bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

Immunophenotyping of PBMC from SLE patients (n~40) indicated significantly reduced numbers of CD4+CD25hi Foxp3+ T cells and CD8+Foxp3+T cells (p<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<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<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

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

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 < 1/3rd sample volume. Alternately with similar sample volumes, the Optimiser boasts >10x higher sensitivity, to achieve detection limits of < 1 pg from a 100 µl sample. In all cases, a complete sandwich assay can be completed in < 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

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

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

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

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

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.
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

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

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

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

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

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<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<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<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<0.05) in MSC treated lungs. Long-term MSC (passage >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

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 (>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

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

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

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 >70% of CD138+ plasma cells in flow cytometry assays, as well as >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

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

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

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

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

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.
Expression profiling of B-1 and B-2 progenitors (36.15)
Authors: Tung James , Veys Kristine , Sembrano Daryl , Hall Casey , Ross Christian
Published: January 2023

B-1 and B-2 comprise the two B cell lineages. B-1 and B-2 cells can be distinguished by their surface phenotype and also by their developmental ontology, immunoglobulin repertoire, anatomical localization, and immune functions. Importantly, B-1 cells arise early in fetal liver while B-2 cells are generated in the bone marrow after birth. The appearance of B-1 and B-2 cells is the result of fetal B-cell development versus adult B-cell development. B-1 and B-2 cells are generated from distinct progenitor cells, namely B-1 progenitors and B-2 progenitors. B-1 progenitors express CD19 but do not express B220. In contrast, B-2 progenitors express B220 but do not express CD19. To determine the fate decisions that led to fetal versus adult B cell development, we sorted B-1 progenitor cells from fetal liver and B-2 progenitor cells from adult bone marrow and performed gene expression array analysis. In our poster, we will present the results of the expression array comparison between the two progenitor populations. We will confirm the expression array comparison results by molecular techniques and discuss the potential significance of these differences.
Ikaros null mice demonstrate defects in dendritic cell development. (36.17)
Authors: Brugmann William , Winandy Susan
Published: January 2023

Ikaros is a transcription factor that is expressed almost exclusively in cells of the hematopoietic lineage. Ikaros null mice lack several hematopoietic cell lineages including some dendritic cell (DC) subsets. Additionally, several Ikaros null bone marrow (BM) progenitor populations have a decreased expression of the receptor tyrosine kinase, Flt3. DCs can be generated in vitro by culture of BM with either GM-CSF or Flt3L (the ligand for Flt3). Our studies find that loss of Ikaros leads to a specific defect in the ability of BM precursors to generate DCs in response to culture with Flt3L but not in response to culture with GM-CSF. Those DCs that do develop from the culture of Ikaros null BM with Flt3L have an activated phenotype with higher expression of MHC class II and the costimulatory molecules CD40, CD80 and CD86 than their wild type counterparts. An examination of gene expression in Ikaros null BM progenitors cultured with Flt3L shows an inappropriately low expression of Irf4, Irf8 and Spib, genes encoding transcription factors known to be important for DC development. Studies are currently underway to determine whether enforced expression of Ikaros, Flt3 or one of the above-mentioned transcription factors can rescue DC development upon culture of Ikaros null BM with Flt3L. These studies will provide a potential molecular mechanism for the impaired development of DCs in Ikaros null mice.
Cell intrinsic E47 is required for stem cell self-renewal and differentiation but is dispensable for short-term myeloid development (36.2)
Authors: Borghesi Lisa , Yang Qi , Esplin Brandt
Published: January 2023

The immune system is constantly replenished by a rare population of hematopoietic stem cells (HSC) residing in the bone marrow of adult organisms. E-proteins, the widely expressed basic helix-loop-helix (bHLH) transcription factors, appear to contribute to HSC activity, but their specific cell intrinsic functions remain undefined. In contrast to a recent report, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs. In our quantitative assays, E47 deficient progenitors show competent myeloid production in vitro and in vivo as well as under burden of a pathogen mimic. We also show that long-term myeloid and lymphoid differentiation is compromised due to the progressive loss of the self-renewal potential of HSCs in vivo. Compromised self-renewal of E47 null HSCs is associated with over-proliferation and premature exhaustion under replication stress. These observations suggest that cell-intrinsic E47 is dispensable for the short-term myeloid repopulation activity of HSCs and, by contrast, that E47 plays an essential cell-autonomous role in the self-renewal of HSCs by preventing hyperproliferation-associated exhaustion following replication stress.
CEACAM1 regulates granulopoiesis through inhibition of G-CSFR-Stat3 signaling in an ITIM-SHP-1 dependent manner (36.20)
Authors: Pan Hao , Shively John
Published: January 2023

CEACAM1 (CD66a) is involved in many biological processes, including regulation of lymphocyte activation. Although granulocytes highly express CEACAM1, the role of CEACAM1 in granulopoiesis and granulocyte dependent immune responses is not well understood. We showed here that CEACAM1 expression correlated with granulocytic differentiation and CEACAM1-/- mice developed severe neutrophilia in a cell-autonomous fashion associated with hyper-proliferation of granulocyte progenitors. Also, the regulation of CEACAM1 on progenitor cells was granulocytic lineage specific. We found CEACAM1 acted as a co-inhibitory receptor for G-CSFR, leading to the down-regulation of Stat3 activity. This inhibitory effect was dependent on G-CSF induced phosphorylation of ITIMs on CEACAM1 and the recruitment of SHP-1. Moreover, CEACAM1-/- mice were hypersensitive to Listeria monocytogenes (LM) infection with accelerated mortality due to augmented neutrophil liver infiltration and enhanced cytokine levels. Reintroduction of CEACAM1 restored normal granulopoiesis and host sensitivity to LM infection upon transduction into CEACAM1-/- mice; Mutation of the Tyr residues in its ITIMs abrogated this restoration. Finally, knocking down Stat3 in CEACAM1-/- mice rescued normal granulopoiesis and attenuated host sensitivity to LM infection. Thus, we identified CEACAM1 as a co-inhibitory receptor for G-CSFR regulating granulopoiesis and affecting the host innate immune response to bacterial infections.
ENPP1 is a novel cell surface marker of germinal center and memory B cells and is highly expressed in plasma cells (36.3)
Authors: Wang Hongsheng , Shin Dong-Mi , Gonzalez-Garcia Ines , Abbasi Sadia , Feng Jianxun , Morse Herbert
Published: January 2023

The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), also known as plasma cell (PC) alloantigen 1 (PC-1), is expressed on PCs and many non-lymphoid tissues. ENPP1 hydrolyzes NTP to generate NMP and pyrophosphate (PPi). PPi is an important factor for controlling mineralization of the bone. In addition, ENPP1 directly associates with the insulin receptor and regulates insulin receptor signaling. Abnormal expression of ENPP1 has been associated with diabetes and insulin resistance. Although ENPP1 was first identified in PCs, its expression and function in B lineage cells remains poorly understood. Here we describe a previously unknown expression pattern of ENPP1 in murine late stage B cells. By using an anti-ENPP1 mAb and flow cytometry, we found that the expression levels of ENPP1 in early and mature naïve B cells were relatively low. However, germinal center (GC) B cells, PCs and memory B cells expressed 2-4-fold more ENPP1 than resting B cells. Moreover, gene expression profiling studies of primary lymphomas in NFS.V+ congenic mice revealed high expression levels of ENPP1 in plasmacytomas, immunoblastic lymphomas, and centroblastic-follicular lymphoma types, corresponding with their PC and GC origins. These studies demonstrate that ENPP1 can be a useful marker for identification of stages in GC and post-GC cell differentiation and of lymphomas with a GC or PC origin. The function of ENPP1 in B cells is currently under investigation.