In the current study we sought to understand 1) the response displayed by purified IgG anti-Jo1 autoantibodies derived from serum towards HisRS-FL, one HisRS splice variant and single HisRS domains as well as IgG and IgA reactivity in paired serum and BALF towards the same protein variants; 2) possible associations between clinical manifestations and the pattern of anti-HisRS reactivity in circulation and in BALF both at the time of diagnosis and during the disease course; and 3) the affinity profile of anti-Jo1 autoantibodies against HisRS.
In our study we could demonstrate that purified IgG anti-Jo1 autoantibodies in sera from a time close to diagnosis of IIM/ASS exhibited high and multiple reactivities against the HisRS-FL, splice variant and domains, with a particularly strong reactivity against the WHEP domain and the HisRS-FL. A similar reactivity pattern to the different HisRS variants was observed in most anti-Jo1 positive patients. The ELISA results were confirmed by WB indicating that anti-Jo1 antibodies recognize both conformation-dependent (ELISA) and -independent epitopes (WB). In BALF the highest reactivity of both IgG and IgA anti-Jo1 autoantibodies was also directed towards the HisRS-FL and when compared to matched serum samples there were no signs of anti-Jo1 autoantibody enrichment in BALF.
Despite the overall similar antibody reactivity to the versions of the HisRS antigen seen in our patient cohort, we observed differences in clinical manifestations associated with the different levels of reactivities to the HisRS-FL antigen at the time of diagnosis. Thus, patients with high IgG serum levels towards HisRS-FL at diagnosis were more likely to ever present with ILD and arthritis, but less likely to have skin rash compared to patients with low to moderate anti-HisRS-FL IgG levels or anti-Jo1 negative. Furthermore, IgG anti-WHEP reactivity in BALF correlated with poor pulmonary function. These observations were further strengthened by applying an unbiased multivariate statistical analysis. The significant correlation between antibody reactivity (especially anti-WHEP) in the BALF at diagnosis and poor pulmonary function together with BALF inflammatory content (e.g. eosinophils, alveolar macrophages, and CD4/CD8 ratio), supports our hypothesis of an association between autoantibody reactivity towards the WHEP domain and lung involvement in IIM/ASS.
Anti-Ro52 antibodies, which have been reported to be associated with more severe ILD when present in serum together with anti-Jo1 antibodies (30–32), were more prevalent in the group of patients with higher anti-HisRS-FL reactivity. An unexpected finding in our study was that patients with low to moderate anti-HisRS-FL reactivity presented with worse pulmonary function (lower FEV and TLC%). Maybe this could be attributed to the significantly higher proportion of patients being ever smokers in this sub-group.
In the IgG purified from sera, the highest anti-HisRS reactivity levels were recorded before and at the time of diagnosis, and generally decreasing thereafter. In addition, IgG against HisRS-FL displayed high affinity already at diagnosis. The reactivity to HisRS-FL remained high, up to three years post-diagnosis, whereas the antibody response to the splice variant and HisRS domains decreased within the first year after diagnosis. This observation could explain why a consistent group of patients with IIM/ASS do not enter remission despite immunosuppressive treatment. Thanks to the access to longitudinal serum samples and clinical data collected at the same time points, we could observe that, despite some exceptions, reactivity levels towards HisRS-FL changed over time consistently with the degree of lung disease activity. In fact, longitudinal levels of anti-HisRS-FL increased in line with ILD progression and decreased when recording an improvement of ILD. Taken into consideration the low number of anti-Jo1 positive patients, this might suggest that anti-Jo1 antibodies could serve not only as diagnostic marker of ASS, but also supports a role of anti-Jo1 autoantibodies in the pathogenesis of ILD in this subset of IIIM/ASS. However, functional studies are needed to confirm this hypothesis. Even though the WHEP domain appears to account for the main reactivity displayed by HisRS, the persistent high anti-HisRS-FL antibody levels over the disease course together with the simultaneous reduction in the antibodies reactivity against the tested HisRS domains and splice variant, suggests that antigenic regions other than the WHEP domain might be present (15) or develop at a later stage.
The administration of rituximab did not result in a homogenous reduction of anti-HisRS reactivity towards the different variant/domains or the full-length protein. This is in contrast to the results from the rituximab in myositis (RIM) trial (33), although differences in methods and time point analysis should be considered when interpreting the results. The persisting anti-HisRS reactivity after rituximab treatment could be explained by the known lack of depletion of memory B cells and plasma cells after rituximab infusion and can contribute to the chronicity of disease and the relapsing disease course seen in many patients with anti-Jo1 autoantibodies (34).
WB experiments were performed to address whether, in addition to the binding of naturally occurring folded HisRS variants, anti-Jo1 antibodies could also recognize non-conformational epitopes within HisRS. The folding of the proteins in ELISA plates was assumed since they were added in native conditions. In addition, to reduce the binding of HisRS proteins directly to the plate and hence also avoiding the risk to unfold the protein, we purposefully produced HisRS variants that retain their native folding and employed a biotinylated Avi-tag that binds to streptavidin coated plates. For the WB experiments, the proteins were added under denatured conditions, thus assuming an unfolded conformation. In line with previous studies (13, 16), our results show that anti-Jo1 antibodies bind conformational independent epitopes, as well as conformational-dependent, with no apparent differences among antibodies available at diagnosis or during follow-up under treatment. Among the five HisRS constructs tested, all denatured HisRS fragments (non-conformational) were recognized by IgG anti-Jo1 antibodies with the exception of the CD, which could not be detected by WB (17). This could be because the antigenic reactivity of the CD is dependent on the 3-dimensional structure of the domain, whereas within the other domains the autoantibodies recognize both structural as well as linear epitopes.
In this study we have also developed a method to measure an average affinity of autoantibodies against HisRS-FL, using SPR. Strikingly, the majority of anti-Jo1+ patients presented anti-HisRS-FL antibodies with a high-affinity profile already at the time close to diagnosis. Recently, another study showed that individual anti-Jo1 monoclonal antibodies, selected based on somatic hypermutation using single cell isolation and sequencing, also displayed affinities from low nM KD and below (35). Also, in patients with RA, high affinity circulating autoantibodies (targeting the citrullinated peptide Cit573) have previously been observed (36). Of note, considering that the average affinity is the component being measured, there are some facts to take into consideration; firstly, the analyzed total IgG samples are polyclonal, thereby likely containing a mixture of specific anti-Jo1 autoantibodies of different affinities, most likely present in very different concentrations. Here, an average affinity is measured, and the binding profile of an individual antibody clone within a polyclonal mix may be very different to what we report. For example, it is possible that high affinity antibodies in the sample mask low affinity antibodies. Secondly, the antigen HisRS-FL is a homodimer and this may give rise to an avidity effect, i.e. an apparent increase in affinity. Despite these complicating factors, the analysis was made using the predefined 1:1 Langmuir binding model to get comparable values among the patient samples. Considering the generally high reactivity against all HisRS domains at diagnosis observed in this study, in combination with the fact that high affinity autoantibodies were retrieved from patients with a recent diagnosis of IIM/ASS, one can speculate that affinity maturation of anti-HisRS antibodies through somatic hypermutation together with epitope spreading have already happened before onset of specific symptoms, ultimately leading to diagnosis of IIM/ASS. To confirm this hypothesis, further studies aiming at characterizing anti-Jo1 antibodies before clinical diagnosis are warranted.
A limitation of our study is the use of the Bohan and Peter criteria for classification of IIM as the EULAR/ACR classification criteria (37) for adult and juvenile IIM were not published at the time of patient inclusion. Another limitation is the low number of anti-Jo1+ patients (n = 19). However, since we aimed for an in-depth characterization of anti-Jo1 autoantibodies (29), the current study design was a feasible approach. A strength of our study is that the antibody reactivities were analyzed in purified IgG which would diminish interference of other factors in sera that could influence antigen binding. In addition, this was later confirmed to be necessary since some of the reactivities detected using purified IgG from sera, could not be found when using sera. Furthermore, paired BALF-serum samples as well as available longitudinal sera in the anti-Jo1+ group with clinical data strengthened our observations. Even with the low number of patients included, the results are consistent with a high IgG and IgA reactivity towards the HisRS-FL protein as well as towards the WHEP domain and SV at diagnosis (both in circulation and in the lung) as well as a solid report on the high affinity profile of IgG anti-Jo1 autoantibodies.