bluuebunny/crossref_metadata_2025_split huggingface dataset:
Collaborative Effects of IL-17 and IL-23 in Deletion of Antigen Stimulated CD4+CD8+ Double Positive Thymocytes (143.17)
Authors: Li Hao , Wu Qi , Yang PingAr , Wang John , Kolls Jay , Hsu Hui-chen , Mountz John 
Published: January 2023

Abstract
Increased levels of IL-17 and IL-23 have been associated with autoimmunity,but their effects on thymocyte selection have not been studied.To test this,normal B6 mice were injected with adenovirus expressing either IL-17 (AdIL17) or IL-23 (AdIL-23).There was dramatic reduction of thymus cellularity and increased apoptosis of DP thymocytes in AdIL-23 injected mice.Coinjection of AdIL-17 potentiated the effects of AdIL-23.Coinjection of anti-IL-17 antibody with AdIL-23 partially corrected the effects of AdIL-23 to deplete DP thymocytes by blocking IL-17 induced recruitment of CD11b+ and CD11c+ cells in the junction between cortex and medulla where they exhibit the maximal interaction with DP thymocytes.AdIL-23 but not AdIL-17 or AdLacZ treatment largely increased the expression of IL-23 receptor in DP thymocytes and further lead to 100 fold increased expression of RORgt which is an important DP thymocyte apoptosis inducing molecule.In vitro coculture of thymocytes from 2D2 TCR transgenic mice with MOG35-55 peptide pulsed dendritic cells showed that there were enhanced apoptosis and a lower proliferative response of thymocytes in the presence of IL-23 but not IL-17.Taken together,our results suggest that IL-17 acts to recruit antigen-presenting cells to interact with DP thymocytes,IL-23 induces deletion of Ag encountering DP thymocytes.Thus,interaction of these two cytokines limits the generation of self-reactive thymocytes and does not lead to autoimmunity in normal mice.
Resistance of Cbl-b-/- Teff to Tregs is infectious (143.21)
Authors: O'Conor Catherine , Housley William , Clark Robert 
Published: January 2023

Abstract
The E3 ubiquitin ligase Cbl-b regulates T cell activation, and dysregulation of this protein in both mice and humans has been associated with autoimmunity. Our lab previously proposed resistance to CD4+CD25+ Tregs as one mechanism for the autoimmunity observed in Cbl-b-/- mice. Recently, we confirmed the in vivo resistance of Cbl-b-/- CD4+CD25- T effectors (Teff) to wild type (WT) Tregs using a model of GVHD. Examining the cells ex vivo, we observed a resistance of Cbl-b-/- Teff to suppression of cytokine production. To further characterize this resistance, WT Teff were examined for their ability to be suppressed by Tregs when mixed with Cbl-b-/- Teff in vitro. We found that WT Teff became resistant to Treg-mediated cytokine suppression when co-cultured with Cbl-b-/- Teff. This is the first evidence that resistance to Tregs is “infectious” and can be transmitted to WT Teff. We next determined that in vitro resistance of Cbl-b-/- Teff is only partially reversed by antibodies to IL-2 and IL-4, suggesting that other mechanisms, in addition to increased production of these cytokines, mediate this resistance. The ability of one subpopulation of Teff to induce Treg resistance in another population may have significant implications for mechanisms underlying autoimmune disease. Finally, using Cbl-b-/- Teff to identify mechanisms of infectious resistance may provide important clues in understanding Treg function and in developing novel strategies for treatment of autoimmune disease.
Knock down of kappa light chain RNA induces receptor editing (143.22)
Authors: Shen Shixue , Manser Tim 
Published: January 2023

Abstract
Light chain receptor editing functions as a critical mechanism in maintaining self tolerance among B lymphocytes. Current models for how this process is induced vary with regard to the type of BCR signaling involved. Here we show that in polyclonal and non-autoreactive B cell populations, genetic knockdown of kappa light chain RNA via a shRNA retrovirus efficiently induces BCR receptor editing to lambda light chain expression both in vitro and in vivo. This correlates with increased level of Rag gene expression and retarded development in the B cell compartment. These data demonstrate that BCR engagement of autoantigen is not required for receptor editing and add support to models suggesting a role for “tonic” BCR signaling in the regulation of this process.
Neuropilin-1 is important for proper suppression of inflammation and autoreactivity (143.23)
Authors: Bynoe Margaret , Solomon Benjamin , Chae Wookjin , Mueller Cynthia , Alabanza Leah 
Published: January 2023

Abstract
The surface molecule Neuropilin-1 (Nrp1), was originally identified for its role in neuronal development. Recently, Nrp1 has been implicated in several aspects of immune function, including maintenance of the immune synapse and development of regulatory T cells (Treg). We provide the first evidence for a role of Nrp1 in the pathogenesis of an immune mediated disease, experimental autoimmune encephalomyelitis (EAE), which serves as an animal model for the central nervous system inflammatory disorder multiple sclerosis (MS). Using a mouse cell type specific deletion system, we observed that the lack of Nrp1 on CD4+ T cells results in increased EAE disease severity. These Nrp1 conditional knockout mice exhibit preferential Th17 lineage commitment. Further, CD4+ T cells expressing Nrp1 can suppress Th17 cell expansion and cytokine production both in vitro and in vivo. These findings suggest that Nrp1 is essential for proper maintenance of peripheral tolerance and its absence can result in unchecked autoreactive responses.
A key role for Fas ligand on B cells in unleashing T cell diabetogenicity by suppression of IL-10-mediated islet tolerance (143.34)
Authors: Xiao Zuoxiang 
Published: January 2023

Abstract
A key role for the Fas pathway in promoting organ-specific-autoimmunity is indicated by resistance of mice bearing the lpr mutation of Fas or gld mutation of Fas ligand (FasL) to several organ-specific autoimmune diseases, including autoimmune diabetes and multiple sclerosis. However, the underlying mechanism remains elusive. Here we show that FasL expressed on B cells plays a key role in facilitating activation of diabetogenic T cells in pancreatic lymph nodes (PLNs) and pancreatic islet accumulation resulting in initiation of insulitis in the autoimmune diabetes-prone NOD mice. Expression of FasL on diabetogenic T cells themselves is not sufficient to mediate its diabetogenic role. By contrast, expression of FasL on B cells leads to deletion of localized IL-10-producing B cells, the presence of which in PLNs and pancreatic islets is associated with suppression of diabetogenic T cell priming and islet accumulation, respectively. Consequently, FasL inactivation by gld mutation or antibody blockade preserves the local IL-10-producing B cells and protects islets from diabetogenic T cells by a mechanism that is reversed by IL-10R blockade in vivo. The results provide novel insights into how FasL orchestrates the pathogenic relationship among B cell subsets in its implications for diabetogenic T cells.
Characterization of B cell function in Wiskott Aldrich Syndrome (143.37)
Authors: Castiello Maria Carmina , Bosticardo Marita , Draghici Elena , Sauer Aisha , Schena Francesca , Schenk Ursula , Cassani Barbara , Aiuti Alessandro , Roncarolo Maria Grazia , Grassi Fabio , Traggiai Elisabetta , Villa Anna 
Published: January 2023

Abstract
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency caused by mutations of the gene encoding for WASp, a protein involved in cytoskeletal reorganization of hematopoietic cells. WAS is characterized by microthrombocytopenia, eczema, infections, tumors and high rate of autoimmunity. WASp negative T cells have been extensively studied, while the role of WASp in B cells has been less characterized. Analysis of serum from adult was-/- mice showed increased level of Igs as compared to wt mice, in particular in IgG2a subclass. This result was further confirmed by ELISpot assays performed on sorted Marginal Zone (MZ) and Follicular (FO) B cells from wt and was-/- mice. The presence of autoreactive B cells was supported by the high titers of autoantibodies against dsDNA and tissue Ags in was-/- mice. In in vitro proliferation assays, was-/- B cells are hyper-proliferating to BCR-dependent (IgM) and Toll Like Receptors (TLRs)-dependent stimuli (LPS, CpG) in comparison to wt B cells, although no difference in the expression of TLRs was found. Preliminary results on Ca2+ flux upon CpG stimulus revealed increased Ca2+ mobilization in was-/- MZ B cell as compared to wt MZ B cells. Our data suggest that WASp absence affects B cell function and further studies are ongoing to better characterize BCR and TLR signaling pathways and intracellular trafficking in order to understand the contribution of B cells in autoimmune manifestations found in WAS patients.
Central mechanisms of T cell tolerance to the AChR, the autoantigen in myasthenia gravis, may depend on the level of AChR expression in the thymus (143.40)
Authors: Kraig Ellen , Williams Earlanda , Pasquali Amanda , Krolick Keith , Infante Anthony , Stacy Sue 
Published: January 2023

Abstract
Given that AChR, the autoantigen in MG, is expressed in the thymus and that many MG patients have thymic abnormalities, it seemed likely that thymic selection would be critical in eliciting tolerance to this autoantigen. To test this, we have used two founders from a transgenic mouse model (Tg1 & Tg3) in which the α chain of the Torpedo californica AChR is expressed as a self antigen; TAChRα mRNA expression is 2-4X higher in thymus and muscles of Tg3 mice. Both Tg1 and Tg3 Aire-/- mice are tolerant to TAChRα despite loss of a protein critical for central tolerance to other autoantigens. We also observed a loss of T cell tolerance in both models when Tregs were inactivated supporting a role for peripheral mechanisms. To determine whether peripheral mechanisms were sufficient, Tg1 was bred onto the nude background and thymus transplants were performed to produce progeny expressing TAChRα from Tg1 or Tg3 in the thymus and/or the periphery. Interestingly, in Tg1 nude mice tolerance was seen when TAChRα was expressed only in muscle, but only 2/7 mice grafted with a Tg1 thymus were tolerant. On the other hand, 8/8 mice grafted with a Tg3 thymus were tolerant suggesting higher levels of thymic TAChRα expression as seen in Tg3 mice elicit central tolerance in this model. Additional experiments are underway to determine if the absolute level of thymic TAChRα expression or its location is correlated with central tolerance induction when TAChRα is not expressed in the periphery.
Sex hormones and gender influence the function of regulatory T cells in SLE patients. (143.54)
Authors: Singh Ram , Dinesh Ravi , Hahn Bevra 
Published: January 2023

Abstract
Immunophenotyping of PBMC from SLE patients (n~40) indicated significantly reduced numbers of CD4+CD25hi Foxp3+ T cells and CD8+Foxp3+T cells (p&lt;0.02) as compared to healthy matched controls (n ~20) and autoimmune disease control RA patients (n=30). Numbers of CD4+ and CD8+regulatory T cells are decreased in healthy females compared to healthy males (p&lt;0.01). Both CD4+CD25hi and CD8+CD25hi subsets in males had 3-4-fold higher Foxp3 mRNA compared to females. Stimulation of PBMCs with b-estradiol (30pg/ml) decreases Foxp3 expression in healthy females but not in age matched healthy males. At higher doses (60,150 pg/ml) estrogen has little effect in either sex. Estrogen decreases Foxp3 mRNA and protein expression in both female and male SLE patients’ CD4+CD25- T cells (p&lt;0.05). An Inhibitor of b estradiol (ERa) increased apoptosis in male SLE patients only. These data suggest that estrogen affects the T regulatory compartment.
The nuclear hormone receptor PPARγ regulates T cell activation and survival in lymphopenic autoimmune models. (143.8)
Authors: Housley William , O'Conor Catherine , Clark Robert 
Published: January 2023

Abstract
PPARγ, a nuclear hormone receptor important in regulating fatty acid and glucose metabolism, has been shown to play a significant role in T cell activation, regulatory T cell function, and in the mediation of autoimmunity. Recently, T cells deficient in PPARγ were reported to show increased Th17 differentiation and enhanced Th17-mediated autoimmune disease. In contrast to enhancing autoimmunity, we now report that PPARγ-deficient T cells are defective in causing autoimmune pathology under conditions of lymphopenia. Thus, PPARγ-deficient CD4+CD25- effector T cells (TPPAR Teff), when compared to wild-type (WT) Teff, fail to mediate disease in both an adoptive transfer model of colitis and an acute model of graft versus host disease. Mechanistically, TPPAR Teff have a severe defect in survival after transfer into lymphopenic mice as evidenced by a significantly decreased recovery in the spleen and mesenteric lymph nodes when compared to WT Teff. Moreover, while TPPAR and WT Teff both express activation markers after transfer, TPPAR Teff demonstrate a persistent decrease in IL-7R alpha (IL-7Ra) expression. These results suggest that PPARγ-deficiency in T cells is associated with a defect in IL-7Ra expression and/or an increased state of activation and AICD. Overall, our results suggest a new function for PPARγ in autoimmunity, namely the regulation of T cell activation and survival in lymphopenia-associated autoimmune diseases.
Optimiser™: The next generation of microplates (144.18)
Authors: Puntambekar Aniruddha , Kai Junhai , Lee Se Hwan , Han Jungyoup , Ahn Chong 
Published: January 2023

Abstract
The Optimiser™ microplates combine the power of microfluidics with the 96-well architecture to offer unparalleled performance. The Optimiser consists of an array of wells defined on one face of a substrate connected to microfluidic channels on the opposing face. The channels are sealed by a sealing layer with opening at outlet of the channel, which in turn connects to an absorbent pad. In the Optimiser the microfluidic channel (with a 50% higher surface area and 50x higher surface area to volume ratio) serves as the reaction chamber, allowing for low volume, rapid assay reactions. During use, the first reagent is added to the well, drawn into the channel by capillary forces and transported to the absorbent pad and partially sucked out. The unique design allows the liquid to exit the well but be trapped within the channel for static incubation. Further steps only involve sequential reagent additions which are transported through the channel for a complete assay sequence. The Optimiser has been successfully tested in chemifluorescence mode to demonstrate comparable sensitivity to a 96-well plate with &lt; 1/3rd sample volume. Alternately with similar sample volumes, the Optimiser boasts &gt;10x higher sensitivity, to achieve detection limits of &lt; 1 pg from a 100 µl sample. In all cases, a complete sandwich assay can be completed in &lt; 30 minutes with the Optimiser. The Optimiser conforms to ANSI microplate specs and can be used with all current microplate instruments.
Efficient cryopreservation technique preserves functionality and phenotype of highly purified human blood cell subsets. (144.21)
Authors: Patke Deepa , Marking Christine , Glover Chondra , DiPucchio Tiziana , Manak Mark 
Published: January 2023

Abstract
During the course of infection with pathogens or exposure to immunogens, interactions between different cell subsets lead to efficient innate and/or adaptive immune responses. The availability of pure populations of different cell subsets allows observation of such interactions in vitro, and could provide essential clues to identify precise immune mechanisms such as cell to cell interactions during antigen presentation or CTL and T helper cell interactions. Such information would be valuable in designing strategies for cell therapy, for improved vaccines and for dampening autoimmune responses. We demonstrate that efficient cryopreservation techniques such as use of specialized freezing media, and initial controlled rate freezing followed by stringently monitored cryopreservation under the vapor phase of liquid nitrogen, allow highly purified frozen human blood cell subsets to retain their functional and differentiation capability. Indeed, cryopreserved CD4 and CD8 T cells show similar ability to proliferate and produce IFN-γ compared to fresh purified cell subsets derived from single or different PBMC donors. Other cell subsets such as CD3 cells and Monocytes also retain their functionality and differentiation capacity. Our results suggest that cryopreserved cell subsets can be used as a basic tool for studying cellular phenotype and function in longitudinal clinical studies.
SA-4-1BBL as an immune modulator and vehicle to deliver antigens into dendritic cells for effective cancer immunotherapy (144.25)
Authors: Sharma Rajesh , Schabowsky Rich-Henry , Srivastava Abhishek , Yolcu Esma , Elpek Kutlu , Zhao Hong , Madireddi Shravan , Shirwan Haval 
Published: January 2023

Abstract
The success of TAAs-based vaccines may depend on the nature of adjuvants used. We recently generated a chimeric 4-1BBL with streptavidin (SA-4-1BBL) that exists as tetramers/oligomers with potent activity and can be used as a vehicle to deliver TAAs into dendritic cells constitutively expressing 4-1BB receptor. We demonstrated that SA-4-1BBL can be effectively conjugated to biotinylated ovalbumin (Ova) as a model antigen. Immunization with Ova conjugated to SA-4-1BBL resulted in increased Ova uptake and cross-presentation by DCs, that resulted in potent Ova specific proliferation of CD4+ (OT-II) and CD8+ (OT-I) T cells. The conjugate vaccine also generated significantly higher in vivo specific CTL responses to Ova and HPV E7 oncoprotein than nonconjugate vaccine. Importantly, a single vaccination with HPV E7 oncoprotein or survivin conjugated to SA-4-1BBL resulted in robust and superior efficacy than nonconjugate vaccine in the eradication of established E7 expressing TC-1 and survivin expressing 3LL tumors, respectively. Therapeutic efficacy was achieved in the absence of detectable toxicity and associated with increased CD4+ T and CD8+ T cell effector and memory responses and higher intratumoral CD8+ Teff/CD4+CD25+Foxp3+Treg ratio. In conclusion, potent immunomodulatory activities of SA-4-1BBL combined with its ability to serve as a vehicle to deliver TAAs to DCs further rationalizes the extensive evaluation for the development of therapeutic vaccines against cancer.
Construction of vectors to produce recombinant M. bovis-BCG to enhance the vaccine protection against tuberculosis. (144.27)
Authors: Santos Lorena , Estes Don , Kipnis Andre , Kipnis Ana Paula 
Published: January 2023

Abstract
A third of the world’s population is exposed to the risk of tuberculosis (TB). The only currently available vaccine against TB is the Bacillus Calmette-Guerin (BCG). Development of a more effective vaccine has been considered as a major international public health priority. Some proteins from M. tuberculosis have been shown to be recognized by the immune system and can be correlated with protection against tuberculosis. The aim of this work was to design a recombinant fusion protein to use as subunit vaccine candidates against M. tuberculosis. We used epitopes of three different proteins as Ag85A, MPT-51 and Hsp-X based on their reported antigenicity. The corresponding gene segment of each epitope were cloned into pGEM-T Easy in fusion, and subcloned into the mycobacterial expression vectors, (pLA71, pLA73 and pMIP12). These vectors contain the E. coli and mycobacterial origins of replication and a multicloning site which places the heterologous gene in fusion with the signal sequence or the whole β-lactamase-encoding gene in pLA71 and pLA73, respectively. pMIP12 has a conserved mycobacterial Shine-Dalgarno sequence that may elevate antigen expression, and the native gene is expressed intracellularly. The recombinant plasmids for the development of recombinant BCG vaccine were produced and are now being tested for the fusion protein expression.
Preclinical design of less immunogenic biologics: Tregitopes and Tolerance (144.28)
Authors: Buhlmann Janet , Najafian Nader , Hui Daniel , D'Addio Francesca , Mingozzi Federico , Moise Lenny , De Groot Lesllie , Keegan Achsah , Hui Katherine , Sayegh Mohamed , Scott David , De Groot Anne 
Published: January 2023

Abstract
The contributions of effector T cell epitopes to autoimmunity and to the clinical immunogenicity of protein therapeutics are now broadly accepted. In allergy and autoimmunity, efforts are directed at broad suppression of cell-mediated immune responses. We have explored the effects of pre-treatment with natural regulatory T cell epitopes (Tregitopes) in murine models of autoimmune disease and protein therapeutics treatment. These natural Tregitopes are promiscuous MHC Class II T cell epitopes identified in IgG. The effect of Tregitopes has been studied in vitro and in vivo. Investigations using standard animal models of allergy (OVA), transplantation (MLR, cardiac transplant), protein therapeutics (OVA, FVIII) and autoimmunity (EAE, NOD) have demonstrated that tolerance to a range of antigens can be induced by co-administration of the Tregitopes. Tregitopes specifically activate CD4+CD25+FoxP3+ natural regulatory T cells. In vitro, co-incubation of antigens with Tregitopes leads to suppression of effector cytokine and chemokine secretion, reduced proliferation, and expansion of antigen-specific adaptive Tregs. In vivo, co-administration leads to suppression of T cell and antibody responses to the test antigens. These corroborating studies have now validated the original finding published by De Groot et al. (Blood 2008). Regulatory T cell induction by Tregitopes represents a paradigm shift for the treatment of autoimmune disease and the design of protein therapeutics.
Molecular characterization of the interaction between the rheumatoid arthritis shared epitope and calreticulin (144.7)
Authors: Ling Song , Cheng Andrew , Holoshitz Joseph , Pumpens Paul , Michalak Marek 
Published: January 2023

Abstract
Calreticulin (CRT) is a versatile endoplasmic reticulum protein also known as a cell surface receptor for several innate immune system ligands. We have recently reported that the rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe RA, acts as a ligand that specifically binds to CRT and activates innate signaling in other cells. To gain better insights into the molecular characteristics of SE-CRT interaction, here, we have mapped the binding site of the SE ligand on CRT. Experiments with CRT domain-deleted mutants and a sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate) chemical labeling method localized the SE binding site at the CRT P-domain. Computer-assisted 3D docking predicted the region 217-224 in the CRT P-domain as a potential SE binding site. Finally, site-directed mutagenesis demonstrated involvement of residues Glu217 and Glu223 in both cell-free binding and signal transduction assays. We conclude that the region encompassing amino acid residues 217-223 in the CRT P-domain is a binding site of the RA SE, with critical roles played by residues Glu217 and Glu223, which interact with SE residues Gln70 and Arg72, respectively. The SE binding site is distinct from those previously reported for other CRT ligands. The pathogenic implications of the findings will be discussed.