4-1BB Agonism Combined With PD-L1 Blockade Increases the Number of Tissue-Resident CD8+ T Cells and Facilitates Tumor Abrogation.

Although the milestone discovery of immune checkpoint blockade (ICB) has been translated into clinical practice, only a fraction of patients can benefit from it with durable responses and subsequent long-term survival. Here, we tested the anti-tumor effect of combining PD-L1 blockade with 4-1BB costimulation in 3LL and 4T1.2 murine tumor models. Dual treatment induced further tumor regression and enhanced survival in tumor-bearing mice more so than PD-L1 and 4-1BB mAb alone. It was demonstrated that dual anti-PD-L1/anti-4-1BB immunotherapy increased the number of intratumoral CD103+CD8+ T cells and altered their distribution. Phenotypically, CD103+CD8+ T cells expressed a higher level of 4-1BB and PD-1 than their CD103- counterparts. Administration of PD-L1 mAb and 4-1BB mAb further increased the cytolytic capacity of CD103+CD8+ T cells. In vivo, CD103-CD8+ T cells could differentiate into CD103+CD8+ progeny cells. In a human setting, more CD8+ T cells differentiated into CD103+CD8+ T cells in the peripheral tumor region of lung cancer tissues than in the central tumor region. Collectively, infiltrated CD103+CD8+ T cells served as a potential effector T cell population. Combining 4-1BB agonism with PD-L1 blockade could increase tumor-infiltrated CD103+CD8+T cells, thereby facilitating tumor regression.

Clinical Significance of Dynamics of Programmed Death Ligand-1 Expression on Circulating CD14+ Monocytes and CD19+ B Cells with the Progression of Hepatitis B Virus Infection.

Programmed death-1 (PD-1) expression has been revealed to be upregulated on T cells and contributes to T cell exhaustion in patients with hepatitis B virus (HBV) infection. In this study, we investigated the dynamic expression of programmed death ligand-1 (PD-L1), the ligand of PD-1, on circulating CD14+ monocytes and CD19+ B cells of HBV-infected patients at the stages of chronic HBV (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC), respectively. The results showed that compared with healthy controls, the levels of PD-L1 expression on CD14+ and CD19+ populations were both upregulated in CHB, LC, and HCC groups. Although there was no significant difference of PD-L1 expression on CD14+ population among three disease groups, further analysis demonstrated that the frequency of CD14+PD-L1+ population was negatively correlated with HBV DNA load, the levels of alanine aminotransaminase (ALT), and the levels of aspartate aminotransferase (AST), respectively, at CHB stage, while it did not present significant correlation with such parameters at LC stage and was only positively correlated with HBV DNA load at HCC stage. Similarly, the levels of PD-L1 expression on CD19+ population also did not present much difference among three disease groups. Intriguingly, the frequencies of CD19+PD-L1+ population at CHB and LCC stages were both positively correlated with the levels of ALT and AST, but they were not significantly correlated with HBV DNA load. Thereby, the current study elucidated the dynamics of PD-L1 expression on monocytes and B cells, along with the dynamic regulation of PD-1 on T cells, which had a close relationship during the progression of HBV infection. Collectively, our findings demonstrated that in the course of HBV infection development, PD-L1 expression on CD14+ monocytes and CD19+ B cells varied and significantly correlated with clinical parameters, which could be utilized as a potential clinical indicator.

The increase of circulating PD-L1-expressing CD68(+) macrophage in ovarian cancer.

Tumor-associated macrophages (TAMs) have been characterized as a critical population of immunosuppressive cells in a variety of tumor types. PD-L1 (also termed B7-H1) has been described to exert co-inhibitory and immune regulatory functions. Here, in ovarian cancer, PD-L1 is selectively overexpressed on some TAM compared that of benign ovarian disease. When expanding the data in peripheral blood, the proportion of PD-L1(+)CD68(+) cell among CD68(+) cells and the intensity of PD-L1 staining on CD68(+) cell in healthy group were similar to that observed in ovarian cyst group; instead, these two measures were significantly higher in ovarian cancer group, thereafter related to TNM stage. Interestingly, intracellular levels of IL-10, IL-6, TNF-α, and IFN-γ in PD-L1(+)CD68(+) macrophage were higher than those in PD-L1(-)CD68(+) macrophage, especially IL-6 expression. Based on the PD-L1 receptor PD-1 expression on tumor-infiltrating cytotoxic cells, our data supported that expression of PD-L1 on TAM promoted apoptosis of T cells via interaction with PD-1 on CD8(+)T cells. Taken together, these results suggested that PD-L1-expressing macrophage represents a novel suppressor cell population in ovarian cancer, which contributes immune escape of ovarian cancer.

Cytokine-induced killer cells co-cultured with dendritic cells loaded with the protein lysate produced by radiofrequency ablation induce a specific antitumor response.

Radiofrequency ablation (RFA) causes coagulative necrosis of tumor tissue and the production of local tumor protein debris. These fragments of tumor protein debris contain a large number of various antigens, which can stimulate a specific cellular immune response. In the present study, dendritic cells (DCs) were loaded with tumor protein lysate antigens that were produced in situ by RFA, and were used to treat murine colon carcinoma in combination with cytokine-induced killer (CIK) cells. Subsequent to the treatment of murine colon carcinoma by RFA, the in situ supernatant of tumor lysis was collected and the DCs were loaded with the lysate antigen to generate Ag-DCs. CIK cells induced from the spleen cells of mice were co-cultured with Ag-DCs to generate Ag-DC-CIK cells. The results revealed that the Ag-DC-CIK cells exhibited strong antitumor activity in vitro and in vivo. The morphology and immunophenotypes of these cells were determined using microscopy and flow cytometry, respectively. The cytotoxic activity of Ag-DC-CIK cells was determined using a CCK-8 assay. To establish a mouse model, mice were randomized into Ag-DC-CIK, DC-CIK, CIK and PBS control groups and monitored for tumor growth and survival time. ANOVA was used to compare the trends in the three groups for implanted tumor volumes. The log-rank test was used to compare the survival time. The present findings indicated that DCs loaded with the protein lysate antigens of tumors, produced in situ by RFA, combined with CIK cells may be a novel strategy for cancer treatment.

CD40 signal regulates CXCR4 mediating ovarian carcinoma cell migration: implications for extrapelvic metastastic factors.

Ovarian carcinomas are highly invasive, especially in the peritoneal cavity. SDF-1α and its receptor, CXCR4, play a crucial role in migration of cancer cells. Here, SDF-1α directed HO8910 cell migration, but not SKOV3 cells. After being educated to express CXCR4 in vivo or by treating with sCD40L, SDF-1α reexhibited the ability of directing SKOV3 cell migration, which could be antagonized by CXCR4-neutralizing antibody. Furthermore, concomitant expression of CXCR4/CD40 in ovarian carcinoma tissues had stronger correlation with pelvic metastasis than did each alone. It is suggest that SDF-1α acts through CXCR4 to induce ovarian cancer cell migration, which could be facilitated by CD40 activation. Simultaneously examining the expression of CXCR4 and CD40 will provide valuable diagnosis of pelvic metastasis for ovarian carcinomas.

Silencing of pancreatic adenocarcinoma upregulated factor by RNA interference inhibits the malignant phenotypes of human colorectal cancer cells.

Aberrant expression of pancreatic adenocarcinoma upregulated factor (PAUF), a novel secretory protein, has been reported in several types of cancer. However, in colorectal cancer (CRC), whether PAUF also plays its oncogenic role through the Wnt/ß-catenin pathway and its effect in regulating malignant phenotypes of CRC is unknown. In this study, we detected PAUF and ß-catenin expression levels by immunohistochemical analysis and real-time PCR in CRC tissues, adjacent non-tumor tissues (NATs) and 5 CRC cell lines. The results demonstrated that the expression of PAUF and ß-catenin in tumor tissues was higher than in NATs. Moreover, the expression of PAUF was correlated with the expression of ß-catenin in both tumor tissues and NATs. The HCT116 cell line, which has the highest PAUF expression of the 5 cell lines, was transfected with small interfering RNA (siRNA) targeting on PAUF, which significantly downregulated the expression of PAUF in cancer cells. Successful transfection was confirmed by using RT-PCR and western blot analysis. Further studies demonstrated that PAUF-siRNA inhibited the proliferation of CRC cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. At the same time, PAUF-siRNA inhibited the invasion, adhesion and migration of the tumor cells. In conclusion, this study suggested that PAUF was expressed in CRC at a high frequency. Interference of PAUF may be an effective strategy for regulating malignant phenotypes of CRC through the Wnt/ß-catenin pathway.

Higher numbers of T-bet(+) intratumoral lymphoid cells correlate with better survival in gastric cancer.

In the present study, we studied the expression of T-bet, a key marker for type 1 immune responses, within the tumor microenvironment of gastric cancer, and analyzed its association with clinicopathological parameters. One hundred and fifty-two archival paraffin-embedded gastric tumor tissues were collected, and the expression of T-bet in these cancer tissue specimens was examined by immunohistochemistry. T-bet(+) tumor-infiltrating lymphocytes (TILs) in some gastric cancer tissues were further characterized by flow cytometric analysis. The density of T-bet(+) TILs in gastric cancer tissues in relation to patient's clinicopathological parameters and postoperative prognosis has been analyzed. Herein, we have found significant increases in T-bet(+) lymphocytes in tumor tissues as compared with normal stomach tissues, gastritis tissues or gastric polyp specimens. T-bet(+) cells mainly consisted of CD4(+), CD8(+) and CD56(+) TILs. In addition, lower numbers of T-bet(+) TILs were associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet(+) TILs have longer disease-free survival and overall survival. Thus, our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity. T-bet expression within tumor can serve as a prognostic indicator for gastric cancer and a potential biomarker for immunotherapy.

[Construction of eukaryotic expression vector of muCD40-IgG1Fc fusion protein and its expression in CHO cells].

AIM: To construct an eukaryotic expression vector of human muCD40Ig fusion protein, and to express it stably in Chinese hamster ovary (CHO) cells for obtaining muCD40Ig fusion protein and founding an experimental basis for investigating the soluble muCD40 molecule in vivo. METHODS: Extracellular domain of human muCD40 gene was amplified by RT-PCR from L929/muCD40-transfected cells, and the genes encoding the constant regions of human IgG1 were cloned from human splenocytes. The genes were inserted into eukaryotic expression vector pIRES2-EGFP, respectively. The recombinant vector was transfected into CHO cells by Superfectin. The transfected cells stably secreting muCD40Ig fusion protein was selected with G418 and subcloned. The serum-free culture supernatant of the selected positive clone was subjected to Western blotting and RT-PCR to confirm the expression of the fusion gene. The affinity of muCD40Ig and L929/CD40L was analyzed by flow cytometry (FCM). RESULTS: The eukaryotic expression vector pIRES2-EGFP/muCD40Ig was constructed successfully. PCR and Western blotting showed that the transfected CHO cell strain was able to secret muCD40Ig fusion protein stably. FCM demonstrated a good affinity between muCD40Ig and L929/CD40L. CONCLUSION: A transfected CHO cell strain stably expressing muCD40Ig fusion protein has been obtained, and the muCD40Ig fusion protein can bind to CD40L.

Increase of circulating B7-H4-expressing CD68+ macrophage correlated with clinical stage of lung carcinomas.

Tumor-associated macrophages (TAM) influence diverse processes such as angiogenesis, tumor cell proliferation, and metastasis during tumor progression. In a variety of tumor types, the amount of TAM has been associated with prognosis, but their role in lung cancer has not been fully elucidated. The purpose of this study was to investigate the B7-H4-expressing TAM in a series of 56 cases of lung carcinoma. In peripheral blood, B7-H4-expressing macrophage (CD68(+) cells) was compared among patients with lung cancer, patients with tuberculosis, and healthy donors. B7-H4-expressing macrophage in peripheral blood from lung cancer patients was significantly higher than that from healthy donors and tuberculosis patients. B7-H4-expressing macrophage was thereafter related to tumor size, lymph node metastasis and TNM stage. On the basis of the data obtained from literature, it is suggestd that lung carcinomas increase B7-H4-expressing macrophages, which might favor tumor progression.

Analysis of CD8+CD28- T-suppressor cells in gastric cancer patients.

Host anti-tumor immune responses can be attenuated by suppressor T cells of the phenotype CD8(+)CD28(-) (T(s) cells). In the present study, we investigated the presence of CD8(+)CD28(-) (T(s) cells) in the peripheral blood compartment of gastric cancer (GC) patients. Flow cytometry was used to detect the population of CD8(+)CD28(-) T(s) cells present in peripheral blood in therapy naïve patients with gastric cancer (n = 26), postoperative chemotherapy naïve gastric cancer patients (n = 23), and healthy controls (n = 27). Meanwhile, the clinical data of gastric cancer patients were analyzed. A significant difference in the percentage of T(s) cells was observed when comparing peripheral blood samples from cancer patients to healthy volunteers (27.08 ± 1.60% versus 10.86 ± 0.75%). In the patient group, the percentage of CD8(+)CD28(-) cells among lymphocytes was higher in patients with LN metastasis than those without LN metastasis. The percentage of CD8(+)CD28(-) cells was also related to tumor infiltration and size, but not with the degree of differentiation of cancer cells. Moreover, the percentage of CD8(+)CD28(-) cells was higher in preoperative gastric cancer patients (26.24 ± 1.78%) than in those of postoperation patients (15.79 ± 1.11%). These findings may reflect the possibility of tumor-induced immunosuppression, and they should be complemented with further studies.

Induced expression of B7-H4 on the surface of lung cancer cell by the tumor-associated macrophages: a potential mechanism of immune escape.

B7-homolog 4 (B7-H4), a recently identified homolog of B7.1/2 (CD80/86), has been described to exert co-stimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H4 in lung cancer in vitro and in vivo. Although a lung cancer cell line constitutively expressed B7-H4 mRNA and protein in plasma, primary tumor cell isolated from the transplanted lung carcinoma model expressed B7-H4 on the surface. Interestingly, in transplanted lung carcinoma model, the expression of membrane-bound B7-H4 in tumor cells was increased as prolonging of tumor transformation. Exposure to tumor-associated macrophages strongly induced membrane-bound B7-H4 expression on the lung cancer cell line. To elucidate the functional significance of lung cancer-related B7-H4 expression, we performed co-culture experiments of lung cancer cell with allo-reactive T cells. Lung cancer-related B7-H4 was identified as a strong inhibitor of T-cell effect. Furthermore, B7-H4 mAb had an ability to inhibit tumor growth in vivo. B7-H4 expression may thus significantly influence the outcome of T-cell tumor cell interactions and TAM induced membrane-bound B7-H4 on the lung cancer cell represents a novel mechanism by which lung cancer cells evade immune recognition and destruction.

Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells.

BACKGROUND: To investigate the expression of cyclin-dependent kinase 8 (CDK8) and ß-catenin in colon cancer and evaluate the role of CDK8 in the proliferation, apoptosis and cell cycle progression of colon cancer cells, especially in HCT116 cell line. METHODS: Colon cancer cell line HCT116 was transfected with small interfering RNA (siRNA) targeting on CDK8. After CDK8-siRNA transfection, mRNA and protein expression levels of CDK8 and ß-catenin were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay in HCT116 cells. Cell proliferation was measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide Methylthiazolyl tetrazolium (MTT) assay, and cell cycle distribution and apoptosis were analyzed by flow cytometry analysis (FACS). CDK8 and ß-catenin protein levels were also examined by real-time PCR and immunohistochemistry (IHC) in colon cancer tissues and adjacent normal tissues. RESULTS: After CDK8 specific siRNA transfection, mRNA and protein expression levels of CDK8 and ß-catenin in HCT116 cells were noticeably decreased (P < 0.05). CDK8 specific siRNA transfection inhibited HCT116 cells' proliferation and facilitated their apoptosis significantly (P < 0.05). In addition, the proportion of HCT116 cells in the G0/G1 phase was remarkably increased after CDK8-siRNA transfection (P < 0.05). The expression levels of CDK8 and ß-catenin in adjacent normal tissues were lower than in tumor tissues (P < 0.05). Moreover, the expression of CDK8 was correlated with the expression of ß-catenin in both tumor and adjacent normal tissues (P < 0.05). CONCLUSIONS: CDK8 and ß-catenin were expressed in colon cancer at a high frequency. CDK8 specific siRNA transfection down-regulated the expression of CDK8 in colon cancer cells, which was also associated with a decrease in the expression of ß-catenin Moreover, CDK8 specific siRNA inhibited the proliferation of colon cancer cells, promoted their apoptosis and arrested these cells in the G0/G1 phase. Interference of CDK8 might be an effective strategy through ß-catenin regulation of colon cancer.

Overexpression of B7-H4 in tumor infiltrated dendritic cells.

DCs infiltrated tumors appears to be phenotypically and functionally defective. B7-H4 was highlighted for its inhibitory role in T cell responses. In this study, we showed that B7-H4 was moderately expressed in imDCs, and up-regulated by IL-10, and TNF-α could counteract the up-regulatory effects of IL-10 on expression of B7-H4 in DCs in vitro. Furthermore, tumor infiltrated DCs expressed B7-H4 at high levels. Blockade of B7-H4 expressed in DCs highly resulted in enhanced T cell proliferation and IFN-γ production significantly. Otherwise, the high level of IL-10 and TNF-α was both detected in the tumor, which suggested that TNF-α can not antagonize the effects of IL-10 on expression of B7-H4 in DCs in vivo. These data indicate that tumor environment may condition local DCs to become dysfunctional in the phenotype, and that the high expression of B7-H4 may contribute to the tumor infiltrated DCs to mediate immune invasion.

Increasing the frequency of CIK cells adoptive immunotherapy may decrease risk of death in gastric cancer patients.

AIM: to analyze the correlation between cytokine-induced killer (cik) cells adoptive immunotherapy and cancer-related death in gastric cancer patients. methods: One hundred and fifty-six gastric cancer patients after operation at the Third Affiliated Hospital of Soochow University were enrolled in this study. Their clinical data including demographic characteristics, operation time, tumor size, pathological type and staging, tumor metastasis, outcome of chemotherapy or CIK cells adoptive immunotherapy, survival time or time of death were collected with a standard structured questionnaire. Kaplan-Meier method was used to estimate the median survival time, and the 2- and 5- year survival rates. Hazard risk (HR) and 95% confidence interval (95% CI) of CIK cells adoptive immunotherapy for gastric cancer were calculated using the two-stage time-dependent covariates Cox model. RESULTS: the survival time of gastric cancer patients was longer after CIK cells adoptive immunotherapy than after chemotherapy (χ(2) = 10.907, P = 0.001). The median survival time of gastric cancer patients was also longer after CIK cells adoptive immunotherapy than after chemotherapy (49 mo vs 27 mo, P < 0.05). The 2- and 5-year survival rates of gastric cancer patients were significantly higher after CIK cells adoptive immunotherapy than after chemotherapy (73.5% vs 52.6%, 40.4% vs 23.9%, P < 0.05). A significant difference was observed in the survival curve for patients who received CIK cells adoptive immunotherapy (0, 1-10, 11-25, and over 25 frequencies) (χ(2) = 14.534, P = 0.002). The frequencies of CIK cells adoptive immunotherapy were significantly related with the decreasing risk of death in gastric cancer patients after adjustment for sex and age of the patients, tumor stage and relapse (HR = 0.54, 95% CI: 0.36-0.80) when the first stage Cox model was used to define the subjects who remained alive beyond 36 mo as survivors. However, no correlation was observed between the frequencies of death in CIK cells adoptive immunotherapy and the risk of gastric cancer patients (HR = 1.09, 95% CI: 0.63-0.89) when the second stage Cox model was used to define the subjects who survived for more than 36 mo as survivors. CONCLUSION: the survival time of the gastric cancer patients treated with chemotherapy combined with CIK cells adoptive immunotherapy is significantly longer than that of the patients treated with chemotherapy alone and increasing the frequency of CIK cells adoptive immunotherapy seems to benefit patients more.

[Influence of co-stimulatory molecules B7-H4 expression on the prognosis of patients with gastric cancer treated with cytokine-induced killer cells adoptive immunotherapy].

OBJECTIVE: To investigate the influence of co-stimulatory molecules B7-H4 expression on prognosis of gastric cancer patients treated by cytokine-induced killer cells (CIK cells) adoptive immunotherapy. METHODS: Clinical data of 156 cases of gastric cancer patients were retrospectively analyzed. Patients were divided into chemotherapy group(n=81) and chemotherapy combined with CIK cell therapy group(n=75). B7-H4 expression was detected in the surgical specimens of gastric cancer patients by immunohistochemistry assay. Disease-free survival was compared between the chemotherapy group and the CIK group at different expression levels of B7-H4. RESULTS: The difference was not statistically significant in all clinical and pathological data between the chemotherapy group and the CIK treatment group (P>0.05). The postoperative median tumor-free survival in two groups was 18.0 and 45.0 months, respectively, and the difference was statistically significant (chi(2)=11.631, P=0.001). The postoperative median survival time was 27.0 and 49.0 months, respectively, and the difference was statistically significant (chi(2)=10.907, P=0.001). In 86 patients with low B7-H4 expression, the median tumor-free survival time was 32.0 and 62.0 months, respectively, and the difference was statistically significant (chi(2)=4.663,P=0.03). In 70 patients with high B7-H4 expression, the median tumor-free survival time was 11.0 and 18.0 months, respectively, and the difference was statistically significant (chi(2)=11.971, P=0.001). CONCLUSION: The median tumor-free survival time of patients with gastric cancer may be further improved by chemotherapy combined with CIK cell therapy, regardless of the level of B7-H4 expression.

[Preparation and characterization of a novel functional anti-human CD83 monoclonal antibody].

AIM: To prepare and characterize a novel anti-human CD83 monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD83 transfectant (L929/CD83) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells.The hybridoma cells were screened with CD83 transfectant (L929/CD83 and 293/CD83) by FCM. The biological characteristics of antibody were investigated by rapid isotyping analysis, karyotype analysis, competitive inhibition test and Western blot. RESULTS: One hybridoma cell line named 9D8 was obtained, which had the property of secreting anti-human CD83 monoclonal antibody steadily, This mAb specifically recognized CD83 molecule expressed on human mature DC, activated T cells, and tumor cell line Daudi, myeloma cell line 8226.This mAb can recognize a distinct epitope from commercial mAb (HB15e). CONCLUSION: One hybridoma cell line has been developed successfully, which may lay a foundation for further study on the function of this molecule.

CD40-activated apoptotic tumor cell-pulsed dendritic cell could potentially elicit antitumor immune response: involvement of up-regulation of B7-H3 expression.

Dendritic cells (DCs) initiate and direct immune responses. Previous in vitro and in vivo studies have showed that DCs matured with CD40 linking signal could potentially elicit and boost antitumor immunity, however, its molecular mechanism remain elusive. This study demonstrates that expression of B7-H3 on apoptotic cell-loading DCs is up-regulated markedly by CD40 activation but not by tumor necrosis factor-alpha stimulation. There was no significant difference found with CD40, CD80, or CD86 expressions when activated by CD40 or tumor necrosis factor-alpha stimulation. In tumor-bearing mice, T cells conditioned with B7-H3-blocked on CD40-activated apoptotic tumor cell-pulsed DCs had a decreased ability to inhibit tumor growth. Therefore, it is hypothesized that high levels of B7-H3 expression contributes to the ability of CD40-activated tumor associated DCs in eliciting efficient antitumor immune response, given this fact the potentially significant clinical implications, CD40-activated DCs merit further considerations when preparing DCs for clinical application.

Fine-tuned expression of programmed death 1 ligands in mature dendritic cells stimulated by CD40 ligand is critical for the induction of an efficient tumor specific immune response.

During maturation, murine myeloid dendritic cells (DCs) upregulated the expressions of CD11c, CD25, CD40, CD80, CD86, MHC II and programmed death 1 ligands 1 and 2 (PD-L1 and PD-L2). Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-alpha. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-alpha stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-ligated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation.

Expression of programmed-death receptor ligands 1 and 2 may contribute to the poor stimulatory potential of murine immature dendritic cells.

Recent data have revealed that Ag presentation by immature dendritic cells (imDCs) plays a role in establishing and maintaining T-cell tolerance, but the mechanism remains unclear. PD-L1 and PD-L2, ligands for programmed-death receptor 1 (PD-1), members of the expanding B7 family, were highlighted for their inhibitory role in T-cell responses. Here, we show that blockade of PD-1 ligands on imDCs resulted in enhanced T-cell proliferation, which is perhaps due to the enhancement of IL-2 production from DC-stimulated T cells. PD-1 ligands blockade on mDCs did not show a significant stimulatory effect as markedly as imDCs. The inhibitory effects of PD-1 ligands would be dependent on maturation status of DCs, where attenuated positive costimulatory molecules provided the opportunity for PD-1 ligands to exert their strong capacity. Our data are consistent with the hypothesis that imDCs have an inhibitory bias, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of imDCs.

[Antitumor effects of cocultured dendritic cells and cytokine-induced killer cells on lung cancer in vitro and in vivo].

BACKGROUND & OBJECTIVE: Cytokine-induced killer (CIK) cells have high cytotoxic activity against tumor cells. Dendritic cells (DCs) are the strongest antigen-presenting cells (APC) and could increase the cytotoxic activity of immunologic effector cells against tumor cells. This study was to investigate the changes of phenotype, proliferative activity, and in vitro and in vivo cytotoxicity of CIK cells after in vitro co-culturing with DCs. METHODS: DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells (PBMC), then CIK cells were cocultured with autologous DCs for 5 days at a stimulator-to-responder ratio of 1:10 to prepare immunologic effector DC-CIK cells. Phenotypes of DC-CIK cells were analyzed by flow cytometry; The in vitro cytotoxicity of DC-CIK cells was detected by 3H-TdR incorporation method; the in vivo antitumor activity was evaluated in BALB/c nude mice bearing A549 lung cancer. RESULTS: At the 14th day of culture, compared with CIK cells, DC-CIK cells got a significant increase of proliferation rate [(17.0+/-1.8) times vs. (10.9+/-2.0) times, P<0.05] and CD3+CD56+ expression rate [(36.0+/-4.2)% vs. (25.7+/-2.9)%, P<0.05], and resulted in an enhancement of cytotoxicity to A549 cells (P<0.05) in vivo. At the 51st day after inoculation of tumor cells, the inhibition rate was significantly higher in DC-CIK group than in CIK group (62.9% vs. 41.5%, P<0.05). DC-CIK cells and CIK cells showed significant inhibitory effects on the growth of transplanted tumor cells as compared with control group (P<0.01). CONCLUSION: DC-CIK cells have higher proliferative activity and cytotoxicity in vitro and in vivo against lung cancer in comparison with CIK cells alone.