotics has potential to modulate immune response to food allergens. Those potential could be used to prevent symptoms of allergy to food allergens.
Stromal cells from TNF-transgenic mouse inflamed lymph nodes differentially affect B cell subset maintenance and activation in vitro (IRC11P.429)
Authors: Bottaro Andrea , Kuzin Igor
Published: January 2023

Inflamed lymph nodes undergo major structural remodeling, associated with proliferation and functional alterations of stromal cell components. In recent years, critical roles for stromal cells have emerged in determining these architectural changes, as well as affecting the trafficking and localization of immune cell populations and the outcome of immune responses. We have shown that in the TNF-transgenic (TNF-tg) mouse model of arthritis, development of chronic inflammatory joint disease is associated with dramatic changes in the organization of draining lymph nodes and in the phenotypic and functional features of resident B cells, with characteristic expansion of a unique CD23+, CD21/35-high, CD1d-high B cell population (B-in cells). These changes in turn profoundly affect lymphatic drainage function, contributing to pathogenesis. In the studies presented here, we show that in vitro co-culture of B cells on stromal cells from TNF-tg, but not non-inflamed wild-type lymph nodes results in maintenance of the B-in phenotype, elevated surface CD23 expression, and changes in proliferative and functional responses to activation stimuli. Altogether, these results are consistent with a direct role of lymph node stroma in the B cell alterations associated with TNF-tg pathogenesis, and more broadly demonstrate that chronic inflammation-induced changes to stromal cells significantly affect the functional properties of resident B lymphocyte populations.
Expression of Helios is required for the differentiation of activated/effector T regulatory cells (IRC11P.438)
Authors: Thornton Angela , Sebastian Mathew , Korty Patricia , Lopez-Ocasio Maria , Shevach Ethan
Published: January 2023

Foxp3+ Regulatory T cells (Treg) suppress immune activation and play a critical role in the maintenance of self-tolerance. A subpopulation (70-80%) of Treg cells in both mouse and man express the transcription factor, Helios, but the role of Helios in Treg function is still unknown. Deletion of Helios expression in all T cells (CD4-Cre) or in early progenitors (Vav-Cre) did not result in immune dysfunction. As some studies have suggested that Helios might be expressed in activated T conventional cells, we generated mice with a conditional deletion (KO) of Helios in Treg. The selective deletion of Helios in Tregs led to a slowly progressive, systemic immune activation characterized by a Th1 profile, lymphadenopathy, splenomegaly, increased serum Ig and enhanced lymphoid follicle and germinal center formation in the absence of immune pathology. In vitro suppressive function of Helios KO Treg was normal, but KO Treg failed to differentiate into T follicular regulatory cells in vivo. While the absolute number of Treg in KO mice was enhanced, in competitive settings Helios deficient Tregs were at a striking disadvantage in the periphery. Notably, Helios KO Tregs with an activated (CD44hiCD62Llo) effector Treg phenotype were greatly decreased. Analysis of Helios KO Tregs revealed increased levels of several BH3 domain pro-apoptotic genes. These findings suggest that Helios plays a critical role in the development and/or survival of effector Tregs by repressing pro-apoptotic genes.
Regulatory T cells ameliorate acetaminophen-induced immune-mediated liver injury (IRC11P.439)
Authors: Wang Xuefu , Sun Rui , Tian Zhigang
Published: January 2023

The contribution of innate immune cells to acetaminophen (APAP)-induced liver injury has been extensively investigated. However, the roles of T cell populations among adaptive immune cells in APAP-induced liver injury remain to be elucidated. Herein, we found that distinct CD4+ T cell subsets but not CD8+ T cells modulated APAP-induced liver injury in mice. After APAP challenge, more CD62LlowCD44hiCD4+ T cells appeared in the liver, accompanied by increased IFN-γ. The removal of CD4+ T cells by either antibody depletion or genetic deficiency markedly compromised pro-inflammatory cytokine levels and ameliorated liver injury. Meanwhile, we also found that the frequency and absolute number of Treg cells also increased. Treg cell depletion increased hepatic CD62LlowCD44hiCD4+ T cells, augmented pro-inflammatory cytokines, and exacerbated liver injury, while adoptive transfer of Treg cells ameliorated APAP-induced liver injury. Our investigation suggests that Th1 and Treg subsets are involved in regulating APAP-induced liver injury. Thus, modulating the Th1/Treg balance may be an effective strategy to prevent and/or treat APAP-induced liver injury.
The effect of 2, 3, 7, 8-Tetrachlorodibenzo -p-Dioxin in the human 3’IgH regulatory region and immunoglobulin expression. (IRC11P.440)
Authors: alfaheeda zahra
Published: January 2023

The transcription of Ig heavy chain (Igh) is regulated by numerous regulatory elements including 3’Igh regulatory region (3’IghRR).Several transcription factors are involved in modulating the 3’IghRR and aryl hydrocarbon receptor (AhR). TCDD is an environmental contaminant and a ligand for AhR known to interfere with Immunoglobulins (Ig) expression in animal models. Our lab have identified in previous study the mouse 3’Ig heavy chain regulatory region (3’IgH RR) as a sensitive transcriptional target of TCDD, which inhibits mouse 3’IgH RR activation and induces AhR binding to dioxin response element (DRE) within 3’IgH RR. In human the hs1, 2 enhancer is polymorphic which has been correlated with several autoimmune diseases. We have identified TCDD-induced activation of the hu-hs1, 2 enhancer and a decrease in enhancer activity with deletion of the polymorphic region. Pervious study in our lab suggested that the hu-hs1, 2 enhancer may be a negative regulator of 3’IgH RR activity and Ig expression. The objective of this study is to study the cooperative effect of the Human 3’IgHRR Enhancers in intronic promoters and the possibility of TCDD interfering with this activity in the presence and absence of the stimulation and to study the polymorphic alleles effect in the human 3’IgHRR basal activity. We used human CL-01 B cell for this study and multiple luciferase reporter constructs driven by human Igγ intronic promoter and regulated by the human 3’IgHRR enhancers .
Regulatory T cell differentiation and Th17 T cell function are regulated by mPGES-1-dependent PGE2 (IRC11P.424)
Authors: Maseda Damian , Crofford Leslie , Johnson Elizabeth
Published: January 2023

Prostaglandin E2 (PGE2) is a lipid mediator that regulates inflammatory and immune responses. PGE2 has pleiotropic effects on many cell types, but it is still unclear how it affects T cell differentiation and function. The mPGES1 enzyme is part of the PGE2 biosynthetic pathway and, due to its inducible nature, it controls PGE2 concentrations during activation of the inflammatory program. Absence of mPGES1 causes shunting to alternate prostaglandins and changes the characteristics of the inflammatory response. We identified that, compared to WT, naïve T cells from mice with genetic deletion of mPGES1 express a 4-fold lower level of TFGbR1 and 8-fold lower level of the EP4 PGE receptor. Since TGF-b is a key cytokine for the generation of both regulatory and Th17 cells we addressed the effect of mPGES1 deficiency during in vitro T cell polarization. We observed that mPGES1-/- naïve CD4+ T cells generate less iTreg cells, while Th17 commitment is unaffected. However, when stimulating T cells with anti-CD3/CD28 from mice following immunization with type II collagen (CII), mPGES1-/- CD4+ T cells were unable to secrete IL-17A compared to its WT T cell counterparts. Furthermore, in-vivo antigen-specific Th17 responses from CII immunized mice were also impaired, showing a decrease in IL17A production in mPGES1-/- T cells compared to WT. Hence, endogenous production and sensing of PGE2 modulates T cell differentiation possibly by altering TGF-b responses.
ICER controls Th17 cell differentiation and autoimmune pathology (IRM15P.600)
Authors: Yoshida Nobuya , Mizui Masayuki , Otomo Kotaro , Koga Tomohiro , Bradley Sean , Comte Denis , Rosetti Florencia , Kyttaris Vasileios , Crispin Jose , Tsokos George
Published: January 2023

Inducible cAMP early repressor (ICER) is a splice variant of cAMP-responsive element modulator (CREM). Because ICER lacks transcriptional activation domains, ICER acts as a repressor of various genes involved in immune cell function and inhibits T cell activation by suppressing the induction of pro-inflammatory cytokines including IL-2. However, little is known about ICER involvement in IL-17 production and Th17 differentiation. Culture of murine naïve CD4 T cells under Th17 skewing conditions showed surprisingly high expression of ICER indicating and important role in these cells. T cells from animals in which the CREM gene had been deleted were cultured under these conditions, which revealed less IL-17 than T cells from CREM sufficient mice. Because ICER induction is abrogated in T cells from STAT3-deficient mice, we considered the possibility that IL6-STAT3 signaling plays a crucial role in ICER induction in Th17 T cells. We expanded our observations in vivo. We induced experimental anti-glomerular basement membrane antibody glomerulonephritis (AIGN) and experimental autoimmune encephalomyelitis model (EAE) in CREM-deficient and sufficient animals and found that CREM-deficient had diminished nephritis and encephalomyelitis. Collectively, our results suggest that the transcription factor ICER supports Th17 differentiation and promotes Th17-dependent autoimmune pathology.
Immunological difference in Th1 and Th2 dominant mouse strains in an ICD model. (IRM15P.601)
Authors: Luckett-Chastain Lerin , Calhoun Kaitlin , Kemp Jesse , Gallucci Randle
Published: January 2023

Of reported occupational injury associated with workman’s compensation, contact dermatitis ranks second most prevalent overall. C57BL/6 and BALB/c differ in their immune responses, tending to be either Th1- or Th2-dominant, respectively. Irritant contact dermatitis (ICD) is an acute inflammatory response that can vary among individuals. Therefore; to better characterize ICD, these two mouse strains were utilized. C57 and BALB/c mice were exposed to benzoalkonium chloride, (BKC) or acetone (control) for 3 or 7 days. Multiplex protein analysis showed pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IFN-γ, IL-22, and CSFs (GM-CSF and G-CSF) were all significantly upregulated in C57s at Day 3 as compared to BALB/c. Inflammatory cell chemoattractants such as CXCL1, 2, and 10, as well as CCL3, 4, 5, and 7 also displayed higher expression in C57s. Interestingly, it wasn’t until Day 7 that BALB/c mice showed significantly elevated levels of inflammatory mediators such as IL-2, IL-4, IL-5, IL-31, IL-9, IFN-γ, and CCL2 compared to C57. Cytokine mRNA expression also varied between the two strains, but did not necessarily correlate with protein data. In addition, C57 appeared to have worse inflammation than BALB/c as indicated via histopathology. Overall, these results further confirm previous findings in which BALB/c and C57 exhibit different immune responses and may provide a better understanding of dermopathology and allow for specific immunomodulation for therapy.
CIS inhibits effector programs in regulatory T cells (IRM15P.459)
Authors: Yang Xuexian , Zheng Handong , Dong Chen
Published: January 2023

Regulatory T (Treg) cells play an essential role in immune homeostasis. Under the pro-inflammatory settings such as autoimmune diseases, the function of Treg cells is impaired, in which pro-inflammatory cytokine IL-4 and IL-6 are involved in destabilizing Treg cells. However, the pathways that stabilize Treg cells have not been well understood. We have shown that cytokine-induced SH-2 protein (CIS) inhibits STAT3 and STAT6 activation; our current study indicates that CIS is required for stabilizing Treg cells in a Treg cell-intrinsic manner. Cis deficient Treg cells had impaired suppressive activity in vitro. Cis deficient mice on a T helper 2 (TH2) cells-prone background develop lymphadenopathy, splenomegaly and multiple organ inflammation, which is associated with spontaneous loss of Foxp3 expression and gain of TH2 cytokine expression in Treg cells. In addition to its role in cytoplasm, CIS also translocates into the nuclei in Treg cells which may serve as a second mechanism of surveillance for pro-inflammatory cytokine induced STAT6 (and maybe also STAT3) in the nuclei, and therefore contribute to stabilization of Treg cells.
Translational control of TGF-β-mediated Treg cell differentiation by hnRNP E1 (IRM15P.460)
Authors: Ansa-Addo Ephraim , Hussey George , Howe Philip , Li Zihai
Published: January 2023

CD4+Foxp3+ T cells are generated either in the thymus for natural regulatory T cells, or in periphery for inducible Treg cells (iTregs), which differentiate from naïve T cell precursors. Both Tregs express Foxp3 albeit they differ in their plasticity and in their expression of certain genes. Trans­forming growth factor-β (TGF-β) and interleukin-2 are important for iTreg generation and maintenance, whereas proinflammatory cytokines such as IL-4 and IL-6 impair Foxp3 induction. Much has been learned regarding the transcriptional roles of TGF-β in determining the fate of iTregs; however, the limited focus if any on a translational mechanism by which TGF-β regulates the fate of functional T cells is somewhat myopic. We previously showed a novel translational mechanism for TGF-β regulation of EMT in mouse mammary gland epithelial cells whereby hnRNP E1 (E1) binds to a structural, 33-nucleotide TGF-β-activated translation element in the 3’ UTR of target transcripts and represses their translation. In this study, we found that E1 knockdown (E1KD) in naïve CD4+ T cells induces TGF-β signaling and upregulates expression of multiple TGF-β target molecules implicated in iTreg cell differentiation. Furthermore, E1KD CD4+ T cells bypass the need for TGF-β and express Foxp3. This study adds to our knowledge on a hitherto unrecognized translational mechanism by which TGF-β controls iTreg cell fate.
The regulation and role of L-glutamine in B-lymphocyte activation (LYM7P.618)
Authors: Heyse Shannon , Connolly Timothy , Doughty Cheryl , Chiles Thomas
Published: January 2023

B lymphocyte activation is a highly energetic event that promotes cell survival and growth. Aberrant activation of B lymphocytes can lead to disease states such as lymphomas or autoimmune diseases. To meet the metabolic demands of activation, B cells increase nutrient uptake to provide energy and intermediates for macromolecule synthesis. We have investigated the role of L-glutamine, which is emerging as a critical immunomodulatory nutrient; however, the precise role of L-glutamine in B cell growth responses is poorly understood. Depletion of extracellular L-glutamine results in impaired cell growth and proliferation in response to B-cell antigen receptor (BCR) ligation, although, early activation events such as induction of CD86 are unaffected. We show that L-glutamine uptake is induced in response to BCR ligation. This corresponds to an increase in the surface expression of the high affinity L-glutamine transporter, ASCT2, indicating its involvement in L-glutamine uptake. Further, inhibition of ASCT2 by the small molecule N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) caused impaired B lymphocyte activation similar to that observed in response to L-glutamine depletion. The addition of L-leucine abated some of the effects of GPNA treatment on B lymphocyte growth responses, indicating mTORC involvement. These data provide new insight on the requirement of L-glutamine in B lymphocyte activation and suggest a possible underlying mechanism of growth regulation by mTORC.
Histone methyltransferase Ezh2 is critical for activation-induced CD8 T cell proliferation and survival (LYM7P.615)
Authors: Chen Guobing , Subedi Kalpana , Mi Jenny , Shi Alvin , Weng Nan-Ping
Published: January 2023

Ezh2 catalyzes trimethylation of H3K27 and regulates transcriptional repression. It plays a vital role during stem cell development and cell differentiation. Although it is well documented that epigenetic changes regulate gene expression, the epigenetic mechanism during CD8 T cell differentiation are not fully understood. Here, we assessed the role of Ezh2 in CD8 T cell differentiation using Ezh2 T cells deficient mice (Ezh2-loxp/CD4-Cre, Ezh2 KO). The number of CD8+ T cells in the young Ezh2 KO were similar to wild-type (WT) mice. When infected with listeria-OVA, OVA+ CD8 T cells were not increased in Ezh2 KO compared to WT. This deficiency was further confirmed with the adoptive transfer of the OT-1 CD8 Tn cells into congenic mice with infection. We then examined CD8 T cell differentiation in vivo and in vitro. We found that CD8 Tn cells express activation-induced markers and cytokines similarly between Ezh2 KO and WT mice. However, CD8 Tn cells from Ezh2 KO mice showed impaired proliferation and enhanced apoptosis after activation compared to that of the WT mice. Genome-wide transcription analysis confirmed that Ezh2 KO CD8 T cells expressed significantly higher levels of cell cycle and apoptosis related genes than WT after activation. Collectively, these findings demonstrate that Ezh2 is required for repression of the transcriptional programs involved in cell proliferation and apoptosis during CD8 T cell activation and differentiation.
IL-21 signaling modulates the differentiation, distribution, and abundance of CD8 T cell subsets (LYM7P.616)
Authors: Tian Yuan , Cox Maureen , Kahan Shannon , Ingram Jennifer , Bakshi Rakesh , Zajac Allan
Published: January 2023

The activation of naïve CD8 T cells typically results in the formation of effector (TE) cells as well as memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory (TCM), effector memory (TEM) and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using adoptive transfer studies and mixed bone marrow chimeras in which CD8 T cells either do or do not express the IL-21 receptor (IL-21R), we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals resulted in reduced accumulation of CD8 T cells in various tissues including the small intestine intraepithelial lymphocyte compartment. The reduced fraction of IL-21R-/- CD8 TRM cells in the small intestine also displayed markedly lower levels of the cytolytic effector molecule granzyme B. Notably, IL-21 signaling was associated with the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on circulating CD8 T cells in the mixed bone marrow chimeras. Acute lymphocytic choriomeningitis virus infection overcame the necessity for IL-21 to establish CD8 TE/TEM and TRM subsets. Overall, this study demonstrates that IL-21 modulates the differentiation, accumulation, and function of effector-phenotype CD8 T cells in a context-dependent manner.
Identification of extrinsic and intrinsic factors that specify CD4+ TCM cell fate (LYM8P.626)
Authors: Oestreich Kenneth
Published: January 2023

The expression of the transcriptional repressor Bcl-6 has been commonly linked to the development of both the CD4+ T follicular helper (TFH) and T central memory (TCM) cell types. Here, we demonstrate that in response to decreased interleukin 2 (IL-2) signaling, Bcl-6 expression is increased in effector T helper 1 (TH1) cells. Consequently, this results in the promotion of both TFH and TCM-like gene programs including expression of Il6ra and Il7r. Strikingly, exposure of this potentially bi-potent cell population to interleukin 7 (IL-7) results in a significant decrease in TFH-associated gene expression patterns including that of Bcl6 and Cxcr5, but not in TCM-specific genes including Sell and Klf2. The decrease in the TFH-profile is dependent upon a repressive complex that includes the IL-7 responsive transcription factor Stat5. These data support a model by which IL-7 signaling specifies TCM cell fate by repressing the TFH gene program.
Biomarkers for progression from latent to active tuberculosis identified in multiplexed proteomic assay (SOMAscan™) of human plasma (MPF6P.657)
Authors: Ochsner Urs , Hraha Thomas , Sterling David , De Groote Mary , Wall Kirsten , Janjic Nebojsa , Zak Daniel , Thompson Ethan , Penn-Nicholson Adam , Scriba Thomas
Published: January 2023

Background: Biomarkers of progression from latent to active tuberculosis (TB) would be very useful for targeted treatment or priority enrollment in TB vaccine trials. Methods: A highly multiplexed proteomic assay (SOMAscan) was used to measure over 3000 human proteins in plasma from adolescents infected with M. tuberculosis. We compared proteomic data in longitudinally collected samples (six-month intervals over two years) from 97 individuals, 29 of whom developed active TB (progressors) and 68 matched controls who remained healthy. Bioinformatics algorithms were applied to discover biomarkers of TB risk. Results: Among the top host biomarkers distinguishing progressors from controls were 17 proteins, and these factors were used for model building to predict TB risk. An 8-marker model gave a cross-validated performance of 76% sensitivity and 86% specificity (AUC=0.86). The individual markers had variable responsiveness at time-points closer to TB diagnosis. The model identified progressors up to 540 days prior to TB diagnosis, with elevated logOdds for developing active TB. In contrast, a previously obtained host response signature for active pulmonary TB detected progressors up to 180 days prior to diagnosis. Conclusions: The discovery of TB-risk biomarkers indicates that subjects who eventually progress to TB disease can be identified well before clinical diagnosis of TB, and thus enables potential triage for targeted treatment or priority enrollment in TB vaccine trials.
Loss of chemotaxis gene ΔcheY3 in Borrelia burgdorferi leads to early immune clearance but does elicits a strong antibody response (MPF6P.647)
Authors: Sekar Padmapriya , Novak Elizabeth , Motaleb A. , Wooten Ronald
Published: January 2023

Borrelia burgdorferi (Bb) is a spirochetal bacterium that displays unique endoflagellar motility to efficiently disseminate through dense host tissues, and eventually persist within target tissues to cause Lyme disease. The importance of Bb motility/chemotaxis in dissemination/immune evasion cannot be correctly assessed in vitro, since the complexity of the host tissues cannot be accurately replicated. We assessed the importance of the chemotaxis-associated cheY3 gene on Bb dissemination and immune evasion within intact mouse tissues using our novel intravital microscopy techniques. CheY3-deficient (∆cheY3) Bb possessed similar cellular morphology and growth rates, but moves at lower velocities as wild-type (WT) Bb in murine skin tissues. Notably, GFP-ΔcheY3 were unable to reverse direction, whereas WT display primarily a sequential forward-and-reverse motility pattern. Within skin inoculation sites, ΔcheY3 were cleared by 72-96h post-infection and were not detected in distant tissues ≤day 60 post-infection. Interestingly, ∆cheY3-infected mice generate Bb-specific IgG levels similar to WT-infected animals at 14-28 days post-infection, though levels decrease by day 60. The antibody profile against ∆cheY3 Bb suggests that immunity is generated against many of the same Bb proteins as during WT infection. These findings suggest that cheY3 is essential for Bb persistence and that the attenuated ∆cheY3 Bb may serve as a vaccine to protect against Lyme disease.
Cutaneous leishmaniasis is regulated by Wnt antagonist Dkk-1 from activated platelets (MPF7P.715)
Authors: Bothwell Alfred , Chae WookJin , Ehrlich Allison , Teixeira Alexandra , Goldsmith-Pestana Karen , Maher Stephen , Hwa John , Krause Diane , McMahon-Pratt Diane
Published: January 2023

Cutaneous leishmaniasis is a chronic non-healing skin inflammatory disease present in many countries. Here we show that the Wnt antagonist Dkk-1 is secreted from activated platelets and regulates chronic inflammation to Leishmania (L.) major. We found that L. major infection could rapidly elevate circulating levels of Dkk-1. First, Dkk-1 from activated platelets increased neutrophil-platelet aggregate formation mainly via PSGL-1. Second, the continuously high level of Dkk-1 further directed T cell differentiation into Th2 cells in the draining lymph nodes. Secretion of Dkk-1 was regulated by PKC-alpha and GSK3-beta in human platelets upon soluble leishmania antigen exposure in vitro. Functional inhibition of Dkk-1 using a small molecule inhibitor showed marked inhibition of neutrophil-platelet aggregate formation at early hours of infection and also resulted in decreased parasite burden and amelioration of skin lesions after 6 weeks. In vitro studies demonstrate that Th2 skewing is directed by Dkk-1 via p38 and SGK-1 resulting in elevation of GATA-3 and c-Maf. This coordinated expression of two Th2 cytokine transcription factors resulted in robust production of Th2 cytokines (e.g., IL-4, IL-5, IL-10 and IL-13). Thus, our results demonstrate that platelet-derived Dkk-1 regulates early and late inflammatory responses in cutaneous leishmaniasis.
Deletion of fibrinogen-like protein 2, a novel CD4+ CD25+ Treg effector molecule, leads to improved control of Echinococcus mutilocularis infection (MPF7P.718)
Authors: Lin Renyong , Wang Junhua , Vuitton Dominique , Müller Norbert , Blagosklonov Oleg , Grandgirard Denis , Leib Stephen , Shalev Itay , Levy Gary , Lu Xiaomei , Spiliotis Markus , wen Hao , Gottstein Bruno
Published: January 2023

The proliferation of Echinococcus multilocularis (E. multilocularis) metacestode is dependent on the nature/function of the periparasitic immune-mediated processes. Fibrinogen-like protein 2 (FGL2) was found to be up-regulated after E. multilocularis infection, however little is known about the contribution of this novel CD4+CD25+ Treg effector molecule to the control of a helminth infection. We showed that, as compared to AE-WT mice, AE-fgl2-deficient mice exhibited a significantly decreased parasite load and proliferation activity, associated with increased T cell proliferation in response to ConA, reduced Treg numbers and function, relative Th1 polarization, and increased B cell number and DC maturation. We showed for the first time that FGL2 is involved in negative immune regulation towards a helminth parasite and that IL-17A contributes to FGL2 regulation. By promoting Treg cell activity, FGL2 appears as a key-player in the immune orchestration of the outcome of E. multilocularis infection and measurement of plasma FGL2 levels might be useful to assess disease progression. Furthermore, targeting FGL2 could also be used for the development of novel treatment approaches in alveolar echinococcosis and other diseases caused by parasite pathogens.