bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation and characterization of GIT1 and GIT2 deficient mice (87.18)
Authors: Phee Hyewon , Weiss Arthur 
Published: January 2023

Abstract
The G-protein-coupled receptor (GPCR)-kinase-interacting proteins 1 and 2 (GIT1 and GIT2) are multidomain proteins involved in diverse cellular processes. In fibroblasts, GITs are thought to regulate focal adhesion (FA) turnover, cell spreading, and motility through interaction with various proteins, including Pak-interacting Rac/Cdc42 guanine exchange factor, PIX. In T lymphocytes, GITs interact with alpha and beta PIX and PAK (p21-activated kinase) through their interaction with PIX. Previously, we showed that the GIT/PIX/PAK complex is recruited to the contact site between T cell and antigen presenting cell and GIT and/or PIX seems to play a crucial role in PAK recruitment and T cell activation. However, the physiological roles and activation mechanism of GITs in TCR-activated signal transduction pathways and T cell immune responses have not been addressed thoroughly. GIT1 and GIT2 both are expressed in human and mouse T cells, although the major form expressed in hematopoietic lineage is GIT2.
To determine the role of GIT1 and GIT2 in immune cell activation in vivo, we generated GIT1 and GIT2 deficient mice. Both deficient mouse lines were backcrossed to C57BL/6 and Balb/C backgrounds in order to perform functional immunological studies. In the meantime, we performed biochemical studies that do not require pure background.
Structure-function analysis of the determinants of CD46 polarization in T cells-combined role for polarity proteins,lipid rafts and ERM proteins (87.19)
Authors: Pasam Anupama , Ludford-Menting Mandy J , Sacirbegovic Faruk , Russell Sarah M 
Published: January 2023

Abstract
CD46 is a ubiquitously expressed human cell surface protein that acts as a receptor for complements for various pathogens including the measles virus. We demonstrated that ligation of the immunoregulatory cell surface receptor, CD46, altered T cell polarity and impaired activation and effector function in response to TCR or NK cell receptor signalling. However the molecular mechanisms by which T cell function is inhibited are not known.
We have previously shown that CD46 binds to the polarity protein, Discs large (Dlg), and that this interaction is important for the polarized localization of CD46. Specifically, CD46 localizes to the uropod of T cells, and to the distal pole of T cells undergoing antigen presentation. Polarization of CD46 is partially reduced by mutation of the Dlg-binding site, and is also partially reduced by mutation of the Cysteine residue in the transmembrane domain that is palmitoylated to allow association of CD46 with lipid rafts. However, comparison of CD46 mutants suggests that other determinants are also important in CD46 polarization. We hypothesize that the ERM proteins interacts with CD46, regulates its polarization in T cells, and perhaps play a role in CD46 signal transduction. To assess this we have generated mutations in the ERM-binding consensus sequences of CD46 and expressed these mutants in a uropod-containing T cell line for functional characterization. These studies will help to elucidate the molecular mechanisms by which CD46 exerts its immunoregulatory effects.
Regulation of Bcl10 by serine 231 (87.2)
Authors: Kane Lawrence P. , Delmastro Meghan , Jordanhazy Ryan 
Published: January 2023

Abstract
Bcl10 is a component of the “CBM” complex of proteins, along with CARMA and MALT1. This complex regulates activation of the NF-κB-inducing IKK kinase complex, and is important for integrating signals from antigen receptors and co-receptors on T and B lymphocytes. We previously showed that activation of NF-κB by the Akt kinase in T cells requires an intact CBM complex, and that Akt may interact with - and regulate the phosphorylation of - one or more members of the complex. Using the online ScanSite algorithm, we identified serine 231 in Bcl10 as a possible site of Akt phosphorylation. We generated a form of Bcl10 in which serine 231 has been mutated to alanine (S231A), and assessed effects on Bcl10 function. Thus, the S231A form of Bcl10 activates the NF-κB pathway much less efficiently in T cells, compared with the wild-type protein. Paradoxically, NF-κB activation by S231A Bcl10 is enhanced in fibroblasts. In both cell types, expression levels of S231A Bcl10 are consistently lower than the wild-type protein. Finally, we have examined the role of S231 in the regulation of Bcl10 localization. However, in contrast to its effects on NF-κB activation, mutation of S231 appears to have no impact on sub-cellular localization of Bcl10. These results suggest that phosphorylation of Bcl10 at S231 is important for regulation of NF-κB and that this pathway may operate differently in T cells and fibroblasts.
Supported by the Ovarian Cancer Research Fund.
Rac GTPases are required for restimulation-induced CD4+ T Cell Death (RICD) (87.20)
Authors: RAMASWAMY MADHU , DUMONT CELINE , MUPPIDI JAGAN R , TYBULEWICZ VICTOR L , SIEGEL RICHARD M 
Published: January 2023

Abstract
In activated CD4 T cells, TCR restimulation or antigen leads to FasL mRNA synthesis, expression and eventually apoptosis through cis or trans FasL-Fas interactions, in the process known as restimulation-induced cell death (RICD). Apoptosis requires both FasL synthesis and secretion and a protein-synthesis independent ‘competency to die’ signal that sensitizes TCR-stimulated cells to apoptosis induced by FasL. Recent work in our lab correlated competency signals with translocation of Fas into specialized membrane micro-domains (lipid rafts) in TCR restimulated T cells. However, what signaling pathways underlie the TCR-induced ‘competancy to die’ signal is not known. We have now identified the small Rho GTPase Rac as a modulator of TCR signals that mediate competency. SiRNA knockdown of Rac1 in jurkats and human CD4 T cells, results in specific abrogation of TCR induced ‘competency to die’ via Fas. Transfection of constitutively active Rac1 rescues TCR mediated Fas apoptosis in Vav-1 deficient Jurkat cells, which are devoid of competency signals. Absence of Rac1 does not affect a number of other parameters of early T cell signaling, however, we observe reduction in dephoshorylation of the Ezrin-Radixin-Moesin (ERM) proteins. CD2Cre Rac1 flox/flox mouse T cells as well as Rac2 −/− T cells have deficient Restimulation Induced cell death (RICD), which is more severe in T cells lacking both Rac1 and Rac2 (Rac1−/+/ Rac2 −/−). Further, in Jurkat and mouse T cells, Rac1 deficiency inhibits FasL upregulation upon TCR stimulation. Therefore, Rho GTPase proteins are critically involved in two distinct aspects of restimulation-induced T cell apoptosis and may be a novel target for immunomodulatory therapy.
The Gads adaptor protein regulates proliferation and differentiation of TCRβ+ CD4−CD8− double negative thymocytes (87.3)
Authors: Yankee Thomas M. , Zeng Ling , Dalheimer Stacy L. 
Published: January 2023

Abstract
TCRβ is the first TCR chain expressed and can be detected for the first time at a stage of T cell development that precedes expression of CD4 and CD8. TCRβ expression triggers signaling pathways that promote survival, proliferation, and differentiation into CD4+CD8+ double positive (DP) thymocytes. Despite the importance of survival, proliferation, and differentiation of TCRβ+ CD4−CD8− double negative (DN) thymocytes, the signaling pathways regulating these outcomes are unclear. To investigate the signaling pathways that regulate cell fate in TCRβ+ DN thymocytes, we are using a mouse line that lacks expression of the Gads adaptor protein. Gads is required for optimal TCR-mediated calcium mobilization and Gads−/− mice have a significant defect in T cell development at the DN to DP transition (J. Immunol. 173: 1711). Here, we extended these results by examining the role of Gads in the survival, proliferation, and differentiation of TCRβ+ DN thymocytes. We found that Gads regulates survival and proliferation of TCRβ+ DN3L thymocytes, but not TCRβ+ DN3E cells. In addition, using an in vitro differentiation assay, we found that Gads−/− DN thymocytes differentiated more readily into DP thymocytes than wild-type cells. These data support a model in which Gads regulates the number of cell divisions triggered by TCRβ expression.
This research is supported by NIH grant P20 RR016443.
T-cell activation requires mitochondrial translocation towards the immunological synapse (87.32)
Authors: Schwindling Christian , Quintana Ariel , Wenning Anna Sylvia , Becherer Ute , Rettig Jens , Schwarz Eva C. , Hoth Markus 
Published: January 2023

Abstract
Activation of the adaptive immune response requires the interaction between antigen-presenting cells and T-cells. This cell-cell interaction, called the immunological synapse (IS), facilitates the activation of several T-cell receptor (TCR)-mediated signalling cascades including a rise in the cytosolic Ca2+ concentration ([Ca2+]i) through the activation of CRAC/ORAI1 channels. These channels are opened after depletion of intracellular Ca2+ stores and inactivated by the inflowing Ca2+ itself. We show by epifluorescence, 2-photon and total internal reflection microscopy, that a large fraction of mitochondria was moved to the immediate vicinity of the IS, a process highly dependent on the actin cytoskeleton. Mitochondrial movement to the IS was required to sustain the CRAC/ORAI1-mediated Ca2+ influx. Disruption of the actin cytoskeleton prevented mitochondrial movement and subsequent [Ca2+]i rises. The increased [Ca2+]i signals following interaction of the IS with mitochondria correlated with an enhanced T-cell proliferation. Our results show that actin cytoskeleton-mediated movement of mitochondria into the vicinity of the IS is required to sustain Ca2+ influx and permit efficient T-cell activation.
This project was funded by the Deutsche Forschungsgemeinschaft (SFB 530 and GRK 845) and a grant from the Saarland University (HOMFOR).
The X-linked lymphoproliferative disease gene product SAP associates with PIX and participates in T cell activation (87.37)
Authors: Wu Jun 
Published: January 2023

Abstract
SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is a SH2 domain-containing adaptor expressed in T cells and natural killer cells. Its essential role in immune responses is underscored by the recent finding that mutations in SAP result in a rare but fatal X-linked lymphoproliferative disease (XLP). Although SAP is known to associate with SLAM-family receptors, the exact molecular mechanism by which SAP regulates lymphocyte signaling remains elusive. We here report that in T cells, SAP associates with the PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor specific for Rac/Cdc42 GTPases. Moreover, SAP, PIX, and an activated form of Cdc42 form a complex in mammalian cells. We demonstrate that the SAP-PIX interaction is specific and is mediated by the C-terminal region of the SAP SH2 domain and the PIX SH3 domain. We further show that SAP is required for the recruitment of PIX to the SLAM-family receptors. Interestingly, over-expression of SAP, but not its homolog EAT-2, leads to a synergistic activation of NFAT in combination with a calcium signal in T cells. This SAP-mediated activation appears to be receptor-dependent and can be blocked by a dominant negative form of PIX. Taken together, our data strongly suggest that, in addition to the known SAP-interacting kinase Fyn, PIX may be another key player in SAP-mediated T cell activation.
The Requirement of Reversible Cysteine Sulfenic Acid Formation in T Cell Activation and Function (87.40)
Authors: Michalek Ryan D , Nelson Kimberly J , Yi John S , Stridiron Daya , King S Bruce , Poole Leslie B , Grayson Jason M 
Published: January 2023

Abstract
Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naïve CD8+ T cell activation and proliferation. We observed that within minutes of T cell activation, naïve CD8+ T cells increased ROI levels in a manner dependent upon antigen concentration. With increased ROI, levels of cysteine sulfenic acid were also elevated. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation increasing concentrations of 5, 5 dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, proliferation, and cytokine production of naïve CD8+ and CD4+ T cells. We also found that TNF-α production by effector and memory CD8+ T cells was more sensitive to inhibition of reversible cysteine sulfenic acid formation than IFN-γ. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8+ T cells are able to modulate signaling, proliferation, and function.
Research supported by American Cancer Society- #RSG-04-066-01-MBC to Jason Grayson.
A molecular analysis of diminished expression of regulatory subunit β protein of type I protein kinase A in SLE T cells (87.45)
Authors: Giri Banabihari , Das Maitreyee , Grayson Jason M. , Khan Islam U. 
Published: January 2023

Abstract
The deficiency of type I protein kinase A (PKA-I) isozyme in systemic lupus erythematosus (SLE) T cells is a signaling disorder and occurs with a prevalence of 80%. Our earlier studies established that SLE T cells have a profound deficiency of regulatory subunit β of PKA-I (RIβ) protein. We hypothesized that the reduction of RIβ-subunit protein is due to altered post-transcriptional processing and/or instability of RIβ mRNA in SLE T cells. To test our hypothesis we studied the translational efficiency of RIβ mRNA in T cells in SLE patients and healthy subjects. CD3+ T cells from human blood were enriched by negative selection with magnetic microbeads. The polysomal fractions were isolated from cycloheximide-treated T cells on linear sucrose gradient. Total RNA was extracted from each polysomal fraction followed by RIβ mRNA quantification by quantitative real-time PCR (Q-PCR). The PCR analysis of polysomal fractions revealed that the polysome profile of SLE T cells was consistent with a global translational attenuation, and specifically a paucity of polysome-bound RIβ mRNA. Interestingly, the stimulation of SLE T cells with a combination of anti-CD3 and anti-CD28 antibodies restored the RIβ mRNA profile, consistent with active translation. In conclusion, reduced RIβ-protein expression in SLE T cells is the consequence of the instability of RIβ mRNA and its absence in high molecular weight polysomes.
Supported by NIH RO1-AR39501.
The role of non-stimulatory MHC-peptides in aiding antigen recognition by CD8+ T cells (87.49)
Authors: Gascoigne Nicholas R.J. , Yachi Pia P. , Lotz Carina , Ampudia Jeanette 
Published: January 2023

Abstract
T cells are able to identify minute quantities of antigenic MHC-peptide (MHCp) complexes among a sea of endogenous MHCp presented on an antigen presenting cell. We previously showed that non-stimulatory endogenous peptides can enhance T cell sensitivity to limiting quantities of antigen. Here we extend these findings to different T cell populations, and show that non-stimulatory peptides are most important in antigen recognition by less differentiated cells, such that their ability to enhance antigen recognition is the best in thymocytes, moderate in naïve T cells, and mild in effector T cells. The increased sensitivity to antigen in the presence of non-stimulatory peptides can be seen in the level of effector functions such as cytokine production and killing of target cells, as well as by phenotypic maturation markers, such as CD25, CD69, CD44, CD5 and HSA. The importance of non-stimulatory peptides is inversely proportional to the agonist activity of the stimulatory peptide presented. Each of the endogenous non-stimulatory peptides tested showed similar ability to aid in antigen recognition. This may indicate that the limiting factor in the ability of non-stimulatory MHCp to aid recognition of a class I-restricted antigen is the CD8-MHC interaction, rather than the strength of the TCR-MHCp interaction, which seems to be paramount in class II-restricted systems.
Supported by NIH GM065230.
IFN-γ secretion is polarized at effector immunological synapses (IS) in vivo. (87.52)
Authors: Lowenstein Pedro R , Barcia Carlos , Liu Chunyan , Wawrowsky Kolja , Kroeger Kurt M , Castro Maria G 
Published: January 2023

Abstract
In vitro cytokine secretion by T cells is polarized. For example, IFN-γ secretion is focused towards the immunological synapse (IS), while secretion of TNFα is multi-directional. Whether polarized cytokine secretion occurs in vivo is unknown. We studied the intracellular distribution of IFN-γ within T cells establishing IS with infected astrocytes in vivo. Astrocytes were infected with an adenovirus (Ads) expressing Thymidine Kinase (TK), a marker for infected cells. 30 days later we induced a systemic immune response against Ads, and 14 days after the immunization animals were sacrificed and brains studied by immunohistochemistry and confocal microscopy. Brain sections were stained for TCR, LFA-1, TK and IFN-γ. Confocal analysis revealed that IFN-γ is polarized in T cells establishing close anatomical contacts with Ad infected cells. Polarization was found in cells with or without polarized LFA-1 or TCR. This demonstrates that in vivo IFN-γ is selectively polarized towards the APC, and further suggests that maturation of the IS is not necessary for IFN-γ polarization. In consequence, polarized IFN-γ secretion may precede the maturation of immunological synapses.
This work was funded by grants from NINDS, NIH.
CD8+ T Cells Possess the Potential to Differentiate into Both a High or Low Avidity Effector Cell (87.7)
Authors: Kroger Charles J. , Alexander-Miller Martha A. 
Published: January 2023

Abstract
During an immune response, CTL clones are present that exhibit a wide spectrum of peptide sensitivities. While the property of functional avidity has been well documented, the range of mechanism(s) by which this T cell property can be regulated is unknown. For example, an unresolved question is whether distinct naïve T cell subsets exist that display “inherent” differences in their peptide requirements for activation, i.e. functional avidity. In this scenario distinct naïve T cell clones would selectively respond upon encountering a high vs. low antigen concentration. Alternatively, differences in peptide sensitivity could be an “induced” property within the responding CTL population following encounter with an APC. Although the latter model is consistent with data from previous studies, formal demonstration that a single clone can give rise to both high and low avidity daughter cells is lacking. Using a TCR transgenic model in which both high and low avidity CTL can be generated, we now demonstrate that naïve T cells do not exhibit differences in the amount of peptide required for conjugate formation and subsequent proliferation/activation. Furthermore, we show that individual T cell clones are capable of differentiating into either a high or low avidity CTL. These results provide the first formal demonstration that an individual cell can give rise to both high and low avidity progeny, suggesting that avidity modulation at the level of an individual cell may be an important contributor to the functional avidity of the CD8+ T cell response generated in vivo.
This work was supported by National Institutes of Health grant R01 AI43591 to M.A.A-M.
The dynamic equilibrium between dimeric and monomeric cell surface B7-1 regulates T cell activation and CD28 expression (88.1)
Authors: Bhatia Sumeena , Almo Steven C , Nathenson Stanley G , Hodes Richard J 
Published: January 2023

Abstract
Numerous studies have identified differences between B7-1:CD28/CTLA-4 and B7-2:CD28/CTLA-4 complexes that form at the immunological synapse. Our earlier studies have established that B7-1 is expressed as a mixture of predominant non-covalent dimers and monomers on the cell surface. In order to test the hypothesis that altering the nature of B7-1:CD28 complexes will have functional consequences for T cell activation, we generated a disulfide-linked covalent dimer of B7-1 (B7-1:Cys), as well as mutants of B7-1 (L58D and I68D) that exist as a monomer on the cell surface. We used modulation of cell surface CD28 to assess the outcome of binding of B7-1 mutants to CD28 as one measure of the dynamics of B7-1:CD28 interaction since earlier studies have shown that CD28 is transiently downregulated upon binding to WT B7-1. As expected, L58D and I68D mutants resulted in reduced T cell activation and reduced CD28 downregulation when compared to WT B7-1, potentially due to low avidity interaction between monomeric B7-1 and monovalent CD28. Surprisingly, B7-1:Cys also resulted in less T cell activation than WT B7-1. Moreover, B7-1:Cys also resulted in reduced downregulation of CD28. These data suggest a novel model in which optimal B7-1 function for CD28 downmodulation and T cell activation requires CD28 engagement by non-covalent B7-1 dimers, followed by dissociation of B7-1 to a monomeric state, potentially facilitating disengagement from CD28.
Reciprocal NFAT1 and NFAT2 nuclear localization in anergic T cells is determined by sub optimal calcium signaling (88.15)
Authors: Srinivasan Mathangi , Frauwirth Kenneth 
Published: January 2023

Abstract
Impaired calcium signaling is a hallmark of T cell anergy. Calcium signaling induces nuclear translocation and activation of the NFAT transcription factors by activating the NFAT phosphatase calcineurin. Using an in vivo anergy model, we found that anergic T cells showed defective nuclear localization of NFAT1 after stimulation compared to responsive cells. NFAT2 remained cytosolic in responsive T cells, but showed nuclear localization after stimulation of anergic cells. To understand the mechanism underlying the translocation pattern, we stimulated naïve T cells in the presence the calcium chelator EGTA. NFAT1 was detected in the nucleus at complete extracellular calcium concentrations, but when calcium levels were reduced with EGTA, NFAT2 was nuclear, while NFAT1 was cytosolic. This pattern of translocation was also seen upon cyclosporin treatment, at concentrations that cause partial inhibition of calcineurin. These data suggest that the differential translocation regulation of NFAT1 and NFAT2 in anergic T cells can be correlated to the defective calcium signaling during anergy. This implies that the gene transcription profile of NFAT1 and NFAT2 in anergic T cells could be regulated differently from responsive cells. Ongoing studies are directed at studying transcriptional activity of NFAT1 and NFAT2 in anergic T cells.