Regulation gone wrong: a subset of Sézary patients have malignant regulatory T cells

Abstract

Cutaneous T cell lymphomas are a heterogeneous group of non-Hodgkin’s lymphomas derived from the population of T cells that home to and inhabit the skin. There have been conflicting reports as to whether CTCL may represent a malignancy of regulatory T cells, a particular T cell subset that can suppress local immune reactions. In this issue of the JID, authors Heid et al. present convincing evidence that the malignant T cells in a specific subgroup of Sézary patients are FOXP3⁺ regulatory T cells. Clonal malignant T cells in these patients have increased expression of the Treg associated transcription factor FOXP3, demethylation of the FOXP3 gene locus, and T cells from at least some of these patients could suppress T cell proliferation in vitro.

By virtue of their ability to inhibit T cell activation, proliferation, cytokine production and cytotoxicity, regulatory T cells (Tregs) can suppress immune reactions in their local vicinity (Sakaguchi, 2005). Tregs play a critical role in inducing and maintaining immune self tolerance; humans and mice with mutations in the Treg associated transcription factor FOXP3 gene lack functional Tregs and develop widespread and lethal automimmunity (Bacchetta et al., 2006; Brunkow et al., 2001; Khattri et al., 2003; Powell et al., 1982; Satake et al., 1993). In addition to their normal role in maintaining self tolerance, Tregs are either expanded or locally recruited in many human cancers and act to impair anti-tumor immunity (Baecher-Allan and Anderson, 2006).

Despite their functional importance, identification of Tregs can be problematic. Both Tregs and recently activated T cells express the high affinity IL-2 receptor chain CD25, making it impossible to discriminate Tregs from recently activated T cells using this marker alone. Tregs can be identified definitively by demonstrating their functional ability to suppress T cell proliferation in vitro. However, Tregs are often present in low numbers, making functional assays difficult or impossible. The next most reliable method of identifying Tregs is by demonstrating high and constant expression of the transcription factor FOXP3. Expression of the FOXP3 protein is critical for the development and functional activity of Tregs. As noted previously, mutations in this gene lead to decreased regulatory T cell function in both mice and humans and expression levels of the FOXP3 protein correlate with the ability of Tregs to suppress (Fontenot et al., 2003; Tao et al., 2007). In mice, FOXP3 is expressed exclusively by Tregs. In humans, FOXP3 is transiently expressed by all T cells after activation, although expression levels do not reach those observed in true Treg (Clark and Kupper, 2007; Gavin et al., 2006; Pillai et al., 2007). Before this biology was fully understood, small increases in FOXP3 protein expression or mRNA levels were misinterpreted as indicating a Treg phenotype. One bright spot in the field of Treg biology is the recent finding that demethylation at the FOXP3 promoter site can be used to discriminate true Tregs from activated non-regulatory T cells (Baron et al., 2007; Janson et al., 2008).

There have been conflicting reports regarding the role of Tregs in cutaneous T cell lymphoma (CTCL). A report that malignant T cells from CTCL patients stimulated in vitro with immature dendritic cells (DC) loaded with apoptotic T cell debris adopted a Treg like phenotype led to assertions that CTCL was a malignancy of Tregs (Berger et al., 2005). However, even normal T cells co-cultured with immature DC presenting peptides from apoptotic cells have been shown to adopt a regulatory T cell phenotype (Steinman et al., 2003). This is a major mechanism for the formation of peripheral tolerance, a mechanism that may explain maternal fetal tolerance and is currently being exploited to treat allograft rejection (Adams et al., 2007; Morelli and Thomson, 2007). Subsequent histologic studies found low or negative expression of FOXP3 in the malignant T cells of CTCL skin lesions (Gjerdrum et al., 2007). However, CTCL skin lesions did contain benign infiltrating FOXP3⁺ Tregs and the increased numbers of benign Tregs were actually associated with improved survival. A second study also found no evidence for preferential FOXP3 expression among malignant T cells in the skin lesions of Sézary syndrome and in fact found an overall reduction in FOXP3⁺ Treg numbers in both the skin and blood of Sézary patients (Klemke et al., 2006). Studies of peripheral blood in advanced stage CTCL found no clear differences in blood Treg numbers in CTCL patients versus normal controls but found that Tregs in patients with high tumor burdens had decreased suppressive function (Tiemessen et al., 2006). Taken together, these studies suggest that Treg function is not always a feature of the malignant T cells in CTCL and that maintenance of normal Treg function may actually be a positive prognostic indicator. Recent findings that extracorporeal photopheresis (ECP) increases Treg numbers in patients with CTCL, as it does in other inflammatory diseases, supports this observation (Shiue et al., 2009). However, one recent study did find convincing Treg function in the malignant T cells from a subset of patients with Sézary syndrome. Malignant T cells in these patients produced IL-10 and TGFβ and could suppress T cell proliferation in vitro (Krejsgaard et al., 2008). Interestingly, suppressive function in these cells differed from that of normal Tregs in that it was driven by Jak3/STAT5 signaling as opposed to FOXP3. In short, there are conflicting findings as to whether CTCL represents a malignancy of Treg and indeed, whether Treg function is part of the problem in CTCL or part of the solution.

Authors of the current manuscript have done a thorough job of demonstrating that the malignant T cells in a subset of Sézary patients are true FOXP3⁺ Tregs. Malignant T cells in these patients expressed elevated levels of both FOXP3 mRNA and protein, had demethylation at the FOXP3 promoter locus and most critically, were able to suppress the proliferation of other T cells in vitro.

Ongoing controversies in CTCL aside, most groups would agree that CTCL is a malignancy of T cells that home to and populate the skin. Normal human skin is populated by 20 billion T cells (Clark et al., 2006). 70–80% of these T cells are diverse, polarized effector memory T cells, including Th1, Th2, and Th17 cells, as well as recently described populations of T cells producing IL-13 (Calarese et al., 2009) and TNFα alone (Clark et al., 2009). 5–10% of the T cells in normal human skin are FOXP3⁺ Tregs (Clark and Kupper, 2007). These cells have high and stable expression of FOXP3 and suppress the proliferation on autologous T cells in vitro. Skin resident effector T cells lack expression of the lymph node homing addressins CCR7 and L-selectin, and thus would be expected to be excluded from LN (Clark et al., 2006). Moreover, few are found in blood (Campbell et al., 2001). Elegant studies in mice have recently shown that skin resident effector memory T cells not only survive long term within the skin but also remain in a fixed location (Gebhardt et al., 2009). This characteristic may explain why lesions of fixed drug eruption (which are infiltrated by IFNγ producing T cells) and psoriasis lesions (infiltrated by Th17 cells) remain in fixed locations and tend to recur at the same sites when suppressive therapy is discontinued (Lowes et al., 2008; Teraki and Shiohara, 2003). In contrast to these populations of effector and regulatory T cells, the remaining 20% of T cells in human skin express co-express skin homing addressins (CLA, CCR4) as well as lymph node homing addressins (CCR7 and L-selectin). Co-expression of L-selectin and CCR7 has been used to identify central memory T cells, a type of T cell found in high numbers in blood and thought to represent a long term repository for immunologic memory (Campbell et al., 2001). The addressin expression pattern of these normal skin T cells suggests they are an intermediate phenotype capable of accessing the skin, blood and secondary lymphoid organs.

Human skin therefore contains 20 billion T cells that are exposed throughout our lives to multiple insults, including environmental toxins, UV light and potentially mutagenic viral infections. Studies utilizing spectral karyotyping, comparative genomic hybridization (CGH) and TCR γ chain rearrangements have demonstrated that chromosomally abnormal T cells are present in the skin lesions of even the earliest stages of CTCL and that in later stages in which malignant T cells can be clearly identified, these genetically damaged cells are identical to those bearing the clonal malignant TCR (Kaltoft et al., 1994; Marcus Muche et al., 2004; Padilla-Nash et al., 2007; Whang-Peng et al., 1982). Thus, even in its earliest stages, CTCL is a malignancy of genetically unstable, chromosomally damaged T cells (Thestrup-Pedersen and Kaltoft, 1994). Given the mixed population of T cells in skin, it should not be surprising that one observes a range of phenotypes depending on the characteristics of the transformed cell type. Malignantly transformed cells often maintain some characteristics of their benign precursors. For example, T cells in stable patch plaque CTCL lack expression of CCR7, L-selectin and CD27, all characteristics of polarized effector memory T cells (Campbell et al., 2009). The fact that malignant T cells in MF are found only in skin and often remain localized for decades to fixed plaques is consistent with the sessile nature of skin resident effector memory T cells as demonstrated in mice (Gebhardt et al., 2009). In contrast, malignant T cells in Sézary syndrome express the central memory T cell markers L-selectin, CCR7 and CD27 as well as variable expression of the skin homing addressins CCR4 and CLA (Campbell et al., 2009). In this way, Sézary syndrome T cells resemble the 20% of T cells in normal skin that coexpress CLA, CCR4, L-selectin and CCR7 (Clark et al., 2006). From what we understand of homing receptor expression, these T cells should be capable of entering and migrating through the blood, skin and lymph nodes. Indeed, the malignant T cells in Sézary syndrome migrate throughout the skin, producing erythroderma, and accumulate in the blood and lymph nodes.

Given this view of CTCL, it is logical that the Tregs in skin are equally vulnerable to malignant transformation. When this occurs, a malignancy of T cells is produced that may maintain the ability to suppress T cell activation. Indeed, the suppressive characteristics of Tregs may give lymphomas derived from these cells a survival advantage, allowing them to suppress tumor specific cytotoxic T cells and thereby evade immune destruction. Infection is the most common cause of death in advanced stage CTCL (Axelrod et al., 1992). In the subset of Sézary patients in whom malignant T cells are derived from the Treg lineage, these cells may contribute to immune suppression. However, one does not have to invoke Treg mediated suppression to explain the profound immune defects in patients with other subtypes of advanced CTCL. In most advanced stage patients and even some with earlier stages, there is profound loss of T cell diversity and compensatory expansion of surviving clones (Yamanaka et al., 2005; Yawalkar et al., 2003) suggesting widespread death of T cells on a par with that observed in HIV disease. T cell losses of this magnitude represent a massive loss of critical immune memory accumulated over a lifetime of pathogen exposures. Why T cell death is so widespread in CTCL, particularly in the subset of early stage patients with only limited skin disease, remains unknown.

Human skin is populated by a varied collection of T cells with differing functional, proliferative and migratory capacities. The diversity of clinical presentations in CTCL likely reflects the functional diversity of these T cells. Authors of the current manuscript have identified a Treg subtype of Sézary syndrome. As our understanding of both CTCL and normal skin T cell biology improves, additional functional subtypes of CTCL will likely be identified, for example, Th17 or Th1 CTCL. In this way, we will move from a description of the reaction pattern (folliculotropic, granulomatous) to a description of the pathogenic T cell of origin. This improved understanding of the biology of malignant T cells will in turn allow us to tailor our therapies to address the specific problems raised by each subtype. For example, therapies that suppress cytokine production and effector functions may be most appropriate in stable patch plaque MF, where malignant T cells make up a small but vocal minority of the T cells in skin lesions and primarily cause trouble by engendering and supporting inflammation. In contrast, therapies that aim to kill malignant T cells may be advantageous in Sézary syndrome, where the problem is more the sheer numbers of malignant T cells infiltrating the skin, blood and lymph nodes and the resistance of these T cells to normal mechanisms of T cell death. Until such time, well executed investigations such as the current study shed much needed light on the dimly lit field of CTCL immunobiology.

Abbreviations used

CLA

cutaneous lymphocyte antigen

CCR

CC chemokine receptor

Tregs

regulatory T cells