bluuebunny/crossref_metadata_2025 huggingface dataset:
MECHANISM OF Th-POK GENE REGULATION DURING DIFFERNTIATION OF HELPER-LINEAGE T CELLS (136.10)
Authors: Taniuchi Ichiro , Muroi Sawako
Published: January 2023

CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper lineage cells upon expression of the transcription factor Th-POK, in whose absence they commit to the CD8+ cytotoxic lineage. An understanding of how cell fate determination of DP thymocytes is regulated therefore requires elucidation of the mechanisms that regulate helper-lineage specific expression of the Th-POK gene. In addition to a transcriptional silencer that represses the Th-POK gene during differentiation of MHC class I-specific thymocytes, we have identified two enhancers in the Th-POK gene. While the proximal enhancer is necessary to maintain Th-POK expression at late developmental stages of helper-lineage T cells, the distal enhancer, located adjacent to the silencer, was thought to be involved in initial induction of the Th-POK gene. Here we show that some MHC class II-specific thymocytes lost Th-POK expression and were re-directed into CD4-CD8+SP thymocytes upon loss of the distal enhancer. However, other MHC class II-specific thymocytes that differentiated into CD4+CD8-SP thymocytes expressed almost normal level of the Th-POK gene in the absence of the distal enhancer. These results indicate that Th-POK itself is involved in regulation of its own expression, supporting an auto-regulatory loop model in which direct antagonism by Th-POK of its own silencer amplifies Th-POK expression.
Evidence of epigenetic suppression of genes involved in lineage commitment in naïve CD4+ T cells following severe sepsis (136.17)
Authors: Carson IV William F , Cavassani Karen A , Schaller Matthew A , Ito Toshihiro , Ishii Makoto , Kunkel Steven L
Published: January 2023

Immunosuppression following severe sepsis remains a significant human health concern, as long-term survival rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to epigenetic suppression of IL-12 production by dendritic cells. However, the effect of severe sepsis on subsequent CD4+ T cell responses remains unclear. In the present study, CD4+ T cells from mice subject to an experimental model of severe sepsis (cecal ligation and puncture, CLP) were isolated and subjected to ex vivo restimulation. Naïve CD4+ T cells from CLP mice exhibited reduced proliferative capacity ex vivo and dysregulated cytokine production as compared to CD4+ T cells from sham surgery mice. Also, CD4+ T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing. Additionally, repressive histone methylation marks were evident at promoter regions for the TH1 cytokine interferon-γ (IFN-γ) and the TH2 transcription factor GATA-3 in naïve CD4+ T cells from CLP mice. These results provide evidence that naïve CD4+ T cells from postseptic mice are unable to properly commit to either the TH1 or TH2 lineage, possibly due to repressive histone methylation events associated with genes involved in cytokine production and gene transcription. This work was supported by NIH RO1 HL031237, HL074024, and T32 HL007517.
Characterization of miRNA regulation of T cell differentiation (136.18)
Authors: Bronevetsky Yelena , Ansel Mark
Published: January 2023

During an immune response, antigen-specific CD4 T cells proliferate greatly and differentiate into different sub-populations of effector T cells that act to coordinate the adaptive immune response by selective cytokine production. Major changes in gene expression characterize this transition, and the role that microRNAs play in this process of T cell differentiation remains largely undefined. Effector T cells exhibit decreased expression of numerous miRNAs, as compared to naïve T cells, which is in contrast to the global increase in gene expression that characterizes this differentiation. We have undertaken to investigate the regulation of this miRNA decrease. We have identified differential regulation between different miRNAs at several levels of miRNA biogenesis.
Absence of KIR2DS3, a receptor implicated in NK cell activation is associated with pulmonary sarcoidosis (136.23)
Authors: Luo Lihui , Sharma Surendra K. , Rajalingam Raja
Published: January 2023

By interacting with certain HLA class I molecules, the killer cell immunoglobulin-like receptors (KIR) control the function of NK cells that trigger early immune response against infection and tumors. To determine whether certain KIR and HLA genes are associated with pulmonary sarcoidosis (PS), a chronic inflammatory disease characterized with pulmonary granuloma, we have characterized KIR and HLA genes in 149 PS patients and 186 healthy controls. There was a significant decrease in the frequencies of KIR2DL2 (56.4% vs. 71.5%, P = 0.0041; 95% CI, 0.33-0.81, OR = 0.52), 2DL5 (61.7% vs. 76.3%, P = 0.0041; 95% CI, 0.31-0.80, OR = 0.50) and 2DS3 (28.2% vs. 45.2%, P = 0.0015; 95% CI, 0.30-0.75, OR = 0.48) in PS compared to controls. Interestingly, these three KIR genes 2DL2, 2DL5 and 2DS3 are closely linked together in the cetromeric half of the KIR gene complex, and thus any of these KIRs or other linked loci may contribute to the risk for PS. Particularly, the decrease in activating receptor KIR2DS3 in PS is consistent with the clinical anergy and diminished cellular immunity evidenced in PS. The individuals missing KIR2DS3 may fail triggering early NK cell response to clear antigenic stimuli, which may contribute granulomas formation. This study offers new insights into the molecular mechanisms of granuloma formation in sarcoidosis and provides a framework for developing new therapeutic strategies. This work was supported by UCLA Department of Pathology and Laboratory Medicine to RR.
Recruitment of proteasome catalytic subunit dictates NFkB-mediated transcriptional termination (136.3)
Authors: Cullen Sarah J , Ponnappan Subramaniam , Ponnappan Usha
Published: January 2023

The ubiquitin proteasome pathway plays a vital role in the regulation of transcription. While a biphasic involvement of the proteasome has been demonstrated in the regulation of immune response gene inducer, Nuclear factor Kappa B (NFκB), the precise molecular link between the proteasome and the persistence of NFκB mediated transcription remains to be fully defined.

Using pervanadate, a stimulus that induces nuclear NFκB in the absence of proteasomal proteolysis of IκB-α, akin to reactive oxygen species, ChIP analyses and qPCR, we now present evidence that pervanadate in combination with proteasomal inhibition induces exaggerated expression of NFκB-mediated proinflammatory genes. We also report that both the 19S and 20S components of the proteasome complex appear to play distinct but non-redundant roles in the regulation of NFκB mediated transcription.

Our results provide insight into the role of proteasomal complexes in NFκB transcriptional regulation and uncover a novel role for the proteasome in chronic inflammatory responses and increased inflammation accompanying aging,

This research was supported by grants AG013081 and AG025220 from the NIH.
BCR-induced cell death of B cells from CD22 deficient mice is mediated by a novel ssRNA-directed endonuclease (136.33)
Authors: Poe Jonathan C , Kountikov Evgueni I , Tedder Thomas F
Published: January 2023

B cells from CD22 deficient mice backcrossed onto a C57BL/6 genetic background (B6 CD22-/-) undergo B cell antigen receptor (BCR)-induced apoptosis. By contrast, B cells from parental inbred CD22-/- mice on a mixed B6x129 genetic background (B6/129 CD22-/-) survive and proliferate normally following BCR ligation. Through an extensive reverse genetic screen, a single locus was identified as being responsible for the proliferation defect of B6 CD22-/- B cells. In B6 CD22-/- mice this locus was homozygous for B6 germline DNA (locusB6), and in B6/129 CD22-/- mice this region was homozygous for 129 germline DNA (locus129). In locusB6 CD22-/- B cells, a novel single-stranded RNA (ssRNA)-directed endonuclease was specifically overexpressed compared to locus129 CD22-/- B cells, suggesting a direct role in BCR-induced cell death. Targeted disruption of the endonuclease gene in locusB6 CD22-/- mice restored BCR-mediated B cell survival and proliferation to normal levels. These results identify a novel ssRNA-directed endonuclease as a potent inhibitor of B cell survival when expressed following antigen receptor stimulation. This research was supported by NIH grant R01CA096547.
Mina53, an IL4 repressor controlling TH2-bias (136.42)
Authors: Van Stry Melanie , Okamoto Mariko , Koyanagi Madoka , Chung Linda , Suzuki Yoshie , Ohara Osamu , Kitamura Hiroshi , Hijikata Atsushi , Tamari Mayumi , Kubo Masato , Bix Mark
Published: January 2023

The naïve CD4 T helper (TH) cell can differentiate into several effector TH subsets, depending on the host-pathogen environment. TH1 cells, which express high levels of the cytokine IFNγ, are essential for clearance of intracellular pathogens, while TH2 cells, which express high levels of IL-4, are required for responses to extracellular pathogens. TH2-bias, which occurs when a naïve T cell preferentially differentiates to a TH2 cell, is a genetic trait impacting infectious, autoimmune, and allergic disease susceptibility. Previous studies in our laboratory have identified a quantitative trait locus that regulates TH2 bias in mice (Dice1.2). Here, we have identified Mina53, a JmjC family member, as the genetic determinant of Dice1.2. Mina53 specifically represses transcription from the Il4 promoter in transient reporter assays, while over-expression of Mina53 converts TH2 bias from high to low. Moreover, Mina53 binds to the proximal Il4 promoter, and this binding requires an interaction between Mina53 and nuclear factor of activated T cells (NFAT), a key regulator of cytokine expression. Taken together, these data provide mechanistic insight into the Il4 regulatory pathway controlling effector T cell differentiation and account, in part, for genetic variation in TH2 bias.
The Expression of Inflammatory Mediators and its Association with the Susceptibility to Mucosal Leishmaniasis in Peru (136.6)
Authors: Vargas-Inchaustegui Diego A. , Espinosa Diego , Carlsen Eric D. , Tulliano Gianfranco , Pacheco Rosa , Llanos-Cuentas Alejandro , Arevalo Jorge , Soong Lynn
Published: January 2023

Leishmania braziliensis can cause cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) in humans, and the latter is characterized by excessive T- and B-cell responses. We hypothesized that conversion from CL to ML is partially due to an imbalanced production of proinflammatory and regulatory cytokines, leading to uncontrolled host responses to the parasites. To test this hypothesis, we first examined the -2518 SNP of the MCP-1 promoter because its G/G genotype is associated with autoimmune disorders and infectious diseases. We genotyped 142 Peruvian subjects (18 controls, 65 CL and 59 ML patients) using PCR-RFLP on genomic DNA. Our pilot studies suggested that ML patients showed a relatively frequency for the MCP-1 G/G genotype (28/62, 45%) than the A/A (9/22, 41%) or A/G genotypes (2/58, 38%), respectively. Using dot blot analysis, we detected higher levels of MCP-1, IFN-γ, IL-8, IP10, MIP-1β, -1d, and soluble TNF receptors in the sera of CL and ML patients as compared with the healthy controls. Additional quantitative studies confirmed that sera of ML patients contained significantly higher levels of IP-10, MIP-1β and sTNFRII when compared to CL and healthy controls. This study suggests that the over-production of inflammatory cytokines is a contributing factor to the pathogenesis of mucosal lesions. Studies are ongoing to define the sources of these inflammatory mediators.
Dephosphorylation of Ikaros by a specific phosphatase regulates its function in chromatin remodeling (136.9)
Authors: Dovat Sinisa , Gurel Zafer , Payne Kimberly J , Popescu Marcela
Published: January 2023

Ikaros encodes a zinc finger protein essential for lymphoid development. Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits target genes, resulting in their activation or repression via chromatin remodeling. Ikaros' function is controlled by post-translational modification. Our previous studies show that phosphorylation of Ikaros by CK2 kinase controls its PC-HC localization, and its ability to bind the upstream regulatory region (URE) and regulate expression of TdT during thymocyte differentiation. Here, we demonstrate, by co-immunoprecipitation, that Ikaros interacts in vivo with a specific phosphatase for which it is a substrate. Mutational analysis identified specific residues essential for Ikaros-phosphatase interaction. Mutant Ikaros protein with disrupted phosphatase interaction undergoes CK-2-mediated hyperphosphorylation that inhibits its ability to bind the TdT URE, and its PC-HC localization. Alanine mutations at the CK2-phosphorylated residues restore both Ikaros' DNA-binding ability and PC-HC localization, regardless of its ability to interact with the phosphatase. We propose a model whereby the regulation of TdT expression by Ikaros is controlled by CK2 kinase and interaction with a specific phosphatase and that the balance of these two signal transduction pathways is essential for normal T cell differentiation. Supported by NIH K22CA111392 (SD)
Dendritic Cell Molecules Involved in Self Reactive T cell Activation (137.12)
Authors: Cabrera Rosalyn , Radhakrishnan Suresh , Pease Larry R.
Published: January 2023

Dendritic cells (DC) initiate and shape an adaptive immune response. DC can be stimulated with B7-DC XAb, a novel immunomodulatory IgM that crosslinks B7-DC/PDL2 on the DC surface. Previously, we have shown that B7-DC XAb treated DC (DCXAb) reprogram Foxp3+ Tregulatory cells into IFNgamma+, IL-17+ effectors. Adoptive transfer of neoself antigen (OVA)-pulsed DCXAb induces destruction of OVA-bearing pancreatic islets and autoimmune diabetes in RIP-OVA mice. We hypothesized that DC-derived molecules involved in Th1 or Th17 polarization would be important in autoreactive T cell activation by DCXAb. In spite of their ability to induce a CTL response against OVA-bearing targets, we found that DCXAb deficient in either Tbet, IFNgamma (Th1 polarizing) or CD40, IL-6 (Th17 polarizing) fail to cause autoimmunity. In conclusion, both Th1 and Th17 polarizing molecules on DC are needed to stimulate an autoimmune response.
Autoantibody profile against retinal proteins in patients with age-related macular degeneration and possible role in neovascularization (137.13)
Authors: Morohoshi Kei , Patel Nishal , Ohbayashi Masaharu , Chong Victor , Grossniklaus Hans E , Bird Alan C , Ono Santa J
Published: January 2023

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the United States and in other developed countries. Recent findings indicate that circulating autoantibodies specific for retinal proteins and macular deposits, drusen, are associated with AMD pathogenesis, yet only a few antibodies have been identified in sera from patients with AMD. In order to reveal an autoantibody profile for AMD, we performed an antigen microarray with constituents of drusen and liquid chromatography and tandem mass spectrometry analysis following two-dimensional immunoblot analysis with retinal proteins using serum samples from patients with AMD and healthy controls. Sera from the AMD group contained high levels of IgG autoantibodies against numerous ocular antigens including lipoproteins and collagen when compared to the normal group. Moreover, IgGs purified from sera of AMD patients induced more tube formation on choroidal-retinal endothelial cells and increased VEGF production on monocytes compared to those of healthy donors. Our data suggest that these IgG autoantibodies may be used as biomarkers for AMD and contribute to progression of this disease.
IL-2 mediated regulation of CD103 Expression on CD4+ T cells and its role in Cutaneous and Mucosal inflammation in Scurfy Mice (137.15)
Authors: Sharma Rahul , Sung Sun Sang , Abaya Christian E , Ju Angela C-Y , Fu Shu Man , Ju Shyr Te
Published: January 2023

Scurfy (Sf) mice lack Foxp3+ regulatory T cells (Tregs) and die of CD4+ T cell mediated multi-organ inflammation. Introducing Il2-/- gene into Sf mice (Sf.Il2-/-) prolonged their lifespan and inhibited inflammation in skin and lung but not liver. We studied, the expression of CD103 (ligand of E-cadherin-a major integrin for cutaneous and mucosal organs), on CD4+ T cells in lymphoid and target organs of Il2-/-, Sf and Sf.Il2-/- mice. CD103+CD4+ T cells were low in normal B6 but were significantly high in Sf mice. Importantly, this population was low in Il2-/- and Sf.Il2-/- mice, indicating that this is a dominant trait of Il2-/- associated with spontaneous T cell activation. Interestingly, Tregs CD103 expression was not reduced in Il2-/- mice. Introducing CD103-/- into Sf mice inhibited inflammation in skin and lung but not liver as compared with age-matched Sf mice. Nevertheless, Sf.CD103-/- mice die at 7 wks of age with inflammation in skin, lungs, colon and liver, demonstrating CD103-independent fatal multi-organ inflammation. The data indicate that IL-2 is required for optimal CD103 expression on CD4+ T cells in Sf mice and this contributes to inflammation in organ-specific manner. IL-2 also plays additional roles because the protection of skin and lung inflammation in Sf.Il2-/- mice is life-long and Sf.Il2-/- mice have longer lifespan than Sf.CD103-/- mice that totally lack CD103-dependent inflammation.
GAD65 p546-554-specific CTL mediate β cell death and loss of insulin production (137.22)
Authors: Rasche Sarah S , Quinn Anthony
Published: January 2023

Autoimmunity to pancreatic β cell proteins such as GAD65 are associated with the destruction of insulin-producing β cells and the progression to autoimmune Type 1 Diabetes (T1D) in both NOD mice and in humans. T cell responses to GAD65 are detectable in the spleens of prediabetic NOD mice and in the peripheral blood of humans prior to the onset of overt hyperglycemia. Previous findings from our lab revealed that p546-554-specific CTL are present in naïve NOD mice and are able to induce islet inflammation in NOD.scid recipients. The GAD65 fragment p546-554 is a naturally displayed dominant epitope on GAD65-expressing APC, and professional APC can generate the p546-554 epitope from a soluble GAD65 fragment, p530-554, as well as from GAD65 released by injured β cells in vivo. Here, we report that p546-554-specific CTL can directly interact with pancreatic β cells to reduce insulin production and induce perforin-mediated cell death. These findings support a role for crosspresentation in the priming and enhancement of dominant GAD65-specific CTL responses in T1D.

This research was supported by funds from NIH-NIAID.
A novel pathogenic mechanism of the ER chaperone BiP/Grp78 response in rheumatoid synoviocytes (137.27)
Authors: Kim Wan-Uk , Yoo Seung-Ah , Kong Jin-Sun , Yoon Hyung-Ju , Park Yoon-Jung , Cho Chul-Soo
Published: January 2023

The accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we investigated the role of the ER chaperone BiP/Grp78 in abnormal proliferation of rheumatoid arthritis (RA) synoviocytes, which is the pathologic hallmark of RA. The results showed that BiP protein was highly expressed in RA synovium and synoviocytes. The proinflammatory cytokines, such as TNF-alpha and IL-1beta, and hypoxic stimuli, but not IL-10, increased the BiP expression in RA synoviocytes. RA synoviocytes were more resistant to ER-stress-induced apoptosis as compared to OA synoviocytes. ER stress inducers, thapsigargin and tunicamycin, increased BiP and Bcl-2 expressions in synoviocytes, which were much greater in RA synoviocytes than in OA synoviocytes. A BiP-inducer, BIX, blocked the ER-stress induced synoviocyte apoptosis, possibly by increasing Bcl-2 and BiP expression. In contrast, BiP knockdown in RA synoviocytes, using siRNA, abolished the TNF-alpha- and TGF-beta-induced synoviocyte proliferation and cyclin D1 upregulation. BiP with a higher molecular size than intracellular BiP was found in all (6/6) RA synovial fluids tested. Exogenous BiP strongly increased IL-17 and TNF-alpha production from RA mononuclear cells, but not from mononuclear cells of healthy controls. In summary, we demonstrated first that BiP is crucial to the resistance to apoptosis of RA synoviocytes and synoviocyte proliferation, and it involves the production of IL-17 and TNF-alpha from mononuclear cells, suggesting that ER chaperone BiP response contributes to RA pathogenesis by inducing synovial hyperplasia and Th17 cell generation.
The role of MR-1 restricted MAIT cells in murine models of arthritis (137.28)
Authors: Miyake Sachiko , Tajima Ryosuke , Miyazaki Yusei , Ichikawa Daiju , Chiba Asako , Yamamura Takashi
Published: January 2023

Mucosal associated invariant T (MAIT) cells express an invariant Vα19-Jα33 TCR and are restricted by MR1, a class Ib MHC molecule. Here we investigated the role of MAIT cells in arthritis models. The severity of clinical scores of K/BxN serum transfer arthritis and anti-CII antibody-induced arthritis were reduced in MR1-/- mice compared to control B6 mice. Histological examination revealed that cell infiltration and cartilage and bone destruction were significantly less apparent in MR1-/- mice. The number of infiltrated cells in the synovia was one third lower in MR1-/- mice compared to B6 mice. The levels of transcript of IL-1, TNF-α and IL-6 were reduced in infiltrated cells in the joints of MR1-/- mice. The transfer of NK1.1+CD3+ cells from Vα19Tg mice into MR-1-/- mice exacerbated the severity of arthritis to the similar level of arthritis in wild type B6 mice. Taken together, MR-1 restricted MAIT cells contribute to the pathogenesis of inflammatory arthritis.
Increased Th17 cell activity in patients with early rheumatoid and psoriatic arthritis (137.30)
Authors: Leipe Jan , Thuemmler Katja , Schulze-Koops Hendrik , Skapenko Alla
Published: January 2023

To gain insights into the role of Th17 cells in human autoimmune diseases, we analyzed Th17 cell biology in patients with ongoing inflammatory autoimmune arthritis (e.g., rheumatoid (RA) and psoriatic arthritis (PsA)), and for control in patients with osteoarthritis and healthy individuals. Frequencies of Th17 cells were significantly elevated in the blood of patients with early, treatment-naive RA and PsA. Moreover, upon activation in vitro, CD4 T cells from the patients yielded enhanced Th17 frequencies and elevated levels of IL-17. In contrast, Th1 frequencies and IFNγ production were not different between patients and controls. As the frequencies of Th17 cells and IL-17 production, but not of Th1 cells or IFNγ production correlated with disease activity, early inflammation in RA and PsA is characterized by both, selectively enhanced Th17 cell generation and increased Th17 cell activity. Of interest for the pathogenesis of autoimmune arthritis, CD4 T cells from early RA and PsA patients were partially resistant to the inhibitory capacity of IL-4 and IFNγ on Th17 generation, suggesting a potential mechanism underlying the breakdown of peripheral tolerance at disease onset. However, as effective immunosuppressive treatment was associated with normalization of Th17 cell activity in the patients, the data further suggest that Th17 cells might also contribute to sustained autoimmune inflammation in RA and PsA.
A role for lymphotoxin in primary Sjögren's Disease (137.34)
Authors: Shen Long , Suresh Lakshmanan , Kumar Vijay , Pankewycz Oleh , Ambrus Jr. Julian L.
Published: January 2023

The etiology of salivary gland injury in primary Sjögren's disease is poorly understood. Increased expression of lymphotoxin (LT) has been demonstrated in patients with Sjögren's disease. The current studies were designed to determine whether LT contributes to early salivary gland injury in a murine model of Sjogren's disease, IL-14alpha transgenic mice (IL-14α TG).

Lymphotoxin mRNA and protein is found in the salivary glands of IL-14αTG mice as early as 3 months of age. IL-14αTG.LT-/- mice retained normal salivary gland secretions and failed to develop lymphocytic infiltration of their salivary glands. However, deposition of IgM antibodies in the salivary glands and infiltration of the lungs still occurred in IL-14αTG.LTA-/- mice. B cell subpopulations and responses to T-dependent and T-independent antigens were similar in IL-14αTG and IL-14αTG.LTA-/- mice.

These studies suggest that locally produced LT may cause direct injury to the salivary glands in IL-14α TG mice. There can be discordance between antibody deposition in the salivary glands and salivary gland dysfunction. The mechanisms of salivary gland and lung injury in Sjogren's disease may differ. Further studies are needed to determine the roles of LT in early injury to the salivary glands in IL-14α TG mice and patients with Sjögren's disease.
Effects of DDE on development and progression of autoimmunity (137.36)
Authors: Viselli Susan M. , Lerner Daniel M. , Miaoulis Anthony G. , Rahmani Kaveh , Torski Brian A.
Published: January 2023

DDE, a metabolic breakdown product of the pesticide DDT, persists in the environment and acts as an androgen receptor antagonist. Androgen deprivation by castration of male mice causes changes in immune function and the lupus-like disease of NZB/W F1 hybrid mice is treated by androgens. Therefore, we hypothesized that the anti-androgenic effects of DDE may alter immune function, with consequences for autoimmunity. We treated both normal C57Bl/6 female mice and lupus-prone NZB/W F1 female mice with oil control or DDE, at 200 mg/kg for 3 days at the study's beginning. NZB/W mice were studied over 6 months and C57Bl/6 mice for 18 months. DDE did not alter organ or body weights. Proteinurea was increased in DDE-treated mice compared to age-matched oil-treated controls, as early as one month after treatment with DDE. By histopathology we observed glomerulonephritis sooner in kidneys of NZB/W mice treated with DDE than control. Kidneys of DDE-treated C57Bl/6 mice showed increased glomerular mesangium compared to kidneys of age-matched controls. By immunohistochemistry we saw increased glomerular expression of monocyte chemoattractant protein-1 and increased tubulointerstitial expression of osteopontin in response to DDE; upregulation of both chemokines occurs in murine lupus nephritis. We conclude that DDE impacts development and progression of autoimmune disease and continue to explore the mechanism of action.
Exosomes are associated with pregnancy-induced suppression of experimental autoimmune encephalomyelitis (137.4)
Authors: Williams Jessica L , Song Fei , Smith Kristen , Kithcart Aaron , Mavrikis Gina , Shawler Todd , Whitacre Caroline C
Published: January 2023

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by inflammation, myelin damage, and axonal degeneration. MS disease relapses are markedly decreased during pregnancy with the greatest reduction in disease observed during the third trimester. We have studied pregnancy in the murine model of MS, experimental autoimmune encephalomyelitis (EAE). Disease severity is significantly reduced when pregnancy is induced during established EAE. We also observed a post-partum flare in disease scores, with EAE suppression only occurring during the gestation period. A serum factor has been implicated as the primary mediator of disease suppression. Exosomes found in peripheral blood are able to modulate the immune response by relaying molecular signals from their cell of origin to target cells bearing specific adhesion molecules. We have shown that pregnancy-associated exosomes are able to suppress antigen-specific T cell proliferation. Exosomes are hypothesized to dampen immune responses during pregnancy; thus, we examined exosomal proteins that are known to influence T cell activity such as Fas ligand, MHC II, TGF-β, IL-17, and IFN-γ. Harnessing the mechanism by which exosomes suppress immunity, and consequently EAE, can have remarkable implications for therapy development in MS. (Supported by NIH grants AI 064320 and T32 AI055411, and National MS Society grant RG3272)
Signaling pathway triggered by sBAFF may be impaired in monocytes of the patients with Sjögren’s Syndrome (137.43)
Authors: Yoshimoto Keiko , Tanaka Maiko , Komatsu Sayaka , Setoyama Yumiko , Suzuki Katsuya , Tsuzaka Kensei , Abe Tohru
Published: January 2023

Background and Purpose: B cell activating factor belonging to the TNF superfamily (BAFF) is indispensable for proliferation, differentiation and survival of B cells. Several lines of evidence suggest that elevated production of BAFF seemed to be involved in the development of autoimmune diseases such as Sjögren's syndrome (SS). In our previous study, we investigated that BAFF and IL-6 were abnormally produced in SS monocytes upon stimulation with IFN-gamma. In the present study, we investigated a regulatory mechanism of the production of these cytokines in SS monocytes.

Materials and Methods: Peripheral monocytes prepared from SS patients and normal individuals were stimulated with IFN-gamma or soluble BAFF (sBAFF). The expression levels of sBAFF, IL-6 and transcription factors were measured by ELISA and/or semi-quantitative RT-PCR.

Results and Discussion: IFN-gamma remarkably induced the production of sBAFF and IL-6 in SS monocytes. The production of IL-6 was significantly suppressed by an anti-BAFF antibody. In addition, stimulation of SS monocytes with sBAFF induced robust increase in the production of IL-6. These data suggest that the production of IL-6 in monocytes is induced partly through a signal transduction pathway triggered by sBAFF. We found that the expression levels of several transcription factors were aberrantly elevated in SS monocytes as compared with normal monocytes. These data strongly suggest that sBAFF produced in monocytes acts in an autocrine or paracrine fashion to activate the expression of the IL-6 gene, and that signal transduction triggered by sBAFF may be impaired in SS monocytes. This aberration may underlie the development of SS.
Characterization of CNS antibody-secreting cells, the blood-brain barrier, and autoantibody in the Theiler's model of MS (137.7)
Authors: Li Libin , Brady Jennifer , Lagunoff David , Pachner Andew
Published: January 2023

The determination of the pathogenic role of autoantibody (AuAb) is a challenge in any autoimmune disease, such as multiple sclerosis (MS). This project used Theiler's virus-induced disease to characterize the AuAbs produced in the central nervous system (CNS) by antibody-secreting cells (ASCs).

Mice infected with Theiler's murine encephalitis virus (TMEV) develop an immune-mediated demyelinating disease (TIDD), characterized by progressive weakness, CNS inflammation and demyelination, which represents an excellent model of MS. Flow Cytometry (FACS), ELISpot, and real-time RT-PCR, were used to characterize ASCs and their antibody production in the CNS, and ELISA and immunohistochemistry (IHC) were used to identify autoantibodies and their deposition. We found that ASCs in the CNS were CD138 positive, readily visualized as IgG+ by IHC, and commonly found in clusters of cells in the CNS. AuAbs to CNS antigens were present in the sera, and antibody was deposited in the spinal cord of TIDD mice. The blood-brain barrier, measured by CSF albumin index and Evans Blue injection, was intact.

This study analyzes CNS ASCs and autoantibodies with specificity toward CNS antigens in TIDD, and raises the possibility that AuAb may significantly contribute to disability in this model.

This work was supported by the New Jersey Commission on Spinal Cord Research.
Kinetics of myelin antigen presentation in the CNS during EAE (137.8)
Authors: Murphey Cathi , Forsthuber Thomas G.
Published: January 2023

Despite the plethora of information on immune mechanisms derived from studies of autoimmune experimental encephalomyelitis (EAE), the animal model of multiple sclerosis, little information is available in terms of the kinetics of presentation of myelin antigens by APCs in the CNS. The purpose of this study was to show at which time points and by which APCs myelin antigen (MBP, MOG, PLP) was acquired and presented after induction of EAE. To address this issue, mice were immunized with MOG 35-55 peptide and injected with pertussis toxin on day 0 and 2 and followed for disease. Representative mice were sacrificed every day over forty days and brain tissue was recovered for immunofluorescence analysis. Immunofluorescence and confocal microscopy was used to determine expression of APC cell surface markers and myelin antigens. The results show that T cells or dendritic cells (DC) were rare (<1 cell per 500 fields) in non-immunized mice. In contrast, numbers of CD4+ T cells in immunized mice began to increase at day 3, and numbers of DCs showed a notable increase around day 9. Myelin laden DCs appeared between day 11 and 13. Inflammatory infiltrates peaked between days 15 to day 20, coinciding with peak EAE scores. In conclusion, our data suggest that infiltration of CD4+ T cells precedes substantial uptake and presentation of myelin antigens by CNS APCs, which could have important treatment implications.
Selective expansion of autoreactive T cell clonotypes drives autoimmune diabetes (137.9)
Authors: Li Li , He Qiuming , Wang Bo , Tisch Roland
Published: January 2023

It is well established that T cells are the primary mediators of β cell destruction in Type 1 diabetes (T1D). However, the relative role for different β cell-specific T cell clonotypes in the progression of the diabetogenic response, and the molecular basis for their expansion remain poorly understood. CD4+ T cells expressing the BDC2.5 clonotypic T cell receptor (TCR) and/or which recognize the mimetic BDC (mBDC) peptide are thought to have a key albeit undefined role in β cell autoimmunity of the nonobese diabetic (NOD) mouse. Accordingly, using sIAg7-mBDC multimers, BDC2.5 clonotypic CD4+ T cells were tracked and/or isolated in a temporal manner and analyzed at the single cell level. sIAg7-mBDC-binding (g7-mBDC+) CD4+ T cells were detected in peripheral blood lymphocytes (PBL) and the islets at the onset of β cell autoimmunity in NOD female mice, with the frequency and/or number of g7-mBDC+ T cells increasing with age. A comparison of TCR gene usage by single, g7-mBDC+ T cells isolated from 16 week-old NOD female mice demonstrated dominate usage of TRBV15 and monoclonality based on variable β (Vβ) gene complementary determinant region 3 (CDR3) sequences of BDC2.5 clonotypic CD4+ T cells in the islets but not PBL. Furthermore, the analysis of TRBV15+ g7-mBDC+ BDC T cells revealed the biased TRAV13 Vα gene usage. Furthermore, diabetes could be transferred into NOD.scid recipients by g7-mBDC+ T cells purified from the islets but not from PBL. These data demonstrate that g7-mBDC+ T cells are an early indicator and one of the main mediator of the development of destructive insulitis, and that both clonotypic expansion and preferential usage of TCR characterize islet infiltrating g7-mBDC+ T cells.
Functionality of the Bruton’s tyrosine kinase from Mexican patients with XLA. (138.18)
Authors: Vargas-Hernández Alexander , López-Herrera Gabriela , Rojo-Domínguez Arturo , Mogica-Martinez Dolores , Espinosa-Rosales Francisco J.
Published: January 2023

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency originated from mutations in the gene encoding Bruton's tyrosine Kinase (Btk). Btk is a vital protein in the B cell signaling machinery. L111P and E605G are two recently characterized mutations in Mexican patients suffering XLA; they gave us the basis for a more detailed study of the effects of these mutations in the conformation and functionality of Btk. In silico assays showed that L111 and E605 residues are fundamental for the activations and functionality of the Btk. Btk from the two patients and a healthy control were expressed in the Btk-deficient DT40 cell line, in order to confirm the predictions obtained in silico. The absence of the Btk catalytic activity of the patients was reflected in the lack of the Y551 phosphorylation. This may resulted in a reduced activation of PLC-ã2 that was reflected by reduction of intracellular calcium flux. This work show that L111 and E605 residues are fundamental in the structural conformation of the Btk and that in silico analysis can be a useful tool for the analysis of Btk mutations found in primary immunodeficiency patients.

Supported by CONACyT, project No. 70062
Osteopontin is required for natural killer cell development (138.19)
Authors: Kim Mi Sun , Piao Zheng-Hao , Jeong Mira , Yoon Suk Ran , Chung Jin Woong , Choi Inpyo
Published: January 2023

Natural killer (NK) cells are bone-marrow-derived lymphocytes, which share a common progenitor with T cells. Although a model of NK development from hematopoietic stem cells (HSC) has previously been proposed, the factors and detailed mechanisms driving the differentiation of NK remain to be clearly elucidated. Here we show that osteopontin(OPN) is an important molecule as microenvironmental factor for NK development. The OPN deficient mice evidenced distinct defects in the NK populations in both BM and spleen. However OPN deficient HSC also effectively induced the development of NK cells as control HSC, while the experiment of coculture system and in vivo transplantation system showed that the development of NK cells significantly was defected when HSC culture with OPN absence stromal cells, and transplanted HSC rarely induced into NK cells in OPN deficient mice. Also, the level of OPN expression is much more in stromal cells than the developing NK cells, confirming that OPN is important factor as microenvironment for NK development. The secretion of OPN was induced stimulation with IL15 in the BM stromal cells. The function of IL15, as major factor in NK development, might occurs through OPN which is one of other ways for NK development. . In addition, OPN-driven NK maturation was reduced in T-bet-deficient HPC, suggesting that T-bet is required for OPN-mediated NK development. Collectively, these results show that paracrine OPN signaling drives NK-lineage commitment, thus ultimately promoting NK cell development.
Distinct Lymphoid and Myeloid Progenitors Seed the Thymus and Give Rise to all Thymic Lineages (138.7)
Authors: Serwold Thomas , Ehrlich Lauren I Richie , Weissman Irving L
Published: January 2023

It is unclear whether myeloid lineages develop within the thymus, or whether their presence relies on trafficking of mature cells from the blood. Recent studies suggested that a multi-potent thymus-homing progenitor gives rise to both lymphoid and myeloid lineages. This conclusion is inconsistent with models of hematopoiesis that include a common lymphoid progenitor (CLP) - a cell that has no myeloid potential but gives rise to all lymphoid populations, including B, T, and NK cells.

We have addressed this issue and find that the earliest cells within thymus (commonly referred to as DN1) are lymphoid committed when transplanted in vivo, but when put into certain in vitro conditions, these cells make macrophages and neutrophils. Similarly, CLP, which in vivo are lymphoid committed, when put into these same in vitro conditions, also make myeloid cells at high clonal efficiency. These results bring into question conclusions about the myeloid potential of thymic progenitors that were determined in vitro.

To address the source of myeloid populations within the thymus, we asked whether they developed from an intermediate that expressed the RAG recombinase using RAG reporter mice (SVEX). We found that 20% of neutrophils, but very few or no macrophages or dendritic cells within the thymus come from a RAG expressing progenitor. This suggests that a fraction of RAG-expressing progenitors retain neutrophil potential. However, neutrophils make up approximately 0.0001% of the thymus, suggesting that this potential is very limited. Furthermore, we show that separate myeloid committed and lymphoid committed progenitors home to the thymus, suggesting that the myeloid and lymphoid cells within the thymus are mostly, if not entirely derived from separate seeding cells.
C/EBPb Mediates the Synergistic Upregulation of Gene Expression by IFNg and TNF in Bone Marrow-Derived Mesenchymal Stem Cells (138.9)
Authors: Xu Guangwu , Zhang Yingyu , Zhang Liying , Roberts Arthur I. , Shi Yufang
Published: January 2023

Mesenchymal stem cells (MSCs) are potent immunoregulatory cells that have shown clinical utility in suppressing immunity. IFN-γ and TNF play critical roles in the process of immune response and have been shown to regulate the function of MSCs. However, there is limited data about synergism of IFN-γ and TNF in inducing gene expression in MSCs. Here, we found that IFNγ and TNF synergistically induce the expression of IL-6, and several other immune-related molecules in MSCs in vitro. Knockdown of C/EBPβ by specific siRNA inhibited the expression of the genes induced by IFN-γ alone or TNF alone; importantly, it markedly inhibited the synergistic gene expression induced by the combination of IFN-γ and TNF in MSCs. In addition, IFN-γ alone or TNF alone induced the expression of C/EBPβ in MSCs; and these two cytokines cooperatively induced C/EBPβ expression. This suggests that C/EBPβ is a key transcription factor employed by IFN-γ and TNF in synergistically regulating gene expression. We further found that C/EBPβ similarly mediated synergistic gene induction by IFNγ combined with IL-1β or LPS, suggesting that synergy between IFNγ and other stimuli share C/EBPβ as a common mechanism. Furthermore, while STAT1 is critical in IFN-γ signaling, we found that STAT1 knockdown in MSCs did not affect C/EBPβ expression or the synergistic induction of IL-6 by IFNγ and TNF. Thus, C/EBPβ is not regulated by STAT1. These results will advance our understanding of cytokine interactions in general and their role in MSC immunobiology, which may allow better clinical application of these cells.
Opposing Roles for Lyn and Fyn Kinases in Mast Cell IgG Receptor Signaling (139.4)
Authors: Falanga Yves , Ryan John
Published: January 2023

Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FceRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased histamine and leukotriene C4 secretion, but no change in cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is required for IgG-mediated histamine, LTC4, and cytokine production in mast cells, and has a similar role in basophils and macrophages. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases. Supported by NIH grants 1R01 AI59638 and U19A1077435.
Dissection of the SHP-1 functions in FcεRI signaling (139.8)
Authors: Mizuno Kazuya , Nakata Kazuko , Yakura Hidetaka
Published: January 2023

Although SHP-1, a cytoplasmic protein tyrosine phosphatase (PTP), is shown to be a negative modulator in hematopoietic cells, its precise molecular mechanisms are not well defined. To clarify how SHP-1 regulates FcεRI-mediated signaling in mast cells, we generated bone marrow-derived mast cells (BMMC) from motheaten (me) that lack SHP-1 expression, viable motheaten (mev) that express phosphatase-deficient form of SHP-1, and control (WT) mice and examined downstream signaling events and final outputs following FcεRI crosslinking. FcεRI-induced tyrosine phosphorylation of SLP-76 and LAT, activation of MAPKs, and cytokine production were increased in me- and mev-derived BMMC, in good accordance with the previous implication of SHP-1 as a negative regulator. Since the activity of Syk that is responsible for the phosphorylation of SLP-76 and LAT was comparable in all BMMCs, both molecules might be substrates of SHP-1 in mast cells. In contrast, me-BMMC showed severely impaired PLCγ phosphorylation, FcεRI-induced calcium mobilization, especially extracellular calcium entry, and degranulation. We could not observe these phenotypes in mev-BMMC, thus suggesting that the expression of SHP-1, irrespective PTP activity, is sufficient for the positive regulation of these pathways. The absence of SHP-1 expression disrupted the association between SLP-76 and PLCγ, which might explain, at least in part, the decrease of calcium response in me-BMMC. Collectively, all these results suggest that SHP-1 regulates FcεRI-induced downstream signaling events both negatively and positively by functioning as a PTP and an adaptor protein contributing to the formation of signaling complex, respectively.
The role of Tyrosine kinase 2 in a murine model of allergic asthma (139.9)
Authors: Uebel Caroline , Karwot Roman , Maxeiner Joachim , Soó-Becker Kinga , Lehr Hans-Anton , Finotto Susetta
Published: January 2023

Tyrosine Kinase 2 is a member of the mammalian Janus kinase (JAK) family of protein tyrosine kinases which consists of three more kinases (JAK1-3). Tyk-2 mutation in humans is associated with a completely defective in signaling in response to IFNα and IL-12, abnormal responses to IL-6, IL-10 and IL-23, susceptibility to viral infection and to mycobacterial infection and Hyper IgE Syndrome.

Microscopic analyses showed that asthmatic Tyk2(-/-) mice suffered from extreme airway eosinophilic inflammation as compared to wild type mice.

We found that Th1 differentiation is severely impaired in Tyk2(-/-) mice as shown by decreased IFNγ production by lung CD4+ T cells. By contrast, an increase in Th2 cytokines could be measured in the supernatants of lung CD4+ T cells isolated from Tyk2(-/-) mice.

Preliminary results on T regulatory cell differentiation revealed an ascent of CD4+CD25+Foxp-3+ T regulatory cells in asthmatic Tyk2(-/-) mice by FACS analysis. Consistent with this finding, quantitative Real-Time PCR also showed an increase of the T regulatory cell-specific transcription factor Foxp3 in Tyk2(-/-) mice. As both the T regulatory inducing IL-10-pathway and the T reg inhibiting IL-6-pathway are blocked in Tyk2 deficient mice only TGFβ might be regulating Foxp-3 expression in T regulatory cells in these mice. We next wish to investigate the suppressive function of T regulatory cells in the absence of Tyk-2 in allergic models of asthma.

This work is supported by Graduiertenkolleg 1043 "Antigen-specific Immunotherapy".