Browsing by Subject "Autoimmunity"
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Item Open Access Analysis of TCR Signaling and Erk Activation in T Cell Development and Autoimmunity(2012) Fuller, Deirdre MarieLAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-γ1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LATm/m) mice, have drastically impaired thymocyte development; however, CD4+ T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LATm/m mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre+LATf/m mice). LATf/m mice develop a similar lymphoproliferative syndrome as LATm/m mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity.
Analysis of LATm/mRasGRP1-/- mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LATf/mRasGRP1-/- mice and observed that they developed disease similarly to LATf/m mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LATm/mGads-/- mice had an even more dramatic block in the DN stage of thymocyte development compared to LATm/m controls, although by four months of age CD4+ T cells had expanded. Following deletion of the wildtype LAT allele, LATf/mGads-/- mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads.
In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1d/d mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4+ T cells expanded and auto-antibodies were produced. RasGRP1d/d thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.
Item Open Access Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.(J Autoimmun, 2014-08) Yi, JS; Guidon, A; Sparks, S; Osborne, R; Juel, VC; Massey, JM; Sanders, DB; Weinhold, KJ; Guptill, JTMuscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.Item Open Access Complement C4 inhibits systemic autoimmunity through a mechanism independent of complement receptors CR1 and CR2.(J Exp Med, 2000-11-06) Chen, Z; Koralov, SB; Kelsoe, GThe complement system enhances antibody responses to T-dependent antigens, but paradoxically, deficiencies in C1 and C4 are strongly linked to autoantibody production in humans. In mice, disruption of the C1qa gene also results in spontaneous autoimmunity. Moreover, deficiencies in C4 or complement receptors 1 and 2 (CR1/CR2) lead to reduced selection against autoreactive B cells and impaired humoral responses. These observations suggest that C1 and C4 act through CR1/CR2 to enhance humoral immunity and somehow suppress autoimmunity. Here we report high titers of spontaneous antinuclear antibody (ANA) in C4(-/)- mice. This systemic lupus erythematosus-like autoimmunity is highly penetrant; by 10 mo of age, all C4(-)(/)- females and most males produced ANA. In contrast, titers and frequencies of ANA in Cr2(-)(/)- mice, which are deficient in CR1 and CR2, never rose significantly above those in normal controls. Glomerular deposition of immune complexes (ICs), glomerulonephritis, and splenomegaly were observed in C4(-)(/)- but not Cr2(-)(/)- mice. C4(-)(/)-, but not Cr2(-)(/)-, mice accumulate activated T and B cells. Clearance of circulating ICs is impaired in preautoimmune C4(-)(/)-, but not Cr2(-)(/)-, mice. C4 deficiency causes spontaneous, lupus-like autoimmunity through a mechanism that is independent of CR1/CR2.Item Open Access JunB promotes Th17 cell identity and restrains alternative CD4+ T-cell programs during inflammation.(Nature communications, 2017-08-21) Carr, Tiffany M; Wheaton, Joshua D; Houtz, Geoffrey M; Ciofani, MariaT helper 17 (Th17) cell plasticity contributes to both immunity and autoimmunity; however, the factors that control lineage flexibility are mostly unknown. Here we show the activator protein-1 (AP-1) factor JunB is an essential regulator of Th17 cell identity. JunB activates expression of Th17 lineage-specifying genes and coordinately represses genes controlling Th1 and regulatory T-cell fate. JunB supports Th17 cell identity by regulating key AP-1 complex constituents. In particular, JunB limits the expression of the subset repressor IRF8, and impedes access of JunD to regulatory regions of alternative effector loci. Although dispensable for homeostatic Th17 cell development, JunB is required for induction and maintenance of Th17 effector responses in the inflammatory contexts of both acute infection and chronic autoimmunity in mice. Through regulatory network analysis, we show that JunB is a core regulator of global transcriptional programs that promote Th17 cell identity and restrict alternative CD4+ T-cell potential.AP-1 family transcription factors regulate CD4+ T helper cell differentiation. Here the authors show that the AP-1 member JunB is a nonredundant regulator of transcriptional programs that support Th17 cell identity and restrain alternative Th1 and Treg cell fates in inflammatory contexts of acute fungal infection and chronic autoimmunity.Item Open Access Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection.(PLoS Med, 2009-07-07) Levesque, Marc C; Moody, M Anthony; Hwang, Kwan-Ki; Marshall, Dawn J; Whitesides, John F; Amos, Joshua D; Gurley, Thaddeus C; Allgood, Sallie; Haynes, Benjamin B; Vandergrift, Nathan A; Plonk, Steven; Parker, Daniel C; Cohen, Myron S; Tomaras, Georgia D; Goepfert, Paul A; Shaw, George M; Schmitz, Jörn E; Eron, Joseph J; Shaheen, Nicholas J; Hicks, Charles B; Liao, Hua-Xin; Markowitz, Martin; Kelsoe, Garnett; Margolis, David M; Haynes, Barton FBACKGROUND: The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells. METHODS AND FINDINGS: In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis. CONCLUSIONS: Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.Item Open Access Rapid complete response of metastatic melanoma in a patient undergoing ipilimumab immunotherapy in the setting of active ulcerative colitis.(J Immunother Cancer, 2015) Bostwick, A Doran; Salama, April K; Hanks, Brent AWhile blockade of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) T cell regulatory receptor has become a commonly utilized strategy in the management of advanced melanoma, many questions remain regarding the use of this agent in patient populations with autoimmune disease. We present a case involving the treatment of a patient with stage IV melanoma and ulcerative colitis (UC) with anti-CTLA-4 antibody immunotherapy. Upon initial treatment, the patient developed grade III colitis requiring tumor necrosis factor-alpha (TNF-α) blocking antibody therapy, however re-treatment with anti-CTLA-4 antibody following a total colectomy resulted in a rapid complete response accompanied by the development of a tracheobronchitis, a previously described extra-intestinal manifestation of UC. This case contributes to the evolving literature on the use of checkpoint inhibitors in patients also suffering from autoimmune disease, supports future clinical trials investigating the use of these agents in patients with autoimmune diseases, and suggests that an understanding of the specific molecular pathways involved in a patient's autoimmune pathology may provide insight into the development of more effective novel combinatorial immunotherapeutic strategies.Item Open Access Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration.(Experimental eye research, 2017-02) Iannaccone, Alessandro; Hollingsworth, TJ; Koirala, Diwa; New, David D; Lenchik, Nataliya I; Beranova-Giorgianni, Sarka; Gerling, Ivan C; Radic, Marko Z; Giorgianni, FrancescoWe report on a novel autoantigen expressed in human macular tissues, identified following an initial Western blot (WB)-based screening of sera from subjects with age-related macular degeneration (AMD) for circulating auto-antibodies (AAbs) recognizing macular antigens. Immunoprecipitation, 2D-gel electrophoresis (2D-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), direct enzyme-linked immunosorbent assays (ELISA), WBs, immunohistochemistry (IHC), human primary and ARPE-19 immortalized cell cultures were used to characterize this novel antigen. An approximately 40-kDa autoantigen in AMD was identified as the scavenger receptor CD5 antigen-like protein (CD5L), also known as apoptosis inhibitor of macrophage (AIM). CD5L/AIM was localized to human RPE by IHC and WB methods and to retinal microglial cells by IHC. ELISAs with recombinant CD5L/AIM on a subset of AMD sera showed a nearly 2-fold higher anti-CD5L/AIM reactivity in AMD vs. Control sera (p = 0.000007). Reactivity ≥0.4 was associated with 18-fold higher odds of having AMD (χ2 = 21.42, p = 0.00063). Circulating CD5L/AIM levels were also nearly 2-fold higher in AMD sera compared to controls (p = 0.0052). The discovery of CD5L/AIM expression in the RPE and in retinal microglial cells adds to the known immunomodulatory roles of these cells in the retina. The discovery of AAbs recognizing CD5L/AIM identifies a possible novel disease biomarker and suggest a potential role for CD5L/AIM in the pathogenesis of AMD in situ. The possible mechanisms via which anti-CD5L/AIM AAbs may contribute to AMD pathogenesis are discussed. In particular, since CD5L is known to stimulate autophagy and to participate in oxidized LDL uptake in macrophages, we propose that anti-CD5L/AIM auto-antibodies may play a role in drusen biogenesis and inflammatory RPE damage in AMD.Item Open Access Roles of Id3 and IL-13 in a Mouse Model of Autoimmune Exocrinopathy(2015) Belle, IanWithin the field of immunology, the existence of autoimmune diseases presents a unique set of challenges. The immune system typically protects the host by identifying foreign pathogens and mounting an appropriate response to eliminate them. Great strides have been made in understanding how foreign pathogens are identified and responded to, leading to the development of powerful immunological tools, such as vaccines and a myriad of models used to study infectious diseases and processes. However, it is occasionally possible for host tissues themselves to be inappropriately identified as foreign, prompting an immune response that attempts to eliminate the host tissue. The immune system has processes in place, referred to as selection, designed to prevent the development of cells capable of recognizing the self as foreign. While a great deal of work has been invested in understanding these processes, many concrete answers remain elusive.
Our laboratory, which focuses on understanding the roles of E and Id proteins in lymphocyte development, has established the Id3 knockout mouse as a model of autoimmune disease. Id3 knockout mice develop a disease reminiscent of human Sjӧgren's Syndrome, an autoimmune disease that progressively damages the salivary and lachrymal glands. Continued study of this model has yielded interesting results. These include the identification of CD4+ T cells as initiators of disease as well as the identification of the cytokine Interleukin 13 (IL-13) as a potential causative agent. However, the source of IL-13, its true role as a causative agent of disease, as well as the developmental basis for its elevated expression remained elusive.
To this end, I utilized a reporter gene that enabled me to detect cells producing IL-13 as well as test the effects of IL-13 deletion on disease progression. Using this system, I was able to identify both CD4+ T cells and γδ T cells as major sources of IL-13. I was also able to determine that elimination of IL-13 in Id3 knockout mice was sufficient to block the development of disease symptoms, reinforcing the hypothesis that IL-13 is a causative agent in disease initiation. Finally, I attempted to better characterize the phenotype of cells producing IL-13. These experiments indicated that the T cell receptor (TCR) repertoire of Id3 knockout mice is markedly different than that of wild-type (WT) mice. Furthermore, cells bearing certain TCRs appeared to express IL-13 at dramatically different rates, indicating that certain TCRs may be predisposed to IL-13 particular effector fates.
Item Embargo Th17 Cell Pathogenicity Promoted by Integrin α3 During Autoimmune Neuroinflammation(2022) Park, EunchongAutoimmune diseases are caused by dysregulated immune responses against self. Multiple sclerosis (MS) is one such autoimmune disease in which the central nervous system (CNS) is affected by chronic inflammation, and Th17 cells are critical mediators of disease pathogenesis. While targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic Th17 cell migration. Here, we report integrin α3 as a Th17 cell-selective determinant of pathogenicity in experimental autoimmune encephalomyelitis, a mouse model of MS. CNS-infiltrating Th17 cells express high integrin α3, the expression of which is induced by transcription factors that are required for Th17 cell specification. The deletion integrin α3 in CD4+ T cells or IL-17A-fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 promoted the polarization, proliferation, and transmigration of Th17 cells, and integrin α3-deficiency enhanced the retention of CD4+ T cells in the perivascular space of the blood-brain barrier. Notably, differential RNA-seq expression analysis revealed that Th17 cells continuously depend on integrin α3 to maintain Th17 cell identity and effector function. The requirement of integrin α3 in Th17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.
Item Open Access The Gatekeeper of TCR Signaling: LAT in T cell Homeostasis and Autoimmunity(2015) O'Brien, Sarah ALinker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLCγ1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLCγ1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ αβ T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLCγ1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.
First, we investigated the importance of the LAT-PLCγ1 interaction in γδ T cells by crossing LATY136F mice with TCRβ-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial γδ T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by αβ T cells in LATY136F mice. Development of these hyperproliferative γδ T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ γδ T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLCγ1 signaling may function differently in various subsets of γδ T cells.
In addition to examining γδ and αβ T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ αβ T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ αβ LATY136F T cells demonstrated that NFκB, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLCγ1 signaling in regulating IL6 production by T cells during autoimmunity.
Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLCγ1 signaling in modulating Tfh cell differentiation and BCL6 expression.