Introduction
4-1BBL is a member of tumor necrosis factor (TNF) super family and it can function as a costimulatory molecule. It is a type II membrane protein and expressed on activated antigen-presenting cells (B cells, macrophages, and dendritic cells, and so on) [
1]. Its receptor, 4-1BB, is a member of the TNF receptor family and expressed on activated CD4
+, CD8
+ T cells and dendritic cells [
2,
3]. Co-stimulation
via 4-1BB and its ligand 4-1BBL plays an important role in the activation, proliferation and differentiation of T lymphocytes, especially to the long-term survival and can sustain the immune memory of CD8
+T cells [
4-
6]. This function does not depend on B7/CD28 [
7]. In the previous studies on using 4-1BBL/4-1BB in the treatment of tumor, genes were often transfected into tumor cells to observe the tumor forming rate [
8,
9]. Until now, there were no reports about transferring the genes to normal tissue cells to treat tumors. In the present study, murine
in situ tumors were used as study objects, and several gene transfection techniques were applied to transfer 4-1BBL gene to normal tissue cells to investigate the therapeutic effects of these genes and analyze the possible mechanisms.
Materials and methods
Animals and cells
Female BALB/C mice were purchased from the Center of Medical Experimental Animal of the Hubei Province in China. The BHK cell line, H22 cell line (murine hepatoma cell strain derived from a BALB/C background) and B16 cells (murine melanoma cell strain derived from a C57BL/6 background) were obtained from the China Type Culture Collection (CTCC, Wuhan, China) and were cultured in Dulbecco's minimum Essential medium (DMEM) supplemented with 10% fetal cow serum (FCS), 2 mmol/L L-glutamine, 100 U/mL streptomycin, and 100 mg/mL penicillin (complete medium).
Plasmid vector construction
Total RNA was isolated from spleen cells using TRIzol reagent (Invitrogen Life Technologies, USA). After being treated with DNase I (Promega, USA), RNAs were reverse-transcribed and the cDNAs were amplified by PCR with gene-specific primers at 94°C for denaturing, 54°C for annealing and 72°C for extension. Specific primers were as follows: mouse 4-1BBL sense primer, 5′-GAGTCAAAGCTTATGGACCAGCACA C-3′; antisense primer, 5′-GCGCTGAATTCTCATTCC CATGGGTT-3′. Then, the 4-1BBL full length cDNA was inserted into the Hind III and EcoR I site of the pcDNA3.1. All plasmid vectors were amplified in Escherichia coli and purified in the absence of ethidium bromide or penicillin derivatives by using a commercially available column chromatography method, according to the manufacturer’s protocol (Qiagen, Chatsworth, CA). The quantity and quality of the purified plasmid DNA were assessed by optical density at 260 and 280 nm and also by electrophoresis in agarose gels.
Cell transfection and gene expression detection
The transfection of cells with plasmids was performed by using DOSPER liposomal transfection reagent (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s instructions. For stable transfection with p4-1BBL, the transfected BHK cells were selected on the basis of G418 resistance (800 μg/mL) for 3 wk. Then, the cells were detected for 4-1BBL gene expression using RT-PCR, western blot and cell immunohistochemistry staining methods. The procedure was performed as described [
10-
12]. The antibody employed was rat anti-mouse 4-1BBL monoclonal antibody TSK-1 which was kindly provided by Professor Hideo Yagita of the Juntendo University, Japan.
Spleen lymphocytes stimulated with 4-1BBL
The heat shock protein 70 (HSP70) H22 cells peptide complex was prepared as described previously [
13]. HSP70 and the peptide mixture were mixed to bind each other as described [
14,
15]. For Antigen-specific stimulation, the spleen cells were cultured at the concentration of 1×10
7/mL in RPMI 1640 supplemented with 20 U/mL IL-2 (PeproTech, London, UK) in a 96-well culture plate in the presence of 0.75 μg/mL HSP70-peptide complex. The cells were passed through, and 48 h later cultured for another 5 days, then they were divided into two parts [(fluorescence activating cell sorter analysis) for FACS and lymphocyte activity detection]. For FACS analysis the stimulated cells were previously incubated with phycoerythrin (PE)-labeled antibody 3H3 (rat anti-mouse 4-1BB antibody, gift from Professor Millter, Emory University, USA) for 1 h at 4°C. To detect the change of lymphocytes activities after they were stimulated by 4-1BBL expressed by BHK cells, the positive cells transfected with p4-1BBL were pre-fixed on the dishes with glutaric dialdehyde. Then, the stimulated spleen cells were added (1×10
6/well). The mixture was incubated for 48 h at 37°C in a humidified 5% CO
2 air mixture. After incubation, the suspended spleen cells were collected for MTT (3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide), ELISA (enzyme linked immunosorbent assay) and cytotoxicity analysis.
MTT assay
The proliferation ability of the lymphocytes was determined by MTT stain assay. The detailed procedure was described in the reference [
16]. Three wells were used for each group. The plate was incubated for 48 h at 37°C in a humidified 5% CO
2 air mixture. After addition of 10 μL MTT per well, the plate was incubated for 4 h and then 100 μL of 20% sodium dodecyl sulfate (SDS) were pipetted into each well to dissolve the formazan crystals. After incubation at 37°C in 5% CO
2 air mixture overnight, the microplate was read on a Bio-Rad microplate reader (Model 550) using test wavelength of 570 nm.
Cytotoxicity assay
Cytotoxicity of lymphocytes to tumor cells was determined as described [
17]. The effector cells were the lymphocytes stimulated with 4-1BBL. The target cells were H22 cells and B16 cells. The ratio of effector cells to target cells was 10∶1 and 20∶1. The assay was performed in 96-well microplates, and all cultures were set up in triplicate.
Measurement of cytokine production
Supernatants were obtained after the spleen cells cultured with fixed gene transfected cells. Cytokine concentration in the supernatants was assayed using a mouse IL-2 or IFN-gamma ELISA kit (Dakawa, Shenzhen, China) according to the manufacturer’s instruction.
Anti-tumor effect in vivo
The BALB/C mice (8 mice each group) were inoculated with H22 cells by the injection of 2×105 cells in 100 μL PBS into right hind thigh muscle. Five days later after inoculation, 100 μL (1 μg/μL) plasmid p4-1BBL were injected every other day for 6 times. The mice of control group received an equal volume of saline or equal amount of plasmid pcDNA3.1. Tumor size was measured using calipers. The tumor diameter was calculated by using the formula: a × b2 × 0.5 (‘a’ as the larger diameter and ‘b’ as the smaller diameter), and described with tumor growth curve. Tumors were weighed on the 15th and 25th day after the cell inoculation. After therapy, the tissues around the tumor were harvested to analyze the gene expression of 4-1BBL by muscle cells and CD8+ T cells by using immunohistochemistry staining.
Statistical analysis
All data were analyzed by using the SPSS10.0 software package.
Results
Plasmid construction
Full length DNA sequence coding mouse 4-1BBL was inserted into the plasmid pcDNA3.1 according to the translation frame. The inserted sequence was 930 bp and confirmed by DNA sequencing and enzyme restriction identification (Fig. 1).
Expression of 4-1BBL on BHK cells
After transfection, continuous passages for more than 10 generations were performed to guarantee the stable expression of recombinant protein. Then, cells stably expressing recombinant protein were subcloned and defined. The transcription of recombinant protein was defined by RT-PCR (Fig. 2) and the expression and localization of the protein were identified by western blot and immunochemical staining. Total protein from cell contained recombinant 4-1BBL of expected molecular weight 34 KD (Fig. 3) and protein 4-1BBL mainly localized on membrane and plasma (Fig. 4).
Effect of 4-1BBL expressed by BHK cells on spleen lymphocytes
The result of FACSort analysis showed that more 4-1BB+ spleen lymphocytes developed after the latter was stimulated with HSP70-H22 cell peptide complex. To evaluate whether 4-1BBL expressed by BHK cells (4-1BBL/BHK cells) could affect the proliferation, cytotoxic lysis and cytokine secretion of spleen cells. BHK cells expressing 4-1BBL were fixed, and then the spleen cells were added after the stimulation of the HSP70-H22 cell peptide complex. The results of MTT detection showed that there was a significant increase inducing proliferation of splenic cells following co-culture with fixed 4-1BBL/BHK cells (Fig. 5). Tables 1 and 2 showed the cytotoxicity lysis and cytokine concentrations. These data demonstrate that 4-1BBL expressed by BHK cells also can significantly enhance the spleen cell activities (such as proliferation, cytotoxicity and cytokines secretion) like immune cells.
The inhibitory effect of 4-1BBL expressed by muscle cell on the H22 tumor
The expression of 4-1BBL on the muscle cells was determined by immunohistochemical staining 2 days after transfection of plasmid p4-1BBL (Fig. 6). The result of the tumor growth curve analysis showed (Fig. 7) that the transfection of p4-1BBL plasmid could inhibit the growth of H22 tumor, only a small proportion of mice developed tumor in the group treated with p4-1BBL, and the growth rate of the tumor was significantly lower than the control group, the weight of the maximal tumor was only 0.3 g. As shown in Figure 8, large number of tumor cells and a small number of immune cells infiltrated the stroma in the control group, but in the p4-1BBL treated group, a large quantity of CD8+ T cells infiltrated the peri-tumoral tissues.
Discussion
Co-stimulatory molecules are critical to the initialization, expansion and diminishment of immune response. There are increasing evidences to demonstrate that controlling the co-stimulatory molecules is a good strategy for tumor therapy, for example, suppression of a negative co-stimulatory molecule or augmentation of a positive co-stimulatory molecule can also elicit a strong anti-tumor immune response. 4-1BBL/4-1BB are a pair of co-stimulatory molecules that play a crucial role in sustaining the functions of T lymphocytes, especially CD8+ T cells, and the latter, we all know, are the most important effector cells to kill tumor cells. So, the positive signal pathway of 4-1BBL/4-1BB is necessary for anti-tumor immunity. Until now, a lot of studies have proven that reinforcement of the function of 4-1BBL/4-1BB is an effective method in tumor immune therapy after their genes were transfected into tumor cells or immune cells. In this study, 4-1BBL was selected as a research target, while the normal tissue cells as target expression cells, and 4-1BBL gene was transferred into the normal tissue cells, by which the anti-tumor immune effect was significantly enhanced.
The result of
in vitro experiment showed that 4-1BBL could be expressed on BHK cells (a kind of normal tissue cells), and it could promote the proliferation and activation of T cells which was previously treated with tumor antigen complexes, and could also enhance the secretion level of cytokines such as IL-2 and interferon-γ (IFN-γ) by activated T cells. Cytokines IL-2 and IFN-γ both can augment the cytotoxicity of T cells and NK (natural killer) cells [
18]. It was confirmed in this study that 4-1BBL expressed on the BHK cells could reinforce the immune response (special immune response and non-special immune response) and lead to an immune attack on tumor cells.
On the basis of
in vitro experiments, we further studied the immune therapeutic effect on the tumor of 4-1BBL expressed
in vivo. After naked DNA was injected into muscle tissues, the expression of 4-1BBL on muscle cells was detected, and the latter can interact with its receptor 4-1BB expressed by T cells in the tumor microenvironment. On one hand, the interaction can stimulate T cells to secrete more cytokines (IL-2, IFN-γ, IL-13 and IL-8) [
19-
22]. On the other hand, the interaction of 4-1BBL and 4-1BB can suppress the apoptosis of tumor-specific cytotoxic T lymphocyte (CTL) [
23]. Therefore, 4-1BBL expressed on muscle cells could enhance the cytotoxicity of effector cells to tumor cells. These results confirmed that transfection with p4-1BBL plasmid
in vivo can produce significant therapeutic effect on tumor in mice inoculated with tumor cells.
Our study has confirmed that expression of 4-1BBL by non-immunocytes and non-tumor cells (BHK cells, muscle cells) can reinforce lymphocyte activities and lead to a stronger anti-tumor effect. This possibly provides a new conclusion that expression of positive co-stimulatory molecules by normal cells in the tumor microenvironment can enhance the proliferation and other functions of T lymphocytes. This therapeutic method can be expected to be a new modality in the tumor biotherapy field.
Higher Education Press and Springer-Verlag Berlin Heidelberg
PDF
(182KB)
