bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination Immunotherapy of Melanoma by inhibiting HSP90 and targeting its client proteins. (TUM7P.934)
Authors: Raveendran Subhara , Rao Aparna , Storkus Walter
Published: January 2023

HSP90 plays a major role in human melanoma progression, by facilitating the over expression of its client proteins, many of which provide growth, survival and migratory advantages to tumor cells and support a pro-angiogenic tumor microenvironment. HSP90-mediated stabilization also limits the proteasome-dependent processing of client proteins and the subsequent presentation of derivative peptides in tumor cell-associated MHC class I complexes which is required for effective host CD8+ T cell recognition of melanoma cells. When considered in the combination of active vaccination, inhibiting HSP90 function might serve as a highly effective anti-tumor therapy. As a consequence, we analyzed the anti-tumor impact of a range of clinically-relevant HSP90 inhibitors (HSP90i; i.e. NVP-AUY922, STA9090, 17-DMAG, Novobiocin) on client protein expression in vitro (human and murine melanoma cell lines) and in vivo (s.c. B16 tumors established in syngenic C57BL/6 mice). We observed that HSP90i promote the proteasome-dependent (i.e. blocked by inhibitors PS-341 and MG-132) degradation of a range of client proteins. We have also identified H-2Kb/Db-presented peptide epitopes derived from these client proteins that may be recognized by Type-1 CD8+ T cells after specific vaccination. We are currently investigating the capacity of NVP-AUY922 and STA9090 to therapeutically sensitize established melanomas to client protein-specific CD8+ T cells in combination therapy approaches.
Mitochondrial-mediated inflammasome activation controls production of Hepatoma-derived IL-1β (TUM7P.942)
Authors: Baseler Walter , O'Connor Geraldine , McVicar Daniel
Published: January 2023

Obesity is emerging as a critical driver of chronic inflammation and inflammation-associated cancers, such as hepatocellular carcinoma. The obesity-associated proinflammatory cytokine IL-1β is upregulated in multiple solid tumors and correlates with poor overall prognosis. Recently, fatty acid-induced inflammasome activation in macrophages has been shown to be dependent on mitochondrial reactive oxygen species (ROS) production. Here we asked whether in addition to traditional sources of IL-1β, the tumor itself might participate in maintenance of the inflammatory state. Stimulation of murine hepatoma line Hep1-6 cells with LPS substantially increased the mRNA level of IL-1β. Addition of LPS and palmitate together significantly elevated Hep1-6 IL-1β mRNA and mature IL-1β abundance. Inflammasome constituents were detectable in Hep1-6 cells with Nlrp3 mRNA content increased in LPS/palmitate treated cells. Cellular ROS levels were significantly increased in the LPS/palmitate treated group. AICAR, a potent activator of AMPK and mitophagy, decreased cellular ROS and mature IL-1β production by LPS/palmitate treated Hep1-6 cells, but did not affect IL-1β mRNA levels. Collectively, our data indicates that fatty acid-induced mitochondrial ROS results in inflammasome activation and cooperates with LPS-driven IL-1β mRNA resulting in tumor cell production of mature IL-1β. Through this mechanism, plasma fatty acids may promote tumor-associated inflammation.
Estrogen receptor α signaling inhibits T cell responses against gynecologic tumors (TUM7P.943)
Authors: Svoronos Nikolaos , Allegrezza Michael , Nguyen Jenny , Perales-Puchalt Alfredo , Rutkowski Melanie , Stephen Tom , Tesone Amelia , Conejo-Garcia Jose
Published: January 2023

Estradiol is a pleiotropic steroid hormone that regulates homeostasis, cell growth, and the immune system. In estrogen receptor positive breast cancer, anti-estrogen therapies are used to inhibit tumor cell growth. However, estrogen receptor α is also found on non-tumor cells within the tumor microenvironment. Therefore, we hypothesized that estradiol is able to affect tumor progression independent of its direct effect on tumor cells by regulating the tumor microenvironment, specifically immune cells. Using an orthotopic mouse model for ovarian cancer, we show that even in an estrogen receptor negative tumor, estradiol is able to drive tumor progression. Conversely, dependent upon an adaptive immune response, depletion of estrogen impedes tumor growth, as WT but not recombinase activating gene KO (RAG KO) estrogen-depleted mice survive longer than control littermates. This effect is dependent on estrogen receptor α expression in T cells. Mice reconstituted with a 1:1 mixture of WT and estrogen receptor α KO bone marrow reveal that estrogen receptor α KO T cells exhibit greater antitumor effector functions and pro-inflammatory cytokine production in a cell-intrinsic manner. These findings suggest anti-estrogens as a novel adjuvant therapy to augment T cell-dependent immunotherapies, such as adoptive cell transfer and programmed cell death protein 1 (PD-1) inhibition.
Chitinase-3 like-protein-1 (CHI3L1) expression associated with pulmonary inflammation accelerates breast cancer metastasis (TUM7P.960)
Authors: Libreros Stephania , Garcia-Areas Ramon , Iragavarapu Vijaya
Published: January 2023

Disseminated metastasis accounts for a majority of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis in both breast cancer and asthmatic patients. We have combined mouse models of breast cancer and pulmonary inflammation to determine if CHI3L1 associated pulmonary inflammation accelerates metastasis. We found that allergic pulmonary inflammation significantly enhances primary tumor growth in 4T1, 4T07 and 67NR mammary tumors by 10-fold, while decreasing survival. 4T1 tumor bearers with allergic pulmonary inflammation showed a 100-fold increase in metastatic tumor formation. We also assessed CHI3L1 levels and myeloid cells in the lungs of wild type and CHI3L1 knockout mice with allergic pulmonary inflammation and 4T1 mammary tumors. CHI3L1 levels were higher in the lungs of mammary tumor bearers with allergic pulmonary inflammation and correlated with increased metastasis. Wild type mammary tumor bearers with allergic inflammation had higher numbers of myeloid cells in the lungs in comparison to CHI3L1 knockout tumor bearers with allergic pulmonary inflammation. CHI3L1 knockout mice tumor bearers had significantly fewer myeloid cells in the lungs, decreased tumor growth and metastasis, along with increased survival. We propose that increased CHI3L1 in the lungs attracts myeloid cells that promote tumor growth and breast cancer metastasis.
TREM2 differential expression is associated with BAL cells that express high levels of ARG1 (VET2P.1044)
Authors: Hiremath Jagadish , Binjawadagi Basavaraj , Ouyang Kang , Dhakal Santosh , Manickam Cordelia , Dwivedi Varun , Gourapura Renukaradhya
Published: January 2023

Lung immunopathology is the major cause of influenza induced morbidity and mortality. DAP12 (DNAX-Activating Protein of 12kDa) is a membrane adaptor protein associated with varied surface receptors, and it is known to regulate influenza induced lung immunopathology. DAP12 associated receptor expression and their association with phenotype of bronchoalveolar lavage fluid (BAL) cells during swine influenza virus (SIV) infection in pigs is unknown. Since pig is a suitable large animal model for influenza research, our aim was to understand the effects of zoonotic SIV H1N1 infection on expression of DAP12 associated MDL-1, TREM-1 and TREM-2 receptors in pig BAL cells through qRT-PCR profiling. Results indicated that DAP12, MDL-1 and TREM-1 were constitutively expressed, but TREM-2 was upregulated. Stimulation of uninfected pig BAL cells in vitro using the cytokines IFNgamma and IL4 revealed that upregulation of TREM-2 was associated with enhanced expression of ARG1, but not with high levels of expression of TNFalpha and iNOS. ARG1 is a phenotype of alternatively activated alveolar macrophages that are known to protect lung tissue; hence TREM2 upregulation in pig BAL cells with similar phenotype suggests the possible beneficial role of these molecules in preventing the lung immunopathology. In conclusion, DAP12 and associated receptors are differentially expressed in pig BAL cells and TREM2 appears to have a beneficial role in regulating the lung immunopathology in SIV infection.
Polyfunctional CD4 T cells in the response to bovine tuberculosis. (VET2P.1034)
Authors: Fernanda Maggioli Mayara , Palmer Mitchell , Vordermeier H. Martin , Whelan Adam , Waters Wade Ray
Published: January 2023

Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) and play relevant roles in several chronic infections, including human TB and HIV. However, the assessment of this response in bovine infections was not feasible due to the lack of monoclonal antibodies that recognize bovine IL-2. Recently, an antibody specific for bovine IL-2 enabled the evaluation of polyfunctional T cells in cattle. The objective of the present study was to access antigen-specific polyfunctional ex vivo responses after aerosol Mycobacterium bovis infection of cattle. Peripheral blood mononuclear cells (PBMCs) were collected from infected cattle and stimulated with rESAT-6:CFP-10 (E:C), media or pokeweed (PWM) for 16 hours. After stimulation, the expression of CD4, CD45RO, CCR7, IL-2, IFN-γ, TNF-α and cell viability were evaluated. The ex vivo response to E:C consisted of 63% effectors (CD4+ CD45RO- CCR7-), 32% effector memory (CD4+ CD45RO+ CCR7-), and 9% central memory (CD4+ CD45RO+ CCR7+) phenotypes. In regard to the cytokine profile, 70% of cells producing cytokines expressed both IFN-γ and TNF-α, 31% expressed all three cytokines, 6% expressed both TNF-α and IL-2, and 3% expressed IL-2 and IFN-γ. Cells producing only IFN-γ, IL-2, or TNF-α represented, 5%, 8% and 1%, respectively. These findings demonstrate that the polyfunctional response consists mainly of effector cells co-producing IFN-γ and TNF-α.
Serologic markers of Epstein-Barr virus (EBV) reactivation in the conditions of viral encephalitis in young patients (VIR7P.1063)
Authors: Goleva Olga , Murina Elena
Published: January 2023

Viral encephalitis is one of the most severe disorders that is accompanied by the processes of tissue demyelization. It can be connected with both significantly causative viruses and a potential activation of latently persisting viruses of herpes group. The impact of persisting viruses on the activation of viral encephalitis is unclear. We analyzed the blood samples from 1-14 year-old patients diagnosed with acute viral encephalitis who had a history of Epstein-Barr virus (EBV) infection. Interestingly, there were low levels of IgM to early antigen (EA), nuclear antigen (NA) and capsid antigen (VCA) but high levels of IgG as determined by ELISA. Next, we used specific stripes with the immonobilized extract of EBV antigens (Euroimmun, Germany). The analysis of IgG antibodies in all blood samples demonstrated the presence of the late phase infection markers: VCA and NA. Importantly, the early antigen EA (areas: p43, p45, p93) that is induced within 3 weeks after the initial infection, was clearly present as well. These results suggest that EBV reactivation could be one of the favorable factors that accompanies the processes of tissue demyelization of central nervous system.
Escherichia coli Nissle 1917 colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model (VIR7P.1064)
Authors: Kandasamy Sukumar , Vlasova Anastasia , Chattha Kuldeep , Fisher David , Shao Lulu , Kumar Anand , Rajashekara Gireesh , Saif Linda
Published: January 2023

Microbiota regulate gut immune homeostasis, but the role of gram positive (G+)/negative (G-) commensals/probiotics in modulating human rotavirus (HRV) diarrhea and immunity is largely undefined. Gnotobiotic (Gn) piglets, the only animal model susceptible to HRV diarrhea, were colonized with G- Escherichia coli Nissle 1917 (EcN), G+ Lactobacillus rhamnosus strain GG (LGG) or LGG+EcN and challenged with virulent HRV (VirHRV) to assess probiotic effects on HRV pathogenesis and immunity. EcN+VirHRV piglets had significantly lower mean peak virus shedding titers and significantly reduced mean cumulative fecal scores and duration of diarrhea compared to LGG+VirHRV or control (uncolonized)+VirHRV piglets. LGG+VirHRV pigs had lower, but statistically similar fecal scores compared to control+VirHRV pigs. Combining LGG+EcN did not moderate VirHRV shedding or diarrhea more than EcN alone. Coinciding with reduced HRV infection, EcN+VirHRV piglets had significantly lower small intestinal (SI) HRV IgA antibody titers and HRV IgA and IgG antibody secreting cells (SC) compared to control+VirHRV piglets. Though not significantly different, SI HRV IgA antibody titers were 2-4-fold lower in the LGG+EcN+VirHRV and LGG+VirHRV piglets. Notably EcN+VirHRV piglets had higher SI total IgA levels and IgA SC compared to LGG+VirHRV piglets. Our results indicate that EcN alone was more effective than LGG or LGG+EcN in ameliorating HRV diarrhea and modulating B cell responses in a Gn piglet disease model.
Pivotal pathways that control host responses to lentivirus infection. (VIR7P.1053)
Authors: Mellor Andrew , Huang Lei , Li Lingqian , Lemos Henrique , Pacholczyk Gabriela , King William
Published: January 2023

Lentiviruses such as Human Immunodeficiency Virus establish persistent infections but it is unclear how lentiviruses evade host immunity. To elucidate pivotal pathways driving tolerogenic host responses to lentiviral infection we used the murine leukemia virus (LP-BM5) infection model, which causes persistent infections leading to splenomegaly, immunosuppression, and increased risk of leukemia. LP-BM5 infection induced indoleamine 2,3 dioxygenase (IDO) enzyme activity in a subset of dendritic cells expressing the B cell marker CD19 that regulate T cells. Different mouse strains exhibit variable resistance to LP-BM5 infection but all mice succumb to persistent LP-BM5 infection. In contrast, mice with genetically-induced, functional defects in Foxp3-lineage regulatory CD4 T cells (Tregs) cleared LP-BM5 infections ~4-6 weeks post infection. LP-BM5 clearance did not occur unless mice were homozygous for defective Foxp3 alleles, indicating that wild-type Tregs mediate dominant tolerogenic responses to LP-BM5 infection. Depleting CD8 T cells prevented LP-BM5 clearance in Treg defective mice, and LP-BM5 resistance correlated with diminished IDO induction, suggesting that Tregs block CD8 T cells via IDO to promote virus persistence. Thus Tregs are pivotal in promoting tolerogenic responses to LP-BM5 infection, and interfering with pathways that activate Tregs and mediate responses to activated Tregs may enhance host resistance to lentivirus infections.
Multiple paths to immunodominance: dominant epitopes are captured by MHC II prior, or post antigen proteolysis, and are enriched by HLA-DM (APP2P.103)
Authors: Sadegh-Nasseri Scheherazade , Kim AeRyon , Hartman Isamu , Cuidad M. Teresa , Song Nianbin , Boronina Tatiana , Caspi Rachel , Jaraquemada Dolores
Published: January 2023

Immunodominance is defined as the responsiveness of T cells to few selected peptide epitopes from complex antigens. To understand the mechanism of epitope selection from antigenic proteins for helper T cells, we used a cell-free antigen processing system composed of five purified proteins: HLA-DR, cathepsins B, H, S, and HLA-DM. We find that immunodominant epitopes are selected differently if derived from self proteins or from pathogens. Sensitivity to cathepsins and resistance to DM-mediated dissociation are equally contributing factors. Self-derived epitopes appear to be less susceptible to cathepsin digestion, whereas pathogen-derived epitopes are sensitive. Cathepsin sensitivity necessitates capture of epitopes by MHC II prior to processing by cathepsins. Resistance to DM-mediated dissociation increases longer epitope survival. We propose that most peptides generated from proteins do not gain immunodominance because they are sensitive to both DM and enzymatic digestions. Thus, immunodominance is established differently for different antigens incorporating functions of DM, cathepsins and MHC II, altogether help to ascertain higher abundance of the immunodominant epitope. Supported by grants from NIAID, R01AI063764 and R21 AI101987 to SS-N.
Evaluation of the role of type I interferon to the development of type 1 diabetes. (BA3P.131)
Authors: Carrero Javier , Calderon Boris , Ferris Stephen , Unanue Emil
Published: January 2023

Our goal is to identify the earliest immunologically relevant events that trigger autoimmune diabetes. The first detectable inflammatory signature in the NOD mouse is the upregulation of type I interferons and an interferon-inducible gene signature. We reason that understanding the early interferon signature may provide targets for the prevention, treatment, or diagnosis of autoimmune diabetes. The two ubiquitous interferon receptors (IFNAR and IFNGR) upregulate hundreds genes, some shared and some unique to each receptor. We backcrossed the type I interferon receptor onto NOD background to determine the relative contribution of IFNAR-inducible transcripts in type I diabetes induction. In our specific pathogen-free colony, NOD.IFNAR-/- mice developed diabetes at a decreased rate and penetrance from littermate controls. This reduction in incidence was gender independent and NOD.IFNAR heterozygous mice developed diabetes normally. There was a reduction in the number of the immunological infiltrates in the islets of Langerhans of NOD.IFNAR-/- mice from 4-8 wks of age as detected by flow cytometry and immunofluorescence. Concomitantly, there was a reduction in inflammatory gene expression in NOD.IFNAR-/- when compared to controls. Additionally, analysis of the NOD.IFNGR-/- mice showed a decrease in penetrance and kinetics of type I diabetes. Based on this data, we hypothesize that IFNAR and IFNGR induce a common genetic signature that is required for type I diabetes to proceed.
Apics deficiency reveals a role for a long noncoding RNA in dendritic cell function and autoimmunity (BA3P.203)
Authors: Brodnicki Thomas , Ashton Michelle , Elso Colleen , Mackin Leanne , Chu Edward , Payne Natalie , Ford Sharon , Tan Iris , Papenfuss Anthony , Kitching A Richard , Bernard Claude , Shortman Ken , Morahan Grant , O'Keefe Meredith
Published: January 2023

Although in vitro observations indicate that long noncoding RNAs (lncRNAs) regulate innate immune responses, their effect upon immune tolerance in vivo has not been defined. We have identified a novel gene of unknown function for which sequence variation is associated with autoimmune diabetes in the nonobese diabetic (NOD) mouse strain. Bioinformatics and expression analyses indicate this gene encodes a lncRNA that is induced by Toll-like receptor (TLR) activation, localizes to the nucleus and cytoplasm of dendritic cells, and binds to proteins within these cellular compartments. Moreover, sequence variation for this gene is associated with altered TLR-mediated cytokine production. Hence this gene was named Apics for Attenuator of Pattern recognition receptor-Induced Cytokine Secretion. To further investigate the function of this lncRNA, we established a C57BL/6 (B6) knockout mouse strain for Apics and discovered that Apics-deficient dendritic cells exhibit enhanced TLR-mediated cytokine production. Apics-deficient B6 mice also exhibit increased susceptibility to autoimmunity in two disease models: experimental autoimmune encephalomyelitis and experimental anti-neutrophil cytoplasmic antibody-associated vasculitis. Our study suggests that lncRNAs, such as Apics, can serve as TLR-inducible repressors that regulate the magnitude of innate immune responses to reduce the risk for developing autoimmune disease.
Disease activity in lupus correlates with expression of the transcription factor ARID3a (BA2P.119)
Authors: Ward Julie , Rose Kira , Montgomery Courtney , Adrianto Indra , Smith Kenneth , James Judith , Merrill Joan , Webb Carol
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with striking clinical, immunologic and genetic heterogeneity making development of targeted treatment strategies challenging. Previously, we showed transgenic mice with constitutive expression of the DNA-binding protein ARID3a/Bright in all B lymphocytes spontaneously produced autoantibodies. Therefore, we asked if ARID3a expression differed in SLE versus healthy control B lymphocytes. Of 114 SLE patients, 43% had expanded numbers of ARID3a+ peripheral blood B cells. A longitudinal study revealed that numbers of ARID3a+ B cells fluctuated with time, but were highly associated with increased disease activity. ARID3a expression was not limited to B cell subsets that normally express it, but was also apparent in large numbers of naïve B cells in many SLE patients. Therefore, we hypothesize ARID3a+ naïve B cells are precursors of autoantibody producing B cells. Antibody repertoire analyses of ARID3a+ and ARID3a- naïve B cells are in progress. Together, these findings implicate ARID3a as a potential biomarker for disease activity in SLE.
A balance between BCR and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels (BA2P.120)
Authors: Luo Wei , Mayeux Jessica , Gutierrez Toni , Getahun Andy , Müller Jennifer , Tedder Thomas , Chen Shu-Hsia , Rickert Robert , Nitschke Lars , Cambier John , Satterthwaite Anne , Garrett-Sinha Lee Ann
Published: January 2023

Signaling through the B cell receptor (BCR) can drive B cell activation and contribute to B cell differentiation into antibody-secreting plasma cells. The positive BCR activation signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. Here, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in B cells. We found that Ets1 is down regulated in B cells by BCR or TLR signaling through a pathway dependent on PI3 kinase, Btk, IKK2 and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase SHP1 or membrane receptors CD22 or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings suggest that the maintenance of Ets1 expression is an important function of the inhibitory Lyn -- CD22/SiglecG -- SHP1 pathway in B cells.
Dosage of X-linked Toll-like receptor 8 determines gender differences in the development of systemic lupus erythematosus (BA4P.216)
Authors: Umiker Benjamin , Andersson Shauna , Fernandez Luis , Korgaokar Parimal , Larbi Amma , Pilichowska Monika , Weinkauf Craig , Wortis Henry , Kearney John , Imainshi-Kari Thereza
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock-in genes encoding an auto-reactive anti-RNA antibody, we investigated how expression of Toll-like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8 or Tlr9. Autoantibody was produced in female mice with the combined deletions of Tlr7 and 9, but not in male mice. Autoantibody was not produced in the combined absence of Tlr7 and 8 in either sex, indicating that TLR8 contributes to the break in tolerance in females. Furthermore, TLR8 was sufficient for the loss of B cell tolerance, the production of class-switched autoantibody, heightened granulopoiesis, and increased production of type I interferon (IFN-I) by neutrophils as well as glomerulonephritis and death. We show that dosage of X-linked Tlr8 plays a major role in the high incidence of disease in female mice. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and IFN-I production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of SLE, suggesting new targets for treatment.
Rapid migration of lymphocytes via murine spleen (CAM1P.226)
Authors: Ganusov Vitaly
Published: January 2023

Spleen is the major secondary lymphoid organ in mammals playing an important role in initiation of adaptive immune response to blood-borne pathogens. Yet, the kinetics of lymphocyte recirculation via spleen in resting and inflammatory conditions remain poorly defined. By perfusing blood and lymphocytes via murine spleen in vitro Ford (Cell Tissue Kinetics 1969) measured accumulation of lymphocytes in circulation over the time of perfusion. I developed a novel mathematical model of lymphocytes recirculation via spleen and fitted that model to Ford's experimental data. The model explains well the majority of experimental data and predicts the average residence time of thoracic duct lymphocytes in the murine spleen of 3 hours. The model predicted that antigenic stimulation of the spleen induces increased migration into the spleen (or increased retention in the spleen) of blood lymphocytes, while cells that recently exited the spleen were nearly not affected. Interestingly, the model was not able to accurately explain the kinetics of lymphocyte migration via the irradiated spleen suggesting that irradiation impacts lymphocyte migration kinetics in a not easily predictable way. Taken together, our analysis provides important insights into kinetics of migration of lymphocytes via spleen.
IL-12p35 single chain subunit antagonizes IL-6 mediated STAT1 and STAT3 activation and prevents expansion of Th17 cells in experimental autoimmune uveitis (CCR1P.246)
Authors: Dambuza Ivy , Yu Cheng-Rong , Wang Ren-Xi , Kim Sung-Ye , Mahdi Rashid , Dolinska Monika , Sergeev Yuri , Wingfield Paul , Egwuagu Charles
Published: January 2023

The Interleukin 12 (IL-12) family of cytokines is comprised of heterodimeric proteins including IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (IL27p28/Ebi3) and IL-35 (p35/Ebi3). IL-27p28 and IL-12p40 have recently been shown to antagonize IL-27 and IL-12 functions, respectively, suggesting that single chain IL-12 family members may act as dominant-negative inhibitors of IL-12 family heterodimeric cytokines. Here, we have genetically engineered IL-12p35 (rIL-12p35) and examined whether IL-12p35 has biological activities independent of its partner, IL-12p40 or EBI3. We show that rIL-12p35 blocked IL-6 mediated activation of STAT1 and STAT3 pathways of TCR-activated CD4+ T cells and inhibited the expansion of Th17 but not Th1 cells. Of particular significance, rIL-12p35 protected mice from autoimmune uveitis by inhibiting pathogenic Th17 cell expansion. Our study demonstrates that IL-12p35 can be used to modulate IL-6-mediated signaling pathways and treat a CNS autoimmune disease.
How can high school students combine computational biology tools with a laboratory model to analyze changes in the human gut microbiome? (EDU1P.247)
Authors: Cobb Andrea , Gillevet Patrick
Published: January 2023

The human gut microbiome is an organ of the immune system. Early in life, an infant and its microbiome interact in poorly understood ways to develop gut-associated lymphoid tissues and to distinguish self from non-self. The gut microbiome produces metabolites used by the host and protects the host from colonization by pathogens. Gut mucosal microbiome composition is fairly constant, but lumen microbiome varies with environmental input. As an AAI Teacher Fellow in Dr. Gillevet’s lab, I used computational tools to determine correlations in alcohol intake, liver health and microbiome composition with changes in inflammatory cytokines and chemokines. How might my students engage in microbiome research? They will construct a gut microbiome model to generate data for computational analysis using Winogradski columns. The columns are simple containers containing wet soil, sulfur and carbon sources. With incubation, a bacterial gradient is established modeling a gut cross section -from lumen to mucosa-column bottom to top -low [O2] and high [S-2]. Students will add an environmental change into columns (for example, alcohol, fiber, fats or antibiotics). After column equilibration, students will collect visual data, then remove column samples to isolate sample DNA and perform 16SrRNA LHPCR and sequencing. Students will use computational analysis of the LHPCR data and (if available) sequence data to generate hypotheses relating environmental input and observed microbiome changes.
STAT5 antagonizes the pre-BCR-dependent NFκB and FOXO tumor suppressor pathways in acute lymphoblastic leukemia (HEM2P.261)
Authors: Katerndahl Casey , Willette Mark , Heltemes Lynn , Ramsey Laura , Koeuth Thearith , Gillespie Emily , Farrar Michael
Published: January 2023

Mice expressing a constitutively active form of STAT5b (Stat5b-CA mice) develop pre-B leukemia with low frequency (~1-2%). Microarray analysis of these leukemias shows decreased expression of genes involved in pre-BCR signaling. Stat5b-CA mice crossed to mice with defects in a coherent signaling pathway downstream of the pre-BCR involving BLNK, BTK, or PKCβ developed pre-B leukemia in > 75% of progeny. These leukemias exhibited reduced expression of a subset of NFκB target genes, which was important for transformation as Stat5b-CA x Nfkb1-/- mice also developed pre-B leukemia. Our results point to an unexpected role for NFκB1 as a tumor suppressor in pre-B cells. Microarrary analysis revealed that STAT5 activation resulted in reduced expression of several NFκB and FOXO target genes, thereby antagonizing these tumor suppressor pathways. To examine this regulation in further detail, we performed ChIP-seq analysis, which showed that STAT5 binds 38% of NFκB target genes that are expressed in pre-B cells. We also observed binding of STAT5 to several FOXO targets including: Foxo1, Cdkn1a, Cdkn2d, Blnk, Vegfa, Cxcr4, Bim, and Dgka. We have validated STAT5 binding to the FOXO targets Foxo1 and Cdkn2d and the NFκB targets Nfkb1, Rel, Irf4, Irf8, Ikzf1, and Ikzf3 by ChIP-qPCR. Our findings demonstrate that STAT5 activation antagonizes the pre-BCR-dependent NFκB and FOXO tumor suppressor pathways that limit pre-B cell proliferation and promote pre-B cell differentiation.
The transcriptional repressor NKAP is required for iNKT cell development and maturation (HEM3P.274)
Authors: Thapa Puspa , Seaburg Lauren , Chen Meibo , McWilliams Douglas , Tangen Sarah , Heibert Scott , Sant' Angelo Derek , Shapiro Virginia
Published: January 2023

Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.
CCR2+ monocytes contribute to hematoma clearance and long-term functional recovery after experimental intracerebral hemorrhage (HUM1P.324)
Authors: Hammond Matthew , Taylor Roslyn , Ai Youxi , Sansing Lauren
Published: January 2023

CCR2+ inflammatory monocytes worsen early neurological injury in mouse models of intracerebral hemorrhage (ICH), but their potential roles during recovery are unclear. The purpose of this work was to determine how CCR2+ monocytes influence repair and recovery after ICH. All mice in this study were subjected to the blood injection ICH model, whereby 25 μl of blood were injected into the striatum, a brain region involved in motor coordination. To first determine if monocytes have the capacity to phagocytose debris in the brain, we added 1-μm carboxylate beads to the injected blood. Using an ImageStreamX MarkII imaging flow cytometer, we found that CCR2+ monocytes engulf more beads than resident microglia at day 1 (4.35 ± 2.23 vs. 0.23 ± 0.45 beads, p<0.0001), suggesting they are phagocytically active in the brain after ICH. Next, Ccr2-/- bone marrow was transplanted into CD45.1 hosts to create chimeras with normal microglia, but few circulating monocytes. Controls received C57BL6 marrow. Ccr2-/- chimeric brains contained significantly more residual hemoglobin 7 days after ICH than controls (8.92 ± 1.03 vs. 3.96 ± 2.80 μl, p<0.05), suggesting a role for CCR2+ monocytes in ICH clearance. Importantly, Ccr2-/- bone marrow chimeras exhibit severe motor deficits following an LPS challenge 14 days after ICH, whereas controls exhibit minimal deficits. Together, these data suggest CCR2+ monocytes are important regulators of hematoma clearance and are necessary for functional recovery.
Thy1 (CD90) regulates adipogenesis and is decreased by the environmental obesogen tetrabromobisphenol-A (HUM2P.339)
Authors: Flores ELissa , Woeller Collynn , Phipps Richard
Published: January 2023

Obesity has risen drastically over the last 30 years and occurs through excessive adipogenesis (adipocyte formation) or increases in adipocyte size. Adipocytes produce cytokines such as IL-6 that promotes inflammatory disease. Environmental toxicants termed obesogens such as Tetrabromobisphenol-A (TBBPA) disrupt the endocrine system and increase adipogenesis; however the mechanisms remain unknown. Thy1 (CD90), a cell surface protein, is a member of the immunoglobulin family. We have previously shown that fibroblasts are heterogeneous for Thy1 and only Thy1-/low fibroblasts form adipocytes. We hypothesized that TBBPA promotes adipogenesis by decreasing Thy1 expression. We used mouse 3T3-L1 pre-adipocytes as a model to determine if TBBPA decreases the expression of Thy1 to promote fat cell formation. We measured Thy1 mRNA levels by qPCR and Thy1 protein levels by western blot. Adipogenesis was measured by inspection of lipid droplets and expression of the key adipocyte marker, FABP4. Our data show that TBBPA decreased the expression of Thy1 in 3T3-L1 cells. Furthermore, TBBPA treated cells also expressed higher levels of FABP4, supporting the concept that loss of Thy1 alters cell fate. TBBPA exposure for one week before adipogenic induction resulted in a further reduction of Thy1 and additional increases in FABP4. Thus, prolonged or developmental exposure to TBBPA may affect Thy1 levels and alter physiology long after exposure, which may contribute to the obesity epidemic.
Transgenic mice whose γ 1 and κ genes have been replaced by human ε and κ genes (HYP3P.402)
Authors: Lu Chien-Sheng , Hung Fu-Hsin , Lin Chien-Jen , Chen Jiun-Bo , Chen Christina , Shiung Yu-Yu , Tsai Ching-Yen , Chang Tse-Wen
Published: January 2023

IgE is a primary mediator in most allergic diseases. Human IgE specific for various allergens are very important reagents in diagnostic assays for the management of allergic diseases. However, allergen-specific human IgEs are present in patients’ blood at very low levels and hence extremely difficult to prepare. We have now constructed a transgenic homozygous mouse strain, referred to as HεκKI strain, whose γ1 constant region gene segment has been replaced by human ε gene constant region segment and whose κ constant gene replaced by human κ constant gene. In those HεκKI mice, the human IgE levels are about 10 times of mouse IgE. Upon immunization with an allergen papain, both human IgE and mouse IgE were increased, with human IgE increased to about 30 folds of mouse IgE. We have prepared a number of hybridomas secreting monoclonal human IgE (the variable regions being mouse) specific for papain. Cells of a basophilic leukemic line pulsed with such human IgE could be activated by papain to undergo degranulation process. These results indicate that the transgenic mouse strain HεκKI could be employed to prepare polyclonal and monoclonal human IgE specific for various allergens.
Role of mast cells in a chronic murine asthma model (HYP3P.348)
Authors: Song Chang Ho , Zhang Xin , Chai Ok Hee , Shon Dong-Hwa , Lim Jeong Min , Kim Ik Seong , Han Eui-Hyeog , Kim Hyoung Tae
Published: January 2023

Mast cells are thought to be important in the cause of allergic diseases, but the role of mast cells in the airway remodeling of chronic long-term asthma remains controversial. In this study, using the mast cell “nock in” strategy to get the mast cell-deficient WBB6F1-W/Wv mice (W/Wv) selectively reconstituted bone marrow-cultured mast cells (BMCMCs) from normal congenic wild-type mice (+/+) (+/+ BMCMCs→W/Wv mice), mice were employed to study the roles of mast cell in a murine asthma model. The mice were sensitized with ovalbumin (OVA) and challenged with OVA for 14 weeks. The mice were assessed airway reactivity by PenH. The lungs and trachea were stained with H-E for pathologic alteration, toluidine blue for mast cell, congo red for eosinophil, PAS for goblet cell and Masson’s-trichrome for fibrosis. Mast cells were not observed in the lungs of control and OVA-sensitized and challenged W/WV mice, but OVA-sensitization and challenge increased mast cell number in +/+ mice and +/+BMCMCs→W/Wv mice. OVA-sensitization and challenge could enhance airway reactivity, pathologic alteration, eosinophil infiltration, goblet cell hyperplasia and fibrosis in +/+ mice. The pathologic changes of OVA treated W/Wv mice were less than that of +/+ mice, but +/+BMCMCs→W/Wv mice could restore the reaction to OVA-sensitization and challenge of +/+ mice. These results indicate that the mast cell play an important role in the OVA-induced chronic murine asthma model.
Inhibition of Jak/STAT signaling synergizes with CTLA4-Ig to promote transplant acceptance (IRC2P.454)
Authors: Raimondi Giorgio , Khalifian Saami , Oh Byoung , Lee W.P. Andrew , Brandacher Gerald
Published: January 2023

Transplant tolerance induction through conventional costimulation blockade (CoB) remains an elusive goal. Recent evidence suggests that inflammatory cytokines (IC) contribute to the activation of alloreactive T cells in a CD28- and CD40-independent manner. We aimed to understand the mechanisms underlying this process and delineating the possible synergism between CD28 blockade and inhibition of IC signaling via Jak/STAT inhibition. Murine T cell activation was assessed by quantification of IL-2 secretion (at 24h post TCR/CD28 engagement). CTLA4-Ig diminished the proportion of IL-2+ cells. However, the concomitant addition of the supernatant (MATSup) from maturing dendritic cells completely abolished this effect. This suggested an early impact of IC on T cell activation. In a CFSE-based proliferation assay, MATSup counteracted the anti-proliferative effect of CTLA4-Ig on both CD4 and CD8 T cells. Inhibition of Jak/STAT signaling through addition of Tofacitinib (Tofa) restored CTLA4-Ig regulation in presence of MATSup. Finally, in a BALB/c to B6 skin transplant model a short course of Tofa (d0-12) synergized with CTLA4-Ig doubling skin allograft survival (MST untreated: 9.5d, CTLA4-Ig only: 13d, Tofa+CTLA4: 18d). Overall, our results indicate that the stimulatory effect of IC counteracts the efficacy of CoB. However, their redundant activity can be neutralized through concomitant inhibition of Jak signaling - a promising strategy for promotion of transplant acceptance.
Anti-TIM-4 mediates long-term engraftment of islet allografts by promoting IL-10 expression by TIM-1+ Bregs and inhibiting IFNγ expression by proinflammatory “Be1” B cells (IRC3P.464)
Authors: Ding Qing , Yeung Melissa , Xiao Sheng , Najafian Nader , Kuchroo Vijay , Rothstein David
Published: January 2023

We previously defined TIM-1 as an inclusive marker for IL-10+ regulatory B cells (Bregs). Moreover, α-TIM-1 mAb prolongs islet allograft survival (GS) through induction of TIM-1+ Bregs. We recently noted that TIM-4, a TIM-1 ligand, identifies B cells enriched for IFNγ (Be1). We therefore hypothesized that TIM-4 blockade should also prolong GS and that this may involve B cells. In fact, α-TIM-4 (RMT4-53) treatment resulted in long-term GS in BALB/c recipients of B6 islets (MST >100d). Moreover, α-TIM-4 increases Th2 cytokines (IL-4, IL-10) and Foxp3+Tregs, while reducing both Th1 cytokines (IFNγ) and CD4 proliferation. However, these α-TIM-4-mediated changes and long-term GS were B cell dependent, and did not occur in either B cell deficient (JHD) or depleted (α-CD20) recipients. To address whether α-TIM-4 directly targets (TIM-4+) Be1 cells, we showed that adoptive transfer of WT, but not TIM-4-/- B cells, into μMT recipients, reconstitutes long-term GS mediated by α-TIM-4. We also found that α-TIM-4 inhibits IFNγ expression by TIM-4+ B cells by 40%. Bregs were also required for α-TIM-4 induced transplant tolerance. Transfer of B cells from TIM-1Δmucin (mucin domain deletion) mice, which exhibit a defect in Bregs, is unable to prolong GS in B deficient mice. In wt mice, α-TIM-4 increases TIM-1+ IL-10+ Bregs by ~100%. Taken together, our data reveal that targeting TIM-4 enhances IL-10 expressing Bregs and also reduces inflammatory Be1 B cells to promote allograft tolerance.
CD86-dependent FoxP3+ regulatory T cells promote resolution of disease after influenza virus infection (IRC4P.492)
Authors: Moser Emily , Hufford Matthew , Braciale Thomas
Published: January 2023

Influenza A virus (IAV) infection in the respiratory tract triggers a robust immune response, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of lung disease occurs during a distinct recovery period. The costimulatory molecule CD86 can modulate T cell activity during the initiation and effector stages of the host response to IAV infection, but the role of CD86 during recovery is unknown. We found that antibody-mediated CD86 blockade after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and innate cytokines in the bronchial alveolar lavage fluid (BAL), but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into α-CD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Direct depletion of Tregs late after infection using DEREG mice mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Finally, we identified neutrophils as a target of Treg suppression, since neutrophil depletion in Treg-depleted DEREG mice reduced excess BAL cytokines. These results show that CD86-dependent Tregs contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.
Development of a new mouse/human FoxP3 antibody using rabbit recombinant antibody technology (IRC4P.477)
Authors: Goetz Christine , Bonnevier Jody , Bjordahl Ryan , Houchins J. , Munshi Cyrus , Aggeler Birte
Published: January 2023

Tregs play a key role in immune system suppression during autoimmunity and tumor development. Tregs are classified as CD4+CD25+FoxP3+, and exist as either nTregs originating from the thymus, or iTregs derived from CD4+ effector T cells in the periphery. Reduction in Treg numbers leads to autoimmunity; this is clearly shown in scurfy mice where FoxP3 has been deleted, Treg numbers are severely reduced, and mice succumb to autoimmunity early in life. In contrast, Tregs play a debilitating role in cancer biology when the Treg numbers are elevated, and the immune response is dampened allowing cancer cells to evade the immune system. Thus, the correct balance of Tregs is essential for maintaining immune homeostasis. FoxP3 has been widely used as a marker of Tregs, and many antibodies are available on the market that distinguish the Treg population in human or mouse cells. We developed a novel FoxP3 antibody and Fix and Perm buffer system that is comparable to current clones, but recognizes both mouse and human FoxP3 equally well, eliminating the need for separate antibodies. The ms/hu FoxP3 antibody was generated using our Rabbit Recombinant antibody technology, which allows for selecting high specificity, high affinity antibodies, lot to lot consistency and detection of both mouse and human nTregs and iTregs. This allows researchers to extend their mouse models into clinical studies without a change in the antibody clone eliminating a variable and streamlining the process.
DNA methylation profiles at the human Th2 locus in BAC transgenic mice point to novel putative regulatory elements (IRM6P.720)
Authors: Pivniouk Vadim , Rosenbaum David , Pivniouk Oksana , Herrell Amanda , Miller Susan , Sprissler Ryan , Lyons Eric , Vercelli Donata
Published: January 2023

Most cis-regulatory elements in the mouse Th2 cytokine locus were initially identified based on their evolutionary conservation. In contrast, the regulatory landscape of the human Th2 locus, which is ~40 kB larger than the mouse syntenic region, remains poorly defined because recently arisen putative regulatory elements are less readily identifiable by bioinformatics and cannot be functionally interrogated in humans. To address these limitations we created BAC transgenic (TG) mice carrying 160 kB of the human Th2 locus including RAD50, IL13, and IL4. These mice appeared normal and demonstrated faithful transcriptional regulation of human Th2 cytokine genes. Because dynamic changes in chromatin architecture typically mark functional non-coding sequences, we used next generation bisulfite sequencing to compare CpG methylation of DNA isolated from TG naïve CD4+ T cells and in vitro polarized Th2 or Th1 cells. All major regulatory elements previously characterized in mice were clearly identifiable by their CpG methylation profile in the human TG Th2 locus. Moreover, we identified at least three other non-coding regions (conserved highly in primates and moderately in species of the clade Laurasiatheria) that were differentially methylated in Th2, Th1 and naïve T cells, and are located in the RAD50/IL13 and IL13/IL4 intergenic areas. Our data suggest these regions contain novel cis-regulatory elements involved in the transcriptional regulation of human Th2 cytokines expression.