bluuebunny/crossref_metadata_2025 huggingface dataset:
ISS (ImmunoStimulatory Sequences) and Iscomatrix boost vaccine-generated HBV-specific immunity in mice (B202)
Authors: Marshall Jason D , Gesner Marianne L , Heeke Darren S , Livingston Brian , Van Nest Gary
Published: January 2023

We have investigated the adjuvant activity of both CpG motif-containing ImmunoStimulatory Sequences (ISS) and the saponin/phospholipid-based Iscomatrix (IMX) on HBV-specific humoral and cell-mediated immunity in C57BL/6 mice. Mice were immunized at weeks 0 and 2 with several formulations that incorporated HBV surface antigen (HBsAg), HBV core antigen (HBcAg), ISS, and IMX. ISS was added to antigen in soluble form or conjugated with HBsAg or HBcAg. Bleeds were performed 2 weeks post 1st immunization (2wp1) and 2wp2 and sera analyzed for anti-HBV IgG1 and IgG2a levels. Mice were also splenectomized at 2wp2 and HBV-specific cell-mediated immunity was examined. HBsAg + HBcAg + 1018 ISS + IMX resulted in high levels of HBsAg-specific and HBcAg-specific IFN-g induction that exceeded that induced by either adjuvant alone. Conjugates of HBV surface or core protein to ISS ODNs also provided for higher levels of antigen-specific IFN-g production and CTL activation. These results indicate that HBV therapeutic formulations that incorporate ISS and/or IMX may stimulate for enhanced levels of anti-HBsAg antibody and augmented anti-HBcAg CMI (cell-mediated immunity) and are promising candidates to test in human clinical trials.
Subcutaneous 21-day Administration of 17alpha-ethinyl estradiol or 17beta-estradiol alters the thymus of 8-week-old C57BL/6 Mice (B217)
Authors: Brummer Tyson Peter Thomas , Phillips Rebecca A , Kerr Richard P , Gogal Robert M , Ahmed Ansar
Published: January 2023

Exposure to exogenous estrogens occurs via multiple sources (i.e. environmental contamination, contraceptives). Presently, little data exist regarding the immune effects of the synthetic estrogen 17α-ethinyl estradiol (EE) and its relative potency compared to 17β-estradiol (E2). In this study, we investigated the immune effects of daily subcutaneous injections of either EE or E2 on intact juvenile male and female C57BL/6 mice for 21 days with concentrations of 0.04 μg/25 g BW, 0.4 μg/25 g BW, or 4.0 μg/25 g BW. In male mice, thymus weight and thymus to BW ratio were significantly decreased at 4.0 EE dose, whereas in female mice significant decreases were noticed at 0.4 and 4.0 doses of both E2 and EE. Thymocyte absolute numbers were significantly diminished at both 4.0 E2 and EE dosing levels in males, and decreased at the 0.4 and 4.0 doses of E2 and EE in females. Decreasing trends in CD4+/CD8+ expression were observed in EE-treated mice, reaching significance at 4.0 EE in the females. The male spleen NK 1.1 cell surface marker was significantly increased at 0.04 and 4.0 EE treatments, as well as at 0.4 E2. In summary, results of these data suggest that a 21-day subcutaneous exposure of either E2 or EE is sufficient to significantly alter thymic endpoints. With the exception of the NK 1.1 subset, splenic endpoints did not yield any remarkable results. These findings suggest that the thymus is a more sensitive target than the spleen with regard to subacute exposure of 17α-ethinyl estradiol.
Stochastic simulation of B cell signaling reveals affinity discrimination through B cell synapse formation (B36)
Authors: raychaudhuri subhadip , tsourkas philippos , simon scott i. , baumgarth nicole
Published: January 2023

We have developed a Monte Carlo based stochastic computer simulation model to investigate the mechanisms of differential B cell signaling upon ligation with antigens of varying affinity. Recent experiments have revealed that B cell signaling is modulated through the immune synapse upon ligation with membrane bound antigens. The immune synapse is an ordered structure consisting of segregated BCR-antigen and LFA1-ICAM1 molecules that is formed at the cell-cell contact area. Our computational model elucidates affinity and diffusion dependent biophysical mechanisms of such B cell immune synapse formation. For a given antigenic affinity, our numerical experiments gave rise to stochastically varying levels of activated signaling molecules such as phosphorylated BCR Igα/Igβ and Syk molecules. Such stochastic outcome of B cell signaling determines how affinity discrimination is achieved by signaling through immunological synapses. For soluble antigens, a diffusion-dependent coarse-grained model of receptor clustering (capping) at the B cell surface yields similar stochastically varying levels of signaling molecules and results in affinity discrimination through receptor cap formation.
Generation of MHC class I-presented peptides in cells over-expressing a dominant interfering ubiquitin (B43)
Authors: Huang Lan , Marvin Julie , Golovina Tania , Eisenlohr Laurence C
Published: January 2023

The participation of the proteasome in MHC class I-restricted antigen processing is well documented, but the degree to which targeting of antigenic proteins to this multicatalytic protease is ubiquitin-dependent remains unclear. To address this question, we over-expressed wild type ubiquitin (Ub-WT) and lysineless ubiquitin (Ub-K0) in mammalian cells using an inducible vaccinia system. Compared to Ub-WT, the Ub-K0 potently abrogated the conjugation of ubiquitin to protein substrates, and subsequently attenuated total protein degradation. In co-infection assays, the Ub-K0 significantly inhibited specific epitope presentation from cytosolic N-end Rule substrates and an exocytic model antigen, but had little effect on antigen presentation from a stable cytosolic antigen. Interestingly, the inhibitory effect on antigen presentation by Ub-K0 decreased when the exocytic model antigen is directed to the cytoplasm, and increased when the stable cytosolic antigen is directed to the endoplasmic reticulum (ER). Despite similar levels of surface MHC class I molecules, different peptide profiles bound to surface class I molecules were observed from cells over-expressing Ub-WT and Ub-K0. The results suggest that cytosolic proteins and glycoproteins may be targeted to proteasome through different pathways to generate MHC class I peptides.

This study is supported by NIH grant AI39501.
Tumor Associated Ascites Inhibits CD1d-Mediated Antigen Presentation (B44)
Authors: Webb Tonya J , Rogers Ophelia , Giuntoli Robert L. , Schneck Jonathan , Oelke Mathias
Published: January 2023

Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. Recently, it has been shown that NKT cells can both directly and indirectly mediate anti-tumor immunity. However, it is thought that the anti-tumor effects mediated by these cells are being hampered because the number and function of NKT cells is reduced in cancer patients. Moreover, it has been shown that tumor associated glycolipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. In these studies we examined antigen presentation by CD1d following treatment with ascites from cancer patients. It was found that pretreatment with ascites inhibited the ability of CD1d molecules to present endogenous antigen to NKT cells in a dose-dependent manner. We found that this inhibition does not require antigen processing, as canonical (Vα14+) and noncanonical NKT cells, which do not require trafficking through endocytic compartments, were both inhibited. Also, ascites treatment of artificial Antigen Presenting Cells (aAPC) preloaded with α-GalCer, abrogated their ability to present antigen to NKT cells, which further suggests that the inhibition is antigen processing independent. In addition, our data demonstrate that treatment with ascites does not inhibit antigen presentation to classical CD8+ T cells. Together, these data suggest that glycolipid shedding in ascites may be a mechanism of immune evasion specifically utilized used by cancers to inhibit CD1d- mediated activation of NKT cells.
The RING E3 ubiquitin ligase Trim13 (Rfp2) influences αβ and γδ T cell development (B5)
Authors: Narayan Kavitha , Kang Joonsoo
Published: January 2023

αβ and γδ T cells arise from a common precursor in the thymus, but the mechanisms involved in this lineage specification remain poorly defined. We identified Tripartite Motif 13 (Trim13) as a γδ lineage biased gene. TRIM13, an RBCC protein, is an ER/nuclear membrane localized RING E3 ubiquitin ligase involved the ER-associated degradation pathway. Similar to other γδ lineage biased genes, the expression of Trim13 in γδ T cells is regulated in part by trans priming signals from αβ lineage DP thymocytes, and Trim13 is also expressed in some unconventional αβ lineage subsets. Interestingly, Trim13 is only expressed in about half of γδ thymocytes, suggesting that Trim13 may mark a subset of γδ T cells distinguishable by different thymic selection requirements, different stages of maturity, and/or functional competence. Preliminary results indicate that ectopic Trim13 expression is detrimental to αβ T cell development, but is conducive for enhanced γδ T cell generation and B cell development and/or expansion. These results suggest that Trim13 may play a role in T and B cell development, and that its expression may segregate specific subsets of T cells. Given its role as a nuclear membrane E3 ubiquitin ligase, it is possible that TRIM13 functions, in part, by controlling the relative abundance of transcription factors involved in lineage differentiation or maturation, resulting in different developmental outcomes.
NF-κB is transported into the nucleus by a subset of importin α molecules (B51)
Authors: Fagerlund Riku , Melen Krister , Kinnunen Leena , Julkunen Ilkka
Published: January 2023

NF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus, where they bind to the promoter elements of their target genes. The molecular mechanisms of nuclear import of various members of the NF-κB family of transcription factors have remained relatively poorly studied. By using E. coli-expressed GST-importin α isoform constructs we previously identified importin α3 and importin α4 as the main importin α isoforms binding to TNF-α activated p50-p65 heterodimers. Altogether 6 different importin α isoforms have been identified in humans. NF-κB p50 and p65 protein binding to importin α3 was found to be mediated by the previously identified nuclear localization signals (NLSs) of these proteins. Baculovirus-expressed importin α5 was also found to bind activated p50-p65 heterodimers. We also found that TNF-α or Sendai virus infection-activated alternative NF-κB pathway components, p52 and RelB bound to baculovirus-produced importin α3, α4 and α5 isoforms. Importin α5 was found to directly bind to NLSs of NF-κB p52, RelB and c-rel proteins. Our findings suggest that the nuclear import of both the classical and alternative NF-κB pathway molecules is a regulated process mediated by a subset of importin α isoforms.
CXCL10 is required for resistance to dengue virus infection independent of effects on leukocyte recruitment (B54)
Authors: Ip Peng Peng , Liao Fang
Published: January 2023

CXCL10 is an interferon-inducible chemokine ligand for CXCR3, a receptor that is expressed on lymphocytes and that CXCL10 shares with CXCL9 and CXCL11. Previously, we found that CXCL10−/− mice were more susceptible to dengue virus infection as compared with wild-type controls. In this study, we explored the mechanisms underlying this enhanced susceptibility. Viral loads were higher in the brains of CXCL10−/− mice vs. control animals. Presuming a defect in lymphocyte migration, we next investigated whether recruitment of effector T cells and antibody-secreting cells (ASC) to the infected tissues were impaired in CXCL10−/− mice. Unexpectedly, CXCL10−/− mice showed only slightly lower numbers of infiltrating total T cells, higher numbers of CXCR3+ T cells, and higher numbers of ASC in the brain as compared with control mice. These results demonstrate that

CXCL10 is not required for recruitment of CXCR3+ T cells to the brain in dengue virus-infected mice, andthe enhanced susceptibility of CXCL10−/− mice to dengue virus infection is not due to a defect in recruitment of effector lymphocytes, but may be due instead to an anti-viral activity of the chemokine.
Norepinephrine-dependent activation of the neuroimmune system is counter-regulated by IL-1RA (B59)
Authors: Park Min Jung , Johnson Daniel R , Martin Jonathan , Freund Gregory G
Published: January 2023

Stress negatively impacts well-being and is linked to a variety of diseases including heart disease and cancer. It is known that stress modulates the immune defense system, but the mechanisms involved are not well established. Here we report that norepinephrine (NE) is a rapid activator of the neuroimmune system as measured by a loss of interest in the social environment. C57BL/6J mice were injected with 1.5 mg/kg of NE (I.P.) and social exploration (SE) was measured over a 24-hour period. Peak decrease in SE was observed 30 min after NE injection (74.1 ± 4.7 % reduction in SE) and full recovery was observed 8 hours following NE treatment. This decrease appears linked to a brain-based up-regulation of IL-1 alpha (370 %) and TNF alpha mRNA (260 %). Additionally, Kineret®, a derivatized recombinant IL-1RA used in the treatment of rheumatoid arthritis, hastened recovery from NE-induced neuroimmune system activation by 31.1%. These results show that NE actives the neuroimmune system, which IL-1RA treatment can ameliorate.
High Affinity Humanized Monoclonal Antibody to Human IL 1β Potently Inhibits Toll-like Receptor (TLR) Agonists in Whole Blood (B61)
Authors: Reznikov leonid , Solinger Alan , dinarello charles
Published: January 2023

XOMA 052, a Human Engineered™ monoclonal antibody, was developed to the mature form of human IL-1β following selection from multiple phage display libraries. This antibody has a binding constant for human IL-1β of 300 fM. Human blood cultures were used to evaluate the biological effects of this antibody. The cultures were stimulated with TLR ligands. After 24 hours, the cultures were lysed with Triton®-X to extract membrane and intracellular cytokines. XOMA 052 inhibited the production of IL-1β induced by Staphylococcus epidermidis by 50% at 0.1 pM and by 75% at 3 pM; IL-1β induced by LPS was reduced by 75% at 10 pM. At 100 pM, inhibition was 100% whether induced by Staphylococcus epidermidis or LPS. LPS-induced TNFα was reduced by 75% at 100 pM. Interferon-γ (IFNγ) was induced by Staphylococcus epidermidis and by LPS. XOMA 052 reduced IFNγ induced by Staphylococcus epidermidis by 75% at 100 pM and LPS-induced IFNγ by 75% at 100 pM.We conclude that the data from the whole blood cultures are consistent with the high 300 fM affinity of XOMA 052 for IL-1β.
Immunomodulatory effects of CMDB, the component of Cordyceps militaris mycelium, by the enhanced production of cytokines in macrophages (B64)
Authors: Kim Kyungjae , Kim Hyunyul , Han Shinha , Kim Kwanghee , Kwon Jeunghak , Lee Seungjeong
Published: January 2023

Cordyceps militaris (CM) have been reported to modify the immune and inflammatory responses both in vivo and in vitro. Macrophages play important roles in the innate immunity through the phagocytosis of antigens. This study examined the effects of CM on the activation of murine macrophage RAW264.7 cells. The filtered components of the draff broth (DB) cultured with mycelium of CM, CMDB, CMDBW, CMDB5P, CMDB25P, were purified by propyl alcohol extraction and HP 20 column chromatography. All the components of CM produced significant amounts of nitric oxide (NO). In particular, CMDB produced much more NO in RAW264.7 cells and primary macrophages than the other fractions of CM. CMDB increased significantly the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 dose-dependently in RAW264.7 cells. To examine whether the CMDB effects on cytokine production were attributable to its influence on the expression of mRNAs, semi-quantitative RT-PCR experiments were performed for the transcripts. Examination of the gene expression level showed that the enhanced production of cytokines was correlated with the up-regulation of i-NOS expression, cycloxygenase (COX)-2 expression, IL-1β and IL-6 expression, and TNF-α gene expression. Western blot analysis also confirmed that CMDB enhances the expression level of these cytokines. These results suggest that CMDB has immunostimulatory effects on macrophages through the enhanced production of cytokines.
Homing molecule expression on skin tropic herpes simplex virus type-2 (HSV-2)-specific CD8+ T-cells: role of fucosyltransferase-VII (B68)
Authors: Hensel Michael , Caka Selin E , Wald Anna , Koelle David M
Published: January 2023

Homing of pathogen specific T-cells is an important component of the host immune response. HSV-2-specific T-cells express high levels of cutaneous lymphocyte-associated antigen (CLA) and the related E-selectin ligand (ESL). Control CMV- or EBV-specific T-cells express low levels of CLA/ESL. CLA/ESL expression and lymphocyte homing to skin are dependent on fucosyltransferases-IV and –VII (FT-IV and FT-VII). We used real-time PCR to compare levels of FT-VII mRNA in isolated CD8-pos T-cells specific for HSV-2, EBV, and CMV from un-manipulated blood using tetramer (tet) staining and cell sorting. Within a group of subjects, circulating HSV-2-specific cells had higher levels of FT-VII mRNA than did EBV-specific cells (p=0.02) but not CMV-specific cells (p=0.29). Using within-subject comparisons, HSV-2 tet-pos cells had higher levels of FT-VII mRNA than did CD8-pos tet-neg bystander cells (p=0.02). In contrast, FT-VII mRNA levels within EBV or CMV tet-pos cells were not higher than levels in CD8 tet-neg bystanders (p=0.31 and p=0.44, respectively). Ongoing studies using HSV-2-specific cells to study skin-homing at the protein and mRNA levels are in progress.

Research support provided by: STD/AIDS Research Training Grant NIHT32 AI07140 and UW Royalty Research Fund 65-0875
Activation and Nuclear translocation of p65 NF-kappaB are sufficient for VCAM-1, but insufficient for ICAM-1, expression in TNF-stimulated primary smooth muscle cells: differential requirement for PARP-1 expression and interaction (B73)
Authors: Boulares Hamid , Suzuki Yasuhiro , Zerfaoui Mourad , Naura Amarjit , Hans Chetan , Nichols C
Published: January 2023

Although nuclear translocation of NF-κB and subsequent binding to promoters of ICAM-1 and VCAM-1 have been shown to be decisive for their expression, a number of discrepancies in the expression pattern of these adhesion molecules have been reported in cell culture systems and in disease settings including atherosclerosis, asthma, and auto-immune diseases. Here we show that while p65 NF-κB nuclear translocation in TNF-treated smooth muscle cells (SMC) was sufficient for the expression of VCAM-1, ICAM-1 expression required the expression of PARP-1. I-κBα phosphorylation and subsequent degradation were virtually identical in both TNF-treated wild type and PARP-1−/− SMC. VCAM-1 expression in TNF-treated PARP-1−/− SMC was confirmed to be NF-κB-dependent given that PDTC inhibited its expression. The expression of VCAM-1 and ICAM-1 were associated with a transient interaction between PARP-1 and p65 NF-κB when examined in the airway epithelial cell line A549. Such interaction was confirmed using fluorescence resonance energy transfer (FRET) assay. Protein acetylation activity, mediated by p300/CBP, was required for VCAM-1 and ICAM-1 expression in TNF-treated SMC, however, interaction of PARP-1 with p300 was not required for VCAM-1 expression. These findings indicate that p65 NF-κB translocation can be in some situations insufficient for some genes (e.g. ICAM-1) yet in the same time can induce the expression of others (e.g. VCAM-1), thus providing a new concept on the role of NF-κB in driving its target genes. Further, the data suggest a differential requirement for PARP-1 expression in inflammatory processes.

Support: NIH-HL072889 and 1P20RR18766 to HB.
Signal transduction blockade with imatinib mesylate prevents and treats autoimmune arthritis (B76)
Authors: Paniagua Ricardo , Sharpe Orr , Ho Peggy , Chan Steven , Chang Anna , Tomooka Beren , Lee David , Genovese Mark , Utz Paul , Steinman Lawrence , Robinson William
Published: January 2023

For over two decades researchers have speculated on the potential use of kinase inhibitors to treat autoimmune diseases. To date, investigations of kinase inhibitors have been largely limited by low therapeutic indices that result in unacceptable toxicity profiles. We show that imatinib mesylate, a tyrosine kinase inhibitor FDA-approved to treat chronic myeloid leukemia and gastrointestinal stromal tumors, has the potential to provide benefit for autoimmune diseases such as rheumatoid arthritis (RA). We demonstrate that imatinib, at a dose equivalent to 200–400mg/day in humans, prevents murine autoimmune arthritis and treats established disease. We further show that submicromolar imatinib concentrations inhibit tyrosine kinases and cellular responses that are central to RA pathogenesis, including PDGF-mediated proliferation of fibroblast-like synoviocytes, c-Fms activation and macrophage production of TNF-alpha, c-Kit-mediated mast cell release of proinflammatory cytokines, and B cell immunoglobulin production. Inhibition of tyrosine kinase pathways by imatinib represents a potent and promising therapeutic approach to treat RA and other autoimmune diseases.
Avidity of CD8 T-cells sharpens immunodominance. (B9)
Authors: Dzutsev Amiran , Belyakov Igor , Isakov Dmitry , Margulies David , Berzofsky Jay
Published: January 2023

In the course of viral infection, the immune system exploits only a fraction of the available CTL repertoire and focuses on a few of a myriad of potentially antigenic peptides. This phenomenon, known as immunodominance, depends on a number of factors, including antigen processing and transport, MHC binding, competition for antigen presenting cells, availability of the CD8 T-cell repertoire and other mechanisms that function largely by restricting the immune response. Here we elucidate a novel mechanism that increases the immunodominance of the epitope rather by enhancing the immune response. Using a peptide-specific MHC-restricted monoclonal antibody and functional assays of CTL activation, we show that T cells with high avidity for the immunodominant, H-2Dd-restricted, P18-I10 epitope expand rapidly following immunization, and this expansion in turn determines the level of the P18-I10 epitope immunodominance. This proliferation has little dependence on the number of MHC-peptide complexes. Since most self-reactive T-cells of high avidity are depleted in the thymus, the selection of immunodominant epitopes based on the expansion of high avidity T-cells in the periphery avoids the potential for autoimmunity.
Note on the Quantitative Relations of Antigen and Antibody in Agglutination and Precipitation Reactions
Authors: Zinsser Hans
Published: January 2023

One of the things that has puzzled bacteriologists and has been particularly difficult to harmonize with our conception of the unity of the sensitizers giving the different antibody reactions with one and the same antigen has been the fact that in doing agglutination reactions against bacteria it has been possible to dilute the serum often several thousand times before extinction of the reaction, whereas in doing precipitation reactions the antigen can be diluted several thousand times, but if the serum is diluted its reaction fades out, usually at dilutions of 1:10, rarely extending beyond dilutions of 1:50.

The explanation for this phenomenon seems relatively simple if one considers it from the point of view of the mechanics of flocculation. It is likely that bacterial agglutination by a specific antiserum takes place as follows: The antibody, which is inseparable from certain globulin fractions of the serum and, therefore,—physically considered—is globulin, coats the exposed particles, the coating taking place specifically by reason of chemical affinities between this antibody globulin and the antigen.
An intrinsic role for the alternative NF-kappa B pathway in the T-cell response to virus (35.21)
Authors: Rowe Alexander M , Murray Susan E. , Parker David C
Published: January 2023

Regulation of NF-κB mediated signaling is controlled by two systems: the classical pathway and the alternative pathway. The alternative pathway refers to the processing of p100 to p52 regulated by an enzyme called NF-κB inducing kinase (NIK), and is essential for the normal function of B-cells, macrophages, and dendritic cells; however, little is known about its function in T-cells. Preliminary experiments determined that activation of the alternative pathway is necessary for CD4 mediated graft versus host disease. Based on those findings, we hypothesized that the broader T-cell response to a viral infection is also dependent on the alternative NF-κB pathway. Because of the dominant effects of deficiencies in the alternative pathway on B-cells, macrophages and dendritic cells, we could not discretely assess the T-cell response to pathogen in alternative pathway mutants. Therefore, we generated mixed bone marrow (BM) chimeras using BM from wild-type mice and alymphoplasia (aly) mice that lack a functional NIK protein or BM from p100 knockout mice. In these chimeras, 15-20% of the T-cells, less than 2% of the B-cells and less than 2% surveyed myeloid cells, possessed the mutant genotype. This allowed us to assess the function of T-cells lacking the alternative pathway in an otherwise normal immune system. Chimeras were infected with lymphocytic choriomeningitis virus (LCMV) and the immune response was analyzed 8 days post-infection. The percent of responding CD8 and CD4 T-cells was significantly reduced among aly/aly and p100-/- populations compared to wild-type cells, as measured by the percent of IFNγ positive cells following ex vivo stimulation with LCMV peptides and activation marker expression. These data demonstrate a T-cell intrinsic role for the alternative NF-κB pathway in the immune response to a viral infection.
A single-chain MHC-class I system to investigate the role of non-stimulatory peptides in T-cell recognition (35.39)
Authors: Hoerter John , Ampudia Jeanette , Yachi Pia , Connolly Janet , Hansen Ted , Gould Keith , Gascoigne Nicholas
Published: January 2023

Recent work has demonstrated that endogenous or non-stimulatory peptides can enhance T-cell recognition, a finding that is unexplained by the classical model of CD8, TCR, and MHC interaction. To investigate this phenomenon, we have developed a tetracycline-inducible CHO cell system where cognate single-chain MHC-peptide is expressed at low levels and non-stimulatory single-chain MHC-peptide is constitutively expressed. Mutations in the CD8 binding site of stimulatory or non-stimulatory MHCp enable us to distinguish between different binding models in this system. Preliminary results indicate that CD8 binding to the non-stimulatory MHC is necessary for efficient activation.

Supported by NIH R01 GM065230
CD3 molecules in a conformational equilibrium that is altered by TCR engagement (35.41)
Authors: Gil Diana , Kruger Travis , de la Cruz Javier
Published: January 2023

Previously we and others observed that the signaling module of the TCR, the CD3 complex, undergoes a conformational change at the level of its cytoplasmic tails when T cells are stimulated. At that time, we interpreted this data as proof that antigen recognition modifies the conformation of CD3 as part of the receptor triggering mechanism. In absence of specific structural information, the induced conformation has been referred to as "open CD3". Here we present new evidence that thymocytes display detectable amounts of "open CD3" even in the absence of antigenic stimulation. However, treatment of thymocytes with stimulatory anti-CD3 antibodies significantly increases the amount of "open CD3". We conclude that ligand binding causes many CD3 molecules to adopt the "open CD3" conformation. We propose that CD3 is expressed in an equilibrium between two distinguishable conformations, which may reflect "on" and "off" states that are perturbed by TCR engagement. The accumulation of a certain level of "open CD3" might trigger T cell activation.

Research support: Diana Gil, Travis Kruger and Javier de la Cruz were supported with start-up funds from the Mayo Foundation.
Positive effects of the middle layer of Phyllostachys nigra var. henonis (PN), Bambusae Calulis in Taeniam, on asthma murine model through down-regulated ICAM-1 of BMMC (36.12)
Authors: Ra Jehyeon , Lee Sanggon , Ahn Hyunjong , Kim Jinju
Published: January 2023

The middle layer of Phyllostachys nigra var. henonis (PN), Bambusae Calulis in Taeniam, has been used for cough and sputum for a long time. Cough is one of the major symptoms of asthma. However, there were few of scientific investigation about PN. To investigate the positive effect of PN on asthma, we measured the production of IL-4 of bronchoalveolar lavage fluid (BalF), Immunoglobulin E (IgE) level of serum and immune cell infiltration at lung tissue by HE stain in ovalbumin (OVA) induced asthma murine model (Balb/C, female, 6 weeks). In asthma model, there was IL-4 of BalF decreased by PN, however, it was not significantly. The IgE induced by OVA in serum significantly decreased by PN (40 mg/kg). Furthermore, we observed the decrease of immune cell infiltration up-regulated by OVA in lung tissue. We focused on decrease of cell infiltration by PN. Intercellular adhesion molecule-1 (ICAM-1) and L-selectin regulate IgE production by controlling mast cell accumulation at sites of inflammation. And then we investigated the mRNA and protein levels of ICAM-1 and L-selectin in bone marrow mast cells (BMMC). BMMC were incubated with 10 ng/ml of TNF-alpha fnand IL-3 plus 1, 10 and 100 £gg/ml of PN for 4 hours. The mRNA and protein level were measeured by using RT-PCR and Fluorescence activated cell sorter (FACS). PN had down-regulated both mRNA and protein of ICAM-1, however, no difference at mRNA and protein of L-selectin in BMMC. Together all things, PN has anti allergy effects on asthma, based on down-regulation of ICAM-1.

Acknowledgements: This study was supported by a grant from Seoul R&BD Program, Republic of Korea.
Various fungi isolated from asthmatic children’s homes induce asthma like allergic lung disease in mice. (36.2)
Authors: Porter Paul C , Corry David , Delclos George L , Vaidya Swapnil , Kheradmand Farrah
Published: January 2023

Fungal exposure has been associated to allergic lung diseases (including asthma) for over a century. The current asthma model holds that fungal exposure acts merely as antigens triggering long developed pre-existing sensitivity. We challenge this assumption and demonstrate A. niger inhalation can lead to the development of asthma-like allergic lung disease at high doses and at lower doses could act as a Th2 adjuvant allowing develop of allergic responses to a secondary benign antigen. Demonstrating that one fungi could illicit a profound allergic response, we wanted to investigate the allergic lung responses of other fungi. 13 fungi were isolated from the homes of asthmatic children and used to intra-nasally challenge normal C57Bl/6 mice. Select fungi were chosen for dose-dependent analysis and fungal clearance. All fungi analyzed induced at least minor allergic lung disease however the levels varied widely. Fungal clearance appeared to decrease with an increase in conidia size. Our results suggest that the general response to fungal conidia inhalation is the development of allergic lung responses.
Dissecting the role of Tyk2 in IL-12 signaling (38.11)
Authors: Maxwell Sarah Sollid , Mozaffarian Afsaneh , Kugler David , Arnett Heather , Gabel Christopher
Published: January 2023

Tyrosine kinase 2 (Tyk2), a member of the Jak family of non-receptor associated kinases, has been implicated in transducing signals mediated by IL-12, IL-23 and IFNα. A strain of mice (B10.Q/J) bears a missense mutation in the pseudokinase domain of Tyk2 which renders the kinase nonfunctional. These mice were initially identified by their heightened resistance to collagen-induced arthritis (CIA) and impaired response to IL-12 and type I IFNs. Following in-house confirmation of disease resistance to CIA, B10.Q/J and wild-type (WT) mice were injected with PBS, IL-12, IL-18, or IL-12+IL-18. Sera were collected at 2 and 24 hours post-injection, and levels of various polypeptide mediators were determined by multi-analyte profiling and ELISA. Compared to WT mice, the absence of functional Tyk2 in the B10.Q/J mice lead to a decrease in circulating IFNγ following IL-12+IL-18 administration. Confirmation that IFNγ output was dependent on Jak involvement was established using the Jak antagonist CP-690,550. This inhibitor was orally administered to WT mice prior to injection of IL-12+IL-18. Sera and tissues collected 2 hours following cytokine injection demonstrated reduced levels of IFNγ and phosphorylated-STAT4. Our results suggest that antagonism of Tyk2 may provide therapeutic benefit in those diseases in which disregulation of cytokines contributes to disease development.
The impact of defective gp130/SOCS3 signaling on the astrocyte response to hyper-IL6 (38.17)
Authors: Frausto Ricardo F , Denyer Gareth , Scheller Jurgen , Rose-John Stefan , Jenkins Brendan J , Ernst Matthias , Campbell Iain L
Published: January 2023

Astrocytes derived from Y757F mutant mice defective in gp130-SHP2/SOCS3 signaling were investigated into their ability to respond to IL-6. Compared with WT astrocytes, Y757F astrocytes treated with hyper-IL6, had higher and more sustained activation of STAT3, while the levels of pY-SHP2 and pERK remained unchanged. Gene expression was investigated by Affymetrix gene chip analysis. At 2 hr, 306 genes were upregulated in WT astrocytes and of these, 28 did not increase in Y757F astrocytes. Of 238 genes upregulated in Y757F astrocytes, 9 were not upregulated in WT astrocytes. Some 99 genes were downregulated in WT astrocytes and of those 55 were not decreased in Y757F astrocytes. In WT astrocytes after 12 hrs the level of expression of many genes was reduced back to or near levels seen in the untreated cells, however, in Y757F astrocytes 109 genes either maintained their 2hr upregulated levels or were further increased. A number of candidate genes upregulated by hyper-IL6 in WT and Y757F astrocytes were not upregulated in astrocytes lacking STAT1. In conclusion, hyper-IL6 stimulated gp-130 SHP2/SOCS3 signaling in astrocytes not only moderates the gp-130-STAT pathway but also directly regulates the transcriptional activity of a number of genes many of which are STAT1-dependent. Support: NSW Spinal Cord Injury and Other Neurological Conditions Project Grant.
IL-15 Receptor alpha increases the stability and promotes secretion and bioactivity of the short signal peptide variant of IL-15 (38.3)
Authors: Bergamaschi Cristina , Jalah Rashmi , Kulkarni Viraj , Rosati Margherita , Valentin Antonio , Zhang Gen-mu , Alicea Candido , Zolotukhin Andrei S. , Felber Barbara K. , Pavlakis George N.
Published: January 2023

Two isoforms of interleukin-15 (IL-15) are known, containing a long signal peptide (LSP) or a short signal peptide (SSP), respectively. The two forms are produced by alternatively spliced mRNAs. It has been proposed that SSP IL-15 remains exclusively intracellular and its function is not known. We show that, similar to LSP IL-15, SSP IL-15 is stabilized and secreted efficiently upon coexpression of IL-15 Receptor alpha (IL-15Ra). Co-injection of mice with SSP IL-15 and IL-15Ra expressing plasmids showed increased plasma levels of bioactive IL-15 and mobilization and expansion of NK and T cells. Therefore, SSP IL-15 is secreted and bioactive when produced as a heterodimer with IL-15Ra in the same cell, though its apparent stability is lower compared to LSP IL-15, due to different intracellular processing. When both LSP IL-15 and SSP IL-15 are produced in the same cell they compete for the binding to IL-15Ra, and result in lower levels of bioactive IL-15. Therefore, co-expressed SSP IL-15 acts as competitive inhibitor of LSP IL-15. This suggests that usage of alternative splicing is an additional level of control of IL-15 activity. Expression of both SSP and LSP forms of IL-15 appears to be conserved in many mammals, suggesting that SSP may be important for expressing a form of IL-15 with lower magnitude and duration of biological effects.

These results support and expand our conclusions (Bergamaschi, J Biol Chem. 2008; 283:4189) that IL-15Ra needs to be co-expressed in the same cell with IL-15 for biological activity, and that IL-15Ra should be considered a part of the heterodimeric IL-15 cytokine, rather than part of the receptor.
Investigating the functional biology of IL-25 during helminth infection (38.6)
Authors: Saenz Steven A , Zaph Colby , Mohrs Markus , Budelsky Alison , Artis David
Published: January 2023

Recent studies have demonstrated key functions of interleukin (IL) -25 in TH2 cytokine-mediated host protective immunity and exacerbation of allergic airway inflammation, however the cell lineages targeted by IL-25 to elicit such responses remains unclear. We show that IL-25 acts through an innate cell mechanism to drive TH2 cytokine responses. Utilizing IL-4/eGFP reporter mice that express green fluorescent protein (GFP) under the IL-4 loci, we demonstrated that IL-25 specifically induced a c-kit+ cell population in the mesenteric lymph nodes. This population was also present in the Peyer's patches, cecal patch, and peritoneum of treated mice. Despite displaying a cell morphology similar to classical mast cells, the IL-25-induced c-kit+ cell population lacked expression of FcεR1 and surface bound IgE. Additionally, IL-25 elicited a TH2 cytokine response and induced this c-kit+ cell population independently of IL-3 and SCF signaling, pathways known to regulate mast cell development. Furthermore, we demonstrated that in mice deficient in the receptor for IL-25 or IL-17A, IL-17Rb and IL-17Ra respectively, the IL-25-mediated induction of this c-kit+ population was lost. Thus, these data suggest that IL-25 acts through a mast-cell independent mechanism to promote TH2 cytokine responses, which is dependent on both IL-17Ra and IL-17Rb.
Contrasting roles for IL-22 in DSS-induced colitis (38.7)
Authors: Smith Carole L , Rottman James B , Maxwell Joseph R
Published: January 2023

Interleukin (IL)-22 is a multifunctional cytokine with diverse biological activities which include involvement in the inflammatory response, tissue repair and pathogen defense. Recently, IL-22 was shown to be protective in two models of inflammatory bowel disease (IBD) in mice. In order to confirm those results and develop a reagent that could serve as a therapy, we generated IL-22-Fc which had an increased half-life relative to recombinant IL-22 (rIL-22). The effects of rIL-22 versus IL-22-Fc were compared in dextran sulphate sodium (DSS)-induced colitis. Prophylactic, but not therapeutic, administration of either rIL-22 or IL-22-Fc prevented disease and was significantly more efficacious than granulocyte macrophage colony stimulating factor (GM-CSF), a cytokine that has been used clinically in human IBD patients. The mechanism of the protective effect of IL-22 was in part driven by reduced circulating levels of inflammatory cytokines and enhanced proliferation/regeneration of the colonic epithelium. However, IL-22-Fc exacerbated weight loss and induced anemia. Thus, although prophylactic IL-22R stimulation protected animals from DSS-induced colitis, metabolic effects such as iron deficiency and weight loss complicate the benefit received. These data demonstrate that IL-22 can simultaneously play beneficial and detrimental roles in inflammatory diseases.
Lung Th17 inflammatory responses augment polymeric immunoglobulin receptor expression by the airway epithelium and promote B cell recruitment and increase in secretory IgA levels (39.10)
Authors: Jaffar Zeina , Ferrini Maria , Herritt Lou A. , Roberts Kevan
Published: January 2023

Polymeric immunoglobulin receptor (pIgR) is a central player in mucosal immunity that mediates delivery of polymeric IgA and IgM to the apical surface via transcytosis across the type I mucosal epithelium. Emerging evidence suggests that Th17 cells, characterized by their production of IL-17, not only mediate autoimmunity but also play key roles in mucosal host defense against extracellular microbes. We demonstrate that adoptive transfer of OVA-specific CD4+ Th17 cells into BALB/c mice, in addition to causing neutrophilic inflammation, mediated a pronounced influx of CD19+ B cells into the lungs following OVA inhalation. Coincident with the cellular recruitment, were the striking elevation in pIgR expression by the airway epithelium and the subsequent increase in IgM and secretory IgA levels. Intranasal administration of IL-17 revealed a key role for this cytokine in the upregulation of pIgR expression by the epithelium. Lung mucosal Th17 responses were markedly regulated by the transfer of antigen-specific CD4+CD25+Foxp3+ regulatory T cells whose suppressive function was restricted to the site of inflammation. These findings support a key role for IL-17-producing Th17 cells in mediating pulmonary immune defense against respiratory pathogens by promoting the expression of pIgR and subsequent transport of mucosal IgA and IgM into the airway lumen.
The impact of obesity on intestinal γδ T cell function (166.17)
Authors: Cheung Kitty , Jameson Julie
Published: January 2023

Populations of people struggling with obesity are reaching critical levels, spanning across the age and economic spectrum. Deterioration of the gut has been implicated in the exacerbation of obesity and metabolic diseases due to its role as the major tissue responsible for the absorption of nutrients. γδ T cells serve an important role in intestinal maintenance by repairing the gut epithelium through production of keratinocyte growth factor and regulating inflammation. We want to investigate the role of γδ IELs in the gut homeostasis and how their functions are altered due to obesity and chronic inflammation. Various obesity models were utilized including leptin receptor deficient mice (db/db), mice fed on a high fat diet (HFD), and γδ T cell deficient mice. Both FACs analysis and CD3 immunofluorescent staining show an overall decrease in T cell numbers in the gut of HFD mice. Surprisingly, αβ and γδ T cell populations are differentially susceptible to the effects of obesity. Through BrdU incorporation, we found that γδ T cells are better able to maintain homeostatic proliferation than αβ T cells. Phenotypic analysis of these IEL subsets may hint at further differences. These data confirm that obesity adversely affects T cell populations in the gut. We are currently investigating how IEL function is impacted by the chronic inflammatory environment and how dysfunctional IELs exacerbate the diabetic condition.
Novel method for the development of regulatory T cells (166.22)
Authors: Ebeling Cory , Ilarraza Ramses , Adamko Darryl
Published: January 2023

Background: Regulatory T cells (Treg) are important in the inhibition of inflammatory disorders, like asthma. Unfortunately, current protocols used to develop them in vitro for study rely on complicated cloning methods that may not represent biologically relevant Treg. LPS-free ovalbumin (OVA) given intranasal (i.n.) can induce mucosal tolerance in mice. Therefore, we hypothesized that Dendritic cells (DCs) taken from the draining lymph nodes (LNs) of mice given LPS-free OVA would induce Treg in vitro. Methods: Mice were given i.n. LPS-free OVA. 24 h later cervical and bronchial LNs were excised and DCs were isolated (~98% purity). DCs were cultured in vitro with transgenic OVA-specific T cells for four days. The development of Treg was assessed by flowcytometry (Foxp3 expression), ELISA (IL-10 production), and inhibition of T cell proliferation (CFSE staining/BrdU uptake). Finally, we studied the effectiveness of these cells to inhibit the development of AHR in a mouse model of asthma. Results: T cells from mice treated with LPS-free OVA showed high levels of Foxp3 expression, high IL-10 production, and the ability to inhibit OVA specific T cells in vitro. compared to control mice. Furthermore, T cells from LPS-free OVA treated mice prevented the development of AHR when given to mice before OVA challenge (i.n.). Conclusion: We believe our protocol could represent an alternative method to develop Treg cells for study.
Unique pathways of purinergic P2X7 receptor-mediated colonic mast cell activation in the intestinal inflammation (166.9)
Authors: Kurashima Yosuke , Amiya Takeaki , Kunisawa Jun , Kiyono Hiroshi
Published: January 2023

Accumulating evidence has demonstrated that purinergic signals by ATP and ADP induced the activation of immune cells through a wide variety of purinergic receptors and purine-metabolic ecto-enzymes. Here we show the P2X7 receptor-mediated activation of colonic mast cells in the induction of mast cell-mediated intestinal inflammation. Intestinal inflammation was ameliorated in MC-deficient KitW-sh/W-sh mice, and reconstitution with wild-type, but not P2x7-/-, MCs resulted in recovered susceptibility to inflammation. Intriguingly, colonic mast cells expressed both ATP-reactive P2X7 receptor and ADP-reactive P2Y receptors, but inhibition of P2Y receptors did not suppress ADP-mediated mast cell activation. Besides, inhibition of P2X7 receptor as well as adenylate kinase, an ecto-enzyme to convert ADP to ATP, reduced ADP-induced mast cell activation. These results suggest that mast cells have potential to produce ATP from ADP by adenylate kinase to enhance P2X7 receptor-mediated inflammatory response in the gut.
Implications of gut bacteria/dendritic cells interactions in development of Th17 lymphocytes and Type 1 Diabetes resistance (167.12)
Authors: Lau Kenneth , Ardissone Alexandria , Wilson Tenisha , Collins Erin , Lorca Graciela , Wasserfall Clive , Neu Josef , Atkinson Mark , Shatz Desmond , Triplett Eric , Larkin, III Joseph
Published: January 2023

Recent literature has suggested that Type 1 Diabetes (T1D) onset is negatively correlated to the presence of TH17 cells. Consistent with this notion, we observed that total lymph nodes isolated from NOD mice (which spontaneously develop T1D) produced lower levels of IL17 in vitro than from diabetes resistant NOR or C57BL/6 mice. Significantly, increasing evidence shows that gut bacteria are vital in the proper development of TH17 cells. Oral feedings of Lactobacillus johnsonii (LjN6.2), a strain isolated from the gut, has been shown to mediate T1D resistance and a TH17 bias. We used LjN6.2 to stimulate diabetes prone and resistant strains, measuring cytokine production and cell populations via flow cytometry. When equal numbers of T cells and antigen presenting cells (APCs) were incubated with LjN6.2, it was observed that NOD T cells were capable of producing abundant IL17. Because APCs in NOD mice are reduced in number and defective, we next analyzed whether this IL17 production was due to restored APC function by LjN6.2. APCs responded to LjN6.2 by producing extensive IL6 and upregulated MHCI, CD86, and CD80. We next tested if a footpad injected vaccine of LjN6.2 pulsed BMDCs could cause a TH17 bias in vitro. Indeed, splenocytes isolated from treated NOD mice showed increased production of IL6 and IL17. These studies present a possible role for gut flora in biasing T lymphocyte effector functions away from autoimmune diseases like T1D through dendritic cell modulation.