Artificial cytokine storm combined with hyperthermia induces significant anti-tumor effect in mice inoculated with lewis lung carcinoma and B16 melanoma cells.

In cancer immunotherapies combined with hyperthermia, one or two cytokines have been tested to augment the anti-tumor effect. However, the therapies have not shown sufficient improvement. The aim of this study is to find a new potent tumor immunotherapy in order to augment antitumor effect of hyperthermia by the cytokine cocktails in vivo. We used a combination therapy of local hyperthermia (LH) and various cytokine cocktails composed of IFNs (IFN-alpha, -beta, and -gamma), Thl cytokines (IL-2, -12, -15, and -18), a Th2 cytokine (IL-4), inflammatory cytokines (IL-lalpha and TNF-alpha), and dendritic cell-inducible cytokines (IL-3 and GM-CSF). These cytokines in a proper combination augmented the anti-tumor effect of LH and prolonged survival time in Lewis lung carcinoma or B16 melanoma significantly. Moreover, the 12-cytokine cocktail suppressed B 16 metastasis to the lung and lymph nodes, and complete regression of the tumors without regrowth occurred in 3 of 5 mice. In the cured three B16 mice, there was hyperplasia of lymphatic organs with many CD3-positive T lymphocytes. The most effective cytokine combination should be able to augment the anti-tumor effect of other therapies besides hyperthermia that induce the necrosis of tumor cells.     

GM-CSF基因修饰肺癌细胞疫苗的抗肿瘤活性及其与化疗的协同作用

目的评价粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰肿瘤疫苗的抗肿瘤活性,探讨其与化疗联合应用的抗肿瘤效果。方法以小鼠肺癌Lewis细胞系接种C57BL/6小鼠建立动物模型。利用含GM-CSF基因的重组质粒GM-CSF-pIRES2-EGFP,转染小鼠肺癌细胞系Lewis和LA795细胞,分别制备自体(Lewis细胞)和同种异体(LA795细胞)肿瘤疫苗。小鼠皮下接种Lewis细胞(1×107个)后5 d,采用GM-CSF分泌性肿瘤疫苗接种3次,同时设PBS组和单纯疫苗组作为对照,检测疫苗治疗前后脾细胞对Lewis细胞杀伤活性的变化,以及血清白介素4(IL-4)和γ干扰素(IFN-γ)的水平。观察GM-CSF分泌性肿瘤疫苗接种及联合化疗对小鼠生存期的影响。结果GM- CSF分泌性自体或同种异体肿瘤疫苗,均可诱导小鼠脾细胞对Lewis细胞杀伤活性增高,第3次接种后分别为(42.0±2.5)%和(39.6±7.3)%;同时血清中Th1类细胞因子IFN-γ的水平升高,而Th2类因子IL-4的水平无显著变化。GM-CSF分泌性肿瘤疫苗治疗组小鼠生存期与对照组比较,差异无统计学意义(P>0.05);而化疗联合疫苗组小鼠生存期较单独治疗组及对照组明显延长(P<0.05)。结论GM-CSF基因修饰的肿瘤疫苗可刺激机体产生特异性免疫反应;化疗的联合应用有助于提高肿瘤疫苗的治疗效果。     

The designer cytokine hyper-IL-6 mediates growth inhibition and GM-CSF-dependent rejection of B16 melanoma cells

The low immunogenic B16 melanoma cell line was transfected with a mammalian expression vector containing the complementary DNA for a sIL-6R/IL-6 fusion protein, termed Hyper-IL-6 (H-IL-6), which was shown to have biological activities at 100-1000-fold lower concentrations than IL-6 in combination with sIL-6R. The secreted p84 glycoprotein was detected in the supernatant of transfected cells and was fully active on BAF3/gp130 cells, which respond to IL-6/sIL-6R but not to IL-6 alone. Administration of recombinant H-IL-6 to C57BL/6 mice resulted in a prolonged acute phase protein gene expression indicating long systemic persistence of the fusion protein. Transfected B16 cells (B16/H-IL6 cells) showed morphological alterations in combination with a dramatic growth inhibition in vitro. Subcutaneous injection in C57BL/6 mice resulted in an almost complete rejection of B16/H-IL6 cells. This effect was partially abolished in FVB/BL/6 mice transgenic for a GM-CSF receptor antagonist, indicating a GM-CSF-dependent rejection of H-IL-6 transfected B16 cells. These results demonstrate that the anti-tumor effect of cytokines like IL-6 which are secreted by transfected melanoma cells at least in part depends on GM-CSF activity.     

Effects of interleukin 2 treatment combined with local hyperthermia in mice inoculated with Lewis lung carcinoma cells

Recombinant human (rhu) interleukin 2 (IL-2) was evaluated alone and in combination with local hyperthermia (LH) in mice inoculated s.c. with 5 x 10(5) Lewis lung carcinoma cells. Four treatment regimens were begun 6 days postinoculation at a time when the tumor had grown to approximately 8.0 mm in diameter. Treatments were: group 1, saline injected as control; group 2, LH; group 3, rhuIL-2; or group 4, LH combined with rhuIL-2. LH utilized hot water circulation by a Brann Thermomix 1420. The intratumor temperature was maintained at 43 +/- 0.2 degrees C for 30 min each on days 6 and 10 and rhuIL-2 was given s.c. at 5 x 10(4) units twice a day for 5 days. Thirty mice in each group were sacrificed 28 days after tumor inoculation. An additional 20 mice in each group were observed for survival time. The size of primary tumor and the number of lung metastases were reduced and the survival time was prolonged in mice treated by either LH or IL-2. However, a greater antitumor effect in Lewis lung carcinoma tumor-bearing mice was observed using IL-2 therapy combined with LH. Tumor growth was associated with increased splenic granulocyte-macrophage progenitor cells and an abnormal L3T4+/Lyt-2+ lymphocyte subset ratio (less than 1.0). Splenic granulocyte-macrophage progenitor cell numbers and the L3T4+/Lyt-2+ ratio returned to normal in the group treated with combination therapy, the best responder group. The L3T4+/Lyt-2+ ratio did not change in the groups treated with single therapy. These results suggest the efficacy and possible clinical relevance of combined therapy with rhuIL-2 and LH for certain metastatic tumors.     

皂角刺提取物对荷瘤小鼠肿瘤生长及细胞因子的影响

目的探讨皂角刺提取物对荷瘤小鼠肿瘤生长及细胞因子水平的影响。方法建立小鼠肉瘤S180及小鼠肝癌H22移植性肿瘤模型,观察皂角刺提取物对荷瘤小鼠肿瘤生长的抑制作用;采用ELISA法测定白介素-2(IL-2)、白介素 6(IL-6)、白介素 12(IL-12)、肿瘤坏死因子-α(TNF-α)的水平,探讨皂角刺提取物对荷瘤小鼠细胞因子的影响。结果皂角刺提取物在50、100 mg/kg时对S180的生长抑制率分别为44.59%、53.38%,对H22生长抑制率分别为39.88%、46.63%,且能提高荷瘤小鼠的脾指数和胸腺指数;皂角刺提取物能提高荷瘤小鼠细胞因子IL-2、IL-6、IL-12、TNF-α的表达水平。结论皂角刺提取物有较强的体内抗肿瘤活性,且能促进细胞因子的表达,增强机体免疫功能。     

Granulocyte-macrophage colony-stimulating factor and interleukin-2 fusion cDNA for cancer gene immunotherapy

Genetic engineering of tumor cells to express both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 can induce synergistic immune antitumor effects. Paradoxically, the combination has also been reported to down-regulate certain immune functions, highlighting the unpredictability of dual cytokine use. We hypothesized that a GM-CSF and IL-2 fusion transgene (GIFT) could circumvent such limitations yet preserve synergistic features. We designed a fusion cDNA of murine GM-CSF and IL-2. Protein structure computer modeling of GIFT protein predicted for intact ligand binding domains for both cytokines. B16 mouse melanoma cells were gene modified to express GIFT (B16GIFT), and these cells were unable to form tumors in C57bl/6 mice. Irradiated B16GIFT whole-cell tumor vaccine could also induce absolute protective immunity against challenge by live B16 cells. In mice with established melanoma, B16GIFT therapeutic cellular vaccine significantly improved tumor-free survival when compared with B16 expressing both IL-2 and GM-CSF. We show that GIFT induced a significantly greater tumor site recruitment of macrophages than combined GM-CSF and IL-2 and that macrophage recruitment arises from novel chemotactic feature of GIFT. In contrast to suppression by GM-CSF of natural killer (NK) cell recruitment despite coexpression of IL-2, GIFT leads to significant functional NK cell infiltration as confirmed in NK-defective beige mice. In conclusion, we demonstrated that a fusion between GM-CSF and IL-2 can invoke greater antitumor effect than both cytokines in combination, and novel immunobiological properties can arise from such chimeric constructs.     

Dietary D-glucarate effects on the biomarkers of inflammation during early post-initiation stages of benzo[a]pyrene-induced lung tumorigenesis in A/J mice

Previous studies showed that dietary calcium D-glucarate (CG) inhibited benzo[a]pyrene (B[a]P)-induced A/J mouse lung tumorigenesis, suppressing cell proliferation and chronic inflammation and inducing apoptosis during late post-initiation stages. The present study aimed to investigate changes in the homeostasis of cytokines in blood serum, as well as alterations in biomarkers of inflammation and apoptosis in lung tissue caused by dietary CG during early post-initiation stages of B[a]P-induced lung tumorigenesis. Two doses of 3 mg of B[a]P were given intragastrically to A/J mice 2 weeks apart. CG administration in the AIN-93G diet (2 and 4%, w/w) commenced at 2 weeks following the second dose of B[a]P. The levels of interleukin (IL)-6, IL-10 and tumor necrosis factor α (TNFα) in blood serum were investigated by FCAP array analysis. Two weeks after the second dose of B[a]P, approximately 8- and 28-fold increases of TNFα and IL-6, respectively, occurred in the blood serum and an approximately 16% decrease of IL-10 levels compared to the untreated control group was noted. At 4 weeks after the second dose of B[a]P and after 2 weeks of CG administration in the diet, the 2 and 4% CG diets significantly reduced the levels of IL-6 and TNFα (by 70 and 33%, respectively). In a dose-related manner, the diets also increased the level of anti-inflammatory cytokine IL-10 compared to the B[a]P group. At 6 weeks after the second dose of B[a]P, the cytokine levels in the serum continued to show a decrease in the CG-treated groups. These events are accompanied by an increased level of cleaved caspase-9 product with a molecular weight of 37 kDa. In conclusion, dietary D-glucarate decreases the level of proinflammatory cytokines, increases the level of the anti-inflammatory cytokine IL-10 during early post-initiation stages of B[a]P-induced lung tumorigenesis in A/J mice and affects apoptotic induction.     

胸腔热灌注治疗对肺癌胸水患者Th1/Th2免疫反应状态的影响

背景与目的:胸腔热灌注术是治疗肺癌胸水的独特方法,辅助性T细胞亚群(Th1/Th2)的平衡消长是反映机体抗肿瘤免疫功能的重要指标。本研究评价胸腔热灌注治疗肺癌胸水的临床综合疗效,同时观察胸腔热灌注治疗对肺癌胸水患者Th1/Th2免疫反应状态的影响。方法:在电视胸腔镜辅助下用43℃温盐水循环胸腔灌注60min,治疗24例肺癌胸水患者,观察随访胸水控制效果、副作用和生存时间。收集热疗前后患者外周血和胸水,用酶联免疫吸附法(ELISA)检测Th1型细胞因子白细胞介素2(interleukin-2,IL-2)、γ-干扰素(interferon-gamma,IFN-γ)和Th2型细胞因子白细胞介素4(IL-4)、白细胞介素10(IL-10)的浓度,同时用RT-PCR方法检测这些细胞因子mRNA的表达。结果:无手术死亡,无明显并发症。全组胸水控制总有效率达100%,完全缓解率为95.8%(23/24),部分缓解率为4.2%(1/24)。热疗后无胸水复发,生活质量有明显提高。全组中位生存期达18.3个月,1年生存率91.7%,2年生存率16.7%。与热疗前比较,热疗后肺癌胸水患者外周血或胸水中Th1型细胞因子浓度和mRNA阳性率均显著升高,而Th2型细胞因子则相反(P<0.05)。结论:电视胸腔镜辅助下胸腔热灌注术是一种安全、有效、微创的肺癌胸水治疗方法。胸腔热灌注治疗可促使肺癌胸水患者Th2免疫反应优势向Th1方向逆转。     

Increased expression of pro-inflammatory cytokines as a cause of lung toxicity after combined treatment with gemcitabine and thoracic irradiation.

BACKGROUND AND PURPOSE: Preclinical evidence suggesting gemcitabine potentiates the anti-tumor effects of irradiation has resulted in clinical trials to evaluate the treatment efficacy of gemcitabine and concurrent thoracic irradiation in non-small-cell lung cancer (NSCLC). Although these studies demonstrated favorable tumor response, this combined treatment modality was accompanied by severe treatment-related toxicities predominantly of the lung. In an attempt to elucidate the determinants of lung toxicity for gemcitabine, we analyzed the expression of the pro-inflammatory cytokines TNF-alpha, IL-1alpha and IL-6 in the lung tissue of mice treated with gemcitabine and concurrent thoracic irradiation. MATERIALS AND METHODS: Four study groups were defined: C57BL/6J mice that received neither irradiation nor gemcitabine (NT-group), those that received gemcitabine (120 mg/kg intraperitoneal, i.p.) but no irradiation (GEM-group), those that underwent thoracic irradiation (12 Gy) without gemcitabine (XRT-group), and those that received both gemcitabine (120 mg/kg i.p., 2 h before irradiation) and thoracic irradiation (GEM/XRT-group). The mice were sacrificed at 1 h, 1 and 3 days, 1, 2 and 4 weeks post-treatment (p.t.). The mRNA expression of TNF-alpha, IL-1alpha and IL-6 in the lung tissue was quantified by competitive RT-PCR. The cellular origin of the cytokine expression was identified by immunohistochemistry. The cytokine expression was correlated with histopathological alterations. RESULTS: The TNF-alpha, IL-1alpha and IL-6 expression in the lung tissue of the GEM/XRT mice was clearly higher at all assessment time points compared to the NT mice (statistically significant at 1 h, 1 and 3 days, 1, 2 and 4 weeks p.t.), XRT mice (statistically significant at 1 week p.t.) or GEM mice (statistically significant at 1 h, 1 and 2 weeks p.t.). Maximal treatment-induced cytokine expression in the lung tissue of the GEM/XRT mice occurred already at 1 week p.t. (TNF-alpha: 30.9 +/- 5.3/IL-1alpha: 28.3 +/- 5.0/IL-6: 4.9 +/- 0.1 times basal level), and coincides with pathohistologically discernable interstitial pneumonitis. The elevated levels of TNF-alpha and IL-1alpha have been found to correlate with immunohistochemical staining of the bronchiolar epithelium and predominantly of inflammatory cells. CONCLUSIONS: Our data provide evidence that the increased expression of pro-inflammatory cytokines and the induction of a cytokine-triggered inflammatory response may be a determinant of the observed elevated lung toxicity after concurrent treatment with gemcitabine and thoracic irradiation.     

Vernonia cinerea Less. inhibits tumor cell invasion and pulmonary metastasis in C57BL/6 mice

The effect of Vernonia cinerea Less. extract on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. V cinerea extract significantly (P < .001) inhibited lung tumor formation (78.8%) and significantly increased the life span (72.5%). Moreover, lung collagen hydroxyproline, uronic acid, and hexosamine and also serum sialic acid, γ-glutamyltransferase (GGT), and vascular endothelial growth factor (VEGF) levels were found to be significantly (P < .001) lower in treated animals compared with untreated controls. Histopathological analysis of the lung tissues also correlated with these findings. V cinerea treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix of the Boyden chamber. V cinerea also inhibited the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. It downregulated the production and expression of proinflammatory cytokines such as TNF (tumor necrosis factor)-α, IL (interleukin)-1β, IL-6, and GM-CSF (granulocyte monocyte colony-stimulating factor). V cinerea extract administration could suppress or downregulate the expression of matrix metalloproteinase (MMP)-2, MMP-9, lysyl oxidase, prolyl hydroxylase, K-ras, extracellular signal-regulated kinase (ERK)-1, ERK-2, and VEGF and also upregulate the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP-1), and TIMP-2 in the lung tissue of metastasis-induced animals. It also inhibited the protein expression of MMP-2 and MMP-9 in gelatin zymographic analysis of B16F-10 cells. These results indicate that V cinerea could inhibit the metastatic progression of B16F-10 melanoma cells in C57BL/6 mice by regulating MMPs, VEGF, prolyl hydroxylase, lysyl oxidase, ERK-1, ERK-2, TIMPs, nm23, and proinflammatory cytokine gene expression in metastatic lung tissue.     

Inhibition of tumor metastasis by adoptive transfer of IL-12-activated Valpha14 NKT cells

A unique lymphocyte lineage, the Valpha14 NKT cells, expresses both NK1.1 and an invariant antigen receptor encoded by Valpha14 and Jalpha281 gene segments. Valpha14 NKT cells play crucial roles in various immune responses, including autoimmune diseases, allergic reactions and anti-tumor immunity. Valpha14 NKT cells were demonstrated to be essential for anti-tumor effect of IL-12 in vivo. Here, we report that adoptive transfer of IL-12-activated Valpha14 NKT cells prevents hepatic metastasis of B16 melanoma. The injection of large amounts of IL-2, IL-4, and IFN-gamma, which are cytokines produced by activated Valpha14 NKT cells, exhibited no significant inhibition of the metastasis of this melanoma. The cells prepared from the liver of IL-12-injected mice expressed a potent cytotoxic activity on B16 melanoma cells in vitro. Although the adoptive transfer of IL-12-activated Valpha14 NKT cells prevents hepatic metastasis of B16 melanoma, activated NK cells from IL-12-injected RAG-1-/- mice failed to inhibit the metastasis of this melanoma. Thus, the anti-tumor effect of IL-12 can be replaced by adoptive transfer of IL-12-activated Valpha14 NKT cells but not by IL-12-activated NK cells, suggesting a minor role of NK cells for the IL-12-mediated anti-tumor effect in this experimental system. Moreover, our studies have suggested the involvement of direct cytotoxic mechanisms rather than cytokine-mediated immune responses at the effector phase of the Valpha14 NKT cell-mediated anti-tumor activity.     

小细胞及非小细胞肺癌CD+4细胞因子水平的观察

 目的 观察Ⅲb期及Ⅳ期小细胞肺癌（SCLC）及非小细胞肺癌（NSCLC）患者辅助性T淋巴细胞亚群（Th1、Th2）细胞因子表达的特点，探讨不同病理类型肺癌的免疫学特点。方法 选择TNM分期为Ⅲb期和Ⅳ期的肺癌患者，分别按照SCLC、NSCLC分为两组，采取静脉血标本，用Th1、Th2细胞因子标记流式细胞技术检测干扰素（IFN-γ）、肿瘤坏死因子（TNF-α）、白细胞介素（IL）-2、-4、-6、-10，比较相同分期的SCLC与NSCLC患者外周血CD+4细胞分泌细胞因子水平的变化。结果 Ⅲb期SCLC患者周围血CD+4细胞表达TNF-α明显高于NSCLC患者[（10.57±2.94）%、（7.03±3.06）%]（P＜0.05），而IL-4明显低于NSCLC患者[（2.48±0.55）%、（4.32±1.74）%]，Ⅳ期SCLC患者外周血CD+4细胞表达Th1／Th2（IFN-γ／IL-4）低于Ⅳ期NSCLC患者。结论 SCLC中Th2细胞因子的高表达及IFN-γ／IL-4的降低可能预示预后不良，在NSCLC中，TNF-α的增高对预后更加有意义。     

Blood classical monocytes phenotype is not altered in primary non-small cell lung cancer

AIM: To evaluate the M1 and M2 monocyte phenotype in patients with non-small cell lung cancer (NSCLC) compared to controls. Also, to examine the expression of Th1 and Th2 cytokines in plasma of NSCLC vs controls.METHODS: Freshly prepared peripheral blood mononuclear cells samples were obtained from patients with NSCLC (lung adenocarcinoma and squamous cell lung carcinoma) and from non-cancer controls. Flow cytometry was performed to investigate M1 and M2 phenotypes in peripheral monocytes (classical monocytes CD14+, CD45+ and CD16-) using conventional surface markers. Th1 and Th2 cytokine production was also analysed in the plasma using cytometric bead array technique.RESULTS: There were no significant difference in expression of M1 (HLA-DR) and/or M2 markers (CD163 and CD36) markers on classical monocytes in patients with NSCLC compared to non-cancer controls. Expression of CD11b, CD11c, CD71 and CD44 was also shown to be similar in patients with NSCLC compared to non-cancer controls. Th1 and Th2 cytokines [interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 (p70), tumor necrosis factor (TNF)-α, TNF-β, and interferon-γ] analysis revealed no significant difference between patients with NSCLC and non-cancer controls.CONCLUSION: This study shows no alteration in peripheral monocyte phenotype in circulating classical monocytes in patients with NSCLC compared to non-cancer controls. No difference in Th1 and Th2 cytokine levels were noted in the plasma of these patients.     

Neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity

BACKGROUND: Sustained intratumoral cytokine release using poly-lactic acid microspheres (PLAMs) can induce a systemic immune response, shifting immunotherapy to the neoadjuvant setting. METHODS: C57BL6 mice with established B16 melanomas underwent a single intralesional injection of IL-12, TNF-alpha or GM-CSF PLAM, alone or in combination. Tumor draining lymph nodes (TDLN) and spleens were assessed for a specific anti-tumor response by IFNgamma release assay and ELISPOT. RESULTS: Intralesional injection of TNF-alpha, alone or in combination, resulted in significant tumor ablation. The induction of tumor specific reactive T-cells in the TDLN was greatest with IL-12 and TNF-alpha. Only mice treated with IL-12 and TNF-alpha demonstrated a substantial T-cell response in cultured splenocytes. This combination resulted in a significant reduction in new tumors after re-challenge. Adjuvant therapy, using irradiated B16 cells in combination with equivalent doses of IL-12 and TNF-alpha, failed to generate a similar T-cell response or prevent re-challenge. CONCLUSIONS: Intratumoral IL-12 and TNF-alpha loaded PLAM leads to both local eradication of tumor and the induction of specific anti-tumor T-cells in the lymph nodes and spleens, resulting in memory immune response. Neoadjuvant treatment was significantly superior to postoperative autologous cellular vaccines using IL-12 and TNF-alpha PLAM.     

Inhibition of Metastasis of B16F-10 Melanoma Cells in C57BL/6 Mice by an Extract of Calendula Officinalis L Flowers

Aim: To determine the effect of a Calendula officinalis flower extract on lung metastasis by B16F-10 melanomacells in C57BL/6 mice. Materials and Methods: Male mice were injected with B16F-10 melanoma cells throughthe tail vein and simultaneously treated with C.officinalis flower extract. Parameters studied were lung tumornodule count, life span of animals, gamma glutamyl transpeptidase activity, sialic acid, TNF-α, IL-1β, IL-6, IL-2,GM-CSF, VEGF and TIMP-1 levels in serum, and lung hydroxyproline, uronic acid and hexosamine levels, aswell as histopathological features. Effects of C.officinalis on the expression of various genes involved in metastasislike matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), prolyl hydoxylase, lysyloxidase, nm23, and proinflammatory cytokines were also investigated. Results: Simultaneous administrationof C.officinalis extract to tumor bearing C57BL/6 mice reduced the lung tumor nodules by 74% with 43.3%increase in life span. Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and γ-glutamyltranspeptidase in the metastatic controls were found to be significantly lowered in the C.officinalis treated animals.The extract also inhibited expression of MMP-2, MMP-9, prolyl hydroxylase and lysyl oxidase and activatedTIMP-1 and TIMP-2 and downregulated proinflammatory cytokines. Conclusions: The present investigationindicated antimetastatic effects of Calendula officinalis flowers through the inhibition of key enzymes involvedin processes of metastasis.     

Lentivirally engineered dendritic cells activate AFP-specific T cells which inhibit hepatocellular carcinoma growth in vitro and in vivo

α-fetoprotein (AFP), a tumor-associated antigen for hepatocellular carcinoma (HCC), is an established biomarker for HCC. In this study, we created a lentivirus expressing the AFP antigen and investigated the anti-tumor activity of AFP-specific CD8+ T cells, with and without CD4+ T cells, which were activated by either AFP peptide-pulsed or Lenti-AFP-engineered Dendritic cells (DCs) in vitro and in vivo. AFP-specific T cells could efficiently kill HepG2 HCC cells, and produced IL-2, IFN-γ, TNF-α, perforin and granzyme B, with minimal production of IL-10 (a negative regulator of T cell activation). Both strategies activated AFP-specific T cells, but the lentiviral strategy was superior by several measures. Data also support an impact of CD4+ T cells in supporting anti-tumor activity. In vivo studies in a xenograft HCC tumor model also showed that AFP-specific T cells could markedly suppress HCC tumor formation and morbidity in tumor-bearing nude mice, as well as regulate serum levels of related cytokines and anti-tumor molecules. In parallel with human in vitro T cell cultures, the in vivo model demonstrated superior anti-tumor effects and Th1-skewing with Lenti-AFP-DCs. This study supports the superiority of a full-length antigen lentivirus-based DCs vaccine strategy over peptides, and provides new insight into the design of DCs-based vaccines.     

淋巴瘤与实体瘤患者Th1/Th2漂移状况比较

目的 比较淋巴瘤患者与实体瘤患者Th1/Th2的漂移状况,建立淋巴瘤生物治疗过程中免疫功能有效检测指标.方法 采集患者全血,淋巴瘤10例,实体瘤172例(其中上消化道恶性肿瘤36例;结直肠癌64例;肺癌43例,其他恶性肿瘤29例,健康对照30例.检测首先用刺激剂刺激细胞,增加细胞内因子表达,用荧光标记的特异性抗细胞因子单克隆抗体,特异性抗原抗体结合,最后应用流式细胞仪分析特异性细胞因子表达水平.结果 淋巴瘤与健康人外周血CD+4T细胞表达IFN-γ、IL-4阳性百分比以及IFN-γ/IL-4比值,差异均具统计学意义(P<0.05).淋巴瘤患者Th1/Th2(CD:胞内细胞因子INF-α/IL4)比值较上消化道肿瘤(食管和胃)明显降低差异具统计学意义(P=0.023);淋巴瘤与其他实体瘤(结直肠癌、肺癌、肾癌、乳腺癌及其他肿瘤)差异无统计学意义,但有下降趋势.结论 淋巴瘤患者免疫功能较实体瘤患者有降低趋势,免疫治疗是辅助化疗的必要途径,Th1/Th2(INF-α/IL4)比值有望成为有效的检测指标.

Abstract：

Objective To compare Th1/Th2 drift situation in patients with lymphoma with that in patients with solid tumors, and establish the effective immune function detectable criterion of lymphoma in biological treatment process. Methods The whole blood samples of 10 patients with lymphoma, 202 patients with solid tumors including 36 patients with upper digestive tract cancer, 64 colorectal cancer, 43 lung cancer and 20 the other malignancies, and 30 healthy persons as controls were collected. Stimulation agent was used to stimulate the cells in order to increase cell factor expression and fluorescent labeled specific anti-cytokine monoclonal antibody was used to bind with specific antigen. The expression of specific cytokines was detected by flow cytometry. Results Positive percentages of IFN- γ and IL-4 and ratio of IFN- γ /IL-4 in CD+4 T cells of human peripheral blood had statistically differences between in patients with lymphoma and controls (P < 0.05). The ratio of Thl/Th2 (CD+4 intracellular cytokine INF-α/IL-4) in patients with lymphoma was lower than that in patients with upper digestive tract cancers (esophagus and stomach cancers) (P = 0.023), however, had no statistical differences with that in patients with other solid tumors (colorectal, lung, kidney, breast and other tumors), but had a downward trend. Conclusion Immune functions in patients with lymphoma are lower than those in patients with solid tumors and immune treatment is a necessary to adjuvant chemotherapy. The ratio of Th1/Th2 (INF- α/IL-4) is expected to become effective detection criterion.