bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autophagy is essential for ER homeostasis and calcium mobilization in T lymphocytes (35.13)
Authors: Jia Wei , He You-Wen 
Published: January 2023

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.