The paraffin-embedded lymph node samples were collected from 35 newly diagnosed cases of T-cell lymphoma (21 males and 14 females; age range, 19–81 years; median age, 53 years) and 20 normal lymph nodes. Histological diagnoses were established according to the WHO classification (15). Normal peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by Ficoll centrifugation (TBD Science, Tianjin, China). T cells were isolated with Nylon Wool Fiber Columns. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University. All samples were obtained with informed consent in accordance with the Declaration of Helsinki.

Jurkat and Molt-3 (T-cell acute lymphocytic leukemia cell lines) were available from Typical Culture Preservation Commission Cell Bank (Chinese Academic of Science, Shanghai, China). MyLa 3676 (cutaneous T-cell lymphoma, lymphoblast, Sezary syndrome) was retained by our laboratory. Karpas 299 (ALK⁺ ALCL cell line) was obtained from Shanghai Bioleaf Biotech Co., Ltd. All the above cell lines were maintained in PRMI-1640 (Gibco, Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum (FBS; Gibco, Life Technologies) and 1% penicillin/streptomycin mixture.

Lentivirus vectors either encoding Klotho (LV-KL) or an empty lentiviral vector (LV-Con) were generated by GeneChem (Shanghai, China). Lentivirus transfection of T-cell lymphoma cells were performed according to the manufacturer's instructions. Infection efficiencies were assessed by green fluorescent protein (GFP) fluorescence through flow cytometry. The stably transfected cells were selected with 5 µg/ml puromycin (Sigma-Aldrich, USA).

IHC was carried out with primary rabbit anti-Klotho (Abcam, Cambridge, USA). 4-µM-paraffin sections were deparaffinized and hydrated. Antigen retrieval was performed in 0.01 M sodium citrate (pH 6.0) buffer under high-pressure. Endogenous peroxidase was blocked with 3% H₂O₂ for 15 min, followed by incubation overnight at 4°C with primary anti-Klotho (1:100). Then the tissue sections were treated with the second antibody from SP reagent kit (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 30 min at room temperature. Bound antibody was detected by secondary antibody and diaminobenzidine (DAB) kit (Zhongshan Golden Bridge Biotechnology). The stained slices were counterstained with hematoxylin and mounted. Negative control was performed with the primary antibody replaced by PBS. IHC staining was scored by two independent observers who were blinded to the patient clinical data. Cases with ≥10% positive cells were considered as positive.

For the qRT-PCR, total RNA was extracted with RNAiso Plus (Takara, Dalian, China). The mRNA level was detected by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription reaction was performed with reverse transcription reagents (Takara). Expression level of mRNA was quantified using SYBR Green Premix Ex Taq II kit (Takara) in LightCycler 480 real-time quantitative PCR system (Roche, Basel, Swizerland). Primers for qRT-PCR are listed in Table I. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and relative quantification was determined by the 2^−∆∆Ct method.

For western blot analysis, cell lysates were extracted by radio-immunoprecipitation assay (RIPA) buffer (Shenergy Biocolor, Shanghai, China) together with phosphatase inhibitor cocktail (PhosSTOP; Roche, Mannheim, Germany). The BCA assay (Shenergy Biocolor) was performed to detect protein concentration. Proteins (30 µg) were electrophoresed on SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in Tris-buffered saline. After incubation with primary antibodies, membranes were incubated with HRP-conjugated secondary antibodies (Zhongshan Golden Bridge). Proteins were visualized by electro-chemi-luminescence detection reagents (Amersham Imager; General Electric, USA). The following antibodies used for western blotting were purchased from Cell Signaling Technology: p-IGF-1R, t-IGF-1R, p-AKT, t -AKT, p-ERK1/2, t-ERK1/2, Mcl-1 and caspase-3. Klotho antibody was purchased from Abcam. The experiments were performed in triplicate with GAPDH (Zhongshan Golden Bridge) as endogenous control.

The influence of Klotho on viability of T-cell lymphoma cells were assessed by Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Briefly, T-cell lymphoma cell lines stably transfected with LV-KL or LV-Con were seeded into 96-well plates (5,000 cells/100 µl/well, respectively) with PRMI-1640 medium supplemented with 10% FBS. Then the cells were incubated with 10 µl/well of CCK-8 and cell viability was detected by light absorption at 450 nm by Multiskan GO Microplate Reader (Thermo Scientific, Rockford, IL, USA).

Apoptosis of T-cell lymphoma cells were tested by Annexin V-PE/7-aminoactinomycin (7AAD) kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions. Cells with designed treatment were harvested and washed twice in clod PBS and resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml, then 100 µl was taken and incubated with 5 µl Annexin V-PE and 5 µl 7AAD for 15 min at room temperature in the dark for each tube. Afterwards, 400 µl of 1X binding buffer was added and cell apoptotic rates were detected by Navios Flow Cytometer (Beckman Coulter, CA, USA).

T-cell lymphoma cells with designed treatment were seeded onto glass slides by liquid thin layer cell smear (Xiaogan Aohua, Xiaogan, China). Thereafter, the T-cell lymphoma cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100. The cells were blocked with normal goat serum for 1 h. Then the slides were incubated with primary antibodies at 4°C overnight. After washing with PBS, the DyLight 488-conjugated secondary antibodies (Abbkine, CA, USA) were added. Slides were washed and mounted with 4′6-diamino-2-phenylindole (DAPI; Boster, Wuhan, China). Images were examined and recorded by Nikon IX73 fluorescent microscope.

All statistical analyses were performed by using statistic software SPSS17.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation (mean ± SD) from three separate experiments. Differences between groups were analyzed by one-way analysis of variance (ANOVA) or t-tests. P<0.05 was considered statistically significant.

We examined Klotho expression for primary T-cell lymphoma samples including NK/T-cell lymphoma (n=12), peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS, n=7), angioimmunoblasic T-cell lymphoma (AITL, N=6), cutaneous T-cell lymphoma (CTCL, n=6), and enteropathy associated T-cell lymphoma (EATL, n=4). Compared with normal lymph nodes, expression levels of Klotho were significantly lower in T-cell lymphoma tissues (Fig. 1A). Klotho positive rate was 14% (5 of 35) in T-cell lymphoma tissues whereas 75% (15 of 20) in normal lymph nodes. Cases with ≥10% positive cells were considered as positive. More detailed data are summarized in Table II.

We next confirmed the expression of Klotho in T-cell lymphoma cell lines. As shown in Fig. 1B, T-cell lymphoma cells, exhibited remarkably lower mRNA levels of Klotho compared to the peripheral blood T lymphocytes from healthy donors. Decreased protein levels of Klotho expression were also noted in T-cell lymphoma cell lines (Fig. 1C).

To explore the function relevance of Klotho in the progression of T-cell lymphoma, T-cell lymphoma cell lines (Jurkat, Molt-3, Karpas 299 and MyLa 3676) were stably transfected with either Klotho-overexpression lentivirus vectors or empty vector control (Fig. 2A). Upregulated level of Klotho mRNA was confirmed by quantitative PCR (Fig. 2B). CCK-8 assay was employed to examine the viability of the above cells. Significantly decreased cell viability was noted in T-cell lymphoma cells transfected with LV-KL, compared with those transfected with LV-Con (Fig. 2C). These results indicate that Klotho inhibits the proliferation of T-cell lymphoma cells.

To further investigate the mechanisms underlying the suppression of Klotho in cell proliferation by ectopic Klotho expression, Annexin V-based apoptotic assays were performed in Jurkat and Karpas 299 cell lines with stable LV-KL or LV-Con transfection. Cells transfected with LV-KL exhibited enforced apoptosis rates in T-cell lymphoma cell lines (Fig. 3A). In addition, apoptotic promotion effect of Klotho was confirmed by western blot analysis. Remarkable reduction of anti-apoptotic protein Mcl-1 and total caspase-3 protein was observed in Jurkat and Karpas 299 cell lines (Fig. 3B and C).

Having elucidated that Klotho could interfere with the viability and apoptosis of T-cell lymphoma cell lines, we next explored the involved molecular mechanisms responsible for the function of Klotho. The IGF-1R signaling plays significant role in the development of T-cell lymphoma and proliferation of T-cell lymphoma cell lines could be induced by IGF-1 exploration. CCK-8 assay was performed to determine the effect of Klotho overexpression on IGF-1-induced cell proliferation. T-cell lymphoma cell lines stably transfected with either Klotho overexpressing vector or empty control vector were incubated with IGF-1 or vehicle control in 0.5% FBS culture medium for 48 h. In the group without IGF-1 treatment, LV-KL transfection induced declined viability of T-cell lymphoma cell lines compared to the empty-vector group. Furthermore, we noted that the Klotho-induced inhibition of T-cell viability was less reversed by IGF-1 treatment. With IGF-1 addition, cell proliferation of LV-Con treated cells increased by up to 50%, whereas the only up to 30% enhancement of cell proliferation was found in cells with Klotho overexpression (Fig. 4A).

We next investigated the modulation effect of Klotho on IGF-1R signaling in T-cell lymphoma cell lines. Jurkat and Karpas 299 cells transfected with LV-KL revealed significantly decreased phosphorylation level of IGF-1R. Moreover, the downstream targets of IGF-1R signaling, including AKT and ERK1/2, were also inhibited by Klotho overexpression (Fig. 4B and C).