Decreased ex vivo production of interferon-gamma is associated with severity and poor prognosis in patients with lupus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that results from a disruption in immune tolerance to self-antigens, leading to inflammation of multiple organs [1]. T cells play a major role in SLE pathogenesis, amplifying inflammation by the secretion of pro-inflammatory cytokines, helping B cells to generate autoantibodies, and maintaining the disease through the accumulation of autoreactive memory T cells [2]. Interferon gamma (IFN-γ) is predominantly produced by T cells and natural killer cells [3], and it plays a critical role in lupus [4]. Previous studies have shown that IFN-γ mRNA expression is increased in peripheral blood mononuclear cells (PBMC) [5], and that serum levels of IFN-γ are elevated in patients with SLE [6, 7]. In addition, in vivo experiments in murine models of SLE have shown that elevations in IFN-γ mRNA levels are correlated with disease progression [8, 9].

Although there is no clinical laboratory test available to examine serum IFN-γ, the IFN-γ-releasing assay (IGRA) is a diagnostic test used to detect Mycobacterium tuberculosis (TB) infection by measuring the IFN-γ production by T cells stimulated with TB antigens [10]. Since biologics such as tumour necrosis factor alpha (TNF-α) inhibitors make patients susceptible to reactivation of TB, screening of latent tuberculosis is recommended in the field of rheumatology [11, 12]. IGRA is composed of three tubes (nil, mitogen, and TB antigen) for measuring IFN-γ. The nil tube is a negative control used to measure baseline IFN-γ production, whereas the mitogen tube is a positive control used to measure IFN-γ production with phytohemagglutinin (PHA). Therefore, IGRA results of negative and positive control tubes yield IFN-γ produced at baseline and by the activated T cells, respectively.

IFN-γ production by T cells has not been used to assess disease activity in SLE before; therefore, we evaluated ex vivo IFN-γ production following stimulation with PHA in patients with lupus by analysing the results of IGRA.

Although IFN-γ mainly mediates host defence against microbial invasion, it is known to have a pivotal role in SLE. The inhibition of IFN-γ has shown benefits in reducing disease activity in murine models of SLE [16, 17] and a therapeutic monoclonal antibody against IFN-γ is being developed for the treatment of SLE [18, 19]. In this study, we evaluated IFN-γ production in patients with SLE, using IGRA results obtained during the screening of latent TB prior to immunosuppressive treatment. Our data demonstrated that ex vivo production of IFN-γ is decreased in patients with active disease compared to patients with inactive disease. The results of our study are different from those of previous publications that have reported increased IFN-γ production or IFN-γ-related gene expression in SLE, because our study evaluated IFN-γ production after PHA stimulation in whole blood [6, 7, 20].

However, our data are not contradictory to previous findings, in that baseline IFN-γ production (nil results) in patients with active SLE is increased compared to that in patients with inactive SLE, in patients with RA, and in healthy controls. While baseline IFN-γ production increased, ex vivo IFN-γ production after stimulation with PHA decreased in patients with active SLE. In a previous study by Hagiwara et al., ex vivo experiments using enzyme-linked immunospot (ELISPOT) assay demonstrated that IFN-γ-producing T cells were decreased in patients with active lupus [21], which is similar to our finding. Our study used ELISA, which can measure the total amount of ex vivo IFN-γ production in whole blood, excluding baseline production. Since ELISA is known for its high sensitivity and wide dynamic range, measuring ex vivo IFN-γ production by ELISA can be a suitable biomarker for disease activity in lupus. Decreased ex vivo IFN-γ production could be associated with T cell exhaustion, which is a non-functional state that occurs under conditions of antigen persistence, such as those that arise during various infections and cancers [22, 23]. Similar to infection and cancer, T cell exhaustion has also been described in SLE [24]. Excessive auto-antigen exposure in active SLE may lead to T cell exhaustion. Alternatively, T cells may become unresponsive due to a negative feedback mechanism during periods of overwhelming inflammation. PD-1, an inhibitory T cell marker, is increased in SLE [25]. Likewise, critically ill patients who are admitted to the intensive care unit have been shown to demonstrate a high proportion of indeterminate results on the IGRA due to unresponsiveness to mitogen stimulation [26].

Our observations have raised an important issue regarding the limitations of the IGRA in SLE. Nearly half of all patients with active SLE had indeterminate results from the IGRA, while none of the patients with inactive SLE had an indeterminate IGRA result. The IGRA results are dependent on IFN-γ production by TB antigen-specific T cells. However, when IFN-γ production is hampered by defective T cells, the sensitivity of the IGRA may be reduced. Unreliable IGRA results in patients with T cell defects have been reported in HIV infection [27]. Our study suggests that the interpretation of the IGRA requires caution in patients with active SLE.

More importantly, decreased ex vivo IFN-γ production correlates well with SLEDAI-2 K scores, and the poor hospitalisation outcome was more frequent in patients with ex vivo IFN-γ production ≤ 0.40 IU/mL. The findings of our study imply that monitoring ex vivo IFN-γ production could aid in assessing disease activity and predicting clinical outcome in SLE. So far, no test is available to evaluate T cell reactivity for SLE disease activity and prognosis, and our data provide the possibility of developing a diagnostic test to measure ex vivo IFN-γ production. Furthermore, we demonstrated that decreased production of IFN-γ in patients with active SLE recovers when SLE becomes inactive following immunosuppressive treatment. Similar to our findings for SLE, decreased IFN-γ production in patients with RA has been shown to recover after TNF-α inhibitor treatment [28]. Decreased ex vivo IFN-γ production was likewise noted in our group of patients with active RA as compared with healthy controls. However, decreased ex vivo IFN-γ production was more pronounced in active SLE than in active RA in our study.

The strength of our study is that we included a large number of subjects who had undergone IGRA, an ex vivo method to estimate IFN-γ production in patients with SLE. However, the present study has several limitations. First, the clinical and laboratory data and IGRA results of patients were collected by reviewing the medical records. Since the IGRA is not routinely performed in patients with SLE, there could have been patient selection bias, resulting in the selection of patients with more severe SLE in our study. Therefore, our study findings should be validated in future prospective studies. Second, the effect of immunosuppressive treatment was not thoroughly controlled. It is possible that immunosuppressive treatment affects the decrease in ex vivo IFN-γ production. However, the proportion of patients with active SLE treated with glucocorticoids or immunosuppressive agents was lower than that of patients with inactive SLE, as most patients with SLE had undergone IGRA before initiating potent immunosuppressive treatment. Therefore, the effect of immunosuppressive treatment on active lupus may not be significant. Third, we did not evaluate CD4 T cell numbers. Although we demonstrated that ex vivo IFN-γ production ≤ 7.19 IU/mL was an independent predictor for discriminating active and inactive lupus regardless of leukopenia and lymphopenia, there is a possibility that the level of ex vivo IFN-γ production is associated with CD4 T cell numbers.