bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct roles of STAT4α and STAT4β in the pathogenesis of experimental allergic encephalomyelitis (B99)
Authors: Mo Caiqing , Chearwae Wanida , O’Malley John T , Kaplan Mark H , Bright John J
Published: January 2023

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model of multiple sclerosis (MS). Interleukin 12 (IL-12) and IL-23 are heterodimeric cytokines, which share common IL-12p40/IL-12Rb2 subunits and play distinct roles in the pathogenesis of EAE. Signaling through JAK-STAT pathway, IL-12 induces Th1 cell development, while IL-23 enhances ThIL17 cell differentiation. We and other have shown earlier that therapeutic intervention or targeted disruption of STAT4 was effective in ameliorating EAE. Recent studies have identified the expression of a full-length STAT4α and a truncated STAT4β, which lacks 44 amino acids at the C-terminus. In this study we show that the STAT4β transgenic mice, induced to develop EAE by immunization with MOGp35-55 peptide, develop an exacerbated EAE than the wild type littermates. However, the STAT4α transgenic mice remain resistant to EAE, as seen in STAT4 knockout mice. Further analyses, by real-time PCR, ELISA and Western blot, show that STAT4β transgenic mice express higher levels of IFNγ, IL-17, IL-12, IL-23 and IL-27 in the brain and spleen than STAT4α transgenic mice, which correlates with the clinical outcome of EAE. Although the distinct roles played by STAT4α and STAT4β in IL-12 and IL-23 signaling in EAE is not known, this study validates the use of STAT4β as a novel therapeutic target in the treatment of MS and other autoimmune diseases.
Incidence of Blood Groups among the Maya Indians of Yucatan
Authors: Goodner Kenneth
Published: January 2023

Two hundred and twenty-three pure Mayas and 202 people of mixed Maya and Spanish blood were grouped. A high percentage incidence of Group O was found, in the case of the Mayas amounting to 97.7 per cent. This points to the racial purity and antiquity of the Maya stock.
Hypersensitiveness to Diphtheria Bacterial Products
Authors: Neill James M. , Fleming William L. , Sugg John Y. , Avery Roy C. , Richardson Lurline V. , Kane Bernard E.
Published: January 2023

The hypersensitive man (individual C) previously reported reactive with toxin-containing and non-reactive with non-toxin-containing test materials, was studied further by a comparison of the hypersensitive reactivity or non-reactivity of the filtrates of highly and of weakly toxicogenic strains of diphtheria bacilli. With a constant test dose (3.0 × 10−4 cc.) of filtrate, each of the 18 highly toxicogenic strains produced a moderate to strong immediate skin reaction; in contrast, none of the 14 weakly toxicogenic strains gave any reaction at all. This sharp demarcation was acceptable proof that the agent of this man's hypersensitive reaction was consistently present in higher concentration in the filtrates of highly toxicogenic than in the filtrates of weakly toxicogenic strains.

When dealing with complex mixtures like bacterial filtrates, it is difficult to assign hypersensitive reactions to any one particular constituent. However, in the present instance, significance must be attached to the regular association between toxin and the agent of C's hypersensitiveness, particularly because of the biological assays of the filtrates for their contents of two other diphtheria constituents (the agent of the delayed “pseudo” reaction and the agent of the immediate reaction on individual A). The fact that some of the weakly toxicogenic strains non-reactive with C were just as potent as any of the highly toxicogenic strains in respect to other diphtheria bacterial substances was important because it showed that the consistent association between the toxin content and content of the agent of C's reaction was not due simply to a greater concentration of bacterial substance in the toxicogenic filtrates by virtue of more luxuriant growth or of consistently greater autolysis on the part of the toxicogenic strains.

While the evidence is of indirect nature, it strongly suggests that diphtheria toxin is the substance involved in the described hypersensitive immediate skin reaction.
The Toxicity of Human Serum for the Guinea Pig. Serum in Various Clinical Conditions
Authors: Ramsdell Susan G. , Davidsohn I.
Published: January 2023

Summary and Conclusions

When normal horse serum was used in large amounts as an antigen in the human, the phenomenon of a toxicity of the serum of such treated persons for the guinea pig was found to occur more regularly and to continue for a longer period of time than had been shown to be the case in our former experiments with sera of persons treated with various immune sera. This increase is attributed to the relatively large amounts of serum with which the patients were treated.

Certain cases were found where the serum taken at a pretreatment bleeding contained the factor for toxicity. This was usually, but not always, accompanied with a higher-than-normal (heterophilic) agglutinin titer for sheep red cells. Such sera tended to become more toxic subsequent to treatment of the patient along with an increase of the agglutinin and hemolysin titers. When the sera of a group of seriously ill patients were tested, the phenomenon of toxicity was found with considerable frequency, especially in those suffering from syphilis and acute infectious processes. Of the sera of the last, the toxicity was correlated with the presence in the serum of heterophilic antibody (sheep cell agglutinin in a titer above that normally found) in 50 per cent of the cases.

It is evident that the toxicity of human serum for the guinea pig must be ascribed to two or more different factors in the serum: one of these its content in heterophilic antibody, the other as yet undetermined.
The role of Akt in NF-kappaB mediated lymphocyte gene regulation in activated T cells (35.11)
Authors: Cheng Jing , Kane Lawrence P
Published: January 2023

Akt is a serine/threonine kinase that is activated by growth factor and antigen receptors. Although NF-kappaB is one of several downstream targets for Akt, it is not known what role NF-kappaB plays in Akt-dependent biological process. The NF-kappaB-dependent gene expression during T cell activation is analyzed and a subset of genes requires Akt for upregulation is established in this study. Duplicate samples of D10 T cells are left unstimulated, stimulated with antibodies to T cell receptor (TCR) and CD28 for 2h, 6h or 12h, with or without Akt inhibitor (or SiRNA against Akt). RNAs are made and hybridized to NF-kappaB-enriched microarray which contains oligonucleotides corresponding to 113 genes that are known targets and regulators of NF-kappaB signaling. We show that of the many genes that are upregulated after T cell activation only a subset is affected by Akt inhibition. Further studies are underway to analyze these genes for transcription factor binding sites with the goal of ascertaining which transcription factors are affected among TCR/CD28-responsive genes that require Akt. Our results may be useful for identifying new targets for therapy of tumors with constitutive Akt and/or NF-kappaB activity.
Apoptotic alkalinization induced by loss of a growth signal is mediated by phosphorylation of the Na+/H+ Exchanger isoform 1 at Ser726 and Ser729 (35.12)
Authors: Khaled Annette R , Zhang Ge , Ruppert Shannon , Boohaker Rebecca , Khaled Amr S
Published: January 2023

Apoptosis is a complex process essential for cellular homeostasis. Early physiological changes that initiate cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to loss of a growth factor experienced a rapid rise in cytosolic pH. We found that the protein responsible was the Na+/H+ Exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of p38 MAP kinase (MAPK). In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to apoptosis, and expression of which protected cells from serum-withdrawal induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Serine phosphorylation of NHE1 during apoptosis decreased upon mutation of S726 and S729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of cell death.
Autophagy is essential for ER homeostasis and calcium mobilization in T lymphocytes (35.13)
Authors: Jia Wei , He You-Wen
Published: January 2023

The evolutionarily conserved intracellular bulk degradation pathway, macroautophagy, is involved in eliminating intracellular pathogens, endogenous antigen presentation in MHC-II pathway and T lymphocytes homeostasis. However, the underlying mechanisms by which autophagy regulates T lymphocyte function remain unknown. Here we used autophagy related protein 7 (Atg7) conditional knockout mice to investigate these mechanisms. Comparing to wild type T cells, Atg7-deficient T cells displayed impaired calcium influx upon TCR triggering or lower concentration of ionomycin stimulation. However, the proximal signaling of TCR in Atg7-deficient T cells was intact. The calcium influx defect in Atg7-deficient T cells may be related to an abnormal expansion of endoplasmic reticulum (ER) and higher intracellular calcium stores. We found that higher amount of ionomycin or the ER sarco/endoplasmic reticulum Ca2+ATPase (SERCA) pump inhibitor, thapsigargin, could rescue the calcium influx defect in Atg7-deficient T lymphocytes. Atg7-deficient T cells failed to efficiently proliferate and were blocked at S phase when stimulated by TCR or PMA plus ionomycin respectively. In contrast, Atg7-deficient T cells were fully activated and capable of secreting IL-2. Our results suggest that an important role for autophagy in T cells is to maintain ER homeostasis.
Contribution of HSP90 to the proteasomal dysfunction accompanying immune senescence (35.14)
Authors: Cane Stefania , Das Rupali , Ponnappan Subramaniam , Ponnappan Usha
Published: January 2023

The ubiquitin proteasome pathway (UPP) modulates a variety of cellular processes ranging from antigen processing to cell cycle progression, including the regulation of transcription factor NFκB within the immune system. Functional decline in UPP has been associated with age-related degenerative diseases. We have identified specific impairment of proteolytic activity of the proteasome during aging in memory and naïve human T lymphocyte subsets. Besides significant decline in specific subunits of the proteasome we now present evidence implicating a role for lowered HSP90 association with the proteasome as a significant contributor to the decline occurring during advanced age. This decline in association between the chaperone HSP90 and the 26S multi-catalytic complex could be mimicked by exposure to mild oxidative stress in T lymphocytes from young donors. Additionally, using geldanamycin, an inhibitor of HSP90, we demonstrate a functional impairment of 26S catalytic activity in assays employing peptide substrates and in reporter assays for the induction of transcription factor NFκB. Finally, exogenous addition of rHSP90 to purified proteasome preparations of T cells obtained from elderly donors partially upregulated catalytic activity of the proteasome. Thus, altered association of HSP90 with the 26 S proteasome contributes to proteasomal dysfunction during aging and/or oxidative stress.

This research is supported by funds from NIH AG 030599, AG 013081 and AG025220
Dual requirement for adapters SKAP55 and ADAP in controlling T cell receptor dependent LFA-1 function (35.22)
Authors: Burbach Brandon J , Cornwell Melissa A , Mitchell Jason S , Shimizu Yoji
Published: January 2023

T cell antigen receptor (TCR) engagement by an antigen-presenting cell (APC) promotes rapid activation of T cell LFA-1 integrins and stable T:APC conjugate formation. The T cell cytoplasmic adapter proteins ADAP and SKAP55 are critical intermediates for efficient LFA-1 function, but their specific contributions are unclear. Constitutive interaction between the N-terminal proline-rich domain of ADAP and the SKAP55 C-terminal SH3 domain is required for SKAP55 stability, resulting in loss of SKAP55 protein from ADAP-/- mice. To identify minimal requirement(s) for SKAP55 and/or ADAP in controlling LFA-1 function, we used adenovirus to express fragments or chimeras of SKAP55 and ADAP in naïve D011.10/hCAR/ADAP-/- T cells and monitored T:APC conjugate formation. Expression of the N-terminus of SKAP55 and/or the C-terminus of ADAP was insufficient to rescue T:APC conjugate formation. In contrast, a construct fusing these SKAP55 and ADAP fragments into a single chimera restores adhesion in ADAP-/- T cells independent of endogenous SKAP55. Surprisingly, a chimera fusing the Rap1 effector RIAM directly to ADAP failed to rescue adhesion, suggesting a key role for SKAP55. Further mutation of the SKAP/ADAP chimera indicates that tyrosine residues in SKAP55 are not required for conjugate formation, but that tyrosine residues in the ADAP C-terminus may be important for recruiting the Rap1 exchange factor C3G/CrkL complex to ADAP. NIH-R01AI038474 and AHA-0725934Z.
Quantitative analysis of immunological synapse formation using ImageStream cytometry (35.27)
Authors: Friend Sherree L , Spurkland Anne , Sundvold-Gjerstadt Vibeke , Burbach Brandon , Matta Pieta , Batista Facundo , George Thaddeus
Published: January 2023

Adaptive immune responses require antigen presentation to antigen-specific effector cells. Early events in the interaction between these two cells involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact forming an immunological synapse (IS). Evaluation of molecular recruitment to the IS is performed by directly imaging cell conjugates. The number of cells that form an IS in any given experimental system may be low and traditional image acquisition and quantitative image analysis are time consuming processes. Here we describe an improved method for quantifying cells with protein localized to the IS using the ImageStream imaging flow cytometer, which automatically and rapidly collects large numbers of images and provides quantitative image analysis tools to evaluate subcellular localization of molecules. Immunofluorescently-stained samples were acquired on the ImageStream and analyzed using a combination of photometric and morphometric features to 1) specifically distinguish and quantify cells participating in conjugate formation and 2) measure the accumulation of actin or adaptor proteins recruited to the IS. Data from systems that activate T cells using APC presenting specific antigen or using artificial APC coated with anti-CD3, as well as that activate B cells with ligand-coated beads are all presented.
KLRG1, Cadherins and their Interaction (35.29)
Authors: Banh Cindy , Tessmer Marlowe S , Fugere Celine , Brossay Laurent
Published: January 2023

The killer cell lectin-like receptor G1 (KLRG1) is a unique inhibitory receptor expressed on a phenotypically mature subset of resting NK cells as well as effector memory CD8+ T cells. KLRG1 is a C-type lectin inhibitory receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. KLRG1 ligands have been recently identified and are members of the broadly expressed cadherin family, N-, R-, and E-cadherin. In this study, we attempt to elucidate the consequences of cadherin engagement by KLRG1 as well as KLRG1 engagement by cadherin. We found that E-cadherin engagement by KLRG1 influences the dynamics of cell adhesion. Along this line, we showed that E-cadherin and KLRG1 interaction regulates the signaling properties of E-cadherin. These data are supported by the fact that recombinant extracellular domain of N-cadherin blocks KLRG1 tetramer binding to cadherin expressing cells. In addition, we show that recombinant extracellular domain of N-cadherin functions to regulate KLRG1 signaling. We also demonstrated ex vivo that KLRG1 engagement by E-cadherin induces a significant inhibition of TCR signaling. Notably, upon analysis of E-cadherin mutants, we discovered that E-cadherin's cytoplasmic domain regulates its cell surface expression. Taken together, our data suggest that immune receptor interaction with cadherin may have an impact not only on immune cells such as NK cells but also on cells expressing cadherin such as epithelial cells. Supported by NIH grant AI 58181.
Determination of Cytoplasmic Tyrosine Residues of Tim-3 Involved in Regulation of TCR signaling (35.30)
Authors: Lee Judong , Su Ee Wern , Phuah Jiayao , Hainline Sarah G , Kane Lawrence Patrick
Published: January 2023

T-cell immunoglobulin and mucin 3 protein (Tim-3) is a type-I transmembrane protein expressed on murine T-helper type 1 (Th1) cells which regulates Th1 cell proliferation and development of tolerance. Tim-3 has six tyrosine residues in the cytosplasmic tail, two and three tyrosines residues are clustered in proximal and distal region of the cytoplasmic tail respectively. Using a motif scan program, we have predicted that two tyrosine residues from each cluster may be phosphorylated by Src family kinases, and three of the five residues may interact with SH2 domains. We generated serial deletion and point mutation constructs of Tim-3 and demonstrated that deletion of the cytoplasmic tail region containing the distal tyrosine residues (Y250, Y251, Y253) slightly increased NFAT-Luciferase reporter activity, while deletion of the region containing distal and proximal (Y235, Y242) tyrosine residues abolished the reporter activity to basal level in Jurkat cells. Point mutation of both Y235 and Y242 decreased NFAT-Luc reporter activity close to basal level while single point mutation of Y235 or Y242 did not result in significant changes in reporter activity. Therefore we conclude that phosphorylation of Y235 and Y242 residues of Tim-3 are responsible for potentiation of signal transduction initiated by activation of TCR, upstream of NFAT/AP1 pathways, in a redundant manner.
Cytoskeleton dependent T cell polarization modulates calcium signals (35.31)
Authors: Junker Christian , Quintana Ariel , Kummerow Carsten , Schwindling Christian , Hoth Markus
Published: January 2023

The objective of the study was to analyze the role of T cell polarization and T cell shape changes for calcium (Ca) signals and Ca dependent T cell activation. We have used sophisticated live cell imaging technology to quantify cytoskeletal components, mitochondria and other organelle rearrangements during and after formation of the immunological synapse (IS) in parallel with Ca signals. We found that T cell polarization after formation of the IS was paralleled by drastic cell shape changes. These changes were dependent on the actin cytoskeleton and they induced a very intimate proximity between mitochondria and the plasma membrane at the IS. The mitochondrial translocation to the IS was Ca dependent but did not require local Ca influx at the IS. The shape change-dependent mitochondrial translocation to the plasma membrane prevented ORAI1 channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the cell shape to control Ca influx. Our results suggest that cell shape changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca dependent T cell activation. Organelle polarization during IS formation is an important mechanism to modulate Ca dependent T cell activation.

This project was funded by the Deutsche Forschungsgemeinschaft (Graduate Research School 1326, SFB 530, project A3) and a HOMFOR grant from the Medical Faculty.
Human CD4+ memory T cells are pre-sensitized to Fas-mediated apoptosis due to increased receptor localization to lipid raft microdomains (35.38)
Authors: Cruz Anthony C , Ramaswamy Madhu , Siegel Richard M
Published: January 2023

Fas-FasL interactions play a critical role in TCR-induced apoptosis of activated CD4+ T cells. However, naïve T cells activated through antigen stimulation expand and contract independent of the presence of the death receptor Fas. Fas deficiency promotes accumulation of CD44+ memory phenotype T cells in mice, suggesting Fas may play a critical role in memory T cell apoptosis. The differential sensitivity of memory versus naïve CD4+ T cells to Fas-induced apoptosis has not been investigated. We compared the sensitivity of purified memory versus naïve human CD4+ T cells, both directly ex vivo and after in vitro activation and expansion, to undergo Fas-mediated apoptosis.

Interestingly, effector memory phenotype (CD27- CCR7-) cells were the most sensitive to non-crosslinked anti-Fas induced cell death. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis. Memory cells have increased lipid raft-associated Fas protein versus naïve cells, which could explain the increased Fas sensitivity. In both primary human CD4+ T cells and transformed cell lines, addition of a competitive inhibitor to palmitoylation abrogates Fas-mediated cell death. These results suggest that effector memory cells are intrinsically more sensitive to Fas-induced apoptosis, possibly due to post-translational palmitoylation and lipid raft localization of Fas.
A chemical-genetic approach to inhibit Csk activates T cells independently of the T cell receptor (35.4)
Authors: Schoenborn Jamie R , Zhang Chao , Shokat Kevan M , Weiss Arthur
Published: January 2023

Src family kinases are normally inhibited by phosphorylation of their C-terminal tail by the tyrosine kinase Csk, and are positively regulated by the phosphatase CD45 and by receptor stimulation. Attempts to study the role of Csk in T cell receptor (TCR) signaling have been hampered by early embryonic lethality of Csk-/- mice and by perturbed thymocyte development in LckCre x Cskfl/fl mice. We have established a small-molecule inhibitor system to determine the effects of Csk localization and catalytic function on the regulation of TCR signaling. A novel analog-sensitive allele of Csk, CskAS, maintains normal function, but can be rapidly and specifically inhibited by 3IB-PP1, an analog of the PP1 kinase inhibitor. The function of wild-type Csk is unaffected by 3IB-PP1. We show that membrane-targeted CskAS, but not cytoplasmic CskAS, blocks TCR signal transduction. Using 3IB-PP1 to inhibit CskAS leads to rapid activation of proximal T cell signaling events including phosphorylation of CD3ζ and ERK1/2, increased cytoplasmic calcium, and downstream upregulation of CD69 expression and downregulation of the TCR. Together, these data suggest that introduction of CskAS alleles are sufficient to alter the basal steady state of Src family kinase activity in T cells. Inhibition of CskAS thus releases cells from tonic inhibition, resulting in rapid T cell activation, even in the absence of stimulation through the antigen receptor. This chemical-genetic approach of specific Csk inhibition is a useful means of studying the dynamic inhibition of TCR signaling and may be useful for manipulating the function of T cells.

Research support: JRS, NIH T32 Training Grant; CZ, NF1 Foundation; KMS, HHMI; AW, HHMI and the Rosalind Russell Medical Research Center for Arthritis
Myosin 1G is a hematopoietic-restricted protein highly enriched in lymphocyte plasma membrane/microvilli whose deficiency impairs lymphocyte activation (35.40)
Authors: Patino Lopez Genaro , Ostap E. Michael , Shaw Stephen
Published: January 2023

Class I myosins regulate diverse aspects of locomotion, vesicular traffic, and peripheral process architecture in amoeba and in mammalian cells. Mass spectrometric analysis of lymphocyte fractions enriched for plasma membrane/microvilli identified myosin 1G (Myo1G) as abundant and enriched from human peripheral blood T-cells and Myo1G plus Myo1C as abundant and enriched from a mouse pre-B-cell line. To understand its structure and function, we have investigated Myo1G by immunofluorescence, by immunoblot, by transfection of wt and mutant constructs and by creation of a knockout mouse. Myo1G is abundant in multiple hematopoietic lineages, but absent in most other cell types. Endogenous Myo1G is highly enriched at the plasma membrane and in microvilli/ruffles of lymphocytes (as is transfected Myo1G in Jurkat T-cells). Structure-function analysis with truncation and point mutants show that Myo1G localization at the membrane, like Myo1C, requires a conserved "PH-like" domain present in the tail. But unlike Myo1C, it also depends on presence of its actin-binding motor domain. Knockout mice have no obvious defect in hematopoietic cell development. However preliminary studies shown that there are defects in activation of splenic T cells after stimulation with anti TCR. Studies are in progress to elucidate the underlying mechanism by which Myo1G contributes to T-cell activation.
A Novel Nanomedicine Platform for Controlling Mast Cell Activation (36.14)
Authors: Dellinger Anthony L. , Zhou Zhiguo , Lenk Robert , Collins Sheila , Macfarland Darren , Kennedy-Norton Sarah , Conrad Dan , Kepley Christopher L.
Published: January 2023

Mast cells play a critical role in the initiation and propagation of several inflammatory diseases and are well known for their function in the allergic response. Fullerenes are a class of novel carbon allotropes with a wide variety of applications in biotechnology and medicine. Here we use a fullerene-based chemistry platform to develop and test several different fullerene derivatives to evaluate on cellular viability, anti-IgE receptor-mediated degranulation, and cytokine release from human skin mast cells (MC). The biological response depended on the properties of the addition group; some fullerene structures were effective inhibitors of degranulation and also blocked cytokine release, while others were ineffective. None were toxic to the cells using concentrations up to 100 µg/ml. Further, we demonstrate that mitochondrial membrane potential (MMP) closely paralleled IgE-mediated SMC degranulatory responses and this response was greatly inhibited by fullerene pre-incubation. Additionally, the fullerene inhibition was due to the inhibition of FcεRI-mediated MAP kinase activation. These findings suggest the biological properties of fullerene derivatives depends on the derivatization of the carbon cage which can be manipulated to compounds which may control those diseases associated with MC activation.
The expression and regulation of amphiregulin on human basophils and CD4 T cells (36.16)
Authors: Qi Yilin , Operario Darwin J. , Oberholzer Chris M. , Kobie James J. , Mosmann Tim R.
Published: January 2023

Amphiregulin (AR), a member of the Epidermal Growth Factor family, is expressed during some allergic (type 2) responses by activated mouse Th2 cells, or human mast cells. AR produced by mouse hematopoietic cells contributes to the elimination of a nematode infection by a Type 2 effector response. We therefore tested whether human peripheral blood mononuclear cells produced AR in response to T cell activation. Unexpectedly, the expression of AR mRNA and protein was found in a non-T cell population. The AR-producing cells were basophils, as judged by histological staining and expression of CD203c and CD123 (the IL-3 receptor alpha chain). AR expression by basophils in response to anti-TCR stimulation required IL-3 produced by T cells, and IL-3 alone induced high levels of AR expression by purified basophils. Mouse basophils also produced AR upon activation. Although human CD4 T cells stimulated through the TCR alone did not express AR, we found that a combination of TCR stimulation and intracellular cAMP upregulation induced human CD4 cells (particularly Th2 cells) to express AR. In contrast, most other cytokines were downregulated by elevated cAMP levels. AR expressed by human basophils, mast cells and CD4 T cells may contribute to tissue remodeling during type 2 immune responses such as asthma.

Supported by a Sandler Program in Asthma Research grant to TRM.
Basophils and Macrophages both Contribute to IgG-Mediated Anaphylaxis (36.7)
Authors: Khodoun Marat V. , Mercatili Philip , Orekov Tatyana , Finkelman Fred D.
Published: January 2023

Studies of anaphylaxis in mice revealed the importance of an alternative pathway that depends on IgG, FcγRIII, and platelet activating factor (PAF). Initial reports identified macrophages (MP) as the critical PAF source, but more recent publications identify the critical source as basophils (BA). Because these studies differed in mouse strain and experimental model used, we evaluated whether either influences the relative importance of BA vs. MP. Studies in which BA are depleted with anti-CD200R3 mAb (Ba103) and MP are depleted with clodronate liposomes or desensitized with gadolinium now show that both mouse strain and anaphylaxis model strongly influence the relative importance of BA and MP. MP depletion always has a much greater effect on anaphylaxis in BALB/c than C57BL/6 mice, while the reverse is true for BA depletion. MP are also relatively more important when mice are sensitized to goat IgG by immunization with goat anti-mouse IgD Ab (GaMD) and challenged with goat IgG than when mice are primed passively with mouse IgG1 anti-TNP mAb and challenged with TNP-ovalbumin, or when unprimed or GaMD-primed mice are challenged with anti-FcγRIII mAb (2.4G2). In addition, C57BL/6 mice are considerably more sensitive than BALB/c mice to PAF. Taken together, these results suggest that increased PAF secretion by FcγRIII-activated MP in BALB/c mice may compensate for the reduced sensitivity of this strain to PAF.
CD4+ T cells from allergic individuals recognize epitopes from the common household allergens Der p 1 and Alt a 1 and secrete Th2 cytokines (36.8)
Authors: LaFond Rebecca E , Robinson David M , Kwok William W
Published: January 2023

Allergy is initiated by adaptive Th2-mediated responses; however, little is known about CD4+ T cell epitopes within many common allergens. The objectives of this study were to utilize MHC class II tetramers to identify CD4+ T cell epitopes within household allergens and to isolate allergen reactive T cells for further phenotypic analysis. PBMCs were isolated from the blood of subjects with dust mite and mold (Alternaria) allergies and from non-allergic control subjects, and CD4+ T cells were assayed for responses to sets of allergen-derived peptides. Dust mite allergen epitopes within the Der p 1 protein were mapped for DRB1*0301, *0401, *0701, and *1501 individuals; Alternaria allergen epitopes within the Alt a 1 protein were mapped for DRB1*0301, *0401, *0701, and *1101 individuals. Tetramer-positive responses to allergen epitopes were frequently seen in CD4+ T cells from allergic subjects. A high proportion of tetramer-positive cells from allergic individuals secreted IL-5 while TNF-α, IL-10 and IFN-γ secretion occurred less frequently, indicating that the cytokine response to these allergens is predominantly Th2 polarized. Identification of CD4+ T cell epitopes and characterization of T cell responses to allergens will further our understanding of allergic disease and may prove helpful to improve specific immunotherapy.
Administration of Melatonin Inhibits the development of DNFB-induced Dermatitis in NC/Nga Mice (36.9)
Authors: Kim Tae-Ho , Jung Jung-A , Cho Jeong-Je , Park Cheung Seog
Published: January 2023

Epicutaneously administered chemical haptens like 2, 4-dinitrofluorobenzene (DNFB) evoke an Atopic dermatitis (AD)-like dermatitis reaction in NC/Nga mice under Specific Pathogen Free (SPF) conditions. Melatonin (N-Acetye-5-methoxytryptamine, MA), synthesized by pineal gland, has different physiological functions, including control of seasonal reproduction, immnune system modulation, anti-aging effect, and function as a free radical scavenger. In the present study, we examined whether treatment with MA suppress AD-like skin lesions in NC/Nga mice treated with DNFB under SPF conditions. Intraperitonealy (i.p.) administration of MA to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. IL-4 and IFN-γ production by activated CD4+T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by MA treatment. Moreover, total IgE level in serum was decreased by MA treatment. Our findings suggest that MA may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing total IgE in serum, IL-4 and IFN-γ production of activated CD4+T cells.
Applying Immunological Methodology to Diagnosis (37.2)
Authors: English Rita
Published: January 2023

This is a high school unit investigating the role of proteins in our immune systems. Students are introduced to the basic structure of proteins and how that structure is related to antigen - antibody reactions. Immunohistochemistry, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation techniques are discussed. Students work through a virtual ELISA lab to see its importance in medical diagnosis. Detection of antibodies is explained by introducing students to Western Blotting. Students perform gel electrophoresis to see how proteins are isolated in samples. Students research Western Blot procedure and apply that knowledge in a diagnostic exercise. Students presented their information and data to their peers in poster and lab report format.
Integrating Immunology into a Clinical Presentation-Based Medical School Curriculum (37.4)
Authors: Piskurich Janet F
Published: January 2023

Immunology can be a difficult discipline for first and second year medical students. As a teacher or learning facilitator it is sometimes hard to decide where to start. Even different text books adopt a variety of approaches. Clearly there is no one best way to begin to study the complex adaptive system that comprises our ability to respond to a wide variety of microorganisms, many of which have developed medically relevant means to evade the immune system. A further problem unique to the educational process of medical students is how to provide a relevant framework to enhance their retention of the concepts of basic immunology and their ability to apply these concepts later as they begin to practice medicine. Paul L. Foster School of Medicine is an emerging medical school that has adopted a clinical presentation-based curriculum. In this curriculum, students learn the basic science that is relevant to clinical science in the context of 120 specific common patient presentations. Novel approaches and the pros and cons of the integration of the basic science of immunology into a clinical-presentation based curriculum will be illustrated.
Indirect, Competitive Enzyme-Linked Immunosorbent Assay determination of Secretory Immunoglobulin A levels in saliva. (37.9)
Authors: Mwangi David
Published: January 2023

The amount of specific antibodies in the body can influence one's ability to fight or succumb to disease. Saliva provides the first line of defense against pathogens that enter the body through the mouth from the air or from ingested food. The primary antibody in saliva is salivary IgA. The students will complete a class activity that mimics the path taken by sIgA from its synthesis by plasma cells to its release from the mucosal membrane. In the first lab, students will construct a standard curve obtained from color intensity readings of serially diluted soft drink. This curve will be used to determine the concentration of soft drinks purchased from various sources (movie theatres, restaurants etc). This lab introduces students to the concept of dilution series and the use of standard curves in determining concentration of unknown samples.

In the second lab, the students will use an indirect competitive ELISA technique to determine the level of sIgA in unknown samples. It is desirable to complete this lab in collaboration with a local immunology lab to give the students a feel of medical research and make use of materials and equipment that may not be available in the school. Students will calculate the flow rate and corrected sIgA levels and use this information to determine the sIgA quantity in saliva samples. Students will use a combination of their own data and individual research to complete a research paper.
Role of micro-RNA and AU-rich elements in post-transcriptional regulation of interferon gamma (38.1)
Authors: Savan Ram , Yalamanchili Rajesh , Hakim Shakeeb , Young Howard A.
Published: January 2023

Interferon gamma (IFN-g) is an important immunomodulatory cytokine that plays a significant role in innate and adaptive immune responses of the host. The tightly-regulated expression of this pleiotropic cytokine provides robust host protection from various infections and tumors while limiting the pathological damage to the host from the inflammatory response. Similar to many inflammatory cytokines, the IFN-g 3'UTR harbors AU-rich element (ARE), that is subjected to ARE-mediated decay (AMD). The destabilization of the mRNA by AMD results in a rapid decrease in IFN-g levels. MicroRNAs (miRNAs) are another class of RNA which have regulatory potential to impact post-transcriptional regulation of gene expression. Our study has identified a miRNA-29 binding site (BS) located at the 3' end of the 3'UTR of IFN-g. Several 3' IFN-g-UTR-mutant reporter constructs have been generated by mutating the ARE and miRNA-BS, revealing competing effects between the ARE and miRNA affecting the stability of the message. Although ARE and miRNA-BS individually inhibit translation of IFN-g, mutations of both the ARE and miRNA-BS does not result in any synergistic effect. From these results, we propose that the AMD and RISC pathways competitively interact in the post-transcriptional control of IFN-g gene expression.
Externalization of the leaderless cytokine IL-1F6 occurs in response to LPS/ATP activation of transduced bone marrow derived macrophages (38.10)
Authors: Martin Unja , Gurgel Jesse , Scholler John , Renshaw Blair , Gabel Chris
Published: January 2023

Interleukin (IL)-1F6, like many members of the IL-1 cytokine family, lacks a signal sequence. Moreover, IL-1F6 does not contain an obvious prodomain or a caspase cleavage site but is somehow released to act on target cells. The mechanism of cellular export of IL-1F6 was compared to that employed by IL-1β. In the case of IL-1β, release of the mature processed cytokine is induced by treating lipopolysaccharide (LPS)-primed monocytes/macrophages with an agonist of the P2X7 receptor, such as adenosine 5'-triphosphate (ATP). Mouse bone marrow derived macrophages (BMDMs) were selected as the model system because they express the P2X7 receptor and because they can be engineered via retroviral transduction to express genes of interest. Stable populations of BMDMs transduced with retroviral vectors encoding full length IL-1F6 or green fluorescent protein (GFP) constitutively expressed IL-1F6 and GFP but did not constitutively release these polypeptides to the medium. Evidence is presented that IL-1F6 can be externalized rapidly in response to sequential LPS and ATP activation via a non-selective process. Therefore, externalization of IL-1F6 from cells engaged in the synthesis of this cytokine proceeds in vitro via a stimulus-coupled mechanism similar to that employed by IL-1β.
Characterization of IFNλ1 (IL-29) gene regulation and analysis of inter-individual variation of IFNλ1 levels (38.18)
Authors: Siegel Rachael , Eskdale Joyce , Gallagher Grant
Published: January 2023

Type III interferons are a relatively new class of anti-viral cytokines comprised of three members: IFNλ1, 2 and 3. Through an uncharacterized mechanism, levels of IFNλ1 are reduced in bronchial epithelial cells of rhinovirus-infected asthmatics. In this study, we have examined the regulation of IFNλ1 transcription by utilizing BEAS-2B bronchial epithelial cells as a model system. We have identified the IFNλ1 minimal promoter region that is responsive to viral infection. Using fluorescent DNA footprinting of epithelial cells and purified peripheral blood mononuclear cells (PBMCs) stimulated with various mitogens, we are mapping the occupancy of transcription factor binding sites up to 4kb from the IFNλ1 transcription start site. Furthermore, we have identified key regulators such as NFκ-B that are involved in control of IFNλ1 expression in epithelial cells. Data obtained from a sample set of PBMCs stimulated with HSV has shown inter-individual variation in IFNλ1, both at the level of mRNA and protein production. These findings have been correlated with analysis of selected SNP haplotypes within the IFN-λ1 promoter. Genotyping of the IFNλ1promoter has led to identification of three major haplotypes, which may relate to variation in IFNλ1 levels. The data presented in this study represent an initial characterization of IFNλ1 gene regulation that can now be applied to understanding pathogenic regulation of IFNλ1.
Generation of IL-17A reporter mice to track IL-17A expression in vivo (38.19)
Authors: Price April E , Liang Hong-Erh , Locksley Richard M
Published: January 2023

IL-17A plays a crucial role in host defense against a variety of pathogens and also contributes to the pathogenesis of autoimmune diseases. Various studies have suggested that IL-17A can be produced by a multiple cell types, including CD4+ T cells, CD8+ T cells, γδ T cells, NKT cells, dendritic cells, macrophages and granulocytes. However, since most studies rely on ex-vivo restimulation to assay IL-17A production, it is unclear which cell types are the predominant sources of IL-17A during the actual course of an immune response in vivo. To address this issue, we generated IL-17A reporter mice in which IL-17A-expressing cells are marked by the presence of a surface marker. We verified the ability of these reporter mice to mark the activation of IL-17A-producing Th17 cells in vitro. We then used these mice to gain a better understanding of the temporal and cell-type specific patterns of IL-17A expression in the mouse model of experimental autoimmune encephalomyelitis (EAE). We demonstrated that a small subset of CD4+ T cells and a larger fraction of γδ T cells express IL-17A in the CNS of mice with EAE, and that IL-17A expression levels peak in the CNS before the onset of paralysis. We have thus far been unable to detect IL-17A expression during infection with bacterial or fungal pathogens, which raises questions as to the actual contribution of IL-17A in these models.
IRS-2 phosphorylation and association with p85 and Grb2 after engagement of type I IL-4 receptor (IL-4R). (38.2)
Authors: Heller Nicola M , Qi Xiulan , Junttila Ilkka S , Shirey Kari Ann , Vogel Stefanie N , Paul William E , Keegan Achsah D
Published: January 2023

We previously showed that IL-4 elicited stronger tyrosine phosphorylation of insulin receptor substrate (IRS)-2 than IL-13. This IRS-2 phosphorylation difference was observed even at high concentrations of IL-13 and was due to the exclusive ability of IL-4 to bind to type I IL-4 Rs (IL-4Rα + γC). Furthermore, IL-4 but not IL-13, significantly augmented expression of a subset of alternatively-activated macrophage (AAM) genes, arginase I, found in inflammatory zone (FIZZ)1, and Ym1 in WT bone marrow-derived macrophages (BMM). In this study, we further analyzed signaling differences between IL-4 and IL-13 downstream of the IRS-2 pathway. IL-4 stimulation resulted in enhanced association of the p85 subunit of PI-3' kinase with IRS-2 as compared to IL-13, while only IL-4 induced the association of Grb2 with IRS-2 in WT BMM. Since IRS-2 activation leads to PI-3' K activation, we investigated the effect of wortmannin on induction of the AAM genes. IL-4-induced expression of FIZZ1 was suppressed by wortmannin to the levels induced by IL-13. Arginase I and Ym1 expression was unaffected by wortmannin. These data suggest that the type I IL-4R can direct site-specific phosphorylation of IRS-2, resulting in enhanced recruitment of adaptor molecules and augmented expression of a subset of AAM genes. In addition, they suggest these genes are differentially affected by the IRS-2/PI-3' K pathway. (PHS AI38985)
Development of an Enzyme-Linked Immunosorbent Assay for Quantitative Determination of TSLP in Cell Culture Medium and Human Serum (38.20)
Authors: Conklin John D , Sun Bingang , Leetola Catherine , Kato Atsushi , Ji Shaoquan
Published: January 2023

Human thymic stromal lymphopoietin (TSLP) is a 24 kDa monomeric glycoprotein produced primarily by non-hematopoietic cells such as fibroblasts, epithelium, and stromal cells. TSLP induces myeloid cells to release T cell chemoattactents while enhancing maturation of CD11c+ dendritic cells that in turn promote a Th2 immune phenotype. We have developed a sandwich ELISA to quantitatively measure human TSLP concentrations in culture media and human serum. The capture and detection antibodies consist of antigen affinity-purified goat anti-human TSLP polyclonal antibodies generated from recombinant human TSLP as the immunogen. The assay has a linear detection range from 31 to 250 pg/ml, and a threshold of detection at approximately 2 pg/ml. Serum spike recovery averaged 92.6% for four human serums (2 male, 2 female) for spike concentrations from 31.25pg to 500pg/ml inclusive. Assay robustness was further demonstrated by acceptable linearity of sample dilutions, intra-assay and inter-assay precisions. When primary normal human bronchial epithelial cells were stimulated with dsRNA and/or IL-4, TSLP concentration in the culture supernatant of the double stimulated cells increased significantly (53pg/ml, sd 3.37) compared with that from dsRNA stimulated cells (22pg/ml, sd 1.5, p=0.001). Unstimulated cell media had no detectable TSLP. This assay will provide a valuable resource for the study of role of TSLP in normal and pathophysiological conditions.
Soluble Receptor for Advanced Glycation Endproducts (sRAGE) Induces Human Monocyte Survival and Differentiation (38.23)
Authors: Wang Yijie , Wang Hongmei , Piper-Hunter Melissa G , McMaken Sara , Mo Xiaokui , Opalek Judy , Schmidt Ann Marie , Marsh Clay B.
Published: January 2023

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor of the immunoglobulin super family or is the soluble portion of extracellular domain of RAGE (sRAGE). sRAGE is a competitive, negative regulator of transmembrane RAGE. RAGE and its ligands are overexpressed in a wide variety of diseases. However, the biological function of RAGE and sRAGE on human mononuclear phagocytes is unclear. Using flow cytometry, we found that sRAGE directly bound to human monocytes to mediate their survival and differentiation into macrophages, events that were dependent on cellular adhesion. In both monocytes and MDM, sRAGE treatment activated Akt, Erk and NFk-B signaling, and decreased caspase-3 activity. Interestingly, while sRAGE bound the surface of suspended pre-monocytic leukemic THP1 cells, it failed to stimulate intracellular signaling or cellular differentiation. Furthermore, we found sRAGE induced monocytes chemotaxis in vitro and recruited monocytes to the lung in vivo. Our data suggest a novel role for sRAGE in mononuclear phagocyte survival, differentiation, and cell recruitment of human monocytes. These observations suggest a potential role for sRAGE to modulate organ-based inflammation.