Anti-interferon autoantibodies in autoimmune polyendocrinopathy syndrome type 1

Abstract

Background: The autoimmune regulator (AIRE) gene influences thymic self-tolerance induction. In autoimmune polyendocrinopathy syndrome type 1 (APS1; OMIM 240300), recessive AIRE mutations lead to autoimmunity targetting endocrine and other epithelial tissues, although chronic candidiasis usually appears first. Autoimmunity and chronic candidiasis can associate with thymomas as well. Patients with these tumours frequently also have high titre immunoglobulin G autoantibodies neutralising type I interferon (IFN)-alpha and IFN-omega, which are secreted signalling proteins of the cytokine superfamily involved in both innate and adaptive immunity.

Methods and findings: We tested for serum autoantibodies to type I IFNs and other immunoregulatory cytokines using specific binding and neutralisation assays. Unexpectedly, in 60/60 Finnish and 16/16 Norwegian APS1 patients with both AIRE alleles mutated, we found high titre neutralising immunoglobulin G autoantibodies to most IFN-alpha subtypes and especially IFN-omega (60% homologous to IFN-alpha)-mostly in the earliest samples. We found lower titres against IFN-beta (30% homologous to IFN-alpha) in 23% of patients; two-thirds of these (from Finland only) also had low titres against the distantly related "type III IFN" (IFN-lambda1; alias interleukin-29). However, autoantibodies to the unrelated type II IFN, IFN-gamma, and other immunoregulatory cytokines, such as interleukin-10 and interleukin-12, were much rarer and did not neutralise. Neutralising titres against type I IFNs averaged even higher in patients with APS1 than in patients with thymomas. Anti-type I IFN autoantibodies preceded overt candidiasis (and several of the autoimmune disorders) in the informative patients, and persisted for decades thereafter. They were undetectable in unaffected heterozygous relatives of APS1 probands (except for low titres against IFN-lambda1), in APS2 patients, and in isolated cases of the endocrine diseases most typical of APS1, so they appear to be APS1-specific. Looking for potentially autoimmunising cell types, we found numerous IFN-alpha(+) antigen-presenting cells-plus strong evidence of local IFN secretion-in the normal thymic medulla (where AIRE expression is strongest), and also in normal germinal centres, where it could perpetuate these autoantibody responses once initiated. IFN-alpha2 and IFN-alpha8 transcripts were also more abundant in antigen-presenting cells cultured from an APS1 patient's blood than from age-matched healthy controls.

Conclusions: These apparently spontaneous autoantibody responses to IFNs, particularly IFN-alpha and IFN-omega, segregate like a recessive trait; their high "penetrance" is especially remarkable for such a variable condition. Their apparent restriction to APS1 patients implies practical value in the clinic, e.g., in diagnosing unusual or prodromal AIRE-mutant patients with only single components of APS1, and possibly in prognosis if they prove to predict its onset. These autoantibody responses also raise numerous questions, e.g., about the rarity of other infections in APS1. Moreover, there must also be clues to autoimmunising mechanisms/cell types in the hierarchy of preferences for IFN-omega, IFN-alpha8, IFN-alpha2, and IFN-beta and IFN-lambda1.

Figure 1. Anti-IFN Neutralisation Titres in APS1 Patients at Different Sampling Times

(A) Anti-IFN neutralisation titres in the first and last available serum samples from 51 AIRE-genotyped Finnish patients. We use arrowheads to mark the unusual APS1 patients A–D (detailed in Table 3), and numerals for patients whose samples neutralised both IFN-β and IFN-λ1. Bars indicate geometric mean titres. From first to last available samples (average interval = 16 y), titres were substantially increased, unchanged, or decreased in 19%, 43%, and 38% patients, respectively. (B) Anti-IFN neutralisation titres in Finnish APS1 patients against IFN-α2, IFN-ω, and IFN-β in the first available sera in relation to the time of diagnosis of CMC. Results are grouped for patients sampled 1 yr prior to diagnosis of CMC (−1), at diagnosis (0), or within the indicated number of years thereafter (0–4, 4–7, 7–10, 10–19, or >19 y). This figure excludes four otherwise typical APS1 patients who never developed overt CMC but who also had high titres against IFN-ω (see text); it includes 63 AIRE-genotyped and 11 untyped patients. For IFN-β, only the positive patients are shown (for clarity). Arrowheads mark unusual APS1 patients: patients A–E (detailed in Table 3); patient x, who currently has only one detectable AIRE mutation; patients y and z, who are two of the three Finnish patients with no detectable AIRE mutations; and patient E, who meets the criteria for APS1 (Table 3), but for whom no neutralising autoantibodies or AIRE mutations have yet been found.

Figure 2. Typical and Unusual Neutralisation Profiles against IFN-α Subtypes and IFN-ω

Patient (Pt) X, a Finnish R257X homozygote, has a typical profile (A and B); patients B (C and D) and D (E and F) have unusual profiles (also detailed in Table 3).

Figure 3. Paraffin Sections of Normal Child Thymus or Reactive Tonsil Double-Labelled for IFN-α and MxA

(A) In the thymic cortex (C) to the left, there are scattered IFN-α⁺ cells (green) with almost no nearby MxA⁺ cells (red), but MxA⁺ cells are much more abundant in the medulla (M) to the right, especially around the Hassall's corpuscle at the edge. Results were similar in two other child thymi, though one showed fewer IFN-α⁺ cells in the cortex. The very dense packing precludes precise phenotyping of medullary IFN-α⁺ cells in paraffin sections. (B) Similar staining in a reactive tonsil shows a germinal centre (to the left of the arrows) with several IFN-α⁺ cells and punctate labelling for MxA. Throughout the adjacent T cell area (right), MxA is expressed strongly by a variety of cell types, as already reported [38], whereas IFN-α⁺ cells are concentrated along a small blood vessel.

Figure 4. Expression of IFN-α2 and IFN-α8 by Immature DCs and Monocytes Cultured from an APS1 Patient Relative to Two Healthy Blood Donors

Expression by immature DCs (A) and monocytes (B). The APS1 donor has typical clinical features of APS1 and high anti–type I IFN titres (see footnote of Table 1). RT-PCR reactions were performed in parallel with HPRT as a “house-keeping” gene; GADPH gave very similar results when tested in one of the healthy donors, C1 (for IFN-α8). The relative gene (mRNA) expression levels were calculated using the comparative Ct (ΔΔCt) method (according to Applied Biosystems) to yield 2^−ΔΔCt, where Ct represents the threshold cycle. Every sample was run at least three times in three parallel reactions. Results are depicted in histogram format as ratios of the expression levels in cells from the APS1 donor (which have been given a nominal value of 1.0) to those in the cells from the controls, C1 and C2. The bars represent the standard error of the mean of each ratio.