bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Kinetics of Transduced Cells in Peripheral T Cell-Directed Gene Therapy: Role of CD8+ Cells in Improved Immunological Function in an Adenosine Deaminase (ADA)−SCID Patient
Authors: Kawamura Nobuaki , Ariga Tadashi , Ohtsu Makoto , Kobayashi Ichiro , Yamada Masafumi , Tame Atsushi , Furuta Hirofumi , Okano Motohiko , Egashira Masayuki , Niikawa Norio , Kobayashi Kunihiko , Sakiyama Yukio
Published: January 2023

We previously reported successful peripheral T cell-directed gene therapy in a boy with adenosine deaminase (ADA)−SCID. In the present study, to better understand the reconstitutive effect of this gene therapy on his immunological system, we investigated the in vivo kinetics and functional subsets of T cells in PBL. Apparent immunological improvements were obtained after infusion of transduced cells at more than 4 × 108 cells/kg/therapy/3 mo. Frequency of ADAcDNA-integrated cells in PBL, ADA activity in PBL and clinical improvement showed good correlation, even though CD8+ cells gradually became predominant in PBL. On the basis that polyethylene glycol (PEG)-ADA was maintained at the same dosage as before gene therapy, we consider that his immunological improvement resulted from the gene therapy itself. Most CD3+ cells in PBL after gene therapy expressed TCRαβ. Analysis of TCR repertoire based on TCR V region usage revealed no expansion of limited clones in his PBL. The T cell subset cells CD8+CDw60+ and CD8+CD27+CD45RA−, which are reported to provide substantial help to B cells, were maintained throughout the gene therapy. Furthermore, his reconstituted peripheral T cells helped normal B cells to produce substantial IgG in vitro. Expression of both Th1- and Th2-type cytokine genes was induced in his reconstituted T cells at the same comparably high level as in normal subjects. Collectively, these results provide evidence of persistent and distinct functions of transduced cells in this patient’s PBL after gene therapy.
Phosphorylated Peptides Can Be Transported by TAP Molecules, Presented by Class I MHC Molecules, and Recognized by Phosphopeptide-Specific CTL
Authors: Andersen Mads Hald , Bonfill Jordi Espuny , Neisig Anne , Arsequell Gemma , Søndergaard Ib , Neefjes Jacques , Zeuthen Jesper , Elliott Tim , Haurum John S.
Published: January 2023

CTL recognize short peptide fragments presented by class I MHC molecules. In this study, we examined the effect of phosphorylation on TAP transport, binding to class I MHC molecules, and recognition by CTL of peptide fragments from known phosphorylated oncogene proteins or virus phosphoproteins. We show that phosphopeptides can be efficiently transported from the cytosol to the endoplasmic reticulum by the TAP. Furthermore, we show that phosphorylation can have a neutral, negative, or even a positive effect on peptide binding to class I MHC. Finally, we have generated phosphopeptide-specific CTL that discriminate between the phosphorylated and the nonphosphorylated versions of the peptide. We conclude that phosphopeptide-specific CTL responses are likely to constitute a subset of the class I MHC-restricted CTL repertoire in vivo.
Two Ancient Allelic Lineages at the Single Classical Class I Locus in theXenopusMHC
Authors: Flajnik Martin F. , Ohta Yuko , Greenberg Andrew S. , Salter-Cid Luisa , Carrizosa Ana , Du Pasquier Louis , Kasahara Masanori
Published: January 2023

Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and α2 peptide-binding domains, and there are highly diverse alleles (defined by the α1 peptide-binding domain) in each lineage. Surprisingly, the α3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.
Intracellular IL-1 Receptor Antagonist Is Elevated in Human Dermal Fibroblasts That Overexpress Intracellular Precursor IL-1α
Authors: Higgins Gloria C. , Wu Yong , Postlethwaite Arnold E.
Published: January 2023

Cultured dermal fibroblasts from systemic sclerosis patients express higher levels of intracellular IL-1α than fibroblasts from healthy controls. In this study, we found that systemic sclerosis dermal fibroblasts also express higher levels of the intracellular isoform of IL-1 receptor antagonist (icIL-1Ra) than normal fibroblasts after stimulation with IL-1β or TNF-α. A possible relationship between elevated precursor IL-1α (preIL-1α) and elevated icIL-1Ra was investigated by transducing normal dermal fibroblasts to overexpress preIL-1α, preIL-1β, or icIL-1Ra. Fibroblasts that overexpressed icIL-1Ra did not have elevated levels of IL-1α. On the other hand, fibroblasts that overexpressed preIL-1α had at least 4-fold higher basal levels of icIL-1Ra than control fibroblasts and 4-fold higher levels of icIL-1Ra after induction with IL-1β or TNF-α. Fibroblasts overexpressing preIL-1β did not exhibit elevated icIL-1Ra. The differences in icIL-1Ra protein levels were reflected in differences in mRNA. In contrast, IL-1-stimulated levels of MCP-1 and IL-6 were not different in control and preIL-1α-transduced fibroblasts. Addition of neutralizing anti-IL-1α Abs to fibroblast cultures did not diminish basal or stimulated levels of icIL-1Ra in the preIL-1α-transduced cells, supporting an intracellular site of action of preIL-1α. This is the first report of an association between intracellular levels of these IL-1 family members. We hypothesize that intracellular preIL-1α participates in the regulation of icIL-1Ra.
Identifying the long-term role of iNOS in contusive spinal cord injury (SCI) using a transgenic mouse model (100.14)
Authors: SINGH AMANPREET , Lopez Michael M , Lovera Lillie C , Maggio Dominic M , Flora Govinder , Dietrich Dalton W , Pearse Damien D
Published: January 2023

The inflammatory mediator iNOS is thought to play an important role in the pathophysiology of SCI. Specific inhibition of iNOS is beneficial after acute SCI, but little is known about its role over longer post-injury periods. The current study was carried out to assess the role of iNOS after contusive SCI through the use of chronic iNOS inhibition by a knockout mouse approach. Wild type (WT) and iNOS(−/−) mice were injured and then subjected to behavioral testing (BMS) and histological evaluation of healthy white and gray matter tissue volumes. Behaviorally, iNOS(−/−) mice displayed a significantly faster recovery of function compared to WT in open-field locomotion during the first 4 wk (WT 2.2±0.2, iNOS(−/−) 3.38±0.43; p < 0.05).At the endpoint of the experiment (8wk) however, iNOS(−/−) mice showed a trend towards worsened functional outcome. Histological analysis revealed that i-NOS(−/−) mice had a significant decrease in the volumes of both healthy gray (53%) and healthy white matter (15%) as compared to WT. The present study shows that acute inhibition of iNOS could be behaviorally beneficial after SCI, however, chronic inhibition likely hampers important wound healing functions of NO, leading to a lack of efficacy at later stages.

FUNDING: The Miami Project to Cure Paralysis, The Buoniconti Fund, The US Army Medical Research and Material Command
Adoptive therapy with allogeneic CD4+CD25+ Foxp3+ Treg cells induces transplantation tolerance (102.10)
Authors: Adeegbe Dennis O , Levy Robert B , Malek Thomas R
Published: January 2023

Alloantigen primed Treg cells offer the potential for non-toxic suppression of transplant rejection reactions. If they are not rejected by the recipient, MHC-mismatched Treg cells might also establish transplant tolerance to the MHC molecules that the Treg cells were selected on during their development. In this regard, we have shown that the adoptive transfer of allogeneic Treg cells into neonatal IL-2Rβ−/−mice, which lack Treg cells and develop rapid lethal autoimmunity, lead to long-term engraftment by the donor Treg cells, effective prevention of this autoimmunity, and simultaneous long-term tolerance to the skin grafts expressing alloantigens shared by the Treg cells. This tolerance depended upon active suppression by Treg cells, not deletion or anergy. Mice tolerant to allogeneic C57BL/6 skin grafts were also tolerant to MHC class II-deficient C567BL/6 skin grafts, but rejected bm3 and bm12 skin grafts. These latter findings demonstrate that Treg cells are not required to directly recognize alloantigens within the skin grafts and that a single neo-antigen was sufficient to break tolerance. Collectively, these data demonstrate induction of dominant donor-specific transplantation tolerance by allogeneic Treg cells provided that the MHC antigens of both the Treg cells and transplant are matched.

(Supported by the NIH)
Mutations to Bid cleavage sites protect hepatocytes from apoptosis following ischemia/reperfusion injury (102.23)
Authors: Krams Sheri M , Nagasaki Kazuhito , Esquivel Carlos O , Martinez Olivia M , Riddle Erica
Published: January 2023

Apoptosis of hepatocytes occurs during graft rejection, and ischemia/reperfusion injury (IRI). Hepatocytes require mitochondrial disruption to execute apoptosis, a process that is controlled by proteins of the Bcl-2 family. Bid is a proapoptotic Bcl-2 family member that is cleaved to its active form, tBid, by caspase 8 and granzyme B.

We demonstrate that FaO hepatoma cells, transduced with recombinant adenoviruses expressing Bid with mutations to the caspase 8 (D60E) and granzyme B (D76E) cleavage sites (AdBidEE), are significantly protected from both TNF-induced and cell-mediated apoptosis. Furthermore, rats expressing cleavage-resistant Bid in the liver display significantly less hepatocyte apoptosis and improved liver function as compared to control (AdLacZ) rat livers following warm IRI. All untreated and LacZ-expressing animals died within the first 24 hours post-IRI. In marked contrast, rats expressing AdBidEE (n=8) had significantly improved survival following IRI compared with AdLacZ-expressing rats (p = 0.0079, n=6), and have normal liver function at seven days.

We demonstrate that administration of AdBidEE protects hepatocytes from apoptosis and preserves liver function. Moreover, we propose that administration of AdBidEE may be useful in preventing apoptosis associated with warm ischemia in donors after cardiac death and thus increase the pool of deceased donors for transplantation.
Fas mediated cell death promotes cardiac allograft acceptance in T cell-NF-κB impaired mice (102.24)
Authors: Molinero Luciana Lorena , Wang Ying , Zhou Ping , Yagita Hideo , Alegre Maria-Luisa
Published: January 2023

The transcription factor NF-κB is critical for T cell activation and survival. We have shown that mice expressing a T cell-restricted NF-κB superrepressor (IκBαΔN-Tg) permanently accept heart but not skin allografts. Overexpression of the prosurvival factor Bclx-L in T cells restored heart rejection, suggesting that graft acceptance in IκBαΔN-Tg mice was due to deletion of alloreactive T cells. Accordingly, transplantation of OVA-expressing skin grafts resulted in division of adoptively transferred DO11.10/IκBαΔN-Tg T cells, whereas heart grafts induced T cell death. In vitro, Fas was responsible for the increased death of IκBαΔN-Tg T cells upon TCR stimulation compared to WT T cells. Similarly, Fas played a key role in cardiac allograft acceptance by IκBαΔN-Tg mice as both genetic and antibody-mediated inhibition of Fas signaling restored cardiac allograft rejection. Rejection correlated with graft infiltration by T cells and splenic production of IFN-γ upon allostimulation. These results indicate that T cell inhibition of NF-κB results in cardiac allograft acceptance because of increased susceptibility to Fas-mediated cell death.

Research support: NIH AI52352-03.
Synergistic control of T cell activation, regulatory T cell function, and autoimmunity by Diacylglycerol Kinases alpha and zeta (128.26)
Authors: Zhong Xiao-Ping , Guo Rishu , Carpenter Jeffery H
Published: January 2023

T cell receptor (TCR) signaling plays a critical role in T cell development, activation of conventional T cells, and induction of suppressive function of regulatory T cells (Treg). Abnormal TCR signaling can skew the T cell repertoire, induce dysregulated T cell activation, and lead to autoimmune diseases. How TCR signals control intrathymic T cell development, T cell activation, and Treg function is still not fully understood. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that catalyze the conversion of DAG to phosphatidic acid (PA) through phosphorylation. Both DAG and PA are second messengers involved in signaling from many receptors and both are generated following TCR stimulation. DAG and PA exert their activities by associating with and modulating the activities and subcellular localizations of numerous effector molecules. In T cells, both alpha and zeta isoforms of the DGK family are expressed. We have recently demonstrated that DGKalpha and zeta negatively regulate T cell activation, and that deficiency of either DGKalpha or zeta results in T cells that are hyper-responsive to TCR stimulation and resistant to anergy induction. Our current studies using DGKalpha and zeta doubly deficient mice have further demonstrated that these two DGK isoforms synergistically regulate thymic selection, T cell homeostasis, and Treg function to prevent autoimmune disease.
The defect in CD4+CD25− cell conversion into CD4+CD25+ regulatory cells in NOD mice can be rescued by treatment with "normal" antigen presenting cells (128.3)
Authors: Zhao Yuan , Alard Pascale , Parnell Sarah , Gross Eleanore , Kosiewicz Michele M
Published: January 2023

NOD mice are unable to convert CD4+CD25− cells into functional CD4+CD25+ regulatory cells in vivo. To analyze this defect, we reciprocally transferred CD4+CD25− cells into NOD and B6.g7 (B6 mice expressing NOD I-Ag7) mice. In NOD recipients, transferred B6.g7 CD4+CD25− cells were unable to convert, while in B6.g7 mice, transferred NOD CD4+CD25− cells converted as effectively as controls, suggesting that it may be the NOD APC that are defective in the conversion process. To test this, we co-transferred B6.g7 DC (APC) along with NOD CD4+CD25− T cells into NOD mice. Providing NOD mice with non-defective APC rescued their ability to convert. Interestingly, although Foxp3 expression did not, granzyme B expression did correlate with regulatory ability in converted CD4+CD25+ cells: NOD CD4+CD25− cells that upregulated CD25 after transfer into NOD mice expressed comparable Foxp3, but lower levels of granzyme B and were unable to regulate, whereas converted CD4+CD25+ cells from B6 controls or NOD mice treated with B6.g7 DC expressed high levels of granzyme B and were able to regulate. Taken together, these data suggest that the failure of NOD CD4+CD25− cells to convert into functional CD4+CD25+ regulatory cells in NOD mice may be due to a defect in the ability of NOD APC to induce granyme B expression in the converted cells.

Supported by NIH grant AI064421 and the KY Challenge for Excellence Fund.
A model to study the role of MHC-II dependent events in the suppression mediated by antigen specific CD4+ CD25+ FoxP3+ regulatory T cells (128.5)
Authors: Mmanywa Faith , Koni Pandelakis
Published: January 2023

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) play an important role in maintaining peripheral tolerance and preventing autoimmunity. In order to exert their suppressive functions, Tregs require TCR stimulation, both in vitro and in vivo. Upon stimulation they are able to suppress both CD4+ and CD8+ T cells in an antigen non-specific manner. The mechanism of suppression by Tregs is still not fully understood. Here we employ conditional MHC class II (MHC-II) deficient mice to test the hypothesis that MHC-II dependent events are involved in the suppressive function of antigen specific Tregs. To this end, we employ Foxp3+ CD4+ cells from ovalbumin-reactive OT-II TCR transgenic mice as Tregs and ovalbumin-reactive CD8+ OT-I TCR transgenic cells as responder cells. We demonstrate that antigen specific Tregs mediate suppression of CD8+ T cell proliferation and effector function in a MHC-II dependent manner. Therefore in this system the suppressive function of Tregs is MHC-II dependent.
Role of Foxp3 in CD8+ T regulatory/inhibitory cells ability to suppression of autoimmunity defined by mircoarray analysis and silencing of target genes after tolerization of BWF1 lupus mice with pConsensus peptide (128.6)
Authors: Singh Ram P , La Cava Antonio , Ebling Fanny M , Tamayo Alejandro M , Elashoff David , Sven De Vos , Patel Divyan , Rooney Robert J
Published: January 2023

NZB/NZW Fl female (BWF1) mice, a model of SLE, after tolerization with an artificial peptide (pCONSENSUS, pCons) that is 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, both of which suppress anti-DNA Ig production. In the present study, we analyzed 45,000 murine genes in a one- to- one comparison of white blood cells (WBC), CD4, and CD8 cell subsets from tolerized vs. non-tolerized mice. Results showed 448, 174 and 60 genes, respectively, that were differentially expressed by at least twofold. From the CD8+ T cells, we confirmed up regulation of several genes in cells from tolerized mice, including Foxp3, Trp-53, CCR7, IFI202B, bcl2, and IFNar1. Using tranfection of CD8+ T cells with siRNA for Foxp3, Trp-53 and CCR7, silencing of Foxp3 eliminated 60–100% of the suppressive capacities of CD8+ T cells from tolerized mice, as well as their ability to secrete TGFb. There was significant correlation between expression of Foxp3 and ability of CD8+ Ti cells to secrete TGFb. In contrast, silencing of Trp-53 and CCR7 did not abrogate suppressive capacity of these cells. In summary, suppressive capacity of CD8+ Ti in this system of tolerance depend in large part 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.
Viral infection enables CD4+CD25+ regulatory T cells to protect from autoimmune diabetes (128.9)
Authors: von Herrath Matthias , Oldham Janine , Christen Urs , Martinic Marianne , Togher Lisa
Published: January 2023

While viral infections may be capable of triggering autoimmune diabetes (T1D) in genetically susceptible individuals, accumulating evidence indicates that viruses can also prevent T1D. However, the underlying mechnisms are only partially understood. Here, we show that activation of CD4+CD25+ Tregs by acute lymphocytic choriomeningitis virus (LCMV) infection provides these cells with the capacity to prevent both spontaneous and virally induced T1D in the mouse. We found that CD4+CD25+ Tregs activated by LCMV in vivo or in vitro produce the regulatory cytokine TGF-b and are capable of protecting NOD and RIP-LCMV mice from spontaneous and LCMV-induced diabetes, respectively. Our results suggest that LCMV-modulated CD4+CD25+ Tregs act in a bystander fashion independent of beta-cell antigens and suppress TNF-a production by autoreactive CD8+ T cells, leading to prevention of T1D. These findings provide a novel mechanistic explanation for the previously reported ability of viral infections to prevent autoimmune diabetes. Furthermore, our results support a model explaining the differential ability of viral infections to modulate diabetes. Activation of Tregs during viral infections may be a general feature accounting for the ability of viruses to prevent T1D as well as other autoimmune disorders.
Differential production of reactive oxygen intermediates by TCR stimulation in SLE T cells (129.10)
Authors: Khan Islam U. , Michalek Ryan D. , Giri Banabihari , Grayson Jason M.
Published: January 2023

Systemic lupus erythematosus (SLE) is a protean autoimmune disease characterized by a wide array of intrinsic T cell defects. Reactive oxygen intermediates (ROI) modulate T cell activation, cytokine production, and proliferation at multiple levels. The purpose of this study was to measure T cell proliferation and ROI production in SLE versus healthy control T cells. CD4+ and CD8+ T cells were isolated by negative selection magnetic micro-beads. We characterized the memory phenotype and activation markers on the cells from both groups by flow cytometry. CD62Lhigh and CCR7high were the most abundant subsets in CD8+ T cells from healthy subjects. The frequency of CCR7highCD8+ T cells in SLE patients was significantly lowered compared to healthy subjects (p=0.0001). For proliferation and ROI studies, cells were stimulated with anti-CD3 and anti-CD28 antibodies. CFSE staining following activation revealed that SLE CD8+ T cells had decreased division in response to TCR-mediated stimulation as compared to healthy subjects. Both subsets of SLE T cells showed increased ROI in response to activation. Interestingly, among the SLE subjects, the maximum increase in H2O2 production was recorded in the CD8+ T cells of one patient with severe SLE disease. In conclusion, we demonstrate that T cells from SLE patients produce increased levels of ROI upon stimulation implicating ROI in SLE pathogenesis.

Supported by NIH RO1-AR39501
The Yaa locus Confers Apoptosis, NF-κB and Rapamycin Signaling Resistance Through Multiple Receptors in C57BL/6J Mice (129.12)
Authors: Brown Aaron C , Bubier Jason A , Sproule Thomas J , Christianson Greg J , Roopenian Derry C
Published: January 2023

BXSB/MpJ mice develop a spontaneous SLE-like disease that is remarkably accelerated in males, due to the presence of the Y-linked-autoimmune accelerator locus (Yaa). Recent insights into the molecular nature of the Yaa mutation were provided when an additional copy of 17 distally located X chromosome genes were discovered on the Y-chromosome of Yaa mice. Included within this region is the gene TLR7, a toll receptor which binds ssRNA and is functionally expressed on B-cells and monocyte derived lineages. Previous reports have suggested that the SLE-like disease phenotype conveyed by Yaa is due to the duplication of TLR7, which causes a shift toward anti-nucleolar antigens in B6.FcγRIIB(−/−). We show here that B6, BXSB and MRL mice that carry Yaa rarely display the anti-nucleolar antibody pattern. We found that B cells of Yaa mice display novel phenotypes consistent with increased rapamycin and NF-κB signaling through receptors other than TLR7, including TLR4, TLR9, and the BCR. In addition, Yaa B cells are resistant to anti-IgM induced apoptosis. These results demonstrate

that the copy number of a single receptor can influence the strength of signaling through other receptors and/orthat additional genes within the duplicated region contribute toward the Yaa phenotype.

Funded by NIH R21DK074463 and DK56597 to DCR, Arthritis Foundation to JAB.
LOSS OF RIP2 EXACERBATES MURINE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (129.16)
Authors: Fairhead Todd , Fenutria Rafael , McCully Michelle L. , Lemke Caitlin D. , Davis Brianne , Lozano Francisco , Strejan Gill , Madrenas Joaquin
Published: January 2023

Receptor interacting protein 2 (RIP2), a putative serine-threonine kinase, is a modulator of the activation of both innate and adaptive immune responses. RIP2-deficient mice have impaired NF-κB signaling and defects in Th1-mediated immune responses. Since activated NF-κB signaling in hematopoietic tissue and in the central nervous system (CNS) has been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we hypothesized that deficient expression of RIP2, a regulator of the NF-κB pathway, would affect the course of EAE.

To test this hypothesis, we induced EAE with MOG35-55 peptide, in C57Bl/6 wild type (WT) and RIP2−/− mice. We found that RIP2−/− mice exhibited exacerbated EAE as compared to WT mice, with both a higher clinical score and increased demyelination. Disease induction in both genotypes was T cell-dependent as demonstrated by adoptive T cell transfer. However, EAE in RIP2−/− mice did not have an accelerated course and did not show increased T cell priming ex vivo. Rather, the exacerbated EAE in RIP2−/− mice was associated with a higher percentage of IL-17-producing T cells, both in the periphery and in the CNS, while the percentage of IFN-γ-producing cells was the same in WT and RIP2−/− mice.

Our data demonstrate that RIP2 is a key in vivo regulator of Th17-associated autoimmunity in the CNS. Such a role is likely due to its activity on the NF-κB pathway.
Reduced expression of NKp46 on CD56dim natural killer cells from MS patients (129.21)
Authors: Edwan Jehad H , Bielekova Bibiana
Published: January 2023

Natural killer (NK) cells possess innate immune responses and play a crucial role in regulating autoimmune T cells. NKp46 is a natural cytotoxicity activating receptor exclusively expressed on NK cells. NKp46 plays a pivotal role in NK cell functions because it mediates the lysis of many human tumor cells lines, as well as infected cells. We investigated the expression of NKp46, CD62L and CD44 on NK cells from Multiple Sclerosis (MS) patients and healthy donors (HD). Apheresis blood samples were obtained from MS patients and HD, PBMCs were separated by density gradient centrifugation with lymphocytes separation medium. NK cells were negatively selected using MACS NK isolation kit, and cultured with or without 1 μg/ml influenza vaccine (Flu HA) for 4 hours. Using 6 color flow cytometry, we examined the CD56+ CD3− cells for the expression of NKp46, CD62L and CD44. We observed that MS patients’ NK cells express unusually low frequency of CD56dim CD44+ NKp46+ cells compared to HD. The levels of CD56bright cells expressing CD44+ CD62L+ NKp46+ were comparable among MS patients and HD. Four hours after incubation with Flu HA, the two CD56dim and CD56bright populations expressing NKp46+ completely disappeared leaving one population expressing only CD56dim CD44+. Our findings suggest that CD56dim CD44+ NKp46+ might be involved in the protection against development of MS.
IFNγ-dependent mechanisms of activation-induced cell death control the progression of autoimmune myocarditis (129.32)
Authors: Barin Jobert G. , Cihakova Daniela , Kimura Miho , Talor Monica V. , Rose Noel R.
Published: January 2023

Interferon gamma (IFNγ) exerts a suppressive effect on experimental autoimmune myocarditis (EAM) in mice, as well as other models of organ-specific autoimmune disease. We have sought to determine the mechanisms by which IFNγ mediates this effect, which may involve the apoptosis of autoreactive lymphocytes by activation-induced cell death. IFNγ knockout and wild type control BALB/c mice were immunized with the cardiac myosin-derived peptide MyHCα614-629 to induce EAM. At day 21 post-immunization, we observed a marked decrease in the activity of Caspase 8 in splenic CD4+ T cells in IFNγ−/− mice with EAM. We further found that this decrease in CD4+ cell Caspase 8 activity could be recapitulated in an antigen-specific manner in vitro by stimulation of spleen cells from IFNγ−/− mice with the MyHCα614-629 peptide. These differences in Caspase 8 activity are paralleled by decreases in the ability of MyHCα614-629-reactive CD4+ cells from IFNγ−/− mice with EAM to apoptose after in vitro stimulation with the peptide. These data support the hypothesis that IFNγ acts through regulating apoptotic mechanisms in autoreactive lymphocytes to limit the progression of autoimmune disease.

(Supported by 2R01 HL67290)
CD24 inhibits thymic deletion of myelin antigen-specific T cells (129.34)
Authors: Carl Joseph W , Joshi Pramod , Whitacre Caroline C , Liu Yang , Bai Xue-Feng
Published: January 2023

CD24 is a glycosylphosphatidylinositol (GPI) anchored cell surface glycoprotein that is expressed in hematopoietic cells and cells of the central nervous system (CNS). We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model of human disease multiple sclerosis. In CD24−/− mice, normal levels of myelin oligodendrocyte glycoprotein (MOG) specific T cell were primed, however these T cells were non-pathogenic. To understand this issue, we bred CD24−/− mice with 2D2 TCR transgenic mice, which bear TCR specific to MOG, and generated 2D2 TCR transgenic mice with or without CD24. Here we show that 2D2 TCR transgenic mice with CD24-deficiency (2D2/CD24−/−) have remarkably withered thymus. In peripheral lymphoid organs, transgenic T cells from 2D2/CD24−/− mice have an immature phenotype (CD4−CD8−), do not respond to MOG peptide stimulation, and fail to cause autoimmune inflammation in the CNS and optical nerves. In contrast, OT-2 TCR transgenic mice with CD24 deficiency (OT-2/CD24−/−), which bear TCR specific to chicken ovalbumin (OVA), have normal thymus and their peripheral T cells have a normal response to OVA peptide. These data suggest that CD24 inhibits thymic deletion of myelin antigen, but not foreign antigen-reactive T cells.
The LFA1-ICAM2-ezrin-Akt pathway in mediating fibroblast-like synoviocyte proliferation in rheumatoid arthritis (129.40)
Authors: SINGH KARNAIL , COLMEGNA INES , HE XIAOWEN , WEYAND CORNELIA M , GORONZY JORG J
Published: January 2023

In rheumatoid arthritis (RA), the synovium is infiltrated by mononuclear cells that influence the proliferation and activation of fibroblast-like synoviocytes (FLS) through soluble mediators as well as cell-cell contact. To identify receptor-ligand pairs involved in this cross-talk, we cultured T cells with FLS lines isolated from RA synovium. T cells induced increased phospho-Akt (Ser473) in FLS, leading to downstream phosphorylation of GSK-3, FoxO1/3a and MDM-2 and enhanced in vitro FLS proliferation. Akt phosphorylation and enhanced FLS proliferation did not require MHC/antigen recognition, but were inhibited by antibodies against ICAM-2, 3 and CD11a. Antibodies to ICAM-1 were not effective. The selective role of the LFA-1 - ICAM-2/3 pathway was confirmed by the finding of increased ezrin phosphorylation at Tyr353 which is known to be downstream of ICAM2/3 and supports cell survival through Akt activation. CD28- T cells, which were present in significantly higher frequencies in RA patients than in healthy controls, had higher CD11a cell surface expression than naïve or regular memory T cells and were more potent to induce Akt phosphorylation in FLS. These findings identify ICAM-2/3 as therapeutic targets in RA allowing for a more selective intervention than the LFA-1 inhibition explored in clinical studies.

(Supported by NIH grants AR41974, AR42527 and AI44142).
Immunohistochemical detection of autoantibodies in the plasma of patients with Autism to neural cells of the non-human primate (129.42)
Authors: Wills Sharifia , Cabanlit Maricel , Bennett Jeff , Ashwood Paul , Amaral David , Van de Water Judy
Published: January 2023

Autoantibodies directed to various ‘self’ neural proteins have been reported in a subgroup of subjects with autism spectrum disorders (ASD) and are proposed to contribute to abnormal neurodevelopment. Our group sought to identify the presence of autoantibodies to brain protein(s) in the plasma of subjects with ASD enrolled in a population based case controlled study. Groups examined include patients with ASD, typically developing age-matched controls, siblings of children with ASD, and children with other developmental disabilities. Immunohistochemical staining of coronal brain sections obtained from the cynomolgus monkey (Macaca fascicularis) was performed to identify specific cellular targets against which the ‘self’ reactive antibodies are directed. Immunohistochemical analysis has thus far been confined to the cerebellum. Strong immunoreactivity was noted in the plasma from 20% of children with autism towards what appear morphologically to be golgi cells in the granular layer of the cerebellum. Such staining was absent in plasma from the typically developing controls, but present in two siblings and one disease control. Identification of the brain-specific autoantigens will help illuminate which brain structures and/or neurodevelopmental processes may be affected in patients with ASD.

[These studies were funded by NIEHS grant 1 P01 ES11269-01 and the M.I.N.D. Institute.]
Activated Insulin-Reactive CD8 T cells in NOD mice Cause Diabetes. (129.46)
Authors: Wong F. Susan , Siew Lai Khai , Thomas Ian J. , Chapman Stephen , Viret Christophe , Wen Li
Published: January 2023

We have previously generated highly diabetogenic insulin-reactive CD8 T cell clones designated G9C8 from the NOD mouse. The aim of our study was to investigate T cell selection and activation of CD8 T cells from a TCR transgenic mouse expressing the cloned TCRαβ receptor of the G9C8 clone. The mice were crossed to TCR.Cα−/− mice so that all the T cells express the clonotypic TCR. There was a predominance of CD8+ single positive thymocytes and peripheral CD8 TCR transgenic cells, compared with CD4 T cells. Peripheral lymph node T cells express a naïve phenotype (CD44lo, CD62Lhi). They proliferate to the insulin B15-23 peptide, produce interferon-γ and are cytotoxic to targets coated with the insulin peptide. In vivo, the TCR transgenic mice developed insulitis but surprisingly spontaneous diabetes did not occur. However, the TCR transgenic mice can be induced to develop diabetes on immunization. They will also cause diabetes on adoptive transfer after activation. In summary, these mice provide a good model for studying selection and activation of insulin-reactive CD8 T cells in the NOD mouse.
Bim−/−Faslpr/lpr Mice Exhibit Accelerated Lymphadenopathy (130.13)
Authors: Weant Ashley E , Khan Islam U , Holbrook Beth C , Willingham Mark C , Grayson Jason M
Published: January 2023

Apoptosis plays a critical role in the development and homeostasis of the immune system. To determine whether loss of genes in both extrinsic and intrinsic cell death pathways would exacerbate defects in immune homeostasis we created Bim−/−Faslpr/lpr mice. Mice were born at the expected Mendelian ratios and appeared to develop normally. However, by 14 weeks of age they developed a massive lymphadenopathy compared to either single mutant or wildtype animals. Their kidneys contained massive lymphocyte infiltration compared to the other genotypes. We determined the apoptotic sensitivity of T cells from these animals and found that naïve T cells cultured in vitro were resistant to growth factor deprivation similar to the Bim mutants and activated T cells were resistant to activation induced cell death similar to the Faslpr/lpr mice. We next determined the ability of these cells to divide following TCR stimulation. Upon stimulation, CD8+ T cells from these animals had slightly decreased proliferation than cells from the other genotypes. However, the percentage of cells surviving through division is increased in the double mutant animals. In conclusion, although T cells from Bim−/−Faslpr/lpr mice exhibit similar in vitro survival as either parental strain, in vivo the number of lymphocytes is synergistically increased resulting in massive lymphadenopathy.

Supported by ACS- #RSG-04-066-01-MBC to Jason Grayson.
Activation of hepatic stellate cells during fibrosis: Comparison of the CYP2D6 model for autoimmune hepatits and CCl4 injection (130.2)
Authors: Christen Urs , Hintermann Edith , Christen Selina , Bayer Monika
Published: January 2023

Only little is known about the mechanisms of periportal fibrosis in the pathogenesis of human autoimmune hepatitis. We used the virus induced CYP2D6 model system to investigate the activation of hepatic stellate cells (HSC) and the kinetics of fibrosis in comparison with the CCl4-induced fibrosis model. CYP2D6 transgenic mice express the human Cytochrome P450 in the liver and develop liver damage upon Adenovirus-CYP2D6 infection. In the CYP2D6 model we found mostly subcapsular fibrosis resulting in the fusion of individual lobules (Sirius Red, Collagen I). At 10–12 weeks post-infection, weak periportal fibrosis became apparent. In contrast, in CCl4-treated mice the kinetic of extracellular matrix deposition was accelerated resulting in periportal fibrosis after 3–4 week of CCl4 administration. At later times, fibrosis was much more pronounced in CCl4-treated mice compared to virus-infected CYP2D6 mice but subcapsular fibrosis was not as dominat. Activation of HSCs could be detected by staining for α-smooth muscle actin (αSMA) in liver sections of both CCl4-treated mice and virus-infected CYP2D6 mice. In addition, isolation of HSCs revealed an enhanced activation status (decreased amount of oil droplets, de novo αSMA expression) in CCl4-treated mice and virus-infected CYP2D6 mice. Our data indicate that virus-infected CYP2D6 mice display subcapsular and periportal fibrosis that correlates with an activation of HSCs similar to CCl4-induced fibrosis. Thus, the CYP2D6 mouse is a good model system to further investigate the molecular mechanisms involved in fibrotic events during autoimmune hepatitis.
Chronic low-grade exposure to bacterial superantigens elicits sustained T cell expansion and multiorgan inflammation. Implications for autoimmunity. (130.22)
Authors: Rajagopalan Govindarajan , Smart Michele K , Grande Joseph P , David Chella S
Published: January 2023

Bacterial superantigens (BSAg) are produced by Staphylococcus aureus and Streptococcus pyogenes. BSAg bind directly to the MHC class II molecules without undergoing any processing and subsequently activate T cells in an antigen non-specific manner by interacting with the TCR Vβ region. BSAg have been implicated primarily in acute diseases such as food poisoning, toxic shock syndrome, etc. Since carrier strains of S. aureus and S. pyogenes can elaborate superantigens, there is a distinct possibility of chronic exposure to low doses of BSAg and it is imperative to study its outcome. As BSAg bind more avidly to human (HLA) than to murine (H-2) MHC class II, we delivered low concentrations (total of 10 μg/mouse) of BSAg to HLA-DR3 [staphylococcal enterotoxin (SE) B] or HLA-DQ8 [streptococcal pyrogenic exotoxin (SPE) A] transgenic mice through subcutaneously implanted miniosmotic pumps for 7 days.

Continuous exposure to low concentrations of BSAg resulted in a significant protracted expansion of CD4+ T cells expressing TCR Vβ8 (for SEB); Vβ8 and Vβ14 (for SPEA) with a concomitant decrease in percentage of CD8+ T cells expressing these TCR. T cells bearing TCR Vβ6 showed an inverse correlation (slight decrease in CD4+ and increase in CD8+ compartments with SEB and SPEA). Histological examinations of major organs showed intense perivascular infiltration in lungs, kidneys and liver along with inflammatory infiltrates in the cardiac valvular endothelium in mice bearing pumps delivering BSAg but not PBS. In conclusion, we have shown the immunological outcome of chronic exposure to low levels of BSAg using the HLA class II transgenic mouse model.
Generation of T-cell Hybridomas Specific for Bile Duct and Rhesus Rotavirus Antigens in a Murine Model of Biliary Atresia (130.27)
Authors: Tucker Rebecca , Gill Ronald , Mack Cara
Published: January 2023

Rhesus rotavirus (RRV)-inoculated neonatal BALB/c mice develop an immune mediated inflamation of both extra- and intraheptatic bile ducts which progresses to biliary obstruction and death by 3 weeks of age. The livers of diseased animals demonstrate increased numbers of T-lymphocytes along with elevated expression of Th1 type cytokines at both one and two weeks. Previous work in our laboratory has demonstrated that the liver infiltrating T-lymphocytes are both RRV-reactive and autoreactive to bile duct antigens. In addition, transfer of T-cells from diseased animals leads to inflamation of bile ducts in SCID mice. This inflamation occurs in the absence of virus, suggesting that bile duct antigens alone are capable of soliciting this response. In order to identify the antigens being targeted, and to determine if the autoreactivity arises via an epitope spreading or molecular mimicry mechanism, we have generated T-cell hybridomas from the livers of 2 week old diseased mice. Initial analysis of these hybridomas reveals four classes of hybridomas.

TCR positive but non-reactive to tested antigens,RRV-specific,bile duct-specific, anddual reactive to both RRV and bile duct antigens.

Further study of these hybridomas should clarify what mechanism is occuring and assist in identification of autoantigens.
Reduction in TCR expression limits the response of myelin specific T cells (131.11)
Authors: Wasserman Heather Anong , Evavold Brian D
Published: January 2023

Previous studies have found that a 95% reduction in TCR expression does not adversely affect response to foreign antigen specific T cells. Since self-reactive T cells may have low affinity for self-antigen, we investigated whether myelin reactive T cells have lower affinity TCR for their specific ligand and are sensitive to a reduction in TCR expression. To test this concept, a MOG-specific Vα3.2 and Vβ11 TCR transgenic mouse model (referred to as 2D2 mice) is employed in our in vitro studies. Autoreactive MOG specific CD4+ T cells pre-incubated with Vα3.2 Ab have a dramatic dose-dependent reduction in proliferation. Quantitation of T cell receptor expression found that there are a maximum of 25,000 TCR per cell. A reduction of TCR expression by 31% led to a 50% reduction in proliferation, whereas a 68% TCR expression reduction correlates in a 67% reduction in proliferative response. Additionally, 2D2 cells cultured with Vα3.2 Ab and MOG 35-55, and then restimulated with MOG 35-55 also have a dose-dependent reduction in proliferation, suggesting that Ab treatment may lead to a possible phenotypic change resulting in T cell anergy. Therefore, TCR expression may provide a potential therapeutic window, which one can use to distinguish foreign antigen specific T cells from self-reactive T cells and allow for inhibition of autoimmunity without inducing global immune suppression.

This research was supported by NIAID (RO1 AI056017) and NMSS (RG 3683) grants
Expansion of GAD65–Specific Immunoregulatory Effector Cells by Biolistic-Mediated Gene Delivery Prevents Autoimmune Diabetes in NOD Mice (131.14)
Authors: Goudy Kevin , Li Li , Tisch Roland
Published: January 2023

Intramuscular (i.m.) vaccination with plasmid DNA (pDNA) encoding beta cell autoantigens such as glutamic acid decarboxylase 65 (GAD65) is an approach to suppress ongoing Type 1 diabetes (T1D) in NOD mice. However, protection is dependent on co-treatment with pDNAs encoding IL-4 and/or IL-10, and the subsequent induction of type 2 CD4+ T effectors. To enhance the therapeutic efficacy of pDNA vaccination, we explored epidermal delivery of pDNA via “gene gun”. Notably, reports have shown induction of a preferential type 2-like CD4+ T cell response when pDNA encoding antigen-only is delivered to the epidermis via gene gun. We hypothesized that biolistic delivery of pDNA would preferentially induce type 2 CD4+ T effectors and suppress established beta cell autoimmunity in NOD mice. Groups of 10 wk-old NOD female mice were treated four times with pDNA encoding GAD65-Ig (pGAD65) administered i.m. or delivered by gene gun on 1.6 uM gold particles. Whereas i.m. injection of pGAD65 resulted in preferential induction of GAD65-specific CD4+ Th1 cells and no protection against T1D, the majority of NOD mice treated with gene gun delivered pGAD65 remained diabetes-free and protection correlated with increased GAD65-specific IL-4 secreting CD4+ T cells. These results demonstrate that gene-gun delivered pDNA encoding beta cell autoantigen-only is an effective strategy to induce immunoregulatory CD4+ T cells and suppress T1D.
Pre-Ligand Assembly Domain (PLAD) is an important therapeutic target in inflammatory arthritis (131.21)
Authors: Deng Guo-Min , Lenardo Michael
Published: January 2023

Tumor necrosis factor (TNF)-α is an important proinflammatory cytokine and plays a crucial role in pathogenesis of inflammatory arthritis such as rheumatoid and septic arthritis. TNF-α exerts its effect by binding to the extracellular domain of TNF receptor (TNFR) 1 and 2. The amino-terminal portion of the TNFR extracellular domain, known as cysteine rich domain 1 (CRD1), or the pre-ligand binding assembly domain (PLAD), plays an important role in the TNFR signaling pathway. Our studies demonstrate that soluble TNFR1 and TNFR2 PLAD proteins could block the actions of TNF-α, the TNFR1 PLAD protein could significantly inhibit inflammatory arthritis such as arthritis induced by TNF-α, bacterial DNA, lipopolysaccharide and collagen. The TNFR1 PLAD protein could inhibit nuclear factor (NF)-κB activation and osteoclastogenesis triggered by TNF-α. The TNFR1 PLAD protein also could suppress expression of receptor activator of NF-κB (RANK), RANK ligand and TNFR1 in arthritic joints. To understand the mechanism of the PLAD protein inhibited inflammatory arthritis, we further studied how TNFR PLAD protein blocks the effect of TNF-α and inflammatory arthritis. Our data demonstrate that the TNFR PLAD may directly bind to TNFR and block relationship of TNF-α binding to TNFR. The TNFR1 PLAD protein could suppress the expression of matrix metalloproteinase and inducible nitric oxide synthase in collagen-induced arthritis. Our research findings indicate the TNFR1 PLAD protein may offer a new approach to inhibit the pathogenic effects of TNF-α, and may be a good prototype for a new generation of pharmaceuticals against TNFRs that could be useful in treatment of inflammatory arthritis.
Sphingosine 1 phosphate (S1P) -Type 1 Sphingosine 1-Phosphate G Protein-Coupled Receptor (S1P1) axis promotes peripheral generation of CD4+CD25+ regulatory T cells (131.5)
Authors: Wang Wengang , Goetzl Edward J
Published: January 2023

Regulatory T cells (Tregs) are a CD4+CD25+ subset of T cells that control normal immunity and autoimmunity by suppressing diverse functions of other immune cells. S1P regulates many immune functions via S1P1 and occasionally through another of its five receptors. The generation of Tregs from CD4+CD25− T cells in non-thymic lymphoid organs may be evoked by T cell activation and the actions of certain immune regulators, such as TGF-beta and IL-4. Using flow cytometry, we found there were1.4–1.8 fold more Tregs in CD4+ T cells of spleen, lymph node, and blood from S1P1 TG mice than in wild-type (WT) mice. In contrast, the numbers of Tregs in the CD4+ T cells from thymus of WT and S1P1 TG mice were the same. Cell proliferation assays using CD4+CD25− effector T cells as targets indicated that overexpressed Tregs in S1P1 TG mice had similar suppressive effects per T cell as Tregs in WT mice. In vitro assays showed around 5% of isolated CD4+CD25− T cells from S1P1 TG mice were converted to CD4+CD25+ Tregs by TCR stimulation or antigen activation in the presence of 10−7 M S1P. Real time PCR and flow cytometry assay showed the induction of FoxP3 mRNA and protein expression in these peripherally generated Tregs. Increased peripheral generation of Tregs by the S1P-S1P1 axis may alter the level of immune and autoimmune reactions sufficiently to make this system a therapeutic target for immunomodulatory drugs.