bluuebunny/crossref_metadata_2025 huggingface dataset:
Microsomal prostaglandin E synthase-1 genetic deletion results in reduced incidence and severity of collagen-induced arthritis associated with a marked reduction of anti-collagen antibodies (130.47)
Authors: Kojima Fumiaki , Kapoor Mohit , Yang Lihua , Crofford Leslie J
Published: January 2023

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of prostaglandin (PG)H2 to PGE2, most prominently in inflammatory conditions. However, the role of mPGES-1 in the immune response has not yet been full elucidated. In this study, we demonstrate the effect of genetic deletion of mPGES-1 on initiating immune responses as well as clinical arthritis development in bovine collagen-induced arthritis (CIA), a well established model to study pathogenic mechanisms relevant to rheumatoid arthritis. mPGES-1 null and heterozygous mice exhibited decreased paw edema, arthritis severity and incidence compared to wild-type mice in a gene dose-dependent manner. Histopathological changes further showed a significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared to their wild-type littermates. In addition, mPGES-1 genetic deletion showed attenuated mechanical allodynia in a gene dose-dependent manner. The reduction of inflammatory features observed in mPGES-1 null mice were associated with significantly lower levels of IgG including IgG1, IgG2a, IgG2b, IgG2c and IgG3 against type II collagen (CII). These results suggest that deficiency of mPGES-1 inhibits the development of CIA, at least in part, by blocking development of the anti-CII antibody response.

Grant Support: Arthritis Foundation Biomedical Sciences Grant and NIH/NIAMS AR49010.
OX40 blockade delays disease progression in NOD by inducing qualitative changes in Tregs (131.1)
Authors: Honkanen-Scott Minna Maria , Nettles Adam , Bansal-Pakala Pratima
Published: January 2023

Costimulatory interaction between T cells and APCs is crucial for eliciting an immune response. By blocking costimulatory pathways it is possible to prevent or slow disease progression in many autoimmune diseases. We previously investigated the role of OX40/OX40L blockade in T1D. We demonstrated that OX40 blockade suppressed T1D progression when given at late stage but before T1D symptoms. These data are consistent with the role of OX40 as secondary costimulatory molecule. In the present study we investigated the mechanisms by which OX40 blockade suppresses T1D progression in NOD.

OX40 blockade marginally reduces expression of CD80 on DCs. There is a reduction in frequency of islet-reactive effector T cells in spleen and LN. Migration of effector T cells to pancreas may be inhibited since greatly reduced lymphocytic infiltrate was observed in the pancreas. Interestingly, OX40 blockade did not enhance Treg numbers in spleen, draining LN or the pancreas. We did not observe a reduction in Tregs in control animals, contrary to previous reports. In addition, FoxP3+ cells were present in and around infiltrated islets, suggesting that the number or location of Tregs may not be defective in NOD. Preliminary data suggests that OX40 blockade induces qualitative changes in Tregs by changing the regulatory mechanisms. Our data suggests that changes in function, rather than number, of Tregs may be crucial for suppressing T1D.
ARALAST or ANTI-TNF-a TREATMENT PREVENTS AUTOIMMUNE DESTRUCTION OF ISLET GRAFTS IN NONOBESE DIABETIC (NOD) MICE (131.23)
Authors: Koulmanda Maria , Hoffman Lauren , Qipo Andi , Shi Hang , Fan Zhigang , Flier Jeffrey F. , Strom Terry B.
Published: January 2023

The onset hyperglycemia in NOD mice coincides with loss of a critical portion of the islet cell mass. Thus, restoration of a portion of the lost islet cell mass should be sufficient to create euglycemia if a deficit in insulin producing tissue is the sole cause of disease. It is notable that many potent T-cell tolerizing therapies have failed to enable restoration of euglycemia in new onset T1D NOD mice with or without transplantation of islets. Hence, we have searched for other potentially complicating features, e.g. insulin resistance, that may serve as an additional barrier to restoration of euglycemia. We now report that new onset T1D is associated with inflammation induced insulin resistance created by faulty phosphorylation of crucial elements of the insulin signaling pathway (insulin receptor and IRS-1). To test the hypotheses that inflammation, insulin resistance and T1DM disease expression are linked we have tested the effects of select anti-inflammatory agents in T1DM NOD mice. We now report that treatment for 15 days with alpha-1-antitrypsin (AAT) or 20 days with anti-TNF-a (i) permanently restores a euglycemic state in 14 of 16 or 22 of 24 treated new onset diabetic NOD mice (ii) ablated insulin resistance and (iii) produces specific tolerance to syngeneic, not acceptance of allogeneic, islets that is manifest long after cessation of treatment of new onset diabetic NODs while preserving other immune responses. In short, the restoration of euglycemia, self-tolerance, and of the faulty insulin receptors and IRS- 1 phosphorylation patterns can be corrected by treatment with ATT, an acute phase reactant with potent anti-inflammatory activity, and/or anti-TNF-a.
CD4+CD25+Foxp3+ T regulatory cell stimulation by high dose gp96 is costimulation and APC independent (131.3)
Authors: Nevius Erin , Binder Robert , Srivastava Pramod
Published: January 2023

The immunogenicity of heat shock protein-peptide complexes (HSPs) against tumors and pathogens is initiated when the HSP engages its receptors, CD91 and maybe others, on antigen presenting cells (APCs). This allows APCs to obtain antigen in the form of peptides, and acquire signals that indicate for them to mature, express co-stimulation and migrate (1). However, high doses of HSPs tend to negate ongoing T cell responses (2), an effect shown to involve generation of a CD4+ suppressor response (3). This immune suppression has been demonstrated to protect mice from autoimmune conditions including experimental autoimmune encephalomyelitis (EAE) and the NOD model of diabetes (3). Here we provide evidence that highly enriched CD4+CD25+Foxp3+ T regulatory cells proliferate up to 3 fold more in the presence of the HSP gp96 and IL-2 compared to IL-2 alone. LPS or albumin were used as controls and did not support proliferation. This effect is APC and co-stimulation independent and may occur via CD91 which is expressed on T regulatory cells or by another receptor. Overall CD4+CD25+Foxp3+ T regulatory cells are stimulated directly by HSPs and may be the mechanism by which high doses of HSPs induce a suppressor response.

This study was supported by NIH grant CA84479-02 and by a research agreement with Antigenics Inc.
Application of humanized FcRn mouse model for in vivo testing of therapeutic antibodies (131.37)
Authors: Petkova Stefka B , Meng Yu-Ju Gloria , Sproule Tomas J , Christianson Gregory J , Roopenian Derry C
Published: January 2023

IgG antibodies modified for high binding affinity for their receptor FcRn have emerged as a major treatment for a wide array of both neoplastic and autoimmune diseases. The goal of this study was to analyze the effect of human IgG1 antibodies with enhanced in vitro binding to FcRn on their in vivo half-life in mice deficient in mouse FcRn and expressing human FcRn. Two mutants (N434A and T307A/E380A/N434A) of the humanized monoclonal antibody Hu4D5, directed against epidermal growth factor receptor 2 (p185 HER2), show increased pH-dependent binding to human FcRn in vitro. They also have an extended half-life in vivo compared to wild-type antibody in FcRn transgenic mice but not in mice expressing endogenous mouse FcRn. When injected into FcRn-humanized mice at a concentration sufficient to partially saturate human FcRn, the mutants with an extended serum half-life were most effective in reducing the half-life of a tracer antibody. Finally, we use a serum-transfer model of arthritis and FcRn-humanized mice to test whether mutant antibodies could have enhanced therapeutic potential by promoting accelerated clearance of arthritogenic antibodies. Results indicate that the engineered mutant T307A/E380A/N434A is most effective in vivo in ameliorating the inflammatory arthritic lesions.

In summary, FcRn-humanized mice are a promising animal model for human IgG therapeutic development.
Performance Evaluation of an Enhanced-Sensitivity Interferon-gamma ELISpot Assay Kit (132.5)
Authors: Moskowitz Keith A , Lathey Janet L , Jeffrey Lisa , Branch Rodshawn , Hickman Scott , Moshkoff Dmitry , Manak Mark
Published: January 2023

Human Interferon-gamma (IFN-y) ELISpot assays are designed for the detection of IFN-y secreting cells at the single cell level. We have developed and validated an ELISpot assay with enhanced sensitivity (SC) and compared its performance relative to a common commercial kit (CK). Three manufactured lots of ELISpot kits (SC1-3) and one CK were analyzed in cellular titration experiments using PHA stimulation of cryopreserved PBMC from four subjects. The minimum quantity of cells required to elicit a single spot-forming cell (SFC) per well was 25,000 for CK compared to 7,000 for SC. In many cases, > 50,000 cells were required to measure a response using the CK. Slopes of log/log plots were 2.3 for CK versus 1.7 for SC, with no overlap at the 95% CI level. At the upper detection limit (200,000 cells/well), the CK averaged 108±46 SFC/well, while SC 1-3 measured 264±79, 377±120 and 231 ±95 SFC/well, respectively, demonstrating a broader linear range. Differences in sensitivity and range are possibly due to spot size, SC produced spots 9 x 10-3 mm2 (95% CI 7-11) while CK averaged 19 x 10-3 mm2 (95% CI 17-21), P < 0.001. The same PBMC were also analyzed using CEF (CMV, EBV and Influenza A – specific peptide pools) and CMV peptide stimulants and media controls. For CEF, the mean values for SC 1-3 were 100.0, 90.0, and 86.6 SFC/well, vs. 77.7 for CK. The CMV responses were 104.3, 80.2 and 74.4 SFC/well for SC 1-3 vs. 77.1 for CK. PBMC added with no stimulus, gave backgrounds of <2 SFC/well in all cases. The average within run coefficients of variation (CV) were similar- 10.2, 14.1 and 18.9 for SC 1-3 and 13.8 for CK when stimulated using CEF and 20.8 to 26.4 with CMV. No significant differences were observed for means or variability between CK and SC 1-3. In summary, SeraCare has developed and validated an IFN-y ELISpot kit having an enhanced dynamic range and sensitivity.
Using Immunology as a Model to Teach Cell Communication. Including: A Practical ELISA for High School Biology (34.1)
Authors: Brokaw Ann , Cobb Brian
Published: January 2023

This educational unit is developed to illuminate the inherent connections between cell communication and immunology through analysis of cell signaling in the context of an introduction to vertebrate immune responses. The students will gain knowledge of the two interrelated fields through a variety of lectures, written assignments, and a computer lab simulation, culminating in a final laboratory experiment entitled, “Determination of Antibody Concentrations by Modified ELISA”. Both the educational unit and the laboratory exercise are developed for a typical advanced high school AP Biology or second year biology course setting, budget, and equipment. Students will complete a macro-scale ELISA (Enzyme-linked Immunosorbent Assay) to provide hands-on experience with one of the most fundamental immunological assays used in modern research laboratories. Upon conclusion of this curriculum, students will have developed a deeper understanding of not only immunology and cell communication pathways, but also an appreciation for common laboratory tools in the fields of immunology and medicine.
CSI Ann Arbor: A Middle School WebQuest Look at PCR Technology (34.2)
Authors: Lundy Philip Alan , Lukacs Nicholas W.
Published: January 2023

A WebQuest is a journey of understanding using information found on the Internet. Students are placed in a situation and guided through their quest, finishing with an individual or group project. At the Middle School level students are being introduced to the concepts of genetics and DNA. This curriculum is designed as an extension to a student’s understanding of DNA and an introduction to the polymerase chain reaction (PCR), an essential technology used in immunological research. Every piece of DNA contains areas called introns that seem to provide no genetic information. In 1984, Dr. Alex Jeffreys of the University of Leicester found that some of these areas contain repeated sequences, ranging from 20 to over 100 base pairs. These repeating sections of DNA are called Variable Number Tandem Repeats or VNTRs. In forensics, several different VNTRs from the same individual are amplified using PCR to provide a DNA fingerprint that is unique to that individual.

For this WebQuest, students take on the role of an undergraduate student paying his or her way through college as a midnight watchman in a Crime Scene Investigation lab. Someone in the lab is constantly stealing the watchman’s chewing gum, (a middle school crime of the highest level), and he/she decides to take matters into their own hands to find the guilty party. The student begins his/her web research with a review of basic DNA information. The student continues on with an introduction to PCR technology and demonstrations of real world examples. Students collect this information and record their results in their lab notebook. Following this information search, they use their acquired knowledge to collect evidence and run a DNA fingerprint to determine the guilty party. Finally the students present their findings to their classmates via PowerPoint, video, or poster.
Our Body’s Response to Infectious Diseases: A Middle School Unit on Infectous Diseases and the Immune System (34.3)
Authors: Paul Debra L. , Budd Ralph C.
Published: January 2023

This unit, written for grades 5 – 8, gives students an introduction to infectious diseases and the immune system. Students will discover the source of the cholera epidemic that broke out in London in 1854, learn how a pathogen can enter the body and how infectious disease spread. They will conduct experiments involving the washing of their hands and about how the skin acts as a barrier, particpate in an activity showing how antigens and antibodies fit together, and develop skits that show each of the body’s lines of defense. Guest speakers from the medical center will talk with the students about infectious disease and and how stress affects the immune system. The unit will culminate with a visit to a DNA Analysis Facility, Microscopy Imagining Department and Flow Cytometry Department.
The Autoimmune Lymphoproliferative Syndrome (ALPS) Associated with a Novel Mutation in Exon 6 of the FAS (TNFRSF6) Gene (35.14)
Authors: Schaffer Frederick M , Frankel Bruce M , Spicer Eleanor K , Cavalier MaryEllen
Published: January 2023

ALPS is the result of dysregulated lymphocyte FAS-mediated apoptosis and is characterized by lymphadenopathy, autoimmune phenomena, the expansion of a CD3+/CD4−CD8− T cell population, hypergammaglobulinemia, and an increased risk for lymphoma. We present the laboratory and clinical studies of a 6 y/o female with a history of adenopathy, severe neutropenia, microcytic anemia, and recurrent systemic infections. She has hypergammaglobulinemia (IgG = 2020 mg/dL; IgM = 413; IgA = 695 mg/dL), an elevated RF, an elevated CD3+/CD4−CD8− lymphocyte fraction, normal in vitro lymphocyte proliferation and normal titers to tetanus, diphtheria and pneumococcal antigens. Bone marrow aspirate and biopsy were unremarkable. We suspected a diagnosis of ALPS; therefore, exons 1–9 of the FAS (TNFRSF6) gene were PCR-amplified from genomic DNA. Bidirectional sequencing demonstrated a novel heterozygous mutation, I184V, an A>G change resulting in a substitution of the normal isoleucine codon (ATT) with a mutant valine codon (GTT) in exon 6. This is a previously unpublished mutation in association with ALPS. The presence of the I184V mutation was confirmed by restriction fragment analysis using the Bsr1 endonuclease. This A>G mutation appears to create a splice acceptor site that could strongly compete with the actual acceptor site located 5′ to exon 6. This mutation further causes an in-frame 15 amino acid (AA) deletion (which includes a cysteine) which most likely effects the folding of the resultant protein. Studies to investigate and assess the novel splice adaptor site and the effects of the 15 AA deletion on FAS expression and on in vitro apoptosis are currently in progress.
Porcine MHC Class II Haplotype Divergence (35.15)
Authors: Luetkemeier Erin , Schook Larry B
Published: January 2023

The swine major histocompatibility complex (MHC) located in SSC7, is a gene dense region with a role in antigen presentation and controlling immune responsiveness. Although highly polymorphic, serological approaches to identify and define alleles have been disappointing. To determine the extent of MHC polymorphisms of class II genes, DNA from divergent world breeds of unrelated pigs was analyzed for SNPs. Primers targeting exon 2 of DQB and DRB were shown to be locus specific and amplified both loci for each animal. Expression of exon 2 PCR amplicons for both genes was verified through sequence analysis revealing 30 haplotypes comprised of over 20 different alleles among the 9 breeds. Located within the two genes were a total of 24 polymorphic sites, 22 of these were located within the antigen recognition site (DRB had two in the alpha helix) where 17 of those were nonsynonymous substitutions (7 synonymous). The breeds analyzed were selected to represent geographic areas in order to determine if haplotype divergence correlated with location of origin. The first group was identified as “Asian” and included Meishan, Erhualian and Lanyu breeds. The second group represented the “British Isles” (Hampshire, Yorkshire and Berkshire breeds) and the third group was identified as “Continental” (Duroc, Large White, and Landrace breeds). Fewer breed specific alleles were observed in breeds selected for lower fat and longer carcasses. SNPs in DRB appear to be under positive Darwinian selection with a dN/dS value of 13 as opposed to DQB with a value of 1 (no selective pressure or purifying selection) To confirm expression of the PCR products and eliminate the possibility of pseudogenes, RT-PCR with primers from intron-exon boundaries were utilized and verified the absence of pseudogene amplification and revealed highest RNA expression in DRB especially in heart, bone marrow, lung, small intestine and lymph node.
NK cell inhibition receptor genes and HLA –DRB1*1201 in Spontaneous Clearance of Hepatitis Virus Infection. (35.16)
Authors: Romero Viviana A , Zuñiga Joaquin , Azocar Jose , Clavijo Olga P , Chung Raymond T , Amos Christopher I , Yunis Edmond J
Published: January 2023

Host genetic factors play an important role in the evolution of hepatitis C virus (HCV) infection. The aim of this study was to analyze the MHC-KIR combinations associated with the susceptibility and clinical outcome of HCV infection in drug users with Puerto Rican American ethnic background. A total of 160 HCV infected drug users (121 with chronic viremia (CV) and 39 with spontaneous clearance (SC)). Frequencies of KIR inhibitory and activating receptors and MHC class I ligands in HCV infected patients were analyzed using DNA methods. We also used a novel approach to rule out genetic stratification. We report a significant association between HLA-C1/C1 and KIR2DL3/KIR2DL3 with SC was detected confirming previous studies in Caucasian and African Americans. In addition we found MHC class II associations with clinical outcome different to those reported in Caucasians. Our results support the existence of interactions between HLA-C1 and KIR2DL3 genes in HCV viral outcome consistent with loss of inhibition mediated by HLA-C1 and inhibitory functions of KIR2DL3 together with the presence of HLA-DRB1*1201 consistent with negative epistasis.
Transcriptional regulation of L-selectin (35.19)
Authors: Dang Xitong , Raffler Nikolai A , Ott Kenneth R , Francis Joshua , Mirmira Raghavendra G , Ley Klaus
Published: January 2023

L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules. To study the transcriptional regulation of L-selectin, we cloned 5′-flanking sequences of the murine L-selectin gene 1084 bp upstream of the translational start site. Analysis of the 5′-flanking sequence shows no TATA box, but an Initiator-like element present around position −104. Multiple transcription initiation sites were mapped between −119 and −15 by 5′RACE. Transfection of serially truncated 5′-flanking regions linked to a reporter gene in Jurkat and EL-4 cells identified that nucleotides −2 to −284 contained the elements to drive high level gene expression. Transfection of the constructs into COS 7 cells, which lacks L-seletin, showed negligible luciferase activity. Within this region we identified several cis-elements, including IRF-2, Sp1, Ets-1, MZF-1. Site-directed mutagenesis of the putative binding sites showed that both Sp1 and Ets binding sites were positive regulatory elements. EMSA and CHIP confirmed the specific binding of Sp1, MZF-1, and Ets1. These results provide a molecular basis for further investigation of the involvement of L-selectin in regulating lymphocyte tethering and rolling.
Regulation of MHC Class II Transcription by the 19S Proteasomal ATPase S8 (35.25)
Authors: Bhat Kavita P , Turner Jonathan D , Greer Susanna F
Published: January 2023

The 26S proteasome is a multi-subunit complex involved in the turnover of polyubiquitinated proteins. Increasing evidence in yeast shows that individual ATPase subunits of the 19S proteasome lid play non-proteolytic roles in transcription regulation and histone modification. To determine if 19S ATPases play similar roles in mammalian transcription, we studied the role of the 19S ATPase S8 in the transcriptional regulation of Major Histocompatibility Complex (MHC) class II genes. Following IFN-γ stimulation, S8 associated in vivo with both the MHC class II master regulator, the Class II transactivator (CIITA), and the MHC proximal promoter. In the absence of S8, both MHC II promoter activity and MHC gene expression were decreased. Although CIITA expression remained constant in the absence of S8, CIITA association at the MHC class II promoter was drastically reduced and this reduction was not affected by proteasome inhibition. Finally, although S8 rapidly associated with the MHC class II proximal promoter following IFN-γ stimulation, the 26S proteasome demonstrated a substantial delay in association. Together, these data demonstrate that S8 plays a crucial but non-proteolytic role in the transcriptional regulation of MHC class II genes and illustrate involvement of 19S proteasome in mammalian transcription.

This work is supported by grants from the NMSS, The Georgia Cancer Coalition and GSU (to S.F. Greer).
Monocyte Polarization is Partly Mediated through Epigenetic Changes which Modify Gene Expression (35.31)
Authors: Sullivan Kathleen E , Garrett Stacey
Published: January 2023

Monocytes and macrophages respond to multiple changes in their milieu by altering their gene expression. Monocytes treated with γIFN alter their morphology and have an enhanced capacity for the production of inflammatory mediators. In contrast, exposure of monocytes to IL-4 dampens the inflammatory response. We utilized gene expression arrays to characterize both the direct effects of γIFN, αIFN, and IL-4 polarization as well as to define the effect of pretreatment with these cytokines on subsequent responses to a common pro-inflammatory stimulus, immune complexes. Each cytokine pretreatment COMPLETELY altered subsequent responses to immune complexes. For example, only 10% of the chemokines detected as upregulated after immune complexes in the absence of cytokine pretreatment were seen when any of the cytokines were utilized as pretreatment. γIFN induced a set of changes which were not completely reversed after a washout and rest period nor after re-treatment with IL-4. This suggested that γIFN induced a type of change in the cell which semi-permanently altered the expression of some genes. To explore the mechanism of this aspect of polarization, chromatin immunoprecipitation assays were performed. These assays demonstrated that γIFN induces increased H4 acetylation at the TNFα promoter. Together, these data suggest that polarization can be long lasting and may act in part by altering the epigenome.
Ets-1 is a negative regulator of Th17 differentiation (35.34)
Authors: Moisan Jacques , Grenningloh Roland , Bettelli Estelle , Oukka Mohamed , Ho I-Cheng
Published: January 2023

IL-17 is a proinflammatory cytokine that plays a role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. IL-17 is produced mainly by T lymphocytes and a newly characterized subset of T helper (Th) cells producing IL-17 has recently been described and termed Th17. Although the role of Th17 cells in the pathology of autoimmune diseases is well established, the transcription factors regulating the differentiation of this subset remain poorly characterized. We report here that the Ets-1 transcription factor is a negative regulator of Th17 differentiation. Naïve CD4 T cells from Ets-1 knockout (KO) mice expressed increased levels of IL-17 and other Th17-specific genes, including IL-17F, IL-22, and IL-23 receptor, following differentiation to the Th17 pathway with TGFβ and IL-6. The increased IL-17 production in Ets-1 KO Th17 cells appeared to be cell intrinsic and was probably caused by altered expression of RORγT, a transcription factor that is essential for the differentiation of Th17 cells. Ets-1 KO mice displayed increased mucus production in their lungs that was associated with increased expression of IL-17, IL-17F, but not IFNγ. Overall, our data demonstrate a role for Ets-1 in inhibiting the differentiation of Th17 cells and broadens the knowledge of the transcriptional program regulating Th17 cell lineage commitment.

The work was supported by a Senior Investigator Award from the Crohn’s and Colitis Foundation of America and a RO3 grant from NIH.
Genetic control of variegated KIR gene expression: polymorphisms of the bi-directional KIR3DL1 promoter are associated with distinct frequencies of gene expression (35.37)
Authors: Anderson Stephen K , Pascal Veronique , Martin Maureen P , Carrington Mary , Li Hongchuan
Published: January 2023

The recent discovery of the bi-directional nature of the KIR gene promoters suggests that the relative strength of competing sense and antisense promoter elements might affect the probability of gene expression. Analysis of a panel of donors has revealed the presence of several functionally relevant promoter polymorphisms, clustered mainly in the inhibitory KIR family members, especially the KIR3DL1 alleles. The percentage of peripheral blood NK cells expressing either KIR3DL1 or KIR3DS1 was evaluated in individuals heterozygous at this locus. The KIR3DL1*001 and KIR3DS1 alleles were found to be expressed on a significantly higher percentage of NK cells than all other KIR3DL1 alleles. High frequency of expression was associated with a specific promoter polymorphism at a Sp1 transcription factor-binding site, and this change increased the relative promoter activity in the sense orientation. The KIR2DS4 promoter region is nearly identical to the KIR3DL1 promoter, and several polymorphisms are present, however none of the KIR2DS4 polymorphisms affect promoter activity in vitro and no association was observed between promoter polymorphisms and frequency of expression.

This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract N01-CO-12400
Association of genetic variations in inflammatory and fibrogenic genes with progressive massive fibrosis in coal miners (35.51)
Authors: Yucesoy Berran , Johnson Victor J , Fluharty Kara , Kashon Michael L , Slaven James , Kissling Grace , Germolec Dori , Vallyathan Val , Luster Michael I
Published: January 2023

Progressive massive fibrosis (PMF) is a chronic interstitial lung disease with a complex etiology. Cytokines, growth factors and cell-surface adhesion molecules play crucial roles in the pathogenesis of pulmonary fibrosis by mediating inflammation, fibroblast proliferation, angiogenesis and collagen synthesis. We examined the influence of functional single nucleotide polymorphisms (SNP) in interleukin-1 and 6, tumor necrosis factor-α, transforming growth factor-β, vascular endothelial growth factor, epithelial growth factor and intercellular adhesion molecule-1 genes on the development of PMF. A total of 648 ex-coal miners from the National Coal Workers Autopsy Study were examined in a case-control study. Genotype analysis was performed on genomic DNA using 5′ nuclease PCR assay. There were no significant differences in the frequencies of individual SNPs between PMF and control groups. However, gene-gene interactions were found between TNFα −308/−238, VEGF+405 / ICAM-1+241 and VEGF+405 / ICAM-1+241 / IL-6+174 (p<0.05) variants. These data suggest that genetic variant of cytokine and fibrogenic genes may play role in individual susceptibility to PMF initiated presumably by coal dust.

Supported in part by the NIEHS-IAG (Y1-ES-0001).

The findings have not been formally disseminated by the NIOSH and should not be construed to represent any agency determination or policy.<
Anergy is a Survival Advantage for the Maintenance of Memory CD4+ T Cells (36.10)
Authors: Dalai Sarat Kumar , Sadegh-Nasseri Scheherazade
Published: January 2023

The hallmarks of specific T cell immunity include proliferative expansion, and acquisition of effector function. The outcome is resolution of the infection and the generation of memory T cells (Tmem). Although majority of Tmem cells die following clearance of infection, a fraction survives through lifetime and is protective on recall. It is not clear how Tmem decline with the fall of antigenic load and survive so long in the absence of antigen. Using CpG mixed with antigen to mimic an infection, we have shown that CD4+ Tmem cells undergo anergy upon encountering suboptimal doses of antigen, a condition that associates with the fall of antigenic load expected post resolution of an infection. We demonstrate that follicular B (B2) cells, and not DCs, induce anergy in CD4+ Tmem by upregulating CTLA-4 expression. These findings suggest that B cells bearing high affinity antigen receptor regulate responsiveness of Tmem by capturing the lowest levels of antigen likely present after the clearance of an infection, and signal Tmem to undergo anergy. We also show that the anergized cells are activated by stimulatory signals generated by innate immunity at the onset of a subsequent infection. Our study suggest that anergy is a mechanism to regulate long-term survival of Tmem in the absence of antigen by preventing unnecessary proliferations that lead to death.

Supported by R01GM53549 and R01AI063764 grants to SS-N.
Exosome-mediated B cell activation (36.11)
Authors: Saunders Ute , Kearney John F
Published: January 2023

The mature B cell population is divided into splenic follicular (FO) B, marginal zone (MZ) B and B1 B cells by phenotype, localization, and function. MZ B and B1 B cells participate in the early immune response against blood-borne T-independent (TI) antigens to bridge the temporal gap between the innate and adaptive immune response. We previously described the capture and transport of bacteria to the spleen by blood-derived CD11c immature dendritic cells (DCs), which are responsible for initiating immune responses to TI-2 antigens. However, the molecular basis of the DC-B cell interaction is complex, since supernatant (SN) from bacteria-primed DCs alone initiates antigen (Ag)-specific MZ B cell differentiation in vitro. Therefore, we propose that bacteria-primed DCs release factors able to initiate Ag-specific plasmablast generation. Recent findings that DCs secrete exosomes, which have the ability to stimulate T-dependent (TD) immune responses, support our hypothesis. To determine whether exosomes are involved in TI immune responses, we isolated exosomes from SN of bacteria-primed DCs (BMDC) and cocultured them with splenic B cells from M167 heavy chain transgenic (tg) mice expressing a dominant B cell clone, reactive with phosphorylcholine (PC). Using this system, we show that exosomes isolated from SN of Streptococcus pneumoniae primed BMDCs can specifically activate the M167 B cell clones, but not B cells from hen-egg-lysozyme (HEL)-specific MD4 tg B cell mice. These results suggest an involvement of exosomes in Ag-specific MZ B cell differentiation.
MHC Class II Expression and Antigen Presentation Function of Mouse Developing-B Cells (36.12)
Authors: Chen Xinjian , Chen Leo C , Gu Yapeng , Jensen Peter E
Published: January 2023

Developing-B cells (d-BC) in bone marrow (including pro-, pre- and immature B-cells) express MHC class II (MHC-II) for reasons not yet understood. We have studied mouse d-BCs for their MHC-II expression and antigen presentation function. Similar to human d-BCs, mouse d-BCs express MHC-II in the absence of co-stimulatory molecules or CD40, and do not express H2-O. Surprisingly, however, no H2-M expression is identified using antibody specific for H2-Mb2, despite the presence of H2-M activity as indicated by lack of cell-surface CLIP that are identified on H2-M-deficient d-BCs. Real-time PCR reveals only H2-Mb1 but not Mb2 mRNA, suggesting that H2-M activity in the d-BCs is derived from H2-Mb1. In vitro, d-BCs can present both peptide and protein antigens. In mixed lymphocyte cultures, d-BCs only stimulate allogeneic CD8 but not CD4 T cells to proliferate, and the d-BC-exposed CD4 T cells loss their rigor to respond to subsequent stimulation by similar allogeneic dendritic cells. Our results indicate that mouse d-BC selectively express components of the MHC-II antigen processing pathway, can present antigens and appear to be tolerogenic to CD4 T cells.
B220− murine splenic natural killer dendritic cells (NKDC) can acquire MHC II expression and become potent stimulators of T cells (36.14)
Authors: Chaudhry Umer Iqbal , Plitas George , Kingham T. Peter , Raab Jesse , DeMatteo Ronald P.
Published: January 2023

Introduction: NKDC (CD3−NK1.1+CD11c+B220+/−) are novel immune cells with characteristics of NK cells and DC. As NKDC exhibit broad cell surface expression of B220, we explored the functional and phenotypic differences between B220+ and B220− NKDC.

Methods: Phenotype analysis was performed on wild-type (WT) and Flt3L expanded B6 splenic NKDC. WT NKDC were FACS purified based on B220 expression and used in various assays. T cell activity was measured by mixed leukocyte reaction (MLR). Supernatant IFN-γ levels were measured with a cytometric bead array. TLR9 ligand CpG (ODN 1826) and IL-18 were added to some wells. NK cells and DC were used as controls.

Results: B220+ NKDC accounted for 33% of total NKDC, were larger in size, and expressed a higher level of MHC II by flow cytometry. After 24 hr culture in media alone, however, B220− NKDC gained high expression of MHC II and other co-stimulatory molecules (MHC I, CD40, CD80, CD86). Additionally, Flt3L expansion, which activates NKDC, led to a similar high level expression of MHC II on B220+ and B220− NKDC. When cultured in IL-18 and CpG for 72 hr, B220+ NKDC secreted higher levels of IFN-γ (73.3±7.4 vs. 50.1±4.1 ng/ml). Compared to B220+ NKDC, B220− NKDC caused lower allostimulation of T cells in an MLR. However, 2 hr activation with CpG prior to culture lead to an increase (and equalization) in allostimulatory capacity of both NKDC subtypes.

Conclusion: B220− NKDC are an immature subtype of NKDC that possess the capacity to gain MHC II expression and become potent stimulators of allogeneic T cells.
Mycobacterium tuberculosis heat shock protein 70 preferentially activates and delivers antigen to CD11b+ mouse dendritic cells (36.18)
Authors: Guerrero Faimola O. , Jund Michelle D. , Goudy Brian D. , Ingulli Elizabeth G. , Pennell Christopher A.
Published: January 2023

Stress proteins are intracellular chaperones that also function as immune adjuvants, linking innate and adaptive immune responses. Some stress proteins, such as heat shock protein 70, deliver their chaperoned peptides to professional antigen presenting cells and enhance ensuing immune responses up to four orders of magnitude more than peptide alone. The net result is T cell-mediated protection in animal models of viral and tumor immunity. To gain insights into how stress proteins elicit immune responses in vivo, we have tracked mouse dendritic cells activated by a fusion protein containing ovalbumin linked to heat shock protein 70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). We find that lymph node resident and migrant CD11b+ dendritic cells preferentially internalize OVA.TBhsp70, leading to their activation and enhanced ability to cross-present antigen to antigen-specific CD8+ T cells. We will present data regarding the nature of the dichotomy between the responses of CD11b+ and CD8alpha+ dendritic cells to OVA.TBhsp70.

(Supported by NIH grants T32CA009138 (F.O.G.) and R01CA113576 (C.A.P.).
IncuCyteTM, a New Multi-well, Intra-incubator Imaging Technology: Time Lapse Photography of T Cell/Stromal Cell Interactions and Apoptosis. (36.31)
Authors: Lundy Steven K. , Memarsadeghi Sohiel , Neagle Bradley D. , Schroeder Kirk S. , Fox David A.
Published: January 2023

IncuCyteTM, a recently developed digital imaging platform, fits inside cell culture incubators and captures phase contrast images at timed intervals. The design allows for imaging from multiple tissue culture vessels while maintaining the cultures in a stationary position. Interactions of T cell subsets with fibroblast-like synoviocytes (FLS) in the presence or absence of superantigen, SEA, were observed at 10 minute intervals over a 6 day period using IncuCyteTM. Beginning on the first day of co-culture, both CD4+ and CD8+ T cells displayed a close association with FLS only when SEA was present. T cell morphology was enlarged and spread. The T cells were highly active in crawling on the surface of the FLS. Individual T cells appeared to maintain association with the same FLS throughout the duration of observation, and a modest proliferation or accumulation of T cells on the FLS surface resulted in 4–10 T cells bound to each stromal cell. Between 24 and 60 hours, FLS cultured with purified CD8+ T cells and SEA underwent apoptosis with profound morphologic changes being observed within a 30 minute time period for individual cells. Purified CD4+ T cells also induced FLS apoptosis, but the timing was delayed compared to CD8+ T cells. This is the first reported visual demonstration of T cell-mediated apoptosis of fibroblast-like synoviocytes.

Research support from the Dryer Foundation.
Transforming Growth Factor β Inhibition of CD1d-mediated Antigen Presentation (36.5)
Authors: Bailey Jennifer C. , Gourapura Renukaradhya J. , Brutkiewicz Randy R.
Published: January 2023

CD1d is an antigen presenting molecule structurally similar to MHC class I and presents lipid antigens to natural killer T (NKT) cells. We have previously shown that the mitogen-activated protein kinases (MAPK), p38 and ERK1/2, reciprocally regulate CD1d-mediated antigen presentation--p38 activation leads to its inhibition, whereas ERK1/2 activation leads to its promotion. Transforming growth factor β (TGF β) is a strong regulator of immune responses as well as cell proliferation, apoptosis and differentiation, and can induce the activation of p38. Our studies have shown that CD1d-mediated antigen presentation is inhibited by TGF β in murine LMTK fibroblasts expressing CD1d. Similar inhibition occurs after TGF β treatment of bone marrow-derived dendritic cells (BMDC). Our studies are aimed at dissecting the mechanisms by which TGF β influences CD1d-mediated antigen presentation.

This work was supported by NIH grant PO1 AI056097 to RRB.
Antigen presenting cells modified with polyunsaturated fatty acids evade T cell mediated lysis by extracellular release of lipids (36.6)
Authors: Shaikh Saame Raza , Edidin Michael
Published: January 2023

Polyunsaturated fatty acids (PUFAs) can be used as adjuvant immunosuppressants to alleviate symptoms of inflammation associated with obesity, heart disease, autoimmune disorders, and the metabolic syndrome. However, the targets and molecular mechanisms by which PUFAs affect pathways not directly linked to inflammatory processes are poorly understood. In the present study, we tested the effects of the ω-6 PUFA arachidonic acid (AA) and the ω-3 PUFA docosahexaenoic acid (DHA) on antigen presentation through the MHC class I pathway. Treatment of human B lymphoblasts (APCs) with AA and DHA lowered their susceptibility to alloreactive CD8+ T cell lysis. Similar effects were observed for mouse APCs. Peptide specific lysis of PUFA-modified B lymphoblasts by CD8+ T cells transgenic for the 2C TCR was lower than lysis of controls. We found that extracellular release of lipids by PUFA-modified APCs was responsible for the reduction in lysis by cognate T cells. Our data suggest that T cell uptake of molecules from lipid modified APCs may be a mechanism by which T cell responses are regulated in response to dietary intake of fatty acids. In addition, elimination of pathogen-derived peptides could be compromised by using PUFAs as immunosuppressants.
Differential T cell responses to glycoantigens (36.7)
Authors: Neaga Andreea , Cobb Brian A.
Published: January 2023

Adaptive immune responses to proteins derived from extracellular pathogens are mediated by MHCII, leading to CD4+ T cell activation. Recently, it was demonstrated that zwitterionic polysaccharide molecules (glycoantigens), such as PSA from the capsule of B. fragilis, are also presented by MHCII and induce T cell activation. However, unpublished anecdotal data has suggested that some individuals fail to respond to glycoantigens in T cell activation assays. In the present study, blood samples were drawn from volunteers to measure T cell responsiveness as measured by proliferation and cytokine production. The data indicates that glycoantigen presentation can induce the development of a TH1 response characterized by robust production of IFNγ and T cell proliferation as previously reported in the literature. Interestingly, T cells from some individuals fail to proliferate in response to glycoantigen, yet produce high concentrations of IFNγ in a T cell-dependent fashion. These data represent the first documented evidence of differential glycoantigen-mediated T cell responses in humans and lead to questions of restriction and allelic specificity during antigen presentation. A complete understanding of the presentation and recognition of glycoantigens during T cell activation could lead to advances in vaccine design using carbohydrate antigens.

This work was supported by a grant to B.A. Cobb (AI062707).
Successful inhibition in contact hypersensitivity by topical painting of CD86 siRNA oligo (37.14)
Authors: Azuma Miyuki , Ritprajak Patcharee , Hashiguchi Masaaki
Published: January 2023

Induction of a costimulatory molecule CD86 on dendritic cells located in peripheral tissues is a critical event for inducing antigen-specific T cell responses. We here investigated therapeutic effects of transdermal administration of siRNA directed against CD86 on skin allergic diseases. In a hapten-induced contact hypersensitivity model, topical painting of cream-base ointment containing a CD86 siRNA oligo without any special delivery system efficiently inhibited the ear swelling. This effect was dose-dependent and comparable to that of intraperitoneal injection of antagonistic anti-CD86 mAb. Histological analyses of the ears revealed that the numbers of infiltrating cells and CD86+ or MHC class II+ cells in the dermis were markedly decreased in the CD86-siRNA -painted ears. The total cell numbers and the percentages of hapten-carrying CD11c+ dendritic cells (DCs) in the regional LNs 24 h after challenge were significantly decreased by the CD86 siRNA treatment. In addition, the expression of MHC class II as well as CD86 on DCs in the regional LNs was also decreased. These results suggest that the silencing of CD86 in local dendritic cells may induce a marked inhibition of allergen-specific T cell responses in the regional LNs and result in the reduction of peripheral inflammatory responses. The use of CD86 siRNA that targets cutaneous DCs may provide great advantages for clinical treatment for skin allergic diseases.
Aqueous allergenic extracts of Parietaria judaica contain non proteic compounds critical for DC maturation and production of Th2-related cytokines independently from specific sensitization. (37.17)
Authors: De Palma Raffaele , D’Aiuto Elena , Abbate Gianfranco , Fontana Angelo , Gorski Jack
Published: January 2023

The pollen of Parietaria judaica (Pj) is a main source of allergenic sensitization in Mediterranean area and in other countries. Recently, many evidences suggests that pollens are a complex mixture of different compounds able to affect the immune response. Here we show that aqueous extracts of whole pollen, but not protein extracts or purified allergens of Pj, induce maturation of human DC prepared from peripheral blood from both sensitized and non sensitized donors. As assessed by flow cytometry, these cells expressed high levels of HLA-DR, CD80, CD54 and CD86 and were able to strongly stimulate allogenic T cells in MLR. Moreover, the whole-pollen extract induce a strong production of IL-10 and IL-13 and expression of several chemokine receptors. The protein extract, as well as the purified allergen failed to induce DC maturation and stimulated only a weak production of IL-4. These findings were independent from the sensitization status of the donors. Taken together, these results further indicate that pollens should not to be considered merely as a carrier of allergenic proteins but, rather, they are complex antigens and affect directly the immune response in sensitized and non sensitized individuals. Therefore, we propose to reconsider the importance of pollens as complex antigens and to further exploit their ability to elicit different immune responses other than classical IgE-mediated reactions.
Dual functionality of B7-DC-expressing dendritic cells in lung draining lymph nodes: Effects on Th1, Th2, and Th17 (37.8)
Authors: Oriss Timothy B , Arora Meenakshi , Dixon-McCarthy Barbara , Ray Prabir , Ray Anuradha
Published: January 2023

The B7 costimulatory family member B7-DC is expressed on activated, but not resting DCs. It is known to inhibit T cells via binding to PD-1, but in PD-1-deficient animals it reportedly delivers a stimulatory signal via an unidentified receptor. We found that it is differentially expressed on lung lymph node DCs from animals undergoing airway inflammation, and examined the functional consequences of this. One day after 3 daily intranasal immunizations, we observed that a majority of lung lymph node myeloid DCs expressed B7-DC. However, expression rapidly diminished only to be re-expressed by 5 days post-immunization. DCs sorted based upon B7-DC expression 1 day following immunization enhanced T cell cytokine production compared to DCs lacking expression, whereas those isolated 5 days post-immunization stimulated less cytokine production. This effect was evident will all three types of T helper cells (Th1, Th2, and Th17) using several different antigen/adjuvant systems. The results could not entirely be explained by differential expression of PD-1 since similar data was obtained with naïve T cells, which do not initially express PD-1, and with activated T cells, which do. These data support a dual role for B7-DC and further suggest that mature myeloid-type DCs can be both stimulatory or inhibitory based upon B7-DC expression.

(RO1 HL77430 to A.R. and RO1 HL60207 to P.R.)