Interferon-α (IFN-α) is the only drug approved for adjuvant therapy of malignant melanoma and is also used in the treatment of hematological and solid tumors. Along with its proven clinical efficacy, IFN-α produces several side effects, particularly with regard to autoimmune disorders. Curious about symptoms of autoimmunity during IFN-α therapy, we asked whether IFN-α directly impacts on immune tolerance. We found that IFN-α does alter the function of tolerogenic dendritic cells (DC) as well as of induced and naturally occurring T-regulatory cells (nTregs). IFN-α blocks the tolerogenic phenotype of DC by inducing maturation and thus preventing the induction of inducible Tregs by DC. It also has direct effects on nTregs. IFN-α reduces cAMP in Tregs via ERK/phosphodiesterase-mediated pathways. Since cAMP is essentially involved in suppression by nTregs, the IFN-α-dependent reduction of cAMP levels abolishes the suppressive capacity of nTregs. Therefore, Tregs are incapable of suppressing the activity of effector T cells and natural killer cells, resulting in tumor rejection. Thus, IFN-α overcomes immunological tolerance processes, leading to an improved immunostimulation and efficient tumor rejection, but also increases the risk of autoimmunity.
The treatment of malignant melanoma still represents a major challenge. While BRAF inhibitors and anti-CTLA-4 antibodies produce significant improvement in therapeutic outcome in patients with progressive disease and distant metastases, in the adjuvant setting interferon-α (IFN-α) is still the only drug approved in Germany for this indication . Apart from immunotherapy of malignant melanoma, IFN-α is commonly used to treat a variety of hematological and solid tumors, such as cutaneous T-cell lymphoma and Kaposi sarcoma; it also is widely used in the treatment of viral hepatitides .
Interferon-α belongs to the group of type-I interferons and is characterized by immune-stimulating, pro-apoptotic and anti-neoplastic effects [3, 4]. Its inhibition of tumor cell growth and blocking of angiogenesis as well as its capacity to promote immune stimulation (for example by activation of dendritic cells initiating an efficient immune reaction against the tumor or by stimulating natural killer cells which destroy tumor cells via cytotoxic effects [2, 5]) are decisive for its therapeutic benefit in tumor therapy. Apart from these desired anti-neoplastic effects, therapy with IFN-α is accompanied by multiple side effects, particularly with regard to autoimmune disorders [2, 5], and in some patients the induction of autoimmunity necessitates discontinuation of therapy (Figure 1). Generally induction of autoimmunity is initiated by the body's own immune cells being misrouted against autologous tissue. A healthy organism manages to differentiate between „self“and „foreign“and thus can prevent the development of autoimmunity. Moreover, over-exuberant immune reactions against self or against exogenous substances such as pathogens or allergens may be controlled via processes of peripheral tolerance (for example anergy and deletion of T cells) or by effects of T-regulatory cells (Tregs). Dysfunction of Tregs and thus, failure of the mechanisms producing peripheral tolerance results in autoimmune phenomena .
Generally, two types of Tregs can be differentiated: naturally occurring Tregs generated in the thymus (nTregs) and peripherally induced Tregs (iTregs). Tregs manage to establish an immunological balance between an adequate immune response (against viruses and against pathogens) and conservation of self-tolerance, thus preventing the development of autoimmune disorders. In case of malignant diseases (for example malignant melanoma) the tumor takes advantage of the regulatory effects of Tregs on tolerance . Along with Tregs, tolerogenic DC play an important role in tumor-associated tolerance mechanisms, which sometimes are directly induced by the tumor itself. Normally, the immune system detects the body's own tumor cells as „foreign“. By stimulating Tregs or tolerogenic DC, the tumor establishes an evasion strategy, which weakens or even inhibits the anti-tumor immune response and thus enables it to withstand the attacks of the immune system. The increased activation of tolerance processes in terms of Tregs and tolerogenic DC in cancer patients is definitely unfavorable for a successful therapy outcome, so modulating or inhibiting tolerance processes is thus an important starting point for tumor therapies .
Aim of the study
Interferon-α has a proven clinical efficacy in the treatment of several hematological and solid tumors and to date it is the only therapeutic drug being approved in Germany in the adjuvant treatment of malignant melanoma. Unfortunately, treatment with this type-I interferon is associated with multiple side effects; particularly with regard to the induction of autoimmune disorders. This observation raises the question of whether IFN-α has an influence on tumor associated tolerance processes and by which mechanisms this cytokine mediates its effects. The aim of our study was to analyze the influence of IFN-α on tumor-associated tolerance processes with regard to the characteristics of human tolerogenic DC and Tregs as well as in view of the intracellular pathways being induced by IFN-α.
State of research
Previous research data already show that IFN-α amplifies the activation of T cells and prolongs their survival time [2, 5]. Our study group observed that IFN-α directly impacts on tolerance by abrogating the effect of tolerogenic DC . Following treatment with IFN-α, these human DC are no longer capable to induce iTregs but activate effector T cells, which have no suppressive capacity (Figure 2a). Administration of IFN-α, however, has no direct effect on already induced human iTregs, so that these iTregs still can fulfill their regulatory function . Similar data obtained in mouse model studies demonstrate that IFN-α has an indirect influence on the induction of Tregs by effecting antigen-presenting cells .
Moreover, the question arises as to whether IFN-α has a direct effect on human, naturally occurring CD4+CD25+FOXP3+ Tregs. For this reason we performed tests with human nTregs. Analyses concerning the phenotype of Tregs considering the expression of functionally important surface molecules (CTLA-4, GITR, PD-1, CD127, CD39, CD62L, ICOS, CCR7, HLA-DR), the cytokine profile (for example IL-10, TGF-β) or their anergic condition showed no modification following treatment with IFN-α. Moreover, the expression of Foxp3 (which is an essential transcription factor for Tregs) or the methylation status of a certain sequence in the FOXP3 locus, which is decisive for the stability and function of the FOXP3 molecule, was not modified by IFN-α. All in all, IFN-α has no impact on the differentiation program of human Tregs .
We then looked at the intracellular signaling pathways in Tregs following administration of IFN-α. Regulatory T cells have to get in contact with target cells in order to carry out their suppressive function. This communication occurs via gap junctions . These channel-like molecule complexes transport adenosine monophosphate (cAMP), which is essential for the suppressive effects of Tregs [12-14]. Through the exchange of cAMP between Tregs and target cells, the expression of the T-cell growth factor interleukin 2 (IL-2) is suppressed, leading to a decreased proliferation of these cells (Figure 3a). The cAMP effect is mediated by different transcriptional control elements in the nucleus [15, 16]. We managed to demonstrate that under the influence of IFN-α Tregs are no longer capable of suppressing co-cultured effector T cells. At the same time they show a significantly reduced level of intracellular cAMP (Figure 2b) . Thus, it is reasonable to assume that Tregs have reduced suppressive properties following IFN-α treatment, due to a dysfunction of the adenosine-dependent signal pathway. The enzymatic control of the intracellular cAMP level is catalyzed by the activity of the cAMP-synthesizing adenylate cyclase as well as by cAMP-degrading phosphodiesterases (PDE). To prove that IFN-α acts via cAMP-dependent mechanisms on Tregs, we performed suppressive tests with PDE-blocking inhibitors with IFN-α- and inhibitor-treated Tregs (Figure 3b). We were able to show that the effect of IFN-α on the suppressive activity of Tregs is dependent on functional PDE and mainly on PDE4, which is active in T cells . This mechanism is not controlled by STAT-mediated but by MAP kinase ERK-controlled signal transduction pathways (Figure 3b). In tests with natural killer cells we were able to show that by inhibition of the suppressive properties of human Tregs by IFN-α, these immune cells regain their cytotoxic function and thus are again capable to destroy tumor cells efficiently.
With our study we could demonstrate that IFN-α mediates one of its most relevant mechanisms of action via its blocking effects on tolerance processes (especially on human Tregs), which is essential for the anti-neoplastic effect of this cytokine (Figure 2b). The results of these experiments are in accordance with other studies showing that treatment with IFN-α in patients with malignant melanoma or renal cell carcinoma lead to a reduction of the number and function of Tregs, whereas reduced levels of IFN-α in tumor patients seem to provoke a reduction of Tregs worsening the patients prognosis and outcome [17-19]. On the other hand, several studies prove that the risk of autoimmunity increases following treatment with IFN-α. In several autoimmune diseases, such as lupus erythematosus or psoriasis, patients display a specific expression pattern of interferon-dependent genes in their leukocytes, the interferon signature, and that increased levels of IFN-α may also be associated with modified tolerance processes, including Tregs .
Conclusion for clinical practice
Treatment of tumor patients with IFN-α produces good anti-tumor effects. However, therapy is associated with an increased rate of side effects, especially autoimmune disorders. In order to improve therapy, our data seen as well as study results of other groups concerning the inhibiting effects of IFN-α on tolerance processes clearly demonstrate the need to find a balance between the knock-out of Tregs as part of the tumor evasion strategy and the conservation of peripheral tolerance at the same time in order to prevent autoimmunity. Detailed knowledge of the effects of IFN-α, for example as we managed to show with regard to the control of cAMP-dependant pathways, may offer a potential therapeutic target.
About the authors
Verena Raker is doing her post-doctoral work at the Department of Dermatology of the University Medical Center Mainz, being member of the working group of Kerstin Steinbrink. Currently, she is working on the role of tolerance processes and inflammation in scleroderma and contact allergies. During her doctoral thesis she was working on the role of unconventional regulatory T cells in allergic respiratory diseases and in the high zone tolerance.
Kerstin Steinbrink was chairwoman and member of the ADF Board and is currently on the board of the European Society for Investigative Dermatology. She is vice chair of the Department of Dermatology, University Medical Center Mainz with a focus on dermato-oncology and autoimmune diseases. In recent years she has concentrated on several individual projects and networks on the research of immunological tolerance processes and the translation of these results into clinical practice.