Soluble interleukin-2 receptors in sickness and in health

Soluble interleukin-2 receptor: a biomarker for immune activation

The quantification of soluble interleukin-2 receptors (sIL-2R) in serum or plasma in adults has become an extremely useful tool for clinicians to assess immune function in vivo for the investigation, management or outcome prediction of a broad spectrum of diseases. However, the measurement of sIL-2R is simple in comparison with the complex story behind this biomarker, which has become so central to our study of immune-mediated diseases.

Why measure sIL-2R levels?

Interleukin-2 (IL-2) is a key signaling molecule in the human immune system. It is a cytokine: one of a group of small, secreted proteins released by cells that have a specific effect on interaction and communication between cells. As such, IL-2 regulates the activities of the white blood cells that are responsible for immunity, forming part of the body’s natural response to infection and helping it to discriminate between foreign (‘non-self’) and ‘self’.1

IL-2 mediates its effects by binding to IL-2 receptors (IL-2R), which are expressed by lymphocytes. The major sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.1 The sIL-2R is secreted – or ‘shed’ – on T cell activation, meaning that elevated concentrations of sIL-2R are found in patients suffering from any one of an extensive range of conditions associated with an ongoing immune response, including sarcoidosis, multiple sclerosis, biliary cirrhosis, chronic immune activation in common variable immunodeficiency (CVID), and hemophagocytic lymphohistiocytosis (HLH). 2-6

The convenient implication of this association is that sIL-2R could hypothetically be used as a generic biomarker to monitor/predict disease activity and treatment response for these diseases, and many others.7,8 However, in order to be certain that we are measuring something that is clinically relevant, we must delve a little deeper into how the IL-2/sIL-2R system functions.

IL-2 and sIL-2R in immune activation: unravelling the twisted plot

Binding of IL-2 to sIL-2R can either enhance or reduce immune response, depending on the target cell being involved in immunity or selftolerance. 9,10 The history of the sIL-2R – also called sCD25, sTAC and IL-2RA – goes back to 1985, when it was first described as being actively released by activated peripheral blood T cells via proteolytic cleavage of the cell surface IL-2R.11 In 1990, Rubin and colleagues were the first to demonstrate that, after in vitro activation, T lymphocytes enhanced cellular IL-2R expression and released sIL-2R(α). The study also showed that sIL-2R binds IL-2 efficiently and, based on in vitro experiments, it has been proposed that sIL-2R may limit activation and proliferation of T lymphocytes by sequestration of available IL-2.13 However, conflicting data has been reported.14 Alternatively, sIL-2R complexing with IL-2 may prolong IL-2 half-life, possibly enhancing the immune-stimulatory properties of IL-2, even by activation of low affinity dimeric IL-2R.15

– similarly to cellular IL-2R expression – the release of sIL-2R required de novo protein synthesis rather than cellular proliferation.12 However, despite this long-recognized association between immune activation and increased sIL-2R release under pathological conditions, the biological actions of this molecule are still far from understood. Several mechanisms of action, ranging from immune-inhibitory to immunostimulatory effects, have been proposed.

It is possible that IL-2 can be presented to CD4+ T lymphocytes through sIL-2R, which then induces differentiation into regulatory T cells (Tregs) – rather than differentiation into T helper cells (Th1 or Th17 lymphocytes) – which can subsequently suppress immune activity.16 On the other hand, there are reports to support observations that sIL-2R may promote (auto)immune processes in association with enhanced Th17 generation, which involves sequestration of the IL-2 that normally inhibits early Th17 differentiation.17 Despite the obvious complexity and lack of agreement so far regarding the exact mechanism(s) of action of different configurations of sIL-2R, as well as their in vivo occurrence and final biological effects, the data currently available supports the role of sIL-2R in regulating IL-2dependent cell function.

What makes sIL-2R an appealing biomarker?

An increased level of sIL-2R in the blood is considered to indicate an ongoing immune response, and this could theoretically be used to monitor a huge range of immune-mediated diseases. This is a good starting point, even if the generic nature of the response might initially be of concern. 9 Generally speaking, conditions that are characterized by excessive production of lymphocytes – so-called lymphoproliferative disorders – show high sIL-2R levels compared to healthy controls. This also applies to granulomatous diseases, such as sarcoidosis, in which T cell activation is a typical hallmark. The relative stability of sIL-2R levels throughout adult life, and the minimal gender-related differences, are further useful attributes that help make sIL-2R an attractive biomarker.12,18 That said, individual differences in age, gender, lifestyle and general health may need to be taken into account when considering reference values and ranges.19 sIL-2R levels are generally measured using immunoassays – either enzymelinked immunosorbent assay (ELISA), or chemiluminescent immunoassay (CLIA). There is very good correlation between the results of both assay types, although absolute values of ELISA (pg/ml) are about 7-8 times higher than those obtained by CLIA (U/ml). Commercially available ELISAs should be calibrated against the international reference standard NIBSC 97/600.

How do we measure sIL-2R?

ELISAs for sIL-2R are typically designed for batch analysis, and have a measuring range up to 10,00020,000 pg/ml, based on a standard serum dilution of ~1:5. Further dilutions will, of course, enable the quantification of higher sIL-2R levels. This process can be streamlined by the use of automated liquid handling systems, so that multiple dilutions can be analyzed simultaneously.

In clinical practice, laboratories will need to use different criteria for determining the upper limit of normal (cut-off) for sIL-2R levels; there is such a broad range of values that distinct cut-off values may be required for each situation. These will depend on the disease that is being diagnosed or monitored, and on the stage at which sIL-2R levels are measured for that disease.

There are many research use only (RUO) ELISA kits available for measuring sIL-2R levels. However, in order to be able to use a kit for diagnostic purposes, it must comply with applicable regulations. In the European Union, this means that kits used for IVD must conform to IVDR (Regulation (EU) 2017/746 for in vitro diagnostic medical devices).

The principal characteristics of sIL-2 kits might include, but are not limited to:

• A ll kit reagents and standards should be ready-to-use – no dilution necessary

• C alibrated against NIBSC 97/600 Standard Preparation

• I nternal kit controls provided

• E asily automatable

Sarcoidosis and sIL-2R: a deep dive Sarcoidosis, also known as BesnierBoeck-Schaumann disease, is a rare condition that causes small patches of swollen tissue – called granulomas – to develop in the organs. 24 It often affects the lungs and lymph nodes, and can also affect the skin. The symptoms of sarcoidosis depend on which organs are affected, but typically include tender bumps on the skin, shortness of breath and a persistent cough.

It is impossible to predict how sarcoidosis will affect a person, as the condition can affect any organ, and the symptoms vary widely depending on which organs are involved.

Sarcoidosis may be acute or chronic, and some people do not have any symptoms at all, so that their condition might only be diagnosed after an X-ray carried out for another reason.

Elevated sIL-2R levels have been reported in numerous studies in sarcoidosis patients and, as such, sIL-2R is already an established biomarker for the disease. 2,25 Some studies suggest the measurement of sIL-2R as a marker of therapy success. 26 Sensitivity of serum sIL-2R as a diagnostic biomarker for sarcoidosis lies around 79 percent and, in patients with uveitis, the sensitivity of elevated sIL-2R levels to establish underlying sarcoidosis is around 81-98 percent, with an AUC of 0.76 (fair) and 0.96 (excellent). 27

Interestingly, patients with extrapulmonary involvement have been shown to have relatively high levels of serum sIL-2R, suggesting value as a staging and/or severity biomarker. Patients with more advanced radiographic stages and progressive disease also show higher levels of sIL-2R. Serum sIL-2R tests have the highest ability to determine pulmonary severity in comparison to soluble angiotensin-converting enzyme (sACE). Furthermore, in contrast to sACE, an advantage of sIL-2R measurement is that interpretation is not confounded by the use of drugs or immunosuppressants. 27

When using sIL-2R levels as a biomarker for sarcoidosis, it should be noted that an increase in serum sIL-2R values is not always disease specific; elevated values can also be found in other conditions, including hematologic malignancies, other granulomatous diseases, various autoimmune disorders and posttransplantation. Renal insufficiency may also have a major impact on sIL-2 levels, which could lead to misinterpretation of test results. 27

However, even given these caveats, and especially when considering the broader clinical picture of the individual patient, serum sIL-2R measurement is a very useful prognostic marker. High serum sIL-2R levels can predict the need for therapy in sarcoidosis patients, and high sIL-2R at initiation of therapy has even shown value as a predictor of relapse after therapy with infliximab. 27 Changes in concentration of serum sIL-2R have also been shown to be related to clinical changes, correlating well with changes in pulmonary function parameters and radiological abnormalities. Finally, the serial measurement of serum sIL-2R during disease follow-up has proven useful for assessing the evolution of disease activity in sarcoidosis. 27 In conclusion, when it comes to establishing a credible tool for diagnosing, monitoring and treating sarcoidosis, sIL-2R measurement is here to stay.

The future of sIL-2R measurement

We have seen that sIL-2R measurement can be an extremely useful tool for clinicians to assess immune function in vivo across a broad spectrum of diseases, and established its key role in the investigation and management of sarcoidosis. The adoption of sIL-2R measurement as standard in the clinical environment will, of course, depend on the development of IVDR-compliant assays. However, much of the groundwork in preparation for their debut in the clinic is complete, and sIL-2R ELISA kits are already available for research. We must now begin to home in on how we can use this ubiquitous and complex biomarker to help in the diagnosis or therapeutic monitoring of autoimmune diseases.

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