bluuebunny/crossref_metadata_2025 huggingface dataset:
RNAi-mediated silencing in immune cells - From single gene analysis to screening applications (132.2)
Authors: Zumbansen Markus , Altrogge Ludger , Lenz Dietmar , Müller-Hartmann Herbert
Published: January 2023

RNAi-mediated gene knockdown is a powerful tool that is successfully applied to identify gene function and to elucidate biological pathways. RNAi experiments and large-scale siRNA screens require the efficient delivery of highly functional and specific nucleic acid substrates into the appropriate cell system. Cell types relevant for immunological research, such as primary T cells and several suspension cell lines, are poorly accessible using reagent based transfection approaches.

Nucleofection® is an established method for the effective, non-viral transfection of nucleic acids into primary and hard-to-transfect cell types. We expanded the method to the 96-well format making high throughput applications such as siRNA-library screenings possible in cell types highly relevant for immunological research.

We here present data showing the efficient RNAi-mediated gene knockdown in various cell lines and primary cells using 96-well nucleofection.

For example, data about the knockdown of Fas-mediated apoptosis in Jurkat cells will be presented. These studies were expanded to PLK1, which is a key regulator of mitotic progression in mammalian cells. Knockdown of PLK1 resulted in impaired proliferation and increased apoptosis. Starting from these single gene analyses RNAi-library screens are set up for the identification of anti-proliferative and pro-apoptotic genes.
Validation of an ELISPOT Based T cell Biomarker Assay for Monitoring Effects of Immune Modulating Agent (132.3)
Authors: Wullner Danika , Yue Erica , Swanson Steven , Jawa Vibha
Published: January 2023

Enzyme linked immunospot (ELISPOT) assays are widely used for detection of rare antigen specific cytokine producing T cells. These T cells are present in circulation during an infection or vaccination. A quantitative assessment of immunogen specific T cell responses may prove to be a meaningful surrogate marker of functional immunity in patients. To be an effective biomarker assay in a clinical trial evaluation, the assay needs to be evaluated in terms of its robustness and precision. This study describes the steps towards validating a T cell ELISPOT assay.

Validation experiments included assessment of variability during cell harvest and freezing procedures, and shipping and handling of cells from clinical site to the laboratory. A variability of below 5% (cell viability and cell count) and 9.3% was observed in ELISPOT counts throughout shipping experiments. A series of mitogens were screened to select the appropriate positive control for T cell activation. PHA and tetanus were selected as positive controls based on the consistently high spot counts in the ELISPOT. An increase in variability was observed with an increase in concentration of cells added to the ELISPOT wells. The optimal cell number to be plated was set at 300,000 cells/ well. Inter and intra assay variability was less than 20% for a PHA mitogen stimulated control. Analysis of variability results enabled the development of cut point for each stimulant. This was helpful in setting criterions for an actual response over false positives/negatives. Based on these validation results, a reliable ELISPOT assay was developed to quantify antigen specific T cell immunity and consequently used as a biomarker for monitoring the effects of immunomodulatory drugs.
Intracellular localization and trafficking using the ImageStream imaging flow cytometer (132.6)
Authors: Friend Sherree , Venkatachalam Vidya , George Thaddeus C , Hall Brian E , Morrissey Philip J.
Published: January 2023

Specific ligands or antibody-conjugated drugs can mediate their cellular effects by gaining entry into cells via receptor mediated endocytosis. Once internalized, molecules differ in their preferential endocytic pathway, and drug efficacy is highly dependant on the route of entry and interaction with cellular components. Evaluation of internalization and intracellular molecular trafficking events are traditionally performed using microscopy. These analyses are limited, because manual image acquistion and quantitative image analysis are time consuming processes. Here we describe a method for measuring internalized events using the ImageStream imaging flow cytometer, which automatically collects large numbers of images per data set and provides qunatitative image analysis tools. In addition, localization of internalized probes to endosomes and lysosomes is quantified in several model systems. Because the ImageStream collects multiple fluorescent images per cell, internalized marker colocalization to different cellular compartments can be done simultaneously in a quantitative manner.
The forkhead transcription factor Foxq1 enhances NF-kB activity and is critical for robust T cell activation and autoimmunity (35.22)
Authors: Bupp Melanie R. Gubbels , Peng Stanford L.
Published: January 2023

Essential immunoregulatory functions have recently been ascribed to several members of the forkhead transcription factor family. For example, Foxo3a and Foxj1 inhibit NF-kB activity and Foxp3 regulates lineage commitment and function of regulatory T cells. Here, we report that another family member, Foxq1, is critical for robust helper T cell activation. CD4+ T cells from mice bearing a mutant allele of Foxq1 displayed a significantly reduced proliferative response with impaired secretion of IL-2 and IFN-gamma after TCR ligation as compared to wild-type (WT) T cells. This defect is largely rescued by the addition of recombinant human IL-2, suggesting that Foxq1 is required for optimal production of IL-2 and consequently, robust T cell proliferation. Accordingly, in in vitro luciferase assays, Foxq1 augmented transcriptional activity of the IL-2 transactivator NF-kB while mutant Foxq1 did not retain this function. Finally, Foxq1-mutant mice exhibited reduced susceptibility to the progression of experimental autoimmune encephalomyelitis than their WT counterparts. Thus, Foxq1 is essential for helper T cell activation and autoaggressive T cell responses in vivo, likely via its ability to potentiate NF-kB activity and IL-2 production.
The epigenetic regulation of major histocompatibility complex class II (MHC-II) genes by the 19S proteasome (35.24)
Authors: Koues Olivia I , Greer Susanna F
Published: January 2023

Epigenetic modifications confer the accessibility of chromatin to transcription factors. Histones provide a scaffold for DNA to wind around and post-translational modifications to their N-terminal tails determine the open or closed state of chromatin. Essential to the adaptive immune response are MHC-II molecules which present antigenic peptides to CD4+T cells, thus initiating the immune system to protect against invading pathogens. Although much is known about the transcription factors necessary for MHC-II expression, there is little information as to the epigenetic histone modifications and the requisite enzymes needed to provide these transcription factors access to DNA. Yeast studies have shown the ATPases that comprise the19S component of the 26S proteasome target histone acetyltransferases (HATs) to active promoters. This targeting increases the level of acetylated histone H3 and opens the chromatin. To investigate the role of the 19S proteasome in the epigenetic regulation of MHC-II genes, we utilized siRNA to knockdown the 19S ATPase, S8. S8 knockdown decreased both HAT recruitment and levels of acetylated histone H3 at the MHC-II promoter. Our current study strongly implicates the 19S proteasome in modifying histones to initiate MHC-II transcription and provides novel insights into the regulation of mammalian transcription.

Research supported by NMSS, Georgia Cancer Coalition and GSU.
Transcriptional regulation of the human FCGR2B gene mediated by promoter polymorphisms associated with Systemic Lupus Erythematosus (35.38)
Authors: Olferiev Mikhail , Masuda Emi , Blank Marissa C , Pricop Luminita
Published: January 2023

The inihibitory Fc gamma receptor IIb is an important negative regulator of peripheral tolerance. The [−343 G to C] SNP in the human FCGR2B promoter is associated with Systemic lupus erythematosus (SLE). The mutant promoter [−343C/C] hasdecreased transcriptional activity. Analyses by electromobility shift, chromatin immunoprecipitation and promoter pull-down assays demonstrated binding of AP1 factors to the FCGR2B promoter. The binding of AP1 factors was decreased with the mutant FCGR2B promoter compared with the common promoter. The −343 G to C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with AP1 for binding at the −343 site, and contributed to the repression of the mutant FCGR2B promoter activity. The [−343 C/C] genotype was found to be in linkage disequilibrium with the [−77 T to A] SNP relative to the transcription initiation site. The [−77 A/A] promoter constructs displayed increased activity compared with [−77 T/T] promoter constructs in luciferase assays, suggesting a role for the −77 SNP in the transactivation of the FCGR2B promoter. Allelic variations at positions −343 and −77 identified in the human FCGR2B promoter differentially modulate the transcriptional activity and may play a role in the expression and progression of SLE disease.
Norepinephrine mediated PKA-dependent regulation of Forkhead transcription factors in murine S49 thymoma cells (35.42)
Authors: Davis Daniel Hal , Graf Lloyd H , Cohen Rhonna L , Chambers Donald A
Published: January 2023

Previous investigations have revealed the presence of a norepinephrine/β2-adrenergic receptor/cAMP/PKA-dependent mRNA decay system in T lymphocytes. The Fox family of transcription factors has been implicated in regulating lymphocyte physiology. Foxm1b is reported to be a master gene related to cellular proliferation and Foxo1 has been associated with the suppression of proliferation and promotion of apoptosis. Therefore, we explored whether a PKA-selective cAMP analogue (8Br-cAMP) and NE could regulate BALB/c murine thymocyte cell lines, S49wt and S49kin- (PKA deficient), by affecting the genetic expression of Foxm1b and Foxo1. Real-time PCR studies revealed a 2–3 fold upregulation of Foxo1 mRNA in S49wt, but not kin- cells with 150uM NE or 600uM cAMP as early as 2 hrs after treatment. Foxm1b mRNA was downregulated in S49wt cells by greater than 2 fold by 8Br-cAMP after 6 hrs (at 24 hrs). Treatment with an Epac-selective cAMP (500uM) analogue resulted in no change in Foxo1 or Foxm1b expression in S49 wt or S49 kin- cells. These results associate PKA in modulating both increased Foxo1 mRNA expression and decreased Foxm1b expression and consequent fox gene-mediated regulation of lymphocyte physiology.

Supported by NIH.
Identification a novel NF-kB regulator by tandem affinity purification combined with mass spectrometry (35.44)
Authors: Wan Fengyi , Zheng Lixin , Anderson D. Eric , Lam Lloyd T. , Hegde Vijay , Deutsch Walter A. , Staudt Louis M. , Lenardo Michael J.
Published: January 2023

The molecular events involved in NF-kB signal transduction from the cell surface to the nucleus have been extensively studied over the last two decades. However, the mechanism of recruitment of NF-kB to target genes and NF-kB-induced transcriptional events after recruitment remains enigmatic. The p65-p50 heterodimer is the most abundant form of active NF-kB. The crystal structure of the p65-p50 heterodimer bound to DNA revealed that p65 binds to the 3′ half of kB sites where most of the kB sequence variation occurs, while p50 binds to the relatively invariant 5′ half. In an effort to identify novel regulatory factors of NF-B, we used p65 as bait protein and performed tandem affinity purification combined with mass spectrometry on mammalian cell extracts. Through this approach, we identified a novel regulatory protein that specifically interacts with p65. RNA interference experiments revealed that knockdown of such protein specifically inhibited p65-mediated transcription of various target genes. This finding may shed light on the mechanism of p65 regulation and provide a new insight into NF-kB specificity.
Physical interaction between the IgH 3′ regulatory region and its target, the expressed VH gene (35.47)
Authors: Ju Zhongliang , Birshtein Barbara K.
Published: January 2023

Immunoglobulin heavy chain (IgH) gene expression is regulated by the intronic enhancer (Eμ) and a complex regulatory region located 3′ of the IgH gene locus (3′RR). One mechanism by which the 3′ RR may act is by physical interaction between 3′RR enhancers and the targeted VH sequence. Using the chromosome conformation capture (3C) assay, we detected interactions between the 3′RR and the expressed VH gene in MPC-11 plasmacytoma cells. The interactions did not occur in MEL cells in which the IgH locus is silent and did not extend to the hole gene, which is the nearest downstream non-Ig gene, nor to the calreticulin gene located on another chromosome. Eμ is not required for this interaction because we observed loops in 9921 cells, an MPC-11 variant in which Eμ has been deleted. Furthermore, we found that 3′RR regulators interacted with each other. The translocated c-myc gene in MPC-11 interacted with the 3′RR, but not with Eμ located on the other, i.e. IgH expressed, allele. This implies that the VH-3′RR interaction occurred in cis but not trans. We observed a similar VH-3′RR interaction spanning ~200kb in resting splenic B cells. We conclude that the 3′RR physically interacts with its target VH gene in both splenic B cells and plasma cells. We predict that this interaction provides a scaffold on which the 3′RR can act on otherwise linearly distant target sequences to influence IgH expression.

Supported by NIH AI13509.
A Large-scale survey of human Ig Heavy Chain genes in Genbank: Structural Correlates of Autoreactivity and Statistical Signatures of Molecular Mechanisms (35.5)
Authors: Volpe Joseph M , Kepler Thomas B
Published: January 2023

There are over 20,000 DNA sequences from human immunoglobulins represented in Genbank. This wealth of DNA sequence information provides an opportunity to study the structural correlates of immunoglobulin function, including autoreactivity and to infer molecular mechanism from statistical signatures. After careful control for removal of clonally related genes, we analyzed approximately 9,400 productively rearranged sequences in order to establish baseline statistical characteristics, such as gene segment usage, n-nucleotide and mutation frequency, and the biochemical properties of CDR regions. We then analyzed 181 autoreactive and 64 anti-DNA Ig sequences against this baseline to discern statistical differences that might distinguish structurally these functionally distinct genes. Our data reveal that there are indeed distinctive biases in gene segment usage, CDR3 length, and mutation frequency that differ from one group to another. We also fit data on n-nucleotide frequency to a negative binomial model, which reveals striking differences between the DJ and VD junctions. These results have an appealing interpretation in terms of differential access of terminal deoxynucleotidyl transferase in the developing junctions.
Characterization of MHC-encoded genes in the chicken governing NK cell responses (35.52)
Authors: Zhang Lei , Goto Ronald M , Goebel Thomas , Yokoyama Wayne M , Miller Marcia M
Published: January 2023

Little is known about the molecular interactions that control NK cell responses in avian species. To define NK cells and their interactions in the chicken we are starting with a gene (Blec2) found within the MHC-B region that encodes a putative inhibitory c-type NK cell receptor. We prepared reporter cells expressing a transfected chimeric receptor molecule having the ectodomain derived from Blec2 (BAZ cells) to interrogate a panel of cell lines. We found that BAZ cells were reliably stimulated by only two cell lines. LMH cells induce a robust BAZ response that is inhibited in a dose response manner by pretreatment with tunicamycin. BAZ cells are also stimulated by RP9 cells expressing high levels of a transfected non-classical MHCI allele (YF 7.1) originating from MHC-Y. In contrast RP9 cells expressing two different classical MHCI alleles (BF2*13 and BF2*21) failed to stimulate the BAZ reporters. In cytotoxicity assays the YF7.1 allele also inhibits RP9 cell killing by IL-2-stimulated CT8+ NK cells isolated from embryonic spleens. Hence non-classical class I molecules encoded in MHC-Y may be functionally active ligands for NK cell inhibitory receptors. N-linked carbohydrate may contribute to the recognition process. These results support the idea that MHC-Y and MHC-B are linked in cooperative interactions governing NK cell responses.
Evidence for Ku70/Ku80 association with full length RAG1 (35.6)
Authors: Raval Prafulla , Kumar Sushil , Kriatchko Aleksei N. , Swanson Patrick C.
Published: January 2023

Antigen receptor genes are assembled by a form of site-specific DNA rearrangement termed V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at recombination signal sequences (RSSs), and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present biochemical evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1 and can become integrated into a stable RAG-RSS complex assembled with full-length RAG1 and core RAG2, but not core RAG1 (aa 384–1040) and either core or full-length RAG2. Formation of this complex minimally requires residues 211–1040 of RAG1. These results provide a biochemical link between the two phases of V(D)J recombination.

This research is supported by National Institutes of Health grant 1R01 AI055599 to P.C.S.
RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination (35.8)
Authors: Matthews Adam Goon Wai , Kuo Alex , Ramon-Maiques Santiago , Han Sunmi , Gallardo Mercedes , Yang Wei , Gozani Or , Oettinger Marjorie
Published: January 2023

A wide range of studies suggest that V(D)J recombination is regulated by modulation of the chromatin structure of antigen receptor loci during lymphoid development. Recent work has analyzed the pattern of histone modifications present at the immunoglobulin heavy chain (IgH) locus during various stages of B cell development. However, mechanisms linking alterations in histone modifications to the function of the RAG recombinase have remained elusive. Here, we show that the RAG2 plant homeodomain (PHD) finger specifically interacts with histone H3 trimethylated at lysine 4 (H3K4me3). The RAG2 PHD finger is necessary and sufficient for H3K4me3 peptide binding. A high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals that the trimethyl-K4 moiety is bound in a groove of three hydrophobic residues. Mutations that abrogate recognition of H3K4me3 severely impair V(D)J recombination in vivo without affecting V(D)J cleavage in vitro. Notably, a conserved tryptophan residue present within the RAG2 PHD finger and mutated in patients with immunodeficiency syndromes is critical for RAG2’s recognition of methylated H3. Together our results identify a novel function for histone methylation in DNA recombination. In addition, we provide the first evidence that disrupting the read-out of histone modifications can cause an inherited human disease.

This work was supported by NIH grant R01-GM48026. A.G.W.M. is a Howard Hughes Medical Institute Predoctoral Fellow.
Liver plasmacytoid dendritic cells stimulate antigen specific T cells in vivo (36.16)
Authors: Kingham T. Peter , Plitas George , Chaudhry Umer I. , Stableford Jennifer , DeMatteo Ronald P.
Published: January 2023

Introduction: Liver plasmacytoid dendritic cells (pDC) are considered to be weak stimulators of in vitro T cell responses. We sought to determine their ability to prime an antigen specific T cell response in vivo.

Methods: C57BL/6 murine spleen and liver pDC (CD11c+B220+NK1.1−CD19−CD11b−) were expanded in vivo with the use of an adenovirus encoding murine Flt3L cDNA and isolated by fluorescence activated cell sorting. CD45.2+ CFSE-labeled OT-I T cells were injected in the lateral tail vein of congenic recipient CD45.1 mice, pDC loaded with OVA257–264 (1μg/ml) or TRP-2181–188 (1μg/ml) were subsequently injected into the footpad. Three days later popliteal nodal lymphocytes were analyzed by flow cytometry for dissolution of CFSE fluorescence among CD3+CD8+CD45.2+cells.

Results: Flt3L expanded OVA257–264 loaded liver pDC and spleen pDC caused 70% and 79% respectively of the transferred T cells to divide. TRP-2181–188 loaded pDC induced no T cell division.

Conclusion: Liver pDC can initiate antigen specific T cell responses in vivo.

This work was supported by grant DK068346.
Functional dichotomy of plasmacytoid dendritic cells: Production of type I interferon versus specific activation of T cells (36.17)
Authors: Jähn Peter Siegfried , Schmitz Jürgen , Dzionek Andrzej
Published: January 2023

Here we describe a remarkable functional dichotomy of plasmacytoid dendritic cells (PDC): dependent on stimulation, PDC produce either type I interferon (IFN-I) and express the programmed cell death receptor 1 ligand 1 (CD274) or efficiently present antigen (Ag) to T cells. Upon culturing, PDC spontaneously redistribute MHC class II molecules from non-lysosomal to lysosomal compartments, resembling the so-called MHC-II-enriched compartments (MIIC). Cytomegalovirus (CMV)-Ag that is targeted to PDC using a CD303 (BDCA-2) mAb is rapidly endocytosed and traffics via early sorting endosomes to emerging MIIC. Type B CpG oligonucleotide (CpG-B)-stimulated PDC efficiently present CMV-Ag and are thus capable of stimulating CMV-specific T helper cells. CpG-A-stimulated PDC produce IFN-I and express CD274. Induction of Ag presentation by CpG-B inhibits induction of IFN-I production by CpG-A. IFN-I itself induces CD274 expression in PDC, indicating that induction of CD274 expression by CpG-A is indirectly mediated by induction of IFN-I production. Our results suggest that Ag presentation and production of IFN-I are mutually exclusively induced. This may be critical for controlling adaptive immunity, maintaining self-tolerance and preventing autoimmunity.
Chronic ethanol ingestion induces peripheral tolerance by dendritic cells (36.21)
Authors: Fan Ji , Schlueter Annette J
Published: January 2023

As initiators of immune responses, dendritic cells (DCs) are required for antigen specific activation of na·ve T cells in the defense against infectious agents. The immune dysfunction in chronic alcoholics could be due to impaired immunostimulatory function or enhanced regulatory function of DCs by exposure to ethanol. Using a murine model of alcoholism, we previously assessed the ability of ethanol exposed DCs to stimulate T cell proliferation. (In this model, C57Bl/6 mice were administrated 20% ethanol in their drinking water, and maintained for 4 or 16 weeks without evidence of stress reactions.) DC chronically exposed to EtOH for 4 or 16 weeks were found to have decreased DC antigen presenting ability and induced hyporesponsiveness of allogenic T cells in mixed leukocyte reaction. In order to understand what mechanism was behind this phenomenon, we investigated whether regulatory T cells could be induced by DC from the same murine model of alcoholism. CFSE labeled splenocytes from OTII mice were transferred to syngeneic recipient mice on day 1. On day2 fresh splenic DC were purified on autoMACS, loaded with OVA peptide 329–337, and transferred to the same recipient mice. On day 4, the CD4+Foxp3+ T cell compartment in CFSE labeled (OTII) splenocytes was defined using flow cytometry. The results showed regulatory T cells were increased in response to contact with EtOH exposed DC. Meanwhile IL10 production by OTII CD4 T cells increased and IFN-gamma production decreased. We also found in EtOH exposed DC that PIR A/B expression (a regulatory DC marker) was increased, which might explain why EtOH exposed DC could induce peripheral tolerance as shown above. Additional phenotypic and functional characterization of these EtOH induced rDC subsets are in progress.

Supported by NIH RO1 AA014405 and AA014406.
P2X7 Activation Induces Novel Dendritic Cell Filopodia (36.23)
Authors: Thomas Louis Michael , Chu Jessica , Watkins Simon C. , Salter Russell D.
Published: January 2023

Dendritic cells (DC) express multiple surface purinergic receptors that bind extracellular adenosine triphosphate (ATPe) resulting in complex changes in cell morphology and function. We demonstrate that ATPe induces rapid extension of filopodia from cultured DC that are distinct from pre-existing dendritic processes. These processes were not observed in other cell types analyzed and require the activity of P2X7 purinergic receptors, based on pharmacologic studies using receptor agonists and antagonists, and on loss of filopodia formation in a mutant DC cell line selected to be deficient in P2X7 expression. Phosphatidylinositol 4-kinase III α, which associates with P2X7, participates in filopodia formation based on inhibition by wortmannin at high concentrations that inhibit enzyme activity. Over time, filopodia often attain a beaded appearance with subsequent detachment of microvesicles from the cell. We hypothesize that P2X7 stimulation by ATPe could facilitate release of cell-associated antigens within microvesicles that might be internalized by phagocytes including DC, and therefore represent a novel cross-presentation pathway.
A method for rapid isolation of highly purified human monocytes using fully automated negative cell selection (36.25)
Authors: Yuan Ning , Guilbault Benoit G , Milton Graeme T , Fadum Jodie , Damen Jackie E , Eaves Allen C , Thomas Terry E , Wognum Albertus W
Published: January 2023

The preparation of highly purified monocytes for experimentation has traditionally been difficult, requiring multiple steps and many hours of work. We report the successful development of a rapid negative selection method that enables the preparation of highly purified monocytes from human peripheral blood using EasySep® column-free immunomagnetic cell separation technology. A cocktail of monoclonal antibodies incorporated into tetrameric antibody complexes was used to crosslink unwanted cells in the sample to EasySep® magnetic particles. The tube containing the labelled cell suspension was then placed in an EasySep® magnet for 2.5 minutes. Unlabelled cells were recovered by pouring off the cell suspension while labelled unwanted cells were held to the walls of the tube by the magnetic field. The whole procedure was completed in 30 minutes and yielded CD14+CD16− monocyte fractions that were on average 90% pure with an average recovery above 60%. Stimulation of purified monocytes with GM-CSF, IL-4 and LPS led to efficient differentiation into dendritic cells (DC) as identified by the expression of the DC markers CD1a and CD83, and the loss of the monocyte marker CD14. Differentiated DC induced robust allogeneic CD4+ T cell proliferation, confirming that the isolated monocytes were fully competent to differentiate into functional DCs. This method was also fully automated using the RoboSep® cell separator.
Tracking antigenic intermediates throughout the antigen processing pathway during Listeria monocytogenes infection (36.3)
Authors: Shugart Jessica Alis , Shastri Nilabh
Published: January 2023

Prior to their appearance on the cell surface where they are presented by MHC I molecules to T cells, antigenic peptides are created by a complex array of intracellular processing events. Using conventional T cell assays, only the final peptide can be efficiently detected on the cell surface. In order to detect the full range of antigenic intermediates that exist intracellularly throughout the antigen processing pathway, members of the Shastri lab have successfully developed the KOVAK system. KOVAK is a modified form of ovalbumin which contains enzymatically cleavable residues flanking the antigenic peptide, SHL8, thus allowing it to be liberated from the context of any antigenic intermediate and detected by SHL8-specific T cells following HPLC fractionation of cell extracts. Over the past year, we have utilized this system to track antigenic intermediates in the processing of KOVAK expressed by Listeria monocytogenes. Because Listeria enters the cell phagosomally and then injects itself into the cytoplasm, Listeria infection provides an excellent model to investigate the roles of various components in both the exogenous and endogenous pathways, and where these pathways merge intracellularly. So far we have successfully used this model in combination with knockout mice and inhibitors to delineate the roles of phagosomal proteases, cytoplasmic proteases and the proteasome, chaperones, TAP transport, and ERAAP in the processing of Listeria-derived antigens in infected macrophages. We will also investigate the roles of macrophage and dendritic cell maturation in the processing of Listeria-derived antigens.
In vitro vaccination site: a novel test bed for immunopotentiators (36.30)
Authors: Ma Yifan , Pawar Santosh , Sanchez-Schmitz Guzman , Poisson Louis , Byers Anthony , Schanen Brian C , Warren William L , Mishkin Eric M , Drake Don R , Higbee Russell G
Published: January 2023

Cytokine-derived dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) are widely used for adjuvant validation. However, the functional limitations reduce their predictive value. A 3D endothelial cell/collagen culture called “vaccination site (VS)” has been characterized as a novel in vitro system, which resembles in vivo physiological conditions and autonomously generates DCs. We used the VS model to investigate the immunopotency of TLR agonists (Poly IC, Gardiquimod, CpG 2006, and LPS). The administration of TLR agonists in the VS for 24 hr induced a wide range of cytokines, including IL-1 alpha/beta, IL-6, IL-8, IL-10, and TNF. The adjuvant-elicited cytokine profiles suggested a relative immunopotency: LPS> Gardiquimod> Poly IC≥ CpG 2006. Compared with the PBMC culture, the VS produced 2–100 fold more cytokines, indicating its enhanced sensitivity in response to TLR agonists. In addition to inducing cytokine production, TLR agonists (e.g. Gardiquimod) increased the generation of VS-derived DCs and elevated CD86 expression. Moreover, VS-derived APCs treated with TLR agonists facilitated T-cell proliferation and augmented Tetanus toxoid-specific antibody production. These data indicate that TLR agonists improved the antigen-presenting capability of VS-derived APCs. Overall the VS model is a more efficient test bed than the traditional PBMC culture to evaluate adjuvant candidates.

(Work supported by DARPA Rapid Vaccine Assessment Program)
Antigen Processing of pneumococcal-CRM197 glycoconjugate vaccine: The polysaccharide component co-localizes with the carrier protein and MHC II on the APC surface (36.8)
Authors: Lai Zengzu , Schreiber John
Published: January 2023

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI), induce no immunological memory and low Ab titers in infants. Conjugation of the Pn PS to the protein carrier CRM197 induces PS-specfic Ab in infants and memory similar to T dependent (Td) antigens. Glycoconjugates induce a Td PS immune response via antigen processing and presentation of carrier with MHC II, but the fate of the PS attached to the carrier is unclear. To determine the location of the PS of PnPS-CRM197 in the APC, we separately labeled PS and protein and tracked their location in APC. The PS of 19F-CRM197 was labeled by Alexa Fluor ® 594 hydrazide (labels PS red). The CRM197 was separately labeled in a reaction that did not label PS. Labeled 19F PS, 19F-CRM197 and CRM197 were incubated with 1x106 APC for 3–12 hours. APC were fixed and incubated with anti- HLA-DR Ab labeled by Alexa Fluor® 488 (green), followed by confocal microscopy. Labeled CRM197 was taken up and presented on the APC surface co-localized with MHC II (yellow; 42% positive cells). Labeled 19F PS was not internalized or presented on the surface. PS labeled 19F-CRM197 was internalized and co-localized on the surface with MHC II (23% positive). Brefeldin A and chloroquine blocked both CRM197 and 19F-CRM197 from co-localizing on the surface. These data suggest that PS of the 19F-CRM197 glycoconjugate enters the endosome, travels with CRM197 peptides to the APC surface and co-localizes with MHC II.
IgE production is enhanced by novel signaling intermediates activated by stimulation of a G-protein coupled receptor on a B cell (37.12)
Authors: McAlees Jaclyn W. , Sanders Virginia M.
Published: January 2023

The level of a Th2/IL-4-dependent antibody response is regulated by extrinsic homeostatic signals that stimulate a G-protein coupled receptor, the beta2-adrenergic receptor (β2AR), which is expressed on a B cell. We find that stimulation of the β2AR on a CD40L- and IL-4-activated B cell increases the rate of IgE mRNA transcription and the amount of IgE produced per cell, without affecting class switch recombination. Our data also show that β2AR stimulation increases IgE production in a p38 MAPK−, CD23−, and CD21/CD19-dependent manner above that induced by CD40L/IL-4 alone (J. Immunol., 177:2926). However, the mechanism(s) by which β2AR stimulation on a B cell leads to an increase in p38 MAPK activity remains unknown. Our findings suggest that the β2AR-induced increase in p38 MAPK activity occurs in a PKA-dependent, CREB-independent manner that may involve hematopoietic protein tyrosine phosphatase (HePTP) regulation as determined using gene-deficient and -transgenic mice, enzyme activation assays, co-immunoprecipitation, western blot analysis, and pharmacological inhibitors. We also show that p38 MAPK activation is necessary for a release of CD23 from the cell surface (sCD23), which may be mediated by an increase in ADAM8/10 activity. Thus, the β2AR-induced increase in IgE may depend on a p38 MAPK-induced increase in the level of soluble CD23.

Supported by NIH RO1 AI37326.
IL-12 treatment alleviates allergic lung inflammation in T-bet−/− mice (37.19)
Authors: Durrant Douglas Matthew , Metzger Dennis W.
Published: January 2023

Allergic asthma is a chronic airway disease characterized by lung inflammation and reversible airway obstruction. Little is known about the underlying mechanisms in the development of asthma, but it is well appreciated that dysregulated CD4+ T helper (Th) cells initiate and perpetuate the disease. To elucidate the potential role of IL-12, a Th1-inducing cytokine, in the treatment of asthma, wild-type and T-bet−/− mice were used in a murine model of ovalbumin (OVA)-induced allergic lung inflammation. T-bet is a Th1-specific T box transcription factor that controls the expression of interferon (IFN)-γ. Thus, T-bet−/− mice have little endogenous IFN-γ production and subsequently exhibit spontaneous lung inflammation. IL-12 treatment reduced pulmonary inflammation in WT mice as expected. Surprisingly, T-bet−/− mice treated with OVA and IL-12 also demonstrated reduced bronchoconstriction compared with T-bet−/− mice treated with OVA only. IL-12 treated T-bet−/− mice also demonstrated reduced airway hyperresponsiveness after methacholine challenge. Intracellular cytokine staining demonstrated the presence of IFN-γ+ CD8+ T cells in the lungs of T-bet deficient mice treated with IL-12. Also, we observed a marked decrease in the levels of the pro-inflammatory cytokine IL-17 in IL-12 treated T-bet−/− mice compared to wild-type, suggesting a role for Th17 cells in this murine model of allergic lung inflammation. Our hypothesis is that the pulmonary immune response is pre-disposed towards a Th17 type in the absence of T-bet regulation and that IL-12 treatment suppresses Th17 cells through a T-bet-independent, yet an IFN-γdependent mechanism. The results of this study will broaden the understanding of pulmonary inflammatory reactions.

Supported by NIH grant RO1 AI41715
The Immunomodulating Effect of AS101 on Interleukin-10 Production and the Involvement of MAPK Signaling Pathway in Atopic Dermatitis (37.3)
Authors: Sredni-Kenigsbuch Dvora , Shohat Michael , Shohat Batia , Ben-Amitai Dan , Chan Chi-Chao , David Michael
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Recently it was reported that interleukin 10 (IL-10) is also overexpressed in skin lesions of atopic dermatitis (AD) patients and this ectopic expression is believed to be an important factor in the pathogenesis of the disease. Thus the regulation of IL-10 production is a potential solution for immunotherapeutic intervention in AD. In this study, we determined whether the tellurium immunomodulating compound, ammonium tri-chloro(dioxoethylene-O,O′-) tellurate (AS101) may inhibit IL-10, and thereby affect the profile of cytokines produced by T cells of AD patients. Further, we investigated the possible correlation between changes in IL-10 levels and the expression of mitogen-activated protein (MAP) kinases. We show here that the IL-10 level was higher in AD patients compared to healthy donors, while their IL-2 and IFNγ levels were reduced. The addition of the AS101 inhibited the production of interleukin-10, while increasing the production of IL-2 and IFNγ. These changes correlate with the inhibition of p38 MAP kinase. The immunomodulatory effect of AS101, together with its excellent clinical safety profile in humans, suggests that AS101 has potential as a therapeutic agent for AD.
Characterization of allergen driven type-2 cytokine response in an adjuvant-free mouse model of tree nut allergy: Critical role of IL-4 (37.9)
Authors: Gangur Venu , Parvataneni Sitaram , Birmingham Neal , Fyolek John , Kelly Caleb , Hassan Hanem
Published: January 2023

Food allergy is a growing problem of serious proportions with potential for fatality. Specific role of type-2 cytokines in food allergy is not completely understood. Our laboratory has previously developed an adjuvant-free mouse model to study food allergy using hazelnut as a model tree nut. Using this model, the present study was conducted to elucidate the role of selected type-2 cytokines in this model. BALB/c mice were transdermally sensitized with hazelnut protein and systemic IgE antibody responses were confirmed. Subsequently, using short term ex vivo spleen cell culture, allergen driven type-2 cytokine (IL-4, IL-5 and IL-13) responses were studied using ELISA. Here, robust allergen driven, dose dependent, recall IL-4, IL-5 and IL-13 responses were observed. Further, all three type-2 cytokine responses could be largely blocked using anti-CD4 antibodies. Furthermore, BALB/c Stat6 knockout (KO) mice lacked an IL-4 but not IL-5 or IL-13 recall response to hazelnut. In addition, BALB/c Il4 KO mice did not elicit an IgE response to transdermal hazelnut exposure and were resistant to systemic anaphylaxis following oral hazelnut challenge. These data together suggest that:

transdermal exposure to hazelnut results in activation of IL-4, IL-5 and IL-13 by CD4+spleen cells; andonly IL-4, which is STAT-6 dependent, is required for food allergy phenotypes studied in this model.

Funding: Michigan State University, Michigan Agricultural Experiment Station.
Endotoxin Tolerance Abrogates Eotaxin-2 Production and Eosinophil Recruitment While Increasing Airways Hyperresponsiveness In Murine Asthma (39.1)
Authors: Natarajan Sudha , Kim Jiyoun , Remick Daniel G.
Published: January 2023

Studies suggest that exposure to high levels of endotoxin (E) at sensitization results in reduced inflammation upon allergen challenge. We hypothesized that persistent E exposure results in endotoxin tolerance (E-T), modulates allergen sensitization and results in reduced inflammation. E-T was induced by exposing female BABL/c mice to 4 consecutive days of 1μg E intratracheally. Non-E-T mice received PBS. 24h later, mice were sensitized with 2μg cockroach allergen, rechallenged 14 and 21 days later and sacrificed 24h post final challenge. Pulmonary Eotaxin-2 levels in E-T mice (92pg/mL) were significantly reduced compared to non-E-T mice (1965pg/mL) and pulmonary eosinophils in E-T mice (13,800) were significantly reduced compared to non-E-T mice (92,800). A significant reduction in pulmonary levels of TNFα (E-T vs. non-E-T 44pg/mL vs.65pg/mL), IL-17 (82 vs. 217) and KC (145 vs. 299) was also seen. Interestingly, airways hyperresponsiveness (AHR) was increased in E-T mice (215% above baseline) compared to non-E-T mice (83%). Reduced inflammation along with increased AHR suggests that these pathways are modulated by divergent mechanisms. Our data indicates that E-T significantly blunts the inflammatory response to allergen challenge, thereby providing protection against severe asthmatic inflammation. NIH Grant ES13528.
Complex role of STAT6 in allergic airway inflammation (39.11)
Authors: Chapoval Svetlana P , Kelly-Welch Ann E , Smith Elizabeth , Keegan Achsah D
Published: January 2023

STAT6 plays a critical role in Th2 cell differentiation and in allergic lung inflammation. Using a chimeric mouse model, we observed alternative lung pathology in STAT6 KO mice even when WT bone marrow or Th2 cells were provided. Thus, we hypothesized that STAT6 contributes to inflammation in a complex manner. To detail STAT6 function, WT and STAT6 KO mice were subjected to OVA priming and challenges. Broncho-alveolar lavage (BAL) cell composition, lung histology, and FACS analysis of digested lungs were assessed 48h after the last challenge. As expected, eosinophils composed a majority of BAL cells in WT mice and less than 2% in STAT6 KO mice. The OVA-induced inflammation in STAT6 KO lungs was composed mainly of macrophages with small fractions of neutrophils and lymphocytes. The OVA-treated WT lungs showed strong although divergent expression of F4/80, Mac-2, and CD11b molecules; this was significantly reduced in STAT6 KO mice. However, the numbers of dendritic cells, B cells, CD4+ and CD8+ T cells in the lungs of OVA-treated mice were unaffected by STAT6 deficiency. Interestingly, STAT6 KO mice showed enhanced basal airway reactivity to methacholine and numbers of Mac-2+ cells as compared to WT mice, suggesting that STAT6 deficiency altered lung homeostasis. Taken together, our studies demonstrate STAT6-dependent and –independent features of asthma phenotype which may impact treatments targeting STAT6. (AI38985)
ICOS/ICOSL interaction is required for CD4+ NKT cells function and for the induction of airway hyperreactivity (39.4)
Authors: Akbari Omid , Meyer Everett H , Freeman Gordon J , Sharpe Arlene H , DeKruyff Rosemarie H , Umetsu Dale T
Published: January 2023

Asthma is a major public health problem that has increased markedly in prevalence in the past two decades. In several mouse models, natural killer T cells (NKT) have recently been found to be required for the development of airway hyperreactivity (AHR), a cardinal feature of asthma. Moreover, in patients with persistent severe asthma, a significant number of NKT cells are present in the lungs. While T cell receptor signaling is required to activate NKT cells, the costimulatory requirements for NKT cell activation and function are not clear. In this study we found that ICOS was highly expressed on both naïve and activated NKT cells and that expression of ICOS was much higher on the CD4+ NKT than CD4− NKT cells. The function of NKT cells from ICOS−/− mice was impaired, as adoptive transfer of these cells into NKT cell deficient Ja18−/− mice failed to restore AHR, whereas transfer of wildtype NKT cells fully restored AHR in the Ja18−/− recipients. The failure of ICOS−/− NKT to induce AHR may be due to a great extent to the absence of cytokine production by ICOS−/− NKT cells. Additionally, ICOS−/− mice challenged with α-GalCer did not develop AHR, but adoptive transfer of WT NKT cells into ICOS−/− mice restored AHR. Finally, ICOS may be required for CD4+ NKT cell survival in the periphery, as the number of CD4+ NKT cells was significantly lower in spleen and liver of ICOS−/− mice. These results indicate that costimulation by ICOS is required for the function and homeostasis of CD4+ NKT cells, which play a major role in development of AHR.
Stem cell factor and recruitment of bone marrow derived fibroblasts in chronic asthma airway remodeling. (39.5)
Authors: Dolgachev Vladislav A , Ullenbruch Matthew R , Lukacs Nicholas W , Phan Sem H
Published: January 2023

Stem cell factor (SCF) is recently shown to be important in airway remodeling in asthma. Moreover bone marrow progenitors contribute to the lung fibroblast population in response to injury. To assess whether SCF is involved in the recruitment and differentiation of these bone marrow derived progenitor cells, GFP bone marrow chimera mice were created. These mice were then sensitized and chronically challenged with cockroach antigen (CRA) to induce the chronic airway disease model. As previously shown these mice exhibited significant airway remodeling with increased numbers of fibroblasts or fibroblast-like cells. Fluorescence microscopy revealed the presence of significant numbers of GFP expressing fibroblasts in these airways, indicative of their bone marrow origin. This was confirmed by flow cytometric analysis, which showed the presence of GFP and collagen I double positive cells. Interestingly the GFP positive cells did not express alpha-smooth muscle actin, thus suggesting that myofibroblasts were not derived from the bone marrow. Furthermore >20% of these cells expressed c-kit, which represented >60% of all bone marrow-derived c-kit+ cells. Most of the latter cells (>90%) were also positive for telomerase reverse transcriptase. Treatment with neutralizing antibodies to SCF significantly reduced airway remodeling and suppressed recruitment of these bone marrow-derived cells to the lung. Thus SCF may play a significant role in airway remodeling by promoting the recruitment of bone marrow-derived fibroblast precursors into the lung.
Smooth muscle responsiveness to IL-13 is sufficient for IL-13 induction of airway hyperresponsiveness (AHR) (39.6)
Authors: Finkelman Fred D. , Perkins Charles , Orekov Tatyana , Potter Crystal , Smulian George , Gildea Lucy , Wills-Karp Marsha , Brombacher Frank
Published: January 2023

IL-13 is the primary inducer of AHR in mouse models of asthma and increases contractility of isolated muscle cells in vitro and the contractility of intestinal smooth muscle in worm-infected mice. Consequently, we evaluated whether smooth muscle responsiveness to IL-13 is sufficient for development of AHR in mice inoculated intratracheally with IL-13. The smooth muscle-specific promoter SMP8 was used to generate mice that express IL-4Rα only on smooth muscle cells (SMonly mice, produced by crossing SMP8-IL-4Rα transgenic mice to IL-4Rα-deficient mice). SMonly mice failed to develop goblet cell hyperplasia or BAL eosinophilia in response to intratracheal IL-13 but developed AHR to inhaled methacholine to the same extent as wild-type mice when tested by barometric plethysmography with a Buxco apparatus and significant, albeit reduced AHR to injected acetylcholine when anesthetized, intubated and evaluated by the airway time-pressure index (APTI) technique. Thus, smooth muscle expression of IL-4Rα is sufficient for IL-13 induction of AHR and this is not dependent on goblet cell hyperplasia or pulmonary eosinophilia. Studies designed to determine whether smooth muscle expression of IL-4Rα is necessary for IL-13 induction of AHR are in progress.

Supported by the US Dept of Veterans Affairs and NIH.