{'fraction_non_alphanumeric': 0.051469456839513775, 'fraction_numerical': 0.03531312509616864, 'mean_word_length': 4.681381118881119, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Clinical Relevance of Autoantibodies against Interleukin-2 in Patients with Systemic Lupus Erythematosus

Background: Increased serum autoantibodies against interleukin-2 (anti-IL-2 autoantibodies) were reported in patients with systemic lupus erythematosus (SLE) and in patients receiving IL-2 therapy. This study aimed to explore the clinical relevance of serum anti-IL-2 autoantibodies and the interactions between low-dose IL-2 therapy and serum anti-IL-2 autoantibodies. Methods: Serum samples were collected from 152 SLE patients and 100 age- and gender-matched healthy controls (HCs). Among them, 75 SLE patients were followed up for 10 weeks, and all of them were treated with corticosteroids, antimalarials, and/or immunosuppressants. Forty-six out of the 75 SLE patients received low-dose IL-2 therapy additionally. Clinical and laboratory parameters were collected at baseline and week 10. Serum anti-IL-2 autoantibodies were determined by enzyme-linked immunosorbent assay. Results: Compared with HCs, median levels and positive rates of serum anti-IL-2 autoantibodies were higher in SLE patients (32.58 [23.63, 45.23] arbitrary unit [AU] vs. 37.54 [27.88, 60.74] AU, P = 0.006, and 5.0% vs. 18.4%, P = 0.002, respectively). Compared to those without the corresponding disorders, serum anti-IL-2 autoantibody was increased in patients with alopecia (49.79 [36.06, 64.95] AU vs. 35.06 [25.40, 58.46] AU, P = 0.033), but it was decreased in those with lupus nephritis (31.71 [22.60, 43.25] AU vs. 44.15 [31.43, 68.52] AU, P = 0.001). Moreover, serum anti-IL-2 autoantibody was positively correlated with serum IgA (r = 0.229, P = 0.005), total IgG (r = 0.327, P < 0.001), and total IgM (r = 0.164, P = 0.050). Treatment with exogenous IL-2 was not significantly associated with serum anti-IL-2 autoantibody. In addition, no significant difference was found in serum anti-IL-2 autoantibody between responders and nonresponders to low-dose IL-2 therapy. Conclusions: Serum anti-IL-2 autoantibody was increased and associated with disease severity in SLE. Exogenous low-dose IL-2 did not significantly induce anti-IL-2 autoantibody production.

patients has not been clearly elucidated. The anti-IL-2 autoantibodies were reported in healthy individuals and patients infected with human immunodeficiency virus. [7][8][9] Previous studies also presented an increased production of serum anti-IL-2 autoantibodies in SLE patients, as well as lupus mice. [10,11] However, the clinical relevance of serum anti-IL-2 autoantibodies and its association with low-dose IL-2 therapy in SLE patients are not well studied. Here, this study detected serum anti-IL-2 autoantibodies and analyzed its clinical significance. The impact of low-dose IL-2 treatment on anti-IL-2 autoantibodies was also investigated in SLE patients.

Ethical approval
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University People's Hospital. Informed written consent was obtained from all patients prior to their enrollment in this study.

Patients and specimens
In total, 152 SLE patients satisfying the 1997 revised classification criteria of the American College of Rheumatology [12] were enrolled from the Department of Rheumatology and Immunology, Peking University People's Hospital, between September 2015 and May 2017. A total of 100 age-and gender-matched HCs were recruited.
Seventy-five patients were followed up for 10 weeks. All the SLE patients were treated with corticosteroids, antimalarials, and/or immunosuppressants. In addition, 46 out of them received exogenous low-dose recombinant human IL-2 (rhIL-2, SL Pharma, Beijing, China) therapy. For low-dose IL-2 therapy, rhIL-2 was administered at a dose of one million units every other day for 2 weeks subcutaneously, followed by a 2-week break. [5] In total, three cycles of low-dose rhIL-2 therapy were admitted consecutively. Serum samples at baseline and week 10 were collected with separation gel coagulation tubes and were frozen at −80°C until measurement, and repeated freeze-thaw cycles were avoided.

Clinical and laboratory evaluation
Patients' clinical and laboratory parameters, as well as Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), were collected at baseline and week 10.
The normal reference ranges of serum anti-IL-2 IgG were determined according to the 95% confidence interval in HCs, and the patients with serum anti-IL-2 IgG above the upper limit of normal (ULN) were defined as serumelevated anti-IL-2 IgG.

Statistical analysis
Statistical analyses were performed using SPSS software version 20.0 (IBM, Armonk, New York, USA). Continuous variables were expressed as mean ± standard deviation (SD), and comparison between the two groups was performed with the Student's t-test or Student's paired t-test. Discrete variable data were expressed as median (Q1, Q3), and comparison between the two groups was made by Mann-Whitney U-test. Categorical data were compared using Chi-square test or Fisher's exact test. Spearman's correlation analysis was applied to analyze relationship between the two groups. A P < 0.05 was considered statistically significant.

Patient characteristics
A total of 152 SLE patients were recruited in this study. The median age and disease duration of SLE patients were 31.0 (25.0, 43.8) years and 48.0 (8.5, 109.5) months, respectively. One hundred and forty out of these 152 SLE patients (92.1%) were female. The median SLEDAI score of these SLE patients was 9 (6,12), and the actual median dose of corticosteroids was 15 (10, 45) mg/d. The 100 healthy individuals with a median age of 32.0 (26.0, 43.8) years and 83 females were also enrolled as the control group.

Influence of low-dose interleukin-2 therapy on anti-interleukin-2 autoantibodies in systemic lupus erythematosus
To further analyze the possible influence of low-dose rhIL-2 therapy on serum anti-IL-2 IgG, serum anti-IL-2 IgG at baseline and week 10 was compared in patients with exogenous low-dose IL-2 supplement (n = 46) and patients with conventional therapy (n = 29), respectively. The characteristics of SLE patients with low-dose rhIL-2 therapy and those with conventional immunosuppressive therapy are shown in Table 2. Treatment with exogenous IL-2 did not lead to significant increase both in the levels and positive rates of serum anti-IL-  Figure 3]. Both serum anti-IL-2 IgG levels and positive rate in SLE patients with low-dose IL-2 therapy were similar to SLE patients with conventional therapy.

Baseline serum anti-interleukin-2 autoantibody levels did not affect low-dose interleukin-2 therapeutic effects
In an attempt to determine whether serum anti-IL-2 IgG affects the therapeutic effect of low-dose IL-2 therapy or not, we divided SLE patients who received low-dose IL-2 therapy (n = 46) into two groups: patients with elevated serum anti-IL-2 IgG (n = 7) and patients with normal serum anti-IL-2 IgG (n = 39), according to the serum anti-IL-2 IgG levels at baseline. As an assessment of disease activity improvement, changes of SLEDAI scores (△SLEDAI) for general disease activity between week 10 after treatment and the baseline were calculated. In comparison to patients with normal serum anti-IL-2 IgG, no significant difference of disease activity improvement (△SLEDAI) was observed in patients with elevated serum anti-IL-2 IgG (5.86 ± 2.73 vs. 5.41 ± 4.59, t = −0.249, P = 0.805). In addition, when we grouped these patients into responders (△SLEDAI ≥4, n = 18) and nonresponders (△SLEDAI <4, n = 28) on the basis of therapeutic responses of low-dose IL-2 therapy, there was no difference of serum anti-IL-2 IgG levels at baseline between these two groups (39.25 ± 17.66 AU vs. 44.74 ± 20.23 AU, t = 0.942, P = 0.352) as well.
Since a notably higher serum anti-IL-2 IgG was present in SLE patients with alopecia than those without alopecia, we explored the difference of anti-IL-2 IgG levels at baseline according to their therapeutic response of low-dose IL-2  therapy, in order to investigate a possible impact of serum anti-IL-2 IgG in the improvement of different disease manifestations of SLE. As shown in Table 3, no difference of baseline serum anti-IL-2 IgG was found between responders and nonresponders with alopecia, as well as other clinical manifestations such as rash, lupus nephritis, decreased C3, decreased C4, and anti-dsDNA antibody.

dIscussIon
In the recent years, anticytokine autoantibodies were found to involve in the mechanism of diseases. For example, anti-tumor necrosis factor-α autoantibodies were detected in patients with rheumatoid arthritis. [13] Autoantibodies against granulocyte-macrophage colony-stimulating factor were found in patients with idiopathic pulmonary alveolar proteinosis. [14] Anti-IL-8 autoantibodies were reported in patients with ovarian cancer. [15] Anti-IL-2 autoantibodies were reported to play roles in the regulation of IL-2 cytokine network. [7,16] In previous studies, it was demonstrated that the presence of anti-IL-2 autoantibodies could contribute to the reduction of serum IL-2 concentrations and could impact the biological effect of IL-2 in vivo. [17] The induction of anti-IL-2 autoantibodies also had a negative influence on IL-2-mediated expansion of lymphocytes and could compromise regulatory T (Treg)-cells' fitness in vivo. [10,17] Moreover, reduced frequency of CD16, CD56, and CD25 lymphocytes and decreased lymphokine-activated killer cell activity were detected in patients with anti-IL-2 autoantibodies. [18,19] In fact, in the recent years, a dual role of anti-IL-2 autoantibodies including immunoenhancement and immunosuppression was reported. The function of anti-IL-2 autoantibodies depends on their target-binding sites on IL-2. For example, some anti-IL-2 autoantibodies binding to an IL-2 epitope that is crucial for interaction with CD25 could enhance immune response, while some other anti-IL-2 autoantibodies occluded IL-2 binding to CD122 and suppressed immune responses. [20,21] It was reported that the injection of anti-IL-2 monoclonal antibody or anti-IL-2/IL-2 immune complexes could reduce the viral load of Friend retroviral and herpes virus in light of increasing the proliferation of natural killer cells and memory-like CD8 + T-cells. [22][23][24][25] However, the exact role of anti-IL-2 autoantibodies on infections in SLE is still elusive.
In this study, a significantly increased serum anti-IL-2 IgG level was observed in SLE patients. The reason for the rise of autoimmune reaction against IL-2 is unknown, and it was probably due to the defective immune tolerance in SLE patients. The anti-IL-2 antibodies might bind to IL-2 and result in the formation of IL-2/anti-IL-2 immune complex, which probably interfered with the physiological functions of IL-2. However, except for serum immunoglobulin, no correlation between serum anti-IL-2 IgG and other laboratory parameters such as specific autoantibodies or complement was detected. It should be helpful to identify the targeting epitopes of anti-IL-2 autoantibodies in future studies. Interestingly, beyond our expectation, significantly decreased serum anti-IL-2 IgG and total serum IgG (data not shown) were observed in patients with lupus nephritis in this study.  In light of previous studies by Yang et al. [26] and Yap et al., [27] which reported that the serum total IgG was negatively correlated with proteinuria and urinary IgG, we speculated that the leakage of protein from urine or the deposition of IgG in the kidney might be responsible for the low serum anti-IL-2 IgG levels in patients with lupus nephritis.
Various factors, including the dose regimen, cumulative dose, duration of therapy, and route of administration of recombinant IL-2, have been shown to account for the incidence of anti-IL-2 autoantibody formation. Although the treatment-induced autoantibodies to IL-2 have been reported for years, [19,28] anti-IL-2 autoantibody was barely detected in our patients receiving low-dose IL-2 therapy. Of note, this suggested that the approach of low-dose IL-2 therapy to SLE patients was incapable of inducing increased production of serum anti-IL-2 autoantibodies.
In this study, patients with certain manifestations and nonresponders to low-dose IL-2 therapy presented higher level of anti-IL-2 autoantibodies, but no significant difference was observed. This was probably owing to the limited number of patients receiving low-dose IL-2 therapy. It is necessary to further investigate the role of IL-2 autoantibody in the pathogenesis of SLE. Furthermore, as the application of low-dose IL-2 in SLE is becoming routinely used, the influence of anti-IL-2 autoantibody on its efficacy should be studied in a larger cohort of patients.
In conclusion, increased serum anti-IL-2 autoantibodies were associated with disease severity in SLE. Short-term application of low-dose IL-2 did not significantly induce anti-IL-2 autoantibody production.