bluuebunny/crossref_metadata_2025_split huggingface dataset:
An allelic series of CD45 dosages uncouples basal and inducible TCR signaling during thymic development (84.13)
Authors: Zikherman Julie , Watson Susan , Jenne Craig , Raschke William C. , Goodnow Christopher , Weiss Arthur 
Published: January 2023

Abstract
CD45 is a receptor-like tyrosine phosphatase expressed at very high levels on all nucleated hematopoietic cells. CD45 serves as a critical positive regulator of TCR signal transduction by dephosphorylating the inhibitory tyrosine of the Src family kinases, Lck and Fyn. Previously, CD45 deficient animals have revealed a critical positive regulatory role in beta-selection and positive selection during thymic development.
Here we describe a novel allele of CD45, lightning, in which a single residue substitution in the extracellular domain produces low protein expression levels but normal isoform splicing. An allelic series of CD45 dosages reveals differential requirements for CD45 in basal and inducible TCR signaling during thymic development. While low levels of CD45 are sufficient to rescue T cell development and TCR inducible signaling, high doses of CD45 are required to fully reconstitute beta-selection and basal signaling. Furthermore, a negative regulatory role for CD45 is identified during inducible but not basal signaling. Hyperphosphorylation of the activation loop tyrosine of Lck but not Fyn at intermediate doses of CD45 correlates with hyper-responsive inducible signaling and demonstrates differential regulation of the src family kinases. Further, distinct thymic subsets exhibit differential requirements for CD45, Fyn and co-receptor-associated Lck in TCR signaling.
c-Myb is a novel regulator of Bcl-xL in the CD4+ and CD8+ double positive stage of T cell development (85.2)
Authors: Yuan Joan , Crittenden Rowena B , Rajewsky Klaus , Bender Timothy P 
Published: January 2023

Abstract
The lifespan of quiescent CD4+ and CD8+ double positive (DP) thymocytes regulates the composition of the peripheral T cell repertoire. However, the molecular mechanisms controlling DP thymocyte survival remain poorly understood. The c-Myb transcription factor is highly expressed in quiescent pre-selection DP thymocytes. Using a conditional deletion mouse model, we demonstrate that pre-selection DP thymocytes lacking c-Myb undergo premature apoptosis due to decreased expression of the key anti-apoptotic factor Bcl-xL. Moreover, c-Myb deficient DP thymocytes display a skewed TCRα repertoire, with a bias towards 3' Jα?usage, as a consequence of premature apoptosis occurring shortly after the initiation of TCRα rearrangements in vivo. Finally, re-introduction of c-Myb to c-Myb deficient DP thymocytes restored control levels of Bcl-xL mRNA and protein. Taken together, our study identifies c-Myb as a novel regulator of Bcl-xL expression in the DP stage of T cell development. We demonstrate that c-Myb acts through a novel pathway, independently of RORγt and TCF-1, two transcription factors previously shown to promote Bcl-xL expression in DP thymocytes. This study sheds new light on the regulation of the survival of pre-selection DP thymocytes and provides a novel mechanism by which c-Myb guards the balance between life, death and differentiation during hematopoiesis.
Id1 attenuates Notch signaling and impairs T cell commitment by elevating Deltex1 expression (85.3)
Authors: Wang Hong-Cheng , Sun Xiao-Hong 
Published: January 2023

Abstract
Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double negative 1 (DN1, CD44+CD25-) thymocytes to the DN2 (CD44+CD25+) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44-CD25+) stage, which was accompanied by enhanced Notch1 expression in T cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased whereas GATA3 levels increased in Id1 transgenic thymocytes, and GATA3 expression up-regulated Deltex1 transcription. Thus, T cell commitment may be controlled by the interplay among E proteins, Gfi1b and GATA3 transcription regulators, which influence Notch function through Deltex1 expression.
This work was supported by grant from the Nation Institute of Health to X.-H. S. (CA77553).
The CD3 ε subunit of the T cell receptor contains a basic-rich stretch important for multiple T cell functions (85.4)
Authors: Watts Laura M , Tassin Tara C , Becker Amy M , Medeiros Jennifer J , Albanesi Joseph P , Love Paul E , Wülfing Christoph , van Oers Nicolai S C 
Published: January 2023

Abstract
The TCR is a multimeric complex, consisting of a ligand-binding α/β heterodimer and four associated signaling chains (CD3γ, δ, ε, and ζ). The CD3 subunits each possess one or more copies of a signaling motif termed an ITAM, which is rapidly phosphorylated on tyrosine residues following TCR engagement. The cytoplasmic tails of CD3 ε and ζ also contain one or more clusters of basic amino acids. We report that these basic-rich stretches (BRS) can complex a subset of acidic phospholipids, including PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. To determine how the BRS of CD3 ε contributes to T cell functions, we generated several CD3 ε constructs in which the positively charged residues of the BRS were eliminated. Analysis of these constructs in transfected cells revealed a role for the BRS in regulating both ITAM phosphorylation and TCR translocation to the immunological synapse. In transgenic mice, the introduction of modifications to the CD3 ε BRS resulted in a significant reduction in surface TCR expression and thymic cellularity. Furthermore, T cells from the BRS-mutant mice exhibited a diminished tyrosine phosphorylation of several signaling intermediates following TCR ligation. Based on these findings, we propose a model whereby electrostatic interactions between the BRS and phospholipids expressed on intracellular membranes regulate multiple T cell functions, including TCR expression and signaling.
Foxn1 is Required for Thymic Vascularization (86.7)
Authors: Manley Nancy R , Bryson Jerrod L , Griffith Ann V , Takahama Yousuke , Richie Ellen R 
Published: January 2023

Abstract
Development of a functional thymic microenvironment depends on crosstalk between multiple cell types. Recent studies suggest that crosstalk between developing vasculature and thymic epithelial cells (TEC) actively contributes to the complex thymic architecture, but little is known about initial immigration of endothelial precursor cells (EPC) into the thymus or subsequent vasculature formation. In a Foxn1 hypomorphic allele, Foxn1ΔΔ, TEC differentiation is arrested and cortical and medullary regions fail to form. We used wild-type, Foxn1Δ, and nude mice to define a role for Foxn1 in thymic vascularization. We show that EPCs first enter the wild-type thymus at E13.5, PDGFR-β + NCC perivascular mesenchyme follows at E14.5, and the vasculature is functional at E15.5 by the detection of intrathymic FITC-dextran after intraocular injection. EPC entry was delayed by one day in Foxn1ΔΔ mice, associated with down regulation of VEGF-A, PDGF-β, and CCL25 by 28%, 49%, and 98% at E13.5 compared to controls. In Foxn1Δ/nu mice, EPC entry was delayed until E15.5, and VEGF-A, PDGF-β, and CCL25 were more severely down regulated. In nude mice, endothelial cells could not be detected in the thymus rudiment. These data suggest that Foxn1 is required in a dose-dependent manner in TECs to regulate VEGF-A and PDGF-β expression necessary for normal thymic vascularization, and CCL25 for recruitment of LPCs in the embryonic thymus.
Bcl3 protects against lung graft injury by limiting emergency granulopoiesis (87.7)
Authors: Sugimoto Seiichiro , Kreisel Daniel , Carmody Ruaihdrhi , Gelman Andrew E 
Published: January 2023

Abstract
Excessive granulocyte accumulation in solid organ grafts leads to severe tissue injury and dysfunction. However, the mechanisms that differentially regulate emergency granulopoiesis from homeostatic granulopoiesis are poorly understood. The IκB family member Bcl3 is thought to mainly control cell survival and toll-like receptor mediated inflammatory gene expression. Here we report Bcl3 expression is required to protect against lung graft injury in a mouse orthotopic lung transplantation model by attenuating emergency granulopoiesis. Notably, Bcl3 was not required for homeostatic granulopoiesis as mice reconstituted with Bcl3-/- bone marrow (B6 (Bcl3-/-)) had equivalent numbers of granulocytes as compared to control wildtype B6 (B6) mice but following lung transplantation B6 → B6 (Bcl3-/-) recipients accumulated significantly more intragraft and peripheral blood granulocytes which exacerbated graft injury relative to B6 → B6 (B6) recipients. However, graft injury and granulocyte accumulation could be significantly attenuated with G-CSF neutralizing antibodies administered to B6 (Bcl3-/-) recipients. Moreover, G-CSF but not GM-CSF or IL-3 administration to resting B6 (Bcl3-/-) mice produced 2-fold more peripheral blood granulocytes than control wildtype B6 (B6) mice. Analysis of common myeloid progenitors revealed that Bcl3 negatively regulates the proliferation and the capacity of these cells to support emergency granulopoiesis following lung transplantation as well as following G-CSF but not GM-CSF or IL-3 stimulation. These data demonstrate a critical negative regulatory role for Bcl3 in G-CSF mediated emergency granulopoiesis and suggest that modulating signals that control the differentiation of myeloid progenitors can help prevent solid organ injury.
Cytokine treatment of macrophage suppression of the Mls response (88.3)
Authors: Swider Adam , Kozlowski Megan , Silberman Daniel , Bucknum Amanda , Matlack Robin , Orlowski Michelle , Riggs James 
Published: January 2023

Abstract
Components of the immune system essential for curbing tumor progression are often found within the tumor. High macrophage to T cell ratios (Mö:T) are hallmarks of the tumor microenvironment. We have shown that such conditions suppress the immune response to the retroviral superantigen Mls by IFN ã -triggered production of the Arg- and Trp-consuming enzymes iNOS and IDO. In attempts to reverse suppression by treatment with pro-inflammatory cytokines, IL-6 improved the T cell response to Mls, IL-1 and TNFá had little effect, and the pro-hematopoietic cyokines IL-3 and GM-CSF increased suppression. GM-CSF treatment increased Mö expression of CD80, a significant ligand for the immunosuppressive B7H1 and CTLA-4 receptors. These results reveal avenues for reversal of the suppression of cell-mediated immunity characteristic of the high Mö:T cell ratios found in many tumors. Supported by NIH AREA R15-AI060356-01.
Induction of peripheral Tregs by homotypic T cell interaction (89.3)
Authors: Thuemmler Katja , Ramming Andreas , Leipe Jan , Schulze-Koops Hendrik , Skapenko Alla 
Published: January 2023

Abstract
The mechanisms by which regulatory T cells are generated in the periphery are incompletely understood. Here we show that homotypic T cell interaction results in the generation of T cells with a potent regulatory capacity.
CD4 memory T cells that were cultured together with activated effector T cells proliferated and produced various cytokines depending on the nature of the activated effector T cell. Whereas Th1 cells promoted the development of IL-10 and/or IFNγ producing cells, T cells cultured with Th2 cells produced IL-4. The interaction between the adhesion receptors ICAM-1, 2, 3 and LFA-1, respectively was crucial for T-T cell communication with regard to proliferation and cytokine production. Blocking of ICAM-1 diminished IFNγ production, whereas ICAM-3 was important for IL-4 secretion. Interestingly, the resulting cells strongly inhibited proliferation of CD25- T cells in a dose dependent manner independent of their cytokine secretion profile. Moreover, in vitro generated T-T cell induced T cells with a TCR specific for OVA prevented the expansion of OVA specific effector T cells in Balb/c mice in vivo upon antigen challenge.
The results indicate that homotypic T cell interaction induces the generation of T cells with a regulatory phenotype, providing a novel negative feedback mechanism to control sustained T cell driven immunity.
Induction of granzyme B expression in human regulatory T cells requires TCR and CD28 triggering but not IL-2 and is suppressed by inhibitors of the PI3K-mTOR pathway. (89.40)
Authors: Kelley Todd W. , Efimova Olga 
Published: January 2023

Abstract
Regulatory T cells (Tregs) can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Granzyme B may also be involved in the suppression of anti-tumor immunity in the tumor microenvironment. In spite of its central importance, a detailed study of granzyme B expression patterns in Tregs has not been performed. Using enriched cultures of CD4+,CD25+,FOXP3+ T cells freshly isolated from the peripheral blood of normal adults, we demonstrate that natural Tregs lack granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering express high levels of granzyme B but CD3 stimulation alone fails to significantly induce such a response. IL-2 stimulation alone also fails to induce granzyme B and there is no co-requirement for exogenous or endogenous IL-2 for granzyme B expression. We find that treatment of Tregs with the mTOR inhibitor rapamycin or the PI3 kinase (PI3K) inhibitor LY294002 markedly suppresses granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibits Treg proliferation or induces significant cell death in TCR/CD28/IL-2 stimulated cells. Rapamycin, but not LY294002, selectively favors the expansion of Tregs over conventional CD4+ T cells. Thus, we conclude that TCR/CD28 mediated activation of mTOR is necessary for granyzme B expression but not proliferation in regulatory T cells. These findings may have implications for the biology of regulatory T cells generated in the clinical setting of rapamycin treatment.
The Effect of Phosphatidylserine on T Cell and Dendritic Cell Function (90.2)
Authors: Xia Changqing , Gangrade Abhishek , Peng Ruihua , Clare-Salzler Michael J 
Published: January 2023

Abstract
Phosphatidylserine (PS) is a phospholipid of plasma membrane. PS is located in the inner leaflet of plasma membrane but turns to outer leaflet when cell death program is initiated. It has been demonstrated that PS on apoptotic cells plays an important role in modulating immune responses. However, whether apoptotic cells release soluble PS which may subsequently affect immunocytes is not known. Recently, we found that apoptotic cells released soluble PS to the culture media which could block the binding of anti-PS antibody to apoptotic cells. To address how soluble PS influences T cell and dendritic cell function, we examined the effect of water-soluble PS (Avanti Polar Lipids, &gt;95% purity with no endotoxin) on T cell proliferation, and monocyte-derived dendritic cell (DC) cytokine and chemokine production. Our results demonstrated that PS suppressed T cell proliferation in a significant dose-dependent manner. However, PS appeared not affecting T cell early activation based on the normal up-regulation of CD69. On DCs, PS did not induce any cytokine or chemokine production. However, PS significantly altered LPS-stimulated DC cytokine and chemokine producing profiles. We found that PS enhanced LPS-induced IL-1beta, IL-alpha, GM-CSF and IL-6, but suppressed IL-10, IL-12p40 as well as most chemokines tested. There was no significant influence on DC maturation. Our findings indicate that the apoptotic cells may modulate immune responses through releasing soluble PS. The mechanisms of PS effect on T cells and DCs are being investigated.
Thy-1 signaling in murine T cells promotes cytokine polarization (90.20)
Authors: Furlong Suzanne J , Hoskin David W 
Published: January 2023

Abstract
Glycosylphosphatidylinositol-anchored proteins (GPI-AP) that provide activating signals to T cells have been implicated in T cell-mediated immune responses. The effect of GPI-AP signaling on cytokine production and the development of effector T lymphocyte phenotypes remains unclear. In this study, highly pure resting T cells were stimulated with either anti-Thy-1 or anti-TcR monoclonal antibody (mAb) along with costimulatory signals provided by LPS-matured dendritic cells. At concentrations of mAb that induced optimal proliferation, Thy-1-activated T cells proliferated ~2-fold less than TcR-activated T cells. Analysis of cytokine production using cytokine arrays and ELISA showed that Thy-1 signaling induced significantly less IL-2 and IFN-γ synthesis than TcR signaling. Interestingly, Thy-1-stimulated T cells produced significantly more IL-17 and IL-4 than TcR-stimulated T cells. Cytokine polarization was unique to Thy-1, as T cells stimulated with mAb against the GPI-AP Ly6A/E did not produce appreciable levels of IL-17 or IL-4. These results support previous reports that Thy-1 signaling differs from TcR signaling and suggest that Thy-1 may provide polarizing signals that promote the development of Th17 and Th2 cells. Thy-1 and other GPI-AP proteins require further study to elucidate their possible roles in influencing the nature of T cell-mediated immune responses. Research was funded by NSERC
Combining PDL-1 blockade with agonistic antibodies against 4-1BB enhances T cell restoration after chronic LCMV infection (90.22)
Authors: Penaloza MacMaster Pablo , Vezys Vaiva , Ha Sang-Jun , Konieczny Bogumila , Barber Daniel , Mittler Robert , Ahmed Rafi 
Published: January 2023

Abstract
The PDL-1:PD-1 pathway is involved in T cell exhaustion after chronic viral infection. PD-1 can bind to either PDL-1 or PDL-2, whereas PDL-1 can bind to either PD-1 or B7.1. We tested the effect of combining standard aPDL-1 blockade with a4-1BB (3H3 agonistic antibody) treatment to determine T cell restoration effects in a model of chronic infection with lymphocytic choriomeningitis virus (LCMV). We chose 4-1BB because it has been shown to be an important costimulatory pathway. It appears that a4-1BB treatment enhances the effect of aPDL-1 blockade if given in a low dose (50 μg, once). Conversely, if a4-1BB is given in a high dose (200 μg, five times), it results in sudden increase of antigen-specific T cells by day 7, but massive T cell death by day 14 post treatment. These results show the importance for 4-1BB costimulation in aPDL-1 mediated T cell restoration.
Selective induction of robust antibody responses following antigen targeting to dendritic cell subsets (90.34)
Authors: Chappell Craig P , Draves Kevin E , Clark Edward 
Published: January 2023

Abstract
Dendritic cells (DCs) are essential for the initiation and regulation of immune responses. CD8α+ DCs in the mouse spleen express DEC-205 (CD205), while many splenic CD8α- DCs express DC-inhibitory receptor-2 (DCIR2/33D1). Delivering antigen (Ag) via mAbs to either DEC-205 or DCIR2 was shown by other groups to preferentially induce CD8+ and CD4+ T cell responses, respectively. However, the ability of these two DC subsets to induce primary Ab responses is not well defined. Using the conventional hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to mAbs that recognize DEC-205 or DCIR2, we demonstrate that DCIR2+ DCs, but not DEC-205+ DCs, are able to induce robust anti-NP Ab responses following intravenous injection of soluble Ag. These Ab responses were restricted to the IgG1 subclass with little to no IgM or IgG2a production. Surprisingly, the DCIR2-induced Ab response did not require the addition of conventional adjuvants. These responses were both TCR and CD40-dependent, indicating that T-B cell collaboration is required. Finally, we found that CD22 expression by B cells was also required for IgG1 anti-NP responses, suggesting that DC-B cell interactions may be important during early B cell priming via this route of administration. Together these results demonstrate that, similar to the selective priming of T cells by these DC subsets, DCs are also specially equipped to selectively prime B cells and Ab responses. This project was supported in part by NAIAD training grant T32AI007411 and by NIH grant AI52203 and does not necessarily represent the official views of the NAIAD or the NIH.
Differential effects of negative costimulatory molecules BTNL2 and PD-L1 on the activation of T cells when using MHC class I tetramer, MHC class II tetramer, or anti-CD3 immobilized on beads (90.6)
Authors: Swanson Ryan , Viney Joanne L 
Published: January 2023

Abstract
Immunologists have identified a growing list of negative costimulatory molecules in the B7 family of proteins. These molecules are defined in vitro by their capacity to inhibit purified T cell proliferation and cytokine production only when immobilized proximally to anti-CD3. We are interested in whether the negative costimulatory molecules behave differently when activating T cells in an antigen specific manner. The required proximal immobilization with TCR agonist can be achieved on beads loaded with MHC class I or class II tetramers and subsequent culture with appropriate transgenic T cells. In this poster, we show evidence that CD4 and CD8 T cells are inhibited differently when stimulated with peptide/MHC molecules compared to activation with anti-CD3. The two negative costimulatory molecules we are currently studying, BTNL2 and PDL1, block activation under different circumstances. Although both molecules can inhibit T cell responses to anti-CD3, PDL1 is more effective at inhibiting CD8 responses to MHC class I than CD4 responses to MHC class II. Conversely, BTNL2 inhibits CD4 T cells in the same system, but cannot block the CD8 response as well as PDL1. These studies imply that different negative costimulatory molecules may have cell specific roles and provide a system to investigate regulatory mechanisms that result in differential inhibition of CD8 and CD4 T cells.
IL-15 produced and trans-presented by DCs underlies homeostatic competition between CD8 and gd T cells under lymphopenic conditions (90.7)
Authors: Do Jeong-su , Min Booki 
Published: January 2023

Abstract
Naive T cells undergo proliferation in lymphopenic environments. However, cellular mechanisms controlling the proliferation and the maintenance of T cells under these conditions remain elusive. Here, we compared naive T cell proliferation and homeostatic maintenance under different lymphopenic conditions: TCRβ-/- and Rag1-/-. Both CD4 and CD8 T cells fully diluted CFSE content under these conditions. While CD4 and CD8 T cells equally proliferated in Rag1-/- mice, enhanced CD4 and impaired CD8 T cell expansion was found in TCRβ-/- mice. B cells played little role in the differential expansion of T cell subsets. However, depletion of γδ T cells in TCRβ-/- mice was sufficient to restore the impaired expansion of CD8 T cells. Similarly, adoptive transfer of γδ T cells into Rag1-/- recipients altered expansion of T cell subsets similar to that within TCRβ-/- recipients. DCs in Rag1-/- mice expressed higher levels of IL-15Rα as well as of membrane bound IL-15 than DCs in TCRβ-/- mice. Adoptive transfer of wild type DCs into TCRβ-/- recipients enhanced CD8 T cell expansion, yet such enhancement was absent following IL-15-/- DC transfer. Collectively, our findings demonstrate that γδT cells suppress naive CD8 but not CD4 T cell expansion under lymphopenic conditions by competing IL-15 trans-presented by DCs, revealing previously unrecognized homeostatic mechanisms between different T cell subsets in vivo.