Loss of B cell Tolerance is TCR Dependent [amplicon-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE229921 | GEO | 2023/12/29
REPOSITORIES: GEO
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Project description:Introduction: Autoreactivity to histones is a pervasive feature of several human autoimmune disorders including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. Methods: We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. Results: We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the IgG and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Conclusions: Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified. Serum samples from 20 systemic lupus erythematosis patients were run on the Human Epigenome Microarray Platform V1.0 (HEMP; a single-color platform), in order to profile their autoantibodies against a library of post-translationally modified histone peptides. These 20 samples were randomly selected from a larger cohort previously profiled (data not shown) on the Utz Lab Whole Protein Autoantigen Array V2.0 (a single-color platform), where 14 were histone-reactive and 6 were histone-nonreactive. Control sera from 9 healthy adults and a positive control comprising a mixture of autoimmune sera with defined reactivities, were also run on HEMP V1.0. Together, these samples comprise the data appearing in Figures 1 and S1 (IgG and IgM isotype reactivity profiles, respectively), identifying IgG reactivity to 9 peptides that significantly distinguish histone-reactive from -nonreactive sera among 96 peptides profiled. For data appearing in Figure 5, serum samples from a total of 6 Balb/c mice, consisting of two treatment groups, NETs (Neutrophil Extracellular Traps) and NETs + CRAMP (cathelicidin-related antimicrobial peptide) were collected monthly over a 3-month period, along with a zero time point. These samples were compared with a positive control consisting of serum collected from a MLR/lpr mice exhibiting lupus-like symptoms, and a negative control with no serum. The 0, 1 and 2 month time points were profiled on the Utz Lab Whole Protein Autoantigen Array V2.0 and are shown in Figure 5A-B, while the 1 and 3 month time points were profiled on HEMP V1.0 arrays and shown in Figure 5E. All samples were run once with no replicates.
2012-01-26 | E-GEOD-32542 | biostudies-arrayexpress
Project description:Introduction: Autoreactivity to histones is a pervasive feature of several human autoimmune disorders including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. Methods: We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. Results: We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the IgG and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Conclusions: Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified. Serum samples from 20 systemic lupus erythematosis patients were run on the Human Epigenome Microarray Platform V1.0 (HEMP; a single-color platform), in order to profile their autoantibodies against a library of post-translationally modified histone peptides. These 20 samples were randomly selected from a larger cohort previously profiled (data not shown) on the Utz Lab Whole Protein Autoantigen Array V2.0 (a single-color platform), where 14 were histone-reactive and 6 were histone-nonreactive. Control sera from 9 healthy adults and a positive control comprising a mixture of autoimmune sera with defined reactivities, were also run on HEMP V1.0. Together, these samples comprise the data appearing in Figures 1 and S1 (IgG and IgM isotype reactivity profiles, respectively), identifying IgG reactivity to 9 peptides that significantly distinguish histone-reactive from -nonreactive sera among 96 peptides profiled. For data appearing in Figure 5, serum samples from a total of 6 Balb/c mice, consisting of two treatment groups, NETs (Neutrophil Extracellular Traps) and NETs + CRAMP (cathelicidin-related antimicrobial peptide) were collected monthly over a 3-month period, along with a zero time point. These samples were compared with a positive control consisting of serum collected from a MLR/lpr mice exhibiting lupus-like symptoms, and a negative control with no serum. The 0, 1 and 2 month time points were profiled on the Utz Lab Whole Protein Autoantigen Array V2.0 and are shown in Figure 5A-B, while the 1 and 3 month time points were profiled on HEMP V1.0 arrays and shown in Figure 5E. All samples were run once with no replicates.
2012-01-26 | E-GEOD-32543 | biostudies-arrayexpress
Project description:We used microarrays to determine how the quality and quantity of peptide-MHC impact TCR-induced gene expression in vivo. Adoptively transferred 5CC7 T cells were stimulated in vivo with different doses if two peptides: MCC peptide is a strong agonist and 102S peptide is a weak agonist for the 5CC7 TCR. After 48hours later, T cells were purified and gene expression was assessed using microarray.
2015-05-15 | E-GEOD-68893 | biostudies-arrayexpress
Project description:Naïve T cells constantly experience TCR signals in response to self-peptides presented by MHC (pMHC) in vivo. The intensity of these basal or tonic TCR signals can be indirectly read out by expression of surrogate markers of tonic TCR signaling, including the Nur77-GFP transgene and Ly6c. The strength of tonic TCR signaling varies broadly across the naïve CD4+ T cell population and is correlated with functional heterogeneity.Cells that experience the most basal TCR signaling (Nur77-GFP hi, Ly6c lo) are hyporesponsive to peptide-MHC stimulation. Here we examine whether tonic TCR stimulation is associated with differences in mRNA expression.
2022-08-22 | GSE206073 | GEO
Project description:Naïve T cells constantly experience TCR signals in response to self-peptides presented by MHC (pMHC) in vivo. The intensity of these basal or tonic TCR signals can be indirectly read out by expression of surrogate markers of tonic TCR signaling, including the Nur77-GFP transgene and Ly6c. The strength of tonic TCR signaling varies broadly across the naïve CD4+ T cell population and is correlated with functional heterogeneity.Cells that experience the most basal TCR signaling (Nur77-GFP hi, Ly6c lo) are hyporesponsive to peptide-MHC stimulation. Here we examine whether tonic TCR stimulation is associated with differences in chromatin accessibility.
2022-08-22 | GSE206072 | GEO
Project description:Antigen-specific regulatory T cells (Tregs) suppress pathogenic autoreactivity and are potential therapeutic candidates for autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis is associated with autoreactivity to the Smith (Sm) autoantigen and the human leucocyte antigen (HLA)-DR15 haplotype; hence, we investigated the potential of Sm-specific Tregs (Sm-Tregs) to suppress disease. We identified a novel HLA-DR15 restricted immunodominant Sm T cell epitope using biophysical affinity binding assays, then identified high-affinity Sm-specific T cell receptors (TCRs) using high-throughput single-cell sequencing. Using lentiviral vectors, we transduced our lead Sm-specific TCR onto Tregs derived from patients with SLE who were anti-Sm and HLA-DR15 positive. Compared with polyclonal mock-transduced Tregs, Sm-Tregs potently suppressed Sm-specific pro-inflammatory responses in vitro and suppressed disease progression in a humanized mouse model of lupus nephritis. These results show that Sm-Tregs are a promising therapy for SLE.
2023-11-28 | GSE242152 | GEO
Project description:Peptide exchange technologies are essential for the generation of pMHC-multimer libraries for probing diverse, polyclonal TCR repertoires in various settings. Here we report, using the molecular chaperone TAPBPR, a robust method for the capture of stable, empty MHC-I molecules, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange ‘goldilocks’ peptides with high-affinity peptides of interest in an overnight reaction. Using the same system, we describe high-throughput assays to validate binding of multiple candidate peptides on the empty MHCI groove. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment.
2020-03-12 | GSE146826 | GEO
Project description:The nature of autoantigens that trigger autoimmune diseases has been much discussed but direct biochemical identification is lacking for most. Addressing this question demands unbiased examination of the self-peptides displayed by a defined autoimmune MHC-II molecule. We have examined the immunopeptidome of the pancreatic islets of the NOD mouse that spontaneously develops autoimmune diabetes based on its MHC-II, the I-Ag7 variant. The relevant peptides that induced early CD4 T cells derived from insulin and C-peptide; these were also found in the MHC-II peptidome of the pancreatic lymph node and spleen. The insulin-derived peptides followed a trajectory from internal processing in beta cells to exocytosis, uptake, and presentation in islets and peripheral sites. Such process not only generated conventional epitopes but also resulted in presentation of post-translationally modified peptides, including deamidated and fused sequences. These analyses reveal the key features of a restricted component in the self-MHC-II peptidome causing autoreactivity.
2020-03-12 | PXD015408 | Pride
Project description:The immune response against tuberculosis relies, at least in part, on CD4+ T cells. Protective vaccines require the induction of antigen-specific CD4+ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. We have purified MHC class-I and MHC-II peptides and analysed them by mass spectrometry. We have successfully identified 97 mycobacterial peptides presented by MHC-II and 54 presented by MHC-I, from 76 and 41 antigens, respectively. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cells from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying novel candidate antigens.
2020-01-08 | E-MTAB-8057 | biostudies-arrayexpress
Project description:Activation of CD8+ T cells depends exquisitely on the affinity of the T cell receptor (TCR) for a peptide MHC (pMHC) ligand complex. Here, we activated OT-I transgenic CD8+ T cells with pure peptide and examined early activation responses by single-cell RNA-sequencing. T cells were activated with the high affinity OT-I cognate peptide (N4=SIINFEKL) for 1, 3 or 6 hours, or with reduced affinity peptides (T4=SIITFEKL and G4=SIIGFEKL) or the non-binding peptide (NP68=ASNENMDAM) for 6 hours. Cells were then sorted into 96-well plates by FACS and RNA was sequenced following an adapted Smart-Seq2 protocol.
2018-06-25 | E-MTAB-6051 | biostudies-arrayexpress