Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities

Abstract

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2^-/- and μMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn^-/- IgD^+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire^-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Keywords: PhIP-seq; antibody reactivity; autoimmunity; mouse proteome.

Figure 1.. Design and validation of murine PhIP-seq library.

A. GRCm38.p5 annotated proteins were downloaded from Refseq and 62 amino acid (aa) tiles were chosen to cover the 76,217 proteins with 482,672 peptides with a 19 aa overlap. The tiles contained necessary cloning sites for expression in T7 phage display system. B. Representation of designed oligos after oligo synthesis and cloning. C. Sum of all fold changes above the mean read counts in mock-IP in each experimental sample (mock IP) or mouse strain (Rag2^−/−, μMT, OB1, B6, and Lyn^−/− IgD^+/−) by PhIP-seq. Exact p-value is reported, each dot corresponds to a mouse or mock-IP replicate; Kruskal Wallis test with Tukey HSD post-hoc.

Figure 2.. Identification of autoreactive epitopes recognized by ovalbumin-specific BCR transgenic (OB1) mice.

A. Log₁₀ fold change and z-score over murine background model (mean of Rag2^−/−, μMT, and mock IP) of peptides enriched by PhIP-seq in OB1 mice colored by alignment score to known epitope and essential FGD motif. B. Multiple-sequence alignment of top OB1-enriched peptides C. Logoplot of multiple sequence alignment of 193 peptides enriched by OB1 sera. D. Sum log₁₀ fold change over MBM of OB1 peptides enriched by sera from Rag2^−/−, μMT, OB1, B6, or IgD^+/− Lyn^−/− mice. Exact adjusted p-value is reported, each dot corresponds to a mouse; Kruskal Wallis test with Tukey HSD post-hoc.

Figure 3.. Autoreactivity to nuclear and apoptotic antigens in Lyn^−/− IgD^+/− mice.

A. Heatmap of nuclear proteins enriched in Lyn^−/− IgD^+/− versus B6 mice. B. Sum log₁₀ fold change over mean background (left) and heatmap of log₁₀ fold change (right) of small ribonucleoproteins in B6 or Lyn^−/− IgD^+/− mice. C. Sum log10 fold change over mean background (left) and heatmap of log10 fold change (right) of E3 ubiquitin ligases in B6 or Lyn^−/− IgD^+/− mice. Peptide enrichments were identified by PhIP-seq in A-C. Exact adjusted p-value is reported, each dot corresponds to a mouse; Kruskal Wallis test with Dunn post-hoc.

Figure 4.. Autoreactivity in Aire^−/− mice.

A. Percent of peptides enriched in NOD.Aire^−/− mice by PhIP-seq compared to orthologues of previously reported APS1 reactivities in humans (11) and genes under the control of Aire in mTEC (40). Sum log₁₀ fold change over murine background model of B. Plin1, C. Muc5b in Aire^−/− versus control mice. D. Logistical regression coefficients of top 10 proteins for classifying Aire^−/− versus control mice colored by orthologous APS1 reactivity and/or Aire-dependent mTEC expression. E. Heatmap of perilipin 1 positional sum log₁₀ fold change over background in Aire^−/− or wildtype mice by PhIP-seq annotated with domain positions (top). Fold change of antibody index in Aire^−/− over wildtype mice by SLBA (bottom). F. Inguinal fat pads stained with H&E, higher magnification below. G. Immunohistochemistry of F4/80 (top) or CD4 (bottom) in inguinal fat pads in Aire^−/− versus NOD mice. Arrowhead indicates positive cells. Peptide enrichments were identified by PhIP-seq in A-E; SLBA in E (bottom). Exact adjusted p-value is reported, each dot corresponds to a mouse; Kruskal Wallis test with Dunn post-hoc.