Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: (A) Radioligand binding assay for detection of anti-PLIN1 antibodies. Radiolabeled PLIN1 protein was incubated with sera from healthy controls (HC, n = 54) or from APS1 patients with or without lipodystrophy (APS1 +/- LD, n = 1/68). Dotted line indicates mean + 3SD of healthy controls. (B) Validation of autoantibodies to PLIN1 in orthogonal cell-based assay. Fixed stomach tissue of mice was mounted and immunostained with sera from Case report patient 1 and commercial antibody to PLIN1. A mix of secondary antibodies anti-Human IgG-alexa547 and anti-Rabbit alexa488 at 1:2000 were used to visualize Human IgG and PLIN1 antibodies, respectively. A Merge is provided on the right of individual images. Images taken at 20X magnification. Note yellow in Merge indicating colocalization of PLIN1 (Green) and Human IgG (RED). DAPI is blue and indicates nuclei. Scale bar indicates 100 microns.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Radio Ligand Binding Assay, Incubation, Cell Based Assay

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Autoantibodies to PLIN1 in sera from a patient with autoimmune AGL following cancer immunotherapy. (A) Radioligand binding assays screening for PLIN1 antibodies in sera from all time points from index patient 2 as well as checkpoint-treated control patients without lipodystrophy (n=7), as done for case report 1. Dotted line indicates mean + 3 std. dev. of healthy controls (n = 11). Numbers to the right of circles indicate the time point series, for reference in panel B. The left panel describes the clinical timeline corresponding to timepoints T0-T4. CPI was discontinued at 34 cycles (16 months) due to progressive weight loss and elevated LFTs, with futher work-up over subsequent months. (B) Validation of autoantibodies to PLIN1 using immunohistochemistry on mouse enteric tissue, as done in . Sera was used from either a control (immunotherapy but no AGL) or case report 2 (Immunotherapy with AGL) with various time points. Each column represents an individual sample, from left to right ; sera from checkpoint control pre-treatment, sera from checkpoint control post treatment with no autoimmunity, Case report 1 pre-therapy, Case report 1 post-therapy-1, Case report 1 post-therapy-3, Case report 1 post-therapy-4. Images are 600×600 pixel insets from original 40X image. Scale bar represents 100 microns. CP = Checkpoint patient controls.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Binding Assay, Immunohistochemistry

Journal: medRxiv

Article Title: Perilipin-1 autoantibodies linked to idiopathic lipodystrophy in the setting of two distinct breaks in immune tolerance

doi: 10.1101/2021.09.24.21263657

Figure Lengend Snippet: Discovery and validation of autoantibodies to Perilipin-1 in Aire -/- mouse sera. (A) PhIP-Seq analysis in Aire -/- (“KO”) and Aire +/+ (“WT”) mice. Aggregated Plin1 PhIP-Seq data from Aire -/- (KO, n=4) and Aire +/+ (WT, n = 3) mice. (B) Whole cell lysates generated from 293T cells expressing full-length mouse PLIN1 were incubated with sera from Aire -/- (KO, n=4) or Aire +/+ (WT, n=3) mice. Antibodies were immunoprecipitated using AG beads and IP elutions were subject to SDS-PAGE immunoblotting. AG lane indicates an IP using AG beads only, no sera. Input lane indicates loading of whole cell lysate with and without (-) transfection of PLIN1-myc-flag plasmid. IP elutions and Input were immunostained with either anti-Flag IgG to identify positive anti-Plin1 signal, or anti-mouse IgG to show qualitative capture of IgG from sera. (C) Representative image of immunohistochemistry on mouse enteric tissue showing positive colocalization of antibodies from Aire -/- sera (KO) and commercial anti-Plin1 IgG. Primary antibodies from Aire -/- mouse (KO) sera and commercial antibody to PLIN1 were visualized with secondaries to Mouse IgG (Alexa-567, RED) and Rabbit IgG (Alexa-488, GREEN), respectively. DAPI (BLUE) stains nuclei.

Article Snippet: Full-length mouse pCMV6-Plin1-myc-flag (Origene, RC206292) was sequenced verified and used for transfection in 293T cells.

Techniques: Generated, Expressing, Incubation, Immunoprecipitation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Immunohistochemistry