bluuebunny/crossref_metadata_2025 huggingface dataset:
Treg Development and Suppression of Experimental Allergic Encephalomyelitis (129.28)
Authors: Haymaker Cara , Yu Ping , Ellis Jason , Divekar Rohit D , Jain Renu , Tartar Danielle , Lee Hyun-Hee , Hardaway John , Hoeman Christine , Guloglu F. Betul , Zaghouani Habib
Published: January 2023

Many autoimmune disorders have been linked to a lack in T regulatory cells. As such, an understanding of how these cells develop and mature is critical. While thymic T cell selection has been described, this process remains largely undefined for Tregs. Since only the DM20 form of proteolipid protein (PLP) is expressed in the thymus during fetal and neonatal life, thymic selection against PLP139-151 which is missing in DM20 is not operative during those periods. PLP139-151 (designated PLP1) as well as PLP1 derived altered peptide with degenerate affinity to PLP1-specific T cells were expressed on immunoglobulin (Ig) and the resulting chimeras were used to cross the maternal placenta, deliver the peptides from mother to fetus, and restore peptide selection during the fetal and neonatal period. This model was then used to determine whether Tregs are subject to thymic selection and to gauge the affinity required for such selection. The findings indicate that selection of functional Tregs requires high affinity. Indeed, Ig chimeras harboring high avidity peptides facilitated development of Tregs that conferred protection against experimental allergic encephalomyelitis (EAE) while chimeras incorporating low affinity peptide did not drive selection of Tregs and the animals remained susceptible to EAE.
Role of MAPK signal pathway in the regulation of IL-23p19 expression in human microglia (129.29)
Authors: Li Yonghai , Chu Niansheng , Ju Jack , Gran Bruno , Rostami A.M. , Zhang Guang-Xian
Published: January 2023

Activated microglia can release a variety of proinflammatory cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). IL-23 is a novel proinflammatory cytokine that shares the p40 subunit with IL-12 and is required for the induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, IL-23 expression and regulation in human microglia is poorly understood. Here we detected IL-23p19 mRNA expression on human microglia by northern blot and protein level by double-fluorescence immunostaining analysis. When the intracellular signaling pathway for p19 regulation was determined, we found that IL-23p19 expression in human microglia was significantly reduced by blocking the p38 MAP kinase pathway with either neutralizing antibodies or dominant-negative p38 transduction. Thus, human microglia inducibly express p19, and the p38 MAP kinase signaling pathway plays a major role in regulation of p19 expression.
IFN-γ receptor signaling in the CNS, but not periphery, is critical for the induction of experimental autoimmune encephalomyelitis (129.33)
Authors: Elhofy Adam , Dogan Rukiye Nazan , Karpus William J
Published: January 2023

Activation of microglia and astrocytes in experimental autoimmune encephalitis (EAE) has been associated with chemokine induction in the CNS resulting in attraction of inflammatory infiltrate. The role of IL-17 in autoimmune disease has called into question the role of IFN-γ in EAE. In this report we investigate the necessity of IFN-γ receptor signaling in disease induction. To test this we made IFN-γR−/− radiation bone chimeras with animals expressing IFN-γR in the CNS, animals deficient for IFN-γR in the CNS but able to signal in the periphery and a wild-type control (WT). We found IFN-γR expression on astrocytes and microglia was critical for clinical disease symptoms in the adoptive transfer model of EAE. Without IFN-γ activation in the CNS there were no infiltrating leukocytes while in WT and in animals capable of signaling in the CNS there were CD4, CD8, and CD11b infiltrating cells. CCL2, CCL5 and CXCL10 were significantly decreased in CNS homogenates of animals devoid of IFN-γR on astrocytes and microglia compared to WT and mice expressing IFN-γR on peripheral leukocytes. These results argue that IFN-γ activation in the CNS is essential for clinical EAE disease induction.

Supported by NIH R01 NS034510.
Il-23 enhances diabetes induction in the wild type but not in INF-gamma −/− mice (129.36)
Authors: Lukic Miodrag L , Shahin Allen - , Mensah-Brown Eric - , Al-Hakim Ahmed -
Published: January 2023

C57BL/6 male mice developed delayed progressive and sustained hyperglucemia, insulitis and loss of β cells when injected with 5 (40 μg/ kg b/w) daily doses of streptozotocin (STZ). C57BL/6 INF-γ −/− mice treated with same regimen develop the disease at the later stage (by day 28) but reach the same level of functional impairment by day 52 after disease induction. RT PCR analysis of the pancreatic lymph nodes revealed the presence of TNF, INF-γ, and IL-17 and in the “wild type” mice and only TNF-α and IL-17 in INF-γ −/− mice. Four injections of STZ did not induce hyperglycemia neither in the “wild type” nor INF-γ −/− mice. Concomitant treatment with IL-23 (10 x 40 mg/daily) dramatically enhanced the effect of this subdiabetogenic treatment in “wild type” mice, accompanied with the expression of TNF, INF-γ and IL-17 in the pancreatic lymph node while INF-γ −/− mice did not develop disease. We assume that INF-γ is required for the initial activation of antigen presenting cells in suboptimal conditions for diabetes induction but not for the expansion and activation of effector cells in this model of toxin induce autoimmune diabetes which is IL-23-IL-17 dependent (European Journal of Immunology, 36: 216–23, 2006).
A novel H2A−E+ transgenic model susceptible to human but not mouse thyroglobulin in autoimmune thyroiditis: identification of a mouse pathogenic epitope (129.37)
Authors: Brown Nicholas Korten , McCormick Daniel J. , Brusic Vladimir , David Chella S. , Kong Yi-chi M.
Published: January 2023

Experimental autoimmune thyroiditis (EAT) is a model for Hashimoto’s thyroiditis which is characterized by mononuclear cell infiltration and follicular destruction. We previously reported on an Eα-transgenic strain (H2A−E+) that is highly susceptible to human (h) thyroglobulin (Tg)-induced EAT, but strongly tolerant to mouse (m)Tg. After mTg and adjuvant, traditional, resistant strains do not develop thyroiditis, nor detectable primed T cell proliferation, but do make anti-mTg Abs. By contrast, this H2Eb+ strain does not even produce mTg Abs. To identify what epitopes in the mouse thyroid hTg-primed, thyroiditogenic T cells recognize to evoke EAT, we first confirmed that hTg-primed cells do not respond to mTg, nor to other thyroid Ags. We next selected the three immunogenic epitopes on hTg known to be processed and presented by H2Eb, as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and generate moderate thyroiditis. Thyroiditis incidence was increased after adoptive transfer of in vitro activated, mTg409-primed cells. Moreover, cells primed to hTg410, the corresponding, pathogenic hTg peptide, cross-reacted with mTg409, and vice versa. This mTg autoepitope, identified by using thyroiditogenic hTg epitopes, helps to explain the severe thyroiditis seen in this novel H2E+ transgenic model.

(Supported by NIH grant DK45960)
Collagen-specific T cells are not downregulated following treatment with 17β-estradiol in CIA (129.39)
Authors: Latham Kary , Whittington Karen , Rosloniec Edward
Published: January 2023

Increases in estrogen levels in rheumatoid arthritis (RA) patients result in amelioration of RA symptoms. Similarly, administration of estrogen (17β-estradiol, or E2) decreases incidence of collagen-induced arthritis (CIA), a well-established murine model for RA. Our laboratory established that this decrease was associated with a change in the isotype of antibodies produced to type II collagen (CII) following immunization with CII from predominantly IgG2a to IgG1. In order to determine if E2-driven changes in the T cell response regulate this isotype switch, we focused on E2’s effect on the function of CD4+ CII-specific T cells. After immunization with CII, analysis of CII-specific T cells using MHC class II tetramers showed that their numbers increased slightly in E2-treated mice at 10 days post-immunization compared to controls. There were no significant differences in expression of activation markers (CD69, CD44, or CD62L) by CII-specific T cells between groups. Surprisingly, analysis of cytokine expression by qPCR following cell sorting of the tetramer+ cells indicated that the CII-T cells from E2-treated mice expressed higher levels of IL-17 mRNA than did CII-T cells from controls. Levels of other proinflammatory cytokines such as IL-2, IFN-γ, and IL-6 were similar to those found in tetramer+ cells from controls. These results suggest that CII-T cells do not directly mediate E2’s protective effect in CIA.
Contribution of VH Replacement Products to the Generation of Autoreactive Antibodies in Systemic Lupus Erythematosus (129.4)
Authors: Fan Run , Su Kaihong , Majors Ashley M , Yu Zhihong , Zhao Evan , Kimberly Robert P , Zhang Zhixin
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by the production of high affinity autoantibodies against various nuclear antigens. To gain insight into the mechanisms of autoantibody generation during the course of SLE, we performed single cell PCR analysis and obtained about 200 IgH genes with paired Igκ or Igλ genes from the plasma cells (CD19lowCD27high) and naïve B cells (CD19+CD27−) of three SLE patients. The SLE derived IgH genes have longer CDR3 regions with enriched Arg residues, which are the common features of autoantibodies. SLE plasma cells derived IgH and IgL genes are highly mutated, indicating their positive selection and active involvement in the abnormal immune responses. Interestingly, analyses of the IgL and IgH genes derived from SLE patients showed signs of excessive secondary recombination when compared with those from healthy donors, such as elevated usages of downstream Jκ4, Jκ5, and Jλ3 genes in the IgL genes and concurrently, significantly elevated frequencies of VH replacement products in the IgH genes derived from SLE patients. All the VH replacement “footprints” preferentially contribute charged amino acids into the IgH CDR3 regions. Initial testing of 12 recombinant antibodies derived from SLE plasma cells showed seven of them recognize peri-nuclear antigens. Importantly, the identified VH replacement products directly encode autoantibodies. These findings revealed for the first time that excessive secondary recombination occurred on both IgL and IgH genes in SLE patients and the enriched VH replacement products in SLE plasma cells directly encode autoantibodies.
IGRP-reactive T cells can be detected directly ex vivo in the blood of type 1 diabetes patients (129.45)
Authors: Jarchum Irene , Nichol Lynn , Trucco Massimo , DiLorenzo Teresa P.
Published: January 2023

Both CD4 and CD8 T cells are required for the development of type 1 diabetes (T1D) in NOD mice and several antigens are known to be involved in the development of the disease. Among them is IGRP (islet-specific glucose 6 phosphatase catalytic subunit related protein), which has emerged in recent years as an important antigen in NOD mice. IGRP-reactive CD8 T cells constitute up to 30–40% of islet infiltrating T cells and these cells can be detected directly ex vivo in islet infiltrates and peripheral blood. To determine whether IGRP is a CD8 T cell antigen in humans, we created a panel of 4 HLA-A2 restricted IGRP epitopes (IGRP211-219, IGRP215-223, IGRP222-230, and IGRP265-273) to be screened directly ex vivo by IFN-γ ELISPOT. We performed the study with unfractionated PBMC from 21 recent-onset T1D patients (mean age 9.2 ± 4.3 years old) and 12 healthy controls (29.7 ± 5.7 years old) with the candidate peptides. Our results show that 66% of patients and 8% of healthy controls are positive for at least 1 IGRP peptide and 57% of patients versus 0% of controls are positive for 2 IGRP peptides. These data provide evidence that IGRP is a CD8 T cell antigen in humans, and that IGRP-reactive T cells can be detected ex vivo in the blood of T1D patients. This contributes to the understanding of the underlying disease process in humans as well as to future directions for diagnosis and intervention in T1D patients.
The role of Lipocalin 2 in the pathogenesis of Lupus Nephritis (LN) (129.7)
Authors: Pitashny Milena , Qing Xiaoping , Zhao Zeguo , Putterman Chaim
Published: January 2023

There is strong experimental evidence that anti DNA antibodies are instrumental in LN pathogenesis. We found that exposure of murine mesangial cells to pathogenic anti DNA mAbs induces overexpression of Lipocalin 2, a protein known to be upregulated following kidney insult. In this study we explored the upregulation of Lipocalin 2 in the context of murine lupus nephritis.

Kidney mesangial and tubular cells from MRL/lpr lupus prone mice were treated with the pathogenic anti DNA mAb 1A3F; consistent upregulation of Lipocalin 2 was detected. Similarly, IL1β, an inflammatory cytokine secreted by epithelial cells and macrophages, and agonists of TLR2 and TLR4 significantly stimulated the expression of Lipocalin 2. Interestingly, we found that 1A3F binds to HMGB1, an endogenous ligand for TLR 2/4.

Old MRL/lpr mice displayed higher kidney RNA expression of Lipocalin 2 than BALB/c mice (p<0.01). In addition, serum Lipocalin 2 was elevated in old as compared to young MRL/lpr mice (p<0.01), and compared to old MRL/+ and BALB/c mice (p<0.05). Importantly, kidney and serum Lipocalin 2 significantly correlated with anti DNA titers.

We conclude that Lipocalin 2 is upregulated in lupus kidneys, although whether this is only due to injury or plays a role in pathogenicity is yet unknown. Nevertheless, antibody deposition mediates the upregulation of Lipocalin 2 in intrinsic kidney cells with possible involvement of TLR 2/4 signaling.
Heritability plots of levels of autoantibodies in thyroiditis and type 1 diabetes using ROMP (129.8)
Authors: Outschoorn Ingrid Maive , Burek C. Lynne , Talor Monica V. , Hoffman William H , Rowley Merrill J , Mackay Ian R , Jones Timothy W , Rose Noel R
Published: January 2023

Few simple methods exist to study the genetic control of autoimmunity. We plotted ROMP (Regression of Offspring on Midparent) for hundreds of trios and compared affected and unaffected siblings. Heritability of autoantibodies (AAbs) to thyroglobulin and thyroperoxidase in chronic lymphocytic thyroiditis (CLT) and thyrotoxicosis (TT), and to GAD65 in type 1 diabetes (T1D) were assayed. AAbs were significantly correlated in TT (R 2 = 0.569, p = 0.001), but not in CLT or T1D. Positive results in TT were found in patients of African American origin, suggesting which quantifiable parameters may determine the sequential appearance of more than one type of antibody, as in T1D, in predicting disease onset in other family members. Parental effects can be determined separately, and data extended to epitope spreading and disease progression. Such a screening method requiring small patient numbers enable extensive studies to be rapidly carried out simultaneously and retrospectively on various AAbs.
PKCδ nitrosylation impairs ERK pathway signaling in lupus T cells (129.9)
Authors: Gorelik Gabriela , Wu Ailing , Richardson Bruce
Published: January 2023

T cells from lupus patients or treated with hydralazine have defective PKC-ERK pathway signaling, causing decreased DNA methyltransferase levels, DNA hypomethylation and overexpression of methylation sensitive genes contributing to autoimmunity. We asked what causes this defect. Immunoblotting mapped the defect to impaired PMA stimulated PKCδ phorphorylation in lupus and hydralazine-treated T cells (49.6 ± 4.3% decrease, p≤0.001 and 28.3± 3.2% decrease, p≤0.05 respectively). Total PKCδ levels were not affected. Rottlerin, a selective PKCδ inhibitor, decreased PMA-induced ERK pathway signaling and increased CD70 expression by demethylating its promoter similar to lupus and hydralazine treated T cells. PDK-1, which activates PKCδ, was not modified in lupus or hydralazine treated cells, and lupus serum did not inhibit PKCδ phosphorylation. This suggested an intrinsic PKCδ defect. Tyrosines in lupus proteins are aberrantly nitrosylated, so PKCδ phosphorylation was studied in peroxynitrite treated cells. Peroxynitrite caused a dose-dependent decrease in PKCδ phosphorylation (p<0.05). Similarly, phospho-PKCδ was barely detectable in anti-nitrotyrosine immunoprecipitates of PMA stimulated lupus T cells, suggesting prior nitrosylation. These results suggest that defective PKCδ activation due to oxidative damage contributes to the development of lupus.

This work was supported by grants from NIH and Veterans Administration
Adoptive transfer of regulatory T cells can delay disease progression in a congenic murine model of SLE (130.12)
Authors: Cuda Carla Marie , Morel Laurence
Published: January 2023

Previous studies have shown that adoptive transfer of regulatory T cells (Tregs) can prevent disease onset in murine organ-specific autoimmune disease models. Our lab studies a congenic murine model of systemic lupus erythematosus containing three susceptibility loci from the NZM2410 spontaneous model bred onto a normal B6 background, called the B6. Sle1Sle2Sle3 triple congenic strain. This strain presents with a decreased number of Tregs, yet on a per-cell basis, these Tregs are equally as capable of suppressing effector T cell (Teff) proliferation as those isolated from normal controls. To determine whether the deficiency in Treg number contributed to disease, we adoptively transferred six million isolated CD4+CD25+ Tregs or CD4+CD25− Teffs from triple congenic donors into young unmanipulated triple congenic recipients. A delay in the production of anti-nuclear antibodies as well as a decrease in spleen weight, proteinuria level, and activation of effector populations were observed four months post-transfer in those mice which received Tregs. Here we show that a single injection of unmanipulated Tregs is sufficient to ameliorate observed disease phenotypes in a systemic autoimmune disease model.

This research is supported by NIH R01 AI1045050, T32 AR007603
Genetic dissection of Sle1c (130.16)
Authors: Perry Daniel James , Morel Laurence
Published: January 2023

The Sle1c locus, derived from the NZW mouse strain, is a ~6.5Mb interval located at the telomeric end of chromosome 1. Previous work has shown that when Sle1c is congenically bred onto a B6 background to yield B6. Sle1cw, a mild lupus-like disease occurs in aged mice (>8mo.), indicated by low to moderate levels of anti-nuclear antibodies (ANA) and low incidence of glomerulonephritis (GN). Further phenotypic analyses of this strain reveal decreased T-dependant (TD) immune responses, abnormal germinal centers (GC), increased T cell activation and proliferation, amplification of chronic graft-versus-host disease (cGVHD), and decreased number of regulatory T cells (Tregs).

Cr2 is located on the telomeric end of Sle1c. It encodes Complement receptor 1 (CR1) and 2 (CR2) thorough alternative splicing has previously been described as a candidate gene for Sle1c. It was shown with subcongenic strains of Sle1c that cr2w results in the abnormal GC and decreased TD immune response phenotypes. However, cr2w does not account for the other phenotypes. We have created a series of subcongenic strains on the centromeric end of Sle1c that map the T cell activation phenotype to <1Mb. Screening is ongoing for the enhanced cGVHD. This should lead to the identification of the corresponding gene(s).

This research is supported by NIH R01 AI1045050
CD8+ T Cell- mediated Suppression of Autoimmunity in a Murine Lupus Model of Peptide-Induced Immune Tolerance Depends on Foxp3 Expression (130.19)
Authors: Singh Ram P , La Cava Antonio , Wong Maida , Tamayo Alejandro M , Ebling Fanny M , Hahn Bevra H
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by autoantibodies including IgG anti-DNA. NZB/NZW Fl female (BWF1) mice, a model of spontaneous polygenic SLE, tolerized with an artificial peptide (pCONSENSUS, pCons) based on anti-DNA IgG sequences containing MHC Class I and Class II T cell determinants, develop regulatory CD4+CD25+T cells and inhibitory CD8+T cells (CD8+ Ti), both of which suppress autoAb production. In the present study, using various cellular and molecular approaches, we show that inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 weeks and at all times depends on expression of Foxp3. Both CD28− positive and –negative CD8+ T cells contain inhibitory cells, but the expression of Foxp3 and of mRNA for TGFb is higher and lasts longer in the CD28− subset. In vitro addition of TGFb (in the presence of IL-2) induces Foxp3 expression in a dose response manner. Gene inhibition or blockade with siRNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ helper T cells. Moreover, we found a significant correlation between expression of Foxp3 and ability of CD8+ Ti cells to secrete TGFb. Therefore, CD8+ Ti in this system of tolerance is depend on expression of Foxp3, and there may be a bi-directional Foxp3/TGFb autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.

Supported by NIH grants AI63515,AI 63515, AR53239 and Tina C Foundation.
Activation of NKT cells exacerbates environmentally induced autoimmunity (130.23)
Authors: Vas Jaya , Mattner Jochen , Bendelac Albert , Howell Amy , Richardson Stewart , Ndonye Rachel , Monestier Marc
Published: January 2023

Administration of subtoxic doses of mercuric chloride to genetically susceptible mice induces an autoimmune syndrome with anti-nucleolar antibody production, polyclonal B cell activation and nephritis. We investigated the role of natural killer T (NKT) cells in the modulation of this autoimmune model. Activation of NKT cells by synthetic variants of α-galactosyl ceramide worsened manifestations of mercury-induced disease. Exposure to heat killed Sphingomonas capsulata which contains NKT cell ligands in its cell wall also worsened disease manifestations. We used this model to further elucidate the interplay between NKT cells and other immune cell types. Mice can be tolerized to mercuric chloride by administration of a very low dose of the chemical. Tolerance establishment requires antigen presentation in the steady state by dendritic cells to elicit CD4+ CD25+ regulatory T cells. Activation of NKT cells by a synthetic ligand was able to completely prevent induction, but was by itself unable to break already established tolerance. Dual stimuli (NKT cell activation and TLR 9 ligation) were required to break established tolerance. This study demonstrates that NKT cell activation worsens chemically- induced autoimmunity and also shows a synergy between NKT and TLR ligands in potentiating disease manifestations.

Supported by a NIH grant.
Invariant NKT cells exacerbate liver injury in a TGF-β receptor II dominant-negative mouse model of primary biliary cirrhosis (130.24)
Authors: Chuang Ya-Hui , Lian Zhe-Xiong , Yang Guo-Xiang , Shu Shang-An , Moritoki Yuki , Gershwin M.Eric
Published: January 2023

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune liver disease characterized by the presence of anti-mitochondrial antibodies (AMA) and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of invariant natural killer T (iNKT) cells were increased in the liver of patients with PBC. To define a specific role of iNKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic iNKT cells in our TGF-β receptor II dominant-negative (dnTGFβRII) mouse model of PBC. We report here that these dnTGFβRII mice demonstrate a massive increase of hyperactive iNKT cells within the hepatic tissues. We generated CD1d−/−and CD1d+/− dnTGFβRII mice and performed a comparative study of liver immunopathology, demonstrating that the CD1d−/−dnTGFβRII mice, lacking iNKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared to CD1d+/−dnTGFβRII mice. Interestingly, there was a significant increase in the production of IFN-γ in hepatic iNKT cells activated by α-galactosylceramide (α-GalCer) in young but not older dnTGFβRII mice, suggested an age dependent role of iNKT cells. These data demonstrate that iNKT cells in dnTGFβRII mice are a critical factor in liver injury.
INCREASED EXPRESSION OF IFN-ALPHA AND IL-6 BY HYPERACTIVE PLASMACYTOID DENDRITIC CELLS IN BXD2 AUTOIMMUNE MICE (130.25)
Authors: Wang John Hsien , Hsu Hui-Chen , Yang PingAr , Wu Qi , Job Godwin , Chen Jian , Timares Laura , Mountz John D.
Published: January 2023

BXD2 is a mouse model which exhibits several features of human SLE and arthritis. IFN-α is a known cytokine which promotes B-cell maturation and antibody CSR and SHM. ELISA results of in vitro culture of spleen cells demonstrates that IFN-α expression reaches a peak at 2 months of age (4-fold increase compared to nonautoimmune mice; p<0.005), suggesting that IFN-α can serve as an early trigger for B-cell autoimmunity. Immunofluorescent staining for CD11c+ antigen-presenting cells identifies an increased population (2–4 fold; p<0.005) of plasmacytoid dendritic cells (pDCs). Flow cytometry results demonstrate splenic pDCs are the primary cells which express IFN-α (>80%) and IL-6 (>96%) in BXD2 mice. In vivo treatment of 2 m-old BXD2 mice with DOTAP/CpG resulted in a 200-fold increase in IFN-α compared to a 4-fold increase in B6 mice. This is associated with high levels of autoantibody titers and CSR. In conclusion, BXD2 mice exhibit a significantly increased population of pDCs that express IFN-α at a young age which is associated with increased humoral response.
CD8+ T cells are critical for collagen induced arthritis disease progression (130.46)
Authors: Bucks Christine M , Katsikis Peter D
Published: January 2023

Despite extensive study of T cells in Rheumatoid Arthritis (RA), relatively little is known regarding the role of CD8+ T cells in RA progression. Here, we use the collagen induced arthritis (CIA) model, and deplete, prior to disease onset, CD8α+ T and dendritic cells (DCs) or CD8β+ T cells in collagen immunized DBA mice. Beginning 23 days post immunization, mice received repeated injections of 100μg anti-CD8α(53-6.7), or anti-CD8β(H35-17.2) or IgG2a isotype. Depletion of CD8αor CD8β cells did not affect the prevalence of disease; suggesting that CD8+ cells are not necessary for disease initiation (isotype=87%, anti-CD8α=78%,and anti-CD8β=78%, n=9/group). However, there was a significant reduction in arthritis duration with CD8α depletion (anti-CD8α= 4.9±2.0 vs. control= 8.6±1.1 arthritic days, p<0.01) and with CD8β depletion (anti-CD8β= 8.1±1.7 vs. control=13.5±1.9 arthritic days). Disease severity was also reduced in CD8α and CD8β depleted mice. Depleted mice had maximum mean paw scores of 2.4±1.1 (CD8α depleted) and 2.5±1.0 (CD8β depleted) compared to 4.5±1.8 in controls, p< 0.03. This was accompanied by reduced mononuclear cell infiltrate, pannus formation and joint destruction, as observed by H&E staining. We have shown that CD8+ T cells are necessary for CIA progression and are critical contributors to disease severity.
HLA-DQ6 (DQB1*0601) molecule modulate PLPp91-110 induced EAE in DR3 (DRB1*0301) mice through apoptosis. (130.6)
Authors: Mangalam Ashutosh K. , Rodriguez Moses , David Chella S
Published: January 2023

The HLA class II region on chromosome 6p21 accounts for majority of familial clustering in MS and is by far the major susceptibility locus. However due to multiple class II genes, high polymorphism and linkage disequilibrium, it has been difficult to understand the role of individual class II genes and interaction between class II genes in the disease process. Previously, we have shown that PLP91-110 induced classical EAE in DR3.Abo but not in DQ6.Abo transgenic (tg) mice, suggesting that DR3 is a disease susceptible gene in the context of PLP. In this study, we have generated double (tg) mice expressing HLA-DR3/DQ6 to simulate heterozygous haplotype of MS patients. Introduction of DQ6 onto DR3 tg mice led to decrease in disease incidence on immunization with PLP91-110 suggesting a protective role of DQ6. We further characterized that this protective effect was due to expression of high level of IFNγ by DQ6 restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis in antigen specific T cells. Our IFNγ blocking experiments confirmed an anti-inflammatory role of IFNγ. Our study suggests that DQ6 modifies the PLP91-110 specific T cell response in DR3 through IFNγ mediated activation induced cell death. Thus our double tg mouse model provides a novel tool to study linkage disequilibrium and explain how HLA-DQ molecule might modulate disease in MS-susceptible individual.
The role of T cell memory and apoptosis in the resistance of Drak2-deficient mice to autoimmunity (130.8)
Authors: Hernandez Jeniffer B. , Ramos Stephanie J. , Walsh Craig M.
Published: January 2023

Autoimmune disease often occurs via the escape of autoreactive T cells from apoptosis, implying a breakdown of central or peripheral tolerance mechanisms. We have recently demonstrated that thymocytes and T cells lacking the serine/threonine kinase DRAK2 have a diminished activation threshold, suggesting that this kinase serves as a quiescence factor to maintain tolerance to weak self-antigens. Drak2−/− T cells exhibit enhanced sensitivity to TCR mediated stimulation and produce higher levels of the cytokines IL-2, interferon gamma and IL-4. Surprisingly, Drak2−/− mice are resistant to experimental autoimmune encephalomyelitis (EAE). EAE is a disease induced in mice by immunization with MOG35-55 peptide, which elicits multiple sclerosis-like symptoms due to T cell activation and recruitment into the CNS. We have observed enhanced apoptosis in T cells derived from Drak2−/− mice, suggesting that DRAK2 serves as both a quiescence factor in naïve T cells and a survival factor in effector T cells. In order to understand the mechanism of resistance of Drak2−/− mice to EAE, we have employed adoptive transfer strategies to evaluate the T cell intrinsic requirements for DRAK2 in maintaining the survival of MOG-specific effector and memory populations during the course of EAE. These results demonstrate that DRAK2 is a key factor that regulates both central and peripheral tolerance.
Suppression of autoimmune diabetes by peptide-MHC class II dimers (131.13)
Authors: Li Li , Goudy Kevin , Pham Alexander , Tisch Roland
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease mediated by pathogenic beta cell-specific T cells. The use of antigen-specific based immunotherapies has been one strategy to selectively target beta cell-specific T cells leaving the remainder of the immune system intact. Administration with peptides derived from beta cell autoantigens such as glutamic acid decarboxylase 65 (GAD65) to NOD mice prevents T1D. However, as beta cell autoimmunity progresses the conditions for peptide treatment to suppress T1D become highly stringent. To enhance the efficacy of peptide treatment, recombinant IAg7-immunoglobulin (Ig) dimers covalently linked to GAD65 peptides (217–236, 286–309) or the mimetic BDC2.5 epitope (sBDC) were tested. Twelve week-old NOD female mice with established beta cell autoimmunity received a short course of IAg7-Ig dimers intravenously. Surprisingly, treatment with IAg7-Ig:sBDC accelerated the onset of diabetes in NOD mice due to expansion of sBDC-specific CD4+ Th1 cells. In marked contrast, the majority of NOD mice treated with IAg7-Ig:GADp217 or IAg7-Ig:GADp286 remained diabetes-free. Protection correlated with an increased frequency of IL-10 secreting immunoregulatory CD4+ T cells that blocked diabetes in a co-adoptive transfer model. These results demonstrate that treatment with a short-course of IAg7-Ig-GAD65 peptide dimers is an effective approach to suppress T1D.
Interferon Beta-1a Modulates the Innate Immune-response-mediated Regulation of Adaptive Immunity in Early Multiple Sclerosis (131.15)
Authors: Zhang Xin , Tang Y , Montes M , Jin J , Ramgolam V S , Markovic-Plese Silva
Published: January 2023

We performed Affymetrix gene arrays (HU133, ~38,500 genes) using RNA from the in vitro activated PBMCs from patients with clinically isolated syndrome suggestive of multiple sclerosis (CIS) (n=15) and matched healthy controls (HC) (n=8). Results were confirmed and further supported by RT-PCR and RayBio Cytokine Arrays. Affymetrix data revealed that IFNB-1a induced significant (p<0.05) up-regulation of 711, and down-regulation of 587 genes in CIS patients, and 446 and 543 in HC, respectively. CXCL11 and CXCL10, the ligands of CXCR3 were significantly increased in CIS patients and HC, whereas proinflammatory chemokines CXCL2, CXCL3 and CXCL5 were decreased in CIS patients. CCL18 and CCL19 were increased in CIS, while CCL22 and CCL17 were decreased in CIS and HC. Those chemokine gene changes reflect the complex effects of IFNB-1a that likely affect differentiation/ maturation and chemotaxis of DCs and other cells bridging the innate and adaptive immune response. The anti-inflammatory cytokine IL-10 was increased in CIS, consistent with the previous findings in multiple sclerosis, while IL1A, IL1R1 and IL-16 were decreased in CIS and HC. IL-1 is an acute inflammatory phase cytokine whose inhibition may decrease differentiation of IL-17 secreting T-cells that is critical in the development of autoimmune response. SOCS7 and SCOS5 were decreased in CIS and HC that may result in impaired Th1 responses. TLR7 and its signal adapter MYD88 were increased in CIS and HC. IRAK4 and TRAF6, the components of MYD88-dependent pathway were also increased in CIS. IRF2 and IRF7 that may be involved in MYD88-independent TLR signaling pathway were increased in CIS and HC. Such effects on TLRs signaling pathways may inhibit DCs maturation and alter the innate immune response mediated regulation of adaptive immunity.
Genomic-based high throughput screening and identification of small molecule inhibitors targeting IFN α signaling pathways (131.17)
Authors: Chen Bo , Cibotti Ricardo , Zong Qin , Morris Chad , Castaneda Juana , Naiman Brian , Liu Derong , Glodek Anna , Soppet Dan , Horrigan Steve , Kiener Peter , Coyle Anthony , Audoly Laurent
Published: January 2023

Multiple lines of evidence suggest that inhibition of Type I Interferons (IFNs), including IFN(, may provide a therapeutic benefit in treating a number of autoimmune diseases such as Systemic Lupus Erythematosus (SLE). Herein, we describe the generation and use of AvalonRx-HITS®, a proprietary genomic based high-throughput screen (HTS), followed by immunological assays in primary human cells to identify inhibitors of the IFN( pathway. In the HTS step, THP1 cells were stimulated with IFN( and 268 defined small molecule inhibitors targeting 41 different intracellular signaling pathways were screened for their ability to block a specific IFN(-evoked gene signature. Four classes of compounds emerged from the HTS: NF-(B, JAK/STAT, Ubiquitin/Proteasome and HDAC inhibitors. In secondary assays, these compounds blocked both IFN(-induced human monocyte activation and differentiation into dendritic cells, and IFNs-associated gene signature induced by SLE serum in human monocytes. In addition to their dose-dependent anti-inflammatory effects, inhibitors targeting NF- (B or JAK/STAT signaling did not modulate IFN( anti-viral effects in an in vitro HSV-1 replication assay. In summary, we have developed a novel and robust platform using genomic, immunological and viral assays that may lead to the identification of small molecule inhibitors for the treatment of IFN(-associated autoimmune disorders.
Determination of affinity constants of adalimumab, infliximab and etanercept for soluble and membrane forms of TNF using kinetic exclusion assay (131.22)
Authors: Sakorafas Paul , Bose Sahana , Kaymakcalan Zehra
Published: January 2023

TNF neutralizing biologics adalimumab (Ada), infliximab (Inf), and etanercept (Eta) have distinct clinical efficacy profiles. It is not known whether their binding characteristics to TNF, especially their affinities for soluble (sTNF) vs transmembrane TNF (mTNF) plays an important role in different clinical outcomes. Previously, BIAcore and radioimmunoassay methods have been used to measure affinities for sTNF and mTNF, respectively. The affinity for mTNF as measured by RIA was much lower than the sTNF for all three biologics. In this study, we employed kinetic exclusion assay (KinExA) to measure overall affinities of unlabeled Ada, Inf and Eta for both sTNF and mTNF. A fixed amount of anti-TNFs were incubated with varying amounts of sTNF or cells transfected with a TNF deletion mutant that expressed a stable form of mTNF to reach to equilibrium. The overall affinities of Ada, Inf and Eta for sTNF were determined as 8.6, 4.2 and 1.2 pM; and for mTNF as 7.9, 7.0, and 11.2 pM. When measured by the same instrument, affinity for the membrane form of TNF was similar to soluble TNF for all three anti-TNFs. Eta had a higher affinity for sTNF, followed by Inf and Ada. Ada, Inf and Eta had similar affinities to mTNF as presented on the transfected cells.
Antagonizing HMGB1 inhibits proteinuria in a murine model of lupus-like disease (131.24)
Authors: Mao Su-Yau , Xu Yan , Zhang Jing , Schifferli Kevin , An Ling-Ling , Leininger Joel , Kiener Peter A , Wu Herren , Coyle Anthony J , Audoly Laurent P
Published: January 2023

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts like an inflammatory cytokine when it is released by dying or stimulated cells. Elevated levels are detected at sites of inflammation in a number of autoimmune disorders including SLE. In addition, HMGB1 has been shown to contribute to the activation of plasmacytoid dendritic cells as well as B cells, cell types that are implicated in the pathogenesis of SLE. To address the role of HMGB1 in experimental lupus, we investigated the protective efficacy of a fully humanized mAb (IA-4, which recognizes both mouse and human HMGB1) in an IFNα-accelerated lupus model. In this model, adenovirus IFNα-treated NZB/W F1 mice exhibit 90–100% proteinuria incidence at 4–6 weeks post-treatment. The functionality of IA-4 has been demonstrated in vitro by its ability to inhibit rHMGB1-induced IL-6 secretion in human PBMC (IC50 = 0.9 nM). Prophylactic administration of the anti-HMGB1 mAb IA-4 at 10 mg/kg significantly inhibited the proteinuria severity score at seven weeks (65% inhibition; p<0.05 compared to an isotype control). Consistent with the proteinuria data, histopathological assessment of the kidney also revealed protection with IA-4 treatment. Despite its protection in nephritis, IA-4 treatment showed no detectable effect on serum levels of IFN-α and autoantibodies against dsDNA or SSA/Ro. Taken together, these data suggest that HMGB1 is a critical effector molecule in lupus nephritis and that anti-HMGB1 treatment may provide a potential therapeutic benefit for the treatment of SLE.
Anti-BR3 antibodies – a new class of B cell immunotherapies combining cellular depletion and survival blockade (131.25)
Authors: Lin WeiYu , Gong Qian , Seshasayee Dhaya , Lin Zhoughua , Ou Qinglin , Ye Shiming , Suto Eric , Shu Jean , Lee Wyne Pun , Lee Vivian , Fuh Germaine , Yeh Sherry , Deforge Laura , Danilenko Dimitry , Thompson Jeffrey S. , Ambrose Christine , Balazs Mercedesz , Starovasnik Melissa , Martin Flavius
Published: January 2023

Pathogenic B lymphocytes removal through therapy with depleting monoclonal antibodies results in clinical benefit in both oncology and immunological indications. Incomplete rates of clinical response and the realization that B cell depletion following immunotherapy is incomplete fuels the need for better therapies. BAFF/BLyS signaling through its BAFFR/BR3 receptor have a critical role on B cell proliferation and survival in the mouse and to some extent in non-human primates and humans. In order to combine B cell depletion with the blockade of BAFF mediated B cell survival, we generated depleting, BAFF-blocking, non-agonistic anti-BR3 monoclonal antibodies. The results from this study indicate that in mice anti-BR3 monoclonal antibody reduces B cells to a higher degree when compared to anti-CD20 or BAFF-blockade through BR3-Fc. Anti-BR3 depletes B cells with a slower kinetics than anti-CD20 but showed high B cell depletion and a decrease in bone marrow plasma cells which was not seen with anti-CD20. Anti-BR3 treatment of NZB/W lupus mouse model results in significant survival benefit and reduction of established nephritic clinical symptoms indicating potential therapeutically value for human disease. In conclusion, in vivo comparison in mice of anti-BR3 antibodies with other B cell immunotherapies (anti-CD20 and BR3-Fc) shows characteristics in B cell subset reduction that can guide translation to treatment of human disease.

This work was supported by Genentech Inc.
Beneficial effects of Vitamin D receptor agonists on retinal autoimmunity (131.30)
Authors: Tang Jun , Zhu Wei , Uskokovic Milan , Sliver Phyllis , Su Shaobo , Chan Chi-Chao , Adorini Luciano , Caspi Rachel
Published: January 2023

1,25-Dihydroxyvitamin D3 (calcitriol) is a seco-steroid hormone of multiple bioactivities that regulates the differentiation, growth and function of a broad range of cells, including cells of the immune system, by binding to the nuclear Vitamin D receptor (VDR). BXL-35 is a synthetic analog of calcitriol with reduced effects on calcium metabolism. We examined their ability to inhibit experimental autoimmune uveoretinitis (EAU), an autoimmune disease mediated by Th1/Th17-type response, which serves as a model for human posterior uveitis. B10.RIII mice were immunized with an uveitogenic regimen of 8 μg IRBP in CFA and treated orally with calcitriol or with BXL-35, throughout (prevention) or starting 7 days after EAU induction (reversal). Calcitriol at 0.5 μg/kg and BXL-35 at 10 μg/kg prevented EAU and reduced Ag specific IFN-γ and IL-17 production by primed lymph node cells. In addition, BXL-35 but not calcitriol was able to reverse EAU at sub-toxic doses. Mechanistic studies revealed that dendritic cells from mice treated with calcitriol had reduced production of IL-1, TNF-α, IL-6 and

IL-12/23p40. Furthermore, calcitriol directly inhibited the ability of purified CD4+T cells to produce IL-17 in response to CD3/CD28 engagement under conditions favoring Th17 polarization. In contrast, calcitriol had no effect on the ability of T cells to adhere to endothelial cell monolayers. Our study suggests that the protective effects of VDR agonists on retinal autoimmunity involve direct and indirect inhibition of priming and function of autopathogenic T cells.
The protease inhibitor, Bowman-Birk inhibitor, suppresses experimental autoimmune encephalomyelitis (131.33)
Authors: Ciric Bogoljub , Touil Tarik , Gupta Anjali , Shindler Kenneth , Rostami Abdolmohamad
Published: January 2023

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is enriched with BBI, but is predominantly made up of other ingredients. In our previous study we found that oral administration of BBIC to Lewis rats with EAE resulted in significant clinical and histological suppression of disease. In the present study we investigated the effect of BBI using mouse EAE models and pure BBI. We found that both I.P. and oral treatment with BBI (typically 1 mg/day BBI) in SJL/PLP139–151, or C57BL/6/MOG35–55 models, significantly improved clinical and histological parameters of EAE (disease onset, severity, weight loss, inflammation and demyelination). This was true for different treatment regimens as regards the day of treatment initiation relative to immunization for EAE induction. In most experiments antigen-specific proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. In the SJL model of optic neuritis BBI significantly reduced the incidence of neuritis, inhibited inflammation and prevented loss of retinal ganglion cells. Using Boyden’s chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. These results indicate that BBI is an excellent candidate for oral therapy in multiple sclerosis.
Prevention of Type 1 diabetes in NOD mice by helminth infection (131.36)
Authors: Liu Qian , Sundar Krishnan , Mousave Gity , Bleich David , Gause William
Published: January 2023

Parasitic helminth infection has been shown to modulate pathologic inflammation associated with allergy and autoimmune disease. The aim of this study was to examine whether infection with a helminth parasite could down-regulate the onset of type 1 diabetes in NOD mice and to examine the mechanisms involved in this protection. At 5 weeks of age, NOD mice were infected with Heligmosomoides polygyrus(Hp). At 36 weeks of age, these mice were compared with uninfected mice for the onset of type 1 diabetes (T1D). 70% of uninfected NOD mice became diabetic but only 10% of Hp infected mice developed T1D. Moreover, Hp infection also significantly reduced the development of the more aggressive cyclophosphamide-induced T1D in NOD mice with 5% developed in Hp-infected group compared to 78% in uninfected group. Further studies showed that infection with Hp in NOD mice:

markedly inhibited insulitis in the pancreas;elevated IL-4 and IL-13 levels in mesenteric and pancreatic lymph nodes as well as in spleens;increased the frequency of CD4+ CD25+ Foxp3+ Tregs in mesenteric and pancreatic lymph nodes;led to a consistent, massive accumulation of mononuclear cells in the regions of pancreatic fat.

These findings suggest that infection with Hp significantly inhibits the onset of T1D in NOD mice by influencing lymphocyte trafficking to the β-islet cells in the context of increased Th2 and T regulatory cell populations.
High Throughput Cell-Based Immunoassays Using Filtration Plates (132.1)
Authors: Park Jun , Kellard Libby , Lynch John , Sharma Ajay
Published: January 2023

Cell-based immunoassays require time consuming centrifugation/wash steps which can cause a substantial cell loss. We describe here automation compatible cell-based immunoassays using 96 well filtration plates. The method is rapid and robust, requiring no centrifugation based washing steps, and enables easy yet rigorous removal of unbound antibodies with little cell loss (<20%) even after extensive filtration (>10x).

We tested the method for detecting surface and intracellular proteins via a high throughput flow cytometer. In all cases, reproducible results with excellent signal to noise ratio were detected.

For surface protein detection, Jurkat cells were stained for CD45 and MFI (mean fluorescence intensity) of 504 +/-19 (CD45) and 12 +/-2 (isotype control) were obtained. For intracellular protein detection, anisomycin treated Jurkat cells and INF-γ treated U937 cells were stained for phospho-p38 (pT180/pY182) and phospho-Stat1 (pY701), respectively. MFI was 19.6 +/-1.8 (induced) and 5.2 +/-0.4 (not induced) for phosph-p38, and 44 +/-0.8 (induced) and 4.8+/-0.6 (not induced) for phospho-Stat1.

In conclusion, reported method allows for sample incubation/preparation in the same plate, while removing the rate limiting centrifugation/wash steps. While we demonstrate its use in flow cytometry, the method is suitable for other cell-based immunoassays such as chemiluminescent protein detection.