bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Phosphoproteomic Dissection of the role of Zap-70 in T Cell Signaling Using Mass Spectrometry (144.9)
Authors: Salomon Arthur , Nguyen Vinh , Cao Lulu 
Published: January 2023

Abstract
Although many details of immune cell signaling have been described previously, a systematic, quantitative wide-scale analysis of the global tyrosine phosphorylation events that are triggered by activation of the immune cell receptors is lacking. Modern phosphoproteomic methods in mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation through a variety of activation states or perturbed at specific pathway proteins or phosphorylation sites. We have combined genetic analysis with quantitative proteomic methods to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase deficient and reconstituted T cells. The promise of this unique approach is illustrated in phosphoproteomic studies of T cells with a disrupted receptor proximal protein tyrosine kinase, Zap-70. The expected T cell signaling pathway structure was replicated and 96 novel phosphorylation events were discovered. These novel phosphorylation sites are located both on proteins previously associated with the T cell pathway as well as functionally uncharacterized proteins. Quantitative phosphoproteomic signatures indicative of downstream inhibition, downstream activation, and mutant compensation are revealed. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.
CHOP mediates PKC-theta (PKCθ) deficient T cell apoptosis (145.1)
Authors: Yin Dengping , Gao Qiang , Gao Xiudan , Geevarghese Sunil , Kelly Burnett , Wright John , Chari Ravi 
Published: January 2023

Abstract
Endoplasmic reticulum (ER) stress, a signaling pathway, is activated when unfolded proteins accumulate in the ER. Sustained activation of ER stress triggers proapoptotic signals via CCAAT/enhancer-binding protein homologous protein (CHOP) that elicits cell apoptosis. PKCθ deficient T cells have revealed spontaneous and TCR/CD28-induced apoptosis, reduced NF-kB activation, and unresponsiveness to cardiac allografts. However, whether PKCθ deficiency-induced tolerance is associated with CHOP expression remains unknown. Here we show that CHOP expression was increased in the cardiac allografts, spleens, and thymus of PKCθ-/- mice. Increased CHOP expression is associated with increased PKCθ-/- T cell apoptosis (Annex V positive cells) and Bax expression. There were no significant changes of other ER stress proteins, such as calnexin and IRE-1α, and chaperones in PKCθ-/- mice. LC3-I to LC3-II conversion represents an activation of autophagy and can be induced by ER stress; however, there was no increased LC3-I to LC3-II conversion in PKCθ-/- mice. The cardiac allografts were accepted in PKCθ and CD28 double knockout (PKCθ-/-/CD28-/-) mice, and activation of NF-kB by TLR engagement with CpG ODNs inhibited CHOP expression and triggered allograft rejection. Our data suggest that PKCθ deficiency stimulates the CHOP signaling pathway, which mediates T cell apoptosis and is negatively regulated by NF-kB activation.
Intrinsic IFN-gamma-T-bet pathway mediates the generation of Th1-like regulatory T cells induced by CD40-activated B cells (145.11)
Authors: Tu Wenwei , Zheng Jian , Liu Yinping , Lau Yu-Lung 
Published: January 2023

Abstract
The plasticity of CD4+ T cell is found to be higher than previously thought and might be modified by the cross-talking among different signal pathways. On the other hand, the role of CD40-activated B cells in the differentiation of CD4+ T cell is still on debate and their underlying mechanism for inducing Tregs remains unknown. In this study, using coculture of human naïve CD4+CD25- T cells with allogeneic CD40-activated B cells without any exogenous cytokines, we have demonstrated that CD40-activated B cells could induce alloantigen-specific CD4hiCD25hi Tregs and Th1 cells from their naïve precursors under weak and optimal activation respectively. Interestingly, the induced CD4hiCD25hi Tregs had Th1 properties with high level of T-bet expression, and some of them produced IFN-gamma. Both IFN-gamma+ and IFN-gamma-CD4hiCD25hi Tregs had a similar alloantigen-specific suppressive capacity. Importantly, we further found that the endogenously produced IFN-gamma and intrinsic T-bet expression were involved in the generation of these CD4hiCD25hi Tregs but did not mediate their suppression by neutralization of IFN-gamma and interference of T-bet expression with siRNA. We concluded that the generation of these Th1-like Tregs was mediated by IFN-gamma-T-bet pathway and the stimulatory or tolerogenic role of CD40-activated B cells in CD4+ T-cell differentiation was dependent on the strength of the activation to T cells.
CD8 Memory T cell Migration to Cardiac Allografts (145.16)
Authors: Walch Jeffrey , Oberbarnscheidt Martin , Camirand Geoffrey , Li Qi , Lakkis Fadi 
Published: January 2023

Abstract
Memory T cells that recognize donor alloantigen jeopardize the survival of life-saving organ transplants. Although recent publications studied the role of chemokines in effector T cell migration to the allograft, the migratory requirements of memory T cells are not known. Phenotyping of CD8 memory T cell populations demonstrated up-regulation of CXCR3 and CCR5 chemokine receptors. Therefore, we investigated the survival of cardiac allografts (BALB/c, H-2d) transplanted into aly mice (alymphoplastic, no secondary lymphoid organs after splenectomy, H-2b) that received sorted CXCR3-/- or wt CD8+CD44+ memory T cells (C57Bl/6, H-2b, recognizing H-2d) via adoptive transfer. Additionally, a group of mice receiving CXCR3-/- cells was treated with anti-CCR5 antibody. No significant differences in allograft survival could be detected between the groups. Furthermore, flow analysis revealed no difference in the migration of adoptively co-transferred, congenic, CFSE labeled CXCR3-/- and wt CD8+CD44+ memory T cells to the cardiac allograft at 20 hours and 3 days after cell transfer (n=6). Our results show that CD8 memory T cell migration to the allograft is independent of CXCR3 and CCR5. Moreover, the migration requirements for CD8 memory T cells clearly differ from those established for effector T cells highlighting the role of memory T cells in immune surveillance and their threat to transplanted organs.
Mesenchymal Stem Cell (MSC) Therapy To Prevent Ischemia-Reperfusion Injury in Lung Transplantation (145.24)
Authors: Salgar Shashikumar , Manning Eddie , Ruiz Phillip , Pham Si 
Published: January 2023

Abstract
Ischemia-reperfusion (IR) injury is an important cause for lung graft loss (~25%). In this study MSC were tested for their ability to prevent lung IR injury. MSC derived from bone marrow and fibroblasts from skin of Lewis rat were expanded ex vivo. Following 120 minutes of left lung ischemia induction, rats received autologous MSC or fibroblasts ( ~2 x 106; i.v.; passage ≤6) or saline. Significant (P&lt;0.01) improvement in lung function as determined by blood oxygenation (PaO2/FiO2 ratio, mmHg) with MSC therapy was observed as early as 4h IR (305 ± 31; n =6) compared to untreated (63 ± 19; n=6) or fibroblast treated (142 ± 96; n=5) controls. By 24h IR blood oxygenation values reached normal (454 ± 59; n=5) in MSC treated group but not in fibroblast or saline treated controls. Histopathology demonstrated reduced (P&lt;0.05) injury score (Grade 1.1 ± 1; n=7) with MSC therapy compared to untreated controls (Grade 2.5 ± 1.4; n=6). ISOL (in situ oligo ligation for DNA fragmentation) & Caspase-3 staining demonstrated reduced (p&lt;0.05) apoptotic cells in MSC treated lungs. Inflammatory mediators (MCP-1, TNF-α, and IL-6) and cells, and wet to dry ratio/microvascular permiability index in the IR injured lungs were reduced (P&lt;0.05) in MSC treated lungs. Long-term MSC (passage &gt;25) improved blood oxygenation marginally by 72h (320±158; n= 6) compared to untreated controls (198±141; n= 6). In conclusion MSC therapy to prevent IR injury and improve lung transplant function seems promising.
Rapamycin promotes tolerance through increased expression of CD4+CD25+Foxp3+ regulatory T cells and impaired development of CD8+CD44+CD62L+ memory T cells. (145.25)
Authors: Urbanellis Peter , Wang Jihong , Shalev Itay , He Wei , Adeyi Oyedele , Phillips M. , Bartczak Agata , Grant David , Levy Gary 
Published: January 2023

Abstract
Long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16 day treatment with Rapamycin led to indefinite graft survival (&gt;100 days) of fully mismatched cardiac allografts (BALB/cJ → C3H/HeJ) whereas untreated hearts were rejected at day 9 + 2 days. Specific tolerance was confirmed by the observation that skin grafts from donor mice were accepted, whereas grafts from a 3rd party were rejected. T-lymphocytes from tolerant mice exhibited lower proliferation and cytotoxicity towards donor but not to 3rd party antigens. CD8+CD44+ memory T cells were reduced in tolerant mice but had increased expression of L-selectin (CD62L). CD4+CD25+Foxp3+ regulatory T cells (Tregs) were increased in tolerant mice. We next examined 23 Treg-related genes by multiplex PCR, and found an increase in expression of Foxp3, Tgfb1, Lag3 and Fgl2 and decreased expression of IFNγ and granzyme B in tolerant hearts, but not in rejecting hearts. Plasma levels of FGL2 determined by ELISA remained elevated in tolerant mice. Here we demonstrate that Rapamycin-induced specific tolerance to fully mismatched cardiac allografts is associated with increased numbers of Tregs and impaired development of memory T cells. Expression of Treg effector molecules may serve as putative biomarkers of tolerance in patients undergoing transplantation.
Transplant-associated autoimmunity to cardiac myosin after cardiac transplantation in non-human primates (145.29)
Authors: Tahir Khola , Ward Alison , Zhang Tianshu , Kelishadi Sean , Fairweather Delisa , Cunningham Madeleine , Pierson Richard , Azimzadeh Agnes 
Published: January 2023

Abstract
Background: Immune responses to autoantigens have recently been described in rodent and human allograft recipients. Here we report that immunity to cardiac myosin (CM), a heart-specific autoantigen, develops after transplantation in primates. Methods: Cynomolgus monkeys received a heart allograft and either: 1) transient CD28 blockade (aCD28), where delayed acute rejection (AR) occurs ≤35 days; 2) cyclosporine A (CsA), where chronic rejection (CAV) occurs ≤90 days; or 3) CsA plus B-cell depletion (CsA+ Rituximab). Antibodies against CM were assayed by ELISA at the time of graft rejection, or graft removal at day 90. Results: AR was consistently associated with increased CM IgM and/or IgG levels (6/6). CsA was associated with moderate to severe CAV and CM Abs were detected in 83% (5/6). When AR and CAV were prevented with B-cell depletion, CM Abs were not detected. Immunity to CM correlated with anti-donor antibody and intra-graft C4d. Conclusion: Detection of humoral immunity to cardiac myosin is closely associated with acute and chronic rejection of cardiac allografts, but not when CAV is prevented. We conclude that antibody against CM may serve as a biological marker for cardiac allograft injury. Whether loss of immunoregulation (tolerance) to the autologous CM protein is an epiphenomenon or pathogenic remains to be determined. If immunity against CM contributes significantly to allograft injury, inducing robust tolerance to CM may help attenuate heart allograft injury.
Critical role for selectin receptor:ligand interactions in the development of IPS following allogeneic BMT (145.32)
Authors: Pierce Elizabeth , Choi Sung , Olkiewicz Krystyna , Chang Doris , Eid Saada , Guardia-Wolff Rocio , Cooke Kenneth 
Published: January 2023

Abstract
Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic BMT and limits successful outcomes. The pathophysiology of IPS involves the release of soluble inflammatory proteins and the recruitment of donor lymphoid and myeloid effectors into the lung. The entry of leukocytes into inflamed tissue is mediated by several molecules that are orchestrated in a sequential manner. We used well-established mouse BMT models to investigate the role of selectin (sel) receptor:ligand interactions in the development of IPS. Firstly, E and P sel ligands are up-regulated on donor T cells when activated by host APCs in vitro. Next, we found that sel receptor (mRNA) and ligand (protein) expression is significantly increased in the lungs of allo-BMT recipients during the development of IPS. BMT using donor cells deficient in E and P sel ligands resulted in a significant reduction in lung inflammation (cellularity, pathology, cytokines), and mixing studies uncovered a contribution to cells of both lymphoid and myeloid lineage. Experiments using isolated MHC class I or class II disparate strain combinations revealed a more significant effect on donor CD4+ vs. CD8+ T cells. Finally absence of E and P sel in BMT recipient mice conferred near complete protection from IPS especially during CD4+ mediated disease. These data demonstrate a critical role for selectin receptor:ligand interactions in the pathogenesis of IPS and uncover novel strategies to regulate disease severity.
B cells differentially regulate acute and chronic cardiac, renal, and skin allograft rejection (145.42)
Authors: DiLillo David , Griffiths Robert , Ruiz Philip , Coffman Thomas , Tedder Thomas 
Published: January 2023

Abstract
T cells contribute to both acute and chronic allograft rejection, but the roles of B cells and alloantibody are less clear. Therefore, CD20 and CD19 mAbs were used to deplete mature or total B cells, respectively, in mice before allografting. Both CD20 and CD19 mAbs effectively depleted mature B cells, but CD19 mAb also depleted &gt;70% of CD138+ plasma cells in flow cytometry assays, as well as &gt;66% of Ab-secreting cells in ELISPOT assays. Consequently, CD19 mAb treatment depleted serum IgG levels by 80% in naïve mice, while CD20 mAb did not. Neither CD20 nor CD19 mAb treatment affected acute cardiac allograft rejection, with all grafts rejected within 7-9 days. However, CD19 mAb treatment prevented the generation of allo-IgG. In a second model of rejection, treatment with CD19 mAb before renal transplant significantly delayed and prevented chronic renal allograft rejection, reduced kidney pathology, and abrogated the development of allo-IgG, while CD20 mAb did not. In additional experiments, CD19 mAb treatment also depleted pre-existing allo-IgG levels in serum. In a third allograft model, pretreatment with CD20 mAb exacerbated acute skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4+ T cells by 2-fold, indicating that B cells negatively regulate acute skin graft rejection and CD4+ T cell responses. Thus, depending on the nature of the allograft, B cells can either positively or negatively regulate allograft rejection.
Establishment of IL-17A/F BAC construct for transgenic reporter mice (34.10)
Authors: Shen Wei , Li Wenqing , Feigenbaum Lionel , Hixon Julie , Durum Scott 
Published: January 2023

Abstract
T cell cytokines are generally rapidly secreted and therefore most of the cytokines cannot be visualized in tissues by immunohistochemistry. Interleukin (IL) 17A and IL-17F are proinflammatory cytokines that could be associated with autoimmunity and many inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. To enhance the visualization of IL-17A and IL-17F inside the producing cells, we are using a method we previously applied to trap a reporter inside the producing cell. A bacterial artificial chromosome was modified to introduce two fluorescent reporter genes into the Il17 locus which includes IL-17A and IL-17F. By homologous recombination, the synthesis of the signal peptide of Il17a/f in the BAC was disrupted and the GFP gene with polyA was inserted immediately after the ATG start site of Il17f, replacing exon 1, while the tdTomato gene with poly A was inserted immediately after the ATG start site of Il17a. BAC-end sequencing and DNA fingerprinting results showed that there is no rearrangement and deletion of the BAC construct with the reporter gene. Mice are being generated that harbor the BAC construct. Confocal microscopy results showed that both GFP and tdTomato were expressed inside the Th17 cells generated from mouse PBMC cells. We will apply this IL-17A/F reporter to determine the cell sources and microenvironmental triggers of IL-17 and to suggest methods of dampening this response in chronic inflammatory diseases.
Up-regulated signaling pathway triggered by sBAFF may induce aberrant production of IL-6 in monocytes of the patients with Sjögren’s Syndrome (34.16)
Authors: Yoshimoto Keiko , Tanaka Maiko , Setoyama Yumiko , Ogata Hideko , Suzuki Katsuya , Abe Tohru , Takeuchi Tsutomu 
Published: January 2023

Abstract
Background and Purpose: Elevated production of BAFF seemed to be involved in the development of autoimmune diseases such as Sjögren’s syndrome (SS). We have demonstrated that BAFF and IL-6 were abnormally produced in SS monocytes upon stimulation with IFN-gamma. In the present study, we investigated a regulatory mechanism of the production of these cytokines in SS monocytes. Materials and Methods: Peripheral monocytes prepared from SS patients and normal individuals were stimulated with IFN-gamma or soluble BAFF (sBAFF). The expression levels of sBAFF, IL-6 and transcription factors were analyzed by ELISA and/or semi-quantitative RT-PCR. Results and Discussion: IFN-gamma induced the production of sBAFF and IL-6 in SS monocytes. The production of IL-6 in SS monocytes was significantly suppressed by an anti-BAFF antibody or rBAFF receptor. In addition, stimulation of SS monocytes with sBAFF induced robust increase in the production of IL-6. Moreover, expression of BAFF receptor was also enhanced in SS monocytes upon stimulation. These data suggest that the production of IL-6 in monocytes is induced partly through a signal transduction pathway triggered by sBAFF. We found that the expression levels of several transcription factors, such as NF-kB2 and NF-IL-6, were aberrantly elevated in SS monocytes. These data strongly suggest that sBAFF produced in monocytes activates the expression of IL-6, and that signal transduction triggered by sBAFF may be impaired in SS monocytes.
Ectopic expression of Interleukin-22 receptors (IL-22R1) on lymphocytes induces multi-organ inflammation and premature death (35.12)
Authors: Savan Ram , Reynolds Della , McFarland Adelle , Feigenbaum Lionel , Ramakrishnan Karthika , Shirota Hidekazu , Klinman Dennis , Donnelly Raymond , Young Howard 
Published: January 2023

Abstract
IL-22 signals through a heterodimeric receptor composed of IL-22R1 and IL-10R2. The IL-22R1 chain of this heterodimeric receptor is not expressed on leukocytes, but aberrant expression of this receptor has been found on ALK+ anaplastic large cell lymphoma. To study the effects of this receptor on lymphocytes, we generated transgenic (tg) mice that express the IL-22R1 gene driven by the CD2 promoter, resulting in T and B-cell expression of a functional IL-22 receptor. Surprisingly, the tg founders succumb to inflammation within 8-12 weeks of age. In IL-22R1 tg mice, we observed constitutive phosphorylation of STAT3 in lymphoid cells. The health of these animals progressively deteriorated showing respiratory distress, rough coat and sluggish movement. Histopathology revealed massive inflammation in the lung, spleen, liver, kidney, gall bladder and colon with corresponding lymphocyte and neutrophil infiltration. The tg animals exhibited a myeloproliferative disorder with an increase in the levels of several Th1 and Th17 cytokines. The neutrophilia that developed in the IL-22R1 tg animals correlated with a marked increase in the levels of circulating IL-17 and G-CSF. Our study shows that the aberrant expression of IL-22R1 on lymphocytes causes multi-organ inflammation with profound effects on the immune system. Thus, aberrant expression of the IL-22R1 chain reveals a novel role for IL-22 in certain inflammatory diseases.
TNF-alpha Decreases Pseudomonas aeruginosa-induced Mortality in Burned Mice through Negative Regulation of IL-1beta (35.2)
Authors: Chen Lee-Wei , Chen Pei-Hsuan , Hsu Ching-Mei 
Published: January 2023

Abstract
Pseudomonas has emerged as an important source of burn wound sepsis. Anti-Tumor necrosis factor (TNF-α) therapy has been failed in decreasing mortality of sepsis patients. To evaluate the role of TNF-α and toll-like receptors in P. aeruginosa infection-induced mortality in thermal injured mice, we injected 109 P. aeruginosa in the back of the wild type (WT) mice, Tnfrsf1a-/- mice, and TLR4-/- mice at 8 hr after 30% total body surface area (TBSA) burn. At 24 hr after burn, animals were killed. Burn with P. aeruginosa injection did not induce lung damage in TLR4-/- mice but increased the mortality; blood and lung bacterial counts; pulmonary microvascular dysfunction; and IL-1β expression in Tnfrsf1a-/- mice compared with WT mice. However, burn with P. aeruginosa injection induced an increase of NF-κB activity of lung in WT mice but not in Tnfrsf1a-/- mice. Injection of Anakinra, a specific IL-1β inhibitor, decreased IL-1β expression, blood and lung bacterial counts; and pulmonary microvascular dysfunction in Tnfrsf1a-/- mice. Collectively, the block of the TNF-α receptor induces an increase of P. aeruginosa-induced IL-1β expression, lung permeability, and mortality in thermal injury through the inhibition of NF-κB. IL-1β inhibition reverses its effect and decreases the pulmonary permeability. Using IL-1β inhibitor to decrease anti-TNF-α therapy-induced lung permeability could be a promising therapeutic strategy in decreasing mortality in sepsis patients.
Dominance of macrophage derived IFN-γ in atheromatous lesions (35.3)
Authors: El Sayed Rania , El Shikh Mohey Eldin , Schenkein Harvey , Tew John 
Published: January 2023

Abstract
IFN-γ activates macrophages that are abundant in atheromas, periodontal lesions, and other sites of chronic inflammation. It is thought that the IFN-γ present in athromas is derived from Th-1 cells. However, macrophages dominate the inflammatory infiltrate in atheromas and macrophages can produce IFN-γ prompting the hypothesis that macrophages are the source of a significant proportion of IFN-γ in established atherosclerotic lesions. To test this, CD14+, CD3+, and CD14- & 3- cells were isolated from endarterectomy tissue and examined for spontaneous production of IFN-γ, IL-1β, IL-6, and TNF-α in supernatant fluids from 48 hr cell cultures. The CD14+ macrophages, including foam cells, were responsible not only for the TNF-α, IL-1β & IL-6 but also for the vast majority of the IFN-γ produced in culture. Moreover, this spontaneous macrophage IFN-γ response was maintained in culture for over two weeks. IL-12 induced IFN-γ responses in macrophages and IL-12 was induced by incubating DCs with A. actinomycetemcomitans and far more dramatically by opsonized A. actinomycetemcomitans. In short, engagement of DCs in the intima of the arterial wall with opsonized microorganisms from the blood could trigger IL-12 production, and the released IL-12 could induce a sustained IFN-γ response by macrophages. This macrophage INF-γ response induced by DC-IL-12 bypasses a need for T cells to produce IFN-γ in established atheromas.