bluuebunny/crossref_metadata_2025 huggingface dataset:
MMP-7 promotes host recovery and lung function to influenza virus infection (VIR2P.1016)
Authors: Sanders Catherine , Thomas Paul
Published: January 2023

Wound healing is a critical aspect of recovery from influenza virus-induced lung damage. However, relatively little attention has been given to the role of host recovery mechanisms in influenza virus pneumonia. MMP-7, or matrilysin, is produced in the airways following damage and is known to promote airway homeostasis and respiratory cell migration and differentiation, possibly via immunomodulatory processes. We hypothesized that MMP-7 is a critical factor in recovery from influenza infection for both airway damage repair and respiratory function. MMP-7-/- mice are more susceptible to PR/8 influenza infection, and, while showing weight loss similar to WT controls, succumb on days 8-13 after infection. MMP-7-/- lungs scored higher than WT lungs in both acute and chronic tissue damage measures on day 7 after infection by histology, demonstrating MMP-7’s ability to reduce tissue damage and limit tissue remodeling at peak disease. Curiously, MMP-7-/- BAL taken day 3 after infection have lower levels of IL-6, KC, and MIG than WT BAL. Plethysmographic respiratory analyses on days 8-11 showed MMP-7-/- mice to have slower breathing rates and exaggerated expiratory functions, suggesting a role for MMP-7 in promoting proper lung function during attempted resolution. This study provides evidence of the impact of a specific tissue remodeling protein on lung function and influenza disease outcome.
Pathogen clearance by engineering of novel innate immune defense (INM3P.414)
Authors: Pardington Paige , Chaudhary Anu , Norvell Meghan , Gupta Goutam , Dandekar Abhaya , Gouran Hossein , Uratsu Sandra , Aguero Cecilia , Feldstein Paul , Bruening George , Civerolo Edwin , Hao Guixia , Duan YongPing , Stover Ed
Published: January 2023

Pathogens circumvent host immune defense to propagate their lifecycle. We have been studying the mechanisms of pathogen-induced host cell death to develop a robust strategy for pathogen clearance. The strategy involves designing and developing a protein chimera with one domain for recognizing conserved pathogen membrane elements and another for lysing the pathogen membrane. This dualistic synergy of recognition and lysis permits rapid pathogen clearance thereby preventing host cell death and disease development. This strategy has been successfully applied to counteract bacterial infections and diseases in grape, citrus, and tobacco. We have engineered a chimera consisting of protease (recognition domain) and cecropin (lysis domain) to prevent Pierce’s disease, a deadly disease in grape caused by Xylella fastidiosa. We have shown that transgenic plants expressing the chimera of protease and cecropin effectively clear Xylella fastidiosa from sites of colonization. We have also designed a protein chimera of thionin (with both recognition and lysis domains) and a synthetic lytic peptide. We have shown that this same chimera prevents citrus canker which is caused by Xanthamonas axonopodis citri and wildfire disease in tobacco which is caused by Pseudomonas syringe pv. tobaci. This protein engineering strategy appears to be an effective therapy against a broad spectrum of plant pathogens. A similar approach is now being evaluated against multiple human pathogens.
Sphingosine 1-phosphate receptor 2 signaling suppresses macrophage phagocytosis and impairs host defense against sepsis (INM3P.416)
Authors: Fang XiangMing , Hou JinChao
Published: January 2023

Background: Sepsis is characterized by an inappropriate systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Sphingosine 1-phosphate receptor 2 (S1PR2) modulates endotoxin induced inflammation in endothelium. However, as a highly expressed S1P receptor in macrophage, its role in regulating macrophage response to bacterial infection remains unclear. Methods: Sepsis was induced in wild-type mice treated with S1PR2 antagonist or S1PR2-deficient mice. The antibacterial ability of cell specific S1PR2 was tested in bone marrow reconstitution or macrophage deletion mice. Signaling molecules responsible for S1PR2 mediated phagocytosis were also measured in bone marrow-derived macrophages. Results: Both genetic deletion and pharmaceutical inhibition of S1PR2 significantly limited bacterial burden, reduced lung damage, and improved survival in experimental sepsis. This protection was attributed to the enhanced phagocytic function of S1PR2-deficient macrophages. Absence of S1PR2 in macrophage inhibits RhoA-dependent cell contraction and promotes IQGAP1-Rac1-dependent lamellipodial protrusion, the different signalings of which depends on the extracellular stimulators. Conclusions: This study implies that S1PR2 as a critical receptor in macrophage, impairs phagocytosis and antimicrobial defense in the pathogenesis of sepsis. Interventions targeting S1PR2 signaling may serve as a promising therapeutic approach for sepsis.
Early type I interferon response correlates with Borrelia burgdorferi dissemination in a mouse model of Lyme disease (INM3P.417)
Authors: Petzke Mary , Brooks Andrew , Iyer Radha , Love Andrea , Spieler Zoe , Wormser Gary , Schwartz Ira
Published: January 2023

Borrelia burgdorferi genotypes differ in their capacity to cause disseminated Lyme disease. Gene array analysis was employed to profile the host transcriptome induced by Borrelia genotypes in the blood of C3H mice during early dissemination following intradermal inoculation. The greatest responses were elicited by Borrelia turicatae, a relapsing fever spirochete that reaches high densities in the blood (313 entities; 170 induced, 143 repressed) and by B515, a B. burgdorferi clinical isolate that causes disseminated infection (129 entities; 52 induced, 77 repressed). Induced transcripts included IFN-responsive genes and genes involved in immunity and inflammation. B331, a B. burgdorferi clinical isolate that did not disseminate, stimulated changes in the fewest number of entities (1 induced, 4 repressed). Transcriptional regulation of type I IFN and IFN-related genes was measured by RT-PCR in mouse skin biopsies collected from the site of infection 24 hours after inoculation. The mean fold change values for Ifnb, Cxcl10, Gbp1, Ifit1, Ifit3, Irf7, Mx1, and Stat2, were found to be significantly elevated in B515-infected mice relative to the control group. In contrast, transcription of these genes was not significantly changed in response to B331 or B31-4, a non-infectious B. burgdorferi mutant, although live B331 spirochetes could be recovered from skin. These results establish an association between B. burgdorferi genotype, dissemination, and early type I IFN induction.
Mycobacterium leprae infection disrupts the vitamin D-mediated antibacterial response (INM3P.362)
Authors: Zavala Kathryn , Amin Sima , Chun Rene , Modlin Robert , Hewison Martin , Adams John , Liu Philip
Published: January 2023

Leprosy is a debilitating disease caused by infection with Mycobacterium leprae (mLEP). Patients diagnosed with leprosy present on a spectrum of disease characterized by disseminated lepromatous leprosy (L-lep) at one end and self-limiting tuberculoid leprosy (T-lep) at the other end. Previous studies have demonstrated the role of the vitamin D (vit D) system in mediating the antibacterial response to mycobacterial infection; however, it is not understood how dysregulation of the vit D system contributes to disease outcomes. The present study investigates the factors that regulate vit D metabolism and activation in leprosy. Monocytes were infected with mLEP and analyzed for vit D system gene expression and metabolism. In contrast to stimulation with synthetic toll-like receptor 2 (TLR2) ligand, mLEP infection did not induce production of the biologically active form of vit D. Furthermore, gene expression of activating enzyme 1α-hydroxylase (CYP27B1) and vit D receptor (VDR) were not induced by infection. Conversely, treatment with irradiated mLEP induced expression of CYP27B1 and VDR. This suggests that infection does not stimulate the vit D intracrine system in the same manner described for TLR2-sensing of mycobacterial peptides. Instead, infection triggers mechanisms that repress vit D metabolism. Moreover, mLEP-infected T-lep-associated macrophages are resistant to this repression. This study highlights the role of the vit D system in the host response to mLEP infection.
The trancriptional factor, Nrf2 is critical for the protection against pneumococcal pneumonia. (INM3P.400)
Authors: Ludewick Herbert , Metzger Dennis
Published: January 2023

Pneumonia is one of the major clinical complications of pneumococcal disease. Innate immunity provides an immediate response to S. pneumoniae that can be protective, but if not controlled, can also lead to deleterious inflammation, extensive tissue damage and mortality. A complete understanding of the regulation of innate immunity and attendant inflammatory responses are critical for clinical control of respiratory bacterial infections. To better comprehend this dynamic process during pneumococcal infection, we specifically focused on the transcriptional factor, Nrf2, which can be both induced by inflammation and critically regulates inflammatory responses. Of interest, acute lung injury (ALI) in humans is correlated with functional polymorphisms in the Nrf2 promoter region. Our studies showed that mice deficient in Nrf2 were highly susceptible to in vivo pneumococcal pneumonia. Nrf2 deficiency was associated with enhanced production of pro-inflammatory cytokines and infiltration of neutrophils into the lungs following infection. However, immunization with the polysaccharide conjugate vaccine completely protected both Nrf2-/- and Nrf2+/- mice against pneumococcal challenge. This study provides evidence that Nrf2 is critical for protection against pneumococcal pneumonia by suppressing deleterious inflammation. In addition, the results suggest that pneumococcal vaccination should be performed to protect humans who have an Nrf2-associated genetic predisposition to develop ALI 
Induction of lipoxin A4 by Francisella tularensis: a mechanism for regulation of pro-inflammatory responses during early-phase tularemia (INM3P.401)
Authors: Singh Anju , rahman tabassum , Bartiss Rose , Musteata Florin , Sellati Timothy
Published: January 2023

During respiratory tularemia, caused by inhalation of Francisella tularensis (Ft), we postulate that bacteria establish a principally anti-inflammatory environment and subvert host cell death programs to facilitate their unfettered replication within various cell types. Here we have explored the role of lipid mediators such as lipoxin A4 (LXA4) and prostaglandin E2 (PGE2) in host inflammatory responses manifested early during infection with Ft. An inverse relationship was observed between the production of LXA4 and PGE2 suggesting that Ft induces the generation of these eicosanoids to modulate cell death and enhance its own survival. This notion is supported by our findings that Ft triggers the release of LXA4, which inhibits apoptosis of macrophages. We also have elucidated the course of tularemia in mice deficient for 5-lipoxygenase (5-LO), leaving them incapable of generating LXA4, and observed elevated levels of PGE2, increased apoptosis, increased neutrophil recruitment and elevated release of pro-inflammatory cytokines and chemokines as compared to their wild-type counterparts. 5-LO deficient mice also were found to be less susceptible to lethal doses of Ft LVS as compared to wild-type mice. Our results suggests that 5-LO activity increases susceptibility to Ft infection, suggesting that targeting this pathway may have therapeutic benefit by limiting bacterial growth in tularemic individuals and perhaps those infected with other virulent respiratory pathogens.
IFNγ-mediated exacerbation of Saccharopolyspora rectivirgula-induced proinflammatory responses is dependent on interferon regulatory factor 1 (INM3P.403)
Authors: Yi Ae-Kyung , Park Jeoung-Eun
Published: January 2023

Saccharopolyspora rectivirgula (SR) is a thermophilic actinomycetes that causes hypersensitivity pneumonitis (HP). Interferon γ (IFNγ) produced after exposure to SR has been demonstrated to be necessary for development of HP. However, the molecular mechanism by which IFNγ enhances SR-mediated inflammatory responses is not completely understood. We investigated how IFNγ alters SR-induced expression of cytokines/chemokines. IFNγ pretreatment substantially enhanced expression of TNFα, IL-6, IL-23, IFNβ, MIP-1α, and MIP-2, without affecting IL-10 expression, in response to SR. Expression of interferon regulatory factor 1 (IRF1) was rapidly increased in cells pre-exposed to IFNγ, and inhibition of IRF1 synthesis abolished IFNγ-enhanced gene expression in response to SR. In contrast, suppression of IRF1 expression did not alter SR-mediated expression of IL-10. SR stimulation led to the binding of IRF1 to the IL-6 promoter region, but not to the IL-10 promoter region in IFNγ-pretreated cells. This indicates that IRF1 expressed in cells pre-exposed to IFNγ is activated by subsequent SR stimulation and contributes to the synergistic expression of certain proinflammatory genes. Our results imply that IFNγ produced after initial exposure to SR may help direct the proinflammatory responses to subsequent SR exposure toward to pathogenic Th1/Th17 development by selectively amplifying expression of certain cytokines/chemokines without affecting expression of regulatory cytokine IL-10.
Novel role for inflammasome-dependent IL-18 in promoting Th2 responses during Leishmania infections (IRC7P.420)
Authors: Gurung Prajwal , Kanneganti Thirumala-Devi
Published: January 2023

Leishmaniasis is a global health disease and is categorized under neglected tropical disease by WHO. Despite tremendous amount of economic burden and morbidity and mortality associated with Leishmania infections, studies to understand these parasitic bugs are nevertheless lacking. As a result, specific targeted treatment options or vaccines to treat Leishmaniasis are still missing. Herein, we sought to understand how inflammasomes are involved in host responses during Leishmania major infection. One of the hallmarks of inflammasome is the activation of caspase-1 and ultimate release of bioactive IL-1β and IL-18. Interestingly, we found that mice lacking components of the NLRP3 inflammasome were resistant to L. major infections in comparison to their WT counterparts. Using in vitro and in vivo approaches, we showed that L. major activates NLRP3 inflammasome. We further demonstrated that NLRP3 inflammasome induced IL-18, but not IL-1β, promoted Th2 responses during L. major infection. Mechanistically, IL-18 enhanced expression of Th2 transcription factors GATA3 and cMAF in T cells. Finally, we were able to show that IL-18 neutralization in WT mice protected from L. major infections. In conclusion, our study uncovered a novel role for inflammasome-induced IL-18 in promoting Th2 responses and Leishmaniasis, which can potentially be targeted for therapeutic benefits during L. major infections.
IFNγ signaling endows DCs with the capacity to control type I inflammation during parasitic infection through promoting T-bet+ regulatory T cells (IRC7P.421)
Authors: Lu Li-Fan , Lee Hyang-Mi , Fleige Anne , Forman Ruth , Khan Aly , Cho Sunglim , Lin Ling-Li , Nguyen Duc , O'Hara Aisling , Yin Zhinan , Hunter Christopher , Muller Werner
Published: January 2023

IFNγ signaling drives dendritic cells (DCs) to promote type I T cell (Th1) immunity. Here, we show that activation of DCs by IFNγ is equally crucial for the differentiation of a population of T-bet+ regulatory T (Treg) cells specialized to inhibit Th1 immune responses. Conditional deletion of IFNγ receptor in DCs but not in Treg cells resulted in a severe defect in this specific Treg cell subset, leading to exacerbated immune pathology during parasitic infections. Mechanistically, IFNγ-unresponsive DCs failed to produce sufficient amount of IL-27, a cytokine required for optimal T-bet induction in Treg cells. Thus, IFNγ signalling endows DCs with the ability to efficiently control a specific type of T cell immunity through promoting a corresponding Treg cell population.
The prostaglandin D2 receptor chemoattractant receptor homologous molecule expressed on Th2 cells regulates accumulation of group 2 innate lymphoid cells in the inflamed lung (IRC7P.426)
Authors: Tait Wojno Elia , Monticelli Laurel , Tran Sara , Alenghat Theresa , Osborne Lisa , Thome Joseph , Willis Cynthia , Budelsky Alison , Farber Donna , Artis David
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) promote type 2 immunity and inflammation, but the pathways that control ILC2 migration into inflamed tissues remain poorly understood. Here, we show that the prostaglandin D2 (PGD2) receptor chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) regulates ILC2 accumulation in the lung in vivo. The frequency of ILC2s that expressed CRTH2 was significantly higher in healthy human and murine peripheral blood than in the lung, suggesting that regulation of CRTH2 expression might be associated with ILC2 accumulation in the lung. Consistent with this, CRTH2-expressing murine ILC2s accumulated in the lung in response to PGD2. Further, CRTH2-deficient mice exhibited reduced ILC2 accumulation and helminth-induced type 2 inflammation in the lung compared to wild-type mice. Critically, adoptive transfer of CRTH2-sufficient ILC2s restored helminth-induced pulmonary inflammation in CRTH2-deficient mice. Together, these data suggest that the PGD2-CRTH2 pathway regulates ILC2 accumulation and type 2 inflammation in the lung in vivo.
Multi-enzyme aminoacyl tRNA synthetase complex component AIMp1/p43 as an important Th1 mediator (IRC7P.428)
Authors: Liang Dan , Halpert Matthew , Konduri Vanaja , Chen Yunyu , Kim Doyeun , Kim Sunghoon , Levitt Jonathan , Decker William
Published: January 2023

Of professional antigen presenting cells, only the dendritic cells (DC) are regarded as initiators of adaptive immune responses. Previously we identified a Th1 polarizing phenotype from DCs loaded with overlapping (homologous) MHC class I and II determinants, including augmented DC IL-12 production, generation of CD8+ cytolytic effectors, and T cell IFNγ secretion, suggesting DC intrinsic mechanisms to recognize and compare antigenic epitopes. AIMp1/p43 is a structural component of the multi-enzyme aminoacyl-tRNA synthetase complex (mARS) and indicated by previous studies to be a pro-inflammatory cytokine. We identified high levels of p43 release correlated with degree of homology between antigenic epitopes on DC class I and II molecules. In vitro studies confirmed p43 deficiency impairs DC costimulatory and Th1 polarizing functions. It also reversely regulates the production of IFNγ and IL4 within T cells. In vivo, p43 deficiency abolishes the ability of DC to mediate effective response against B16 melanoma tumors, and leads to higher susceptibility to influenza viral infections. These data support our hypothesis that p43 is a critical Th1 polarizing effector molecule. We will investigate further into p43 functions in DC and T cell crosstalk, targeting candidate cell surface receptors and intracellular signaling pathways. Additionally, we will better characterize in vivo roles of p43 in anti-tumor and anti-viral models and seek to apply it as a novel vaccination adjuvant.
Bidirectional polarization of T cell function via CD43 (IRC8P.437)
Authors: Modak Madhura , Majdic Otto , Cejka Petra , Waidhofer-Söllner Petra , Jutz Sabrina , Steinberger Peter , Zlabinger Gerhard , Stoeckl Johannes
Published: January 2023

Co-receptors, being either co-stimulatory or co-inhibitory, play a pivotal role in T cell immunity, as they decide the fate of T cell function. CD43 is one of the abundant cell surface glycoproteins expressed on T cells. CD43 has been demonstrated to act as not only a potent co-receptor but also a negative regulator for T cell activation. To further investigate role of CD43 in T cell activation and subsequent function, peripheral blood T cells were activated via two distinct CD43 epitopes recognized by monoclonal antibodies (mAbs) 6E5 (T6E5-act) and 10G7 (T10G7-act) along with TCR signalling. Both the CD43 mAbs could potently induce T cell activation when immobilized but exerted differential downstream effects including differential activation of signalling pathways, T cell cytokine production and also distinct effector function. T10G7-act poorly responded in re-stimulation assay and further acquired suppressive function compared to T6E5-act or to T cells activated via CD28 (TCD28-act). Furthermore, T10G7-act did not directly inhibit responder T cells, but rather exhibited their effect via dendritic cells when added to allogenic mixed leukocyte reaction. Together our data suggests a unique role of CD43 in bidirectional polarization of T cell immunity, a positive co-stimulatory role and a negative role in down-modulation of immune response, depending on its targeted epitope.
Antigen level, OX40 (CD134) and 4-1BB (CD137) costimulation, and IL-33 support CD4 T cell differentiation and functionality (IRC8P.448)
Authors: Morales Crystal , Maxwell Joseph , Adler Adam , Vella Anthony
Published: January 2023

We set out to determine how costimulation induces cytotoxic CD4 T cells. In our in vivo model, CD11c+ cells express varying levels of Eα antigen linked to MHC II. To study CD4 T cell differentiation we transferred in Eα-specific T cells and monitored functionality. Mice were treated with OX40 and 4-1BB agonists, and cytotoxic CD4 T cells were assessed. In high antigen presenters (Eαhi) the T cells became primarily T-bet+ GzmB+ IL-33R++ and clonally expanded, while low antigen presenters (Eαlo) induced T cells that were mostly Gata3+ or Eomes+ KLRG1+ IL-33R+ with reduced expansion levels. Upon ex vivo stimulation with Eα, T cells from Eαhi mice produced higher IFNɣ levels than T cells from Eαlo mice, whereas IL-4 levels were low in both. Since IL-33R was expressed, we showed that higher IL-33R on specific T cells from Eαhi mice conferred greater production of IFNɣ by demonstrating that IL-33 synergized with IL-2, IL-12 and IL-36β independent of TCR signals. The decreased functionality of T cells from Eαlo mice may be due to more Tregs since exhaustion markers were unexpectedly higher on T cells from Eαhi mice. These novel findings demonstrate that 1) cytotoxic CD4 T cells can be induced by costimulation therapy but may be a heterogeneous population, 2) antigen presentation levels have a substantial effect on T cell differentiation programming and 3) IL-33 can act as a Th1 activating cytokine. Thus, this data will greatly impact CD4 T cell immunotherapeutic approaches.
Role of microtubule acetylation in T cell activation and downstream function (IRC8P.450)
Authors: Hu Kenneth
Published: January 2023

Microtubules (MTs) play a number of important roles in antigen-mediated T cell activation and effector functions. Tubulin can undergo a number of post-translational modifications including acetylation of alpha tubulin. This acetylation affects MT growth dynamics and ability of motor proteins to traffic along them. We investigated the role of this acetylation in T cell function by using a mouse bearing deletion of the gene for alpha tubulin acetyltransferase 1 (aTAT1), the main acetyltransferase tubulin. Absence of aTAT1 leads to complete loss of acetylated alpha-tubulin. We found that CD4+ T cells from an aTAT1 deficient mouse showed more proliferation than controls upon stimulation with intermediate levels of antigen. We observed no difference in the level of calcium influx between WT and KO CD4+ T cells upon antigen stimulation. An important function of microtubules in CD8+ T cells is trafficking of cytolytic vesicles. Given that acetylation can affect the movement of motor proteins, we tested whether aTAT1 KO CD8+ and Natural Killer (NK) showed altered ability to kill target cells. These findings indicate that acetylation of alpha-tubulin is important in regulating T cell effector responses.
Mammalian target of rapamycin inhibition with enterically given rapamycin alters immunity and gut flora in young and aged wild type mice and extends life of immunodeficient mice (IRC8P.452)
Authors: Hurez Vincent , Dao Vinh , Liu Aijie , Pandeswara Srilakshmi , Gelfond Jonathan , Sun Lishi , Svatek Robert , Bergman Molly , Padron Alvaro , Drerup Justin , Liu Yang , Dodds Sherry , Hasty Paul , Sharp Zelton , Curiel Tyler
Published: January 2023

mTOR regulates many important immune processes. Interest in immune effects of mTOR inhibitors is heightened by recent findings of ability to boost specific immunity and vaccine response in elderly humans, treat cancers and autoimmunity, and extend life and healthspan in mice, suggesting important new clinical applications. However, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. We comprehensively analyzed immune effects of long-term rapamycin in mice. Gene expression profiling of purified T and B cells (CD4+PD1+, CD4+PD1-, CD8+PD1+, CD8+PD1-, B220+), CD11b+CD11c- myeloid cells and CD11c+ dendritic cells showed numerous, novel changes in genes affecting differentiation, function, homeostasis, migration, exhaustion, cell death and inflammation. Novel effects on Th9, Th22 and Tfh differentiation were seen as was improved function of exhausted PD1+ T cells (supported by human clinical data) and increased numbers and function of innate lymphoid cells. Immune functions relevant to cancer, infections, aging and inflammation, and innate lymphoid cell effects were validated in vitro and in vivo. eRapa prolonged life in RAG KO mice (without T or B cells) >12x and in IFN-γ KO mice >3x over wild type mice, supporting mTOR suppression immune effects mitigating disease as a mechanism for longevity extension. Metagenomic effects were linked to immune outcomes. Our data demonstrate novel mTOR immune effects meriting further investigations.
T cells and dendritic cells release chemokines to recruit innate immune cells after intranasal staphylococcal enterotoxin A exposure (IRC8P.438)
Authors: Svedova Julia , Vella Anthony
Published: January 2023

Staphylococcus aureus, an important cause of life-threatening infections, produces a number of virulence factors including superantigens. Staphylococcal enterotoxin A (SEA) is a superantigen that directly binds to MHC II on antigen presenting cells and then crosslinks with specific T cell receptor (TCR) Vβ chains on T cells (e.g. Vβ3). We hypothesized that SEA-activated T cells guide dendritic cells to release chemokines causing a systemic recruitment of innate immune cells. Here we showed that intranasal SEA exposure in mice caused a rapid influx of neutrophils and monocytes to peripheral blood, spleen, and lymph nodes. This migration was dependent on T cells since TCR βδ-/- mice were unable to recruit these cells unless they received an adoptive T cell transfer. Confocal microscopy imaging demonstrated that the recruited neutrophils localized to the T cell zone of lymph nodes. Increased expression of several neutrophil and monocyte chemotactic factors, including CXCL1, CXCL2, CCL2, CCL3, and CCL4, was detected in lymph nodes as early as one hour after SEA exposure. By sorting specific cell populations we found that these chemokines were differentially expressed. While CXCL1 and CXCL2 were mostly produced by migratory dendritic cells, CCL3 and CCL4 were made by SEA-activated Vβ3+ T cells. Our future studies will focus on determining the mechanism of chemokine release and on describing the consequences of innate immune cell recruitment to lymph nodes.
Effects of leucine metabolism on T cell activation: a potential target for nutritional immunotherapy (IRC8P.439)
Authors: Ananieva-Stoyanova Elitsa , Powell Jonathan , Patel Chirag , Hutson Susan
Published: January 2023

T cells are essential for cell-mediated defense against pathogens and cancer. Recent advances in understanding T cell regulation reveal that cell metabolism can greatly impact the final outcome of the immune response. A primary regulator of cell growth and proliferation, the mammalian target of rapamycin (mTOR), is critical for T cell activation and differentiation. Leucine is a nutritional activator of mTOR and its intracellular concentrations are regulated by the branched chain aminotransferase (BCAT) isoenzymes, cytosolic BCATc and mitochondrial BCATm. We showed recently that activated CD4+ T cells, lacking cytosolic leucine transamination (BCATc KO), had up-regulated mTOR signaling and glycolysis. To explore further the role of BCATc in T cell activation, we measured INFƴ production and proliferation in T cells from BCATc KO and wildtype mice. Loss of BCATc increased INFƴ by 2.8-fold in activated T cells but did not impact T cell proliferation. Knocking out the mitochondrial BCATm had a robust effect on T cell glycolytic rate (3.1-fold increase) upon activation but only mild effects on mTOR and INFƴ. The results suggest that loss of either enzyme is sufficient to increase T cell glycolysis and mTOR signaling but not T cell proliferation. Thus, BCATc and BCATm are critical players in T cell activation by providing negative feedback regulation of mTOR and glycolysis. Manipulating leucine metabolism may be one mechanism to modulate activation and differentiation of T cells.
Differential regulation of alloreactive CD8 T cell responses by inflammation. (IRC8P.440)
Authors: Jangalwe Sonal , Greiner Dale , Brehm Michael
Published: January 2023

Tissue transplantation results in the release of endogenous danger molecules that stimulate inflammation by activating the innate immune system. Inflammatory signals regulate the magnitude and functionality of T cell responses during infection, but the direct impact of inflammation on alloreactive T cell responses has not been extensively studied. Understanding the role of inflammation in regulating the activation of alloreactive T cells will aid in the development of novel therapies to prolong allograft survival. We have used a TCR-transgenic alloreactive CD8 T cell model to study the response of alloreactive T cells in the presence of inflammation. Our results show that stimuli inducing different inflammatory profiles have significant effects on the size, phenotype and quality of alloreactive CD8 T cell responses as compared to stimulation with alloantigen alone. Inflammation induced by exposure to poly(I:C) and LPS increased the number of alloreactive CD8 T cells at the peak of the response. The ability of effector alloreactive CD8 T cells to produce IFN-g was enhanced with poly(I:C) but decreased with LPS, while cytotoxic activity was similar. LPS slowed the contraction phase of the alloantigen response, resulting in higher levels of memory CD8 T cells as compared to poly(I:C). These data indicate that inflammatory signals differentially regulate the kinetics and functionality of alloreactive CD8 T cell responses and may determine susceptibility to immune suppression.
Dysregulation of ERK signaling in CD4-expressing cells induces osteochondromas (IRC8P.441)
Authors: Wehenkel Marie , Corr Maripat , Edwards Benjamin , Calabrese Christopher , Pagès Gilles , Pouyssegur Jacques , Vogel Peter , McGargill Maureen Ann
Published: January 2023

ERK1 and ERK2 are serine/threonine kinases critical for the proliferation and development of T cells. Loss of ERK signaling in T cells, mediated by conditional deletion of Erk2 (CD4Cre) on an Erk1-/- background (DKOCD4), results in a block in the thymic development of T cells. Surprisingly, 80% of DKOCD4 mice spontaneously developed visible osteochondromas by 28 weeks of age. Histological analysis of these lesions revealed excessive accumulation of hypertrophic chondrocytes originating from the growth plates, but no mononuclear infiltrate indicative of inflammation. To determine if these lesions were caused by deletion of Erk in T cells, DKOCD4 mice were bred to Rag1-/- mice. Unexpectedly, osteochondromas still appeared in DKOCD4Rag1-/- mice, indicating deletion of Erk2 in other cell types mediates excess cartilage accumulation. Moreover, the addition of T cells delayed development of osteochondromas, which is a previously unknown level of regulation in cartilage homeostasis. Osteochondromas also appear to be influenced by alterations in the microbiota, as DKOCD4 mice treated with an antibiotic cocktail have a delay in osteochondroma development, which can be accelerated by housing the mice in non-specific-pathogen free conditions. Together these data suggest that Erk plays a critical role in a CD4+ cell type other than T cells, possibly an innate-like lymphocyte, to alter cartilage homeostasis. Finally, we present a novel role for T cells in regulating this process.
Maintenance of immune diversity during an effector T cell response in face of antigen selection (IRC8P.444)
Authors: Devarajan Priyadharshini , Zou Tie , McKinstry Karl , Flies Amanda , Swain Susan , Dutton Richard
Published: January 2023

Self-antigen recognition has been shown to be instrumental for the maintenance of the pool of circulating naïve T cells. While the mechanisms of immune diversity maintenance in the naive T cell repertoire have been explored, how immune diversity is maintained in the face of antigen selection is less understood. Data from several studies show that self-peptide presented by FRCs in the secondary lymphoid organs help maintain the diversity of the naïve T cell repertoire. We propose that an analogous mechanism is in play even during the effector stage of an immune response, which if left unchecked would bias the repertoire towards clones with higher affinity for the foreign antigen. In this study, we report a clone-specific regulation of the diversity in the face of antigen selection. Our data show that CD8+ T cell clones selected on the same self peptide compete for this self-antigen presented by stromal cells even during the clonal expansion phase in an effector response. The supply of self-peptide and hence IL-7 becomes limiting as the clone increases in size and the ability for the clone to further expand is curtailed. Such a mechanism would explain the maintenance of the diversity of the immune repertoire even through an antigen-driven response as T cells transition from being naïve to effectors.
CCR7 modulate the homeostasis of CD8 T cells in response to acute viral infections (IRC8P.445)
Authors: Jung Yong
Published: January 2023

CCR7 is an important chemokine receptor for naive and memory T cells to migrate toward T cell zone of the spleen and lymph nodes where CCL19 and CCL21 are expressed. Although the role of this receptor in the antigen-dependent activation of T cells is well documented, it is still not clear if CCR7 alters the homoestasis of effector and memory T cells during acute viral infections. Here, we report that the deficient of CCR7 expression increased the number of memory CD8 T cells particulary in the lung and bone marrow compared to CCR7 wild-type T cells following LCMV infections. We also demonstrated that CCR7-deficient memory T cells proliferated homeostatically faster than wild type controls. This is presumably a result of increased exposure to IL-15 as an outcome of diminished experience of IL-7 because the numbers of CCR7-deficeint and CCR7-sufficient memory T cells are comarable in IL-15 knock-out mice but not in IL-7 deficient mice. Therefore, IL-15 is suggested to be the dominant factor for the increased homeostasis of CCR7 deficient memory T cell pool. Based on these data, we concluded that CCR7 predispositions memory CD8 T cells into the IL-7-dependent niches rather than IL-15- and this also offers better understandings of the regulation of homeostasis based on the differential CCR7 expressions of memory T cell subsets.
Hyperproduction of IL-6 caused by aberrant TDP-43 overexpression in high-fat diet-Induced obese mice (IRM9P.602)
Authors: Kang Sujin , Lee Sungwook , Park Boyoun
Published: January 2023

Inclusion of Tat-activating regulatory DNA-binding protein-43 (TDP-43) due to hyperphosphorylation or hyperubiquitination is one of the main causes of neurodegenerative diseases. In particular, cellular TDP-43 expression levels are tightly controlled through a negative feedback loop involving its mRNA; however, misregulation of TDP-43 expression is linked to neuropathology. Very recently, we reported that the TDP-43-mediated subnuclear body is an essential site of interleukin -6 (IL-6) pre-mRNA processing. Here, we show that aberrant TDP-43 overexpression is significantly associated with the pathogenesis of obesity. Mice fed a high-fat diet exhibit increased TDP-43 expression in the liver and fat-pad masses along with a prominent increase of IL-6 production. Importantly, overexpression or depletion of TDP-43 significantly increases or decreases, respectively, IL-6 pre-mRNA processing and mRNA production in adipose cells. Furthermore, in vivo depletion of TDP-43 reduces IL-6 production in the liver. Thus, our findings provide evidence that homeostasis of TDP-43 expression influences IL-6 production levels, and thus, the alteration of IL-6 levels by disruption of TDP-43 self-regulation causes a high risk of developing obesity.
ATF4 reprograms T cell metabolism in response to the environmental stress and is required for Th1 immune responses (IRM9P.459)
Authors: Xia Rui , Lu Binfeng , Yang Xi
Published: January 2023

Rui Xia1, Xi Yang1, 3, Binfeng Lu1, 2 1 Department of Immunology, School of Medicine, University of Pittsburgh, EBST E1047, 200 Lothrop Street, Pittsburgh PA 15261, USA. 2 University of Pittsburgh Cancer Institute 3 School of Medicine, Tsinghua University, HaiDian, Beijing P.R. China 100084 Abstract: T cell metabolism is strongly influenced by the extracellular tissue microenvironment such as oxidizing status and amino acid availability. The underlying molecular mechanisms are not well understood. Here, we showed that increased extracellular oxidation and amino acid starvation induced a unique ATF4-regulated genetic program in T cells. ATF4 deficient T cells had a severe defect in metabolism, particularly in the oxidizing as well as amino acid-deprived environments. Lack of ATF4 resulted in T cell autonomous defects in the function of Th1 cells. In vivo, ATF4 deficiency resulted in decreased antitumor immune responses and associated with drastic changes of the tumor microenvironment. Our study establishes that ATF4 plays an important role in T cell metabolism and is important for the Th1 type immune response.
Mesenchymal stromal cell immunomodulation and aryl hydrocarbon receptor activation by tryptophan catabolites (IRM9P.462)
Authors: Lewis Christopher , Chinnadurai Raghavan , Galipeau Jacques
Published: January 2023

The catabolism of Trp by indoleamine 2,3-dioxygenase (IDO) is a key correlate of the immunomodulation by human mesenchymal stromal cells (MSCs). AHR is a cytosolic protein expressed by a variety of cells, that upon activation, initiates transcription at aryl hydrocarbon response elements (AHREs). We show MSCs express AHR basally, and hypothesized that the catalytic activity of IDO and signal transduction via AHR are linked, as intracellular signals which deploy MSC suppressive properties. To test this, we treated MSCs with hydrocarbons known to activate AHR: TCDD or FICZ. Upon treatment of resting MSCs with TCDD or FICZ, we observed 100-fold induction of Cyp1a1/1b1 mRNA, reflective of AHRE activation. We next examined the response of MSCs after 48h treatment with 1MT, Trp, kynurenine (Kyn) or kynurenic acid (KynAc), the latter two being IDO-catalyzed Trp metabolites. We observed 1MT and KynAc both induced Cyp1a1/1b1 expression by 60-fold. These data support the theory that IDO-catabolism of Trp generates endogenous ligands (i.e.KynAc) which can directly activate AHR. We performed T cell suppression assays, and observed Trp catabolites augment the immunosuppressive function of MSCs (independently of PDL1 or IDO). Our findings suggest endogenous AHR signals from IDO catalysis may play a role in MSC immunomodulation. This observation supports a novel paradigm for intracellular signaling from catabolic conversion of Trp to AHR ligands and induction of immunomodulatory properties.
Histone deacetylase inhibitors dampen antibody and autoantibody responses by upregulating selected B cell microRNAs that silence AID and Blimp-1 expression (IRM10P.603)
Authors: Zan Hong , White Clayton , Pone Egest , Lam Tonika , Tat Connie , Li Guideng , Casali Paolo
Published: January 2023

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require AID, and plasma cell differentiation, which requires Blimp-1, are critical for the generation of class-switched and hypermutated (mature) antibody and autoantibody responses. We show that histone deacetylase inhibitors (HDIs) valproic acid (VPA) and butyrate upregulated miR-155, miR-181b and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1 and Xbp-1, thereby inhibiting CSR, SHM and plasma cell differentiation. The selectivity of HDI-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important AICDA/Aicda inducer/modulators), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2 or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell intrinsic epigenetic mechanisms, VPA blunted class-switched and hypermutated T-dependent and T-independent antibody responses in C57BL/6 mice. It also decreased class-switched and hypermutated autoantibodies, ameliorated disease and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline important epigenetic mechanisms of modulation of antibody responses and providing proof-of-principle therapeutics in autoantibody-mediated autoimmunity.
Novel role of B cell receptor signaling in regulation of Tcl1, an oncogene, and Par-4, a tumor suppressor gene, in B cell chronic lymphocytic leukemia (IRM10P.619)
Authors: McKenna Mary , Nooti Sunil , Alhakeem Sara , Gachuki Beth , Byrd John , Muthusamy Natarajan , Rangnekar Vivek , Bondada Subbarao
Published: January 2023

B cell malignancies such as non-Hodgkin’s Lymphoma have a probability of affecting 1 in 43 people in the United States, contributing to 7% of all cancer related deaths. The most aggressive forms of non-Hodgkin’s Lymphomas, which include Chronic Lymphocytic Leukemia (CLL), express elevated levels of T cell leukemia 1 (Tcl1), an oncogene that promotes cell survival. CLL is characterized by clonally expanded population of CD5+CD19+ B lymphocytes with impaired apoptotic mechanisms. The Eµ-Tcl1 mouse, serves as an excellent model for the development of CLL due to Tcl1 overexpression in B cells through the Ig VH promoter and Eµ enhancer. In these CLL cells the B-cell receptor (BCR) signaling pathway is activated as shown by constitutive tyrosine phosphorylation of the Src Family Kinases (SFK) and total expression of Lyn, compared to normal B cells. Treatment of B-CLL cells with dasatinib, an SFK inhibitor, results in decreased proliferation and cell death, a decrease in Tcl1, and surprisingly, a decrease in prostate apoptosis response-4 (Par-4), a pro-apoptotic protein. Knockdown of Lyn with shRNA in B-lymphoma cells, also reduced Par-4 suggesting a specific role of Lyn in Par-4 regulation. Par-4 was also downregulated after inhibition of other non-receptor tyrosine kinases such as Syk and Btk involved in BCR signaling. Presently, we are determining the importance of Par-4 in the growth of B-CLL and the mechanism of novel regulation of Par-4 and Tcl1 through BCR signaling.
T-bet regulates long-lived plasma cell development in an influenza model (IRM10P.604)
Authors: Stone Sara , Ballesteros-Tato Andre , Lund Frances
Published: January 2023

Long-lived plasma cells (LLPC) arise from germinal center B cells (GCB). The transcription factor Bcl6, which is required for GCB cell survival, inhibits LLPC development by repressing the transcription factor Blimp1, which normally initiates LLPC development. To date, it is not clear how these opposing transcription factors are regulated in GCB cells. In T lymphocytes, the transcription factor T-bet modulates the balance between Blimp1 and Bcl6 and controls the subsequent differentiation of the T cells into memory and effector cells. When naïve B cells are activated in vitro with T helper 1 (Th1) cells, the B cells differentiate into antibody (Ab) secreting cells in a T-bet and IFNγ dependent manner. Therefore, we hypothesized that B cell intrinsic expression of T-bet would also be necessary for LLPC development following influenza A infection. We found that T-bet expression increased in GCB cells formed after flu infection and was highest within GCB cells expressing low levels of the PC marker CD138, suggesting that T-bet supports PC differentiation from GCB cells. In chimeric mice in which 50% of cells are T-bet-/-, T-bet-/- GCB cells expressed lower levels of Blimp1 and IRF4 than WT GCB cells. Furthermore, flu-specific Ab titers were attenuated in chimeric animals lacking T-bet selectively within the B lineage cells compared to control chimeras with WT B cells. These results support a role for T-bet in the development of LLPC following influenza infection.
Notch1 is an important secondary stimulator for activated B cells (IRM10P.606)
Authors: Park Sung-Gyoo , Park San-Heon , Kang Jung-Ah
Published: January 2023

The roles of Notch1 and Notch2 in T cell function have been well studied, but the functional roles of Notch in B cells have not been extensively investigated, except for Notch2 involvement in peripheral marginal zone B cell differentiation. This study examined the roles of Notch1 in murine primary B cells. We found that the intracellular domain of Notch1 could substitute for Notch2 in marginal zone B cell differentiation, and that Notch1 expression was important for further activation of B cells. During B cell activation by B cell receptor ligation, Notch1 was up-regulated while Notch2 was not. In addition, Notch1 up-regulation itself did not contribute to the further activation of B cells, but the Notch ligand was important for Notch1-mediated further B cell activation. Moreover, Notch1 increased antibody secretion from activated B cells. These data suggest that Notch1 is more important for B cell activation than Notch2, while Notch2 is more important for marginal zone B cell differentiation.
Regulation of IgM+ memory B cells by the inhibitory FγRIIb (IRM10P.610)
Authors: Papillion Amber , Winslow Gary
Published: January 2023

Our laboratory has described a long-term IgM+ memory population in the spleens of mice infected with Ehrlichia muris (J. Immunol. 191:1240). Among the many surface markers that distinguish the memory cells from conventional B cells is the inhibitory receptor FcγRIIb, which exhibited 4-fold higher expression. We hypothesized FcγRIIb negatively regulates IgM+ memory cells by binding immune complexes present during low-level chronic infection. To investigate this question, we monitored the population in infected FcγRIIb-deficient mice. Thirty days post-infection, the IgM+ memory B cells were increased by 3-fold in frequency in FcγRIIb-deficient mice, compared to control mice. This increase in the frequency of spleen IgM+ memory cells was due to an increase in the number of IgM+ memory cells, and not simply a decrease in the number of other B cells present. Preliminary studies revealed the IgM+ memory population generated in FcγRIIb-deficient mice was identical in phenotype to IgM+ memory cells generated in infected wild-type mice. The increase in the IgM+ memory population was associated with an increase in antigen-specific IgG. These data indicate that FcγRIIb plays an important role in regulating the expansion and/or persistence of IgM+ memory cells. We propose the inhibitory receptor acts to limit the contribution of IgM+ memory cells to recall responses under conditions where antigen-specific IgG is sufficient to control infection.