Gastric diffuse large B-cell lymphoma (GDLBCL) is a common disease with an increasing incidence. However, the regulatory effect of exosomal programmed death-ligand 1 (PD-L1) on the immune microenvironment in GDLBCL is unclear. In the present study, the protein expression levels of exosomal PD-L1 in the supernatants of cultured diffuse large B-cell lymphoma (DLBCL) cells and the plasma of patients with GDLBCL was assessed using immunoblotting. Exosomes derived from DLBCL cells were cocultured with T lymphocytes or injected into tumor xenograft mice by tail vein injection. The relationship between the protein expression level of exosomal PD-L1 in the plasma and the clinical characteristics and immune microenvironmental parameters of GDLBCL was evaluated using immunoblotting and immunohistochemistry. High levels of exosomal PD-L1 were found in the supernatants of cultured DLBCL cells. Exosomes with high levels of PD-L1 promoted growth of tumors formed by DLBCL cells
Primary gastric lymphoma (PGL) is the most common type of extranodal tissue lymphoma of non-Hodgkin's lymphoma, accounting for 30–40% of all extranodal tissue lymphomas worldwide (
Immunotherapy reverses the immunosuppression induced by cancer, which enhances the killing effect of immune cells toward cancer cells. Because they enhance the anticancer effect of adaptive immunity based on effector T cells, inhibitors of immune checkpoints are widely used in cancer treatment, which has opened a new era for immunotherapy (
Exosomes are a type of membranous vesicle with a diameter of 40–200 nm with special expression of protein markers such as cluster of differentiation 9 (CD9), cluster of differentiation 63 (CD63), and tumor susceptibility gene 101 protein (
Cell-intrinsic PD-L1 promotes the occurrence and development of GDLBCL; however, the regulatory effect of exosomal PD-L1 derived from GDLBCL cells on the immune microenvironment is still unclear. Although T cells, natural killer (NK) cells, macrophages and other immune cells are involved the in formation of the tumor microenvironment, immuno-oncology, represented by the inhibitory PD1/PD-L1 signaling, is mainly focused on enhancing T-cell responses (
The DLBCL cell lines U2932 (RRID: CVCL_1896) and OCI-LY8 (RRID: CVCL_8803) were purchased from Nanjing Cobioer Biotechnology Co., Ltd., the human gastric mucosal epithelial cell line GES-1 (RRID: CVCL_EQ22) was kindly provided by the Institute of Oncology, Nanhua University (Hengyang, China), and the human T cell line H9 (RRID: CVCL_1240) was purchased from Shanghai Yiyan Biotechnology Co., Ltd. U2932, OCI-LY8 and H9 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc.) with 10% (v/v) heat inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 units/ml penicillin (Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.). H9 cells were cultured in 75 cm2 tissue culture flasks at 37°C in an incubator with 5% CO2 and 95% humidity, and the culture medium was refreshed every 2–4 days. H9 cells were centrifuged at 125 × g at room temperature for 5–10 min and resuspended in fresh medium at 5×105 cells/ml to remove cell debris and replace the medium. For good growth states, H9 cells were maintained at cell concentrations between 5×105 and 2×106 cells/ml. Passage of U2932 and OCI-LY8 cells was performed when cells reached 80–90% confluence. U2932 and OCI-LY8 cells were sequentially centrifuged at 125 × g at room temperature for 5 min, resuspended in 2 ml fresh medium, seeded into a new 25 cm2 cell culture bottle with 8 ml fresh culture medium. The human normal gastric mucosal GES-1 cell line was cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) with 10% (v/v) FBS, 100 U/ml penicillin (Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.). The FBS used for cultivation of all types of cells was ultracentrifuged at 100,000 × g and 4°C for 10 h to remove exosomes. All kinds of cells were cultured at 37°C in a cell incubator with 5% CO2.
The total cellular RNA isolated using TRIzol® regent (Thermo Fisher Scientific, Inc.) from U2932 cells was used for synthesis of complementary DNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). Sequences used for plasmid construction were obtained using a high-fidelity PCR kit KOD-Plus-Neo (Toyobo Life Science) and cloned into the p3×Flag-CMV-14 vector (MilliporeSigma). The primers used for plasmid construction were as follows: forward, 5′-GGGGTACCATGAGGATATTTGCTGTCTTT-3′ and reverse, 5′-GCTCTAGACGTCTCCTCCAAATGTGTAGT-3′. Gene silencing plasmids for PD-L1 were constructed using the pRNAT-U6.1/neo vector (GenScript) according to the manufacturer's protocols. The target sequence used for constructing the gene silencing plasmid shRNA-PD-L1 was 5′-GCATTTGCTGAACGCATTT-3′. The negative control (target sequence: 5′-ACTACCGTTGTTATAGGTG-3′) was constructed previously (
Cell transfection of plasmids was performed according to the manufacturer's protocols. Briefly, a total of 1.5×106 cells at the logarithmic growth stage were seeded into each well of a 12-well cell culture plate. A total of 1 µg plasmids and 4 µl Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) were mixed in 100 µl of Opti-MEMTM medium (Thermo Fisher Scientific, Inc.), separately. After incubation for 5 min at room temperature, plasmids and Lipofectamine® 2000 were mixed together and incubated at room temperature for 20 min. Then, 200 µl of the mixture was added to each well of the 12-well cell culture plate. After transfection at 37°C for 12 h, the culture medium was replaced with fresh medium. The cells were used for further experiments 24–48 h after transfection.
Exosomes in the supernatants of cultured cells were extracted by ultracentrifugation according to previously reported protocols (
The DLBCL cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) with proteinase inhibitors. After incubation on ice for 15–30 min, lysates were centrifuged at 16,100 × g and 4°C for 15 min. After determination of the protein concentration by the BCA method, 40 µg total protein was sequentially run on 10% SDS-PAGE gels and transferred onto PVDF membranes which were blocked with 5% skim milk at room temperature for 1 h. Membranes were incubated with the primary antibodies at 4°C overnight, washed using TBST buffer with 1% Tween-20, and incubated with the secondary antibodies at room temperature for 2 h. Protein bands were detected using the SuperSignal® West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Inc.). β-actin was used as the internal control for total protein, and CD9 and CD63 were used as the internal controls for exosomes. Densitometric analysis of protein bands was performed using software Image Lab (version 5.2; Bio-Rad Laboratories, Inc.). The antibodies used in the present study were as follows: Rabbit anti-PD-L1 (1:1,000, clone E1L3N, cat. no. 13684, Cell Signaling Technology, Inc.), mouse anti-β-actin (1:5,000, clone AC-15, A5441, Sigma-Aldrich; Merck KGaA), mouse anti-Flag (1:5,000, clone M2, F3165, MilliporeSigma), mouse anti-CD9 (1:200, clone C-4, sc-13118, Santa Cruz Biotechnology, Inc.), mouse anti-CD63 (1:200, clone MX-49.129.5, cat. no. sc-5275, Santa Cruz Biotechnology, Inc.), goat anti-mouse IgG (HRP-linked, 1:5,000, AP124P, MilliporeSigma), goat anti-rabbit IgG (HRP-linked, 1:5,000, AP132P, MilliporeSigma).
Briefly, an average of 1×104 stably transfected DLBCL cells in 100 µl of culture medium were seeded per well of a 96-well cell culture plate. After culturing at 37°C for 24 h, 10 µl of 5 mg/ml MTT solution was added to the culture medium and incubated at 37°C for 4 h. Then, the culture medium was carefully removed and replaced with 150 µl of DMSO. The OD value at 490 nm was quantified using a Synergy HTX microplate reader (BioTek Instruments, Inc.). Experiments were repeated three times.
A total of 12 NOD-SCID mice (age, 6 weeks; average weight, 20 g; Hunan Slack Jingda Experimental Animal Co., Ltd.) were used to perform animal experiments. Mice were fed under standard conditions (25°C and 50% humidity) with free access to sterile feed and water in a pathogen-free environment with a 12 h light/dark cycle at the animal care facility of Hunan Cancer Hospital (Changsha, Hunan, China). The mice were randomly assigned to two groups with six mice per group. An average of 5×106 U2932 cells in 200 µl of sterile PBS were subcutaneously injected into all mice. Seven days after cell injection, 100 µg of exosomes derived from U2932 cells stably transfected with the p3×Flag vector or p3×Flag-PD-L1 in 100 µl of sterile PBS were administered to the mice every four days through the tail vein. Mice were sacrificed when they experienced a sharp decrease in activity, water and diet intake, if the tumors formed under the skin of mice were assessed to be about to reach 15 mm in diameter in any dimension or if a total of 30 days after cell injection was reached. The mice were sacrificed by cervical dislocation immediately after isoflurane anesthesia (induction, 3%; maintenance, 1%). The formed tumors were measured every two days and recorded for further analysis.
All tissue specimens used in the present study were obtained from Hunan Cancer Hospital with the informed consent of patients and the present study was approved by the institutional review boards of Hunan Cancer Hospital (approval no. 2021-012) and was performed in accordance with the Declaration of Helsinki. The blood samples of 26 GDLBCL patients were collected from Hunan Cancer Hospital from January 2017 to December 2020. The inclusion criteria used were as follows: i) the stomach was the primary site, which may have been accompanied by lymph node metastasis in the gastric drainage area; and ii) the pathological diagnosis was DLBCL. Samples that failed to meet both of the above inclusion criteria were excluded. A total of 10 males and 16 females, aged 30–69 years old with a median age of 51.2 years were included. The patients were not treated with antineoplastic therapy before samples were taken. The Lugano stage (2016) (
Tissue sections used for IHC were assessed by two pathologists at Hunan Cancer Hospital (Changsha, Hunan, China), and IHC staining was performed as previously reported (
Tissue slices were viewed under an inverted microscope, and representative images were presented in the figures. Three fields of view per section were analyzed. Positivity for CD5, CD10, CD8, PD-L1 and PD-1 was defined as ≥5% positively stained cells, and positivity for CD20, CD79a and Ki67 was defined as ≥50% positively stained cells. One-fifth of the cases were scored by two observers to assess reproducibility. For cases to be considered suitable for evaluation, ≥25% area of a tissue slice had to be available for morphologic analysis following staining and at least one positively stained tumor-infiltrating macrophage was required as a positive internal control. Analysis of IHC staining of CD3, CD5, CD8, CD10 and CD20 was performed according to the manufacturers' protocols and as previously reported (
Data analysis was performed using SPSS version 22.0 (IBM, Corp.). Statistical graphs were drawn using GraphPad Prism 5.0 (GraphPad Software; Dotmatics). Unpaired, two-sided Student's t-test was used to assess the statistical significance. The relationship between the PD-L1 level in plasma exosomes and the clinicopathological characteristics of GDLBCL patients was determined using the χ2 or Fisher's exact test. Data were presented as the mean ± SD from three independent biological replicates. P<0.05 was considered to indicate a statistically significant difference.
To assess the regulatory role of exosomal PD-L1 in the immune microenvironment of GDLBCL, U2932 and OCI-LY8 DLBCL cells were chosen as experimental cell models and the supernatants of the cell culture medium were collected to extract exosomes by ultracentrifugation. The protein markers CD9 and CD63 detected using immunoblotting indicated the successful extraction of exosomes (
Next, Flag-tagged PD-L1 was overexpressed or PD-L1 expression was silenced using shRNA gene-silencing plasmids in U2932 and OCI-LY8 cells. Assessment of the protein content using immunoblotting, demonstrated that Flag-tagged PD-L1 appeared in exosomes derived from the supernatants of U2932 and OCI-LY8 cells (
To further illustrate the regulatory role of exosomal PD-L1, tumor xenograft and tail vein injection experiments were performed in NOD-SCID mice. One week after injecting wild-type U2932 cells into mice, exosomes derived from the supernatants of U2932 cells overexpressing PD-L1 and the control vector were administered into mice through tail vein injection. Four weeks after cell injection, significantly larger and heavier tumors were observed in the mice injected with exosomes derived from PD-L1-overexpressing cells compared with that of the control (
As exosomal PD-L1 contributes to the immune evasion of cancer cells, it was hypothesized that exosomal PD-L1 might influence T lymphocytes in the immune microenvironment. Therefore, the influence of exosomal PD-L1 from the supernatants of cultured DLBCL cells on the proliferation of H9 human T lymphocytes was assessed. In MTT experiments, exosomes with PD-L1 overexpression or PD-L1 silencing as well as those of controls derived from U2932 and OCI-LY8 cells were added to the culture medium of H9 cells. The proliferation of H9 cells was significantly inhibited by treatment with exosomes with PD-L1 overexpression compared with the control and was significantly promoted by treatment with exosomes with PD-L1 silencing compared with the control (
Exosomes from the plasma of GDLBCL patients and healthy individuals were obtained for analysis. The detection of CD9 and CD63 proteins indicated the successful extraction of plasma exosomes (
The correlation between the protein expression level of exosomal PD-L1 and clinicopathological features of GDLBCL was further analyzed, including gender, age, Lugano stage, IPI score and pathological subtypes of lymphoma (
To evaluate the relationship between exosomal PD-L1 in the plasma and the immune microenvironment of GDLBCL, the protein expression levels of CD20, CD79a, CD5, CD10, Ki67, CD8, PD-1 and PD-L1 were assessed using IHC staining in a series of consecutive slices of GDLBCL tissue specimens (
Immunotherapies targeting PD-1/PD-L1 have been reported to be effective strategies for the treatment of malignances (
Exosomes have lipid bilayer membrane structures, which can provide a protective barrier for vulnerable biomolecules. Based on biofunctions such as transmitting biological information through protein or RNA, exosomes have been regarded as promising drug carriers (
PD-L1 is widely expressed in numerous types of lymphoma tissues and lymphoma cell lines (
Exosomal PD-L1 is positively correlated with head and neck squamous cancer progression and administration of anti-PD-L-1 antibodies inhibits the immunosuppressive function of PD-L1 (
The present study demonstrated the prognostic role of PD-L1 in plasma exosomes in GDLBCL and analyzed the association between the protein expression level of exosomal PD-L1 and the immune microenvironment, which highlighted the importance of exosomal PD-L1 in the development and immune evasion of GDLBCL. The significance and innovations of the present study were as follows: Firstly, high expression of exosomal PD-L1 was demonstrated to be positively related with the malignant transformation and poor prognosis of GDLBCL. Secondly, the upregulated expression of PD-L1 in plasma exosomes was identified as a potential indicator for the immunosuppressive microenvironment of GDLBCL. The present study further demonstrated the significance of the PD-1/PD-L1 axis in the development and therapy of GDLBCL and indicated the possibility of exosomal PD-L1 as a predictor of clinical anti-PD-1 immunotherapy.
Not applicable.
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
CZ and YL conceived and designed the experiments. HZe, JW, BX and HD performed the main experiments and analyzed the data. MP, RD and SS collected tissue and blood samples. QH established the cell lines. JLi and JLin conducted the protein experiments. HZh helped to design the experiments. YL and CZ wrote the manuscript. RD and HZh performed language correction. HZe, CZ and YL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
All tissue specimens used in this study were obtained from Hunan Cancer Hospital with informed consent from patients and the present study was approved by the institutional review boards of Hunan Cancer Hospital (approval no. 2021-012) and in accordance with the Declaration of Helsinki.
Animal experiments were approved by the Animal Care and Experiment Committee of Hunan Cancer Hospital (approval no. SBQLL-2021-050). All procedures performed in the experiments were in accordance with the Animal Care and Experiment Committee of Hunan Cancer Hospital.
Not applicable.
The authors declare that they have no competing interests.
primary gastric lymphoma
gastric diffuse large B-cell lymphoma
diffuse large B-cell lymphoma
nanoparticle tracking analysis
transmission electron microscope
international prognostic index
Immunohistochemistry
programmed death-ligand 1
natural killer
rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone
cluster of differentiation 9
cluster of differentiation 63
germinal center B-cell like lymphoma
Influence of exosomes derived from DLBCL cells on tumor growth in vivo. (A) Immunoblotting for PD-L1, CD9, CD63 and β-actin in exosomes from the supernatants of cultured DLBCL U2932 and OCI-LY8 cells, and human gastric mucosal epithelial GES-1 cells. (B) NTA for the size of exosomes extracted from the supernatants of cultured U2932 cells. Immunoblotting for Flag and PD-L1 in exosomes derived from the supernatants of cultured U2932 and OCI-LY8 cells with (C) overexpression or (D) inhibition of PD-L1. (E) Tumors formed by U2932 cells in NOD-SCID mice. Data were analyzed using unpaired, two-sided Student's t-test and are presented as the mean ± SD. *P<0.05. DLBCL, diffuse large B-cell lymphoma; NTA, nanoparticle tracking analysis; shRNA, short hairpin RNA; PD-L1, programmed death-ligand 1.
Regulatory effect of exosomal PD-L1 on T-cell proliferation. Proliferation of T cells influenced by exosomes derived from DLBCL (A) U2932 and (B) OCI-LY8 cells. A total of 24 h after seeding H9 cells in a 12-well cell culture plate, 2 µg of exosomes derived from DLBCL U2932 and OCI-LY8 cells with differential expression of PD-L1 were added to the supernatant of the culture medium, and the proliferation of H9 cells was then assessed using MTT assays at different time points. Data were analyzed using unpaired, two-sided Student's t-test and are presented as the mean ± SD. **P<0.01 and ***P<0.001. DLBCL, diffuse large B-cell lymphoma; shRNA, short hairpin RNA; PD-L1, programmed death-ligand 1.
Differential protein expression level of PD-L1 in plasma exosomes of patients with GDLBCL and healthy individuals. (A) Immunoblotting for specific markers of exosomes, including CD9 and CD63, and β-actin. (B) Transmission electron microscopy for morphology of exosomes extracted from plasma of a representative patient with GDLBCL. Red arrows indicate the exosomes. (C) Immunoblotting for differential levels of PD-L1 protein in plasma exosomes derived from three representative patients with GDLBCL and three healthy individuals. (D) Scatter plots presenting the relative protein expression levels of PD-L1 in plasma exosomes, compared between 22 healthy individuals and 26 patients with GDLBCL. Data were analyzed using unpaired, two-sided Student's t-test and are presented as the mean ± SD. **P<0.01. GDLBCL, gastric diffuse large B-cell lymphoma; PD-L1, programmed death-ligand 1.
Protein expression levels of protein markers of B cells and the immune microenvironment in tissue specimens of gastric diffuse large B-cell lymphoma. (A and B) Immunohistochemistry images of protein expression levels of the proliferative B-cell markers CD20, CD79a, CD5, CD10, Ki67 and PD-L1 as well as the immune microenvironmental markers CD4, CD8 and PD-1 in two representative tissue specimens of GDLBCL. Scale bar=50 µm. PD-L1, programmed death-ligand 1.
The relationship between the protein expression level of programmed death-ligand 1in plasma exosomes and the clinicopathological characteristics of patients with gastric diffuse large B-cell lymphoma.
Exosomal PD-L1 level | ||||
---|---|---|---|---|
Clinicopathological characteristics | Number of patients | High | Low | P-value |
Gender | 0.6882 | |||
Male | 10 | 4 | 6 | |
Female | 16 | 9 | 7 | |
Age, years | 0.4110 | |||
>60 | 9 | 6 | 3 | |
≤60 | 17 | 7 | 10 | |
IPI score | 0.0414 | |||
0-2 | 16 | 5 | 11 | |
3-5 | 10 | 8 | 2 | |
Pathological type | 0.0183 | |||
Non-GCB | 14 | 10 | 4 | |
GCB | 12 | 3 | 9 | |
Lugano stage | 0.0055 | |||
I + II | 15 | 4 | 11 | |
III + IV | 11 | 9 | 2 |
IPI, international prognostic index; GCB, germinal center B-cell like lymphoma.
The relationship between the protein level of PD-L1 in plasma exosomes and the immune microenvironment of gastric diffuse large B-cell lymphoma.
Exosomal PD-L1 level | ||||
---|---|---|---|---|
Protein level | Number of patients | High | Low | P-value |
CD20 | >0.9999 | |||
+ | 21 | 11 | 10 | |
- | 5 | 2 | 3 | |
CD79a | 0.6447 | |||
+ | 20 | 9 | 11 | |
- | 6 | 4 | 2 | |
CD5 | >0.9999 | |||
+ | 3 | 1 | 2 | |
- | 23 | 12 | 11 | |
CD10 | 0.6914 | |||
+ | 11 | 6 | 5 | |
- | 15 | 7 | 8 | |
Ki67 | 0.3783 | |||
+ | 19 | 8 | 11 | |
- | 7 | 5 | 2 | |
CD8 | 0.0183 | |||
+ | 12 | 9 | 3 | |
- | 14 | 4 | 10 | |
PD-1 | 0.2393 | |||
+ | 13 | 5 | 8 | |
- | 13 | 8 | 5 | |
PD-L1 | 0.0004 | |||
+ | 15 | 12 | 3 | |
- | 11 | 1 | 10 |
PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.