bluuebunny/crossref_metadata_2025 huggingface dataset:
Tolerogenic Vaccination Using IL-12 Gene-Silenced Dendritic Cells for Autoimmune Arthritis (131.9)
Authors: Min Wei-Ping , Zheng Xiufen , Popov Igor , Li Mu , Zhang Xusheng , Sun Hongtao , Vladau Costin , Suzuki Motohiko , Inman Robert D , Garcia Bertha
Published: January 2023

We have recently demonstrated that dendritic cell (DC)-mediated immune modulation and deviation can be accomplished through RNA interference (RNAi), highlighting the therapeutic potential of RNAi-modified DC as antigen-specific tolerogenic vaccines. To date, an RNAi-based vaccine has not been reported. The current study was designed to develop siRNA-modified DC as antigen-specific, tolerogenic vaccines for prevention and intervention of autoimmune arthritis. Using small interfering RNA (siRNA) that specifically targets IL-12p35 gene (IL-12 siRNA), we have generated a type of DC that exhibits multiple tolerogenic characteristics. Immunization with type II collagen (CII)-pulsed and IL-12 gene-silenced DC (CII-pulsed/gene-silenced DC) resulted in antigen-specific nonresponsiveness in T cell responses. Vaccination with CII-pulsed/gene-silenced DC prevented collagen-induced arthritis (CIA) onset in a murine rheumatoid arthritis model. Furthermore, administration of CII-pulsed/gene-silenced DC was sufficient to inhibit progression of CIA. The therapeutic effects were further evidenced by decreased clinical scores, inhibited inflammatory infiltrates, and suppressed T cell and B cell responses to CII. In conclusion, this study is the first to demonstrate the therapeutic utilization of RNAi-modified DC as antigen-specific tolerogenic vaccines for autoimmune arthritis.
Simultaneous Detection and Quantitation of Multiple Antigen Expression in Tissue by Fluorescence Microscopy and Hyper-spectral Imaging (132.4)
Authors: Carpenter Anne , Chatigny Julie , LeFever Mark , Ashworth-Sharpe Julia , Haubein Ned , Vanpatten Kristie , Kosmeder Jerry , Lett Scott , Grogan Thomas , Bieniarz Chris , Miller Philip , Pestano Gary
Published: January 2023

Analyses of biomarkers in tissues represent a critical component in the development of therapeutics. In drug development, entire signaling pathways are profiled with a variety of high-throughput techniques. However, in the field of histology, tissue-based diagnostics are generally dominated by single- or dual-analyte tests. In addition, manual methods for slide processing promote inconsistency. We have developed automated instrumentation for conducting high-throughput IHC and ISH in widely available formalin-fixed paraffin-embedded samples. In this report we describe the utilization of Quantum dots (Qdots®) in advanced antigen detection in intact tissues. Specifically, we developed a novel set of hapten and anti-hapten monoclonal antibodies for use in multi-analyte assays. Spectral imaging software was utilized to deconvolute images generated by fluorescence microscopy on tissue samples. In model systems, we demonstrate simultaneous detection, and quantitation of expression levels and cell numbers, for markers including CD20, CD34, CD45, IgGκ, IgGλ, and Ki67. These reagents provide a growing set of tools that could complement early marker validation. The examples in this study demonstrate the utility of coupling high-throughput, automated histochemistry with quantifiable, multiplexed analyte detection. These findings are reported for individual cells in their anatomic context.
KIR/HLA Compound Genotypes Determine Early Antiviral Response of Natural Killer Cells (35.17)
Authors: Ahlenstiel Golo , Martin Maureen P , Gao Xiaojiang , Carrington Mary , Rehermann Barbara
Published: January 2023

A growing number of genetic epidemiological studies have suggested an influence of the extraordinarily polymorphic KIR/HLA compound genotypes in determining outcome to viral infections, but functional data to explain these genetic data have not been reported to date. Using an in vitro influenza A virus infection model, we here attribute functional differences in human NK cell activity to distinct KIR/HLA genotypes. Using cytokine EIAs and multicolor flow cytometry, we show that NK cells from KIR2DL3+/KIR2DL1+ and HLA-C1 homozygous subjects display a faster response kinetics and release more IFN-γ and TNF-α faster to influenza A virus infection than NK cells from KIR2DL3+/KIR2DL1+ and HLA-C2 homozygous subjects. Subanalysis revealed that the HLA-C-regulated, i.e. KIR2DL3+ NK cell subpopulation of HLA-C1 homozygous subjects responded more rapidly than their HLA-C-regulated, i.e. KIR2DL1+ counterpart in HLA-C2 homozygous subjects. This differential functional response was independent of Bw4/KIR3DL1 interactions. It was also not due to intrinsic difference between NK cell of HLA-C1 and HLA-C2 homozygous subjects, as demonstrated by equal cytotoxicity in the absence of HLA-C. In summary, these results provide first functional evidence for the epidemiological associations between specific KIR/HLA compound genotypes and protection against viral infection observed at the population level.
IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in multiple myeloma cells (35.20)
Authors: Piskurich Janet F. , Zhao Mojun , Flynt Frederick L. , Hong Mei , Chen Han , Gilbert Carolyn A , Briley Nicole T
Published: January 2023

MHC Class II transactivator (CIITA) is the master transcriptional regulator of MHC class II (MHC II) genes. MHC II expression is central to antigen presentation and the immune recognition of tumor cells. The human CIITA gene, MHC2TA, is regulated in a cell-type specific manner by multiple promoters. Regulation by IFN-γ is generally via the type IV promoter (pIV) and involves the binding of IFN regulatory factor (IRF)-1 and IRF-2. The response of pIV to IFN-γ was examined in myeloma cells. Western blot and RT-PCR show that STAT1 is activated by phosphorylation and IRF-1 RNA expression increases in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up-regulates the activity of MHC2TA pIV as does ectopic expression of IRF-1 or IRF-2, and the IRF-E site is required. Binding studies confirm IRF-1 and IRF-2 binding to MHC2TA pIV. Myeloma cells express positive regulatory domain I-binding factor 1 (PRDI-BF1), also called B lymphocyte-induced maturation protein-1 (Blimp-1), which represses both MHC2TA pIII and pIV. Importantly, these cells retain the ability to up-regulate CIITA pIV and MHC II in response to IFN-γ. Although PRDI-BF1/Blimp-1 diminishes the ability of myeloma cells to present antigen by limiting CIITA and MHC II expression, these studies indicate that expression can be restored by the use of cytokines like IFN-γ.

Research support: RO1CA10220, RO1CA114504, MEDCEN Foundation of Central GA
The role of the 19S ATPase S6a in the transcriptional regulation of major histocompatibility class II (MHC II) genes (35.26)
Authors: Truax Agnieszka Dorota , Greer Susanna F
Published: January 2023

MHC II molecules are glycoproteins that present intracellular antigens to CD4+ T cells and play an important role in induction and regulation of adaptive immune responses. MHC II molecules are regulated at the level of transcription by a master regulator, the class II transcriptional activator, CIITA, whose association with the MHC promoter is necessary for initiation of transcription. It is well established that one mechanism of regulating transcription is through degradation of factors by the 26S proteasome. The proteasome is composed of a 19S regulatory particle that recognizes ubiquitinated proteins and a 20S proteolytic core where the tagged proteins are degraded. Previous studies in yeast have demonstrated that the S6a ATPase of the 19S associates with actively transcribed genes, suggesting a non-degradative role for the ubiquitin-proteasome machinery. To further understand these roles in mammalian cells, we have investigated the role of S6a in regulating CIITA mediated MHC II transcription. Our research indicates that S6a is recruited to actively transcribing MHC II and CIITA genes and that RNAi mediated S6a knockdown correlates with decreased expression of MHC II and CIITA. Our study demonstrates a novel mechanism of regulating MHC II expression and will enhance our understanding of mammalian transcription.

Research supported by the NMSS, the Georgia Cancer Coalition and GSU.
Genomic analysis of RAE-1 genes (35.30)
Authors: OGASAWARA KOUETSU , Ishizaki Kazusa , Fujiwara Naruyoshi , Sasazuki Takehiko
Published: January 2023

NKG2D is an activating receptor on NK cells and activated CD8 T cells that has been implicated in tumor immunity, viral immunity and autoimmunity. The ligands of NKG2D are tightly regulated stress-inducible molecules, whose expression is triggered by transformation or infection. In mice, these NKG2D ligands include the retinoic acid early inducible-1 (RAE-1) proteins. However, the mechanisms of transcriptional regulation in RAE-1 genes are poorly understood. We examined what transcription factors regulate RAE-1 expression. We found that RAE-1 delta has six exons and RAE-1 epsilon has seven exons. We isolated genomic DNAs of upstream from exon one which are thought of RAE-1 promoters in RAE-1 delta or RAE-1 epsilon. We found that consensus sequences of several transcription factors are present in RAE promoters. Therefore, these transcription factors may regulate RAE-1 expressions.

This work is supported by Grants-in Aid for Science Research from the Ministries of Education, Culture, Sports, Science, and Technology of Japan, Grants-in Aid for Science Research from the Ministriesof Health, Labor, and Welfare of Japan, Human Frontier Science Program CDA, The Mitsubishi Foundation, The Sumitomo Foundation, Takeda Science Foundation and Uehara Memorial Foundation.
Loading and phosphorylation of RNA polymerase II influence production of secretory-specific Ig heavy chain mRNA in plasma cells. (35.33)
Authors: Milcarek Christine , Shell Scott
Published: January 2023

A crucial step in the development of B cells into plasma cells is the increased production of secreted immunoglobulin which occurs by differential selection of the secretory-specific polyA site. Factors involved in polyadenylation can be coupled with transcription through phosphorylation of the carboxyl-terminal domain (CTD) of RNA pol II. We analyzed loading and phosphorylation patterns of RNA Polymerase II along an identical immunoglobulin heavy chain gene expressed either in B- or plasma cells by chromatin immunoprecipitation using various antibodies. Data indicate that the switch to secreted immunoglobulin heavy chain mRNA production is influenced by increased RNA Polymerase II accumulation and CTD phosphorylation at the promoter in plasma cells. Loading of transcription factors is increased in the plasma cells. This allows for increased access of specific processing factors to the polymerase and nascent RNA.

Supported by NIH-CA-86433 to C.M.
FOXP3 COOPERATES WITH LINKER HISTONE H1b TO REPRESS IL-2 (35.46)
Authors: Mackey-Cushman Stephanie , Su Lishan
Published: January 2023

Regulatory T cells (Treg) are a subset of T cells indispensable for regulating self tolerance by actively suppressing autoreactive T cells. Treg cells are characterized as CD4+CD25hi, which preferentially express the transcription factor FoxP3. FoxP3 is required for Treg lineage development. Ectopic expression of FoxP3 converts CD4+CD25− T cells to Treg cells with suppressed IL2 expression, and mutations within the FoxP3 gene lead to autoimmune diseases in humans and mice. In order to understand the molecular mechanism by which FoxP3 functions in Tregs, we performed a large scale co-immunoprecipitation of FoxP3 followed by mass spectrometry identification of interacting proteins. We identified a physical interaction between FoxP3 and H1b, a specific isoform of histone H1. The leucine zipper region of FoxP3 is required for the interaction with H1b. This interaction is specific to H1b, while other H1 isoforms, H1a and H1e, do not interact with FoxP3. Furthermore, ectopic expression of FoxP3 and H1b in Jurkat T cells synergistically represses IL-2 expression following activation. These findings support a role for H1b in FoxP3-mediated repression of IL-2 expression in Treg cells.
Regulation of the Perforin Locus by IL-2 During CD8+ T-cell Differentiation (35.49)
Authors: Pipkin Matthew Eugene , Cruz-Guilloty Fernando , Lichtenheld Mathias Georg , Rao Anjana
Published: January 2023

The molecular basis of memory and effector CD8+ T cell differentiation is not well understood. Here, we show that the strength of IL-2 signaling determines the transcriptional profile of primary mouse cytotoxic T lymphocytes (CTL) during in vitro differentiation. CD8+ T cells were activated and cultured in either high or low dose IL-2 for 1 week; both groups proliferated equivalently and expressed similar amounts of IFNγ upon restimulation. However, cells cultured in high IL-2 maintained phosphorylated Stat5 throughout differentiation, induced the transcription factor Eomesodermin, expressed robust amounts of perforin and granzyme B and displayed a surface phenotype characteristic of effector cells. In contrast, cells cultured in low IL-2 expressed low amounts of granzyme B, reverted to a surface phenotype characteristic of memory cells and gradually silenced expression of perforin mRNA. DNase I hypersensitivity mapping showed no major differences in chromatin structure in >200 kb surrounding the perforin gene, whereas chromatin immunoprecipitation assays showed that cells cultured in high IL-2 displayed increased density of RNA polymerase II at the perforin promoter. Based on these studies in vitro, we propose that the strength of signaling through the IL-2 receptor regulates commitment to the memory cell lineage in vivo.
Single Nucleotide Variations Between the I gamma 1 and I gamma 4 Promoters Result in Biased Recruitment of Transcription Factors in Response to CD40 Signaling (35.7)
Authors: Sinquett Frank L , Dryer Rebecca L , Covey Lori R
Published: January 2023

IgG subclass expression is highly biased in human IgM+ B cells activated with CD40L and IL-4. This bias is clearly reflected at the level of germline (Iγ) transcription despite extensive homology throughout the four Iγ promoters. Our current aim is to define the underlying mechanism by which Iγ1 transcripts are preferentially expressed relative to Iγ4 transcripts in order to extend our understanding of CD40 mediated humoral responses. Characterization of both promoters using defined regions of the Iγ1 and Iγ4 promoters revealed a marked decrease in Iγ4 transcriptional activity when compared to Iγ1 activity despite similar regulation. Expression studies have further shown that exchanging a previously defined 36 bp region of the Iγ1 promoter that is essential for Iγ1 transcription and binds NF-κB and various co-activators, can elevate Iγ4 transcription. This increase can be attributed to single nucleotide changes in either a putative CREB site or a putative NF-κB site within this region that renders this element CD40 inducible, and increases recruitment of p50, p65, c-Rel, CREB, and p300. Together, these data indicate that a single base pair change in a key regulatory region can contribute to differences in promoter strength. Competition assays and site-directed mutagenesis are currently in progress to further our understanding of the effect of these variances on Iγ transcription targeting.

Supported by AI37081
Role of aryl hydrocarbon receptor activation in antigen-presenting cell functions that result in diminished clonal expansion and differentiation of influenza virus-specific CD8+ T cells (36.27)
Authors: Jin Guangbi , Neumiller Joshua J. , Lawrence B. Paige
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is expressed in cells of the immune system. The AhR binds many different types of exogenous compounds, including pollutants and phytochemicals. The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is the most potent AhR agonist known, and is a well known immune suppressant. However, the precise molecular mechanism remains unclear. We have previously shown that AhR activation by dioxin suppresses the proliferation and differentiation of influenza virus-specific CD8+ T cells by disrupting events within bone marrow-derived cells important for the activation of naïve CD8+ T cells. However, TCDD does not affect CD8+ T cell directly, suggesting that defects in other cells essential for activating CD8+ T cells are affected by AhR activation. In this study, we show that the suppression of influenza virus-specific CD8+ T cell responses by TCDD appears to be independent of CD4+ cells, suggesting that deregulated APC function underlies TCDD-mediated decreasing the clonal expansion of virus-specific CD8+ T cells. To test this, we characterized the phenotype of DC present within lung, and examined their migration and function in secondary lymphoid tissue.

Funded by NIEHS Grants R01ES10619 and K02-ES012409 to BPL.
Fetal/neonatal ethanol exposure irreversibly alters immune parameters in young mice (36.28)
Authors: Fan Ji , Young Betty M , Cook Robert T , Edsen Michelle R , Legge Kevin L , Waldschmidt Thomas J , Tygrett Lorraine T , Schlueter Annette J
Published: January 2023

Chronic ethanol ingestion has been shown to impair immune defensive function of adult hosts to invading organisms. Children born to alcoholic mothers are known to have increased incidence and severity of infections, but EtOHÕs effect on the immune system in the fetus/neonate has not been well studied. We have developed a model of fetal/neonatal EtOH exposure in which female mice are maintained on 10% EtOH in the drinking water during breeding and gestation, and then on 12% EtOH during lactation. Pups have successfully been weaned and showed no significant difference in weights from controls through 42 days of age. These pups have been studied at 20 d (weaning) and 42 d (adolescence) for immunological cell numbers and function. Our data showed decreased splenic T and B cells in EtOH pups, but increased pulmonary leukocyte and dendritic cell (DC) numbers. Splenic DC and epidermal Langerhans cells numbers did not change. However, EtOH decreased baseline costimulatory molecule expression on splenic DC and the ability of these DC to stimulate allogeneic T cells, changes that persisted following cessation of EtOH exposure. These results indicate that mice born to EtOH-consuming mothers have immune deficiencies that are persistent following cessation of EtOH exposure. The dissection of T, B cell and DC dysfunction in EtOH pups is in progress.
Apoptosis-induced inhibition of CD1d-mediated antigen presentation: Different roles for caspases and p38 MAPK (36.4)
Authors: Khan Masood Alam , Sriram Venkataraman , Gourpura Renukaradhya J , Nagala Manjula , Brutkiewicz Randy R
Published: January 2023

The cross presentation of antigens derived from apoptotic cells enhances MHC class I or II-mediated antigen presentation. In the current study, we show that presentation of lipid antigens by CD1d molecules is inhibited by the induction of apoptosis in a p38 MAPK- or caspase-dependent manner. Treatment of murine CD1d1–expressing fibroblasts with PPMP or camptothecin (both of which can induce apoptosis) resulted in the activation of p38, and a p38-specific inhibitor (but not ERK1/2 pathway inhibitor) rescued CD1d-mediated antigen presentation in PPMP- or camptothecin-treated cells. The pan-caspase inhibitor Z-VAD-fmk rescued antigen presentation by CD1d in camptothecin (but not PPMP)-treated cells. Confocal microscopic analysis showed an altered intracellular distribution of CD1d in PPMP- or camptothecin-treated cells, which was corrected by the addition of p38 or caspase inhibitors. These results suggest that the induction of apoptosis compromises with CD1d-mediated antigen presentation.

This work was supported by NIH grant PO1 AI056097 to RRB.
Role of H1 receptors in the regulation of IL-4 production (37.16)
Authors: Khan Manzoor M. , Kharmate Geetanjali , Liu Zhongfeng
Published: January 2023

Histamine alters the Th1/Th2 cytokine balance towards the Th2 cytokine profile and consequently play a role in allergic disease and asthma. This study was designed to determine if the effects of histamine on Signal Transducer and Activator of Transcription(STAT)-6 were direct or mediated via release of IL-4 production.The splenocytes from IL-4 knockout C57Bl/6 mice were pretreated with antibody to IL-13 for 30 minutes followed by treatment with histamine. The cells were then stimulated with PMA + ininomycin for 6 hours under standard cell culture conditions. For the control experiments splenocytes from the wild type C57BL/6 mice were used. The cells were lyzed and the total basal and phosphorylated STAT6 were analyzed using the Western Blot Analysis. Furthermore, splenocytes from C57Bl/6 mice were pretreated with antibodies to IL-4 and IL-13 followed by treatment with histamine and PMA + ionomycin and Western blot analysis were performed to determine the phosphorylated Stat6 levels.In the control group histamine caused hyper-phosphorylation of Y641 tyrosine residues of STAT6. These effects were not observed when splenocytes from the IL-4 knockout mice were used or the wildtype splenocytes were pretreated with antibodies to IL-4 and IL-13. These observations suggest that histamine indirectly affected the heper-phosphorylation of STAT6 via its effects on the secretion of IL-4 and H1 receptors played a role in this process, since H1 receptor antagonists blocked these effects. The clinical significance of STAT6 hyper-phosphorylation in the exacerbation of asthma symptoms has not yet been determined.
On Intrinsic Allergenicity. Using Lipocalins To Probe The Mechanisms Of Allergy. (37.20)
Authors: Logdberg Lennart Erik , Akerstrom Bo , Hair Gregory A , Allhorn Maria , Vikulina Tatyana , Kirshenbaum Arnold S , Sundstrom J Bruce
Published: January 2023

What makes an antigen an allergen? It seems obvious that IgE-epitopes of allergens are necessary but not sufficient to cause initial sensitization and further to postulate that allergens contain separate molecular motifs that allow interaction with functionally important non-IgE molecules of the key cellular effectors. This way they actively influence development of the hypersensitive phenotype through “intrinsic allergenicity” of the allergen molecules.. We propose that comparative studies of lipocalin allergens, a collection of distinct, but structurally and functionally homologous molecules from different species, may shed light on the property of intrinsic allergenicity. Our overall hypothesis is that xenogeneic lipocalins, via interaction with components of the innate human immune system, such as mast cells, dendritic cells, lymphocytes, or progenitors or secreted mediators of these cells, can promote the afferent and/or the efferent phase of the allergic response, stimulating development of IgE-, rather than IgG-responses to their major immunogenic epitopes. Using human cultured mast cells, we have shown that these cells express the lipocalin receptor megalin, that megalin is massively induced by cultivating the mast cells with monomeric IgE, and. report on further investigations into its role in development of IgE-dependent lipocalin allergy.
Effect of IL-4 and IL-13 on the chemotaxis of mouse eosinophils (38.2)
Authors: Heller Nicola , Gwinn William M. , Lee James J. , Constant Stephanie L. , Keegan Achsah D.
Published: January 2023

Eosinophils are a major pathological component of the inflammatory cell infiltrate found in lungs as a result of allergic disease. They migrate into lungs in response to a variety of chemokines. The development and effector phases of the allergic response are dependent on the actions of interleukin (IL)-4 and IL-13 on a wide variety of cell types. However, how these two cytokines directly affect eosinophil signaling, function and chemotaxis is largely unknown. Using mouse eosinophils purified from IL-5 transgenic mice and a transwell migration assay, eotaxin-1 (30nM) induced eosinophil migration 6–11x over background. IL-4 alone was able to induce eosinophil migration 2x over background. More eosinophils migrated in response to eotaxin when IL-14 or IL-13 was also placed in the bottom chamber. IL-4 appeared to be more potent than IL-13. This effect was dependent upon cytokine concentration. Interestingly, when eosinophils were pretreated with IL-4 for 30 minutes prior to chemotaxis, migration to eotaxin-1 was enhanced 4-fold. However, pretreatment with IL-13 did not enhance chemotaxis to eotaxin-1. These data suggest that IL-4 and IL-13 have direct and rapid effects on the chemotactic function of eosinophils with some differences. We are investigating the mechanisms of these effects, as well as other signaling and functional responses of eosinophils to these cytokines. (AI38985 and HLT32007698)
Insights into mast cell signaling from rigid trivalent DNA-based ligands (38.5)
Authors: Sil Dwaipayan , Lee Jong B. , Luo Dan , Holowka David , Baird Barbara
Published: January 2023

Antigen-mediated crosslinking of IgE bound to its receptor, FcεRI, stimulates degranulation and other responses in mast cells and basophils to initiate allergic responses. To investigate possible roles for structural constraints of crosslinked receptors on signaling, we utilized double-stranded DNA spacers to synthesize ligands with tunable lengths and valencies. We produced and characterized a series of symmetric trivalent ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in range of 4.5–13.5 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all crosslink IgE receptor complexes effectively. They initiate length-dependent tyrosine phosphorylation of FcεRI β and γ subunits and the adaptor protein LAT that are ~5–10-fold less for the longest ligand compared to the shortest. Receptor-mediated Ca+2 responses exhibit different kinetics and magnitudes as a function of ligand length, and degranulation responses parallel differences in these Ca+2 responses and in FcεRI tyrosine phosphorylation. In contrast, tyrosine phosphorylation of phospholipase Cγ1 and Ca2+ release from stores are relatively length independent, indicating differences in signaling pathways important for Ca+2 influx and release from stores. The length dependence of FcεRI phosphorylation that we observe is consistent with a transphosphorylation model of receptor activation leading to degranulation, whereas length-independent activation of PLCγ1 reveals unanticipated divergence from conventional signaling pathways for this process.
Regional increases of CD103+ Tregs corresponds with resolution of murine allergic airway disease (39.10)
Authors: McNamara Jeffrey Thomas , Carson William F , Guernsey Linda A , Secor Eric R , Singh Anurag , Schramm Craig M , Thrall Roger S
Published: January 2023

Mice sensitized to ovalbumin (OVA) develop allergic airway disease (AAD) with short-term OVA aerosol challenge (7 days); however, airway inflammation resolves and local inhalational tolerance (LIT) results with long-term continuous aerosol challenge (42 days). We have previously seen that regulatory T cell (Treg) populations that are CD4+CD25+Foxp3+ increase locally in the lung at AAD and expand further at LIT. A recent role for CD4+CD25+Foxp3+ CD103+ Tregs (CD103+Tregs) has been defined in mucosal models of inflammation; this subset of Treg cells has been demonstrated to have greater functionality in suppressing inflammation. The current study was designed to determine if the numbers of CD103+Treg cells in HLN correlate with the development of LIT. HLN and inguinal lymph nodes (ILN) were harvested from groups (n=6) of naive, sensitized, AAD and LIT mice. Flow cytometric analysis on the cells isolated from HLN and ILN demonstrated a significant (p<.001) increase in the numbers and percentages of CD103+ Treg in HLN at LIT (7x103+ 4 HLN; 2x103+0.5 ILN ) compared to AAD (2x103+ 1 HLN; 1x103+0.5 ILN ). There was no significant increase of CD103+Treg in HLN between naïve, sensitized and AAD mice. The increase of CD103+ Tregs was seen regionally in HLN but not in ILN. In summary, the data suggests a novel in vivo role for CD103+Tregs in the subsequent resolution of a Th2 driven inflammatory response.

Funded by NIH/AIRO1 HL-43573
A CD1d-dependent antagonist inhibits the activation of iNKT cells and prevents development of allergen-induced airway hyperreactivity (39.9)
Authors: Akbari Omid , Yang Wen , Meyer Everett , Umetsu Dale T , Wilson S. Brian
Published: January 2023

The prevalence of asthma continues to increase and its optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted iNKT cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in rodents and are associated with severe peristent asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway hyperreactivity disease, mice sensitized with ovalbumin were treated with a CD1d-binding lipid antagonist, DPPE-PEG. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltrates after ovalbumin challenge, but not the development of ovalbumin-specific Th2 responses. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel therapy for iNKT cell-mediated diseases such as asthma.
Intestinal macrophages in human Crohn disease produce excess IL-23 in response to the enteric bacteria (40.7)
Authors: Kamada Nobuhiko , Hisamatsu Tadakazu , Chinen Hiroshi , Kobayashi Taku , Okamoto Susumu , Hibi Toshifumi
Published: January 2023

We have previously reported that murine intestinal macrophages (Mφ) show anti-inflammatory phenotypes, which produced IL-10 but not IL-12/23 and negatively regulated the excess immune response to enteric bacteria. In contrast, intestinal Mφ from IL-10 deficient mice, which spontaneously develop Th1 dominant chronic colitis, produced abundant levels of IL-12/23 in response to bacteria and these abnormal responses of intestinal Mφ contribute to Th1 polarization. In the present study, we examined the pathogenic role of intestinal Mφ in human Crohn disease (CD). We identified CD14+CD33+ Mφ subset in human intestine. These subsets produce larger amount of pro-inflammatory cytokines than CD14−CD33+ intestinal Mφ. The number of CD14+CD33+ intestinal Mφ were increased in CD patients compared to normal controls (NL) and ulcerative colitis (UC) patients. Moreover, these CD14+CD33+ intestinal Mφ from CD patients produced larger amount of IL-12/23p40 and IL-23 in response to enteric bacteria compared to NL and UC. Thus, intestinal Mφ may contribute the pathophysiologies of human CD via excess production of IL-12/23p40 and IL-23 in response to enteric bacteria.
Resident CNS cells functionally express NOD2 (40.8)
Authors: Marriott Ian , Sterka David
Published: January 2023

There is a growing appreciation that resident glial cells such as astrocytes and microglia can initiate and/or augment inflammation following trauma or infection. These cells are uniquely positioned to detect and respond to invading pathogens, and the mechanisms by which resident CNS cells perceive microbial challenges are now becoming apparent. We have previously shown that astrocytes and microglia express NOD2, a member of the nucleotide-binding oligomerization domain (NOD) family of proteins that serves as an intracellular receptor for bacterial peptidoglycan motifs and that can act in a cooperative manner with Toll-like receptors (TLR). In the present study, we have performed experiments to confirm the functional significance of NOD2 expression in resident CNS cells. We demonstrate that exposure of glia to the clinically relevant CNS pathogens, B. burgdorferi and N. meningitidis, elicits interaction between NOD2 and its downstream effector molecule Rip2 kinase as assessed by co-immunoprecipitation. In addition, we have shown that siRNA for NOD2 can diminish the robust constitutive expression of NOD2 in both microglia and astrocytes as assessed by Western blot analysis. Finally, we have demonstrated that the exacerbation of TLR ligand-induced inflammatory cytokine production by microglia in the presence of the NOD2 ligand, MDP, is attenuated by siRNA for NOD2. Taken together, these data indicate that NOD2 is functionally expressed by resident glia and may therefore represent an important component in the initiation and/or regulation of bacterially-induced inflammation in the CNS.
Substance P augments bacterially induced inflammatory cytokine production by resident CNS cells (40.9)
Authors: Chauhan Vinita Singh , Sterka David , Marriott Ian
Published: January 2023

The neuropeptide, substance P (SP), can augment inflammatory responses at peripheral sites such as the gastrointestinal tract and skin. We have recently demonstrated the expression of high affinity receptors for SP (NK-1R) on microglia and shown that this tachykinin can significantly elevate inflammatory prostanoid production by these cells following bacterial challenge. Here, we have investigated the ability of SP to augment the production of inflammatory cytokines by microglia following challenge with B. burgdorferi or N. meningitidis. Furthermore, we have assessed the in vivo relevancy of these findings by directly administering these pathogens in into the CNS in NK-1R knockout mice or normal animals in the absence or presence of the NK-1R antagonist, L703,606. Our results indicate that substance P significantly augments the production of TNF-a and IL-6 by bacterially challenged microglia in vitro. Importantly, we show that in vivo expression of these inflammatory cytokines is significantly diminished in both NK-1R KO animals and mice given L703,606. Together, these results suggest that SP augments the immune responses of resident glia to disparate and clinically relevant bacterial pathogens, which could serve to exacerbate damaging inflammation that occurs following CNS infection.
Induction of mucosal immunity by hsp gp96 (41.2)
Authors: Strbo Natasa , Pahwa Savita , Kolber Michael , Podack Eckhard
Published: January 2023

Targeting immune responses to the mucosal entry site before pathogen dissemination is critical for protection. We have generated a secreted gp96-fusion protein (gp96-Ig) that mediated strong, antigen specific CD8-CTL expansion in vivo.

The aim of this study was to evaluate the effect of the site of gp96 secretion on mucosal immunity.

Expansion and migration of antigen specific CD8 T cells (adoptively transferred gfp-OT-I responder cells) were measured in response to immunization. Following different routes of administration of gp96-Ig secreting cells (EG7/3T3-OVA) the OT-I response was measured in the peritoneal cavity, spleen, mesenteric lymph nodes and gut mucosa.

OT-I home significantly more efficiently to the gut mucosa if the gp96-Ig was delivered by the intraperitoneal route. Vaginal administration of gp96 also mediated mucosal immunity while subcutaneous and intradermal immunization delivered the weakest response. Secreted gp96-ova complexes recruited OT-I to Peyer’s Patches, intraepithelial compartment and lamina propria within five days. Mucosal OT-I express alpha4beta7, alphaEbeta7/CD103, CCR9, granzyme B, IFNgamma and exhibited effector memory phenotype.

Our results indicate that peritoneally or vaginally cell secreted gp96-Ig induces migration of effector memory cells equipped with cytotoxic molecules into the mucosal compartment by inducing selective expression of gut-homing markers.
Interleukin-17 plays a role in successful resolution of Trypanosoma cruzi infection. (43.17)
Authors: Yoshida Hiroki , Wang Sen , Iwakura Yoichiro , Miyazaki Yoshiyuki
Published: January 2023

Interleukin-17 (IL-17) is a proinflammatory cytokine produced mainly by T lymphocytes. Production of IL-17 is activated by IL-23, an IL-12 cytokine family member, and its over-production causes inflammatory disorders such as rheumatoid arthritis. In contrast, signaling through IL-27, another IL-12-related cytokine, and its receptor (WSX-1) participated in suppression of Th17 development and excessive IL-17 production during Trypanosoma cruzi infection. While IL-17 has been reported to be important for resolution of some bacterial infection, its role in defense against infection of intracellular protozoan parasites remains to be elucidated. Therefore, we investigated the effect of IL-17-deficiency on T. cruzi infection. C57BL/6 (wild-type; WT) and IL-17-deficient (IL-17−/−) mice were intraperitoneally injected with the plasma containing trypomastigotes, and the number of parasites in the blood was microscopically counted. IL-17−/− mice showed prolonged higher parasitemia over WT mice. As acute T. cruzi infection causes liver injury, we measured serum levels of liver enzymes, ALT and AST. The levels of ALT and AST increased along with the parasite expansion, and these were higher in IL-17−/− mice as compared with WT mice. Furthermore, mortality of the infected IL-17−/− mice was higher than that of WT mice. These results demonstrated that IL-17−/− mice were more susceptible to T. cruzi than were WT mice. Therefore, it is suggested that IL-17 plays an important role in resolution of T. cruzi infection. Detail functions of IL-17 in regulation of the protective immunity are currently under investigation.
Functional impairment of CD8 T cells following acute virus infections (43.32)
Authors: Fulton Ross Bane , Varga Steven M
Published: January 2023

Following acute respiratory syncytial virus (RSV) infection the virus-specific CD8 T cells from the spleen show a 1:1 correspondence between tetramer staining and in vitro cytokine production after peptide stimulation. In contrast, pulmonary virus-specific CD8 T cells are deficient in IFN-γ and TNF-α production and show impaired ex vivo cytotoxicity compared to CD8 T cells from the spleen. We have studied CD8 T cell functional impairment in viral infections outside the Paramyxoviridae family and show that similar dysregulation of pulmonary CD8 T cells also occurs after respiratory infections by lymphocytic choriomeningitis virus (LCMV) and by vaccinia virus expressing the RSV M2 protein (vvM2). In addition, LCMV and vvM2-specific CD8 T cells in the lungs demonstrate impaired degranulation relative to splenic CD8 T cells as measured by CD107a staining following ex vivo peptide stimulation. Importantly, we show here that pulmonary CD8 T cell IFN-γ and TNF-α production can be recovered by stimulating these cells in vitro using P815 cells pulsed with peptide as a source of exogenous antigen presenting cells (APCs). Our results indicate that the CD8 T cells in the lung are fully functional and that the apparent functional impairment may be caused by the APC milieu within the lung tissue.
A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized following VACV infection (43.56)
Authors: Assarsson Erika , Sidney John , Oseroff Carla , Pasquetto Valerie , Bui Huynh-Hoa , Peters Bjoern , Grey Howard , Sette Alessandro
Published: January 2023

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8+ T cell responses demonstrated that about 1/40 of all possible 9–10 mer peptides were high-affinity A*0201 binders. Peptide immunization and T cell recognition data generated from 91 peptides indicated that about half of the binders were capable of eliciting T cell responses, and that 1/7 immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes about 150 dominant and subdominant epitopes restricted in the context of HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide binding affinity, complex stability, TCR avidity nor amount of processed epitope appeared to strictly correlate with immunodominance status. Further experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that immunoregulatory factors restrict the repertoire of T cell specificities following vaccinia infection by at least a factor of ten.
Length of Antigenic Stimulation During Expansion Dictates Commitment to CD8 T Cell Memory Subsets (43.6)
Authors: Sarkar Surojit , Kalia Vandana , Haining W. Nicholas , Konieczny Bogumila T. , Subramaniam Shruti , Ahmed Rafi
Published: January 2023

There is considerable interest in understanding early events that drive CD8 T cells towards diverse cell fate decisions: differentiation into central memory (TCM), effector memory (TEM) or terminal effector (TE) subsets. Although, a brief antigenic encounter is sufficient to trigger a cell autonomous program of clonal expansion and differentiation of naïve cells into effector and memory cells, how the ensuing differentiation program is impacted by subsequent antigenic stimulation in the setting of an acute infection is unclear. Here we show that commitment to the TCM, TEM and TE subsets occurs early during the induction and expansion phase of CD8 T cell responses. Repetitive antigen encounters during expansion and the extent of proliferation conditioned by it play a major role in dictating this memory subset commitment, such that increasing antigenic encounter favors TE>>TEM>TCM differentiation. Interestingly, early effectors that preferentially differentiate into TCM, exhibit a novel signature; in addition to elaborating characteristic cytotoxic function, they exhibit strong IL-2 production, a property typically ascribed to naïve and TCM cells. Gene profiling of this novel IL-2+ effector subset revealed that they represent a slightly less differentiated effector subset compared to the non-IL-2 producing counterparts, even though their ex-vivo and in-vivo cytotoxic potential were comparable. These studies underscore the importance of the length of antigenic stimulation, in regulating the commitment to various memory lineages, and will likely provide leads upon which tailored vaccines can be designed.

Supported by EGPAIDS Foundation Scholar Awards to SS & VK, & NIH & Gates Foundation Grants to RA
Modulation of the CD8+ T cell response to subdominant epitopes of respiratory syncytial virus by deletion of an immunodominant epitope (43.9)
Authors: Lee Sujin , Mok Hoyin , Wright Daivd , Crowe James E
Published: January 2023

Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. Recently, we identified a new H-2Kd- restricted subdominant epitope in the RSV M2 protein. This finding allowed us to study the frequency of T lymphocytes responding two H-2Kd-restricted epitopes, M282–90 and M2127–135, following RSV infection in both lymphoid and non-lymphoid tissues. The ratio of T lymphocytes during the peak CTL response to RSV infection that were specific for immunodominant or subdominant was approximately 3:1 in the spleen and 10:1 in the lung. This ratio was observed consistently in primary or secondary infection. In addition, we showed that the kinetics of response against the two epitopes was regulated differently; the response to the subdominant epitope was delayed compared to the dominant epitope. In further studies, we demonstrated that modification of residues at positions 2 and 9 in the dominant M282–90 epitope altered the CTL epitope dominance hierarchy following immunization with plasmid DNA encoding M2 or mutant M2 proteins. Immunogenicity of the subdominant epitope M2127–135 was enhanced when the anchor residues of the dominant epitope were mutated, suggesting that the immunodominant epitope induces a suppression of response to subdominant epitopes. Taken together, our findings have implications for understanding the immune response to RSV infection or vaccination and provide insight into methods for the rational design of vaccines that induce CD8+ T cells.
Cholesterol-dependent cytolysins elicit adenosine triphosphate from dendritic cells that acts through the P2X7 purinergic receptor (44.12)
Authors: Chu Jessica , Thomas Louis Michael , Watkins Simon C. , Salter Russell D.
Published: January 2023

Cholesterol-dependent cytolysins (CDCs), a class of gram-positive bacterial exotoxins, bind cholesterol in cell membranes and oligomerize to form pores. We observe that CDCs induce pores in dendritic cells (DC) as well as other cell types in a dose dependent manner, which allows the subsequent release of adenosine triphosphate (ATP). Extracellular ATP can exert stimulatory effects on cells through engagement of surface purinergic receptors, several of which are expressed on DC, including P2X7, which upon ATP binding forms a pore in the plasma membrane. We show here that a mutant DC line selected for resistance to lytic CDC doses loses expression of the P2X7 receptor and also becomes insensitive to ATP-induced pore formation compared to wild type cells. A model is suggested where CDCs form pores in DC membranes, allowing the release of ATP that signals through P2X7 on DC. Chronic ATP stimulation of DC results in skewed DC maturation that favors type 2 immune responses. Thus, CDC-induced ATP release from DC could represent a novel evasion mechanism for gram-positive bacteria.
Mice lacking TLR4 function exhibit enhanced susceptibility to Rickettsia conorii, correlated with decreased NK cell activity (44.14)
Authors: Jordan Jeffrey M. , Woods Michael E. , Walker David
Published: January 2023

Toll-like receptors (TLR) are critical in the initiation and polarization of protective immune responses to a wide variety of pathogens. However, the importance of TLRs in immunity to rickettsial diseases has been poorly defined. We demonstrated in a model of human rickettsiosis that mice lacking TLR4 function exhibited enhanced susceptibility to R. conorii infection compared to mice having functional TLR4 responses. Lack of TLR4 signaling resulted in overwhelming rickettsial growth in the brain and lung—the major pathologic organs. Infected mice lacking TLR4 function had decreased splenic NK cells and an increase in regulatory T-cells in peripheral lymph nodes. Further, splenocytes obtained from infected mice lacking TLR4 activity had decreased NK cell cytotoxicity activity. The lack of TLR4 signaling resulted in significantly reduced serum IL-12, IL-23, and IL-17. Thus, TLR4 is important in initiating early rickettsial immunity, possibly because of enhanced Th1 proinflammatory cytokines and increased NK cell activity.
Notch ligand Delta-like 4 regulates disease pathogenesis during viral infections by modulating Th2 cytokines (44.21)
Authors: Schaller Matthew , Rudd Brian D , Kunkel Steven L , Kelley Lara K , Neupane Rupak , Lukacs Nicholas W
Published: January 2023

Notch has recently been implicated in skewing T cell phenotype during immune responses. It has previously been reported that DCs from MyD88 deficient mice do not express the notch ligand delta-like 4. Our recent data has shown that CD4+ T cells from MyD88 deficient mice are skewed toward a Th2 phenotype during RSV infection. Here we demonstrate that the addition of recombinant dll4 to a dendritic cell/T cell co-culture reduced the amount of Th2 cytokines produced upon restimulation of MyD88 deficient T cells with RSV. In this system there was no change in the amount of IFNγ produced. To further investigate the role of dll4 in viral infection, we developed a polyclonal antibody to dll4 and blocked the protein in vivo during the RSV response. We found that mice receiving anti-dll4 had significantly higher airway hyperreactivity, an increase in the number of CD69+ CD4+ T cells in the lung, and a lower number of CD69+CD8+ T cells. CD4+ T cells isolated from lung draining lymph nodes during RSV infection and restimulated with RSV pulsed DCs produced significantly more IL-4, IL-5 and IL-13, however no change was observed in IFNγ. To investigate the role of dll4 during primary T cell responses, recombinant dll4 was used in co-cultures of DCs and DO11 ova-specific transgenic T cells. The presence of dll4 during a primary ova response decreased IL-4 and IL-13 production and increased IFNγ.