bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1.
Authors: Patel K D , McEver R P
Published: January 2023

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.
Common and Distinct Signaling Pathways Mediate the Induction of TNF-α and IL-5 in IgE Plus Antigen-Stimulated Mast Cells
Authors: Csonga R. , Prieschl E. E. , Jaksche D. , Novotny V. , Baumruker T.
Published: January 2023

A small number of signaling cascades represented by mitogen-activated protein kinases, phosphoinositol-3-kinase, protein kinase C, signal transducers and activators of transcription, Ca2+/calcineurin, and a few other molecules are linked to an incomparably large number of surface receptors. Parallel activation of several of these pathways and the existence of isozymes for a number of signal transmitting molecules generate the required complexity and specificity matching the receptor variety. Here we show that the proinflammatory mediator TNF-α and the growth factor IL-5 are activated along common and distinct signaling cascades in allergically stimulated murine mast cells. Both of them are dependent on Ca2+ influx, activation of calcineurin and nuclear factor of activated T cells as well as a member of the atypical PKC family, most likely PKCμ. Additionally, mitogen-activated protein kinases for TNF-α and members of the classical or nonclassical PKCs for IL-5, respectively, were identified as additional required pathways. Inhibition of the classical and nonclassical PKCs, however, does not abrogate IL-5 induction but instead leads to a switch to mitogen-activated protein kinases, which then become essential. The activated branches of this “salvage” signaling cascade are represented by extracellular signal-regulated kinase 1/2 and c-jun NH2 terminal kinase 1 in allergically stimulated mast cells.
Differences Between Responses of Naive and Activated T Cells to Anergy Induction
Authors: Hayashi Robert J. , Loh Dennis Y. , Kanagawa Osami , Wang Fanping
Published: January 2023

T cell unresponsiveness to Ag stimulation can be induced by several means. The precise mechanism by which this process occurs remains poorly understood. Preincubating T cells with either EDCI-fixed APC or ionomycin is a proven means of inducing T cell anergy with reduced IL-2 production in response to Ag stimulation. Using T cells from mice expressing the TCR transgene DO11.10, which is specific for a peptide (323–339) derived from hen egg OVA, we demonstrate that naive cells obtained directly from the host are resistant to the anergy induction by either fixed APC or ionomycin. TCR transgenic mice also deficient in the recombination-activating gene-2 (RAG-2−/−), preventing the formation of T cells with endogenous TCRs, were immunized with OVA, and in vivo activated T cells with low expression of CD62 were isolated. These primed cells possess the same sensitivity to ionomycin-induced anergy as in vitro activated cell lines. This unresponsive state most profoundly affects Ag-induced IL-2 production, with IFN-γ and IL-3 affected to a lesser degree and no effect observed on IL-4 production. Thus, T cells in vivo can be distinguished phenotypically by their susceptibility to anergic stimuli. Anergy so induced affects selected T cell functions.
Successful Adoptive Cellular Immunotherapy Is Dependent on Induction of a Host Immune Response Triggered by Cytokine (IFN-γ and Granulocyte/Macrophage Colony-Stimulating Factor) Producing Donor Tumor-Infiltrating Lymphocytes
Authors: Nagoshi Makoto , Goedegebuure Peter S. , Burger Ulrike L. , Sadanaga Noriaki , Chang Maximilian P. , Eberlein Timothy J.
Published: January 2023

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and systemic low dose rIL-2 effectively eradicates pulmonary metastases of the murine MCA-105 sarcoma. We described earlier that host CD8+ T cells are critical for tumor eradication and that successful treatment is associated with production of high levels of IFN-γ and granulocyte/macrophage (GM)-CSF by donor TIL in vitro. Here, we propose the mechanism through which adoptively transferred Thy-1.1+ TIL induce a host antitumor response in congenic Thy-1.2+ tumor-bearing mice. Donor Thy-1.1+ TIL were detected at the tumor site 12 h after transfer. These Thy-1.1+ cells produced IFN-γ and GM-CSF in situ. The percentage of Thy-1.1+ TIL at the tumor site increased up to 16.4 ± 4.9% 24 h after transfer but decreased to undetectable levels thereafter. In contrast, the percentages of host cells producing IFN-γ and GM-CSF continued to increase at the tumor site. These increases were significantly higher in TIL + rIL-2-treated mice compared with untreated mice and rIL-2-treated mice 48 h after TIL transfer. The appearance of IFN-γ+ and GM-CSF+ cells was followed by a large influx of host CD4+, CD8+, and Thy-1.2+ TIL and eventually by tumor eradication. This response was tumor specific since TIL obtained from MCA-205 did not induce high levels of IFN-γ and GM-CSF and did not induce tumor eradication of MCA-105 tumor. Coinjection of Thy-1.1+ TIL and anti-IFN-γ or anti-GM-CSF mAb significantly inhibited antitumor efficacy of the TIL + rIL-2 treatment. We conclude that successful adoptive immunotherapy in this model is mediated through cytokine production by adoptively transferred TIL that induce a host T cell-dependent antitumor response.
Distinct Classes of Chaperoned IL-6 in Human Blood: Differential Immunological and Biological Availability
Authors: Ndubuisi MacKevin I. , Patel Kirit , Rayanade Ravi J. , Mittelman Abraham , May Lester T. , Sehgal Pravin B.
Published: January 2023

Transport of IL-6 in blood is fundamental to the biology of this cytokine. In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guérin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both). Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays. Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not. The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6. In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays. Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG. The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.
Models for Antigen Receptor Gene Rearrangement. I. Biased Receptor Editing in B Cells: Implications for Allelic Exclusion
Authors: Mehr Ramit , Shannon Michele , Litwin Samuel
Published: January 2023

Recent evidence suggests that lymphocyte Ag receptor gene rearrangement does not always stop after the expression of the first productively rearranged receptor. Light chain gene rearrangement in B cells, and α-chain rearrangement in T cells can continue, which raises the question: how is allelic exclusion maintained, if at all, in the face of continued rearrangement? In this and the accompanying paper, we present comprehensive models of Ag receptor gene rearrangement and the interaction of this process with clonal selection. Our B cell model enables us to reconcile observations on the κ:λ ratio and on κ allele usage, showing that B cell receptor gene rearrangement must be a highly ordered, rather than a random, process. We show that order is exhibited on three levels: a preference for rearranging κ rather than λ light chain genes; a preference to make secondary rearrangements on the allele that has already been rearranged, rather than choosing the location of the next rearrangement at random; and a sequentiality of J segment choice within each κ allele. This order, combined with the stringency of negative selection, is shown to lead to effective allelic exclusion.
Expression and Function of TNF-Related Apoptosis-Inducing Ligand on Murine Activated NK Cells
Authors: Kayagaki Nobuhiko , Yamaguchi Noriko , Nakayama Masafumi , Takeda Kazuyoshi , Akiba Hisaya , Tsutsui Hiroko , Okamura Haruki , Nakanishi Kenji , Okumura Ko , Yagita Hideo
Published: January 2023

TNF-related apoptosis-inducing ligand (TRAIL), a new member of TNF family, induces apoptotic cell death of various tumor cells. We recently showed that TRAIL mediates perforin- and Fas ligand (FasL)-independent cytotoxic activity of human CD4+ T cell clones. In the present study, we investigated the expression and function of TRAIL on murine lymphocytes by using newly generated anti-murine TRAIL mAbs. Although freshly isolated T, B, or NK cells did not express a detectable level of TRAIL on their surface, a remarkable level of TRAIL expression was induced preferentially on CD3− NK1.1+ NK cells after stimulation with IL-2 or IL-15. In contrast, TRAIL expression was not induced by IL-18, whereas it efficiently potentiated lymphokine-activated killer activity of NK cells. In addition to perforin inactivation and neutralization of FasL by anti-FasL mAb, neutralization of TRAIL by anti-TRAIL mAb was needed for the complete inhibition of IL-2- or IL-15-activated NK cell cytotoxicity against mouse fibrosarcoma L929 target cells, which were susceptible to both FasL and TRAIL. These results indicated preferential expression of TRAIL on IL-2- or IL-15-activated NK cells and its potential involvement in lymphokine-activated killer activity.
α(1,3)-Fucosyltransferase VII and α(2,3)-Sialyltransferase IV Are Up-Regulated in Activated CD4 T Cells and Maintained After Their Differentiation into Th1 and Migration into Inflammatory Sites
Authors: Blander J. Magarian , Visintin Irene , Janeway Charles A. , Medzhitov Ruslan
Published: January 2023

Activated Th1 CD4 T cells bind to P-selectin and migrate into inflamed tissue, whereas Th2 cells do not. We show that α(1,3)-fucosyltransferase VII (FucT-VII) and α(2,3)-sialyltransferase IV (ST3GalIV), which are crucial for the biosynthesis of functional P-selectin ligands, are absent in naive CD4 T cells, but are rapidly up-regulated upon activation. Th1 or Th2 differentiation in the presence of polarizing cytokines leads to down-regulation of FucT-VII mRNA selectively in Th2 but not in Th1 cells. Influencing the differentiation by varying the priming dose of antigenic peptide results in similar FucT-VII down-regulation only in Ag-specific Th2 cells. ST3GalIV levels remain elevated. FucT-VII and ST3GalIV mRNAs are also up-regulated by Th1 cells primed in vivo and recruited into the lymph nodes draining delayed-type hypersensitivity sites. We identify FucT-VII gene expression as a principal difference between Th1 and Th2 cells, and underscore the importance of FucT-VII and ST3GalIV expression for the biosynthesis of functional selectin ligands.
Cross-Reactivity of Myelin Basic Protein-Specific T Cells with Multiple Microbial Peptides: Experimental Autoimmune Encephalomyelitis Induction in TCR Transgenic Mice
Authors: Grogan Jane L. , Kramer Achim , Nogai Axel , Dong Liying , Ohde Manuela , Schneider-Mergener Jens , Kamradt Thomas
Published: January 2023

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ α−). A peptide substitution analysis was performed in which each residue of the MBPAc1–11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1–11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1–11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1–11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.
The Signal Transduction Pathway of CD23 (FcεRIIb) Targets IκB Kinase
Authors: Ten Rosa M. , McKinstry Melissa J. , Trushin Sergey A. , Asin Susana , Paya Carlos V.
Published: January 2023

Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (FcεRIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets IκBα, leading to the activation of the transcription factor NF-κB. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of IκBα at Ser32/Ser36 residues. Overexpression of a dominant-negative IκBα transgene containing mutations at Ser32/Ser36 completely inhibits degradation of IκBα, NF-κB activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NFκB demonstrates that IκB kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of IκBα. Cotransfection experiments with an IKKβ negative dominant completely inhibit CD23 induced NFκB activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, IκBα degradation, and NF-κB nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates IκBα at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-κB activation and gene transcription.
β2-Microglobulin-Deficient Background Ameliorates Lethal Phenotype of the TGF-β1 Null Mouse
Authors: Kobayashi Shigetoshi , Yoshida Kunihiro , Ward Jerrold M. , Letterio John J. , Longenecker Glenn , Yaswen Linda , Mittleman Barbara , Mozes Edna , Roberts Anita B. , Karlsson Stefan , Kulkarni Ashok B.
Published: January 2023

TGF-β1 null (TGF-β1−/−) mice die at 3–4 wk of age and show an autoimmune inflammatory phenotype associated with enhanced expression of both class I and II MHC molecules. To determine the role of MHC class I Ags in the autoimmune manifestations and the inflammation observed in TGF-β1−/− mice, we generated TGF-β1−/− mice in the genetic background of β2-microglobulin deficiency (β2M−/−). TGF-β1−/−;β2M−/− mice had improved survival compared with TGF-β1−/− mice. Histopathological examination showed less severe inflammation, especially in the heart, where Mac-2 reactive macrophages were significantly decreased as compared with TGF-β1−/− mice. In vivo depletion of CD8+ T cells in TGF-β1−/− mice confirmed suppression of inflammation and reduction in the severity of the wasting syndrome. MHC class II mRNA expression in TGF-β1−/−;β2M−/− mice was also lower than that in TGF-β1−/− mice, suggesting reduced systemic inflammation. Autoimmune response as judged by serum Ab titers to ssDNA and 16/6 Id and by immune complex deposits in kidney was reduced in TGF-β1−/−;β2M−/− mice, when compared with that in TGF-β1−/− mice. Our data thus indicate that MHC class I molecules influence the development of the autoimmunity and the inflammation seen in TGF-β1−/− mice and CD8+ T cells may have a contribution to the inflammation in TGF-β1−/− mice.
An HLA-A2 Polyepitope Vaccine for Melanoma Immunotherapy
Authors: Mateo Luis , Gardner Joy , Chen Qiyuan , Schmidt Christopher , Down Michelle , Elliott Suzanne L. , Pye Stephanie J. , Firat Hüseyin , Lemonnier Francois A. , Cebon Jonathon , Suhrbier Andreas
Published: January 2023

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. Here we describe a recombinant poxvirus vaccine that codes for ten HLA-A2-restricted epitopes derived from five melanoma Ags conjoined in an artificial polyepitope or polytope construct. Target cells infected with the melanoma polytope vaccinia were recognized by three different epitope-specific CTL lines derived from HLA-A2 melanoma patients, and CTL responses to seven of the epitopes were generated in at least one of six HLA-A2-transgenic mice immunized with the construct. CTL lines derived from vaccinated transgenic mice were also able to kill melanoma cells in vitro. Multiple epitopes within the polytope construct were therefore shown to be individually immunogenic, illustrating the feasibility of the polytope approach for melanoma immunotherapy. Tumor escape from CTL surveillance, through down regulation of individual tumor Ags and MHC alleles, might be overcome by polytope vaccines, which simultaneously target multiple cancer Ags.
MDP (Muramyl Dipeptide) Limits Anatomical Damage and Neurological Dysfunction in Rat with Contusive Spinal Cord Injury (SCI) (101.16)
Authors: Garg Maneesh S , Patel Monal , Puzis Raisa , Alexander Marcillo E , Pereira Francisco C , Bunge Mary B , Pearse Damien D
Published: January 2023

MDP is the minimum structural unit responsible for the immunoadjuvancy of bacterial cell wall peptidoglycans. Many properties of MDP manifest potential therapeutic efficacy for SCI. These include

stimulation of phagocytosis of immunocompetent cells, thereby facilitating myelin removal with minimal beneficial proinflammatory cytokine alterations,reduced glutamate release,increased intra- and extra-cellular Mg2+, thereby blocking NMDA receptors, andsuppressor cytokine expression, such as IL-10.

The current experiment delivered MDP by injection, 6 hours post T8 moderate contusive spinal cord injury in adult Fischer rats. At 2 wk after injury, histological examination revealed reductions in lesion volume associated with deposits of chondroitin sulfate proteoglycan and more myelinated axons. The activated (ED1+) macrophage/microglia count was higher in the more limited injury site of MDP animals. Concurrently, most were Mac1+, indicating active phagocytosis. Furthermore, MDP treated animals displayed fewer ED1+ cells rostral and caudal to the lesion compared with controls. Behaviorally, MDP animals displayed frequent co-ordination with weight-supported steps (BBB, 13.7 ± 1.6) compared with the occasional stepping in controls (BBB, 10 ± 0.6; n=6, p<0.05). Thus, MDP appears to be a novel therapeutic agent to treat acute SCI.

Supported by CRF, Hollfelder Foundation, and Miami Project
Anti-inflammatory activity of algal oils containing Docosahexaenoic Acid (DHA) and omega-6 Docosapentaenoic Acid (DPAn-6) (101.5)
Authors: Nauroth Julie Marie , Van Elswyk Mary , Liu Ying , Arterburn Linda
Published: January 2023

Background: Studies have shown anti-inflammatory activity associated with fish oil and ascribed this effect to the long-chain omega-3 fatty acids DHA and especially eicosapentaenoic acid (EPA).

Objective: The purpose of this study was to examine the anti-inflammatory effects of DHA-rich algal oils which contain little or no EPA and determine the contribution of their individual constituent fatty acids, particularly DHA and DPAn-6.

Results: The effects of feeding DHA-rich Martek DHA-T™ algal oil and DHA/DPAn-6-rich Martek DHA-S™ algal oil on rat carrageenan-induced hind paw edema was examined. The S-oil was more effective at reducing edema than the T-oil, suggesting that DPAn-6 has anti-inflammatory properties. We next tested the in vitro effects of the individual pure fatty acids on reducing LPS-stimulated IL-1β and TNF-α secretion by human peripheral blood mononuclear cells. The relative potency was DPAn-6 (most potent) > DHA > EPA. All three fatty acids were effective at reducing by >50% neutrophil fMLP-mediated migration in vitro. Further in vivo analyses demonstrated that feeding pure DHA and EPA ethyl esters (EE) reduced paw edema and the combination of DHA/DPAn6-EE which mimics algal oil was more potent than the combination of DHA/EPA-EE which mimics fish oil.

Conclusions: DHA, DPAn-6, and EPA have anti-inflammatory activity in vitro and in vivo. Algal S-oil containing the combination of DHA and DPAn-6 shows potential for use in anti-inflammatory applications.
Human umbilical cord blood (UCB) derived CD133 cells demonstrate homing capability in vasculogenesis and evoke a proliferative response as well as IL-4 and IL-10 cytokine production in vitro (102.3)
Authors: Fanning Laura R. , Finney Marcie R. , Winter Daniel G. , Greco Nicholas J. , Kozik Margaret M. , Laughlin Mary J.
Published: January 2023

Homing, IL-8 production and immunogenicity are aspects of CD133 cells, a population of stem cells, which may augment postnatal angiogenesis therapy. CD133 cells were isolated from UCB mononuclear cells (MNC) by magnetic bead selection (Miltenyi) and analyzed by flow cytometry. CD133 cells showed co-expression of KDR (3.5%), CD105 (22.7%), CXCR4 (8.7%), HLA-DR (57.6%) and HLA-ABC (66.5%), suggesting CD133 cells are capable of presenting alloantigen. We next performed 3H-thymidine incorporation MLR using adult peripheral blood (AB) MNC stimulated by irradiated UCB CD133 (46939 ± 2764 cpm) and MNC (49548 ± 2018 cpm). MLR studies with CSFE-stained responder cells confirmed equivalent rates of cell division. The observed proliferation was not an artifact of CD133 cell selection. Preliminary comparison studies of cytokine production of IL-4 and IL-10 increased when AB MNC were stimulated by CD133 as compared to UCB MNC (IL-4: 141±45.7pg/mL and IL-10: 964±538pg/mL,). Additionally, UCB CD133 production of IL-8 was elevated as compared to MNC (103±380 pg/mL). Transwell migration to SDF-1 (100ng/mL) demonstrated a 1.8 ± 0.7 fold increase compared to basal media, correlating with CXCR4 expression. In summary, UCB CD133 cells demonstrate homing capability, potential cellular recruitment (IL-8 production) demonstrate TH2 skewing and induce AB MNC proliferation relevant to angiogenesis and cellular therapeutics.
TGFbeta1 is not essential for generation and maintenance of natural Treg cells (128.10)
Authors: Bommireddy Ramireddy , Babcock George F , Singh Ram R , Doetschman Thomas
Published: January 2023

Natural CD4+CD25+FOXP3+ Treg cells serve as powerful regulators of autoimmunity. Hence, there has been a great deal of interest in understanding their regulation. TGFβ1 is considered to be required for peripheral maintenance of Treg cells. However, we demonstrate an increase in the percentage of FOXP3+ Treg cells in the spleens and thymi of Tgfb1−/− mice. Despite this abundance of Treg cells, Tgfb1−/− mice develop a marked expansion of activated T cells in their lymphoid organs, suggesting that activated Tgfb1−/− T cells may not be amenable to suppression any more. However, we demonstrate that Tgfb1−/− T cells do remain responsive to the suppressive effect of TGFβ1 in vitro, suggesting that the absence of TGFβ1 production by Treg cells likely leads to their loss of regulatory function. Finally, we show that whereas Tgfb1−/− mice that express a chicken OVA-specific TCR transgene (DO11.10) have an increase in Treg cells, there are no detectable CD4+CD25+ T cells in the spleens of DO11.10 Rag1−/− mice that have no self-reactive T-cells, regardless of whether they express Tgfb1. This suggests that Treg-cell generation is self-antigen dependent and that TGFβ1 is not required for Treg-cell generation and maintenance. We contemplate that TGFβ1 is likely required for the regulatory function of Treg cells to prevent activation of T cells and autoimmunity.
Therapeutic potential and the mechanism of action of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) in established Experimental Autoimmune Myasthenia Gravis (128.13)
Authors: SHENG JIAN RONG , Prabhakar Bellur S , Meriggioli Matthew N
Published: January 2023

Dendritic cells (DCs) are potent antigen presenting cells responsible for priming naïve T cells in an immune response. Distinct subpopulations of DCs play a central role in immune regulation by preferentially inducing immunity or tolerance. Recently, we observed that the selective activation of particular subsets of DCs utilizing fms-like tyrosine kinase receptor 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had profound effects on the induction of Experimental Autoimmune Myasthenia Gravis (EAMG). Treatment with GM-CSF effectively suppressed the induction of EAMG, and down-modulated anti-AChR T cell and antibody responses. These effects were associated with the activation of CD8α- DCs, the enhanced production of suppressive cytokines such as IL-4 and IL-10, and the mobilization of CD4+CD25+ and Foxp3+ regulatory T cells (Tregs). Based on these observations, we hypothesized that the selective activation of DC subsets using GM-CSF can skew ongoing anti-AChR immune responses in favor of a Th2 polarization and mobilize antigen-specific CD4+CD25+ and Foxp3+ Tregs capable of suppressing established EAMG. In the present study, we evaluated the therapeutic potential of GM-CSF by examining its effectiveness in the treatment of established disease, and its ability to suppress ongoing autoimmune responses in active EAMG. We are continuing to understand the mechanisms of Treg induction and modes of action in the GM-CSF induced suppression of ongoing EAMG, with regard to antigen specificity and duration of induced tolerance.
A Novel Role for PTEN as a Regulator of T Cell Tolerance (128.27)
Authors: Buckler Jodi L. , Walsh Patrick T. , Turka Laurence A.
Published: January 2023

The lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a key negative regulator of the PI3K pathway. Recent studies characterizing T cell specific PTEN deficient mice (PTENΔT) suggest that in addition to its role as a tumor suppressor, PTEN also plays a key role in regulating self-tolerance. Our studies have identified a novel mechanism by which PTEN regulates peripheral T cell tolerance. Initial studies revealed that in the absence of PTEN, TCR signals alone, without CD28 costimulation, are sufficient to activate CD4+ T cells both in vitro and in vivo. Importantly, uncoupling T cells from the requirement for costimulation renders them refractory to anergy/tolerance induction. Ongoing studies in which PTENΔT mice were bred onto a TCR transgenic background suggest that the skewing the repertoire away from self-specificities prevents the development of autoimmune serology, suggesting that TCR signals are necessary for the onset of the autoimmune disease observed in these mice. Together, these data suggest that negative regulation of TCR signals by PTEN imposes a requirement for costimulation and in doing so plays a critical role in the maintenance of T cell tolerance.
Activation-induced split anergy: a mechanism of persistence of autoimmune T cells (128.28)
Authors: Dubey Shweta , Wadhone Pallavi , Wang James , Singh Ram Raj
Published: January 2023

Anergy and apoptosis are major pathways of T cell tolerance. Impairments in these mechanisms are believed to contribute to the development of autoimmune diseases such as SLE and arthritis. Here we analyzed features of T cell anergy, activation and activation-induced cell death in lupus-prone and MHC-matched healthy mice. Unexpectedly, lupus mice exhibit reduced calcium flux, IL-2 production and proliferation in spleen cells upon anti-CD3 stimulation. Such T cell hyporesponsiveness is abrogated upon ionomycin stimulation, suggesting a specific impairment in TCR signaling pathway. This suggests that autoimmune T cells exhibit features of anergy, rather than resistance to it. In contrast, autoimmune T cells produce higher levels of pro-inflammatory cytokine interferon-gamma, and exhibit features of activation, i.e., increased activation markers. Consistently, quantitative gene expression analyses show lower Il2, but higher Ifn-g, mRNA in lupus T cells than in controls. Further, lupus T cells exhibit increased constitutive phosphorylation of ZAP-70 and other T cell signaling molecules, which do not increase upon anti-CD3 stimulation. Finally, lupus T cells resist apoptosis upon specific TCR signaling. Thus, autoimmune T cells display features of activation as well as anergy. Such split anergic T cells display resistance to activation-induced cell death. These data lead us to posit that persistent in vivo T cell activation results in subsequent induction of anergy that manifests as impaired responsiveness to TCR mediated signaling and prevents T cells from undergoing activation-induced cell death. Thus, induction of anergy may actually prevent autoreactive T cells from being eliminated. Ongoing experiments will delineate mechanisms underlying this novel mechanism of autoimmune T cell persistence.
Central Histamine H3 Receptor Signaling Negatively Regulates Autoimmune Inflammation (129.31)
Authors: Teuscher Cory , Subramanian Meena , Noubade Rajkumar , Gao Jian Feng , Offner Halina , Zachary James F. , Blankenhorn Elizabeth P.
Published: January 2023

Histamine is a ubiquitous regulator of diverse physiologic processes including inflammation, immune modulation and neurotransmission. Four subtypes of histamine receptors are currently recognized and genetic and pharmacological studies have shown that the H1 and H2 receptors play a role in susceptibility to experimental allergic encephalomyelitis (EAE), the primary autoimmune model of multiple sclerosis. Histamine H3 receptor (H3R), which is not expressed in hematopoietic cells, is a presynaptic auto- and hetero-receptor. Here we show that H3RKO mice develop significantly more severe acute phase clinical disease and neuropathology compared to wild-type controls. In H3RKO mice this is preceded by disruption of the blood brain barrier and increased chemokine/chemokine receptor expression in peripheral T-cells. These data are consistent with inhibition of H3R-mediated neurogenic control of cerebrovascular tone and T-cell function. Additionally, genetic studies indicate that an H3R polymorphism leading to differential expression of H3R isoforms underlies eae8, a locus controlling disease associated weight loss, a phenotype known to be regulated by central H3R activity.
The role of regulatory T cells in the NZB disease (130.10)
Authors: Scaglione Brian , Salerno Erica , Langer Jerome A. , Pan Manjing , Raveche Elizabeth
Published: January 2023

Regulatory T cells (Tregs) play an important role in protection against autoimmune disease. These CD4+ CD25+ Foxp3+ T cells are also known to be potent inhibitors of antitumor immune responses. The NZB mouse is a murine model for both autoimmune diseases such as SLE, since high levels of autoantibodies are present, as well as a model of human CLL, due to the expansion of malignant B-1 cells. In this study, we examined the role of Tregs in the onset of these disease manifestations. Flow cytometric analysis of spleen, lymph node, and peritoneal cells showed increased levels of CD4+ CD25+ Foxp3+ Tregs in 12 to17-month old NZB mice with a B cell malignancy identified by IgM+ CD5+ B220 dull cells as compared to healthy C57Bl/6 controls. Furthermore, ex vivo treatment of cells from these tissues with IFN-α, which has been known to increase autoimmune symptoms in NZB mice, led to a decrease in the levels of Tregs 48 hours post treatment. These results indicate that while the levels of Tregs found in NZB mice may suppress a more severe autoimmune disease, they might also contribute to the development of the B cell malignancy.

(Funded in part by a grant from the Alliance for Lupus Research to JAL).
The role of B-cell subsets in pristane-induced lupus (130.14)
Authors: Barker Tolga , Lee Pui Y. , Weinstein Jason , Nacionales Dina C. , Kelly-Scumpia Kindra M. , Sobel Eric S. , Reeves Westley H. , Satoh Minoru
Published: January 2023

An intraperitoneal (ip) injection of pristane in mice induces a biphasic immune response characterized by an early T-independent phase (increased total IgM, IgM anti-ssDNA peaking at 2 wks) and a later T-dependent phase (increased total IgG, IgG anti-snRNPs, Su, chromatin after 4–6 wks). Although peritoneal B-1 cells are thought to be responsible for the early phase, they disappear after ip pristane. IgG autoantibodies can be produced in SCID mice by transferring peritoneal, spleen, or lymph node cells from pristane-treated mice. To evaluate further the role of B-cell subsets in the two phases of pristane-induced autoimmunity, GFP+ naïve peritoneal, spleen, or bone marrow (bm) cells were transferred ip to T- and B-cell deficient RAG1−/− mice, followed by an ip pristane 3 days later. Autoantibodies and total Ig (ELISA), and the phenotypes of GFP+ cells in the recipients (flow cytometry) were analyzed. At 3 days prior to pristane ip, GFP+ peritoneal B-1 cells (CD19+CD11b+ CD5+ IgM+) were detected in recipients of bm, peritoneal, or spleen cells. Following pristane ip, peritoneal B-1 cells disappeared, as seen in immunocompetent mice. Analysis of GFP+ cells in peritoneal cavity at day 10 revealed the predominance of CD11b+ macrophages in the peritoneal cell recipients whereas T and B cells were predominant in bm or spleen cell recipients. In mice that received GFP+ spleen cells, IgM anti-ss- and -dsDNA dramatically increased at 2–4 weeks after pristane, while the total IgM levels increased in all groups at later time points. We conclude that splenic B cells may be responsible for the early IgM response in pristane model.

Supported by NIH
Intrinsic B cell defect results in abnormal TD antigen response in lupus-prone mice (130.17)
Authors: Niu Haitao , Morel Laurence
Published: January 2023

The response to TD antigen NP-KLH was compared between the lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice and control B6 mice. The amount of anti-NP IgG Abs in TC mice was significantly decreased as compared to B6 mice. In contrast, total anti-IgG Abs and anti-dsDNA Abs were increased compared to B6 mice, indicating that the decreased was specific for the immunization. This corresponds to the known poor immunization response of lupus patients. In addition, germinal center (GC) NP-specific B cells were decreased in TC mice compared to B6 mice, although there was no difference in the total number of GC B cells between the two strains. Co-transfer of purified CD43- B and CD4+ T cells from either B6 or TC mice showed that a TC intrinsic B cell defect was involved. B6.Rag−/− mice transferred with non-T BM cells from immunized TC mice lost anti-NP antibody production unlike transfer with BM cells from B6 mice. In contrast, there was similar Ab production between the two strains when transferred with non-T splenocytes, indicating that long-lived plasma cells in TC mice accumulated in spleen, instead of migrating to BM. In conclusion, lupus-prone mice show a deficient response to NP-KLH, in which NP-specific B cells differentiate differently from autoantigen-specific B cells. This deficiency is due at least in part to a defect in B cell maturation and plasma cell migration. SLE susceptibility loci involved in these defects will be mapped in future.
Novosphingobium aromaticivorans - causative agent or trigger of Primary Biliary Cirrhosis? (130.29)
Authors: Mattner Jochen , Savage Paul B. , Teyton Luc , Wicker Linda , Ridgway William M , Oertelt Sabine M , Gershwin Eric M , Bendelac Albert
Published: January 2023

Undetected bacterial infections elicit strong immune responses that can masquerade into autoimmune diseases. This has recently been proposed for Primary Biliary Cirrhosis (PBC), a liver disease where individuals have signs of chronic immune responses against Novosphingobium aromaticivorans, an ubiquitous xenobiotic-metabolizing microorganism. Humans with PBC exhibit also NKT cell redistribution to the liver. We have obtained evidence that infection of female B6, SJL and NOD congenic mice with Novosphingobium triggers potent and persistent IgG responses to the mitochondrial antigens of PBC and induce biliary duct lesions and portal inflammation resembling PBC in humans. As our previous work has identified microbial glycosphingolipid (GSL) ligands in the cell wall of these Novosphingobium bacteria that trigger NKT cell activation we propose that the striking differences originate from the nature of the innate signals associated with Sphingomonas infection, which recruits NKT cell help for autoreactive B cells, as opposed to TLR signaling alone in the case of other bacteria, which fails to provide similar helper signals.
Vγ4+ γδ T cells act as TH-17 cells in a mouse model of collagen-induced arthritis (130.39)
Authors: Roark Christina , French Jena D. , Taylor Molly A. , Bendele Alison M. , Born Willi K. , O’Brien Rebecca L.
Published: January 2023

We have examined two γδ T cells subsets, Vγ1 and Vγ4, in collagen-induced arthritis. The Vγ4+ subset increases expression of CD44 and loses expression of CD62L after each collagen/CFA injection, suggesting this subset is activated during the disease process. The Vγ1+ subset increases in overall numbers but the surface markers remain unchanged. We depleted the γδ subsets to examine their roles in the disease process. Disease scores and histology revealed a significant reduction in both the severity and incidence of arthritis in mice depleted of Vγ4+cells. Depletion of the Vγ1+ subset had no significant effect. We also noted a significant decrease in the levels of total IgG and IgG2a anti-collagen antibodies in mice that were depleted of Vγ4+ cells. Next, we used intracellular cytokine staining to identify which cytokines were being made by the Vγ4+ cells during the height of their activation. Surprisingly, over 60% of the Vγ4+ cells produce IL-17. In fact, as a population, the Vγ4+ cells contain as many or more IL-17 producers as do all CD4+ αβ T cells together. Thus, depletion of the Vγ4+ subset may reduce IL-17 levels, leading to less severe disease in this model. Finally, preliminary data indicate that the Th-17 Vγ4+ cells in this disease model are preferentially paired with one delta chain and express very limited junctional sequences.

This work is supported by an Arthritis Investigator award from the Arthritis Foundation.
An autoimmune basis in the disease pathogenesis of COPD/Emphysema (130.41)
Authors: Goswami Sangeeta , Barranco Wade , Lee Seung-Hyo , Grudo Ariel , Corry David B , Kheradmand Farrah
Published: January 2023

Chronic obstructive pulmonary disease (COPD) and emphysema are chronic inflammatory diseases of the lung. Chronic exposure to cigarette smoke is the most common cause of COPD/emphysema however the pathogenesis is not fully understood. Innate immune cells like macrophages and neutrophils are thought to be the most critical factor in the disease pathogenesis. However, we have found that lung and peripheral blood T cells in patients with severe emphysema secrete Th1 cytokines and chemokines when stimulated with elastin peptides in vitro and they have increased anti-elastin antibody as compared to controls. Based on these human studies, we tested the hypothesis that elastin peptides can act as self-antigen and induce an autoimmune lung inflammation in mice. Mice were immunized intra-peritonealy with mouse elastin peptides and adjuvants. Like human COPD, we found increased numbers of activated macrophages and neutrophils as well as increased MMP9 and MMP12 in the airway fluid of elastin immunized mice. Hilar lymph node cells of elastin immunized mice produced more IFN-g upon restimulation with elastin peptides. Additionally, elastin immunized mice had increased anti-elastin antibody titer in the serum and increased IFN-g in the lungs. The establishment of the mouse model of elastin-mediated autoimmune lung inflammation will be critical in elucidating the role of adaptive immune system in pathogenesis of COPD/emphysema.
Cardiac Troponin I Induces Severe Autoimmune Inflammation in the Myocardium (130.43)
Authors: Kaya Ziya , Göser Stefan , Andrassy Martin , Buss Sebastian J. , Leuschner Florian , Volz Christian H. , Öttl Renate , Zittrich Stefan , Blaudeck Natascha , Hardt Stefan E. , Pfitzer Gabriele , Rose Noel R. , Katus Hugo A.
Published: January 2023

Cardiac troponins are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. However, little is known about the long-term effects of the released troponins on cardiac function.

In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES, MCP-1, MIP–1, MIP-1ß, MIP-2, TCA-3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening. Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.
PD20, a novel regulator of lymphocyte responses and immune homeostasis. (130.5)
Authors: Sun Honghong , Gong Shunyou , Hilliard Anja , Chen Youhai H.
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS). Using a high-throughput gene microarray technology, we identified a novel gene, designated programmed cell death 20 kD (PD20) from inflamed spinal cord of mice with EAE. PD20 encodes a polypeptide of 184 amino acids with an estimated molecular weight of 20 kD. It is preferentially expressed in lymphoid tissues and inflamed but not normal nervous tissue. Germ line mutation of the PD20 gene induced a systemic inflammatory disease characterized by multi-organ inflammation, splenomegaly, heightened inflammatory cytokine production and eventual death of the animals. The disease can be significantly accelerated by a single injection of endotoxin. In vitro, PD20-deficient T cells are hypersensitive to TCR stimulation but are resistant to activation-induced cell death. Importantly, PD20 is able to interact with caspase-8 and is capable of enhancing Fas ligand-induced cell death. These results establish that PD20 is a novel caspase-8-interacting protein that regulates apoptosis, lymphocyte responses and immune homeostasis.
Inhibitory oligonucleotide (IN-ODN) structure-function relationships: species and backbone differences (131.19)
Authors: Ashman Robert F. , Goeken Adam , Lenert Petar
Published: January 2023

The recognition of CpG motifs in bacterial DNA by Toll-like Receptor (TLR9) is a key function of the innate immune system, announcing the presence of an infection. We have previously developed synthetic oligonucleotide (ODN) inhibitory of TLR9 function with phosphorothioate (S) backbones conforming to the sequence n CC n not C not C nn GGG nnnn where n is any base. Here we show that these sequence requirements generate IN-ODN when the backbone is phosphodiester (O) also. In general, whereas stimulatory O-ODN are 2 logs less potent than their S-backbone congeners IN-ODN are 3 logs less potent. Changes in the critical bases diminish activity with either backbone; however, when the backbone is S, such changes diminish activity of stimulators much less, and inhibitors much more, than if the backbone is O. Certain end truncations enhance activity and even the minimal sequence CCTGGAGGG retains substantial inhibitory activity. Human-mouse relative activity comparisons tend to be similar between critical base variants, but may differ substantially between end truncation variants. HEK cells transfected with mouse or human TLR9 and an NFkB binding luciferase promoter respond to much lower concentrations of IN-ODN than do primary cells of either species. The neutral sequences with S backbones developed for mouse cells are also neutral with O backbones and with human cells.

Supported by AI04737405 from NIH
MDX-1100, a fully human anti-CXCL10 (IP-10) antibody, is a high affinity, neutralizing antibody that has entered Phase I clinical trials for the treatment of Ulcerative Colitis (UC). (131.20)
Authors: Kuhne Michelle , Preston Benjamin , Wallace Steven , Chen Sharline , Vasudevan Geetha , Witte Alison , Cardarelli Pina
Published: January 2023

The chemokine, CXCL10 or interferon γ inducible protein-10 (IP-10) is a chemotactic cytokine for activated T cells and monocytes and plays an important role in migration of cells into sites of inflammation. The receptor for CXCL10, CXCR3, is expressed by activated T cells, eosinophils, NK, and endothelial cells. CXCL10 levels are elevated in ulcerative colitis (UC) amongst other inflammatory diseases. In preclinical animal models of UC, antibodies against CXCL10 have been shown to modify disease progression. Medarex, Inc. has developed a fully human monoclonal antibody (MDX-1100) that binds selectively to CXCL10. This antibody binds to the ligand with high affinity and effectively competes for ligand binding to CXCR3 expressing cells. MDX-1100 blocks CXCL10 induced calcium flux and cell migration with an estimated IC50 in the low nM range. In order to identify potential pharmacodynamic markers for CXCL10 activity, gene chip analysis was performed with RNA purified from CXCL10-stimulated human PBMCs. We identified cell surface receptors, intracellular and soluble markers that are CXCL10 responsive and confirmed the induction of a subset of these genes using quantitative RT-PCR. In summary, MDX-1100, an antibody that binds and neutralizes the activity of CXCL10 is predicted to reduce disease severity in patients with UC and thus a Phase I clinical trial has been initiated.