Elsevier

Clinical Immunology

Volume 128, Issue 3, September 2008, Pages 392-399
Clinical Immunology

CC-4047 promotes Th1 cell differentiation and reprograms polarized human Th2 cells by enhancing transcription factor T-bet

https://doi.org/10.1016/j.clim.2008.04.009Get rights and content

Abstract

The IMiDs® immunomodulatory drugs are an expanding family of compounds under investigation in a broad range of diseases because they exhibit immunomodulatory and anti-tumorigenic properties. Although the molecular targets remain unidentified, the broad activity of select IMiDs® immunomodulatory drugs on cell signaling pathways and transcription regulation has been partly described. One characteristic of these compounds is their ability to act as a co-stimulus of TCR ligation leading to increased IL-2, TNF-α and IFN-γ expression indicative of a Th1 phenotype. Because clinical evidence for this response has been observed in thalidomide and lenalidomide treated patients, we investigated the effect of CC-4047 on T cell activation and differentiation at the molecular level. We used primary human CD4+ T cells as a model and found that CC-4047 enhances the expression of transcription factor T-bet in both naive and pre-polarized Th2 cells. This modulation leads to upregulation of Th1 markers and cytokine production. By increasing the expression of T-bet, CC-4047 promotes the differentiation of naive T-cells to Th1 as well as effectively reverting Th2 cells into Th1-like effector cells in vitro. These findings elucidate a novel mechanism of action of CC-4047 on T cell differentiation, suggesting that certain IMiDs® immunomodulatory drugs may have expanded clinical application in treating both allergic diseases and certain T cell lymphomas where a predominant Th2 phenotype is displayed.

Introduction

Naive T helper (Th) lymphocytes differentiate into two distinct subsets, Th1 and Th2, as defined by their functional ability and cytokine profile. Th1 effector cells produce interferon gamma (IFN-γ) that mediates cell immunity while Th2 effector cells produce IL-4, IL-5 and IL-13 which mediates humoral responses against extra-cellular pathgens. This T cell differentiation paradigm has been well studied and is mainly regulated by transcription factors T-box protein (T-bet) for Th1 cells and GATA-3 for Th2 cells [1], [2], [3], [4], [5]. Cytokines such as IL-12, IL-23, IL-27, type I interferon and type II interferon have been documented to induce T-bet expression via STAT1/STAT3 signaling. In contrast, IL-4 induces GATA-3 expression via STAT6 signaling [6], [7]. The importance of this fundamental paradigm was illustrated by the generation of knockout animals targeting T-bet. T-bet null mice displayed profound defects in the development of Th1 polarized response and were associated with spontaneous airway hyperresponsiveness (AHR), airway inflammation and overproduction of Th2 cytokines. Severe combined immunodeficient mice (SCID) receiving CD4 T cells from T-bet knockout mice also displayed asthmatic symptoms and lung inflammation similar to mice lacking T-bet. Neutralization of IL-13 resulted in amelioration of AHR in airways of T-bet knock out mice. In addition, ectopic expression of T-bet in committed Th2 cells was able to produce high expression of IFN-γ and reverse the Th2 phenotype. Retroviral expression of T-bet in primary human polarized Th2 cells isolated from atopic dermatitis patients exhibited high levels of IFN-γ, IL-12Rβ2 and CXCR3 as well as decreased IL-5 expression while sustained T-bet expression in these cells limited IL-4 production and expressed the Th1 chemokine receptor repertoire, suggesting that T-bet functions to both promote IFN-γ production and inhibit IL-4 production. Moreover, other evidence also suggests that T-bet directly inhibits IL-4 production, for instance, mRNA expression and promoter activity of IL-4 gene were significantly diminished by ectopic expression of T-bet in Jurkat T cells following stimulation [8]. At the molecular level, T-bet has been shown to suppress Th2 lineage commitment via tyrosine kinase-mediated interaction between T-bet and GATA-3 to interfere with the binding of GATA-3 to its target DNA [9].

The IMiDs immunomodulatory drugs are an expanding family of approved and candidate therapies derived from discrete chemical modifications of thalidomide [10]. Lenalidomide (CC-5013, Revlimid®) is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved by FDA for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. CC-4047 (Pomalidomide, Actimid®) is currently in clinical development for the treatment of hematological malignancies [11], [12], [13], [14]. In particular, both CC-4047 and lenalidomide have been shown to co-stimulate T lymphocytes at a level 100 to 1000 times that of thalidomide by elevated TNFα, IL-2, and IFN-γ expression in anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) [15], [16], [17]. The co-stimulatory activity and enhancement of immunity with activation and expansion of CD4+ T cell, NKT cells, γδ T cells and subsequent augmentation of NK cell cytotoxic activity against tumor cells by lenalidomide are considered as a critical mechanism of anti-tumor activity [18]. Although the activation of CD4+ T cells by CC-4047 has been linked to the molecules involved in CD28 signaling in vitro [19], the effect of CC-4047 on naive T cell differentiation is not yet defined. Evidence for a Th1-type immune response triggered by lenalidomide or thalidomide has been reported in clinical trials. For instance, increasing IFN-γ cytokine expression was observed in thalidomide treated scleroderma patients, a disease characterized by excessive fibrosis of the skin and internal organs with elevated Th2 cytokines [20], [21]. Preclinical investigation using a whole tumor cell vaccine showed that CC-4047 enhanced specific anti-tumor immunity in association with enhanced Th1-type cytokine production [22]. And clinical trials with lenalidomide suggest that drug induced T cell differentiation may have an impact on the clinical response. In heavily pretreated patients with advanced mycosis fungoides (MF)/Sézary syndrome (SS), a cutaneous T cell lymphoma (CTCLs) characterized by a dominant Th2 cytokine expression, lenalidomide has shown encouraging clinical response [23]. In contrast, a randomized, double-blind, placebo-controlled trial in moderately severe Crohn's disease, a predominant Th1 cytokine disease, lenalidomide did not show any clinical benefit in treated patients compared to placebo control [24]. Recently a clinical trial with lenalidomide in refractory CLL was preceded by a temporary but acute type I immune response accompanying with tumor lysis syndrome [25].

In order to understand the very early molecular events that certain IMiDs, TNF−α, IL-4 and IL-5 expression during the course of T lymphocyte differentiation in the presence of CC-4047 and anti-CD3 up to 6 days culture. By demonstrating the effect of CC-4047 in T cell differentiation, we provide a cellular and molecular rationale for CC-4047 in treating Th2 cytokine dominated diseases such as cutaneous T cell lymphoma or allergic disorders.

Section snippets

CC-4047 promotes Th1 differentiation via upregulating T-bet

Although it has been reported that patients treated with thalidomide and certain other IMiD compounds bias to a Th1 rather than Th2 response, the molecular mechanisms underlying the polarization of naive T cells have yet to be defined. In initial studies, we hypothesized that the treatment of primary CD4+ T cells with CC-4047 in conjunction with cross linking of T cell receptor (TCR) using plate-bound antibodies to CD3 (anti-CD3) could alter the levels and ratio of two transcription factors,

Discussion

In the present study, we characterized the molecular mechanism of CC-4047 on T lymphocyte activation and initiation of a committed Th1 genetic program. In addition to the anti-tumor and anti-inflammatory properties of certain IMiDs® molecules, the stimulatory effect of this class of compounds on T cells (CD4+, CD8+ and γδ˜ --T), NK cells and B lymphocytes has been documented suggesting an opportunity to expand the clinical use of these drugs. While the specific molecular target(s) remain to be

Isolation of human CD4+ T cells and treatment procedures

CD4+ cells were isolated from human buffy coats by first purifying PBMC by density centrifugation on Ficoll-paque Plus (Amersham Biosciences/GE Healthcare, NJ) and then isolating CD4+ cells by positive selection using anti-CD4 magnetic microbeads (Miltenyi, Germany). CD4+ T cells were washed twice with RPMI complete medium and plated at a density of 2 × 105 cells/mL in 96 well plates or 60 mm dishes. Cells were treated with plate bound anti-CD3 (BD Biosciences, NJ), 20 ng/mL IL-4 (PeproTech, NJ),

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