Anti-UBE2T antibody: a novel biomarker of progressive-brosing interstitial lung disease

Antibrotic therapy has demonstrated ecacy against progressive-brosing interstitial lung disease (PF-ILD); therefore, it has become a priority to identify disease behavior before disease presentation. As autoimmunity is implicated in the pathogenesis of various ILDs, we explored the possibility of a circulating biomarker that can predict the chronic progressive behavior of ILDs. A single-center retrospective cohort study was conducted to investigate a biomarker of PF-ILD. Circulating autoantibodies against 9,483 puried full-length human recombinant proteins of patients with interstitial pneumonia were screened by microarray analysis. The candidate auto-antibodies were veried their existence by multiples solution assay. In addition, enzyme-linked immunosorbent assay (ELISA) was performed in larger sample sets to evaluate accurate sensitivity, specicity and clinical signicance in ILDs. IPF IPF All samples clinical in anti-UBE2T-positive group. We found UBE2T was highly expressed in the lining epithelium of the honeycomb structure, where repeated DNA damage(30) and repair is expected to occur. The higher prevalence of IPF in the anti-UBE2T antibody-positive group may have affected the honeycomb structure frequency observed in the second-year follow-up data. However, considering that early stages of IPF lack the specic honeycomb structure and are often indistinguishable from those of other IIPs, the relation between elevated anti-UBE2T antibody levels and tissue remodeling may represent a breakthrough in identifying the future clinical course.

that are not currently detected by commercially available biomarkers.

Background
Idiopathic interstitial pneumonia (IIP) refers to a heterogeneous group of idiopathic interstitial lung disorders with varying degrees of in ammation and brosis. Idiopathic pulmonary brosis (IPF) and nonspeci c pulmonary brosis (NSIP) are the two major IIPs. Given the e ciency of anti brotic therapies in the treatment of progressive interstitial lung diseases (ILDs), including IIPs, (1,2), it is crucial to predict the clinical course of IIPs at an early stage.
Autoimmunity has been implicated in the pathogenesis of IIPs. Kinder et al. (3) classi ed idiopathic NSIP as an autoimmune disease because, in most cases, it satis es the de nition of undifferentiated connective tissue disease. In contrast, IPF is clinically classi ed as a non-autoimmune disease, with the exception of cases that meet the criteria for interstitial pneumonia with autoimmune features (IPAF) or connective-tissue disease (CTD). Although IPF pathogenesis is likely to be based on wound healing rather than on autoimmune processes, there is still signi cant evidence demonstrating background immune abnormalities in IPF. For example, IPF lung tissues exhibit aggregation of mature dendritic cells, lymphocytes, and IgG in the active brotic site.(4-6) IgG autoantibodies against cellular antigens are found in sera from IPF patients. (6) Considering the autoimmune pathogenesis observed in IIPs, we hypothesized that unknown autoantibodies could have a bearing in terms of clinical outcomes. In this study, we present the discovery of a new circulating autoantibody in progressive IIPs.

Study design and participants
This was a single-center retrospective cohort study conducted at Nihon University Itabashi Hospital in Tokyo, Japan. The institutional ethics committee approved all protocols. Patients (≥20 years of age) who visited Nihon University Itabashi Hospital for regular follow-up were asked to participate. In total, 61 healthy subjects and 87 patients with ILDs enrolled between October 2010 to December 2017, and all participants provided written informed consent for analyzing blood samples and clinical data. Blood samples were centrifuged at 1,800 g (at 4 ℃ for 10 min). The sera were aliquoted and immediately stored at -80 ℃ until use. Lung tissues analyzed in this study were collected between April 1996 and September 2013. Normal lung tissue specimens were obtained from the uninvolved areas of surgically removed lung cancer tissues. IPF lung tissue specimens were obtained during surgical biopsies for the purpose of IPF diagnosis. All samples and clinical data were analyzed from December 2010 to April 2021 for this study.
Interstitial pneumonia was classi ed according to the o cial American Thoracic Society and European Respiratory Society joint statement.(7) Hypersensitivity pneumonitis (HP) and IPF were diagnosed according to the o cial guidelines.(8, 9) Expert rheumatologists diagnosed CTDs according to the American College of Rheumatology criteria. All diagnoses were screened and con rmed by the consensus of two expert respiratory physicians.
To analyze progressive IIPs, IPF and NSIP were reclassi ed into progressive (PF-ILD) and non-progressivebrosing ILD (non-PF-ILD). No o cial de nition exists for PF-ILD. In this study, the criteria for PF-ILD were de ned according to the methods of INBUILD trial (1,10): decline in forced vital capacity (FVC) of at least 10% of the predicted value, relative decline in the FVC (5-10% of the predicted value) and worsening of respiratory symptoms or increased extent of brosis on high-resolution computed tomography (HRCT), or worsening of respiratory symptoms and an increased extent of brosis.
The serum samples were diluted to 1:500. Alexa Fluor®647-conjugated goat anti-human IgG antibody was used as a secondary antibody to detect IgG levels of associated autoantibodies. Arrays were scanned using the Axon GenePix 4000B uorescent microarray scanner (Molecular Devices, LLC., San Jose, CA, USA). GenePix 6.0 software (Molecular Devices, LLC., San Jose, CA, USA) was used for data acquisition. Invitrogen's proprietary ProtoArray® Prospector software (Invitrogen, Carlsbad, CA) was used for analyzing images. This assay was performed by Invitrogen Corporation (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.

Multiplex Solution Assay
ProtoPlex™ (Invitrogen, Carlsbad, CA, USA) is a multiplex solution assay that validates candidate autoantibodies using uorescent bead-conjugated human recombinant proteins. (11) Diluted serum samples (1:200) were incubated with the bead-antigen complex. Biotinylated anti-human IgG was used as a secondary antibody. After incubation with streptavidin R-phycoerythrin, the beads were analyzed using a Luminex® 200™ system (Invitrogen, Carlsbad, CA, USA). By monitoring the spectrum of the beads and the amount of associated R-phycoerythrin uorescence, the presence of autoantibodies against particular antigens was assessed. This assay was performed by the Invitrogen Corporation (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentration of antiubiquitin-conjugating enzyme E2T (UBE2T) antibodies following a standard protocol. Histidine-tagged human recombinant UBE2T protein was incubated overnight on a 96-well plate. Diluted serum samples (1:200) and anti-histidine-tag antibody (diluted gradually from 0 to10 ng/ml for calibration curve, GeneTex, Los Angeles, CA, USA) were applied to the wells, respectively. Biotin-tagged anti-human IgG Fc antibody was used as a secondary antibody. After been incubated with streptavidin-horseradish peroxidase (Biolegend, Inc., San Diego, CA, USA), KPL SureBlue™ TMB Microwell Peroxidase Substrate (Kirkegaard and Perry Laboratories, Inc. Gaithersburg, MD, USA) was added for coloring reaction. The reaction was stopped with hydrochloric acid. The absorbance at 450 nm was measured, and the calibration curve was obtained using the concentration of the anti-histidine antibody and its absorbance.

Immunohistochemistry
Formalin-xed, para n-embedded lung tissues were used for UBE2T protein immunohistochemistry following a standard protocol. Staining with primary antibody against UBE2T (Novus Biological, Inc. Littleton, CO) diluted at 1:100 and with the secondary antibody using Histo ne Simplestain Max-PO® (Nichirei Bioscience Inc. Tokyo, Japan) was performed.
Statistical analysis M-statistics were performed to analyze data from the ProtoArray® and ProtoPlex™ platforms provided by Invitrogen Corporation (Life Technologies, Carlsbad, CA, USA). Otherwise, data were analyzed using GraphPad Prism version 5.04 (GraphPad Software Inc., La Jolla, CA, USA). Comparisons of continuous variables were conducted using Mann-Whitney and Kruskal-Wallis tests. The Fisher's exact test was used to compare categorical variables. Spearman's product-moment correlation was used to investigate correlations between variables. P-values <0.05 were considered signi cant. Data are presented as means ± standard errors.

Discovery and veri cation of the circulating autoantibodies
The work ow of identifying circulating biomarkers in this study falls into three broad categories; discovery method using protein microarray, veri cation by multiplex solution assay, and validation by ELISA.
The discovery method by protein microarray was performed to identify the pro les of circulating autoantibodies. The goal of this method was to select several candidate autoantigens that react with the ILD serum autoantibodies for further study.
Signals arising from the serum-loaded microarrays were evaluated for changes in uorescence intensity relative to each other and to the control array. In total, 127 proteins exhibited elevated interactions with serum autoantibodies in the ILD group. The top 50 proteins were ranked by the ratio of the average signal value for the ILD group divided by the average signal value for the control group (Supplemental Table 1).
Among the 50 candidate autoantigens, UBE2T had the highest average signal ratio in the ILD group.
The second step was the veri cation study using the multiplex solution assay. In this assay, we screened ten potential autoantibodies identi ed during the discovery method using the same serum samples to ensure that the candidate autoantibody could be moved forward into a larger validation study. We examined serum samples from healthy control participants (n=12) and patients with IPF (n=13), NSIP (n=7), and CTD-ILDs (n=3). CTD-ILDs included systemic scleroderma (n=1), rheumatoid arthritis (n=1), and systemic lupus erythematosus (n=1). These samples include the same samples used in the protein microarray method.
UBE2T had the highest prevalence among ILD patients, with the lowest prevalence observed in the control group (Table 1). This veri cation assay demonstrated that anti-UBE2T antibodies constitute potentially useful biomarker. Validation study of anti-UBE2T antibody by ELISA A validation against anti-UBE2T antibodies among various ILDs was performed using ELISA ( Figure 1) as the nal step of this study. This method aimed to test the accurate sensitivity, speci city and quantitatively of this novel autoantibody and evaluate the clinical signi cance in PF-ILDs.

Clinical characteristics of anti-UBE2T antibody
We calculated the accuracy of anti-UBE2T antibody to diagnose PF-ILD using a receiver operating characteristic analysis. The area under the curve was 0.85 (95% con dence interval 0.76-0.93, p<0.01) and yielded a cut-off value of 238.1 ng/mL. Using this cut-off value, the sensitivity was 83.3% and speci city was 85.3%. To compare the clinical characteristics, IIP patients were categorized into anti-UBE2T-positive or anti-UBE2T-negative groups using the cut-off value ( Table 2). There were no statistically signi cant differences in age, sex, history of malignancy, or glucocorticoid or immunomodulator use between these two groups. Additionally, anti-UBE2T status did not correlate with positive results of commercial biomarkers such as C-reactive protein, KL-6, or IPAF. No IIP patients developed CTD within two years of registration. Pulmonary function parameters such as predicted FVC, predicted diffusing capacity for carbon monoxide (DLCO), and predicted forced expiratory volume in 1 second were similar between groups. The ILD-gender age physiology (ILD-GAP) score(12) was signi cantly higher in the anti-UBE2T-positive group than in the anti-UBE2T-negative group (2.9 ± 0.3 vs. 1.7 ± 0.4, p = 0.01).
To assess the progression of IIPs objectively, we analyzed HRCT scans taken on registration and on the second-year follow-up visit. Since a second-year HRCT scan was not available in two patients, we screened 60 HRCT scans (anti-UBE2T-negative group, n=20; anti-UBE2T-positive group, n=40). At registration, there was no difference in the prevalence of honeycomb structures between the two groups (p=0.29). However, after two years of follow-up, the prevalence of honeycomb structures was signi cantly higher in the anti-UBE2T-positive group (p=0.04) (Table 3). Additionally, the prevalence of PF-ILD was higher in the anti-UBE2T-positive group than in the anti-UBE2T-negative group (Table 3).  Among the 62 IIP patients, clinical information for acute or subacute exacerbations was available in 55 patients. At registration, 13 patients had acute or subacute exacerbations, as judged by their treating physician. The average concentration of anti-UBE2T antibodies was higher in exacerbated patients than in stable patients (668±133 ng/mL vs. 308±30 ng/mL, p<0.01); the same result was observed for KL-6 levels (1556±193 U/mL vs. 1098±161 U/mL, p<0.01). Although both anti-UBE2T and KL-6 levels were increased in patients with acute or subacute exacerbations, these two variables did not correlate with each other (Spearman's r=0. 19, p=0. 17), suggesting their presence was independent of the other.

Immunohistochemical staining of UBE2T
We conducted immunohistochemical staining of UBE2T in the lung tissues of IPF patients and control participants. Twelve normal tissues and nine tissues with usual interstitial pneumonia (UIP) patterns were analyzed. In the normal lung biopsies, expression of UBE2T was observed sparsely in the bronchiole epithelium and macrophages (Figures 2A and 2B). Conversely, all lungs with IPF showed a strong expression of UBE2T, not only on the bronchiole epithelium but also on the regenerated type II alveolar epithelium lining inside the honeycomb structure ( Figures 2C and 2D).

Discussion
This study is the rst to describe the existence of anti-UBE2T antibodies and their potential role as biomarkers to predict progression in IIP patients.
We found that anti-UBE2T antibody levels measured using ELISA were increased only in patients with idiopathic PF-ILD and not in those with non-PF-ILD or other ILDs, suggesting that this tool may predict the future progression of IIPs.
In this study, most patients presenting with PF-ILD had IPF (n=30; 73%). Most theories on IPF pathogenesis are based on mechanisms relating to epithelial cell apoptosis, tissue repair, and regeneration, (14) acknowledging little participation of autoimmune in ammatory responses. IPAF criteria do not include UIP patterns in the morphological domain, implying that IPF is far from having an autoimmune nature. However, replicated observations support an abnormal immune response and autoantibody production in IPF. CD3+ T cells, mature dendritic cells, and CD20+ B cells near the brotic foci with collagen deposition have been recognized repeatedly by many researchers(4, 5, 15). Abnormal CD4+ T cells expressing MHC class II and CD154+ proteins show clonal expansions in the sera of patients with IPF, with increased secretion of transforming growth factor-β1, interleukin-10, and tumor necrosis factor-α; 82% of these patients had circulating antibodies against cellular antigens.(6) Regulatory T cells in IPF bronchoalveolar lavage uid and blood are numerically and functionally impaired, indicating a de ciency of immunological self-tolerance.(16) T-cell activation against accessible self-antigens does not occur in healthy people. (17) Given that IPF is more frequent in smokers and in elderly people, the abundant autoantibodies against cellular autoantigens may be due to injury and senescence of the epithelial cells. The anti-UBE2T antibody was found in 76% (n=31) of IPF patients; to our knowledge, this prevalence is higher than that of every commercial autoantibody used in IPF.(18-20) Ubiquitination plays an essential role in proteasome-mediated protein degradation and DNA repair, affecting the cell cycle and regulating signaling pathways. (21,22) The process of ubiquitination involves the sequential action of activating, conjugating, and ligating enzymes that introduce an isopeptide link between the C-terminus of ubiquitin and target proteins. (22) UBE2T is one among the 35 types of ubiquitin-conjugating enzymes identi ed in humans. (22) UBE2T was initially identi ed as the possible cause of Fanconi anemia because it impairs DNA repair. (23) Overexpression of UBE2T has been observed in carcinomas in the lung, prostate, nasopharyngeal tissue, breast, and stomach. (24)(25)(26)(27)(28) UBE2T upregulation promotes epithelial cell proliferation and epithelial-mesenchymal transition in vitro, whereas knockdown of UBE2T attenuates this process. (25,26,29) However, it remains unclear whether the UBE2T antibody has an effect on the epithelial cells, and further investigation is thus required.
In this study, the prevalence of HRCT-diagnosed honeycomb structure increased over time in the anti-UBE2T-positive group. We found that UBE2T was highly expressed in the lining epithelium of the honeycomb structure, where repeated DNA damage (30) and repair is expected to occur. The higher prevalence of IPF in the anti-UBE2T antibody-positive group may have affected the honeycomb structure frequency observed in the second-year follow-up data. However, considering that early stages of IPF lack the speci c honeycomb structure and are often indistinguishable from those of other IIPs, the relation between elevated anti-UBE2T antibody levels and tissue remodeling may represent a breakthrough in identifying the future clinical course. 2 years remained small in both anti-UBE2T-positive and -negative patients (data not shown), PF-ILD was much more prevalent in those with a positive UBE2T status due to worsening of irreversible respiratory symptoms and continuous disruptions of the lung structure as seen on HRCT scans. Considering that predicting disease progression requires a combination of several parameters such as PFTs, HRCTs, or even 6-minute walking tests (which can be too strenuous for patients with exertional dyspnea), measuring circulating anti-UBE2T antibody levels can be an alternative, convenient tool.
Some of the past studies of IPAF have reported that the positivity of IPAF serological domain correlates with better prognosis in IIPs (31,35). However, IPAF serological domain positivity in our data did not correlate with fewer PF-ILD development.
Multiple studies have supported the utility of serum KL-6 as a diagnostic, prognostic, active disease marker for ILDs.(36-38) In our study, KL-6 was elevated in patients with exacerbations and chronic progression, which is consistent with the ndings of past reports.(36-38) High anti-UBE2T antibody levels were associated with the appearance of honeycomb structures that ultimately develop into PF-ILD.
Although anti-UBE2T antibody and KL-6 both predict progression, the values of these two biomarkers did not correlate. Even though the function and origin of anti-UBE2T antibodies remain unclear, anti-UBE2T antibodies and KL-6 may be re ecting different pathological states. Thus, the anti-UBE2T antibody may accurately predict progressions that KL-6 or IPAF serological domain cannot detect.
This study had several limitations. First, clinical data were collected and analyzed retrospectively. Second, clinical data, such as information regarding exacerbations, PFT, HRCT, and treatments were limited in some patients. Third, due to the small sample size, we combined IPF and NSIP cases into the IIP group, which increased the variability of clinical characteristics and prognoses. Fourth, the single-center nature of our study carries a risk of institutional bias. Currently, the concept of PF-ILD includes various lung disorders and pathophysiological processes. In this study, we mainly focused on analyzing limited target groups, especially IIPs. The study of a broader range of participants, such as CTD-ILD patients, is required for future research.

Conclusions
This is the rst report to describe anti-UBE2T antibody, a new biomarker that is signi cantly elevated in progressive IIPs. This new antibody may constitute a sensitive biomarker to detect cases of progressive IIPs that are not currently detected by commercially available biomarkers.  Figure 1 Comparison of serum anti-UBE2T antibody levels in various lung diseases. Anti-UBE2T antibody levels were signi cantly higher in the PF-ILD group than the healthy control group. HP, chronic hypersensitive pneumonitis; PF-ILD, progressive-brosing idiopathic interstitial pneumonia.