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Principal Investigator: John D Mountz
Organization: BIRMINGHAM VA MEDICAL CENTER
Fiscal Year: 2024
Funding agency: Veterans Affairs
The overall goal of this project is to determine if the IKAROS transcription factor regulates a program that
maintains IL-4 receptor (IL-4R)-mediated B-cell quiescence to ribonuclear protein (RNP) autoantigens
(autoAgs). We propose that B cell activation through the type I interferon (IFN)/TLR7 activation pathway is
inhibited by this B-cell quiescence program. We also propose that failure to maintain B cell quiescence is
associated with the development of RNP autoantibody in systemic lupus erythematosus (SLE). Our
preliminary data suggest that in both lupus mice and in human SLE, multiple immune disease phenotypes can
be initiated by loss of quiescence at the earliest stage of B cell development; that is, the transitional B cell. The
key forces that promote quiescence is an IKAROS (or IKZF1)-based program that is sustained through IL-4R
signaling on IgD+CD23+ B cells. The major opposing program that promotes loss of quiescence is mediated
through the type I interferon receptor (IFNAR) signaling, assembly and signaling competency of the TLR7
pathway, and development of B cells that can produce autoantibodies. We hypothesize that early-stage loss
of quiescence leads to a series of B cell development defects after the transitional stage involving progression
from a resting naïve to an activated naïve predominance and then to development of the pathogenic
CD11c+Tbet+IgD−CD27− double negative 2 (DN2) or germinal center (GC) B cells that ultimately lead to the
development of RNP autoantibody producing plasmablasts and plasma cells (PB/PC). In Aim 1, we will use
several knockout and knock-in mouse strategies in lupus prone mice to determine if type I IFN and IL-4 act at
the Tr B cells to regulate autoreactive B cell development and survival. The specific effects of Ikaros in
modulating type I IFN and IL-4 signaling-mediated RNP-reactive B-cell quiescence at the Tr and naïve stage
will also be analyzed. In Aim 2, we will determine if SLE patients exhibit a loss of IL-4R/IKZF1-mediated B cell
quiescence program starting at the Tr stage of B-cell development. We will determine if the loss of this
pathway disrupts B-cell tolerance to type I IFN and TLR7 stimulation, leading to the development of RNP-
reactive DN2 B cells and PB/PC. The innovative scientific basis of this proposal will be its ability to interpret
the broad spectrum of immune and disease characteristics of SLE by understanding the key initiating events of
loss of B cell quiescence at the transitional stage. B cells from mouse models of lupus and from SLE subjects
will be characterized by high dimension flow cytometry and transcriptomics analyses for well-established cell
surface protein antigen markers that define B cell development at all major developmental stages. We have
established a team of coordinators at the BVAMC to facilitate recruitment and have acquired numbers of SLE
subjects necessary for definitive and statistically meaningful results. A state-of-the-art immunologic laboratory
and BVAMC flow cytometry facility that includes the latest FACS analysis and sorting equipment, as well as a
dedicated BVAMC 10X single-cell analysis facility, and analytical pipeline for bioinformatics analysis of the
results are established. Significance: The successful conclusion of these experiments will be the development
of new diagnostic and therapeutic approaches. From a diagnostic approach, the key defining phenotype of
quiescent versus non-quiescent B cells can be economically and easily measured using standard clinical
laboratory flow cytometry equipment with a relatively low number of cell surface (such as IL-4R or IFNAR1) or
intracellular factors (such as intracellular IFNβ, TLR7, or IKAROS). This could be extended to analyze
trajectories of abnormal B cell development that result from the loss of B cell quiescence at the earliest stages
to understand therapeutics that may be beneficial in subjects who have acquired SLE in the past. At the very
minimum, such approaches should enable the studies and types of treatments that will promote the
maintenance of B cell quiescence and in effect, force SLE into a state of remission which can be maintained
once the causes of the immune or environmental factors that disrupt such remission are defined.
Terms: <19S Gamma Globulin><Alpha-Beta-Omega Interferon Receptor-1><Antiviral Protein Alpha Type><Autoantibodies><Autoantigens><Autoimmune Diseases><Autologous Antigens><B Cell-Activating Factor Receptor><B blood cells><B cell><B cell growth factor><B cell receptor><B cells><B-Cell Activation><B-Cell Antigen Receptor><B-Cell Development><B-Cell Differentiation Factor-1><B-Cell Growth Factor-1><B-Cell Growth Factor-I><B-Cell Proliferating Factor><B-Cell Stimulating Factor><B-Cell Stimulating Factor-1><B-Cell Stimulation Factor-1><B-Cell Stimulatory Factor-1><B-Cells><B-Lymphocytes><B-cell><BACH2><BACH2 gene><BCDF-1><BCGF><BCGF-1><BCSF 1><BSF-1><BSF1><BTB and CNC Homology 2><Basal Transcription Factor><Basal transcription factor genes><Basic Leucine Zipper Transcription Factor 2><Beta Cell><Binetrakin><Blood Plasma Cell><CD11c><CD124 Antigens><CDw124 Antigen><Cell Body><Cell Communication and Signaling><Cell Cycle><Cell Division Cycle><Cell Signaling><Cell Surface Proteins><Cell surface><Cells><Characteristics><Clinical><Competence><Coupled><Data><Dedications><Defect><Development><Differentation Markers><Differentiation Antigens><Differentiation Markers><Disease><Disease remission><Disorder><Down-Regulation><Endosomes><Environmental Factor><Environmental Risk Factor><Equilibrium><Equipment><Evaluation><Event><Exhibits><Failure><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profiling><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Germinal Center><Goals><HuIFN-Alpha-Rec><Human><IFN><IFN-Gamma><IFN-g><IFN-γ><IFNAR><IFNAR1><IFNAR1 gene><IFNBR><IFNG><IFNγ><IFRC><IGD receptor><IL-4><IL-4 Receptors><IL4 Protein><IL4 Receptors><ITGAX><ITGAX gene><IgD><IgM><Immune><Immune Diseases><Immune Disorders><Immune Dysfunction><Immune Interferon><Immune System Diseases><Immune System Disorder><Immune System Dysfunction><Immune System and Related Disorders><Immunes><Immunochemical Immunologic><Immunodeficiency and Immunosuppression Disorders><Immunoglobulin D><Immunoglobulin M><Immunologic><Immunologic Diseases><Immunologic Factors><Immunological><Immunological Diseases><Immunological Dysfunction><Immunological Factors><Immunological System Dysfunction><Immunologically><Immunologics><In Vitro><Insulin Cell><Insulin Secreting Cell><Interferon Alpha-Beta Receptor Alpha Chain><Interferon Gamma><Interferon Receptor><Interferon Type I><Interferon Type II><Interferons><Interleukin 4 Receptor><Interleukin-4><Interleukin-4 Precursor><Interleukin-4 Receptor Alpha><Intracellular Communication and Signaling><KI mice><Knock-in Mouse><Knock-out><Knockout><Laboratories><Lupus><Lupus Erythematosus Disseminatus><Lymphocyte Stimulatory Factor 1><MCGF-2><Maintenance><Marker Antigens><Mast Cell Growth Factor-2><Mature B-Cell><Mature B-Lymphocyte><Measures><Mediating><Medical center><Mice><Mice Mammals><Modeling><Modern Man><Murine><Mus><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Phenotype><Plasma Cells><Plasmablast><Plasmacytes><Prognosis><Proteins><RNA Expression><Receptor Signaling><Receptosomes><Remission><Rest><Role><SLE><Self-Antigens><Series><Signal Transduction><Signal Transduction Systems><Signaling><Sorting><Structure of germinal center of lymph node><Systemic Lupus Erythematosus><Systemic Lupus Erythematous><Systemic Lupus Erythmatosus><T-Cell Growth Factor 2><TLR7><TLR7 gene><Techniques><Testing><Therapeutic><Toll-Like Receptor 7><Transcript Expression Analyses><Transcript Expression Analysis><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><activated B cells><analyze gene expression><anergy><autoimmune antibody><autoimmune condition><autoimmune disorder><autoimmunity disease><autoreactive B cell><autoreactive antibody><balance><balance function><bio-informatics pipeline><bioinformatics pipeline><biological signal transduction><cytokine><developmental><diagnostic approach><diagnostic strategy><disease phenotype><disseminated lupus erythematosus><environmental risk><experiment><experimental research><experimental study><experiments><flow cytophotometry><gene expression analysis><gene expression assay><global gene expression><global transcription profile><high dimensionality><ifnar1 gene product><immunoglobulin D receptor><immunologic substance><immunological substance><imprint><improved><in vivo><innovate><innovation><innovative><knockin mice><lFN-Gamma><lupus prone mice><mouse model><murine model><new diagnostics><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><next generation diagnostics><novel><novel diagnostics><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><pathway><peripheral tolerance><plasmocyte><prevent><preventing><programs><recruit><response><self reactive B cell><self reactive antibody><single cell analysis><social role><systemic lupus erythematosis><therapeutic target><transcription factor><transcriptional profiling><transcriptome><transcriptomics><type I IFN receptor><type I interferon receptor><uptake><β-cell><β-cells><βCell>