bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral Infection Modulates Expression of Hypersensitivity Pneumonitis
Authors: Gudmundsson Gunnar , Monick Martha M. , Hunninghake Gary W.
Published: December 2022

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes that cause farmer’s lung disease. The early response to Ag is an increase in neutrophils in the lung, whereas the late response is a typical Th1-type granulomatous disease. Many patients who develop disease report a recent viral respiratory infection. These studies were undertaken to determine whether viruses can augment the inflammatory responses in HP. C57BL/6 mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) for 3 consecutive days per wk for 3 wk. Some mice were exposed to SR at 2 wk after infection with respiratory syncytial virus (RSV), whereas others were exposed to SR after exposure to saline alone or to heat-inactivated RSV. SR-treated mice developed a typical, early neutrophil response and a late granulomatous inflammatory response. Up-regulation of IFN-γ and IL-2 gene expression was also found during the late response. These responses were augmented by recent RSV infection but not by heat-inactivated RSV. Mice with a previous RSV infection also had a greater early neutrophil response to SR, with increased macrophage inflammatory protein-2 (MIP-2, murine equivalent of IL-8) release in bronchoalveolar lavage fluid. These studies suggest that viral infection can augment both the early and late inflammatory responses in HP.
Protection by Group II Phospholipase A2 Against Staphylococcus aureus
Authors: Laine Veli J. O. , Grass David S. , Nevalainen Timo J.
Published: December 2022

Group II phospholipase A2 (PLA2) is an enzyme that has marked antibacterial properties in vitro. To define the role of group II PLA2 in the defense against Staphylococcus aureus, we studied host responses in transgenic mice expressing human group II PLA2 and group II PLA2-deficient C57BL/6J mice in experimental S. aureus infection. After the administration of S. aureus, the transgenic mice showed increased expression of group II PLA2 mRNA in the liver and increased concentration of group II PLA2 in serum, whereas the PLA2-deficient mice completely lacked the PLA2 response. Expression of human group II PLA2 resulted in reduced mortality and improved the resistance of the mice by killing the bacteria as indicated by low numbers of live bacteria in their tissues. Human group II PLA2 was responsible for the bactericidal activity of transgenic mouse serum. These results suggest a possible role for group II PLA2 in the innate immunity against S. aureus infection.
Oligodendrocyte-Specific Protein Peptides Induce Experimental Autoimmune Encephalomyelitis in SJL/J Mice
Authors: Stevens David B. , Chen Kendall , Seitz Robert S. , Sercarz Eli E. , Bronstein Jeff M.
Published: December 2022

Oligodendrocyte-specific protein (OSP) is a recently isolated and cloned, 207-aa, hydrophobic, four-transmembrane protein found in CNS myelin. It represents ∼7% of total myelin protein. The OSP cDNA sequence has no significant homology with previously reported genes, but the predicted protein structure suggests that OSP is a CNS homologue of peripheral myelin protein-22. We previously reported the presence of anti-OSP Abs in the cerebrospinal fluid of relapsing-remitting multiple sclerosis (MS) patients, but not control patient groups. In this study, we tested the ability of a panel of 20-mer peptides with 10-aa overlaps, representing the sequence of murine OSP, to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. SJL mice challenged with murine OSP peptides 52–71, 82–101, 102–121, 142–161, 182–201, and 192–207 exhibited clinical EAE. OSP:52–71 elicited severe relapsing-remitting EAE in some individuals. All other encephalitogenic peptides elicited, at most, a loss of tail tonicity from which the mice most often completely recovered. Mononuclear cell infiltrates and focal demyelination characteristic of EAE were evident. T cell proliferative responses were seen with all encephalitogenic peptides except 142–161 and 182–201. OSP peptides 72–91 and 132–151 did not cause clinical EAE, but did elicit robust proliferative responses. B10.PL and PL/J mice challenged with the same OSP peptide doses as SJL mice did not exhibit clinical EAE. These results in the SJL EAE model, together with the results from MS patient clinical samples, make OSP a promising candidate for autoantigenic involvement in MS.
Human CD28−CD8+ T Cells Contain Greatly Expanded Functional Virus-Specific Memory CTL Clones
Authors: Weekes Michael P. , Carmichael Andrew J. , Wills Mark R. , Mynard Kim , Sissons J. G. Patrick
Published: December 2022

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28−CD8+ T cells has not previously been defined, we studied the contribution of CD28−CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR β-chains. We designed clonotypic oligonucleotides complementary to each β-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28− cells than in the CD8+ population as a whole. Because CD28−CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28− CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28−CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28−CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.
Negative Regulation of Macrophage Activation in Response to IFN-γ and Lipopolysaccharide by the STK/RON Receptor Tyrosine Kinase
Authors: Liu Qing-Ping , Fruit Kristy , Ward Jennifer , Correll Pamela H.
Published: December 2022

IFN-γ primes macrophages for antimicrobial activity, increased killing of intracellular pathogens, and Ag processing and presentation to lymphocytes by cooperating with a second signal (provided by LPS or endogenous TNF-α) to promote increased proinflammatory cytokine production, NO production, and MHC class II expression. Macrophage-stimulating protein (MSP) suppresses NO production by activated peritoneal macrophages in vitro. Furthermore, targeted deletion of the receptor for MSP, stem cell-derived tyrosine kinase receptor (STK/RON), resulted in increased production of NO by activated macrophages both in vitro and in vivo. Here we demonstrate that expression of STK in RAW264.7 cells resulted in suppression of NO production following IFN-γ+/− LPS stimulation in the presence of MSP, reflecting a decrease in the levels of inducible NO synthase (iNOS) mRNA and protein, which was confirmed by decreased trans-activation of an iNOS reporter. The iNOS expression is regulated by the coordinate activity of the inducible transcription factors STAT-1, IFN response factor-1, and NF-κB. The presence of the STK receptor did not significantly alter the expression of the IFN-γ receptor, STAT1 phosphorylation, or the up-regulation of IFN response factor-1 expression following IFN-γ stimulation. However, nuclear translocation of NF-κB following stimulation of RAW cells with IFN-γ and LPS was reduced in the presence of the MSP/STK signaling pathway. These results suggest that the negative regulation of macrophage responses by MSP/STK occurs at least in part via inhibition of costimulatory signals, resulting in NF-κB activation, that cooperate with IFN-γ to promote activation.
Misfolding of HLA-B27 as a Result of Its B Pocket Suggests a Novel Mechanism for Its Role in Susceptibility to Spondyloarthropathies
Authors: Mear John P. , Schreiber Kathy L. , Münz Christian , Zhu Xiaoming , Stevanović Stefan , Rammensee Hans-Georg , Rowland-Jones Sarah L. , Colbert Robert A.
Published: December 2022

The MHC class I protein HLA-B27 is strongly associated with susceptibility to spondyloarthropathies and can cause arthritis when expressed in rats and mice, implying a direct role in disease pathogenesis. A prominent hypothesis to explain this role suggests that the unique peptide binding specificity of HLA-B27 confers an ability to present arthritogenic peptides. The B pocket, a region of the peptide binding groove that is an important determinant of allele-specific peptide binding, is thought to be critical for arthritogenicity. However, this hypothesis remains unproven. We show that in addition to its role in peptide selection, the B pocket causes a portion of the pool of assembling HLA-B27 heavy chains in the endoplasmic reticulum to misfold, resulting in their degradation in the cytosol. The misfolding phenotype is corrected by replacing the HLA-B27 B pocket with one from HLA-A2. Our results suggest an alternative to the arthritogenic peptide hypothesis. Misfolding and its consequences, rather than allele-specific peptide presentation, may underlie the strong link between the HLA-B27 B pocket and susceptibility to spondyloarthropathies.
The CD14 Ligands Lipoarabinomannan and Lipopolysaccharide Differ in Their Requirement for Toll-Like Receptors
Authors: Means Terry K. , Lien Egil , Yoshimura Atsutoshi , Wang Shuyan , Golenbock Douglas T. , Fenton Matthew J.
Published: December 2022

Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.
Syk Activation Initiates Downstream Signaling Events During Human Polymorphonuclear Leukocyte Phagocytosis
Authors: Raeder Evelin M. B. , Mansfield Pamela J. , Hinkovska-Galcheva Vania , Shayman James A. , Boxer Laurence A.
Published: December 2022

We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cδ (PKCδ) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCδ and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCδ and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcγ receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.
Endothelial Cell E- and P-Selectin Up-Regulation in Murine Contact Sensitivity Is Prolonged by Distinct Mechanisms Occurring in Sequence
Authors: Harari Olivier A. , McHale Julie F. , Marshall Diane , Ahmed Saifur , Brown Derek , Askenase Philip W. , Haskard Dorian O.
Published: December 2022

The selectins are adhesion molecules that mediate the tethering and rolling of leukocytes on vascular endothelium. Although E-selectin and P-selectin are known to be expressed by endothelial cells (EC) in response to proinflammatory stimuli, their pattern and mechanisms of expression in immune-mediated inflammation remain poorly understood. By quantifying luminal endothelial selectin expression via i.v. administration of radiolabeled mAb, we detected constitutive expression of P-selectin, but not E-selectin, in mouse skin. Both selectins were transiently up-regulated after intradermal TNF-α, IL-1α, or IL-1β. In contrast, during a contact sensitivity response to oxazolone, expression of both selectins was prolonged, with distinct peaks at 6 and 48 h. Experiments with P-selectin gene-targeted mice showed that the P-selectin measured was exclusively expressed by EC rather than platelets. The early and late phases of selectin expression in contact sensitivity were differentiated in terms of their requirement for prior sensitization, and the action of IL-1. Whereas the early phase was a nonspecific ’irritant’ response to oxazolone, the late phase was Ag specific and was partially IL-1 dependent. Therefore, persistence of both E- and P-selectin expression in vivo can occur as a result of sequential and distinct EC activation processes that appear to be at least partially different from those previously reported as stimulating ICAM-1 and VCAM-1 expression. The further elucidation of mechanisms of EC activation in this model may help determine the relative roles of selectins and ligands for leukocyte integrins in the sequential recruitment of T cells and other leukocyte subsets during ongoing immune-mediated inflammatory responses.
Differential Loss of T Cell Signaling Molecules in Metastatic Melanoma Patients’ T Lymphocyte Subsets Expressing Distinct TCR Variable Regions
Authors: Maccalli Cristina , Pisarra Patrizia , Vegetti Claudia , Sensi Marialuisa , Parmiani Giorgio , Anichini Andrea
Published: December 2022

In this study we tested the hypothesis that loss of T cell signaling molecules in metastatic melanoma patients’ T cells may affect differently T cell subsets characterized by distinct TCR variable regions. By a two-color immunofluorescence technique, expression of ζ-chain, lck, and ZAP-70 was evaluated in CD3+ T cells and in three representative T cell subsets expressing TCRAV2, TCRBV2, or TCRBV18. Partial loss of lck and ZAP-70 was found in CD3+ T cells from PBL of most melanoma patients, but not of healthy donors. The extent of ζ-chain, lck, and ZAP-70 loss depended on the TCRV region expressed by the T cells, and this association was maintained or increased during progression of disease. Coculture of patients’ or donors’ T cell with melanoma cells, or with their supernatants, but not with normal fibroblasts or their supernatants, down-modulated expression of ζ-chain, lck, and ZAP-70 in a TCRV region-dependent way. Immunodepletion of soluble HLA class I molecules present in tumor supernatants, but not of soluble ICAM-1, blocked the suppressive effect on T cell signaling molecule expression. T cell activation with mAbs to a single TCRV region and to CD28 led to significant and TCRV region-specific re-induction of ζ-chain expression. These findings indicate that extent of TCR signaling molecules loss in T lymphocytes from metastatic melanoma patients depends on the TCRV region and suggest that tumor-derived HLA class I molecules may contribute to induce such alterations.
GILT-mediated processing of a self and melanoma antigen in thymic epithelial cells promotes deletion and regulatory T cells (APP3P.113)
Authors: Rausch Matthew , Metzger Todd , Waterfield Michael , Cortez Jessica , Anderson Mark , Hastings Karen
Published: December 2022

Central tolerance is critical to prevent autoimmunity, but limits T cell responses to tumor Ag that are self Ag. We have identified that gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for thymic deletion of CD4+ T cells specific for the self and melanoma Ag, tyrosinase-related protein 1 (TRP1). To define GILT’s role in central tolerance, we show that GILT expression is enriched in thymic APC capable of mediating deletion, medullary thymic epithelial cells (mTEC) and dendritic cells. TRP1 expression is restricted to mTEC. GILT facilitates MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APC. Bone marrow (BM) chimeras demonstrate that GILT expression in TEC is necessary and sufficient for efficient deletion of TRP1-specific thymocytes. In chimeras that express GILT in TEC, TRP1-specific T cells undergo deletion, and chimeras are not protected from melanoma challenge. Although an intermediate level of TRP1-specific T cells develop in chimeras with GILT expression limited to BM-derived cells, only chimeras lacking GILT in both populations are protected from melanoma challenge. Chimeras that express GILT in TECs or in which GILT is limited to BM-derived cells have a substantially higher percentage of TRP1-specific regulatory T (Treg) cells and lower effector:Treg cell ratio. These findings suggest that GILT operates in mTEC to facilitate the presentation of tissue-specific self Ag and promote deletion and development of Treg cells.
Towards the development of artificial antigen-presenting cell (APP3P.116)
Authors: Anikeeva Nadia , Sykulev Yuri , Blanchette Craig , Fischer Nicholas
Published: December 2022

T cell programming into distinct T cell subsets depends in a large extent from the quality and quantity of the signal delivered to T cells by professional antigen presenting cells (APC). However, it is difficult to vary parameters of T cell stimulation using live APC. To calibrate the strength of naïve T cell stimulation, we are developing artificial antigen-presenting cell (APC) that display nanolipoprotein particles (NLPs) presenting pMHC and other ligands incorporated into lipid bilayers built on a glass beads. Soluble analogs of the pMHC assembled on NLPs permits to control the extent of the homotypic ligand clustering and to influence the strength of T cell stimulation. We have found that the apparent equilibrium binding constants of an agonist pMHC/NiNLPs were strongly dependent on pMHC density. A higher density of pMHC on NiNLPs resulted in a much stronger response even though the concentration of the pMHC/NiNLPs at the lower pMHC density was 10-fold higher. We have also demonstrated that bilayers on glass beads were fluid allowing the mobility of pMHC/NLPs on the surface of engineered APC. In a model experiment, T cells concentrated the ICAM-1/pMHC/NLPs conjugates at the contact surface indicating that these conjugates were able to move on the bilayers mimicking direction stimulation of the T cells with live APC. These and other data will be discussed to demonstrate the utility of engineered APC for stimulation of naïve T cells ex vivo.
Neuropsychiatric lupus occurs independently of systemic autoimmunity (BA6P.120)
Authors: Stock Ariel , Wen Jing , Doerner Jessica , Putterman Chaim
Published: December 2022

Neuropsychiatric disease is a common manifestation of systemic lupus erythematosus (SLE). Potential mechanisms underlying neuropsychiatric SLE (NPSLE) include thrombosis, complement deposition, brain-reactive autoantibodies, and cytokine mediated inflammation. However, it is not clear whether the neuropsychiatric manifestations of SLE are secondary to systemic disease, or a primary pathogenic process in the brain. In order to distinguish between central nervous system (CNS) and hematopoietic contributions, we generated bone marrow chimeras between lupus-prone MRL/lpr and congenic control MRL/+ mice. We monitored systemic disease progression, performed extensive behavioral testing, and analyzed brain tissue for evidence of inflammation. MRL/lpr (donor) → MRL/lpr (recipient) chimeric mice showed significantly elevated autoantibodies and marked proteinuria while MRL/+→MRL/+ and MRL/+→MRL/lpr did not, indicating abrogation of the MRL/lpr systemic disease phenotype. Surprisingly, MRL/+→MRL/lpr mice displayed a behavioral phenotype consistent with MRL/lpr→MRL/lpr mice, including depression-like behavior and increased spatial memory deficits. In contrast, MRL/+→MRL/+ mice behaved normally. Additionally, there was elevated RANTES expression, cellular infiltration, and neurodegeneration in MRL/lpr→MRL/lpr and MRL/+→MRL/lpr chimeric mice. The data presented herein indicate that the MRL/lpr CNS is sufficient to mediate NPSLE development absent significant hematopoietic contribution.
33D1+ dendritic cells in the maintenance of Il-2 dependent regulatory T cells (BA6P.130)
Authors: Stolley Michael
Published: December 2022

Regulatory T cells (Tr cells) are potent anti-inflammatory cells that restrain autoimmune and inflammatory disease by dampening immune responses to self- and foreign-antigens. Given their potent immunosuppressive properties, manipulation of the Tr cell compartment is an attractive therapeutic strategy to either boost or inhibit immune responses in a variety of clinical settings. Proper implementation of Tr cell based immunotherapies necessitates a clearer understanding of how these cells are maintained in different anatomical niches under homeostatic conditions. Although it has been recognized for several years that dendritic cells (DC) help control Tr cell abundance and function, the mechanisms of this cross-talk, and a more specific definition of the cellular and molecular components underlying Tr cell maintenance by DCs, has not been elucidated. Here we show Il-2 signaling in Tr cells, which is critical for their proper homeostasis and function, is DC dependent. Intriguingly, CD8α+ DCs are not required for Il-2 activation in Tr cells and their frequencies are in fact elevated in BATf3-/- mice lacking CD8α+ DCs. Instead 33D1+ DCs have a critical yet indirect function in the generation of Tr cell supportive Il-2, and this effect is largely mediated by the micro-anatomical positioning of these cells within T cell zones of secondary lymphoid organs.
Effector T cell resistance to suppression by regulatory T cells as a mechanism of SLE-accelerated atherosclerosis (BA6P.132)
Authors: Wilhelm Ashley , Rhoads Jillian , Moore Jared , Major Amy
Published: December 2022

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T and B cell activation and circulating autoantibodies. Common early causes of mortality include kidney disease and infection, while there is a striking increase in atherosclerosis risk for those surviving more than a year after diagnosis. Despite identification of this increased risk more than 40 years ago, mechanisms for SLE-accelerated atherosclerosis remain elusive. The B6.Sle1.2.3 (B6.SLE) mouse model of SLE contains three genetic loci that confer SLE susceptibility. Transplantation of bone marrow from B6.Sle1.2.3 into LDLr-/- mice results in increased atherosclerosis accompanied by more CD4+ T cells in the lesions, pointing to T cell dysregulation as a contributing factor. We confirmed a central role for CD4+ T cells in this process by demonstrating that transfer of B6.SLE CD4+ T cells into Rag-/-, LDLr-/- mice resulted in increased atherosclerosis compared to B6 CD4+ T cells. In the current study, we hypothesized that acceleration of atherosclerosis in SLE is due to resistance of B6.SLE CD4+ effector T cells (Teff) to suppression by regulatory T cells (Treg). Our data show that, although B6.SLE Treg have normal suppressive capacity, B6.SLE Teff are resistant to this suppression. In co-transfer experiments, results suggested an IL-17-mediated mechanism, by which B6.SLE Teff elicit increased IL-17 production by B6 Treg, much like levels produced by B6.SLE Treg.
NLRP12 negatively regulates autoinflammatory disease by modulating T cell responses and IL-4 production (BA6P.133)
Authors: Lukens John , Gurung Prajwal , Kanneganti Thirumala-Devi
Published: December 2022

Missense mutations in the Nod-like receptor Nlrp12 are associated with periodic fever syndromes and atopic dermatitis in humans, however the cellular and molecular mechanisms involved in NLRP12-mediated regulation of immune responses remain poorly defined. In this study, we focused on identifying the roles of NLRP12 in shaping inflammatory T cell responses and T cell-mediated disease. We found that immunization of NLRP12-deficient mice with myelin antigen to induce experimental autoimmune encephalomyelitis (EAE) provoked the generation of hyperinflammatory T cell responses in the periphery. Surprisingly, NLRP12-deficiency did not cause exacerbated forms of paralyzing EAE disease, instead Nlrp12-/- mice developed atypical neuroinflammatory symptoms that were characterized by ataxia and loss of balance. Non-classical EAE pathogenesis was characterized by altered regulation of canonical and non-canonical NF-kB signaling in the CNS. Furthermore, enhanced IL-4 production was identified to promote the development of atypical EAE disease in Nlrp12-/- mice. Interestingly, Nlrp12 was found to be highly expressed in T cells and purified NLRP12-deficient T cells exhibited hyperproliferation and enhanced NF-kB signaling in response to TCR stimulation. These results define an unexpected role for NLRP12 as an intrinsic negative regulator of T cell-mediated immunity, and identify altered NF-kB regulation and IL-4 production as key mediators of NLRP12-associated disease.
Fn14 deficiency protects lupus-prone mice from cutaneous lesions induced by UVB irradiation (BA6P.135)
Authors: Doerner Jessica , Friedman Adam , Burkly Linda , Putterman Chaim
Published: December 2022

Cutaneous lupus erythematosus (CLE) in one of its various forms is seen in the majority of patients with systemic lupus. Photosensitivity is common in lupus, and sunlight (via UVB irradiation) is a well-recognized trigger of CLE. A TNF superfamily member, TNF-like weak inducer of apoptosis (TWEAK), is a soluble cytokine that binds to its sole known signaling receptor, Fn14. TWEAK and Fn14 are upregulated with inflammation and/or injury, and this pathway is important in critical biologic processes including angiogenesis, cell survival, and local inflammation. Previously, we found that lupus-prone MRL/lpr mice deficient for Fn14 (Fn14 KO) had markedly attenuated skin disease (which develops spontaneously in this strain) as compared to Fn14 wild-type (WT) mice. Here, we demonstrate that absence of the Fn14 receptor is protective against the expected UVB-induced eruption. MRL/lpr Fn14 WT mice developed significantly more severe skin involvement following exposure to UVB when compared to strain, age, and gender matched Fn14 KO mice. Histologically, MRL/lpr Fn14 WT mice had significantly more apoptotic keratinocytes, basement membrane degeneration, and dermal inflammation comprised of macrophages following UVB. RT-PCR revealed higher levels of CXCL9, CXCL10, CXCL11 and RANTES in MRL/lpr Fn14 WT mice. Our data suggests a novel role for the TWEAK/Fn14 signaling pathway in the induction of cutaneous lupus lesions following UVB.
Exposure to inhaled atmospheric particulate matter enhances autoimmunity. (BA6P.125)
Authors: Fechner John , Julliard Walker , O'Driscoll Chelsea , Mezrich Joshua
Published: December 2022

Epidemiological studies have demonstrated a relationship between exposure to atmospheric particulate matter (PM) and autoimmune disease. Multiple mechanisms have been proposed, including our research showing PM exposure in vitro can enhance Th17 differentiation in an aryl hydrocarbon receptor-dependent manner. The current study sought to determine the impact of PM exposure in vivo on immunity outside of the lung. In the 1st experiment, male B6 mice (n = 4 per group) were treated every 3 days with either vehicle or 800ug of SRM1949b, a representative sample of urban dust particles, in 20ul PBS i.n. for a total of 5 doses. One day after the last dose, splenocytes were cultured for 4 days with anti-CD3 and cytokines were measured. Treatment with PM increased IL-17A 3-fold but had no impact on IFNγ expression. In the 2nd experiment, female B6 mice (n = 4 per group) were treated every 3 days with vehicle or PM for a total of 9 doses. On the day of the 5th dose, a standard EAE protocol commenced. Clinical scoring using a 5-point scale began on d7 and ended on d21. All mice in both groups displayed EAE symptoms. Mice treated with PM had significantly higher mean peak score (3.5 + 0.3 vs 2.5 + 0.3 for PBS), trended toward having a higher mean clinical score and had earlier onset of disease. Together, these data suggest that inhaled PM exposure can impact immune responses beyond the lung, and support a mechanism where inhaled pollution aggravates autoimmunity.
Mast cells play a key role in autoinflammatory disease development in PSTPIP2-deficient cmo mice (BA7P.141)
Authors: Craig Andrew , Lee J.H. , Sharma Namit , Chitu Violeta , Stanley E.
Published: December 2022

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory disease characterized by fevers, bone lesions, and skin lesions. A CRMO-like disease develops in cmo mice, which harbor a mutation in proline serine threonine phosphatase-interacting protein 2 (PSTPIP2). Loss of PSTPIP2 in cmo mice has been linked to elevated macrophage and osteoclast activation, and IL-1β-dependent disease progression. Since mast cells were detected in cmo lesions, we have tested the role of mast cells in cmo disease. In diseased cmo mice, increased connective tissue mast cells were observed in both normal skin and within skin lesions. Crossing cmo mice with a transgenic model of mast cell deficiency (Mcpt5-Cre:Rosa26-Stopfl/fl-DTa) resulted in mice (cmo/MC-) that were protected from developing cmo disease, including skin lesions and joint swelling. Fewer osteolytic lesions were also observed in cmo/MC- mice by micro-computed tomography. Within these tissues, cmo/MC- mice had reduced levels of IL-1β, and less activated immune cells in popliteal lymph nodes compared to cmo. Intravital microscopy experiments on young cmo mice revealed increased leukocyte recruitment in response to lipopolysaccharide compared to cmo/MC- and wild-type mice. Together, these results implicate mast cells in promoting leukocyte recruitment to inflamed tissues during autoinflammatory disease progession, and suggest that therapies targeting mast cells or their mediators may benefit CRMO patients.
Immune complex-induced, nitric oxide-mediated vascular endothelial cell death by phagocytes (BA7P.159)
Authors: Shashidharamurthy Rangaiah
Published: December 2022

Immune-complex (IC) mediated vascular endothelial cell death linked to the release of toxic free radicals by activated inflammatory cells. In particular, nitric oxide (NO), a highly reactive free radical, is implicated in many IC-mediated inflammatory disorders. However, the mechanisms of NO-induced endothelial cells death during autoimmune vasculitis have not been clearly understood. Therefore, we investigated the molecular mechanisms of extracellularly secreted NO by IC-activated phagocytes on endothelial cells using in-vitro and in-vivo vasculitis models. When co-incubation of antibody-coated endothelial cells, murine macrophages released significantly high levels of NO (12 folds) into the culture media. In addition exogenously added NO induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in endothelial cells. Further, in vivo studies revealed that circulating ICs deposit and lead to accumulation of inflammatory cells in the capillaries but not in large arteries. In addition, the IC-mediated damages to vascular endothelium are prevented by uncoupling IC and inflammatory cells interaction. In summary these results suggest that, though the complexity of NO-induced cell death signaling exists due to its differential (dichotomous) cellular activities, it is absolutely dangerous for cell survival at sustained and chronic level of production as possibly occurring in IC-mediated autoimmune vasculitis
Heat shock protein 90, a potential biomarker for type I diabetes, is secreted by human pancreatic beta cells in response to cytokine stress (BA7P.161)
Authors: Gardiner Gail , Guindon Lynette , Perez Liliana , Evans-Molina Carmella , Blum Janice
Published: December 2022

Heat shock protein 90 (HSP90) is a molecular chaperone that regulates diverse cellular processes by facilitating the activities of a wide variety of protein clients. Recent collaborative studies from our lab have shown that serum HSP90 levels are elevated in newly diagnosed type I diabetic patients, thus distinguishing HSP90 as a potential biomarker for this autoimmune disease. The clinical onset of type 1 diabetes (T1D) is known to be associated with insulitis, the infiltration of inflammatory cells into the pancreatic islets. Therefore, to test the hypothesis that inflammatory stress results in HSP90 release from pancreatic islets, we treated primary human cadaveric islets from non-diabetic donors with a cocktail of inflammatory cytokines IFN-γ, TNF-α, and IL-1β and found that increased levels of HSP90 are secreted by cytokine-treated islets. Flow cytometry of dispersed pancreatic islets suggests beta cells may be responsible for the release of this chaperone in response to cytokine treatment. Further studies with cytokine-treated human beta cell lines βLOX5 and 1.1B4 support this finding. In contrast, beta cell lines do not secrete HSP90 upon treatment with thapsigargin, suggesting HSP90 is not released in response to beta cell endoplasmic reticulum stress, which is also known to precede the onset on T1D. Studies are underway to determine the exact mechanism by which HSP90 is released in response to cytokine stress as well as to elucidate the role of HSP90 in T1D etiology.
The role of neutrophils in B cell dysregulation in systemic lupus erythematosus (BA7P.146)
Authors: Bird Anna , Rangel-Moreno Javier , Anolik Jennifer
Published: December 2022

Systemic lupus erythematosus (SLE) is an autoimmune disease involving dysregulation of both innate and adaptive immune compartments. The hallmark of SLE is loss of B cell tolerance and production of auto-antibodies, which results in tissue damage and inflammation. Factors driving loss of B cell tolerance are incompletely characterized, but include failure of central tolerance mechanisms, inadequate clearance of cell debris, and alterations in the cytokine milieu, including elevated interferon alpha (IFNα). Our lab and others have shown that SLE neutrophils are a significant source of IFNα as well as self-antigen in the form of apoptotic debris and neutrophil extracellular traps (NETs). However, it is unknown whether neutrophils contribute to B cell activation and tolerance loss in lupus. To address this, we examined both splenic and bone marrow (BM) tissues from lupus prone mice to determine whether neutrophils secrete cytokines that alter the inflammatory milieu. Our data shows that neutrophil numbers and apoptosis are increased in lupus BM. BM neutrophils from lupus prone mice have elevated production of the B cell survival cytokines BAFF and APRIL, as well as IFNα. There was an enrichment of IFNα-producing neutrophils in the spleen in close proximity to both B and T cells. These data suggests that neutrophils likely participate in the creation of an inflammatory microenvironment that sustains auto-reactive B cells and exacerbates SLE pathogenesis in the spleen and the BM.
Induction of autoimmune disease by adoptive transfer of an atypical NK cell subset (BA7P.147)
Authors: Bolland Silvia , Voynova Elisaveta , Skinner Jeffrey , Scott Bethany , Qi Chen-Feng
Published: December 2022

Several mouse models of SLE, including FcgRIIB-KO and TLR7tg mice, develop an expansion of an atypical NK cell subset with functional similarity to cells referred as IKDCs or pre-mNKs in other systems and identified as NK1.1+CD11c+CD122+MHC-II+. These cells belong to the NK cell lineage because they depend on IL15 and express E4BP4. Functionally they are cytotoxic, produce type I and type II interferons upon activation and they are efficient antigen presenting cells both through MHC-II expression and in cross-presentation to CD8s. These atypical NK cells are responsive to TLR stimulation and thus are most abundant in mice with high copy number of the Tlr7 gene. They are highly proliferative as assessed by in vivo BrdU incorporation. Transferring 4 million atypical NKs purified from spleens of SLE-prone mice into WT induces a 2-week-long wave of inflammatory cytokines in the serum, a sustained increase in T cell activation and follicular helper cells for the following months, and a progressive expansion of dendritic cells, monocytes and granulocytes. Furthermore IL15 deficiency, which impedes development of NK cells, ameliorates the autoimmune pathology of TLR7tg mice. These results suggest that cells of the NK lineage can develop into cytokine producing/antigen-presenting cells that affect the priming and progression of systemic autoimmune disease.
CD4+ T cells display increased cross reactivity when primed with peptides of lower affinity for MHC (BA8P.165)
Authors: Blanchfield Jennifer , Evavold Brian
Published: December 2022

Studies propose the degree of T cell cross reactivity for peptide antigens is associated with similarities in the amino acid residues contacting TCR. Here we advance this concept by assessing how the stability of peptide for MHC contributes to cross reactivity in demyelinating autoimmune disease by evaluating two CNS antigens MOG35-55 (myelin oligodendrocyte glycoprotein) and NFM15-35 (neurofilament medium protein). These peptides share identical TCR contact residues yet vary at 3 MHC anchors such that MOG exhibits greater stability than NFM for I-Ab. This difference dictates encephalitogenic potential as well because MOG promotes demyelination and NFM does not. Polyclonal CD4+ T cells primed with MOG or NFM were probed for affinity and cross reactivity using MHC monomers presenting the priming peptides or the encephalitogenic peptides MOG(42P) and MOG(47A), which vary from wild type MOG at TCR contact residues. The 2D micropipette adhesion frequency assay used to assess these parameters showed that NFM and MOG expanded splenic CD4+ T cells that recognized 47A and 42P with similar frequencies and low affinities ~1.8 x10-6 μm4. Interestingly though, NFM specific cells had a greater capacity to recognize multiple peptides than MOG specific cells and this potential was unique to individual clones. Thus, peptide stability for MHC influences the degree of cross reactivity in peripheral tissues and provides a unique point of comparison for studying cross reactivity in the CNS.
The design of an Introductory Immunology course for new graduate and medical students within a systems biology type curriculum (EDU1P.216)
Authors: Price Joseph
Published: December 2022

As a result of a major curriculum restructuring, it became necessary to formulate an introductory Immunology course, 16 class hours plus exam, meeting in a group of 115 students. It would be taken in the Fall semester by all our first year graduate and first year medical (including dual degree) students, many of whom have had no prior Immunology instruction. The course would provide a basic introduction to and foundations of modern human Immunology abstracted to a graduate level, while segregating into later Systems Biology courses selected topics of diseases which significantly impact or involve the immune system. Course organization is presented in a concept map. Course teaching objectives included a focus on: basics with connections to subsequent topics/content, clinical relevancy, and experience with higher order learning. Also included were opportunities for in-class review, and to compare and contrast aspects within and between the systemic and local innate and adaptive immune systems. For each class meeting 3 - 5 learning objectives were developed and used in content development for classes and assessments. Exam questions were prepared before the course, and a small selection (not used later on the exam) were released to the students during the course as practice exercises. Students were very enthusiastic about the course design and classes by the course designer.
iPads as a tool for increasing active learning in an undergraduate immunology class (EDU1P.218)
Authors: Pollock Roberta
Published: December 2022

Immunology is a challenging subject, and active learning techniques can greatly facilitate student mastery of this material. Active learning approaches are particularly important in a rapidly changing field such as immunology, as students develop a sense of the dynamics of this area and acquire flexibility in how they acquire new information. iPads were used in two different immunology classes, taught in the Fall of 2012 and 2014. The iPads were used in numerous ways throughout the course and proved to be a very useful tool. Applications included lectures and note taking, journal article discussions, peer editing of research papers, clicker responses, eBooks, lab manuals, and lab notebooks. Some approaches worked very well, while others were mixed in their effectiveness. The ease with which students mastered the various uses of the iPads changed markedly in the two year period between the two classes; the first class had a very steep learning curve while the second class was immediately comfortable with using them. One of the most striking changes was the creativity shown by students in using the iPads in both lecture and lab.
Orienting incoming PhD students through hands-on training in immunology research methods (EDU1P.220)
Authors: Sweere Johanna , Seong Yekyung , Schaffert Steven , Spitzer Matthew , Martinez Olivia , Butte Manish
Published: December 2022

Students entering graduate study in immunology face a sharp transition into the autonomous world of laboratory research and a steep learning curve for the techniques particular to immunology. Our goal was to expose new graduate students to essential research skills to facilitate the transition into rotation laboratories and eventually a thesis lab. At Stanford University, over the past three years, we incorporated a weeklong intensive orientation (“Startup”) involving a series of guided, hands-on projects and workshops designed to introduce incoming graduate students to experimental methods in immunology research. Our curriculum posed questions in both mouse and human immunology, answered through pre-validated experiments including flow cytometry, quantitative PCR and fluorescence microscopy. For example, cytokines were measured by qPCR and intracellular staining of T cells. Data visualization and analysis were introduced through exposure to R, Flowjo and Fiji. The students attended lunches with senior graduate students, dinners with faculty, and tours of core facilities. Surveys of participants before and after Startup revealed improvements in experimental/technical confidence. Also, students reported that Startup built class cohesion and facilitated the selection of rotation labs. In summary, we provide a refined recipe for introducing students to immunology research through hands-on experimental modules that mitigate the pedagogical barriers of first-year graduate school.
The characteristics of liver hematopoietic progenitor cells (HEM5P.231)
Authors: Peng Hui , Tang Ling , Sun Rui , Tian Zhigang
Published: December 2022

During fetal life, hematopoiesis majorly occurs in the liver. After birth, the site of hematopoiesis shifts from fetal liver to the bone marrow (BM). However, some recent studies have shown that the adult liver also contains stem cell population with potent hematopoietic-reconstitution potential. To investigate the properties of adult liver hematopoiesis, we compared the phenotype of liver lineage (Lin)-negative progenitor cells with that of BM counterparts. We found that Lin- cells in the adult liver are phenotypically distinct from BM Lin- cells. Moreover, transfer of fetal liver cells, but not adult BM cells, can repopulate these liver progenitor cells with unique phenotype. In addition, hematopoietic progenitor cells in the liver and BM exhibit differential transcription factor-expression. Liver Lin- progenitor cells express much higher levels of promyelocytic leukemia zinc finger (PLZF), than their BM counterparts. Liver Lin-PLZF+ progenitors express high levels of Sca-1 and, in contrast to BM Lin-PLZF+ cells, lack c-kit and α4β7. Besides PLZF, another transcription factor, namely T-bet, is also expressed at higher levels by liver progenitors cells than their BM counterparts. Therefore, given the essential roles of PLZF and T-bet in the development of liver-resident natural killer (NK) cells, our data suggest that the unique characteristics of liver hematopoietic progenitor cells may contribute to liver-resident NK cell development.
microRNA-132 regulates hematopoietic stem cell function and survival through FOXO3a (HEM5P.226)
Authors: Mehta Arnav , Baltimore David , Zhao Jimmy
Published: December 2022

A complex network of interactions tightly regulates hematopoiesis to ensure balanced and appropriate output of blood cells, both under normal and stressful conditions. This network includes a novel class of RNA molecules, called microRNAs, that do not code for proteins, but instead negatively regulate the expression of genes. These molecules serve as “fine-tuners” of gene expression, and when dysregulated, can drastically alter the balance of hematopoiesis, potentially leading to cancer. We have identified microRNA-132 to be enriched in certain hematopoietic progenitors, particularly hematopoietic stem cells (HSCs). Ectopic expression of microRNA-132 leads to proliferation HSCs and early progenitors, with eventual exhaustion and extra medullary hematopoiesis. Deletion of microRNA-132 leads to increased survival of HSCs, especially in conditions of continuous inflammatory stress, and an improvement in engraftment potential compared to WT controls. This phenotype is primarily mediated by the microRNA-132 target FOXO3, a key transcription factor in regulating HSC proliferation and protective autophagy of aged HSCs. microRNA-132 deficient HSCs further show altered authophagy and reactive oxygen species accumulation in response to growth-factor starvation when compared to WT controls. We have thus identified microRNA-132 as an important regulator of HSC self-renewal and function, with therapeutic implications in hematopoietic aging and cancer.