Phage Immunoprecipitation-Sequencing Reveals CDHR5 Autoantibodies in Select Patients With Interstitial Lung Disease

Abstract

Objective: Interstitial lung diseases (ILDs) are a heterogeneous group of disorders that can develop in patients with connective tissue diseases. Establishing autoimmunity in ILD impacts prognosis and treatment. Patients with ILD are screened for autoimmunity by measuring antinuclear autoantibodies, rheumatoid factors, and other nonspecific tests. However, this approach may miss autoimmunity that manifests as autoantibodies to tissue antigens not previously defined in ILD.

Methods: We use Phage Immunoprecipitation-Sequencing (PhIP-Seq) to conduct an autoantibody discovery screen of patients with ILD and controls. We screened for novel autoantigen candidates using PhIP-Seq. We next developed a radio-labeled binding assay and validated the leading candidate in 398 patients with ILD recruited from two academic medical centers and 138 blood bank individuals that formed our reference cohort.

Results: PhIP-Seq identified 17 novel autoreactive targets, and machine learning classifiers derived from these targets discriminated ILD serum from controls. Among the 17 candidates, we validated CDHR5 and found CDHR5 autoantibodies in patients with rheumatologic disorders and importantly, patients not previously diagnosed with autoimmunity. Using survival and transplant free-survival data available from one of the two centers, patients with CDHR5 autoantibodies showed worse survival compared with other patients with connective tissue disease ILD.

Conclusion: We used PhIP-Seq to define a novel CDHR5 autoantibody in a subset of select patients with ILD. Our data complement a recent study showing polymorphisms in the CDHR5-IRF7 gene locus strongly associated with titer of anticentromere antibodies in systemic sclerosis, creating a growing body of evidence suggesting a link between CDHR5 and autoimmunity.

Figure 1

PhIP‐Seq identifies novel candidate autoantigens with predictive capacity for ILD. (A) Candidate autoantigens were selected by identifying genes with PhIP‐Seq Z scores greater than 17 in each cohort and excluding genes in the blood bank control samples that had any Z scores greater than 17. Candidates were further filtered as being found 10 times between both cohorts and found at least once in each cohort. A heatmap showing the number of individual patients for each candidate is shown for all 17 candidates. (B) Candidate autoantigens selected in (A) with limited reactivity in the Center 2 non‐ILD group. The screened population including numbers of patients and samples described in Table 1 for panels A‐B. (C‐D) A random forest classifier was trained on PhIP‐Seq data derived from all screened patients and trained either on (C) Center 1 to predict Center 2 or (D) vice versa to distinguish between patients with and without ILD. Patients with known RA without known ILD were excluded from (C‐D) given potential overlap of autoreactivity with patients with RA and ILD. AUC and 95% CI are annotated on the graphs. A total of 181 samples from 169 patients with non‐ILD and 388 patients with known ILD excluding 10 individuals with ILA in (C‐D). 95% CI, 95% confidence interval; AUC, area under the curve; ILD, interstitial lung disease; PhIP‐Seq, Phage Immunoprecipitation‐Sequencing; RA, rheumatoid arthritis.

Figure 2

CDHR5 is a unique candidate autoantigen in a subset of patients with ILD. (A) Network of interactions for peptide data using a Z score threshold of 50 per individual patient. Each edge indicates co‐occurrence of given candidates within screened patients. Each node represents a gene for which peptides were detected. (B) Number of unique peptides for a given autoantigen is plotted against the number of unique patients with peptide autoreactivity. A Spearman's correlation coefficient and P value is annotated. (C) Co‐occurrence of both gene and peptide data by total number of positive patients with darker intensity colors indicating uniqueness (1:2 indicates the designated autoantigen was found with two other autoantigen candidates in a given patient; 1:1 indicates the designated autoantigen was found with one other autoantigen candidates in a given patient; 1 indicates the specified autoantigen was found in the absence of the remaining 16 candidate autoantigens). (D) Predicted structure of CDHR5 protein using AlphaFold with UniProt accession A0A7L0FKT8; per‐residue confidence score (pLDDT) indicated in legend inset. Sites of CDRH5 peptides from Supplementary Table 2 annotated. ILD, interstitial lung disease; pLDDT, predicted local distance difference test.

Figure 3

CDHR5 autoantibodies identify patients with progressive ILD. (A) RLBA results for CDHR5 autoreactivity (anti‐CDHR5 index on y‐axis) plotted by groups. Patients are separated by having RA without known ILD (RA), all other non‐ILD samples as Control, and all patients with ILD in the ILD group (n = 223 Control samples, n = 461 ILD samples, n = 15 samples for patients with RA without known ILD). Wilcoxon test P value annotated for P < 0.05. (B) Patients with autoreactive CDHR5 indicated by the light orange wedge in the pie chart (CDHR5+, left), and the breakdown of these patients based on their original diagnoses is shown in the bar chart (right). (C) Representative images of computed tomography scans from four disparate ILD categories for the patients with CDHR5+. (D) CDHR5 immunohistochemistry from unused donor lung and a patient with CDHR5 Ab+ lung tissue. (E‐H) Kaplan‐Meier curves for survival probability accounting for the outcome of death (E, G) or accounting for the outcomes of death and lung transplantation (F, H). In (E‐H) survival data from 10 patients with CDHR5+ from Center 1 are included; in (E‐F) survival data from 250 patients from Center 1 included in this study; and (G‐H) includes an additional 195 patients with CTD ILD from Center 1. A log‐rank P value is annotated in (E‐H). CTD, connective tissue disease; HP, hypersensitivity pneumonitis; ILA, interstitial lung abnormality; ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; RA, rheumatoid arthritis; RLBA, radioligand binding assay.