bluuebunny/crossref_metadata_2025_split huggingface dataset:
Twist1 impairs inflammatory cytokine production in T helper 17 cells (P1227)
Authors: Pham Duy , Walline Crystal , Blum Janice , Kaplan Mark 
Published: January 2023

Abstract
Subsets of T helper cells are specialized for the production of specific cytokines, and this specificity is controlled by the function of transcription factors in each subset. Twist1, a basic helix-loop-helix (bHLH) transcription factor, is induced by STAT3-activating cytokines and inhibited by TGF-β in Th17 cells. Ectopic Twist1 expression in Th17 cells reduced IL-17A and IL-17F production while Twist1-deficient Th17 cells produced more IL-17A, IL-17F, and other inflammatory cytokines than wild type cells. In a myelin oligodendrocyte glycoprotein (MOGp35-55)-induced EAE disease model, Twist1-deficient mice manifest earlier onset of paralysis compared to wild type mice. Mechanistically, Twist1 controls cytokine production in Th17 cells by directly repressing Il6ra expression, limiting activation of the IL-6-STAT3 signaling pathway. Thus, Twist1 inhibits inflammatory cytokine production in Th17 cells, suggesting a central role for Twist1 in the regulatory network controlling autoimmune disease.
Regulated cytoskeletal control of ICAM-1 mobility on the dendritic cell plasma membrane enhances T cell activation (P1205)
Authors: Comrie William , Boyle Sarah , Li Shuixing , Burkhardt Janis 
Published: January 2023

Abstract
T cell activation involves actin-dependent assembly and centripetal movement of signaling microclusters at the Immune Synapse (IS). Model stimulatory surfaces that limit microcluster dynamics enhance TCR signaling, but the possibility that antigen presenting cells can modulate T cell signaling by regulating ligand distribution is largely unexplored. Dendritic cells (DCs) form a multi-focal IS rather than the concentric pattern seen with freely mobile ligands, suggesting that DCs can control receptor-ligand localization. We have measured the lateral mobility of T cell ligands on the DC membrane, and find that ICAM-1, the key ligand for the integrin LFA-1, is selectively constrained via interactions with the actin cytoskeleton. ICAM-1 mobility decreases further upon LPS-induced DC maturation, concomitant with upregulation moesin and α-actinin 1, two actin-binding proteins that bind the cytoplasmic tail of ICAM-1. ICAM-1 co-localizes with moesin at punctate sites on the DC surface. Mutation of the ICAM-1 cytoplasmic tail or suppression of moesin and/or α-actinin liberates ICAM-1 lateral mobility. Importantly, liberating ICAM-1 from cytoskeletal constraints decreases the ability of DCs to induce the proliferation of naïve CD4 T cells. Taken together, our data show that the DC actin cytoskeleton actively controls ligand mobility and thus enhances T cell activation, and raise the possibility that DCs modulate T cell signaling by actively regulating molecular dynamics at the IS.
FoxP3+ Tregs cells sensitivity to low doses of IL-2 can be rapidly and easily monitored by p-STAT-5 detection in ex-vivo whole blood samples. (P1211)
Authors: Malergue Fabrice , Mallet Valérie , Scifo Caroline , Van Agthoven Andreas , Miloud Tewfik , Montero-Julian Felix A 
Published: January 2023

Abstract
Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2 to 4 hours, 37°C and ice incubations, and 4 to 8 wash steps. The reference methods also uses methanol which is toxic for the user and harmful for the other cell markers, what compromises some multi-parametric studies. The FoxP3 marker is incompatible with “harsh” P-epitopes detection methods, rendering even more challenging the studies of Tregs functions. Methods: Here, we used a new commercially available kit; named PerFix EXPOSE (Phospho Epitopes Exposure Kit; for Research Use Only) based on multiple innovations in the permeabilization, staining, and wash steps. It is devoid of methanol and supports staining of all common P-epitopes with one single procedure of about 1 hour. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimisations. The best conditions were then used to analyse the ex-vivo functionality of FoxP3+ cells: The IL-2 signalling pathway especially. Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and p-STATs. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10 to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.
Enhanced susceptibility of RIPK3 deficient mice to influenza A virus infection (P6196)
Authors: Sharma Shalini , Sturm Oliver , Green Douglas , Thomas Paul 
Published: January 2023

Abstract
Viral infection can activate cytokine expression and trigger cell death. Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense while also regulating the function and survival of professional immune cells, including T cells. Receptor-interacting protein kinase (RIPK3) has been shown to mediate programmed necrosis induced by certain viral infections. Deficiency of RIPK3 results in compromised elimination of pathogen-infected cells and increased viral burdens. We present evidence for this effect in mice acutely infected with influenza A virus (IAV). RIPK3-deficient (RKO) mice have significantly increased body weight loss after both mild IAV x31 infection and severe IAV PR8 infections, with decreased survival in the latter case and significantly increased viral loads in the lungs compared to littermate controls. However these effects were primarily observed after day 7 post infection, indicating that regulation of death in the virally-targeted epithelial cell was not the cause of the observed pathogenic phenotype. Indeed, ex-vivo tetramer and ICCS assays showed significantly diminished virus-specific CD8 T cell responses with a reduced frequency of bifunctional IFNγ+ and TNFα+ cells and a lower MFI of IFNγ production by RKO CD8 T cells suggesting that the increased viral loads and weight loss in RKO animals may be a result of reduced T cell activity. Current studies are addressing the role of RIPK3 in regulating T cell function.
Natural Killer cells in defense against parainfluenza virus (P6178)
Authors: Hendricks Deborah , Kohlmeier Jacob , Lanier Lewis 
Published: January 2023

Abstract
Respiratory viral infections, including parainfluenza virus (PIV), are a significant cause of illness and death worldwide, especially among young children and the elderly. Understanding the immune response to these viruses is important for reducing pathology and developing vaccines. As a first line of defense, Natural Killer (NK) cells provide critical host protection against viruses. Their presence in the lung, a key site of pathogen entry, suggests an important role for these cells in respiratory viral infection. Sendai virus (SeV), a natural mouse pathogen, is closely related to human PIV, and causes respiratory infection similar to that seen in humans. No studies have directly assessed NK cells in host protection during SeV infection. Our research shows that NK cells are recruited into the lung airways by day 2 post infection (p.i.), and continue to accumulate in the lungs. NK-depleted mice have higher viral titers in the lungs on days 3 and 5 p.i. NK cells upregulate the activating receptor NKG2D during infection, and in the absence of NK cells, a higher percentage of CD8 T cells expresses NKG2D. NKG2D antibody blockade results in higher viral titers on day 4 p.i. Our data show that NK cells play a protective role and are an important cell population during PIV infection. These studies will deepen our understanding of NK cell biology, and may also help in the design of new methods to treat, and possibly prevent, these diseases.
Critical role of PD-L2 in limiting pulmonary immunopathology during RSV infection (P6182)
Authors: McDermott Daniel , Varga Steven 
Published: January 2023

Abstract
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract-associated hospitalizations in children under the age of 2. Acute RSV infection induces both Th1- and Th2-associated responses, with elevated airway hyperactivity being highly correlated with increased Th2-associated responses. However, the mechanism for induction of Th1 vs. Th2-associated responses following RSV infection is currently unknown. The inhibitory ligands PD-L1 and PD-L2 serve a critical role in the induction and regulation of Th1 and Th2-associated responses, respectively, during acute infections as well as asthma. Following acute RSV infection, lung epithelial cells upregulate PD-L1 within 24 hours post-infection (p.i.) with peak expression occurring at day 6 p.i.. Furthermore, both lung and mediastinal lymph node (MedLN) CD11c+ cDCs and pDCs upregulate PD-L1 expression early following RSV infection and retain increased PD-L1 expression throughout the course of infection. In contrast, PD-L2 expression is primarily restricted to CD11c+ cDCs. PD-L2 expression peaks at day 6 p.i. on both lung and MedLN CD11c+ cDCs, correlating with increased airway resistance following acute RSV infection. PD-L2-blockade early during RSV infection results in increased airway hyperactivity, whereas PD-L1-blockade exhibited no significant effect. These data suggest that PD-L2 plays a critical role in the regulation of RSV-induced immunopathology.
TCR avidity for thymic and peripheral self peptide-MHC sets and sustains intrinsic CD4+ T cell sensitivity (P1170)
Authors: Persaud Stephen , Weber K. , Allen Paul 
Published: January 2023

Abstract
TCR interactions with pMHC are crucial to T cell development and function. It is not known how the strength of the positively selecting signal impacts peripheral T cell function. To pursue this issue, we utilized the LLO56 and LLO118 TCR transgenic mice, each of which bears CD4+ T cells that recognize the same epitope from Listeriolysin O. During in vivo responses to L. monocytogenes, LLO118 T cells expand better upon primary challenge, whereas LLO56 prevails upon rechallenge. Surprisingly, naive LLO56 T cells showed stronger IL-2 responses than LLO118 to nonspecific in vitro stimulation, associated with greater ERK activation and higher basal phospho-TCRζ levels. The greater basal TCRζ phosphorylation, along with higher CD5 expression, suggested that LLO56 has a higher avidity for self-pMHC, possibly underlying its higher sensitivity. Indeed, the respective sensitivities of LLO56 and LLO118 cells emerged as they matured into CD4SP thymocytes after positive selection, during which LLO56 thymocytes received a stronger signal from selecting self-pMHC. Peripheral self-pMHC was required to actively maintain mature LLO56 and LLO118 T cell sensitivity, as transfer of these cells into MHC II-/- mice rendered them similarly unresponsive to stimulation. In summary, our data demonstrate unanticipated roles for TCR:self-pMHC avidity in establishing T cell sensitivity centrally, sustaining it in the periphery, and affecting CD4+ T cell responses to pathogens.
DLG1 alternative splice variants: molecular conduits specifying proximal TCR signals to distinct CTL functional responses (P1174)
Authors: Silva Oscar , Crocetti Jillian , Humphries Lisa , Miceli M. 
Published: January 2023

Abstract
CD8+ cytotoxic T lymphocytes (CTLs) are a critical component of the adaptive immune system because they mediate contact-dependent cytolysis and produce proinflammatory cytokines. Although both of these CTL activities require T cell receptor (TCR) activation these functions are not always coordinately invoked, suggesting that signaling pathways downstream of the TCR can be selectively activated to guide specific functional outcomes. Here we investigated the role of Discs large homolog 1 (DLG1) alternative splice variants, DLG1 i1A-i1B and DLG1 i1B, in coupling proximal TCR signals to select downstream pathways to specify cytotoxicity and proinflammatory cytokine transcription in CD8+ T cells. We found that DLG1 i1A-i1B, but not DLG1 i1B selectively directs TCR-induced p38/NFAT-dependent proinflammatory cytokine production by binding Lck, promoting alternative p38 activation and downstream NFAT mediated upregultion of IFNγ and TNFα gene expression. Conversely, DLG1 i1A-i1B mediated alternative p38 activation had no effect on actin polymerization, granzyme B gene expression, granzyme B release, or degranulation. Rather, either DLG1 variant expressed in CD8+ T cells promoted antigen-induced degranulation, suggesting that both DLG1 variants have p38-independent activity guiding granule-dependent cytotoxicity. Thus CD8+ T cells contain two distinct DLG1 scaffolds that act as molecular conduits for proximal TCR signals to allow differential CTL functional responses.
mTOR complex signaling inhibits expression of a specific set of microRNAs during T cell activation that regulate G1 cell cycle progression (P1180)
Authors: Podshivalova Katie , Wang Eileen , Salomon Daniel 
Published: January 2023

Abstract
microRNAs are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. T cell activation results in dramatic changes in the expression of individual microRNAs. Mammalian target of rapamycin (mTOR) is activated promptly after TCR stimulation via the PI3K signaling branch and promotes T cell exit from the quiescent resting state into a highly proliferative activated state. Bringing these two important regulatory pathways together, we show that mTOR signals the down-regulation of several specific microRNAs after activation of T cells. Inhibition of mTOR in human T cells by rapamycin or PP242 results in up-regulation of a unique set of microRNAs. Conversely, the same miRNAs are dramatically reduced with T cell activation. Regulation of these miRNAs by mTOR appears to be T cell specific, as their expression does not change in B cells, fibroblasts or epithelial cells treated with mTOR inhibitors. Mechanistically, we show that mTOR-dependent microRNAs negatively regulate proliferation and induce G1 cell cycle arrest by targeting multiple genes, acting either additively or synergistically with mTOR inhibitors. Our data demonstrate that several microRNAs help maintain the quiescent state of resting T cells, are down-regulated with activation due to negative regulation by mTOR and because these microRNAs contribute to the induction of a cell cycle arrest, they may have potential roles as tumor suppressor genes.
Nck proteins are recruited directly to the BCR and regulate PI3K signaling thereby shaping B cell immune responses (P1154)
Authors: Castello Angelo , Engelke Michael , Tucholski Johannes , Oellerich Thomas , Tafuri Anna , Pawson Tony , Batista Facundo 
Published: January 2023

Abstract
Binding of antigen to the B cell antigen receptor (BCR) triggers the activation of different kinases and the initiation of many signaling pathways required for mounting B-dependent immune responses. Scaffolding proteins, recruited downstream of the BCR, act as signal integrators through the recruitment of multiple signaling effectors. Nck is a family of scaffolding proteins best known for linking phosphotyrosine signals to cytoskeleton regulation but until now uncharacterized in the context of B lymphocytes. In our study, Nck was found to be directly recruited to the Igα tail of the BCR and to be important to regulate immune-receptor signaling. Through the generation of a novel Nck1 and Nck2 B cell conditional KO mouse model, we found these adaptors to be essential for mounting T-independent immune response and differentiate into extrafollicular plasma cells during T-dependent responses. The phenotype reported was shown to be due to defective PI3K signaling and Foxo1 downregulation in response to antigen challenge. Our work therefore provides the first compelling evidence for a critical role of Nck in BCR signaling, which is independent of its cytoskeleton regulation yet essential to mount B cell responses.
Micron-scale exclusion of CD45 enhances activation of TCR zeta associated protein of 70 kDa tyrosine kinase. (P1192)
Authors: Lee Joung-Hyun , Dustin Michael , Kam Lance 
Published: January 2023

Abstract
CD45, a transmembrane protein tyrosine phosphatase, is critical for activation of T cells. CD45 is known to modulate T cell signaling by dephosphorylating the inhibitory Tyr 505 of Lck, but at the same time it has been argued that exclusion of CD45 from the vicinity of the TCR is necessary for activation of T cells. While many data support the notion that CD45 exclusion is necessary for triggering of TCR signaling, there is no evidence that CD45 exclusion is sufficient. In this work, we designed substrates to test if local exclusion of CD45 can trigger TCR without aid of TCR ligation. To test this hypothesis, mouse CD4 T cells were seeded onto surfaces micropatterned with an antibody targeting the extracellular domain of CD45. These surfaces were created by first defining 1- or 2-µm diameter features of PLL-g-PEG onto glass coverslips, followed by adsorption of anti-CD45 to the remaining regions. These substrates depleted CD45 from the PLL-g-PEG patterned regions and enhanced production of pZap70 over levels observed on uniformly coated anti-CD45 surfaces as determined by total internal reflection fluorescence. Surprisingly, the areas of higher pZap70 were not localized in the specific regions of CD45 exclusion, but were concentrated in the center of the interface, approximating early stages of signaling in a T cell immunological synapse. These results demonstrate that local control over CD45 distribution can alter T cell signaling, independent of TCR-MHC recognition.
STIM2 drives calcium oscillations through store-operated calcium entry caused by mild store depletion (P1160)
Authors: Thiel Markus , Penner Reinhold , Lis Annette 
Published: January 2023

Abstract
The main source of calcium in T cells is through a mechanism known as store-operated calcium entry (SOCE). STIM1 and STIM2 serve as ER calcium sensors that, upon store depletion, activate calcium release-activated calcium (CRAC) channels (Orai1-3,CRACM1-3) in the plasma membrane. In the absence of sustained calcium influx through CRAC channels, numerous lymphocyte functions are severely compromised, as demonstrated in patients suffering from SCID. Agonist-induced calcium oscillations in many cell types are triggered by calcium release from intracellular stores and driven by SOCE. However, their relative roles in agonist-mediated calcium oscillations remain ambiguous. We here report that while both STIM1 and STIM2 contribute to store-refilling during calcium oscillations in T cells, RBL and HEK cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 selectively suppresses SOCE and agonist-mediated calcium oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in ER calcium levels. We conclude that with increasing agonist concentrations, SOCE is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of calcium entry over a range of stimulus intensities and levels of store depletion.
Sustained Treg expansion by repeated in vivo injection of anti-TNFRSF25 mAb (P4142)
Authors: Zhang Hong , Pahuja Anil , Lundsten Kelly , Li Sky , Lin Xiao , Ransom John , Yang Xi Feng 
Published: January 2023

Abstract
FOXP3+ Tregs play key roles in maintenance of immune tolerance and autoimmune reactions. TNFRSF25 (also known as DR3) is one of the more recently discovered TNFRSF member, which is expressed by mouse Tregs, naïve CD4, CD8, NKT cells and TLR4-induced DC and macrophages. A significant Treg in vivo expansion by TNFRSF25 stimulation has been reported. Here, we show that by single dose i.p. injection of an agonistic mAb, 4C12, Treg expansion starts to be observed on day 3, reached a peak (up to 30-40% of total CD4+ T-cells) on day 5-6, and then returned to baseline (5-7%) by day 10. Upon repeated 3-day interval injections, high level (about 20%) of expanded Tregs can be sustained more than 10 days. As reported by other authors, followed by one dose 4C12 injection, the absolute number of CD8+T cells, B cells, dendritic cells and macrophages was not affected. In the in vitro study, 4C12 antibody did not significantly induce Treg expansion in CD3/CD28/IL-2 culture system. Moreover, we characterized the TNFRSF25 expanded Tregs in vivo, we found that the expression level of CD304, CD152, Helio, GARP, LAP (TGF-b1), FR4, GITR was significantly increased in comparison to the control. These data suggest that multiple injections of 4C12 can long term sustain expanded Tregs at certain level, the biological significance of Treg need to be further studied.
Resistance of diabetes in aged NOD mice is mediated by CD4+CD25+Foxp3+ regulatory T cells (P4144)
Authors: Guo Zhiguang , Liu Bolin , Tian Lei , Tian Bole , Ansarullah A. , Lu Yan , Holstein Martha , Deruyter Brittany 
Published: January 2023

Abstract
We investigated the role of CD4+CD25+FoxP3+ regulatory T cells on diabetes resistance in aged NOD mice that do not develop diabetes in pathogen-free housing. At 60 weeks age, &lt;80% NOD mice developed diabetes. Adoptive transfer of lymphocytes from diabetic mice to aged nodiabetic NOD mice did not significantly increase the onset of diabetes. When 1x107 lymphocytes from age NOD mice were co-transferred with 5x106 lymphocytes from diabetes NOD mice into NOD.scid mice, all mice developed diabetes at 6 weeks. When 2x107 lymphocytes from aged NOD mice were co-transferred, none developed diabetes at 6 weeks and only 50% NOD.scid mice developed diabetes at 12 weeks. When 1x106 CD4+CD25- T cells was cotransferred with 5x106 lymphocytes from diabetic NOD mice, diabetes was developed in all NOD.scid mice. When 1x106 CD4+CD25+ T cells were used, only 17% mice developed diabetes at 12 weeks. The percentages of CD4+CD25+Foxp3+ T cells in the pancreatic lymph nodes, spleen and peripheral blood were 20.1±5.3%, 14.6±4.1% and 9.8±3.5% in aged NOD mice; 5.8±1.5%, 8.6±0.7% and 6.4±1.7% in control diabetic NOD mice. When a low dose of cyclosphamiade was given, diabetes was developed in 75% young NOD mice and in 18% aged NOD mice. However, when the high dose was given, 58% aged NOD mice had diabetes and CD4+CD25+Foxp3+ T cells in pancreatic lymph nodes were 7.0±1.7%. Our data demonstrated that immunoregulation through CD4+CD25+Foxp3+ T cells mediated the resistance of diabetes in aged NOD mice.