Interleukin-2 treatment of tumor patients can expand regulatory T cells

Abstract

Augmented numbers of regulatory T cells contribute to the overall immunosuppression in tumor patients. Interleukin-2 has been widely used in the clinics in anticancer therapy, yet evidence has accumulated that the major drawback, limiting clinical efficacy, is the expansion of regulatory T cells, which aggravates immunosuppression.

Keywords: :

• immunotherapy
• Interleukin-2
• T_reg cells
• tumor

Interleukin-2 (IL-2) has been identified more than 30 years ago and primarily been described as a factor acting on conventional T cells to promote their activation and proliferation.¹ Due to its T-cell activating and expanding properties, IL-2 has been introduced early into the immunotherapy of cancer patients, either as a single agent or in combination with other cytokines or chemotherapy.¹ However, over the last several years it has become clear that IL-2 not only has beneficial properties, but also can expand regulatory T (T_reg) cells.¹

T_reg cells are involved in self-tolerance, immune homeostasis, prevention of autoimmunity, and suppression of immunity to pathogens.² The forkhead transcription factor FOXP3 is essential for T_reg-cell development and function, as mutations in FOXP3 cause autoimmunity in mice and the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome in humans.²

In tumor-bearing individuals, T_reg cells are increased in numbers both in the local tumor microenvironment and in the peripheral blood, and can contribute to the overall immunosuppression.³ In several human tumors, T_reg cells even have prognostic significance and their abundance can be correlated with the stage of disease.³ Depletion of T_reg cells has been suggested as a therapeutic option, with early clinical trials using an IL-2 immunotoxin showing promising results.³

One important and yet unresolved aspect in tumor immunology is how and where T_reg cells in tumor patients develop. Peripheral induction is one possibility, whereby tumor antigen-specific T cells might be converted into FOXP3-expressing T_reg cells with suppressive functions within the tumor microenvironment. An alternative scenario would be the accumulation of T_reg cells generated in the thymus, which would be attracted to the tumor site by specific factors such as chemokines.

Several studies have assessed the abundance and function of human T_reg cells after IL-2 administration, and the overall consensus was that IL-2 augments their frequency.^4-6 Possible explanations for such an increase were peripheral expansion of T_reg cells but also altered migratory activity.⁷ However, none of these studies assessed how IL-2 influenced the thymic output of T_reg cells and whether this would interfere with efficient antitumor immune responses.

In a recent study,⁸ we investigated how IL-2 treatment influences T_reg-cell numbers and function in colorectal cancer patients undergoing a combined immunochemotherapy. We observed increased levels of T_reg cells, as determined by a combined staining for CD4, FOXP3, and CD25 in the peripheral blood of these patients at the start of therapy, confirming previously published observations.³ These cells expressed typical T_reg-cell markers including CTLA-4 and GITR and had normal immunosuppressive functions. Next, we assessed the influence of IL-2 on the number of total T_reg cells after completion of therapy, finding an expansion of the pool of T_reg cells in IL-2 treated patients. This is in line with previously published studies, which also reported elevated numbers of T_reg cells after treatment with IL-2.^4-6

Similar to conventional T cells, T_reg cells can be distinguished into memory and naïve subsets, according to the surface expression of CD45RA. Valmori et al. were the first to report that a subset of naïve T_reg cells exists that is anergic following stimulation in the absence of IL-2, exerts ex vivo cell-cell contact-mediated suppressor functions yet proliferates in response to stimulation with autologous antigen-presenting cells.⁹ These observations indicate that a high proportion of these cells have self-reactive T-cell receptors and hence that they are derived from the thymic T_reg-cell compartment.⁹ The relationship between memory and naïve T_reg cells was further delineated in humans using genomic and functional approaches by Miyara et al., who clearly established that naïve T_reg cells are an important subpopulation of human FOXP3⁺ T_reg cells.¹⁰ Using a similar gating strategy, which included the assessment of CCR7 expression to distinguish between central- and effector-memory T_reg cells, we could detect an increase in naïve T_reg cells before the initiation of immunotherapy. This increase in naïve T_reg cells was even more pronounced after IL-2 administration and assessment of their suppressive function showed immunosuppressive activity comparable to that of memory T_reg cells. One approach to determine the vicinity of T cells to the thymus is to determine the number of T-cell receptor excision circles (TRECs). Assessing TRECs in sorted naïve T_reg cells from healthy donors and patients, before and after therapy, indicated that naïve T_reg cells are enriched in thymus-derived T_reg cells even before therapy, but particularly after the administration of IL-2, suggesting that IL-2 primarily acts on the thymus to produce additional T_reg cells that join those already present in the tumor microenvironment and the peripheral blood of these patients.

To substantiate this observation, we administered IL-2 in a murine model system and could show that the IL-2 treatment results in an expansion of naïve T_reg cells in all immunological cell compartments. This was mainly due to an increased thymic output, as assessed by analyzing TRECs in the sorted naïve T_reg cells from these animals.

Taken together, our data supports an overall increase in T_reg cells in tumor patients with an expansion of newly generated naïve T_reg cells post IL-2 therapy as a major mechanism of the T_reg-cell expansion in IL-2 treated tumor patients (Fig. 1). This finding has implications for the future direction on how to target T_reg cells in tumor patients. Depletion of T_reg cells, e.g., by the administration of T_reg-cell targeting antibodies or immunotoxins, will only result in a short-term depletion of peripheral T_reg cells. Long-term reduction of T_reg cells will warrant therapeutic strategies reducing the thymic output of T_reg cells, thus properly circumventing their immunosuppressive functions in tumor patients.

Figure 1. Interleukin 2 administration results in a preferential expansion of regulatory T (T_reg) cells. Administration of interleukin-2 to tumor patients only results in a weak activation and expansion of potentially tumor-counteracting effector T cells, while memory T_reg cells in the periphery as well as the naïve thymic T_reg-cell pool are consistently expanded.