Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis

Abstract

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Fig. 1

Binding autoantibodies to IFN-α₂ in MG subgroups, and in patients with LEMS, RA/SLE, pemphigoid (PEMPH) or viral diseases (VIRAL) or in healthy controls (NHC). Sera were diluted 10-fold and assayed by ELISA. We consider as positive any values greater than a cut-off 2 s.d. above the mean of the controls’ (this cut-off averaged 0·2 over many assays; dotted line). The percentages of positives for each category are shown in Table 1. In the TMG^– group, the larger triangles represent those cases positive for anti-AChR antibodies.

Fig. 2

Binding autoantibodies in TMG⁺ patients to type I and II IFNs (assayed by ELISA as for Fig. 1). For IFN-α₂, the TMG⁺ patients have been divided into those already receiving immunosuppressive treatment (closed symbols, left) and those sampled at diagnosis, before any immunosuppressive treatment was given (open symbols, right). We consider as positive any values > 2 s.d. above the mean of the controls’ (average absorbance values ranged from 0·2 to 0·3 depending on the cytokine ELISA). Data for NHC are shown in Fig. 1 for IFN-α2 and non-graphically in Table 1 for IFN-β and IFN-ω; none was positive for IFN-ω, IFN-Con1 and IFN-γ (data not shown).

Fig. 3

Neutralization titres against type I IFNs and IL-12 in sera from TMG⁺ patients. For each cytokine (except IFN-β), the patients already receiving immunosuppressive treatment are shown by closed symbols (left) and those sampled before any immunosuppressive treatment by open symbols (right). Titres of 100 or greater are considered to be positive. No positives were found in the NHC category (Table 1 and data not shown)

Fig. 4

Lack of correlation between anti-IFN-α2- and anti-IL-12- binding antibodies in the TMG⁺ group. The arrows signify those few sera that also bind IFN-β, which are widely scattered.

Fig. 5

Binding autoantibodies to IL-12 in the same patient groups as in Fig. 1 (and assayed by ELISA in sera diluted 10-fold). Cut-off is as for Fig. 1 (it averaged 0·23 over many assays; dotted line). The percentages of positives for each category are shown in Table 1. In the TMG^– group, the larger triangles represent those cases which were positive for anti-AChR antibodies.