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Cytotoxic Proteins and Therapeutic Targets in Severe Cutaneous Adverse Reactions

Severe cutaneous adverse reactions (SCARs), such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN), are rare but life-threatening conditions induced mainly by a variety of drugs. Until now, an effective treatment for SJS/TEN still remains unavailable. Current studies have suggested that the pathobiology of drug-mediated SJS and TEN involves major histocompatibility class (MHC) I-restricted activation of cytotoxic T lymphocytes (CTLs) response. This CTLs response requires several cytotoxic signals or mediators, including granulysin, perforin/granzyme B, and Fas/Fas ligand, to trigger extensive keratinocyte death. In this article, we will discuss the cytotoxic mechanisms of severe cutaneous adverse reactions and their potential applications on therapeutics for this disease.

These conditions are rare but could cause significant morbidity and mortality. Here, we discuss the cytotoxic mechanisms and treatment options of severe cutaneous adverse reactions, with a focus on two related diseases, SJS and TEN.
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are considered a spectrum of life-threatening cutaneous diseases, differing only by the degree of epidermal detachment. SJS is defined as the extent of skin detachment involving less than 10% of body surface area (BSA), while TEN is characterized by the detachment greater than 30% of BSA. The severity in-between is categorized as SJS/TEN overlap [1]. Histopathological analyses of skin biopsy specimens from affected areas typically show subepidermal blistering, widespread keratinocyte apoptosis, and full-thickness epidermal necrosis and detachment with a sparse dermal mononuclear infiltrate [2]. The incidence of SJS/TEN is estimated at one to six cases per million inhabitants per year in Europe and the United States [3][4][5][6][7]; however, the mortality rate is 10% for patients with SJS, approximately 30% for patients with SJS/TEN overlap, and almost 50% for patients with TEN [8,9]. Several clinical parameters have been identified as risk factors and incorporated into an illness severity score named SCORTEN (The Severity of Illness Score for Toxic Epidermal Necrolysis), which predicts the mortality [10]. Although microbial infections have occasionally been described as the causes of SJS/TEN, the majority of cases are triggered by drug exposure [1,[11][12][13]. To date, over 100 drugs have been associated with this devastating disease [1], among which aromatic anticonvulsants, allopurinol, sulfonamide antibiotics, oxicam nonsteroidal anti-inflammatory drugs, and nevirapine, show a high relative risk [3,6,14,15].
Although the pathogenic mechanism of SJS/TEN is not fully understood, current pharmacogenomic studies have demonstrated a correlation of human leukocyte antigen (HLA) genes with drug-induced SJS/TEN [16]. With the identification of specific HLA alleles as the predisposing factor to the condition, it becomes clear that drug-induced SJS and TEN are severe hypersensitivity reactions where cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are activated and, subsequently, carry out the cellular immune reactions directed at keratinocytes in a major histocompatibility class (MHC) I-restricted manner. Upon the activation of these immunocytes, various cytotoxic signals, including granulysin, perforin/granzyme B, Fas/Fas ligand, and cytokines/chemokines are launched to mediate the disseminated keratinocyte death in skin lesions ( Figure 1). Other than the immune etiology, bioactivation of the drug and the genetic variations in genes associated with drug metabolism have also been implicated in the pathogenesis of SJS/TEN or other SCARs [17][18][19]. In this review, functional and therapeutic significance of these cytotoxic mediators in the pathomechanism and patient management of SJS and TEN is discussed.

Granulysin
Granulysin is a multifunctional protein present in human CTLs and NK cells, with broad cytolytic activities against a variety of microbes and tumors. It has been studied that granulysin serves as a key molecule responsible for the disseminated keratinocyte death in SJS/TEN [20]. Genome-wide gene expression profiling and immunohistochemical staining of blister cells revealed that granulysin is highly expressed in blister cells of affected individuals. Evidence from experiments using human cells and mice indicated that CD8 + CTLs and NK cells can induce cell death in epidermal keratinocytes through releasing granulysin. Moreover, depletion of granulysin significantly reduced apoptosis of keratinocytes triggered by blister fluids from patients with SJS or TEN. These findings not only support a functional involvement of CTLs, NK cells, and NKT cells in the pathogenesis of SJS and TEN, but also highlight a new cytotoxic mechanism that does not require a direct cellular contact. Figure 1. Cytotoxic proteins and cytokines/chemokines in SJS/TEN. Cytotoxic T lymphocytes (CTLs) are activated through the antigen (drug) presentation by the antigen presenting cell (APC) and subsequently carry out the cellular immune reactions directed at keratinocytes in a major histocompatibility class (MHC) I-restricted manner. Upon the activation of CTLs, various cytotoxic proteins, including granulysin, perforin/granzyme B, Fas/Fas ligand, and cytokines/chemokines, are launched to mediate the disseminated keratinocyte death in skin lesions. These toxic signals in turn regulate trafficking, proliferation, and activation of T cells and other immune cells to exaggerate the reaction. The color of CD8+ CTLs is used to illustrate the presence of multiple T cell clones.
Granulysin belongs to the family of saposin-like lipid-interacting proteins and is localized in the granular compartments along with other pore-forming proteins, perforin and granzymes [21]. Two forms of granulysin protein, 9 kDa and 15 kDa, can be found at equivalent levels in human CTLs and NK cells. The 9 kDa form, which is sequestered in the cytolytic granules, is achieved through a proteolytic cleavage at both the amino and carboxyl ends of the 15 kDa precursor, which is constitutively secreted [22]. Crystallography of recombinant granulysin reveals that it is folded as a five-helix bundle stabilized by two conserved intramolecular disulfide bonds [23]. Based on its cationic ampholytic structure, granulysin is able to lyse bacterial membranes which generally are composed of negatively charged lipids. This structural feature renders a scissor-like motion, allowing granulysin to mediate a broad bactericidal activity [24]. In addition, granulysin can kill human cells targeted by CTLs or NK cells by virtue of its permeabilizing effects on mitochondrial and lysosomal membranes. Upon interfering with the membrane of these cellular organelles, cell death pathways are induced through the perturbation of intracellular ion homeostasis [25] and the release of both cytochrome c and apoptosis-inducing factor (AIF) [25][26][27].
In addition to the cytolytic function, both 9 and 15 kDa granulysin have been shown to act as alarmins, endogenous mediators that can induce recruitment and activation of antigen-presenting cells and consequently promote the generation of immune response [28]. Of note, 9 kDa form of granulysin is found to induce the chemotaxis for a variety of immune cells, including T cells, monocytes, and NK cells, and cause an increase in the expression of chemokines (RANTES/CCL5, MCP-1, MCP-3, and MIP-1α/CCL3) and cytokines (IL-1, IL-6, and IFN-α) [29]. Moreover, a recent study indicates that 15 kDa, but not 9 kDa, granulysin is able to activates monocytes to differentiate into immature dendritic cells [30]. These data collectively demonstrate an effect of granulysin on mediating various aspects of immune responses.
The clinical relevance of multifaceted granulysin is observed not only in SJS/TEN but also in a myriad of diseases [31]. It is reported that granulysin is a vital mediator of damage in a number of cutaneous diseases, including folliculitis [32], psoriasis [33], acne [34], lichen planus [35], and other forms of cutaneous ADRs [36,37]. Noteworthily, numbers of granulysin-positive cells in fixed drug eruptions were similar to those observed in SJS/TEN [36], and serum granulysin levels were elevated in DRESS patients [37]. Furthermore, serum levels of granulysin are markedly heightened and correlated with the severity of graft vs. host disease (GVHD) [38], which may mimic SJS and TEN when it presents as similar dermatological manifestations in bone marrow transplant recipients.

Perforin/Granzyme B
Another hypothetic mechanism underlying the cytotoxicity in SJS/TEN involves perforin and granzyme B, two cytolytic proteins that are released by activated CTLs and NK cells. Increasing levels of perforin, granzyme B, and other toxins, have been observed to be correlated with disease severity of drug hypersensitivity from mild maculopapular rashes to severe TEN [39]. In addition, the inhibition of perforin/granzyme B expression has been shown to attenuate the cytotoxic effect of TEN blister lymphocytes toward keratinocytes, whereas neutralization of Fas/Fas ligand signaling by the anti-Fas monoclonal antibody failed to block cell lysis [40].
Exocytosis of cytotoxic compartments is one of the most potent cell death mechanisms employed by activated CTLs and NK cells. Upon lymphocyte recognition of target cells, cytoplasmic granule toxins, predominantly perforin, a membrane-disrupting protein and a family of structurally related serine proteases (granzymes) with various substrate specificities are secreted and together induce apoptosis of the target cells [41]. Perforin is present as a thin 'key-shaped' molecule and composed of multiple domains, among which a carboxy-terminal C2 domain mediates initial, Ca 2+ -dependent membrane binding [42,43]. Perforin usually synergizes with granzymes to carry out cytotoxicity. Granzyme B remains the most extensively studied granzyme that induces DNA fragmentation and different death pathways [44]. It is believed that perforin creates pores within the cell membranes, through which the granzymes B can diffuse into the target cell and trigger its killing activity [45]. However, numerous lines of evidence indicate that granzyme B may enter the target cells independently of perforin, either through receptor-mediated endocytosis or macropinocytosis, and is rapidly internalized into endosomes-like vesicles. Subsequently, perforin perturbs endosomal trafficking in the target cell through a process known as endosomolysis, thereby allowing the release of granzyme B into cytosol [46][47][48][49]. This hypothesis is still the topic of vigorous debate as the molecular mechanisms by which perforin enables granzyme B to escape the endosomal compartment into the cytosol have not been clarified yet. In addition, significant amount of perforin has not been shown to be present in the endosome or cytoplasm of the target cell. More recently, a combined theory suggesting that perforin permeabilizes the plasma membrane and this somehow potentiates the uptake of granzymes into an endolytic compartments is proposed [50]. Regardless of which mechanism occurs, all hypotheses assign the key role of perforin to its membranolytic properties, facilitating granzyme B to exit to the cell cytosol, where it can access its substrates.
Once reaching the cytosol, granzyme B is known to induce cell death through the activation of caspase-dependent and -independent pathways. Specifically, granzyme B can directly cleave and activate the caspases [51,52], a large family of endogenous cytosolic proteases that act in a cascade to dismantle the cell. Moreover, granzyme B can trigger caspase activation indirectly by processing of a pro-apoptotic protein Bid into an active form, which then translocates to the mitochondria, interacts with other pro-apoptotic proteins, such as Bax and Bak, and induces mitochondrial outer-membrane permeabilization (MOMP) [53,54]. Subsequently, Bid-mediated mitochondrial damage results in the leakage of pro-apoptotic mitochondrial mediators, such as cytochrome c, Smac/Diablo and Omi/HtrA2, into the cytosol to augment the downstream caspase activation [55]. Granzyme B also acts on the mitochondria to induce caspase-independent cell death through the generation of reactive oxygen species (ROS) [56]. Furthermore, granzyme B directly cleaves nuclear lamins, a type of intermediate filaments that mediates the structural function and transcriptional regulation in the cell nucleus, as CTLs induce apoptosis during caspase blockade [57]. This finding provides additional insight into the role of granzyme B in caspase-independent death pathways. Taken together, granzyme B elicits its killing ability through various pathways that may maximize the effectiveness in granule-mediated cell death.

Fas/Fas Ligand
Fas/Fas ligand (FasL) interaction is another reported mechanism that modulates keratinocyte apoptosis in SJS/TEN and is also of paramount importance for CTL-mediated lysis in vitro. The pathophysiological study of Fas and FasL on the epidermal cells of TEN patients revealed that an augmentation of soluble FasL (sFasL) and epidermal FasL expression were observed in the sera and skin biopsy specimens from patients with TEN, respectively, suggesting that sFasL detected in the sera is derived from cleavage of a membrane-bound FasL (mFasL) on the epidermal cells of patients with TEN [58]. Moreover, cell death was abolished by the addition of a FasL-blocking antibody. These data implicate the ligation of FasL expressed by TEN keratinocytes with Fas on adjacent keratinocytes as a critical step in the pathogenesis of SJS/TEN. However, keratinocyte FasL was found to be primarily cytoplasmic in vivo and unable to cause apoptosis in a follow-up study [59]. This proposed mechanism was also challenged by the finding that no membrane-bound FasL expression on keratinocytes in TEN patients or in healthy controls can be found, although elevated levels of sFasL in SJS and TEN were detected [60]. Noteworthily, an alternative source of serum sFasL in SJS/TEN was proposed as sFasL levels increased significantly when peripheral blood mononuclear cells (PBMCs) from TEN patients were cultured with the offending drug. Although the involvement of Fas-FasL interactions in mediating keratinocyte death in SJS/TEN was demonstrated in numerous studies, controversy remains as to whether elevated level of sFasL in the TEN sera results from cleavage of mFasL on the epidermal cells or PBMC, as well as whether TEN keratinocytes express lytically active forms of FasL.
Fas (CD95, also called APO-1) is a trimeric transmembrane protein, belonging to a member of the death receptor (DR) family, a subfamily of the tumor necrosis factor (TNF) receptor superfamily [61]. Ligation of Fas with its cognate ligand, FasL, which is also a TNF related transmembrane molecule [62] and expressed in a far more limited way than the receptor, allows the engagement of receptor and subsequent transduction of the apoptotic signal. Upon the activation, a complex of proteins termed death-inducing signaling complex (DISC) forms and associates with activated Fas [63]. This protein complex encompasses the adaptor, Fas-associated death domain protein (FADD) and pro-apoptotic protease, procaspase-8. The latter is recruited by the former and auto-processed into an active form that is subsequently released from the DISC to the cytoplasm. Activated caspase 8 cleaves various protein substrates in the cytoplasm including procaspase-3 and -7, followed by the activation of nucleases, ultimately leading to the degradation of chromosomal DNA and cell apoptosis [64]. In addition, another Fas-mediated death pathway that is not propagated directly through the caspase cascade has been proposed to be amplified via the mitochondria. In such a paradigm of Fas-induced apoptosis, cleavage of Bid by active caspase-8 mediates the mitochondrial damage, which results in release of cytochrome C [65,66]. Once cytochrome c is released, it interacts with the apoptosis protease activating factor 1 (APAF1) to form the apoptosome, the second initiator complex of apoptosis. The apoptosome unleashes the apoptotic activities by the recruitment and activation of caspase-9, which in turn proteolyzes the downstream effector caspases, caspase-3 and -7, and further triggers a cascade of events, leading to apoptosis [64]. Noteworthily, generation of ROS has also been documented as a key mechanism of apoptosis regulation in Fas-induced cell death and related apoptosis disorders [67].
In addition to the regulation of apoptosis, Fas-FasL interaction has also been shown to play a prominent role in the activation of NF-κB [68,69] and the induction of inflammatory response [70][71][72]. These distinct effects of FasL may result from the functional differences in membrane-anchored and soluble form of this molecule. It is reported that murine sFasL is not apoptotic in vivo [73], and under certain circumstances, sFasL may even antagonize the effects of mFasL [74,75]. These diverse activities of Fas suggest that the pathogenic role of epidermal Fas expression in SJS/TEN may be different from that of elevated sFasL detected in the sera.

Cytokines and Chemokine Receptors
Except for those mentioned above, an overexpression of TNF-α derived from macrophages as well as from keratinocytes was observed in the lesions of TEN, indicating a potential link of TNF-α to extensive necrosis in this disease [76]. TNF-α is a potent cytokine that induces cell apoptosis, cell activation, differentiation, and inflammatory processes [77,78]. Binding of TNF-α to its cell surface receptor triggers apoptosis through DISC-mediated activation of caspase cascade and mitochondrial changes, leading to a series of cytotoxic processes, including generation of free radicals and damage to nuclear DNA by endonucleases [79]. In addition to the apoptotic activities, the pathogenesis of SJS/TEN, in part, is contributed by TNF's effects on inflammatory response. TNF-α appears to be central to the changes in the vascular endothelial permeability and to the interaction between the leukocytes and vascular endothelium [80,81]. In coordination with the expression of specific cell adhesion molecules, TNF-α is also known to recruit different populations of immunocytes [82,83], which fits the observation that the leukocyte infiltrate remains a key histopathological feature of SJS/TEN.
Another key cytokine that has been reported to play a key role in SJS/TEN is interferon-γ (IFN-γ) [84]. Although not transmitting apoptotic signal through a conventional death receptor, IFN-γ orchestrates the cytotoxic activities, to some extent, by induction of ROS [85], which is a shared mechanism connecting the involvement of IFN-γ in SJS/TEN with TNF-α and FasL [86]. Moreover, the apoptotic effects of IFN-γ can also be explained by its transcriptional regulation of a variety of genes that are vital for apoptosis, such as TNF-a receptor, Fas/FasL, caspase-1, -4, and -8 [87][88][89][90]. More strikingly, IFN-γ is known to promote antigen presentation and thus stimulate the cell-mediated immunity by upregulation of MHC molecules [85,91,92], further supporting a pathogenic role in SJS/TEN.

Therapeutic Interventions of SJS/TEN
With the advances in uncovering the genetic markers of SJS/TEN, the incidence has improved over the past few years with the approval of the genetic screening as a guildline-based test before the use of certain offending drugs [97,98]. Unfortunately, treatment for SJS/TEN remains ineffective and only supportive. Supportive care, including protection of exposed dermis and mucosal surfaces, management of electrolyte and energy intake, pain control, sedation, and detection and treatment of infections, was initiated after admission [99,100]. In addition, prompt withdrawal of causative drugs is believed to be a priority of patient management [101], although early diagnosis of the condition and identification of the offending medication remain a real clinical challenge.
Plasmapheresis and hemodialysis that remove the causative drug, its metabolites or other toxic mediators from the circulation have been used to effectively alleviate the manifestations of SJS/TEN [102][103][104][105]. However, due to the stress associated with this treatment and the lack of formal proof, these strategies at the present time are still debated [11,106].
In addition to elimination of toxins from circulation, many therapy modalities that target those toxic mediators mentioned above have been found to be beneficial for practical management of SJS/TEN. A notable example is the use of systemic corticosteroids in patients with TEN. Corticosteroids, often known as steroids, are an immunosuppressant prescribed for a wide range of conditions. Despite the arguments regarding their efficacy and safety on controlling disease progression [107][108][109], systemic corticosteroids have been the mainstay of the treatment of SJS/TEN for long time, which may be explained by the notion that many toxic mediators involved in the pathogenesis possess not only cytotoxic but also inflammatory effects. Other immunomodulators that have been studied in isolated cases of SJS/TEN include cyclophosphamide [110,111], cyclosporine [112][113][114], N-acetylcysteine [115,116], pentoxyfilline [117], and thalidomide [118].
Another therapeutic approach directed against the Fas/FasL interaction to treat SJS/TEN has been proposed by using intravenous immunoglobulins (IVIg) [58]. Treatment of this condition with IVIg has been documented in several case series, with conflicting outcomes. Some support the use of IVIg based on reduced mortality rates [119][120][121][122][123], whereas others consider that their efficacy is questionable [124][125][126][127]. A potential clinical trial that targets Fas/FasL interaction through specific blockade, such as anti-Fas (CD95) antibody may further elucidate the pathogenesis of SJS/TEN.
In addition to targeting the Fas/FasL signaling, several agents have been used to improve the outcome of SJS/TEN through their action on antagonizing the TNF-α pathway. These include TNF inhibitors, pentoxyfilline [117] and thalidomide [118], and an anti-TNF monoclonal antibody, infliximab [128].

Conclusions
In spite of being uncommon, SJS and TEN have an immense impact on public health by virtue of high mortality. Although the incidence has improved with the identification of the genetic predisposing factors, there is unfortunately no treatment guideline, highlighting the inadequacy of current accepted regimens. The rationale for recent therapeutic interventions of SJS/TEN is mainly derived from empirical evidence regarding our understanding of cytotoxic pathways in keratinocytes and lymphocytes elicited by drug exposure. However, until now, no treatment is specifically directed against granulysin, whose role in epidermal necrolysis has been recognized as a breakthrough in the pathogenesis of SJS/TEN. A reagent blocking the effects of granulysin may be of greater therapeutic value and becomes an urgent need. It is conceivable that a novel treatment for combating granulysin-mediated cell death alone or in combination with existing agents will be beneficial to achieve a better outcome in unfortunate people who suffer from this deadly adverse drug reaction.


extfieldsofstudy: Biology Medicine

text:
TRAF3 regulates the effector function of regulatory T cells and humoral immune responses

The induction of follicular regulatory T cells and control of germinal center reactions require the adaptor protein Traf3, in part because of Traf3-induced expression of ICOS.

Foxp3 + regulatory T cells (T reg cells) form a major population of CD4 + immunosuppressive T cells that plays a pivotal role in maintaining peripheral immune tolerance and preventing autoimmune diseases (Sakaguchi et al., 2008). In addition, T reg cells also restrain the immunity against foreign antigens and cancer. The development, maintenance, and function of T reg cells are dependent on the master transcription factor Foxp3 and factors that regulate Foxp3 expression and function (Josefowicz et al., 2012). Genetic deficiencies in these core regulatory factors typically lead to impaired self-tolerance and homeostasis of T cells, coupled with severe autoimmune disorders. Strong evidence suggests that T reg cells represent a diverse cell population, comprising functionally distinct subsets that control different types of immune responses (Campbell and Koch, 2011;Josefowicz et al., 2012). The molecular mechanism that regulates the differentiation and function of the diverse T reg cells subsets remains poorly understood.
Recent studies have identified a specific subset of T reg cells, the follicular T reg cells (T FR cells), which are localized in the B cell follicles and specialized for the control of germinal center (GC) reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). Formation of GCs is essential for various events of a T-dependent humoral immune response, such as antibody class switching, somatic hypermutation, and affinity maturation (Ramiscal and Vinuesa, 2013). The GC reactions depend on follicular T helper cells (T FH cells), a subset of CD4 + T effector cells which provide essential help to cognate B cells for their activation and differentiation in GCs (Linterman et al., 2012).

TRAF3 deficiency partially impaired the in vivo function of T reg cells
To study the function of TRAF3 in T reg cells, we generated T reg -conditional TRAF3 KO mice by crossing the Traf3-floxed mice with Foxp3-Cre mice (Fig. 1 A). Ablation of TRAF3 did not reduce, but even moderately increased, the frequency of T reg cells in the spleen (Fig. 1 B). To examine the effect of TRAF3 deficiency on the immunosuppressive function of T reg cells, we performed in vitro suppression assays using T reg cells purified from the spleen of WT and Traf3 Treg-KO mice. The WT and TRAF3-deficient T reg cells had comparable efficiencies in suppressing the proliferation of naive T cells ( Fig. 1 C), suggesting a dispensable role for TRAF3 in regulating the in vitro function of T reg cells.
We next examined the in vivo function of T reg cells using a well-defined adoptive transfer model, which involved cotransfer of CD45RB hi naive CD4 + T cells with T reg cells purified from the WT and Traf3 Treg-KO mice. As expected, adoptive transfer of CD45RB hi naive T cells into Rag1-KO mice induced severe body weight loss of the recipient mice, coupled with massive expansion of the transferred T cells, and these pathological phenotypes were efficiently blocked when the naive T cells were cotransferred with WT T reg cells (Fig. 1 D). Compared with the WT T reg cells, the TRAF3-deficient T reg cells exhibited a reduced ability to suppress the expansion and pathological activity of the naive T cells (Fig. 1, E and F). Although the mutant T reg cells remained fully functional in inhibiting the production of Th17 cells from the cotransferred naive CD4 + T cells, their ability to suppress the generation of Th1 cells was partially impaired (Fig. 1 F). After the adoptive transfer, a lower number of TRAF3-deficient T reg cells was recovered compared with that of the WT T reg cells ( Fig. 1 G), suggesting reduced expansion or survival of the mutant T reg cells. Furthermore, they showed a reduced level of IL-10 production ( Fig. 1 H). Collectively, these data suggest that T reg cell-specific TRAF3 deficiency does not compromise the homeostasis or in vitro function of T reg cells but partially impairs the in vivo functions of T reg cells.

Altered CD4 T cell homeostasis in Traf3 Treg-KO mice
To assess the function of the TRAF3-deficient T reg cells under nontransferred conditions, we examined the homeostasis of CD4 + T cells in peripheral lymphoid organs. The spleens and lymph nodes of the Traf3 Treg-KO mice contained an enhanced frequency of CD4 + T cells with memory-like markers (CD44 hi CD62L lo ; Fig. 2, A and B). Among the memory-like CD4 + T cells in the peripheral lymphoid organs, there was a significant increase in the frequency of IFN--producing Th1 cells in the Traf3 Treg-KO mice, although the frequency of Th17 cells was generally low and not significantly different between the WT and mutant mice (Fig. 2 C).
T reg cells play an important role in maintaining T cell homeostasis in the intestine, an organ with dynamic interactions between the immune system and microbiota (MacDonald et al., 2011). The T reg cell-specific TRAF3 deficiency resulted The T FR cells resemble the T FH cells in that they express high levels of the chemokine receptor CXCR5, the inducible co-stimulator (ICOS), and the inhibitory receptor PD-1 (Linterman et al., 2012). However, in contrast to T FH cells, the T FR cells express Foxp3 and possess immunosuppressive function. It appears that the T FR cells are generated from CXCR5  T reg cells, instead of naive T cells, in response to T cell-dependent antigens. The T FR cell production requires the transcription factor Bcl-6 and is negatively regulated by the inhibitory receptor PD-1 (Chung et al., 2011;Linterman et al., 2011;Sage et al., 2013). However, the intracellular signaling events involved in the induction of T FR cells are largely unknown.
The TNF receptor (TNFR)-associated factors (TRAFs) form a family of signaling adaptors that mediate signal transduction from both the TNFRs and various other immune receptors (Ha et al., 2009). Among the TRAF family members, TRAF3 is particularly complex in signaling functions, which differ in the context of different receptors in different cell types (Hildebrand et al., 2011). In B cells, TRAF3 functions as a negative regulator of the noncanonical NF-B signaling pathway and B cell survival. TRAF3 physically interacts with the NF-B-inducing kinase (NIK) and mediates continuous degradation of this central component of the noncanonical NF-B pathway (Liao et al., 2004). Thus, loss of TRAF3 causes constitutive activation of noncanonical NF-B, coupled with B cell hyperplasia and aberrant production of antibodies (He et al., 2006;Xie et al., 2007;Gardam et al., 2008). Similar abnormalities have been observed in B cell conditional transgenic mice expressing a stable form of NIK that lacks the TRAF3-binding motif (NIKT3; Sasaki et al., 2008). In contrast to its negative role in B cell homeostasis, TRAF3 has a positive role in mediating TCR signaling and T celldependent immune responses (Xie et al., 2011). Interestingly, the TRAF3 deficiency is associated with an increase in the frequency of T reg cells, although the role of TRAF3 in regulating the homeostasis and function of T reg cells remains unknown (Hildebrand et al., 2011;Xie et al., 2011).
In the present study, we directly examined the function of TRAF3 in T reg cells by generating T reg cell-specific TRAF3 KO (Traf3 Treg-KO ) mice. We show that TRAF3 is dispensable for the homeostasis of the general population of T reg cells. However, TRAF3 has a particularly important role in the regulation of T FR cell generation and GC reactions. The T reg cell-specific TRAF3 deficiency severely inhibited the antigen-stimulated production of T FR cells, coupled with deregulated activation of T FH cells and heightened GC reactions. Consequently, the Traf3 Treg-KO mice have elevated production of high-affinity antibodies of the IgG subtypes. Mechanistically, TRAF3 is required for maintaining the high-level expression of ICOS in T reg cells. We provide evidence that ICOS deficiency compromises the induction of T FR cells and the control of GC reactions. These findings shed light on the signaling mechanism that mediates T FR cell induction and identify TRAF3 as a crucial factor involved in this specific process. (A) T reg (CD4 + CD25 + YFP + ) and non-T reg (CD4 + CD25  YFP  ) T cells were sorted from the WT-R26 YFP (WT) and Traf3 Treg-KO R26 YFP (KO) mice, and TRAF3 expression was assessed by immunoblot. -Actin served as a loading control. (B) The frequency of Foxp3 + T reg cells (among CD3 + CD4 + cells) in the spleen of WT and Traf3 Treg-KO (KO) mice (6 wk old) was assessed by flow cytometry; data are presented as mean ± SD of five mice per group. Data are representative of five independent experiments. (C) Naive CD4 + T cells (T eff ) were labeled with CFSE and activated by anti-CD3 plus antigen-presenting cells (irradiated splenocytes from WT mice, depleted of CD3 + T cells) in the presence of the indicated ratios of WT or KO T reg cells. Naive CD4 + T cell proliferation was measured as dilution of the cytosolic dye CFSE. Data are presented as a representative plot (left) and a summary graph (right) of six mice per genotype in three independent experiments. (D) Rag1-KO mice were adoptively transferred with WT B6.SJL CD45RB hi naive CD4 + T cells (CD45.1 + ) along with either PBS buffer or sorted T reg cells (CD45.1  ), and body weight was monitored. (E and F) CD45RB hi CD4 + T cells (CD45.1 + ) were transferred into recipient mice as in D (on week 5), and the total number (E) and frequency (F) of Th1 and Th17 cells in the spleen of the recipient mice were assessed by ICS. Eight mice in two different experiments were used for each group. (G and H) The frequency of adoptively transferred Foxp3 + T reg cells (G) and IL-10-producing T reg cells (gated on Foxp3 + cells) in the spleen of the recipient mice described in D was determined by flow cytometry (5 wk after adoptive transfer; H). (C-E, G, and H) Mean ± SD is shown. Data are representative of two independent experiments. *, P < 0.05; and **, P < 0.01. obtained when the mice were immunized with another protein antigen composed of the hapten 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to the carrier protein KLH (NP-KLH; Fig. 3 B). ELISA assays using NP9-BSA (for high affinity) and NP26-BSA (for global affinity) suggested that the T reg cellspecific TRAF3 ablation particularly enhanced the production of high-affinity antibodies ( Fig. 3 B and not depicted). In response to immunization by a nonprotein antigen, NP-Ficoll, the production of high-affinity IgG, mainly IgG2b, was only moderately enhanced in the Traf3 Treg-KO mice (Fig. 3 C).
We also examined the effect of TRAF3 deficiency on the basal level of antibodies in different ages of mice. At a young age, the WT and Traf3 Treg-KO mice had comparable serum concentrations of antibodies (not depicted). However, older Traf3 Treg-KO mice (18-20 wk old) had an elevated basal level of serum IgG, particularly IgG1 and IgG2b (Fig. 3 D). These results suggest that expression of TRAF3 in T reg cells is important for controlling the magnitude of humoral immune responses, especially the production of high-affinity IgG antibodies.
To further assess the role of T reg cell-specific TRAF3 in mediating humoral immune responses, we used a model of influenza infection, which is known to trigger strong T celldependent antibody responses (Elsner et al., 2012). As expected, the WT mice experienced bodyweight loss after the intranasal infection with the A/PR8 (H1N1) strain of influenza virus (Fig. 3 E). Importantly, under the same infection conditions, the Traf3 Treg-KO mice had much less bodyweight reduction compared with the WT mice ( Fig. 3 E). Moreover, the Traf3 Treg-KO mice also produced significantly higher levels of antiviral IgG antibodies (Fig. 3 F). These results emphasize the biological significance of the TRAF3-mediated T reg cell regulation.
in a moderate increase in the frequency of CD4 + T cells, but not the CD8 + T cells, in the lamina propria of the small and large intestines (not depicted). Among the CD4 + T cells, there was a significantly increased frequency of Th1 cells in the lamina propria of Traf3 Treg-KO mice (Fig. 2 D). Thus, the loss of TRAF3 in T reg cells altered the homeostasis of T cells, a finding which was consistent with the partially compromised function of the TRAF3-deficient T reg cells (Fig. 1, D-F). It is important to note, however, that the overall phenotype of the Traf3 Treg-KO mice in T cell homeostasis was considerably milder compared with that observed in mutant mice lacking Foxp3 or its major regulators that mediate the development and core functions of T reg cells (Josefowicz et al., 2012). Moreover, the Traf3 Treg-KO mice only displayed relatively mild tissue inflammation at older ages, mostly seen in the lung and the liver (not depicted). These results suggest that TRAF3 may regulate a specific function, instead of mediating the core suppressive activity, of T reg cells.

T reg cell-specific TRAF3 controls antigen-stimulated humoral immune responses
In addition to maintaining T cell homeostasis and preventing autoimmune diseases, T reg cells play an important role in modulating immune responses to foreign antigens. To examine the role of TRAF3 in mediating this latter function of T reg cells, we analyzed the effect of T reg cell-specific TRAF3 deficiency on the induction of antibodies by well-characterized protein and nonprotein antigens. Upon immunization of a protein antigen, sheep RBCs (SRBCs), the young Traf3 Treg-KO and WT control mice produced comparable levels of IgM (not depicted). However, the Traf3 Treg-KO mice produced significantly more IgG and its subtypes (Fig. 3 A). Similar results were  deficiency preferentially promoted the production of highaffinity antibodies of a major secondary isotype, IgG, we examined the role of TRAF3 in regulating GC formation by flow cytometry, based on the typical surface markers of GC B cells. As expected, immunization of the WT animals with SRBCs induced production of GC B cells, which peaked around 1 wk and declined around 2 wk after the immunization T reg cell-specific TRAF3 regulates T FH cell function and GC formation A hallmark of T cell-dependent antibody responses is the formation of GCs, which are required for various events of the humoral immune response, including antibody class switching, somatic hypermutation, and affinity maturation (Ramiscal and Vinuesa, 2013). Because the T reg cell-specific TRAF3

Figure 4. T reg cell-specific TRAF3 regulates T FH cell function and GC formation. (A and B) WT and Traf3
Treg-KO (KO) mice (8 wk old) were immunized i.p. with SRBCs, and the frequency of GC B cells (CD95 + GL7 + ) among B220 + cells was assessed by flow cytometry. Data are presented as a representative plot (A) and mean ± SD based on five mice for each group (B). (C) Mice were immunized as in A and B, and GCs were analyzed by immunofluorescence detection of PNA + cells. Graph shows average GC areas as mean ± SD based on multiple slides. Data are representative of four WT and three Traf3 Treg-KO (KO) mice. Bars, 500 µm. (D) Frequency of W33 to L mutation in the GC B cells of SRBC-immunized WT and Traf3 Treg-KO mice (10 d after immunization), determined by sequencing the cDNA clones constructed using pooled RNA from six WT and six KO mice. (E and F) WT-R26 YFP (WT) or Traf3 Treg-KO R26 YFP (KO) mice were immunized as in A, and 10 d later, the frequency of T FH cells (CXCR5 + PD-1 + ) among CD4 + YFP  T cells was assessed by flow cytometry. Data are presented as a representative plot (E) and mean ± SD (F) based on six mice for each group. (G) T FH cells were sorted from the spleens of the WT and KO mice in E and F, and expression of the indicated genes was assessed by RT-PCR. Mean ± SEM is shown. Data in A-G are representative of three to four independent experiments. *, P < 0.05; and **, P < 0.01. Mitsdoerffer et al., 2010;Linterman et al., 2012). Thus, the perturbed T FH cell activation provided explanations for the aberrant formation of GCs and hyper production of IgG isotypes in Traf3 Treg-KO mice.

TRAF3 is important for T FR cell induction
Recent studies identified a subset of T reg cells, the T FR cells, which controls the function of T FH cells and GC reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). T FR cells are characterized by expression of the chemokine receptor CXCR5, which mediates their migration into the lymphoid follicles (Linterman et al., 2012). In addition, the T FR cells also express high levels of the co-inhibitory molecule PD-1 and the transcription factor Bcl-6, respectively exerting negative and positive roles in T FR regulation (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011;Sage et al., 2013).
The particularly prominent phenotype of the Traf3 Treg-KO mice in T FH cell activation and humoral immune responses prompted us to examine the induction and function of T FR cells. We analyzed the T FR cells based on their surface expression of CXCR5 and PD-1 within the population of GFP + (expressed from the Foxp3-GFP-hCre transgene) T reg cells. As expected, the spleens of the WT mice contained a small population of CXCR5 + PD-1 + GFP + T FR cells, and this population was significantly increased in response to SRBC immunization (Fig. 5, A and B). Under unimmunized conditions, the T FR frequency of the Traf3 Treg-KO mice was only moderately lower than that of the WT mice (Fig. 5, A and B). However, these mutant animals were attenuated in antigen-stimulated production of T FR cells after SRBC immunization (Fig. 5,  A and B). To further confirm this remarkable result, we stained the T FR cells based on their surface expression of CXCR5 ( Fig. 4 A). Importantly, the Traf3 Treg-KO mice produced a markedly higher frequency of GC B cells than the WT mice (Fig. 4, A and B). Parallel immunofluorescence assays also revealed the formation of enlarged GCs in the spleen of the Traf3 Treg-KO mice after SRBC immunization (Fig. 4 C).
Formation of GCs is associated with antibody somatic hypermutations, a mechanism which generates high-affinity antibodies (Allen et al., 1987;Weiss et al., 1992). Given the enhanced GC formation in Traf3 Treg-KO mice, we analyzed the antibody somatic hypermutation frequency in the immunized WT and Traf3 Treg-KO mice based on the tryptophan (W) 33 to leucine (L) hotspot mutation in the Ig heavy chain variable gene V H 186.2 (Allen et al., 1988). Consistent with the elevated GC formation in the Traf3 Treg-KO mice, these mutant animals had a profoundly higher frequency of W33 to L mutations (Fig. 4 D).
GC formation and antibody class switching are dependent on T FH subtype of CD4 + helper T cells. We found that in response to SRBC immunization, the WT and Traf3 Treg-KO mice produced a comparable frequency of T FH cells (Fig. 4, E and F). Interestingly, however, the T FH cells derived from the Traf3 Treg-KO mice displayed hyperexpression of genes encoding several cytokine genes, including IL-4, IL-10, IL-17, and IFN- (Fig. 4 G). Furthermore, the expression of several transcription factors, such as Bcl-6, Gata-3, and RORt, were also elevated in the T FH cells derived from the immunized Traf3 Treg-KO mice (Fig. 4 G). Of note, T FH cells are known to produce different effector cytokines depending on the nature of antigens, which appears to influence the antibody class switching in a humoral immune response (Bauquet et al., 2009;Linterman et al., 2012). Several of these cytokines, IL-4, IL-17, and IFN-, have been shown to mediate B cell help and induction of antibody class switching toward the generation of different IgG subtypes (Bauquet et al., 2009;

NIK overexpression does not inhibit ICOS expression
The signaling function of TRAF3 is complex and may vary among cell types (Hildebrand et al., 2011). TRAF3 is known to bind to NIK and mediate NIK degradation, thereby negatively regulating the noncanonical NF-B signaling (Sun, 2012). Loss of TRAF3 causes accumulation of NIK and constitutive activation of noncanonical NF-B in B cells and T cells (Liao et al., 2004;He et al., 2006;Xie et al., 2007;Gardam et al., 2008). To examine how TRAF3 regulates ICOS expression, we examined the potential involvement of noncanonical NF-B activation. We used a transgenic mouse expressing a stabilized form of NIK lacking its TRAF3-binding motif (NIKT3) under the control of a loxP-flanked stop cassette (Sasaki et al., 2008). By crossing these mice with the Foxp3-GFP-hCre mice, we generated T reg -conditional NIKT3 transgenic (NIKT3 Treg-Tg ) mice. As seen with the Traf3 Treg-KO mice, the NIKT3 Treg-Tg mice displayed a moderate increase in memory-like effector T cells (Fig. 7 A). This result suggested that deregulated noncanonical NF-B activation might partially impair the function of T reg cells. However, the NIKT3 Treg-Tg mice did not show obvious abnormalities in antibody responses (Fig. 7 B) or reduced expression of ICOS and IL-10 in T reg cells (Fig. 7, C and D). In fact, the level of ICOS was even slightly enhanced in these mutant T reg cells (Fig. 7 C), probably reflecting their abnormal activation. Consistent with this idea, another activation marker, CD103, was also enhanced on the NIKT3 T reg cells (Fig. 7 E).

TRAF3-mediated ICOS induction involves activation of ERK and AP1
In conventional T cells, TRAF3 has a role in mediating TCR-stimulated activation of the MAP kinase ERK (Xie et al., 2011). We found that loss of TRAF3 in T reg cells also inhibited the activation of ERK by the TCR and CD28 signals (Fig. 8 A). To test whether attenuated ERK activation contributed to the reduced expression of ICOS in T reg cells, we used a selective ERK inhibitor, U0126. Indeed, the ERK inhibitor severely inhibited the induction of ICOS expression (Fig. 8 B). ICOS promoter contains an AP1-binding site that is important for promoter activation by the TCR/CD28 signals (Watanabe et al., 2012). Consistently, we found that crosslinking the TCR and CD28 in WT T cells led to rapid activation of AP1 that bound to the ICOS AP1 probe (Fig. 8 C). Moreover, the TRAF3 deficiency attenuated the activation of AP1 (Fig. 8 C). These findings suggest that TRAF3 regulates ICOS gene induction via an ERK-AP1 signaling pathway. and intracellular expression of Bcl-6 and Foxp3. This method once again revealed a defect of the Traf3 Treg-KO mice in T FR cell production in response to immunization with SRBCs (Fig. 5, C and D). Thus, although TRAF3 is dispensable for the homeostasis of T reg cells, it is important for the induction of a specific subset of T reg cells, the T FR cells, involved in the control of GC formation and humoral immune responses. This finding explains the aberrant induction of GCs and highaffinity antibodies in the Traf3 Treg-KO mice.

TRAF3 mediates ICOS gene expression in T reg cells
To assess the molecular mechanism by which TRAF3 regulates T reg cell function, we examined the expression of signature molecules on WT and TRAF3-deficient T reg cells. Although the TRAF3 deficiency did not affect the expression of most of the T reg cell surface markers analyzed, it caused an increase in the frequency of CD103 hi T reg cell populations (Fig. 6 A). The increase in CD103 hi T reg cells might be caused by their elevated activation, as expression of this molecule is known to be associated with effector/memory type T reg cells (Huehn et al., 2004). Interestingly, the TRAF3-deficient T reg cells had a profound reduction in the expression level of ICOS ( Fig. 6 A), a co-stimulatory molecule thought to mediate specific T reg cell functions, such as IL-10 gene induction and T FR cell development (Ito et al., 2008;Busse et al., 2012;Redpath et al., 2013;Sage et al., 2013). QPCR analysis further confirmed the defect in Icos gene expression (Fig. 6 B). Moreover, the TRAF3-deficient T reg cells were also impaired in the induction of IL-10 expression (Fig. 6, B-D). These findings further suggest the involvement of TRAF3 in the regulation of specific aspects of T reg cell functions.
Because Traf3 Treg-KO mice had a defect in T FR cell induction, we tested whether TRAF3 was required for antigenstimulated ICOS expression on T FR cells. As seen with the total T reg cells, ICOS expression was reduced on T FR cells under unimmunized conditions (Fig. 6 E). Importantly, the defect of the TRAF3-deficient T FR cells in ICOS expression became much more drastic after immunization of the mice with the protein antigen SRBCs (Fig. 6 E). Furthermore, this defect appeared to be cell intrinsic because it was not detected in the Foxp3  non-T reg cells (Fig. 6 F). A mixed bone marrow adoptive transfer experiment revealed that the TRAF3-deficient T reg cells had reduced ICOS expression even when they were developed in the presence of WT T reg cells in the same recipient mice (Fig. 6 G), thus further suggesting a cell-intrinsic role for TRAF3 in the regulation of ICOS expression in T reg cells.
were sorted from the spleens of three WT and three KO mice and stimulated for 24 h with PMA and ionomycin. IL-10 secretion was measured by ELISA. (E and F) WT or Traf3 Treg-KO (KO) mice (6 wk old; n = 5) were immunized with SRBCs, and 10 d later, ICOS expression in T FR (E) or T FH (F) cells was assessed by flow cytometry. Graph in E shows the mean fluorescence intensity (MFI) of ICOS based on five mice for each group. (D-F) Mean ± SD is shown. (G) Rag1-KO recipient mice were adoptively transferred with 1:1 ratio of mixed bone marrows of WT (B6.SJL, CD45.1 + ) and Traf3 Treg-KO (KO, CD45.2 + ) mice. 6 wk later, mice were immunized with SRBCs, and ICOS expression on gated WT (CD4 + CD45.1 + Foxp3 + ) and KO (CD4 + CD45.2 + Foxp3 + ) T reg cells was assessed by flow cytometry at day 10 after immunization. Graph shows MFI as mean ± SD from four mice for each group. Data are representative of three to four (A-F) or two (G) independent experiments. *, P < 0.05; and **, P < 0.01.

Tcrb/Tcrd double KO (Tcrb/Tcrd-dKO) mice with WT naive CD4 + T cells together with T reg cells purified from WT,
Traf3 Treg-KO , or Icos-KO mice and then challenged the mice with SRBC antigen. The different T reg cells were highly similar in homeostasis after the adoptive transfer ( Fig. 9 A). In contrast, although the WT T reg cells efficiently suppressed the induction of GCs in the immunized recipient mice, the TRAF3-deficient T reg cells were largely defective in this function (Fig. 9 B). The

ICOS expression in T reg cells is required for the formation and function of T FR cells
A recent study suggests that Icos-KO mice have a defect in T FR cell induction, although it is unclear whether this function is cell intrinsic (Sage et al., 2013). We used a T reg cell adoptive transfer model to examine whether the expression of ICOS in T reg cells is required for the development and function of T FR cells. We adoptively transferred the T cell-deficient approach, it is thought that TRAF3 may regulate the development of T reg cells (Hildebrand et al., 2011). Our present study revealed that ablation of TRAF3 in committed T reg cells, using the Foxp3-Cre system, also caused a moderate, but significant, increase in the T reg cell frequency. Our finding is consistent with the previous observation and further suggests that TRAF3 may negatively regulate both the early development and the late-stage maturation or survival of T reg cells. Interestingly, despite their increased T reg cell frequency, the Traf3 Treg-KO mice had impaired conventional T cell homeostasis, characterized by an increase in the percentage of CD4 + T cells with memory/effector surface markers. This result suggests a positive role for TRAF3 in the regulation of T reg cell function. In contrast, the phenotype of the Traf3 Treg-KO mice in T cell homeostasis is considerably weaker than that observed in mice lacking Foxp3 or other factors required for the development and core functions of T reg cells. The Traf3 Treg-KO mice also had only moderate immune cell infiltrations in the nonlymphoid tissues, particularly the lung. Consistently, adoptive transfer experiments revealed that the TRAF3-deficient T reg cells had weakly reduced ability to suppress the expansion and pathogenesis of CD4 + T cells. We observed reduced expression of IL-10 in the TRAF3-deficient T reg cells, which may contribute to their partially impaired function in maintaining immune homeostasis.
Despite the relatively weak phenotype in immune homeostasis, the Traf3 Treg-KO mice had a prominent abnormality in antigen-stimulated GC reactions. In response to T-dependent antigens, these mutant animals formed enlarged GCs in the spleen and produced markedly more GC B cells, coupled with hyper production of high-affinity IgG subtypes. Gene expression analyses suggest the aberrant activation of T FH cells. Of particular interest is the elevated production of various effector cytokines, including IL-4, IL-10, IL-17, and IFN-, by the T FH cells derived from the immunized Traf3 Treg-KO mice. Production of these cytokines by normal T FH cells has been observed similar deficiency was observed with the ICOS-deficient T reg cells (Fig. 9 B).
We next examined the production of high-affinity IgG antibodies in the immunized recipient mice. Consistent with the deficiency of the Traf3-KO and Icos-KO T reg cells in suppressing the GC formation, they also displayed attenuated function in the control of IgG production compared with the WT T reg cells (Fig. 9 C). Moreover, the recipient mice of Traf3-KO and Icos-KO T reg cells contained substantially higher levels of plasma cells (Fig. 9 D) and T FH cells (Fig. 9 E), coupled with reduced frequency of T FR cells (Fig. 9 F). Collectively, these data suggest that TRAF3 and ICOS have a T reg cell-intrinsic role in mediating the generation and function of T FR cells.

DISCUSSION
The Foxp3 + T reg cells represent a functionally diverse population composed of subsets that regulate different immune functions (Campbell and Koch, 2011;Josefowicz et al., 2012). The signaling mechanism that regulates the functional diversity of T reg cells is still poorly understood. In the present study, we have identified TRAF3 as a signaling factor that mediates the effector function of T reg cells, particularly their control of GC formation and antibody responses. Our data suggest that TRAF3 is involved in the induction of the recently defined T FR cells, a subset of T reg cells specialized in the control of GC reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). Consequently, the mice with T reg cell-specific TRAF3 deficiency display heightened responses to antigens in the formation of GCs and production of high-affinity antibodies.
A recent study suggests that TRAF3 ablation in total T cells, by crossing the Traf3-floxed mice with CD4-Cre mice, causes an increase in the percentage of Foxp3 + T reg cells (Xie et al., 2011). Because TRAF3 is knocked out during the double-positive stage of thymocyte development in this cells (CD4 + YFP + CD25 + ) were sorted from WT-R26 YFP and Traf3 Treg-KO -R26 YFP mice and stimulated for the indicated time periods with anti-CD3 plus anti-CD28 using a cross-linking method. Levels of total and phosphorylated ERK1,2 were assessed by immunoblot. Data are presented as a representative blot (left) and a summary graph based on quantification of three independent blots (right). Mean ± SD is shown. (B) ICOS expression on sorted WT T reg cells that were either not treated (NT) or treated with anti-CD3 plus anti-CD28 in the absence (T) or presence (T + U0126) of an ERK inhibitor, 50 µM U0126, for 24 h was assessed by flow cytometry. (C) Nuclear extracts were prepared from WT or TRAF3-deficient CD4 + T cells stimulated with anti-CD3 plus anti-CD28 for the indicated time periods, and EMSA was performed using ICOS AP1 or the control NF-Y oligonucleotide probes. Data are representative of three independent experiments. **, P < 0.01. attenuated in this response. In an adoptive transfer model, the TRAF3-deficient T reg cells also displayed a severe defect in generating T FR cells and suppressing GC responses. Under the transfer conditions, the TRAF3-deficient T reg cells also had significantly reduced ability to suppress the production of T FH cells. These results suggest that TRAF3 may regulate a signaling pathway in T reg cells that is particularly important for their conversion into T FR cells during an immune response.
The generation of T FR cells from Foxp3 + T reg cells is critically dependent on the transcription factor Bcl-6 (Chung et al., previously and thought to regulate B cell proliferation and antibody class switching (Rousset et al., 1992;King et al., 2008;Bauquet et al., 2009;Mitsdoerffer et al., 2010). Thus, deregulated production of these effector cytokines by T FH cells may contribute to the heightened GC formation and antibody responses in the Traf3 Treg-KO mice. The aberrant T FH cell activation was apparently caused by the defect of these mutant animals in producing T FR cells. Upon immunization with a T-dependent antigen, the WT mice had a substantial increase in the T FR cells, whereas the Traf3 Treg-KO mice were severely Figure 9. ICOS is essential for controlling T FH cell function and GC formation. Sorted naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25  ) from WT B6. SJL mice (5 wk old; CD45.1 + ) and sorted T reg T cells (CD4 + YFP + CD25 + ) from WT, Traf3 Treg-KO (Traf3-KO), or Icos-KO mice (6 wk old; CD45.2 + ) were mixed in a 5:1 ratio and intravenously transferred into Tcrb/Tcrd-dKO mice. 2 d later, recipient mice were immunized with SRBCs and sacrificed for experiments 14 d after immunization. (A) Frequency of T reg cells (CD45.1  ) and naive CD4 + T cells (CD45.1 + ) in the spleen of Tcrb/Tcrd-dKO recipients was assessed by flow cytometry. (B) Frequency of GC B cells (GL7 + CD95 + ) among B220 + cells was assessed by flow cytometry. Data are presented as a representative plot (left) and mean ± SD (right) based on three mice for each group. (C) SRBC-specific antibody levels in the sera of Tcrb/Tcrd-dKO recipient mice transferred with WT, Traf3-KO, and Icos-KO T reg cells was assessed by ELISA. Mean ± SD is shown. (D) Frequency of plasma cells (B220  CD138 + ) in the spleens of the Tcrb/Tcrd-dKO recipients was assessed by flow cytometry. (E and F) Frequency of the T FH (CXCR5 + PD-1 + Foxp3  CD4 + ) cells among CD4 + Foxp3  T cells (E) and the frequency of T FR (CXCR5 + PD-1 + GFP + CD4 + ) cells among CD4 + Foxp3 + T reg cells (F) were assessed by flow cytometry. Data are presented as mean ± SD based on three mice for each group and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
T reg cell function and humoral immune responses, but also shed new light onto the signaling mechanism regulating the specific functions of T reg cells.

MATERIALS AND METHODS
Mice. Foxp3-GFP-hCre BAC transgenic mice (Zhou et al., 2008; The Jackson Laboratory) were backcrossed for nine generations to the C57BL/6 background and then crossed with Traf3-floxed mice (C57BL/6 background; Gardam et al., 2008) to produce age-matched Traf3 +/+ Foxp3 GFP-hCre (termed WT) and Traf3 fl/fl Foxp3 GFP-hCre (termed Traf3 Treg-KO ) mice. In some experiments, these mice were further crossed with the Rosa26-YFP Cre reporter (termed R26 YFP ; C57BL/6J background; The Jackson Laboratory) to generate WT and Traf3 Treg-KO mice that express YFP in their T reg cells (termed WT-R26 YFP and Traf3 Treg-KO R26 YFP mice, respectively). The Traf3-floxed mice were also crossed with CD4-Cre (C57BL/6 background; The Jackson Laboratory) mice to produce mice harboring TRAF3 deletion in total T cells (called Traf3 TKO mice). NIKT3 mice (C57BL/6 background; The Jackson Laboratory) express a stabilized form of NIK lacking the TRAF3-binding motif (NIKT3) from the Rosa26 locus under the control of a loxP-flanked stop cassette (Sasaki et al., 2008). These mice were crossed with the Foxp3-GFP-hCre mice (backcrossed to C57BL/6 background) to generate T regconditional NIKT3 transgenic (NIKT3 Treg-Tg ) and internal WT control mice. B6.SJL (expressing the CD45.1 congenic marker), Tcrb/Tcrd-dKO, Icos-KO, and Rag1-KO mice (all in C57BL/6 background) were obtained from the Jackson Laboratory. For all experiments, WT littermates were used as controls. Mice were maintained in a specific pathogen-free facility of the University of Texas MD Anderson Cancer Center, and all animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center.

Influenza viral infection. WT and Traf3
Treg-KO mice were subjected to intranasal infection with a nonlethal dose (25 tissue culture ID 50 units in 25 µl PBS per mouse) of influenza virus A/PR8 (H1N1) under isoflurane anesthesia. Body weight change was monitored every another day for 13 d. On day 13, influenza-specific antibodies in the serum were detected by ELSIA as described previously (Elsner et al., 2012). In brief, ELISA plates were coated with influenza virus overnight at 4°C, blocked with 1% BSA in PBS, and then loaded with serum samples prediluted in 1% BSA. After 3 h of incubation, the plates were washed three times with PBST buffer, and the bound antibody was detected by with biotinylated goat anti-mouse antibody and streptavidin-HRP using TMB as substrate. 2011; Linterman et al., 2011;Wollenberg et al., 2011). Our data suggested that TRAF3 is not required for expression of Bcl-6 or the various T reg cell signature molecules. Instead, TRAF3 appeared to be required for maintaining the highlevel expression of ICOS in T reg cells because the TRAF3 deficiency greatly reduced, although did not completely block, the expression of ICOS at both RNA and protein levels. By using an adoptive transfer approach, we obtained in vivo evidence that ICOS expression in T reg cells is crucial for T reg cell-mediated suppression of GC formation and antibody responses. Like the TRAF3-deficient T reg cells, the ICOS-deficient T reg cells were severely attenuated in the generation of T FR cells and suppression of GC induction in response to immunization. These findings provide a mechanistic insight into the function of TRAF3 in regulating T FR cell production and antibody responses. We believe that ICOS induction is an important, but probably not the only, mechanism by which TRAF3 regulates T FR cell induction and antibody responses.
The signaling function of TRAF3 is complex and differs in different cell types and in the context of different receptors (Hildebrand et al., 2011). One important function of TRAF3 is to mediate degradation of NIK and, thereby, negatively regulate the noncanonical NF-B signaling (Liao et al., 2004). This negative signaling function of TRAF3 is particularly important for maintaining normal homeostasis and function of B cells, although its role in other cell types remains unclear (He et al., 2006;Xie et al., 2007;Gardam et al., 2008). Our data suggest that the activation of noncanonical NF-B might contribute to the moderate defect of the TRAF3-deficient T reg cells in maintaining CD4 + T cell homeostasis because perturbed CD4 + T cell homeostasis was also detected in mice with conditional expression of stable form of NIK (NIKT3 Treg-Tg mice). However, the noncanonical NF-B activation is unlikely responsible for the functional defect of the TRAF3deficient T reg cells in the regulation of GC reactions. Unlike the TRAF3 deficiency, conditional expression of NIKT3 in T reg cells did not cause hyperproduction of IgG or other antibody isotypes. The NIKT3-expressing T reg cells did not show any defect in ICOS expression either. Instead, our data suggest a positive role for TRAF3 in mediating the TCR signaling in T reg cells. As seen in conventional T cells (Xie et al., 2011), the loss of TRAF3 in T reg cells resulted in attenuated activation of the MAP kinase ERK. We obtained evidence that ERK is important for TCR-stimulated ICOS expression in T reg cells.
In summary, we have identified TRAF3 as a signaling factor that mediates the effector function of T reg cells, particularly in the control of humoral immune responses. Although TRAF3 is dispensable for the homeostasis of T reg cells, it is crucial for antigen-stimulated production of T FR cells. Our data suggest that the T reg cell regulatory function of TRAF3 involves induction of the co-stimulatory molecule ICOS, which is the result of impaired ERK activation. Our study has also revealed the requirement of T reg cell-specific ICOS in the induction of T FR cells and the control GC responses. These findings not only establish a novel role of TRAF3 in the regulation of by flow cytometric cell sorting based on the B220 + CD95 + GL7 + surface markers. Somatic hypermutation was analyzed based on the W33 to L hotspot mutation, essentially as previously described (Muramatsu et al., 2000). In brief, V H 186.2 transcripts were amplified by PCR using the V H 186.2 (5-GCT-GTATCATGCTCTCTTG-3) and CH-1 (5-GGTGCTATCCGGTC-CCTGAGT-3) primer pairs, and the PCR products were inserted into the pGEM-T vector by TA cloning. Randomly picked cDNA clones were sequenced for identification of the W33 to L mutations.
Statistical analysis. Prism software (GraphPad Software) was used for twotailed unpaired Student's t tests. P-values of <0.05 or <0.01 were considered significant and very significant, respectively.
Online supplemental material. Table S1 lists the gene-specific primers used for real-time RT-PCR analyses. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20131019/DC1.
Histology and GC analysis. Organs were removed from sacrificed mice, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned for hematoxylin-eosin staining. For GC formation analysis, SRBC-immunized mice were sacrificed after 10 d of immunization. Frozen spleen sections were fixed in methanol/acetone (2:1) for 10 min, followed by blocking for 30 min in a biotin-and serum-free SuperBlock buffer (Thermo Fisher Scientific). The GCs were detected using biotinylated peanut agglutinin (Vector Laboratories) followed by Alexa Fluor 594-conjugated streptavidin (Molecular Probes). The sections were mounted using an antiquenching agent (Vectashield; Vector Laboratories) and analyzed under a fluorescent microscope (DP70; Olympus). Panoramic pictures were taken and the GCs counted. The area and perimeter of each GC from each mouse (n = 3) were obtained using ImageJ software (National Institutes of Health). The area and perimeter are expressed in micrometers. GC B cells were analyzed by flow cytometry after staining with FITC-conjugated anti-CD95, PE-conjugated anti-B220, and APC-conjugated anti-GL7, and plasma B cells were analyzed similarly by staining with FITC-conjugated anti-B220 and PE-conjugated anti-CD138. For detecting T FH cells, the cells were stained with FITC-conjugated anti-CD4, PE-conjugated PD-1, and APC-conjugated CXCR5, followed by permeabilization with a Foxp3 staining kit (eBioscience) and intracellular staining with PE-conjugated anti-Bcl-6. T FH cells were detected similarly except the inclusion of intracellular staining with FITC-conjugated anti-Foxp3 or the GFP marker.

ELISA.
For SRBC or NP-specific antibody detection, Nunc Immuno Plate MaxiSorp plates (Thermo Fisher Scientific) were coated with SRBC lysate, NP9-BSA, or NP26-BSA and incubated overnight at 4°C. After blocking with 1% BSA in PBS, the plates were incubated with serially diluted standard or samples and then with HRP-conjugated detection antibodies. After colorimetric reaction (tetramethylbenzidine; Moss), the absorbance at 450 nm was measured using an ELISA plate reader. For IL-10 detection, Nunc plates were coated with 1 µg/ml anti-IL-10 capture antibody in 50 mM sodium bicarbonate and incubated overnight at 4°C. The coated plates were incubated with supernatant of the T reg cell culture and subjected to colorimetric detection as described above.
Immunoblot and real-time quantitative RT-PCR. Purified T reg and CD4 + T cells were lysed in RIPA buffer supplemented with inhibitors of phosphatases and the proteases. For analyzing ERK phosphorylation, the cells were stimulated with agonistic anti-CD3 and anti-CD28 antibodies using a crosslinking method (Reiley et al., 2007) before lysis. The cell lysates were fractionated by SDS-PAGE and subjected to immunoblot assays with the indicated antibodies. For analyzing RNA expression, total cellular RNA was isolated and subjected to real-time quantitative RT-PCR assays as previously described (Chang et al., 2011). The gene-specific primer sets are listed in Table S1.
In vitro and in vivo T reg cell suppression assays. The in vitro T reg cell suppression assay was performed as previously described (Chang et al., 2012). In vivo T reg cell assays were performed essentially as described previously (Chang et al., 2012). For examining the in vivo roles of T reg cells in the regulation of GC reactions, sorted CD4 + CD62L hi CD44 lo CD25  naive T cells from B6.SJL mice (5-6 wk old) and sorted CD4 + GFP + CD25 + T reg cells from WT, Traf3 Treg-KO , and Icos-KO mice (5-6 wk old) were intravenously transferred into Tcrb/Tcrd-dKO mice. Recipient mice were immunized with SRBC 2 d after adoptive transfer and sacrificed 14 d later for analyzing serum antibodies and lymphocyte populations.
Antibody somatic hypermutation analyses. WT and Traf3 Treg-KO mice were immunized with SRBCs and sacrificed after 10 d for isolating GC B cells


s2fieldsofstudy: Biology Chemistry

text:
Anti-Proliferative and Apoptosis-Inducing Effects of Camptothecin-20(s)-O-(2-pyrazolyl-1)acetic Ester in Human Breast Tumor MCF-7 Cells

Camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester (CPT6) is a novel semi-synthetic analog of camptothecin. In a previous report, CPT6 possessed higher cytotoxic activity in vitro towards human breast tumor MCF-7 cells than topotecan. In this study, the antitumor activity of CPT6 on the human breast tumor MCF-7 cell line was analyzed using the MTT method. The underlying mechanism of CPT6 action was investigated by analyzing the cell cycle distribution, apoptotic proportion, changes in mitochondrial membrane potential, and intracellular Ca2+ concentration using flow cytometry. Nuclear and mitochondrial morphologies were also observed by laser scanning confocal and transmission electron microscopy. DNA damage was observed in MCF-7 cells treated with CPT6. Low-dose CPT6 had a significant cytotoxic effect and could inhibit proliferation and induce apoptosis in MCF-7 cells, possibly through cell nucleus fragmentation and DNA damage. CPT6 thus appears to display potent antitumor activity against human breast tumor MCF-7 cells via the induction of apoptosis, and may be a useful alternative drug for breast cancer therapy.

Introduction
Camptothecin (CPT) is an alkaloid originally isolated from Camptotheca acuminate by Wall and Wani in 1966 [1], and is one of the most important lead compounds in anticancer research. The antitumor activity of CPT is due to its ability to stabilize the reversible covalent DNA-topoisomerase I complex [2,3]. Topoisomerase I act by breaking and then resealing a single strand of DNA, thus relieving the torsional strain that occurs during DNA transcription and replication. CPT derivatives (CPTs) have been shown to exhibit anticancer activity both in vitro and in vivo. CPTs represent an important class of anticancer drugs with a wide spectrum of activities in many solid tumors, including lymphoma, gastric cancer and colorectal cancer. Further evidence indicates that the lipophilic CPTs show improved cytotoxicity and inhibition of topoisomerase I, compared with the water-soluble CPTs [4,5]. In previous studies, we introduced nitrogenous heterocyclic aromatic groups in the 10-and 20-positions of CPT and obtained a series of CPT analogs, some pyridine quaternary salt derivatives of which showed good water solubility and potent antitumor activity both in vitro and in vivo [6,7]. Other nitrogenous heterocyclic aromatic groups introduced in the 10-or 20-postion can enhance the stability of the lactone component and the antitumor activity [6,7]. Derivatives modified by pyrazole in the 10-or 20-position all showed notable antitumor activity in vivo and stability of the lactone component in vitro. The antitumor activity was not only related to the substituted positions, but also to the substituent. Among these derivatives, camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester (CPT6, Figure 1) exhibited good antitumor activity in vitro, and demonstrated stronger cytotoxicity towards the human breast tumor MCF-7 cell line. The antiproliferative activity and the mechanism of action of CPT6 against human breast tumor MCF-7 cells, however, have not been reported. In this study, the antiproliferative activity of CPT6 against human breast tumor MCF-7 cells was analyzed using the MTT method. The mechanism of CPT6 action against human breast tumor MCF-7 cells was also investigated by flow cytometry, laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM).

Antiproliferative Activity of CPT6
The antiproliferative activity of CPT6 was determined in three cancer cell lines: human breast cancer MCF-7 cells, human colon cancer HCT-8 cells, and non-small cell lung cancer A549 cells. As shown in Figure 2, MCF-7 cells were the most sensitive of the three cell lines to CPT6 within 72h. The IC 50 value of CPT6 against MCF-7 cell was 0.46 ± 0.02 nM, which was more effective than CPT (IC 50 = 0.65 ± 0.04 nM) and TPT (IC 50 = 1.64 ± 0.05 nM). The growth of MCF-7 cells was significantly inhibited in a dose and time-dependent manner in the presence of CPT6 (0, 0.125, 0.25, 0.5, 1, 2, 4 nM) at 24, 48, and 72 h (Table 1).

Analysis of Cell Cycle Distribution
The cell cycle distribution was analyzed using Multicycle software (Partec GmbH, Münster, Germany). A significant increase in the percentage of cells in Sub-G1 phase was found after treatment with CPT6, compared with control cells (Figure 3). MCF-7 cells was significantly inhibited in a dose-dependent manner in the presence of CPT6. As shown in Figure3B, when cells were exposed to CPT6 (1 nM) for 48 h, the percentage of Sub-G1population showed a marked increase (32.42%) compared with the control (2.25%). G0/G1 phase were decrease along with the increase of Sub-G1 phase, and the population of cells treated in S phase increased significantly compared with that of control cells. All these data indicate that CPT6 affects the replication of DNA and cause cell cycle arrest in S phase, prevent the cell proliferation. The cell proliferation inhibition causes increased cells entering in Sub-G1 phase, subsequently induces cell apoptosis. So we can deduce that CPT6 may induce apoptosis by causing DNA fragmentation.

DNA Damage
MCF-7 cells were treated with CPT6 (0, 0.25, 0.5, 1, 2 nM) for 48 h and apoptosis was confirmed using the DNA laddering method. Obvious diffusion bands of DNA fragments representing multiples of the internucleosomal DNA length (about 180-200 bp) were detected ( Figure 3C). The apparent densities of the DNA fragment bands were increased in the treated group, compared with untreated cells, along with increasing concentrations of CPT6. Control cells did not exhibit any such DNA fragmentation. Such a pattern corresponds to inter-nucleosomal cleavage, which is characteristic of apoptosis.

Apoptosis/Necrosis Analysis
To elucidate the mode that CPT6 used to exert cytotoxicity, apoptosis or necrosis, experiments were conducted by using MCF-7 cells. The disruption of cell membrane phospholipid asymmetry, evidenced by phosphatidylserine (PS) externalization, was examined by utilizing annexin V-FITC and propidium iodide assay and monitored via flow cytometer protocol. The cell distributions in flow cytometric histograms are as follow: cells in the lower left quadrant (Q3) represented live cells, negative for annexin V and PI; the lower right quadrant (Q4) represented early apoptotic cells; the upper right quadrant (Q2) represented late apoptotic cells; and upper left quadrant (Q1) represent necrotic cells ( Figure 4A). The addition of both early and late apoptotic cells (annexin V-FITC positives) was defined as the total percentage value of apoptotic cells. When MCF-7 cells were treated with CPT6 (0, 0.25, 0.5, 1, 2, 4 nM) for 48 h, the percentage of apoptotic cells increased from 1.80% to 55.60% ( Figure 4B). This indicates that CPT6 was able to induce apoptosis of MCF-7 cells in a dose dependent manner.

Changes in Nuclear Morphology and Chromatin Condensation
CPT6 treatment resulted in many visible morphological changes in the nuclear chromatin of MCF-7 cells. In untreated cells, the cell membrane was orbicular and the nuclei were sharp and regular ( Figure 5A,C). MCF-7 cells treated with 0.5 nM CPT6 for 48 h showed lunate morphology of chromatin and condensed chromatin ( Figure 5B). Moreover, the membranes of the treated cells were shrunken and the nuclei were crescent-or even strip-shaped, which is a typical characteristic of apoptotic cells ( Figure 5D).

Morphological Observation of Cell Ultrastructure by TEM
Untreated cells showed typical ultrastructure characterized by rounded nuclear membranes, regular mitochondria and nuclei containing a nucleolus and euchromatin ( Figures 6A,C). However, in treated cells, the nuclear membrane became unclear, the chromatin condensed and the mitochondria were damaged, and more myeloid body could be seen outside the nuclear membrane ( Figure 6B). Vacuoles appeared in the cytoplasm and the nuclear membrane was umbilicate ( Figure 6D). These early signs of apoptosis were not visible in untreated cells. These observations indicate that 0.5 nM CPT6 induced early apoptosis in MCF-7 cells treated for 48 h.

CPT6-Induced Loss of ΔΨ m in MCF-7 Cells
To evaluate the role of mitochondria in CPT6-induced apoptosis, we investigated the effects of CPT6 on ΔΨ m. Rh123 is available to stain the mitochondria of live cells, and elicit fluorescence. When mitochondria are damaged and ΔΨ m decreases, fluorescence from Rh123 will reduce. So ΔΨ m can be measured by the number of the cells with strong fluorescence. Untreated MCF-7 cells elicited maximal Rh123 fluorescence, reflecting intact functional mitochondria. CPT6 treatment resulted in a rapid dose-dependent dissipation of ΔΨ m, detected by a consequent decrease in mean fluorescence ( Figure 7A,B). ΔΨ m was reduced by 59.24% when the cells were treated with 1nM CPT6. These data showed that CPT6 could induce apoptosis in MCF-7 cells, accompanied by alterations in ΔΨ m.

Effect of CPT6 on Intracellular Calcium in MCF-7 Cells
To investigate the role of Ca 2+ signaling in CPT6-induced apoptosis of MCF-7 cells, we determined the intracellular Ca 2+ concentration using flow cytometry. The fluorescence intensities of the control and treated groups were not coincident ( Figure 8A), and the intensity of the treated group was shifted to the right [8]. WinMDT 2.9 software (Partec GmbH) was used to overlay the absorption peaks and the values of the peak locations were then calculated using CellQuest software. The intracellular Ca 2+ increased from 19.03% ± 2.12% to 64.52% ± 2.48%. The histogram indicated that CPT6 could significantly enhance the intracellular Ca 2+ concentration in MCF-7 cells ( Figure 8B) (* p < 0.05, ** p < 0.01).

Materials
Dulbecco's modified Eagle's medium (DMEM) and RPMI-1640 medium were purchased from HyClone  [7]. Its purity was 98.8%, as determined by high-performance liquid chromatography. CPT6, CPT and TPT were dissolved in 300 μL DMSO at a concentration of 6 μM as stock solutions. Dilutions of the compounds were made up in 2% culture medium before each experiment. The final concentration of DMSO did not exceed 0.1% (v/v) and was nontoxic to the cells.

Cell Line and Culture
The human breast cancer cell line MCF-7, the colon cancer cell line HCT-8, and the non-small cell lung cancer cell line A549 were purchased from the Cell Bank of the Institute of Materia Medica, Chinese Academy of Medical Sciences. All cells were grown in DMEM or a nutrient medium (RPMI-1640) supplemented with 10% FBS, 100 μg/mL of streptomycin and 100 U/mL of penicillin, in a water-saturated atmosphere of 5% CO 2 at 37 °C.

Cytotoxicity and Antiproliferative Activities
Cell proliferation was measured using the MTT assay. Cells in the exponential growth phase were seeded in a 96-well plate (1 × 10 6 cells/well) and routinely cultured for 24 h. CPT6, CPT and TPT was added at serial concentrations (from 0.125 to 4 nM). 2% culture medium (with 0.1% DMSO) alone was used as a control. The cells were then incubated for 24, 48 and 72 h. MTT PBS solution (20 μL of 5 mg/mL) was then added to 100 μL and incubated for 4 h at 37 °C. The supernatant was removed and the formazans were dissolved in 100 μL DMSO per well. After 10 min shaking, the absorbance was read at 492 nm (with 630 nm as a reference) on a microplate reader (Bio-Tek, America). Assays were performed in three independent experiments.

Cell Cycle Distribution
To analyse the cell cycle phase distribution, MCF-7 cells (1 × 10 6 cells/well) were seeded in a 6-well plate for 24 h and then exposed to CPT6 (0, 0.25, 0.5, 1 nM). After incubation for 48 h, the untreated cells and treated cells were washed twice with precooled PBS, and then fixed in 70% ethanol at 4 °C [9]. Fixed cells were stained with 200 μL 4'-6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. The cell cycle distribution of 10,000 cells was determined by flow cytometry (Partec GmbH,) using the FloMax software (Partec GmbH).

Detection of DNA Fragmentation
DNA fragmentation assay was used to identify apoptosis. Briefly, 5 × 10 5 cells were harvested and treated with CPT6 (0, 0.25, 0.5, 1, 2 nM) for 48 h. The supernatant and adherent cells were then collected in a centrifuge tube, washed twice with cold PBS and resuspended in 200 μL PBS. Genomic DNA was isolated using an Apoptosis DNA Ladder Detection kit (Nanjing KeyGEN Biotech. Co. Ltd., Nanjing, China). DNA samples were separated by electrophoresis in a 2% agarose gel for 150 min and visualized by ethidium bromide staining.

Apoptosis Assays
Apoptosis assay was performed using rh Annexin V/FITC Kit (Bender MedSystems GmbH,Wien, Austria), and the fluorescence intensities of FITC-conjugated annexin-V and PI in cells were analyzed using flow cytometry [10,11]. MCF-7 cells were cultured in medium containing different concentrations of CPT6 (0,0.25, 0.5, 1, 2, 4 nM) for 48 h, collected and washed twice with PBS, gently resuspended in 195 μL annexinV-FITC binding buffer (1×) and incubated with 5 μL annexinV-FITC in the dark for 10 min at 25 °C. The cells were then centrifuged at 3000 rpm for 5 min, gently resuspended in 190 μL annexinV-FITC binding buffer (1×) and 10 μL PI was added in an ice bath, followed by immediate analysis by flow cytometry CellQuest software (Partec GmbH).

Morphological Observations
MCF-7 cells were grown on glass coverslips at a density of 2 × 10 4 cells/well in 6-well plates. After adhering for 24 h, the cells were washed twice with cold PBS and treated with or without 0.5 nM CPT6 for 48 h at 37 °C. The cells were then washed twice with cold PBS and stained with AO (20 μg/mL) for 5 min at 37 °C, then immediately viewed by LSCM (Nikon ECLIPSE TE2000-E, Tokyo, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 535 nm [12,13].
Ultrastructural analysis was performed by TEM (Tecnai G 2 , Holland). Cells were treated with or without 0.5 nM CPT6 for 48 h, washed with PBS, collected, and pre-fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4 °C for 2 h. Cells were then washed three times with distilled water and post-fixed in 1% OsO 4 at 4 °C for 1 h. After fixation, cell blocks were dehydrated through graded concentrations of acetone and embedded in Epon812 (SPI Supplies, West Chester, PA, USA). Cell ultrastructure was analyzed in ultrathin sections by TEM after staining with uranyl acetate and lead citrate [14,15].

Assessment of Mitochondrial Membrane Potential (ΔΨ m)
ΔΨ m was measured by flow cytometry with Rh123 and PI double staining [16]. Rh123 and PI (10 mg/L) were dissolved in DMSO and diluted in PBS before use. MCF-7 cells (1 × 10 6 cells/well) were seeded in a 6-well plate. After adherence for 24 h, the cells were cultured in medium containing different concentrations of CPT6 (0, 0.25, 0.5, 1 nM) for 48 h. Cells were then washed twice with PBS and finally harvested in chilled PBS containing Rh123. The samples were incubated at 37 °C for 30 min in the dark. The cells were collected at 3000 rpm for 5 min, resuspended in PBS and 10 μL of PI was added for 10 min in the dark, followed by immediate analysis by flow cytometry.

Statistical Analysis
Results were expressed as mean ± SD. Statistical analysis was performed using Student's t-test. Significance was established at p < 0.05.

Conclusions
Structure-activity studies of CPTs have revealed that positions 7, 9 and 10 of CPT can be modified with no loss of activity [17]. In our previous studies, CPTs modified in the 10-or 20-position by pyrazole all showed notable antitumor activity in vivo, and stability of the lactone component in vitro.
Further, we evaluated that the growth inhibitory activity of CPT6 was associated with the induction of DNA damage and apoptosis in MCF-7 cells.
Due to the critical role of apoptosis in cellular homeostasis, compounds able to induce apoptosis are considered to be potential antitumor agents for cancer treatment [18,19]. Apoptosis is a physiological process that functions as an essential mechanism of tissue homeostasis and is regarded as the preferred way to eliminate unwanted cells [20]. CPT and its derivatives are able to form cleavable drug-enzyme-DNA complexes that inhibit the DNA relegation step [21]. Collision between the DNA replication fork and these complexes has been proposed as a means of explaining the CPT-driven S phase-specific cytotoxicity and the arrest of cells in the G2-M phase of the cell cycle [22]. Apoptosis is related to cell cycle arrest, which is a complicated process that can be regulated by numerous distinct growth signals [23,24]. In the present study, MCF-7 cells treated with CPT6 revealed DNA fragmentation with appearance of the Sub G0/G1 peak indicative of apoptosis, and showed a dose-dependent generation of apoptosis-specific Sub-G1 changes and apoptotic rates. This could be explained by the ability of the CPT6 to induce apoptosis and thus effectively inhibit DNA unwinding and replication [25].
Annexin-V binding is based on the transposition of phosphatidylserine from the inner to the outer face of the cell membrane during the early stages of apoptosis. With fluorescent probe FITC to mark Annexin-V, it directly detected phosphatidylserine valgus which was an important feature of cell apoptosis [26]. CPT6-treated cells clearly showed apoptotic morphologies and the apoptotic rate of the treated cells increased in a dose-dependent manner. The formation of apoptotic bodies, condensed chromatin in MCF-7 cells treated with CPT6 were indicative of apoptosis. CPT6 deformed the cancer cell membranes and caused shrinkage and bleb formation, which are also important morphological characteristics of apoptosis. Disruption of ΔΨ m is one of the earliest intracellular events that occurs following the induction of apoptosis [27,28]. The decrease in ΔΨ m in MCF-7 cells treated with CPT6 was dose-dependent, suggesting that the ΔΨ m may be one significant appearance in MCF-7 cell apoptosis by CPT6. Ca 2+ is an important signal transducer, not only for intracellular communication but also for intercellular communication. Many studies have demonstrated that an increase in intracellular Ca 2+ plays a primary role in triggering the apoptosis pathway [29]. MCF-7 cells treated with CPT6 showed a distinct, dose-dependent increase in intracellular Ca 2+ .
At the same time, both DNA fragmentation and flow cytometry analyses showed dose-dependent increases in representative apoptotic characteristics, such as DNA fragmentation and an increase in cells in Sub-G1 phase, the apoptotic cells were increased along with the concentration of CPT6. These results suggest that higher concentrations of CPT6 induced irreparable DNA damage and apoptosis in MCF-7 cells.
In summary, this study provides the first results regarding the antiproliferative and apoptosisinducing effects of CPT6. CPT6 had a significant cytotoxic effect and was able to inhibit proliferation and induce apoptosis in MCF-7 cells at low doses. CPT6 displayed greater antitumor activities in MCF-7 cells, compared with TPT and CPT at 72h. Although many details of the effects of CPT6 remain to be elucidated, the in vitro findings of the present study provide the basis for future in vitro and in vivo studies and clinical applications.