bluuebunny/crossref_metadata_2025 huggingface dataset:
An in vivo model of the human CD1 antigen presenting system (42.24)
Authors: chen xiuxu , Rajesh Deepika , Roenneburg Drew A. , Zhou Ying , Hegde Subramanya , Cheng Tan-Yun , Brenner Michael B. , Moody B. Branch , Burlingham William J. , Gumperz Jenny E.
Published: January 2023

CD1 molecules are a family of antigen presenting molecules that present lipids and glycolipids for recognition by specialized T cells. The CD1 system has been implicated in microbial infections, autoimmune and allergic disorders, and immunological control of tumors. However, our understanding of its role in human disease remains limited because animal models that resemble the human CD1 system are lacking. Here we report that a severely immune deficient mouse engrafted with human fetal tissues and hematopoietic stem cells develops an intact human CD1 antigen presenting system. Human CD1a, b, c, and d isoforms are highly expressed in the thymic organoid that develops in the engrafted mice, and histological analysis indicates the presence of CD1a+ APCs. Moreover, CD1+ APCs are expressed in peripheral tissues including the spleen and liver, and are capable of presenting bacterial glycolipids to human T cells specific for CD1a, b, or c. Staining with CD1d tetramers detected comparable percentages of human CD1d-restricted T cells in engrafted mice and human blood, and CD1d-restricted T cells isolated from the mice expressed a characteristic Vα24+/Vβ11+ TCR and recognized the glycolipid antigen α-GalCer. These results demonstrate the functional reconstitution of the human CD1 system in a mouse model.
Optical Quantification of Disease Activity using Hydrophobically Modified Glycol Chitosan labeled with Near-Infrared Fluorescence in Experimental Arthritis Model (42.25)
Authors: Kang Young Mo , Park Kyeongsoon , Kang Jin Hee , Sa Keum Hee , Cho Hyung Jeong , Lee Seulki , Kwon Ick Chan
Published: January 2023

Objective. To investigate the quantitative analysis of disease activity using the optical imaging system with hydrophobically modified glycol chitosan labeled with Cy5.5 (HGC-Cy5.5) in animal arthritis model.

Methods. Fluorescence reflectance images were captured two hours after intravenous injection of HGC-Cy5.5 nanoprobe from the paws of collagen-induced arthritis mice. Immunofluorescence staining for F4/80 and Mac-1 molecules were performed to identify the cell types phagocytosing HGC nonoprobe, both in vivo and in vitro.

Results. In raw reflectance images, paws of the active arthritis group showed stronger fluorescence intensity than those of nonarthritis group. According to the progression of arthritis, total photon counts (TPC) increased until 5 weeks after the booster injection in parallel with the clinical arthritis index. The quantitative analysis of fluorescence intensity showed that the TPC had significant differences in methotrexate treated groups compared with an untreated group. Fluorescence microscopy demonstrated considerable accumulation of HGC-Cy5.5 within the synovial tissues, with the localization of the probe within the interstitial and a portion of cells expressing Mac-1 molecule. Macrophages differentiated with the stimulation of GM-CSF revealed conspicuous intracellular NIRF signal after two hours of incubation with the HGC-Cy5.5, with up-regulated expression of Mac-1 on the surface.

Conclusion. The present results indicate that the quantitative analysis of joint inflammation using HGC-Cy5.5 nanoprobe can provide an objective measurement of disease activity in RA.
DNA vaccine encoding spike protein of SARS-coronavirus loaded polyethylenimine elicits antigen-specific immune responses in mice immunized through intranasal route (42.8)
Authors: YUN CHEOL-HEUI , Shim Byoung-Shik , Park Sung-Moo , Han Heung Hyun
Published: January 2023

The spike protein (S) of severe acute respiratory syndrome (SARS) coronavirus-(CoV) is a ~180-kDa glycoprotein, which is essentially required its recognition of and binding to host receptors. It has been known that the receptor-binding motif of spike protein can produce neutralizing antibodies and therefore prevent the infection caused by SARS-CoV. Polyethylenimine (PEI) is one of the most widely used and effective nanoparticles, which have been tested for its use in the drug delivery system including DNA vaccine. In the present study, we examined the immune responses in BALB/c mice after immunized with SARS DNA vaccine in a pci-S/PEI complex form via intranasal (i.n.) route. PEI/pci-S nanoparticles showed a spherical shape with size around 194+/-99nm. The mice intranasally immunized with pci-S/PEI nanoparticles produced significantly (P<0.05) higher S-specific IgA as well as B220+ cells in the lung wash than those in mice treated with pci-S alone. CD80, CD86 and Class II MHC molecules (I-Ad) were increased on pulmonary dendritic (CD11c+) cells after vaccination. The percentage of IFN-gamma, TNF-alpha, and IL-2 producing cells were also higher in PEI/pci-S vaccinated mice than in either pci-S or pci-mock treated mice. Taken together, our results showed that immunization with pci-S/PEI nanoparticles induce antigen specific humoral and cellular immune responses.
PD-1 traffics to the immunological synapse along the plasma membrane to inhibit CTL cytotoxicity and cytokine secretion (44.25)
Authors: Ahmed Fariyal , Friend Sherree , Barteneva Natasha , Lieberman Judy
Published: January 2023

ABSTRACT

Functional impairment of antigen-specific T cells is a defining feature of chronic HIV infection. During HIV infection, the inhibitory receptor programmed death 1 (PD-1) and its ligand PD-L1 are markedly upregulated on 'exhausted' HIV specific CD8 T cells and antigen presenting cells, respectively. PD-1 up-regulation on HIV specific CD8 T cell correlates positively with effector cell exhaustion and plasma viral loads, and inversely with CD4 T-cell count. Blockade of the PD-1/PD-L1 pathway augments some aspects of HIV-specific CD8 T cell activation, including proliferation and cytokine production. However, the underlying biology behind the workings of this described inhibitory pathway is not fully understood. I will investigate the changes in PD-1 expression and localization in response to CD8 T cell activation and study its effect on T-cell receptor signaling and effector cell functions. I hypothesize that PD-1 concentrates in the central region of the immunological synapse and undermines its stability and signaling. To test this hypothesis, I will investigate the effect on T cell signaling, proliferation, cytokine production and cytotoxicity by both over-expressing PD-1 and silencing its ligands, using RNA interference, in primary T cells and macrophages, respectively. Understanding the PD-1/PD-L1 pathway of reversible T-cell impairment may help develop novel therapeutic strategies for rescuing the function of exhausted T cells in chronic HIV infection.
Selective deficiency of STAT1 in CD4+ T cells enhances susceptibility to Leishmania major infection not by preventing induction of Th1 response, but by impairing CD4+ T cell homing (44.26)
Authors: Snider Heidi M , Bhardwaj Neeti , Barbi Joseph , Lezama-Davila Claudio M , Durbin Joan E , Satoskar Abhay R
Published: January 2023

We previously reported that STAT1 signaling is indispensable for host resistance against Leishmania major. In this study, we examined the role of STAT1 in lymphocytes as well as CD4+ versus CD8+ T cells in mediating immunity to L. major by performing adoptive transfers of T cells from WT and STAT1-/- C57BL/6 mice to Rag2-/- C57BL/6 mice. Rag2-/- mice reconstituted with STAT1-/- splenocytes (B cells and T cells) failed to mount an efficient Th1 response following L. major infection, produced more IL-4 and developed large lesions full of parasites. In contrast, Rag2-/- mice reconstituted with WT (STAT1+/+) splenocytes mounted a Th1 response and developed self-resolving lesions. Studies using Rag2-/- recipients that received a combination of purified CD4+ and CD8+ T cells from WT or STAT1-/- mice revealed that STAT1 in CD4+, but not CD8+ T cells was indispensable for immunity against L. major. Further studies using Thy1.1+ congenic recipients receiving WT or STAT1 Thy1.2+ CD4+ cells showed that STAT1 in CD4+ T cells was not required for Th1 differentiation and IFN-γ production during L. major infection, but was critical for migration to regional lymph nodes and the cutaneous site of infection. Together these studies indicate that STAT1 signaling in CD4+ T cells plays a critical role in immunity against L. major by controlling migration of Th1 cells to the site of infection rather than their generation.

Research Support: NIH
Promoter Complexity Model for TRIF-biased Stimulation of TLR4 by Synthetic Monophosphoryl Lipid A (44.28)
Authors: Cekic Caglar , Casella Carolyn R. , Eaves Chelsea E. , Mitchell Thomas C.
Published: January 2023

TLR4 uses both MyD88 and TRIF adaptors to induce inflammation and to enhance antigen presentation. Previously, we reported that the low-toxicity vaccine adjuvant monophosphoryl Lipid A (MPLA) is a TRIF-biased TLR4 agonist compared to LPS. However, not all TRIF-dependent genes were optimally induced after MPLA stimulation. Further complications were that both MPLA and LPS are heterogeneous and have several structural differences compared to each other. We now report that homogeneous preparations of synthetic MPLA (sMLA) selectively induce TRIF-dependent genes such as IFIT2 but not other TRIF-dependent genes such as IFNβ or MyD88/TRIF co-dependent genes such as COX-2 when compared to synthetic diphosphoryl Lipid A (sDLA). Consideration of the promoters for these genes revealed that IFIT2 requires fewer transcription factors for it to be active (IRF-3 and p65), whereas requirements for COX-2 and IFNβ transcription are more complex (p50/p65, AP-1, IRF-3). sMLA weakly induced signaling pathways leading to p50/p65 and AP-1 activation, but activation of IRF3 and p38 was largely unaffected. Thus, low toxicity sMLA may drive Trif-biased signaling because the promoters of its targeted genes are more likely to be 'simple', while those associated with MyD88 are more often complex. Results of this study identify a new model for TRIF-biased stimulation of TLR4 by sMLA.

This work was supported by the NIH/NIAID and the AHA.
The Programmed Death-1 and Interleukin-10 pathways play a down-modulatory role in LP-BM5 retrovirus-induced murine immunodeficiency syndrome (44.9)
Authors: Green Kathy A , Honjo Tasuku , Green William R.
Published: January 2023

We have focused on studying pathology due to the immune system's response to viral infections. In C57BL/6 mice the retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, B- and T-cell immunodeficiency, susceptibility to opportunistic pathogens, and terminal B-cell lymphomas -- a syndrome termed murine AIDS. LP-BM5 infected B6.PD-1-/- mice develop more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice, and are more susceptible to lower virus dose and show exaggerated disease early post infection. Infected PD-L1-/- and B6.IL-10-/- mice, also develop exaggerated LP-BM5-induced disease, again without a significant change in retroviral load, relative to B6 mice. By reciprocal reconstitution experiments, PD-1+ B cells appear crucial in the limitation of LP-BM5 induced disease in B6 mice. LP-BM5 infected B6 mice have a macrophage/granulocyte-like subset of spleen cells which are PD-L1+, CD11b+, F4/80+, GR-1+, and IL-10 expressing; while infected PD-1-/- mice, even though showing a greater expansion of this subset, did not have an equivalent increase in IL-10 production. We conclude that PD-1/PD-L interactions and IL-10 are required in moderating the effects of LP-BM5 induced disease in B6 mice. We are continuing to define the functional role of this unique macrophage/granulocyte subset in LP-BM5-induced disease.
Redemption from Original Antigenic Sin response to influenza virus (45.1)
Authors: Kim Jin Hyang , Jacob Joshy
Published: January 2023

Original antigenic sin (OAS) is an inherent flaw in the immune system. Following sequential exposure to influenza viruses, the antibody response is predominantly against a prior influenza virus strain. This occurs at the expense of a response to the novel, protective epitopes in the current strain. Recently, we showed that sequential infection of mice with antigenically related strains of influenza virus, A/PR/8/34 and A/FM/1/47 led to antibody responses almost exclusively against the original antigen, A/PR/8/34. We also showed that sequential immunization with formalin-inactivated influenza viruses led to less pronounced OAS, however, memory development and recall responses were significantly compromised. The phenomenon of OAS is operational not just in mice but also in rabbits, ferrets and humans. Since OAS dampens efficacy of influenza vaccines, it is critical to develop vaccine strategies that prevent OAS. In this study, we show the host immune response can be redeemed from OAS by (a) using adjuvants such as Bordetella perutssis toxin or CpG-oligonucleotide or (b) by repeated immunization with the vaccine strain. These data indicate that while the induction of OAS might be a potential strategy by which variant influenza viruses subvert the immune system, co-administering adjuvants or expanding the memory pool of the vaccine strain represent novel approaches to prevent the effects of OAS.
Epstein-Barr Virus Latent Membrane Protein 2A protects B cells from MYC-induced apoptosis and accelerates tumor development (45.14)
Authors: Swanson-Mungerson Michelle , Bultema Rebecca , Longnecker Richard
Published: January 2023

Despite its initial isolation from Burkitt's lymphoma (BL) tumors over 40 years ago, the exact contribution of Epstein-Barr Virus (EBV) to BL is undefined. EBV encodes for multiple proteins in latently-infected B cells that affect B cell survival and activation. Due to the ability of one of these latency proteins, latent membrane protein 2A (LMP2A) to protect B cells from apoptosis, we tested if LMP2A protects B cells from apoptosis induced by aberrant c-MYC expression that precedes and dominates BL. We crossed LMP2A-transgenic mice (LMP2A-Tg) in which all B cells express LMP2A to a transgenic mouse that expresses a BL translocation of myc (λ-MYC-Tg mice). LMP2A protects B cells from MYC-induced apoptosis and accelerates the development of B cell lymphomas in LMP2A/λ-MYC-Tg mice. LMP2A also increases the expression of Bcl-XL in both pre-tumor B cells and tumor cells, suggesting a mechanism for LMP2A mediated B cell survival in the presence of MYC. These results support a hypothesis that EBV LMP2A promotes tumor development by protecting pre-tumor B cells that would normally apoptose after the c-myc translocation. This work is supported by Public Health Service grants CA62234 and CA73507 from the NCI.
Self-Reactive CTL Reveal a Strong Focus of the CD8+ T cell Receptor Repertoire on Self MHC (46.12)
Authors: Radhakrishnan Suresh , Hansen Michael , Pease Larry R
Published: January 2023

Mouse CD8+ T cells can be activated to lyse targets expressing self class I molecules more potently than allogeneic targets. Self-reactive CTL express IL-17 and can be enriched following intracellular staining. Such CTL lyse targets in an MHC restricted fashion. This recognition is independent of the bound peptides, as demonstrated by cold-target inhibition studies. Activation of OT-1 transgenic T cells using traditional APC results in peptide specific CTL recognition, stimulation of the same T cells induces peptide independent, yet Kb-restricted recognition, irrespective of whether RAG sufficient or deficient mice were used. Self-reactive CTL efficiently lyse targets expressing wild type Kb or Kb-mutants with amino acid substitutions on the β-strands of the antigen binding groove, but not mutants with substitutions on the α1 and α2 helices, suggesting self MHC recognition is mediated by interactions of the CDR1 and CDR2 regions of the TCR with the α-helices of the class I molecules, and not by bound peptides. The ability of CTL to react in an MHC restricted, yet peptide independent, manner raises important questions about the nature of TCR-MHC interactions. We propose a novel "pre-positive selection" check point in thymic development and in peripheral T cell activation in recruitment of TCR-MHC complexes at the immunological synapse during T cell activation and homeostatic recognition.
A novel subset of T helper cells promotes immune responses by producing GM-CSF (46.13)
Authors: ZHANG JIMIN , Devadas Satish , Liu Catherine , Shi Yufang
Published: January 2023

T helper cells are critical for maintaining proper immune responses. Yet, they have an undefined relationship with one of the most potent immune stimulatory cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF). Although produced locally, GM-CSF can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense and inflammatory processes. By depleting major cytokines during CD4+ T cells differentiation in vitro, we obtained a novel subset of T helper cells, which produce large amounts of GM-CSF, but little of the cytokines produced by other T helper subsets. Through GM-CSF, these cells promoted cytokine production by Th1, Th2, Tc1, Tc2 and naïve T cells. Anti-GM-CSF antibody supplementation abolished the cytokine augmentation properties of the cells. Interestingly, dendritic cells (DC) differentiated with GM-CSF alone significantly upregulated DC-SIGN, a DC receptor that is critical in DC adhesion, migration, and activation of primary T cells. Furthermore, these cells are prone to AICD and express elevated levels of pro-apoptotic proteins, such Bid and Bim. Inhibitors of FasL, TRAIL, caspase 8 or granzymes could not block their AICD. Thus, ThGM cells are a novel T helper population that produces GM-CSF, exhibits great susceptibility to apoptosis, and plays an important role in regulating immune responses.
Development of CD4 T cells from Human Hematopoietic stem Cells in a Lentiviral-engineered Stromal Culture (46.15)
Authors: Patel Ekta  S , Chang Lung Ji
Published: January 2023

Hematopoietic precursor/stem cells (HPCs) are progenitors to all functional immune cell lineages. We established a novel stromal cell line expressing (Delta-like 1) DL1, interleukin-7 (IL-7), and FMS-like tyrosine kinase 3 Ligand (Flt3-L) and demonstrated efficient differentiation and expansion of early T cells from adult CD34+ HPCs. However, continuous IL-7 signaling led to the production of immature CD8+ T lymphocytes at the expense of CD4+ T lymphocytes. It is known that the developing double positive T cells shut down IL-7 mediated signaling and require TCR signaling for survival and positive selection. We hypothesize that IL-7 inhibits the TCR signaling event required for positive selection. We found that by removal of IL-7, followed with CD3/CD28 stimulation, the early T cells were able to differentiate into mature single positive CD4 T cells expressing CD3, CD28, and TCRαβ. This can also be demonstrated by bypassing TCR signaling through stimulation with PMA/ionomycin. When further stimulated, these in vitro derived CD4+ T cells differentiated into various T helper subtypes and produced effector cytokines including IL-2, IFN-γ, and IL-17. This novel culture system can greatly facilitate the study of late T cell development and has potential for immunotherapeutic applications.

Supported by Vectorite Biomedica Inc. and Yongling Foundation.
LFA-1 signaling plays an important role in effector and memory CD8+ T cell survival via TNF receptor/ligand family-activated Bcl-2 apoptosis pathway and Caspase cascade (46.19)
Authors: Xiang Jim
Published: January 2023

LFA-1 signaling is required for CD8+ T cell priming and cytolytic activity. However, its role for effector and memory CD8+ T cell survival in transition and maintenance phase is elusive. We adoptively transferred ovalbumin (OVA)-pulsed dendritic cell (DCOVA)-stimulated effector CD8+ T cells or purified OVA-specific memory CD8+ T cells into wild-type C57BL/6 or CD54 gene knockout (CD54-/-) mice. We assessed CD8+ T cell survival kinetics and molecular mechanisms responsible for LFA-1 signaling defect-induced T cell apoptosis by flow cytometric and Western-blotting analysis. We found that transferred OVA-specific effector and memory CD8+ T cell survival significantly decreased (p<0.05) in transition and maintenance phase in CD54-/- mice due to increased T cell apoptosis, compared to C57BL/6 mice. Increased CD8+ T cell apoptosis is initiated via altered gene expression in TNF receptor/ligand (TNFR/L) (up-regulation of Fas and down-regulation of CD27, CD30 and 41BB), Bcl-2 (down-regulation of proapoptotic Bid, anti-apoptotic Bcl-2 and Bcl-XL and up-regulation of apoptotic Bax) and Caspase (down-regulation of pro-Caspase 8 and up-regulation of cleaved Caspase 3 and 7) families, and in survival signals (down-regulation of IL-15R). Therefore, LFA-1 signaling plays an important role in effector and memory CD8+ T cell survival via TNFR/L family-activated Bcl-2 apoptosis pathway and Caspase cascade.
Stability of peptide:MHCII complexes determines clonal CD4+ T-cell selection (46.4)
Authors: Hausl Christina , Ferrante Andrea , Gorski Jack , Malherbe Laurent
Published: January 2023

Antigen-specific CD4+ T-cells are major regulators of adaptive immune responses. Interactions between TCR and peptide-MHCII complexes are critical for the selection and expansion of these cells. The role of peptide-MHCII stability in CD4+ T-cell selection, however, has not been reported yet. We used altered peptide ligands (APL) which displayed different stabilities in complex with MHCII. Mice were immunized with the APLs and draining lymph nodes were analyzed. Although the accumulation of antigen-specific CD4+ T-cells was similar, we found that the repertoire of T-cells selected was different among the APLs. Single cell sorting of antigen-specific CD4+ T-cells and sequencing of their TCR alpha- and beta-chains revealed that high stability complexes selected a broad and mainly private repertoire of CD4+ T-cells. Low stability complexes on the other hand selected a more focused and public repertoire. Our findings suggest that peptide-MHCII stability is a critical parameter in the selection of CD4+ T-cells during adaptive immune responses.

This study is partly supported by FWF - Austrian Science Fund (Erwin Schroedinger Fellowship).
Resistance of Th17 cells to Activation-Induced Cell Death (47.13)
Authors: Yu Xue-Zhong , Iclozan Cristina , Yang Xuexian , Anasetti Claudio , Dong Chen , Yu Yu
Published: January 2023

Peripheral T cells undergo apoptosis induced by repeated TCR stimulation, known as activation-induced cell death (ACID), which plays an important role in T-cell tolerance. AICD in CD4 T helper (Th) cells including Th1 and Th2 effectors has been extensively studied. Recently, IL-17-producing CD4+ T cells (Th17 cells) have been identified as a unique Th subset, but the susceptibility and underneath molecular mechanism of AICD in Th17 effectors have not been defined. In this study, we found that Th17 cells were significantly less susceptible to AICD upon TCR restimulation compared to Th1 cells. The resistance was confirmed on pure Th17 cells by gating on CD4+RFP+ cells that were polarized from CD4 naïve T cells from il17f/rfp knock-in mice. Furthermore, the resistance of Th17 cells to AICD was also observed when Th17 cells were co-cultured in vitro or co-injected in vivo with Th1 cells. To explore the molecular mechanism, we found that Th17 cells were defective in FasL expression and in caspase activation, but highly expressed anti-apoptotic protein c-FLIP. After knocking down c-FLIP with its specific siRNA, Th17 cells re-expressed FasL and underwent rapid apoptosis upon TCR restimulation. In conclusion, Th17 cells are resistant to AICD likely because the high level of c-FLIP prevents Fas-mediated apoptosis. The resistance of Th17 cells to AICD reveals additional mechanism to explain the high pathogenicity of Th17 cells in autoimmune diseases, and may also provide a rationale to generate tumor-specific Th17 cells for adoptive cell immunotherapy.
Phytohaemagglutinin plus anti-CD28 induce conversion of CD4+CD25- T cells into CD4+CD25+ regulatory T cells (47.17)
Authors: cao mengde , Cabrera Roniel , Xu Yiling , Liu Chen , Nelson David
Published: January 2023

CD4+CD25+ regulatory T cells (Tregs) play a key role in establishing and maintaining immune tolerance via their suppressive activities. The functional analysis, isolation methods, and clinical application of Tregs is impeded by their scarcity in peripheral blood. Large scale in vitro generation of Tregs with stable suppressive property represents an attractive strategy to set up cellular therapies that could prevent and cure T cells-mediated disorders. In this study, we present a novel in vitro approach to generate a large number of inducible Tregs from the conversion of conventional CD4+CD25- T cells by means of PHA and anti-CD28 mAb. The protocol described herein does not involve feeder cells, beads or other cellular compounds that would interfere with in vivo use. These CD4+CD25- - derived inducible Tregs not only have the similar phenotypes of natural Tregs, but also exhibit stronger suppression on in vitro CD4+CD25- T cell activation and proliferation in cell-to-cell contact way. Moreover, due to the limited number of naturally occurring Tregs in the peripheral CD4+ T cell compartment, our strategy might provide a novel basis for convenient therapeutic approaches designed to generate Tregs to known antigens in autoimmune diseases or graft-versus-host reactions for use as targeted immunotherapy. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4+ Tregs. These quickly induced in vitro Tregs can be used for further biological study and developed for antigen-specific cellular immunotherapy.
Involvement of SOCS Proteins in the Inhibitory Effect of IL-27 on Th17 Development (47.21)
Authors: Qin Hongwei , Reynolds Stephanie L. , Cong Yingzi , Elson Charles O. , Benveniste Etty N.
Published: January 2023

Suppressor Of Cytokine Signaling (SOCS) proteins are critical feedback inhibitors of the JAK/STAT signaling pathway and have neuroprotective effects. Recently, several reports have shown that the cytokine IL-27 functions as an inhibitory regulator, and represses the development of Th17 cells associated with autoimmune brain inflammation. Stimulation with IL-27 results in the activation of members of the STAT family, predominantly STAT-1 and STAT-3. In this study, we describe that IL-27 induces SOCS-1 and SOCS-3 gene expression, which is correlated with IL-27 induced STAT-1α and STAT-3 activation in anti-CD3 and anti-CD28 activated CD4+ T cells. Furthermore, we demonstrate that IL-27 induction of SOCS-1 and SOCS-3 gene expression in CD4+ T cells occurs at the transcriptional level as assessed by promoter assays. IL-27-induced SOCS-1 and SOCS-3 expression in CD4+ T cells from WT and STAT-1 deficient mice indicate that induction of SOCS-1 occurs in a STAT-1 dependent manner, and SOCS-3 induction partially depends on STAT-1. The inhibitory effects of IL-27 on Th17 development in CD4+ T cells with specific SOCS-3 deficiency and/or SOCS-1 knockdown will be investigated in the near future. These results indicate that IL-27 induces SOCS-1 and SOCS-3 expression in CD4+ T cells by activation of STAT-1α and/or STAT-3. We propose that SOCS-1 and SOCS-3 may participate in IL-27-mediated suppression of Th17 cell production and autoimmune brain inflammation.
SATB1 regulates expression of multiple genes involved in human T helper cell differentiation (47.23)
Authors: Ahlfors Helena , Limaye Amita , Tuomela Soile , Rasool Omid , Galande Sanjeev , Lahesmaa Riitta
Published: January 2023

Naïve CD4+ Th progenitors (Thp) differentiate to functionally distinct effector T cell subsets including Th1, Th2 and Th17 cells which while being responsible for specific immune functions have also been implicated in pathological responses, such as autoimmunity, asthma and allergy. Special AT-rich binding protein 1 (SATB1) is a global chromatin organizer and a transcription factor induced by interleukin-4 (IL-4) during the early T helper 2 (Th2) cell differentiation. Having established that this upregulation of SATB1 is mediated by STAT6 transcription factor, we became interested in identifying SATB1 target genes to understand its role in human Th cell differentiation. Our results from genome wide transcriptomics analysis after SATB1 knockdown and ChIP-chip studies indicate that SATB1 regulates multiple genes important for Th cell polarization or function. Moreover, several genes are regulated by SATB1 in a Th cell subset specific fashion. Candidate genes of interest have been selected for further studies. The integration of these multiple datasets is in progress and aims at providing a basis for understanding SATB1 regulatory network during the early stages of human Th cell differentiation. This study was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation and Turku University Hospital Fund.
Correlating the phenotype of in vitro-polarized Th17 cells with expression of IL-17A and IL-17F (47.6)
Authors: Schifano Matthew , Konrad Mark A. P. , Li Gangzhou , Araji Soha , Ruiz Natalie , Just Peggy
Published: January 2023

The T-helper 17 (Th17) lineage has surfaced as a topic of great interest in the fields of inflammation and autoimmunity. The secretion of interleukin (IL)-17A and IL-17F has implicated these cells in many autoimmune diseases including rheumatoid arthritis, Crohn's disease, asthma and multiple sclerosis. IL-17A and IL-17F have been shown to form both homodimers and heterodimeric complexes. Characterization of the expression of these Th17 effector molecules together with surface proteins such as CCR6, CD161 and RORγt will be useful for studying further the role of Th17 cells in human disease. In this study, in vitro-polarized Th17 cells were analyzed for surface expression, secretion or intracellular expression of Th17-related proteins. Using an ELISA with minimal cross-reactivity to IL-17A/A and IL-17F/F, IL-17A/F was found to be a major product of in vitro-polarized Th17 cells. Furthermore, IL-17A/A expression was less than or equal to IL-17A/F, while IL-17F/F expression was consistently lower than IL-17A/A or IL-17A/F.
Cytokine-neuroantigen fusion proteins represent a novel therapeutic approach for EAE (48.10)
Authors: Mannie Mark D. , Abbott Derek J. , Blanchfield Lori
Published: January 2023

Cytokine-neuroantigen (NAg) fusion proteins have promise as a new class of antigen-specific therapies for treatment of CNS autoimmune disease. The primary objective of this study was to assess therapeutic efficacy of selected cytokine-neuroantigen fusion proteins in the Lewis rat model of EAE. The neuroantigen domain of each fusion protein was comprised of the major encephalitogenic determinant of myelin basic protein. Three cytokine-NAg fusion proteins were potent inhibitors of EAE. These fusion proteins incorporated the IL-2 cytokine as the N-terminal domain (the IL2NAg fusion protein), the secreted IL-16 cytokine as the C-terminal domain (the NAgIL16 fusion protein), or the IFN-beta cytokine as the N-terminal domain (IFNbeta-NAg fusion protein). Compared to NAg alone, these fusion proteins strongly attenuated the subsequent active induction of EAE. These fusion proteins also inhibited EAE when administered after encephalitogenic challenge during onset of EAE. In conclusion, cytokine-NAg fusion proteins that incorporate IL-2, IL-16, or IFN-beta cytokine domains may comprise an effective tolerogenic therapy for CNS autoimmune disease.
Critical regulation of early Th17 cell differentiation by IL-1 signaling (48.24)
Authors: Chung Yeonseok , Chang Seon Hee , Martinez Gustavo J , Yang Xuexian O , Nurieva Roza , Kang Hong Soon , Watowich Stephanie S , Jetten Anton , Dong Chen
Published: January 2023

Th17 cells have been recently discovered in both mouse and human. Although IL-1 has been shown to be important in human Th17 cell differentiation with little knowledge of the underlying mechanism, its function in mouse is less clear. Here we show that IL-1R1 expression in T cells, which was induced by IL-6, was necessary for Th17-mediated autoimmunity and for early Th17 differentiation in vivo. Moreover, IL-1 signaling in T cells was required in dendritic cell-mediated Th17 differentiation from naïve or regulatory precursors and IL-1 synergized with IL-6 and IL-23 to regulate Th17 differentiation and maintain cytokine expression in effector Th17 cells. Importantly, IL-1 regulated the expression of IRF4 and RORγt during Th17 differentiation; over-expression of these two factors resulted in IL-1-independent Th17 polarization. Our data thus indicate a critical role of IL-1 in Th17 differentiation and this pathway may serve as a novel target for Th17-mediated immunopathology.

The work is supported by research grants from NIH (to CD), an Intramural Research Program of the NIEHS, NIH (to AMJ), Leukemia and Lymphoma Society (to CD) and MD Anderson Cancer Center (to CD and SSW) and the Gillson Longenbaugh Foundation (to SSW). RN is a recipient of a Scientist Development Grant from the American Heart Association. CD is a Trust Fellow of the MD Anderson Cancer Center, a Cancer Research Institute Investigator, a Leukemia and Lymphoma Society Scholar and an American Lung Association Career Investigator.
Physical and Chemical stress induce Changes in the Expression of Ro60, p53, PCNA, Fas-L, Fas-R, Caspase 8, Caspase 3 in skin of Newborn Balb/c mice (48.27)
Authors: Sanchez Rodriguez Sergio  Hugo , Macías-Pérez1 Martha Edith , Reveles Espinoza Alicia Maria , Aguilera Galaviz Luís Alejandro , López-Luna Maria Argelia
Published: January 2023

Systemic Lupus Erythematosus is an autoimmune disease, whose skin lesions are exacerbated by environmental stressors such a UV light and heat. These physical factors modified the expression and localization of Ro60 protein and the homeostasis in the cell cycle. Objective: To better understand the molecular expression profile of Ro60, p53 and PCNA expression, and the possible activation of apoptosis through the Fas-L, FasR, caspase 8, and caspase 3 pathway, we developed an animal model under physical and chemical stress. Methods: Balb/c mice were exposed to UV radiation, heat, HgCl2, H2O2, ethanol, camptothecine and cicloheximide, in absence or presence of caspase 3 inhibitors. Afterwards, a skin extract was made it, proteins were characterized by SDS-PAGE and Western blot, and p53, PCNA, FasL, FasR, caspase 8, and caspase 3 were immunodetected by ECL and densytometric analysis. Results: The Ro60 expression is modified under the influence of physical and chemical stressors, also it is increased mainly with UV light and heat in sharp contrast decreased with caspase 3 inhibitor. It was observed that p53 expression is increased by all the stressors except UV light, using the caspase 3 inhibitor the expression is abolished. In other hand PCNA, is down regulated under the influence of all the stressors but is considerable expressed with caspase 3 inhibitor. The induction of apoptotic pathway using stressors, was monitoring by changes of proteins like FasL, FasR, caspase 8, caspase 3, it was observed an increased levels of this proteins with HgCl2, camptothecine and cicloheximide, however in presence of caspase 3 the expression was decreased.
Chlorpromazine, procainamide and hydralazine produced a murine autoimmune disease resembling human lupus (48.31)
Authors: Wong-Baeza Carlos , España Luis , Tescucano Alonso , Wong Ramírez Carlos , Baeza Isabel
Published: January 2023

Chlorpromazine, procainamide and hydralazine have indeed been implicated in producing a human lupus-like disorder. These drugs induce the formation of a phospholipid arrangement different to the lipid bilayer called non-bilayer phospholipid arrangement (NPA). Since these drugs form NPA in liposomes and cultured cells, it is possible that these drugs can stabilize NPA structures in cell membranes producing an abnormal NPA exposure, perhaps facilitating the subsequent production of specific antibodies. To evaluate this potential role of NPA inductors, the drugs alone or drug-induced NPA were administered to BALB/c mice. We observed that 30-45% of mice treated with NPA inductors and 55-60% treated with drug-induced NPA, produced anti-NPA antibodies. These antibodies appeared 4 weeks prior to anti-cardiolipin, anticoagulant, anti-histone and anti-nuclear antibodies. Mice presented some areas of the face with alopecia, the histopathology of these areas showed similar changes to those found in human lupus skin lesions. Dense inflammatory infiltrate in dermis with deposition of immune complexes in dermal-epidermal junction and also along the basement membrane of glomerular capillaries were observed. This model of lupus might contribute to a better understanding of the immunological basis of human lupus and probably toward the development of better human therapeutic strategies.
IRF5 Genetic Risk Haplotype Influences Host B Cell Gene Responses to Epstein-Barr Virus (EBV) (49.14)
Authors: Templeton Amanda K , Dominguez N , Lu R , Vidal G , Levin J , Sestak A , Kelly J , Kaufman K , Bruner G , Gaffney P , Harley J , James J , Guthridge J , Poole B
Published: January 2023

Both gene and environment interactions play keyroles in the development of systemic lupus erythematosus (SLE). We examined effects of the IRF5 lupus risk haplotype upon the host's B cell response to the binding or infection with EBV, a suspected environmental trigger for SLE. Whole genome microarray expression profile data was collected from cells exposed to EBV for 16 hours. Analysis of gene expression by gene set enrichment analysis revealed that key differences in expression between SLE patients and controls (or individuals carrying the IRF5 risk and non-risk haplotype) were in a subset of interferon response genes. Patients with the IRF5 risk haplotype have a heightened interferon signature under all experimental conditions; whereas, the patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of unrelated, matched controls. Overexpression of interferon pathway genes in B cells following viral exposure in control individuals carrying the IRF5 risk haplotype suggests that the IRF5 risk alleles alone can modulate ones biological response to the environmental insult. Patients carrying either the IRF5 risk or non-risk alleles appear to already be predisposed to having a higher interferon signature even without exposure to virus, suggesting that other genetic factors are also influencing the interferon response, independent of virus. Support by NIH (AI007633 and AI31584).
The Sle2c1 murine susceptibility locus controls T cell homeostasis through defective IL-2 production (49.15)
Authors: Xu Zhiwei , Morel Laurence
Published: January 2023

The function of the Sle2 mouse lupus susceptibility locus and its derived Sle2c1sublocus have been previously shown to be associated with a lower threshold of activation of the BCR leading to an accumulation of B1a cells in NZM2410 model. To explore whether these loci had any effects on other immune components, we introduced lpr as an amplifier into B6.Sle2 and its subcongenic strains, and analyzed systemically their cellular and functional phenotypes. Consequently it was found that the co-expression of Sle2 and lpr on a C57BL/6 background significantly increased lymphadenopathy primarily targeting T cells, as well as induced renal pathology which included heavy lymphoid infiltrates. Comparison of the 3 Sle2 subloci mapped the synergistic interaction with lpr to Sle2c. Further examination of both Fas-deficient and Fas-intact mice showed that Sle2c1 resulted in increased T cell proliferation, a reduced IL-2 production, and a reduced Foxp3+ regulatory T cell compartment in spleen and lymph nodes, phenotypes which are characteristic of lupus in human patients and mouse models. Our results demonstrate that the Sle2c1 locus contains a candidate gene which, in addition to regulate BRC activation threshold, also affects T cells homeostasis through altering IL-2 production and Treg cell balance.
PTPN22 deficiency cooperates with the CD45 E613R wedge allele to break tolerance on a non-autoimmune background (49.18)
Authors: Zikherman Julie , Hermiston Michelle , Steiner David , Hasegawa Kiminori , Chan Andrew
Published: January 2023

Pep and CD45 are tyrosine phosphatases whose targets include the Src-family kinases, critical mediators of antigen receptor signaling. A polymorphism in PTPN22, the gene that encodes the human Pep ortholog Lyp, confers susceptibility to multiple human autoimmune diseases. However, the significance of the R620W risk allele is not clear. We report that misexpression of wild type or R620W Lyp/Pep in Jurkat cells, in the context of their binding partner Csk, unmasks the risk allele as a hypomorph.

Although Pep deficient mice on the B6 background have hyper-responsive memory T cells, they fail to develop autoimmune disease. Mice containing a point mutation in CD45 (E613R) develop a B cell-driven lupus-like disease on the mixed 129/B6, but not on the B6 background. To model in mice how human susceptibility loci such as PTPN22 cooperate to provoke autoimmune disease, we studied the ability of Pep deficiency to act as a genetic modifier of the CD45 E613R mutation on the non-autoimmune B6 background.

Double mutant mice develop a lupus-like disease as well as a lymphoproliferative syndrome. Following antigen receptor stimulation, peripheral B cells in the double mutant mice phenocopy hyper-responsive CD45 E613R B cells, whereas peripheral T cells respond like Pep-/- T cells. These studies suggest that Pep-/- T cells in the context of a susceptible microenvironment can drive hyper-responsive CD45 E613R B cells to break tolerance.
A B cell intrinsic defect initiates autoimmunity in New Zealand Black chromosome 13 congenic mice (49.21)
Authors: Loh Christina , Pau Evelyn , Chang Nan-Hua , Wither Joan
Published: January 2023

Introgression of a New Zealand Black (NZB) chromosome 13 (c13) interval onto a lupus-resistant C57BL/6 (B6) background (denoted, B6.NZBc13) is sufficient to produce many of the hallmarks of lupus. To characterize the immune defects leading to these abnormalities observed in B6.NZBc13 mice, bone marrow (BM) chimeras and BCR transgenic mice were produced. In BM chimeras, transfer of B6.NZBc13 BM cells was sufficient to transfer autoimmunity. Interestingly, in mixed BM chimeras the abnormal T and B cell activation as well as DC expansion was observed in both B6 and B6.NZBc13 derived cells; but with greater B cell activation in B6.NZBc13 derived cells. When an anti-HEL Ig transgene was crossed onto the congenic background disease was abrogated and the abnormal cellular phenotypes normalized. Although tolerance was retained in anti-HEL Ig/soluble HEL double transgenic mice, increased numbers of 'edited' cells were seen in the periphery. B cell function studies revealed altered phosphorylation of signaling molecules downstream of the BCR. These findings indicate the presence of a BM-cell intrinsic defect on NZB c13 that can be localized to a B cell defect, which is necessary to initiate the autoimmune phenotype in B6.NZBc13 mice.
The PTPN22 allelic variant associated with autoimmunity impairs B cell signaling (49.28)
Authors: Habib Tania , Arechiga Adrian F , Funk Andrew J , He Yantao , Zhang Xian , Zhang Zhong-Yin , Buckner Jane H
Published: January 2023

PTPN22 is a gene encoding the protein tyrosine phosphatase (PTPase) Lyp. A single-nucleotide polymorphism (SNP) changing residue 1858 from cytosine to thymidine (1858C/T), encoding the variant Lyp620W, is associated with multiple autoimmune disorders. Studies have demonstrated that Lyp has an inhibitory effect on T cell receptor signaling, and that Lyp620W expression results in a dominant gain of inhibitory function. However, the presence of autoantibodies in all of the diseases associated with the 1858T variant, and recent evidence that Ca2+ flux is altered in B cells of 1858T carriers, strongly suggest that Lyp620W may play a role in B cell signaling and homeostasis. In this study we examined the B cell activation profiles following BCR ligation of healthy subjects who express Lyp620W. We found that B cells from these subjects display impaired B cell signal transduction, characterized by impaired proliferation and a decrease in phosphorylation of key signaling proteins including Syk and PLCγ2. Inhibition of Lyp PTPase activity reversed the signaling defect in B cells expressing Lyp620W, resulting in enhanced BCR activation to the level of LypR620 controls. These findings demonstrate that Lyp620W alters BCR signaling and implicates B cells in the mechanism by which the PTPN22 1858T allelic variant contributes to autoimmunity. This work was supported by funds from the Juvenile Diabetes Research Foundation.
A functional single nucleotide polymorphism in PTPN22 contributes to diabetes susceptibility in the BioBreeding Rat (49.4)
Authors: Sarmiento Janice D. , Wallis Robert , Marandi Leili , Poussier Philippe
Published: January 2023

The C1858T polymorphism in the Ptpn22 gene is associated with increased susceptibility to several autoimmune disorders including Type 1 Diabetes (T1D). This produces a gain-of-function R620W variant with diminished binding affinity to Csk, which, like PTPN22 is an inhibitor of antigen receptor signaling. In the T1D-prone BioBreeding (BB) rat, this gene is located in the Iddm3 locus on chromosome 2, making it a likely candidate gene. Sequencing revealed a non-synonymous SNP causing an A629T substitution C-terminal to the PTPN22 P1 domain that binds to Csk. Co-immunoprecipitations examining the PTPN22-Csk interaction have shown a 2-fold reduction in binding affinity of the BB compared to the ACI variant. Importantly, a genome-wide segregation analysis of an F2(BBxACI.1u.lyp) cohort revealed the dominant effect of the BB allele to confer a 3-fold increased T1D risk compared to the allele carried by the T1D-resistant ACI strain. Homozygosity for the BB allele is also associated with phenotypes indicative of immune dysregulation including increased proportions of activated CD4 T cells in the periphery. Reminiscent of the phenotypes observed in T1D patients carrying the R620W variant, BB-derived T and B cells exhibit hyporesponsiveness to antigen receptor engagement, with a concomitant decrease in IL-2 production by CD4 T cells.

Banting and Best Diabetes Centre, Genome Canada, Ontario Research Foundation and CIHR
A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity (49.6)
Authors: Hsu Lih-Yun , Tan Ying-Xim , Weiss Arthur
Published: January 2023

ZAP-70, a Syk family protein tyrosine kinase, is critical for efficient signaling downstream of the TCR. Our lab and others have previously shown that tyrosine 315 and 319 in the interdomain B positively regulate ZAP-70 function by recruiting Vav1 and Lck, respectively. Surprisingly, in addition to their scaffolding function, we recently discovered that Y315 and Y319 play an important role in the autoinhibition ZAP-70 kinase activity. To address the relative importance of the scaffolding function of these residues versus their autoinhibition function, we generated a knock-in ZAP-70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to ananine residues. These YYAA mice showed many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development, hyporesponsiveness to TCR stimulation, and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Analysis of TCR repertoire revealed that qualitative difference in TCR signaling may skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility.