bluuebunny/crossref_metadata_2025 huggingface dataset:
DISPLAY OF A NOVEL FORM OF FasL PROTEIN ON ALLOGENEIC ISLETS RESULTS IN PROLONGED GRAFT SURVIVAL (102.13)
Authors: Shirwan Haval , Zhao Hong , Lacelle Chantale , Askenasy Nadir , Yolcu Esma S.
Published: January 2023

Manipulation of donor organs, rather than the recipients, for tolerance induction represents an exciting approach. We have recently developed a novel approach designated as ProtEx™ that allows for the generation of chimeric immunological ligands with core streptavidin (SA) and their display on the biotinylated cell surfaces in a rapid and efficient manner. Chimeric proteins have potent immunological activities, possibly due to their ability to form tetramers and higher structures. We generated a chimeric form of FasL molecule (SA-FasL) with potent apoptotic activity and previously demonstrated that this protein can be displayed on the surface of donor splenocytes in a rapid and efficient manner, and such cells when co-transplanted with islets prevented their rejection in allogeneic hosts. We herein tested if islets can directly be engineered to display SA-FasL on their surface and if such islets become refractive to rejection in allogeneic hosts. BALB/c islets were biotinylated, decorated with SA-FasL, and transplanted into syngeneic or allogeneic C57BL/6 hosts with STZ-induced diabetes. SA-FasL-decorated islets had no observed toxicity and normalized blood glucose levels in 100% of syngeneic hosts. Transplantation of SA-FasL-decorated BALB/c islets into C57BL/6 resulted in prolonged graft survival with >20% of grafts surviving indefinetly. All the islets decorated with the control SA protein were rejected in a normal tempo. Engineering pancreatic islets for the display of SA-FasL in a rapid and efficient manner represents a practical and safe alternative to gene therapy for immunomodulation. NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), JDRF (1-2001-328), and ADA (1-05-JF-56.
A Soluble Factor from Mesenchymal Stem Cells Suppress T cell Proliferation by Induction of IL-10 Production from T cells (102.16)
Authors: Yang Seung-Ha , Yun Il-Hee , Park Chung-Gyu
Published: January 2023

Mesenchymal stem cells (MSCs) have immune modulation effect on T cells. The underlying mechanism is not fully understood. In this study, we investigated the mechanism of immune modulation effect of MSCs on T cell proliferation and its effector function. MSCs from BALB/c and C57BL/6 were identified by surface marker (CD106+, CD45−, CD31−, CD34−, CD11c−, and CD14−) and their differentiation capacity into adipocyte and osteoblast. MSCs from BALB/c expressed neither MHC class I nor II molecules whereas those from C57BL/6 expressed only MHC class I molecule. The co-culture of irradiated MSCs in mixed lymphocyte reactions (MLRs) PMA & ionomycin stimulated T cell resulted in the suppression of T cell proliferation in a dose-dependent manner. In addition, the same suppressive effect was observed on priorly activated T cells. The transwell assay, where T cells were separated from MSCs, showed comparable reduction of T cell proliferation suggesting the role of soluble factors from MSCs on the suppressive effect. The inhibition of T cell proliferation by the addition of MSCs culture supernatant in MLRs confirmed a soluble factor as a T-cell-inhibitory effector mechanism ELISPOT assay demonstrated the addition of MSCs supernatant in MLR markedly increased IL-10(+) cells. Furthermore, T cell proliferation was partially recovered by IL-10R neutralizing antibody. Taken together, these results suggest that IL-10 from activated T cells in the presence of MSCs culture supernatant would be one of the mediators of T cell suppression by MSCs.

This study was supported by a grant of the Korea Health 21 R & D Project, Ministry of Health & Welfare, Republic of Korea. (Project No. : A040004)
Male microchimerism in peripheral blood of women awaiting kidney transplantation correlates positively with HLA allo-immunization (102.19)
Authors: Albano Laetitia , Meynet Ophélie , Cassuto Elisabeth , Bernard Alain , Bernard Ghislaine
Published: January 2023

Microchimerism is the persistence of a small population of donor or fœtal cells, in graft recipients or in mothers. It appears after transfusion, pregnancy, transplantation and has been implicated in graft rejection, tolerance and auto-immunity. Anti-HLA Ab can appear under identical circumstances. Why and how such Ab persist for decades is only partially understood. We questioned if male microchimerism in the peripheral blood of women could be one of the mechanisms allowing the maintenance of anti-HLA Ab production. We evaluated 46 women, waiting for a first kidney graft, for the presence or absence of anti-HLA Ab using an ELISA. A Panel Reactive Ab was determined using Luminex® technology. Y chromosomal DNA was identified by nested PCR using specific primers of the SRY region. All patients were pregnant and/or transfused. In the Ab+ group (n=24), all women were sensitized and in the Ab− group (n=22), all women were unsensitized. Thirty-nine percent of all women (18/46) carried a male microchimerism: 54% in the Ab+ group (13/24) vs. 22% in the Ab− group (5/22) with pχ2= 0.029. We found a significant frequency of male microchimerism in the peripheral blood of women with kidney disease, which positively correlated with the presence of anti-HLA Ab.
MyD88 is required for the Differentiation of Recipient Monocytes into Dendritic Cells and Macrophages in Orthotopic Lung Grafts (102.2)
Authors: Gelman Andrew E , Kreisel Daniel
Published: January 2023

In lung grafts ischemia-reperfusion (I/R) signals rapidly induce the recruitment and differentiation of host monocytes into macrophages and dendritic cells. The nature of these I/R signals are antigen-independent but have been hypothesized to initiate Toll-like receptor (TLR)-mediated signaling pathways that are thought to potentiate alloimmune responses. We asked if MyD88, an adaptor molecule critical for most TLR-mediated inflammatory responses, regulated recipient monocyte differentiation in a mouse model of orthotopic lung transplantation. Orthotopic left lung transplants were performed in the following syngeneic combinations: CD45.1+ B6 → CD45.2+ MyD88−/− and CD45.1+B6 → CD45.2+ B6 (WT). 24 hrs later recipient-derived dendritic cell (DCs: CD45.2+ CD11c+CD11b+IAblo–hi) and macrophage (Macs: CD45.2+ CD11c−CD11b+ IAblo) numbers were assessed in bronchial lavage (BAL) by FACS analysis. Compared to the BAL of WT recipients MyD88−/− recipients had lower mean numbers of Macs (535/ml vs. 1419/ml) and DC’s (651/ml vs. 1763/ml) of recipient origin. Moreover, airway DC’s of lung grafts in MyD88−/− recipients expressed lower levels of IAb, CD40 and CD86 than did their WT counterparts. We also analyzed monocyte differentiation in allogeneic lung recipients by comparing the fate of adoptively transferred CD45.2+CD11b+IAb−Ly6ChiGr1− bone marrow monocyte precursors (MPs) from either MyD88−/− or WT mice in CD45.1+B6 mice that received Balb/c lung grafts. As in the syngeneic graft combinations, adoptively transferred MyD88−/− MPs were similarly defective at differentiation into DCs and macrophages in allografts. In conclusion the data demonstrates that MyD88 regulates recipient monocyte differentiation and suggests a mechanism by which TLR signaling pathways control adaptive responses in lung allografts.
Pre-sensitized B-cells prevent anti-CD154 mediated cardiac allograft tolerance (102.21)
Authors: Troutman Audrea Melody , Ma Lianli , Ding Jinwen , Shen Jikun , Chong Anita
Published: January 2023

Memory T-cells have been identified to be a significant cause of costimulatory resistant rejection; however, the role of pre-sensitized B cells has yet to be elucidated. We have developed a heart transplant model that specifically generates allospecific memory B-cells without generating allospecific memory T-cells. Pre-sensitized 3.83 B-cells with dual-specificity for H-2Kk and H-2Kb were generated in 3.83 BCR (BALB/c background) knock-in mice that received C3H (H-2Kk) hearts. To test whether pre-sensitized 3.83 B-cells can prevent tolerance induction by α-CD154 treatment, C3H-primed 3.83 recipients (30–90d post-C3H heart transplant) received a second heart and treatment with α-CD154. Second B/6 hearts (H-2Kb), which can be recognized by the pre-sensitized 3-83 B-cells, were acutely rejected within 10–13 days despite α-CD154 therapy. To confirm the importance of the pre-sensitized B cells, we transferred only B-cells or T-cells from C3H-presensitized 3.83 mice into naïve BALB/c recipients treated with α-CD154. Only transferred B-cells, but not T-cells, abrogated the tolerogenic effects of α-CD154. Histological examination of the rejected grafts from pre-sensitized 3-83 recipients treated with α-CD154 revealed significant numbers of infiltrating T cells. Also, B/6-specific IFNγ-producing T cells were detected in the pre-sensitized 3.83 recipients treated with α-CD154, but not in similarly treated naïve 3-83 recipients. We conclude that pre-sensitized B-cells are sufficient to prevent tolerance induction by permitting the CD40-CD40L independent activation of alloreactive T cells.

Funding source: NIH Immunology Training Grant.
Prolonged Survival of Vascularized Skin Grafts Across a Full MHC Mismatch Using Donor Antigen-Pulsed Tolerogenic GM+Rapa Dendritic Cells (102.22)
Authors: Feili-Hariri Maryam , Horibe Elaine , Sacks Justin , Unadkat Jignesh , Wang Zhiliang , Raimondi Giorgio , Ikeghchi Ryosuke , Thomson Angus , Lee WP , Marsteller Douglas
Published: January 2023

We compared the tolerogeneic properties of BM-derived GM+IL-4 and GM+Rapa host DCs and their potential to induce long-term acceptance of vascularized skin allograft in rats across a full MHC mismatch with transient immunosuppression. Results show that both DCs were CD11b+ and expressed low levels of CD86 and produced low levels of IL-12p70 following TLR activation. GM+Rapa DC produced lower levels of anti- and pro-inflammatory cytokines in response to LPS. Both DCs had low T cell stimulatory capacity in vitro. Interestingly, donor Ag-pulsed GM+Rapa DC (GM+Rapa DCp) induced long-term survival of the vascularized skin grafts and this was significant compare to GM+IL-4 DC and control groups. PBMC from long-term graft survivors that were previously treated with donor Ag-pulsed GM+Rapa DC were hyporesponsive to donor Ag, but proliferated in response to the third-party Ag and produced IL-10 in addition to IFN-gamma. Recipients of the long-term surviving allografts were challenged with full-thickness tail skin grafts. While the third-party grafts were rejected fast the donor graft had delayed rejection, suggesting that this treatment induced a regulatory mechanism that prevented the acute rejection of donor skin grafts. Further experiments demonstrated presence of CD4+ Foxp3+ T cells in the vascularized skin and secondary lymphoid tissues. Taken together, these results demonstrate that the donor Ag-pulsed tolerogenic GM+Rapa DC have ability to induce regulatory T cells and prevent vascularized skin graft rejection across a full MHC mismatch.
The Superdominant Minor Histocompatibility Antigen (MHAg) H60 Recognized by CD8 T Cells Fails to Elicit Lethal Graft Versus Host Disease (GVHD) Regardless of Its Tissue Expression Pattern (102.26)
Authors: Al-Awwami Moheeb , Sproule Thomas , Al-Khabbaz Hana , Roopenian Derry
Published: January 2023

Studies both in mice and humans have argued that immunodominant MHAg eliciting CD8 T cells cause increased risk for GVHD. We have shown that the H60 MHAg is remarkably immunodominant in that an overwhelmingly predominant CD8 T cell response developed after the transfer of T cells and bone marrow (BM) into MHAg-mismatched lethally irradiated recipients is directed against H60. Here we address whether H60 is a barrier to lethal (L) GVHD. Matching of donor cells and recipients for H60 while remaining disparate for all other MHAg failed to result in improved survival, indicating that H60 is not a barrier for LGVHD. Expression of H60 is normally limited to hematopoietic tissues, possibly explaining H60’s inertness in LGVHD. To broaden its expression, we produced transgenic mice expressing H60 driven by a tissue-ubiquitous cassette. We then evaluated whether H60 expressed broadly in recipients resulted in GVHD. Naïve B6 T cells transferred into B6-H60 transgenic or congenic mice failed to show any evidence of GVHD regardless of a robust anti-H60 CD8 T cell response. Augmentation of the response to H60 by preimmunization, resulting on over 50% of the CD8 T cell compartment being H60-specific in GVHD target organs, an failed to result in LGVHD. The overall results are inconsistent with a recent report (B. Kappel, et al. Blood, 2006.107:2045) and clearly indicate that potent CD8 T cell responses to MHAg are remarkably inert in GVHD.
Host conditioning is not required for generation of fibrotic graft-versus-host disease (102.5)
Authors: Askew David , Zhou Lixin , Gilliam Anita
Published: January 2023

Graft versus host disease (GVHD) is a major complication of hematopoietic cell transplant. One major form of GVHD is manifested by destruction of epithelial tissue in skin, liver and gut. This cytotoxic GVHD is thought to be initiated by the conditioning regimen (irradiation and/or chemotherapy) that is required to ablate the host immune system. In a model of GVHD that occurs in a minor histocompatability mismatch (B10.D2 → Balb/c), instead of cytotoxic GVHD, fibroblasts are stimulated to overproduce collagen resulting in fibrosis in the skin and lung. In earlier studies we found cutaneous TNFα-producing CD4+ T cells could be found in mice undergoing cytotoxic GVHD, but not in fibrotic GVHD.

To determine the relationship between host conditioning, TNFα production and type of GVHD generated, we used Balb/c Rag 1 knockout mice (Balb/c Rag1−/−) as recipients to generate GVHD in the absence of host conditioning. We found fibrotic GVHD in both B10.D2 → Balb/c Rag1−/−, and C57BL/6 → Balb/c Rag1−/−. This was surprising as C57BL/6 → Balb/c generates cytotoxic GVHD using standard BMT. Cutaneous TNFα-producing CD4+ T cells were again absent in the skin during GVHD. Thus, in some mouse strain pairs, while host conditioning may influence the development of TNFα-producing T cells that target skin and other organs leading to cytotoxic GVHD, other triggers promote the development of fibrotic GVHD.
Dendritic Cells Modified by Interleukin-10 Gene-Loaded Nanoparticles Expand Allogeneic Foxp3+ T Cells In vitro (102.9)
Authors: Kovacs Jeffrey Ryan , Jia Liang , Zheng Ying , Gawalt Ellen S , Shen Hongmei , Meng Wilson S
Published: January 2023

Interleukin-10 (IL-10) has been used to downregulate allogeneic T cell response in vivo. Herein we investigated two particulate systems to deliver a plasmid encoding murine IL-10 (pIL-10) into dendritic cells (DC). The hypothesis is IL-10 gene-modified DC can expand regulatory T (Treg) cells in allogeneic settings. The carriers were formed by coating on poly-(d, l-lactide-glycolide) (PLGA) or polystyrene (PS) particles pIL-10 condensed with the cationic peptide O10H6 (designated PLGAO10H6-pIL-10 and PSO10H6-pIL-10). These particles have a diameter below 300 nm and the DNA is protected from enzymatic degradation. DC transfected with either particles exhibited a 2-fold increase in IL-10 mRNA (RT-PCR, n=2). Compared to controls, DCs (H-2b) modified by the particles upregulated allogeneic CD4+ T cells (H-2d) Foxp3 mRNA by 1.5 fold (n= 3, p < 0.05). This expansion was not due to changes in CD80 and CD86 expression on DCs for these markers were expressed at similar levels on modified (69.9±2%, 74.3±2%; n=3) and control DCs (68±5%, 71±5%; n =3). In cocultures of IL-10-modified H-2b DC and H-2d T cells, production of IFN-γ and TNF-α were suppressed by 40.1±4% and 43.3±2% respectively compared to those with control DC (n=2). Taken together, these data demonstrate that pIL-10 complexed PLGAO10H6 and PSO10H6 can promote tolerogenic phenotypes in vitro and should be further investigated in allograft transplant therapy.
GM-CSF expanded tolerogenic CD11c+CD8a− dendritic cells can induce autoantigen specific CD4+ CD25+ Foxp3+ T regulatory cells (128.11)
Authors: Ganesh Balaji Baskaran , Cheatem Donald , Vasu Chenthamarakshan , Prabhakar Bellur S
Published: January 2023

Treatment of CBA/j mice with Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), a potent growth factor for dendritic cells can prevent and suppress ongoing experimental autoimmune thyroiditis (EAT). GM-CSF treatment causes expansion of CD11c+CD8a− myeloid dendritic cells (DCs) with a consequent increase in CD4+CD25+ T regulatory cells (Tregs) in an EAT model. In this study, we investigated the significance of the expansion of CD8a− myeloid DCs by GM-CSF and its direct role in inducing CD4+CD25+Foxp3+ T regs. Co-culture of CD4+ T cells with CD8a-ve DCs from GM-CSF treated mice in the presence of mouse thyroglobulin (mTg) induced an increase in the number of CD4+CD25+Foxp3+ T cells. These Foxp3+ T cells produced high levels of IL-10. These T regs could suppress the mTg specific proliferation of effector CD4+CD25− T cells, which was reversed upon addition of anti-IL10R antibodies. In contrast, GM-CSF treated CD8a+ lymphoid DCs failed to show a significant suppression of mTg specific T cell proliferation. In vivo experiments in a SCID mouse model revealed that GM-CSF treated SCID mice could induce increased numbers of Foxp3 and IL-10 expressing CD4+ T cells that were adoptively transferred from wild type CBA/j mice. These results indicate that GM-CSF acts directly on CD8a− myeloid DCs in the absence of T cells and renders them tolerogenic in that they cause expansion of CD4+Foxp3+ Tregs, which suppress mTg specific proliferation in an IL-10 dependent manner. NIH 1RO1AI058190
CD11c+ hepatic dendritic cells induce strong innate immune response with reduced capacity to activate allogeneic T cells (128.14)
Authors: Shu Shang-An , Lian Zhe-Xiong , Chuang Ya-Hui , Yang Guo-Xiang , Moritoki Yuki , Comstock Sarah S , Gershwin Eroc
Published: January 2023

Splenic and bone marrow dendritic cells (DCs) and their subsets have been shown in multiple studies to play a critical role in maintaining tolerance and also in select differentiation of pathways responsive to infectious agents. Comparable studies in the liver have been lacking primarily due to difficulty in isolation and contradictory data. We have taken advantage of our ability to identify and isolate relatively pure DC populations and have compared the phenotype and function of select subsets of DCs in liver compared to spleen in C57BL/6 mice. Importantly, freshly isolated NK1.1−CD11c+ liver DC subsets (conventional and plasmacytoid DCs) can efficiently endocytose dextran as well as induce significant levels of TNF-α, IL-6 and IL-12 p40 in response to toll-like receptor ligand stimulation. These responses are higher than comparable studies of isolated splenic DCs from the same animals. In addition, there is a relatively higher frequency of pDC populations in liver. Our results suggest that hepatic DCs has differential capability in response to innate signals and that in fact CD11c+ hepatic DCs have a greater capacity to respond to innate stimulation but less capable of inducing CpG activated-allogeneic T cells. Our data emphasize not only the critical difference in hepatic DC subsets, but also highlight the need to study individual DC subsets in understanding the modulation of the immune response in the liver.
Th1 and Th17 in tumor Immunity versus autoimmunity (128.15)
Authors: Jacob Jennifer B. , Kong Yi-chi M. , Nalbantoglu Ilke , Snower Daniel P. , Wei Wei-Zen
Published: January 2023

To determine the role of Th1 and Th17 T cells in the reactivity to self antigens in tumor vs autoimmunity, IFN-γ and IL17 secreting T cells to rat neu and mouse thyroglobulin (mTg) were measured in neu transgenic BALB/c (NeuT) and NeuTxDR3 mice, which expressed neu and HLA-DR3. HLA-DR3 is associated with susceptibility to some autoimmune diseases in humans and experimental autoimmune thyroiditis (EAT) in mice. BALB/c and BALBxDR3 served as controls. α-neu response was induced by vaccination with pneuTM encoding neu ECD and TM & pGM-CSF, or by TC-1/neu, heterologous tumor cells transfected with neu, and LPS. α-mTg response was induced by mTg & LPS, i.v. Regulatory T cells (Treg) were depleted by CD25 mAb 9 d before mTg. In BALBxDR3 & NeuTxDR3 mice, pneuTM & pGM-CSF vaccination induced 59 & 1 ug/ml Ab and 592 & 217 IFN-γ secreting cells. Vaccination with TC-1/neu & LPS induced 18 & 41 ug/ml neu Ab and 241 & 187 IFN-γ cells. Neu+ tumors can be regressed, yet no IL-17 was detected in any group. In BALBxDR3 mice depleted of Treg and immunized with mTg/LPS, 51400 U/ml mTg Ab, 119 IFN-γ secreting cells, and 64 IL-17 secreting cells were present, with 40–60% thyroid destruction. Hence, Th1 but not Th17 were activated by neu vaccination in normal or NeuT mice. Both Th1 and Th17 were activated in mTg-induced EAT. Since Neu vaccination & LPS did not induce IL-17 secretion, Th17 activation is unrelated to LPS. Elucidating the precise roles for Th1 and Th17 in tumor immunity and thyroiditis is critical when both are present in an individual. (CA 125680, CA76340, DK45960, St. John Hosp)
Embryonic-like stem cells and mesenchymal cell precursors expand in the bone marrow of lupus mice (128.17)
Authors: Wang James , Chen James Y , Vazquez Brenda , Singh Ram Raj
Published: January 2023

Autoimmune diseases such as lupus are characterized by inflammation and fibrosis in multiple organs. Development of inflammation in these diseases is preceded by abnormal development and functions of various immune cells. These immune aberrations may represent secondary immune changes or intrinsic abnormalities of their precursors, i.e., stem cells (SC). The impairments of SC and how they contribute to these diseases remain unclear. Here, we analyzed SC characteristics and differentiation in autoimmune-prone (NZB/NZW F1, MRL-Faslpr/lpr and MRL-Fas+/+) mice and MHC-matched normal controls. We found that BM cells expressing ScaI, a marker on mesenchymal SC and HSC, expand with age in all mice. Strikingly, the proportion of Sca1+ cells and Sca1 mRNA levels in fresh BM are markedly higher in autoimmune mice than in controls. However, surface expression and mRNA levels of c-Kit, a marker of HSCs, in the fresh BM are not different between normal and autoimmune mice. The expansion of Lin–cKit–Sca1+ cells occurs long before the onset of disease in lupus mice. Interestingly, the bone marrow of lupus mice had a 3-fold increase in the embryonic-like stem cells (ELSC, Lin–CD45–Sca1+ cells) as compared to normal bone marrow. In long-term cultures, BM cells from young mice differentiated into adherent cells, which is dramatically higher in autoimmune mice than in controls. These adherent cells exhibit structural characteristics of adipocytes, stain for oil red O (adipocytes), and express fatty acid binding protein (a marker of mesoderm cell adipocyte), but not nestin (an ectoderm marker), osteocalcin (a bone formation marker) or AFP (an endoderm marker). Thus, BM cells in young autoimmune mice exhibit expansion and preferential differentiation of mesenchymal stem cell lineage cells. The pathogenetic potential of these stem cell abnormalities is under investigation.
Tolerance induction for immature anti-insulin B cells involves impaired calcium mobilization (128.20)
Authors: Henry Rachel A. , Acevedo-Súarez Carlos A. , Thomas James W.
Published: January 2023

B cells that recognize small self proteins are presumed to escape tolerance and reside in a non-tolerant state of “clonal ignorance”. In contrast to this assumption, B cells that harbor anti-insulin Ig transgenes (125Tg) are not clonally ignorant; rather they are maintained in a novel anergic state. Anti-insulin B cells do not proliferate to BCR cross-linking and show impaired Ca2+ and NFAT responses, but unlike the anti-HEL/HEL model, they are not developmentally arrested and show intact tyrosine phosphorylation. To understand the genesis of this novel form of anergy, IL-7 was used to generate naïve anti-insulin B cells from bone marrow cultures. Naïve anti-insulin B cells mobilize Ca2+ upon acute antigen exposure. However, continuous antigen exposure decreased surface IgM expression and lead to impaired Ca2+ mobilization following BCR-dependent and BCR-independent stimuli. These data show that anti-insulin B cells are not clonally ignorant; rather, continuous BCR signals initiate tolerance early in development and maintain anergy in the mature repertoire. This highlights the therapeutic potential of targeting developing B cells in the bone marrow for functional silencing.

Support provided by 5T32 HL069765, 5T32 GM08554, AI051448, and JDRF.
Apoptotic cells: a source of autoantigens for antiphospholipid antibodies. (128.32)
Authors: Kang Sun-Ah , Khan Salar N. , Radic Marko Z. , Monestier Marc
Published: January 2023

Antiphospholipid antibodies (APAs) are detected in various autoimmune diseases, such as antiphospholipid syndrome (APS) and systemic lupus erythematosus. In addition to their binding to negatively charged phospholipids, APAs often cross-react with other molecules. Their biological effects are not fully understood.

Apoptotic cells are a potential source of auto-antigens during systemic autoimmunity. Inefficient clearance of apoptotic cells results in the development of autoimmune manifestations and intracellular antigens such as nucleosomes become accessible during apoptosis.

We examined a panel of monoclonal APAs generated from NZW/BXSB F1, a strain which spontaneously develops autoimmune symptoms reminescent of APS. These APAs did not bind to live cells, but reacted strongly with different structures within apoptotic cells. Further analysis with various inhibitors indicated that the binding of APAs to apoptotic cells depends on specific caspase activities and on the modification of auto-antigens by reactive oxygen species (ROS).

Therefore, apoptotic cells provide a potential source of APA antigens that may not be limited to phospholipids. Our data also indicate that physical accessibility and apoptosis-specific modification of auto-antigens by caspases or ROS are crucial factors for APA-antigen interactions.

NIH, AHA, Lupus Research Institute
Regulation of TLR9 dependent DNA Immune complex mediated cell activation by High Mobility Group Box Protein 1 (HMGB1) and Receptor for Advanced Glycation End products (RAGE) (128.35)
Authors: Tian Jane , Avalos Ana Maria , Chen Bo , Fitzgerald Katherine A. , Sirois Cherilyn , Golenbock Douglas , Marshak-Rothstein Ann , Crow Mary K. , Audoly Laurent , Kiener Peter A. , Latz Eicke , Coyle Anthony J.
Published: January 2023

Stimulation of endosomal associated TLR9 by DNA plays an important role in activation of plasmacytoid dendritic cells (pDCs) and autoreactive B cells and may contribute to the pathogenesis of systemic lupus erythematosus (SLE). HMGB1 is a DNA binding protein that is liberated either from cells undergoing necrosis or following cytokine stimulation. Here we show that HMGB1 forms a high affinity complex with CpG-A oligodeoxynucleotides and augments the production of IFN-a from murine pDCs. We also show that HMGB1 is indeed a component of naturally formed DNA containing chromatin complexes. IFNa induced by either the exogenously formed HMGB1/CpG-A complex or naturally formed complex is TLR9 dependent and is inhibited by HMGB1 and RAGE antagonists. The complex without HMGB1 induces significantly less IFNa. HMGB1-RAGE interactions are required for activation of autoreactive B cells following stimulation with DNA immune complexes and are critical in the regulation of type I interferon gene induction by DNA complexes present in lupus plasma. Our data provide a novel mechanism by which HMGB1 confers potent stimulatory activity to DNA through a RAGE dependent mechanism and may contribute to the pathogenesis of lupus.
A Destabilizing Di-nucleotide Deletion in 3’-Untranslated Region of Human CD24 mRNA Confers Protection against Multiple Sclerosis (129.1)
Authors: WANG LIZHONG , LIU RUNHUA , ZHENG XINCHENG , LIN SHILI , ZHENG PAN , LIU YANG
Published: January 2023

Single-nucleodie polymorphism (SNP) has a profound impact upon one’s susceptibility to autoimmune diseases such as multiple sclerosis (MS). Although it is well established that 3’UTR play a major role in regulating RNA stability, the contribution of SNP in the 3’UTR to the risk of autoimmune diseases has not well characterized. We have proviously reported the contribution of mouse CD24 3’UTR to its mRNA stability, so we investigated the significance of SNPs in the hCD24 3’UTR to MS susceptibility.

Our analysis of the 316 MS patients and 342 controls revealed that a di-nucleotide deletion at position 1580 (P1580) of hCD24 mRNA is associated with significantly reduced risk (odds ratio OR = 0.53 with 95% confidence interval = 0.34–0.81) and delayed progression (p = 0.016). More importantly, using 150 families from two independent cohorts, we have found that the P1580del allele is preferentially transmitted to unaffected individuals (p = 0.003). In heterozygous individuals, the mRNA levels for the the di-nucleotide-deletion allele is 2.5-fold less than that of the wild-type allele (p = 0.004). Our transfection studies revealed that the di-nucleotide deletion dramatically reduced the stability of hCD24 mRNA (p < 0.001).

Our results demonstrate that a destabilizing di-nucleotide deletion in the 3’UTR of hCD24 mRNA substantially reduces the risk and delays the progression of MS.
Visualizing mechanisms of peripheral tolerance against autoreactive T cells (129.35)
Authors: Divekar Rohit D. , Haymaker Cara , Jain Renu , Ellis Jason , Tartar Danielle , Yu Ping , Lee Hyun-Hee , Hardaway John , Hoeman Christine , Guloglu Betul , Franklin Craig , Miller Mark , Zaghouani Habib
Published: January 2023

Where and how T cell tolerance happens remains largely undefined. Here, we used an established in vivo model and investigated the site and the mechanisms that govern APC-mediated inactivation of autoreactive T cells.

Fcγ receptor-deficient (FcγR−/−) mice were induced for experimental allergic encephalomyelitis (EAE), with a myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide and when the disease became apparent the mice were given FcγR+/+ APCs and treated with Ig-MOG, a tolerogenic Ig expressing MOG peptide. Under this regimen the mice were able to reverse their EAE only when given FcγR+/+ APCs that can present Ig-MOG and carry out T cell tolerance. This system was then used to visualize encounter of the APCs with the pathogenic T cells. The results indicate that tolerance occurs in a time dependent fashion when both the T cells and APCs have accumulated in sufficient number at the encounter site but unlike immunity, can take place in the non-draining lymph nodes. Two-Photon microscopy analysis revealed that tolerized T cells exhibit organ-specific changes in motility, pattern of migration and trafficking which could be related to functional matters as tolerance manifest the form of Th2 deviation in the lymph node and anergy in the spleen.

Research supported by grant NS37406 from NIH.
Critical roles of Bcl-3 in type 1 diabetes (129.43)
Authors: Ruan Qingguo , Carmody Ruaidhri , Zheng Shijun , Chen Youhai H.
Published: January 2023

Bcl-3 (B cell leukemia-3) is a member of the inhibitor of nuclear factor kappa B (NF-κB) family. In contrast to other members of this family, Bcl-3 is localized primarily in the nucleus, and selectively interacts with the p50 and p52 homodimers but not their heterodimers. To explore the roles of Bcl-3 in models of type 1 diabetes, we studied cyclophosphamide-induced diabetes in NOD mice and low-dose streptozotocin-induced diabetes in C57BL/6 mice that did or did not express Bcl-3. We found that Bcl-3-deficient mice were more susceptible to autoimmune diabetes and were defective in Th2, but not Th1 responses. Additionally, we found that chemokine production in Bcl-3-deficient mice was significantly enhanced as compared to the control mice. Mechanistic studies reveal that in the absence of Bcl-3, the inhibitory p50 homodimer of the NF-κB family is ineffective in suppressing gene expression because it is rapidly ubiquitinated and degraded upon binding to DNA. Bcl-3 effectively inhibits p50 polyubiquitination and stabilizes its interaction with DNA through the formation of a stable Bcl-3:(p50)2:DNA complex. These results indicate that Bcl-3 is a critical transcriptional inhibitor of autoimmune diabetes.
Mice immunized with EBNA-1 protein produce antibodies that cross-react with double stranded DNA (129.5)
Authors: Spatz Linda Ann , Tran Hoa , Mumbey-Wafula Alice , Chandrasekhar Sharanya , Wei Hoi , Lewis Rita , Gottlieb Paul
Published: January 2023

Systemic Lupus Erythematosus (SLE) is characterized by the production of antibodies to nuclear antigens, in particular antibodies to double stranded DNA (dsDNA). The etiology of these autoantibodies is unknown but one theory suggests that viruses may play a role. One virus that has been shown to have an association with SLE is the Epstein Barr Virus (EBV). We previously demonstrated that mice immunized with an EBNA-1 expression vector or with purified EBNA-1 protein develop antibodies to dsDNA as well as to the viral antigen. We have recently demonstrated that these antibodies are pathogenic and deposit in the kidney and induce moderate proteinuria. We hypothesized that since EBNA-1 is a dsDNA binding protein, it could complex with chromosomal dsDNA in order to render it immunogenic and elicit an anti-dsDNA response. To test this hypothesis we generated recombinant EBNA-1 protein that lacks the 3 known chromosomal binding sites and immunized mice with it. We observed that mice immunized with this mutated EBNA-1 protein still develop an anti-dsDNA response. We have now generated hybridomas producing anti-EBNA-1 antibodies from these mice and observed that 10 out of 18 monoclonal anti-EBNA-1 antibodies actually cross-react with dsDNA. These results suggest that a viral protein determinant can mimic chromatin and elicit an anti-dsDNA response that can lead to autoimmunity. Future studies will analyze the molecular basis of this cross-reactivity.
The CYP450 mouse model for autoimmune hepatitis: Breaking of self-tolerance in transgenic CYP2D6 and wildtype FVB-mice by viral infection (130.1)
Authors: Christen Urs , Holdener Martin , Hintermann Edith , Johnson Eric , Manns Michael , von Herrath Matthias
Published: January 2023

The etiology of autoimmune hepatitis (AIH) is poorly understood although the major autoantigen, cytochrome P450 2D6 (CYP2D6), has been identified. We generated an animal model for human AIH using the natural autoantigen CYP2D6. We infected transgenic mice expressing human CYP2D6 in the liver with an Adenovirus-CYP2D6 vector (Ad-2D6) to break self-tolerance. Upon infection with Ad-2D6 not only transgenic CYP2D6 mice but also wildtype FVB mice showed persistent features of severe liver damage associated with AIH (hepatic fibrosis, fused liver lobules, cellular infiltrations, elevated serum ALT levels, confluent necrosis). Ad-2D6-infected mice (CYP2D6 and FVB) generated high titers of anti-CYP2D6 antibodies. Epitope mapping revealed that anti-CYP2D6 antibodies predominantly recognized the same immunodominant linear epitope recognized by sera of AIH patients (AQPPRD aa265-270). In contrast, mice infected with a control Adenovirus expressing green fluorescence protein did neither develop liver damage nor generated anti-CYP2D6 antibodies. The severity of liver damage as well as antibody formation was enhanced in FVB mice compared to transgenic CYP2D6 mice indicating a stronger tolerance to human CYP2D6 in CYP2D6 mice. In FVB mice, due to the homology of the mouse isoenzymes of the CYP superfamily to human CYP2D6, autoimmune liver damage by Ad-2D6-infection was possibly induced via true molecular mimicry.
CD73-deficient mice are resistant to experimental autoimmune encephalomyelitis (130.30)
Authors: Bynoe Margaret S. , Mills Jeffrey , Thompson Linda F
Published: January 2023

To test the role of CD73-generated adenosine in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we induced EAE in CD73−/− mice with standard protocols. CD73 (ecto-5’-nucleotidase) makes extracellular adenosine from AMP. Adenosine is anti-inflammatory in several settings via triggering cell surface adenosine receptors and CD73−/− mice exhibit a variety of pro-inflammatory phenotypes. However, CD73−/− mice were highly resistant to the development of EAE (mean EAE score of 0.5 vs 2.0 for WT mice). This was not because their T cells failed to be activated by EAE induction. When T cells from CD73−/− and WT mice with EAE were transferred to CD73+/+ TCRα−/− mice, disease activity was much more severe in recipients of CD73−/− T cells (mean EAE score 3.7 vs. 1.7 in WT mice). To determine whether CD73−/− T cells were defective in their ability to cross the blood brain barrier, immunohistochemistry was performed on the brains of CD73+/+ and CD73−/− mice with EAE. Consistent with the lower disease activity scores in the CD73−/− mice, there were many fewer leukocytes in their brains. The higher apparent blood brain barrier function in CD73−/− mice was confirmed with Evan’s blue dye extravasation studies. Thus, our results show that CD73is required for the entry of pathogenic T cells into the brain and suggest a novel role for CD73-generated adenosine in modulation of the blood brain barrier.
Induction of CD8 T cell-mediated EAE by viral infections in a CD8 TCR transgenic mouse model (130.31)
Authors: Ji Qingyong , Perchellet Antoine , Goverman Joan
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Besides CD4 T cells, CD8 T cells have recently been shown to mediate the pathogenesis of EAE. The cause of MS is not clear, but infections have been suggested. In this study, we investigated the roles of viral infections in the development of EAE on a CD8 T cell receptor (TCR) transgenic mouse model. Infection with wildtype vaccinia virus broke tolerance and triggered atypical EAE in a TCR transgenic mouse specific for myelin basic protein 79–87 (MBP79-87). CD8 T cells up-regulated activation markers and trafficked to the CNS in virus-infected mice. Interestingly, CD8 T cells from TCR-transgenic MBP deficient mice also displayed activation markers, and had cytolytic effector function similar to that from TCR transgenic MBP sufficient mice. T cell activation was not due to MBP TCR cross-reactivity against vaccinia virus. Furthermore, preliminary data showed that disease induction by vaccinia virus required endogenous TCRs. Ongoing experiments are extending these observations into another viral infection. In conclusion, the results suggest that viral infection may induce EAE by activation through dual TCR.
CD8 epitope MOG 37-46 induces attenuated form of EAE characterized by reduced CNS infiltration and myeloid activation (130.32)
Authors: Bettini Maria , Evavold Brian D
Published: January 2023

Multiple sclerosis is a predominantly CD4 T cell mediated disease, yet recent observations have indicated a potential contribution from CD8 T cells. We have previously identified MOG 37-46 in the context of H2-Db as the CD8 specific epitope and showed that most CD8 T cells in the CNS of MOG 35-55 immunized mice are specific to MOG 37-46. In an attempt to isolate the contribution of CD8 T cells in a murine model of MS, we immunized mice with MOG 37-46, a truncated version of MOG 35-55. MOG 37-46 does not contain the full T helper epitope (40–48) and shows minimal activation of CD4 T cells. The data presented here show that MOG 37-46 immunization induced disease characterized by lower disease scores, a decrease in CNS infiltration and microglial activation, and reduced demyelination and axon damage. Upon re-stimulation splenic-derived CD8 T cell lines from MOG 35-55 and MOG 37-46 immunized mice were IFNγ+ and TNFα+ (>60%). CNS derived CD8 T cells of EAE mice produced IFNγ and TNFα in response to antigen after ex vivo re-stimulation. Our data indicate that CD8 T cells reactive to MOG 37-46 are pro-inflammatory, and suggest that CD4 T cells are required for myeloid activation in CNS and recruitment of CD8 T cells.

This research was supported by NIAID (R01 AI056017) and NMSS (RG 3683) grants.
Cyclophosphamide treatment of the NOD mouse reveals quasi-public dominant T cell clones (130.34)
Authors: Marrero Idania G , Dai Yang D , Sercarz Eli E
Published: January 2023

To define the major components of the autoreactive T cell repertoire that arise spontaneously in the NOD mouse, we used CDR3 length spectroscopy to

study the T cell repertoire in PLN from pre-diabetic NOD mice to identify public dominant clones; andto identify potentially pathogenic clones in islet-infiltrating cells that persist after Cyclophosphamide (Cyp) treatment.

We found that any public T cell expansions were identified in PLN of female NOD mice at 4 and 10 weeks of age. Two expansions in the rearrangements Vb5.2/Jb2.5 and Vb10/Jb1.5 were found in 60% and 50% of 4 week old NOD mice. These expansions can be useful in the study of how the T cell repertoire can be modified after islet transplantation. After Cyp-treatment, the Vb4, Vb5.2, Vb10 and Vb12 families showed dominant expansions. Interestingly, we found a unique Vb4-"195 nucleotide" peak that was found in 10 out of 11 (91%) Cyp-treated mice. In contrast, this peak was not detected in untreated NOD mouse islets. In the Vb12 family, a "200" peak was prominent in 6 out of 10 (60%) Cyp-treated mice. These expansions may represent the more pathogenic clones. Currently, we are sequencing these expansions to establish whether the Cyp-induced expansions represent truly unique clones. We believe that these Cyp experiments will provide the possibility of distinguishing between regulatory and aggressive clones.

This work was supported by JDRF 3-2005-336.
T cell receptors and I-Ad/peptide interactions in autoimmune gastritis (AIG) (130.40)
Authors: Revilleza Maria Jamela Roxas , Levin Ditza , Mage Michael , Teyton Luc , Shevach Ethan , Margulies David H , Natarajan Kannan
Published: January 2023

We have developed a murine model of autoimmune gastritis by generating two TCR transgenic lines expressing I-Ad restricted TCRs specific for two different peptide epitopes of gastric H/K ATPase. Both lines spontaneously develop AIG but with differing incidence, severity, and kinetics. The Tx23 line develops disease early, severely, with 100% incidence and a predominantly Th1 phenotype. The Tx51 line develops disease only after two months, only 50–80% of the mice are affected. and is predominantly of the Th2 type. To investigate the role of TCR affinity in the disease phenotypes in these mice, we generated recombinant, soluble forms of the Tx23 and Tx51 TCRs, and monomeric and multimeric forms of their cognate I-Ad/peptide complexes. Peptide dose response experiments suggest that the Tx51 TCR is of higher functional avidity than the Tx23 TCR, suggesting that decreased peptide availability in vivo may limit the activation of Tx51 T cells. Surface plasmon resonance (SPR) studies revealed specific and dose dependent binding when multimeric but not monomeric I-Ad/peptide complexes were offered to the TCR, consistent with the relatively weak affinities of these autoreactive TCRs. These experiments exploit strategies that may be employed to detect binding of low affinity TCRs to their MHC/peptide ligands.

This was supported by the Intramural Research Program of the NIAID.
Serum transfer of spontaneous polychondritis from HLA-DQ6αβ 8αβ transgenic mice. (130.42)
Authors: Doll Joseph R , Bradley David S
Published: January 2023

We provide here evidence for a pathogenic role for antibodies in spontaneous polychondritis occurring in HLA-DQ6αβ8αβ transgenic (tg) mice. We have previously reported that HLA-DQ6αβ8αβ tg mice develop spontaneous polychondritis, which occurs at middle age and lacks CII specificity. The onset of disease is associated with elevated serum levels of immunoglobulins compared to non-diseased mice. However, a role for immunoglobulins in disease pathogenesis has not been identified. Serum was collected from HLA-DQ6αβ8αβ tg mice displaying clinical spontaneous polychondritis and serum immunoglobulins were concentrated via density centrifugation. Concentrated serum immunoglobulins were transferred intravenously into young, naïve B10.T(6R) mice, resulting in a transient clinical display of polyarthritis. Gross clinical symptoms of polyarthritis occurred within 48 hours of the initial immunoglobulin transfer and were of a transient nature, symptoms generally reduced by 96 hours. Histological assessment of the limbs obtained from mice receiving antibody transfer identified the presence of cellular infiltrates in the synovium and peri-synovium. Control mice receiving sham immunizations displayed neither clinical nor histological signs of polyarthritis. The studies described here should aid in characterizing and understanding a newly identified transgenic mouse model of human autoimmune polychondritis and may provide insight into new therapeutic avenues for human relapsing polychondritis and related diseases such as rheumatoid arthritis. In addition, these findings may aid in the identification of unidentified target antigens required for spontaneous polychondritis.
DRB1*0402 protects while DRB1*0401 is permissive for inducing collagen-induced arthritis in transgenic mice (130.45)
Authors: Taneja Veena , Behrens Marshall , Griffiths Marie , Luthra Harvinder , David Chella
Published: January 2023

DRB1*0401 has been associated with predisposition to rheumatoid arthritis while DRB1*0402 is associated with resistance. We have generated transgenic mice lacking endogenous H2-A and E molecules (AEo) but expressing DRB1*0401 or DRB1*0402 molecules. All transgenic mice expressed functional DR molecules which could positively and negatively select various Vβ T cell repertoire profile. Transgenic mice were studied in vivo for induction of arthritis following immunization with type II collagen. DRB1*0401 mice are susceptible to collagen-induced arthritis while DRB1*0402 mice are resistant. Presence of DR4 leads to arthritis predominantly in female mice. CIA susceptible AEo.DQ8 mice have been shown to develop arthritis without a gender bias. However, AEo mice expressing DRB1*0401.DQ8 develop arthritis in female mice with a ratio of 3F:1M, similar to that in humans. DRB1*0402 mice did not show any defect in humoral response though they demonstrated a lower cellular response to CII and an increased activation-induced cell death compared to susceptible mice. In vitro T cell response with superantigen staphylococcal enterotoxin B and Concovalin A showed that DRB1*0402 mice are not defective in generating T cell response. The resistant mice showed an increased cell death in thymus suggesting deletion of autoreactive T cells at thymic level might be one reason for protection. The data suggest that DRB1 polymorphism lead to selection of TCR in thymus thus controlling the selection of autoreactive T cells available in periphery. Thus these transgenic mice may be useful to study the reasons for gender-bias observed in human patients develop therapeutic strategies.