PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis

Abstract

Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual's unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.

Figure 1. Enrichment recurrence and multi-epitope targeting

A. Frequency distribution of the 14,604 different peptides enriched above threshold by at least one healthy individual. AAb, autoantibody; TTN, titin; ACVR2B, activin receptor type-2B; MAGEE1, melanoma antigen family E, 1. Number following underscore denotes the 36-residue tile (order is N- to C-terminus). B. Multi-epitope targeting of the CENPC1 protein. Peptides are organized from top to bottom, N- to C-terminus. Peptide enrichments above threshold are colored black. Three individuals exhibit evidence of multi-epitope (antigen driven) responses. The sample category is denoted below the individual’s data. BC, breast cancer; HC, healthy control; MS, multiple sclerosis. C. Frequency distribution of the 7,619 different ORFs enriched above threshold by at least one healthy individual. NEB, nebulin.

Figure 2. Analysis of T1D and healthy control sera

A. Sensitivity of PTPRN (IA-2) autoantibody detection by PhIP-Seq compared to RIA in T1D patients (red boxes) and healthy controls (circles). PhIP-Seq values correspond to the most enriched peptide from the PTPRN ORF. A value of >0.5 is considered positive for RIA. None of the HC sera were positive for PTPRN reactivity by RIA or PhIP-Seq. 27 of 39 (69%) T1D patients were positive for PTPRN reactivity by RIA, and 4 of these 27 were positive by PhIP-Seq (15%). B. Comparison of the total number of unique peptides enriched by individuals of different age groups and disease status. “Youth” individuals are 12 years old or younger; “Adult” individuals are 18 years old or older. Statistical comparisons of the means were performed using the Student’s t test, with one tail.

Figure 3. RA associated peptides and their clusters

A. Permutation analysis of peptide enrichments associated with RA. “Observed” bars indicate the number of peptides associated with RA at a given P-value by Fisher’s exact test. “Expected” bars show the number of peptides expected to have a −log10 Fisher P-value at least that extreme due to chance alone (as determined by permuting sample labels). B. RA1 and RA2 peptide enrichment heat map illustrating nonrandom segregation of above-threshold peptide enrichments and RA patients. −Log10 P-values less than 4 are white and greater than 4 are black. Patients are organized by their seropositivity, and by their RA1/RA2 status.

Figure 4. MS associated peptides share a sequence motif

A. Permutation analysis of peptide enrichments associated with MS. “Observed” bars indicate the number of peptides associated with MS at a given P-value by Fisher’s exact test. “Expected” bars show the number of peptides expected to have a −log10 Fisher P-value at least that extreme due to chance alone (as determined by permuting sample labels). B. Peptide enrichment heat map (as in Figure 1) illustrating nonrandom segregation of MS1 peptide enrichments (rows) and MS patients (columns). −Log10 P-values of enrichment less than 4 are white and greater than 4 are black. Patients are organized by their MS1 status. C. Alignment of the co-segregated peptides reveals a shared epitope. D. MS associated epitope (MS1) motif logo, calculated from the peptides in C (MEME software).

Figure 5. Analysis of MS patient CSF/serum pairs

Scatter plots of matched samples from the same individuals. Each sample was analyzed in duplicate and the average −log10 P-value is plotted for peptides enriched by more than −log10 P value of 3 in both duplicates. A. Patient 9292 peptide enrichments in CSF versus serum, and enrichment of nearly identical peptides from IFN-α5/14 specifically in the CSF. B. Patient 10894 peptide enrichments in CSF versus serum, and CSF-specific enrichment of the JCV motif is illustrated. C. Patient 8911 peptide enrichments in CSF versus serum. D. PhIP-Seq serum profile in patient 8911 taken at two time points.