Fixed-Tumor Vaccine: A Practical Formulation with Cytokine-Microspheres for Protective and Therapeutic Antitumor Immunity

Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragments combined with sustained-releasers of cytokines and a non-toxic adjuvant was developed. C57BL/6J mice were immunized intra-dermally with the vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa 1–6 cells.Results: All of 15 nonimmunized control mice developed the hepatoma. Protection of mice immunized with fixed Hepa 1–6 cells and both of IL-2/GM-CSF microspheres or further mixed with TiterMax Gold reached 80% and 87%, respectively. Mass growth of the established tumors, vaccinated twice at 5 mm in diameter, the tumor of control animals continued to grow. However, 7–10 days after the second injection of the tumor vaccine, the tumor growth was suppressed in 9 of 10 mice and then markedly reduced. Complete tumor regression was observed in 60% (6/10) of mice. Splenocytes from the control mice were not able to lyse target Hepa 1–6 cells and other tumor cells. In contrast splenocytes from the vaccinated mice exhibited a 41% lytic activity against the Hepa 1–6 cells tested at an effector/target (E/T) ratio of 5, whereas they did not exhibited such activity against the melanoma cells (B16-F1), Lewis lung carcinoma cells (LLC), renal carcinoma cells (Renca), and bladder carcinoma cells (MBT-2). The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class I monoclonal antibodies but not with anti-CD4 and anti-MHC-class II antibodies. In the Phase-I clinical trial, vaccination of HCC patients with the autologous vaccine is a well-tolerated treatment and induces fixed tumor fragment-specific immunity.Conclusion: Fixed HCC vaccination elicited protective and therapeutic antitumor immunity against HCC. The tumor vaccine elicited antigen specific CTL response lysis of the target HCC was mediated by the typical MHC-class I restricted CD8+ T cells. Key words cancer vaccine - cytotoxic T lymphocyte - immunotherapy - hepatoma

T-regulatory lymphocytes in peripheral blood of gastric and colorectal cancer patients

AIM: To assess the absolute number of T-regulatory cells (Tregs; CD4+CD25+Foxp3+) in the peripheral blood of gastric and colorectal cancer patients. METHODS: We enrolled 70 cancer patients (33 gastric cancer, 37 colorectal cancer) and 17 healthy volunteers. The CD3+CD4+ lymphocytes and CD4+CD25+Foxp3+ Tregs in the peripheral blood were analyzed with flow cytometry. The absolute numbers of Tregs were calculated based on the CD4+CD25+Foxp3+ cells percent-age of CD3+CD4+ cells and the absolute numbers of CD3+CD4+ cells per microliter. RESULTS: The mean number of CD4+CD25+Foxp3+ cells per microliter in colorectal cancer patients was 15.7 (SD: 21.8), for gastric cancer patients 12.2 (SD: 14.3), and for controls 17.5 (SD: 11.4). The absolute number of Tregs was significantly lower in gastric cancer patients than in controls (P = 0.026). There was no statistically significant difference for gastric vs colorectal cancer or colorectal cancer vs controls. The absolute number of Tregs was also significantly depressed in N+ vs Ncancer patients [22.0 (27.7) vs 10.1 (9.0), P = 0.013], and in the subgroup of gastric cancer patients [30.3 (27.6) vs 9.6 (8.0), P = 0.003]. No statistical difference was observed in the proportion of Tregs in the CD4+ population between the groups. CONCLUSION: The absolute number of Tregs in peripheral blood of gastric cancer but not colorectal cancer patients was significantly decreased in comparison with that in healthy controls.

Preoperative sorting of circulating T lymphocytes in patients with esophageal squamous cell carcinoma: Its prognostic significance

AIM: To elucidate the immunologic parameters for the outcome of patients with malignant tumors, especially esophageal squamous cell carcinoma (ESCC) associated with high malignant potential. METHODS: Clinicopathologic features were compared between patients with lower and higher CD4 and CD8 values as well as CD4/CD8 ratio in peripheral blood. RESULTS: The survival rate of patients with higher CD4 value was significantly better than that in patients with lower CD4 value (P = 0.039). The survival rate of patients with higher CD8 value was significantly worse than that of patients with lower CD8 value (P = 0.026). Similarly, the survival rate of patients with higher CD4/ CD8 ratio was significantly better than that of patients with lower CD4/CD8 ratio (P = 0.042). Additionally, multivariate analysis demonstrated that lower CD8 and lower CD4/CD8 ratio were factors independently associated with worse prognosis of patients. CONCLUSION: All the immunologic parameters can predict the outcome of patients with ESCC.

沙利度胺联合CHOP方案治疗外周T细胞淋巴瘤的效果

目的探讨沙利度胺联合CHOP方案(环磷酰胺+表柔比星+长春新碱+泼尼松)治疗外周T细胞淋巴瘤的临床效果。方法选取2017年1月至2019年1月于聊城市人民医院接受治疗的外周T细胞淋巴瘤患者72例,按随机数字表法分为观察组(36例)与对照组(36例)。对照组采用CHOP方案治疗,观察组则在对照组基础上联合沙利度胺治疗。3周为一疗程,两组均连续治疗8个疗程。比较两组临床疗效与治疗前后的精氨酸酶1(Arg-1)、血管内皮生长因子(VEGF)、诱生型一氧化氮合酶(iNOS)水平及治疗期间的不良反应(胃肠道反应、肝损伤、骨髓抑制、神经毒性、脱发、皮疹)情况。结果治疗后,观察组总有效率(86.11%)高于对照组(63.89%),差异有统计学意义(P<0.05);治疗后,观察组Arg-1、VEGF、iNOS水平[(12.38±2.50)ng/ml、(20.11±10.02)pg/ml、(14.96±3.72)U/ml]低于对照组[(14.29±2.40)ng/ml、(32.72±10.43)pg/ml、(16.77±3.80)U/ml],差异有统计学意义(P<0.05);治疗期间,两组各项不良反应的发生率比较,差异未见统计学意义(P>0.05)。结论沙利度胺联合CHOP方案治疗外周T细胞淋巴瘤的临床效果显著,能有效降低Arg-1、VEGF、iNOS水平,且不会增加不良反应发生率。

Reversing effects of traditional Chinese antitumor medicines on colorectal tumor immunosuppression of natural killer cell and T lymphocyte in vitro

Objective： Reversing effects of traditional Chinese medicines on colorectal tumor immunosuppressions of natural killer （NK） cell and T lymphocyte were analyzed to provide evidence on selecting medicines for patients according to the differ- ent types of tumor immunosuppression. Methods： Six traditional Chinese medicines, including Arsenious acid （AS）, Ligustra- zine hydrochloride （LHC）, Astragalus mongholicus bge （AMB）, Matrine N-oxide （MOX）, Polyporus umbellatus polysaccharide （PUPS） and Artesunate （ART）, were enrolled. The reversing effects on suppression of murine splenocyte transformation and NK killing activity were measured by 3-{4,5-dimethyl-2-thiazolyl}-2,5-diphenyl tetrazolium （MTT）, and the effects on the suppressed expression of intefieukin 2 receptor a （IL-2R（I）, CD3E＊~,＊ and CD3~.-~＊ were detected by flow cytometry （FCM）. The effects on immunosuppressive molecules were measured by enzyme linked immunosorbent assay （ELISA）, including transforming growth factor ~1 （TGF-~I）, vascular endothelial growth factor （VEGF）, interleukin 4 （IL-4）, IL-6, IL-10 and pros- taglandin （PG） E2. Results： （1） The reversing effects of AMB on the inhibition of NK killing and CD3 expression were the most significant; the effect of LHC on inhibition of CD3 expression was the strongest; the effects of AMB, PUPS and ART on inhibition of transformation were the greatest; and the effect of ART on inhibition of IL-2Ra expression was the strongest. （2） The correlated molecules of these medicines that exerted reversing effects on colorectal tumor immunosuppression were TGF-~I and IL-10. AMB had the highest down-regulating effect on the secretion of TGF-~I. AS and ART had the highest effects on IL-10. Conclusion： Reversing tumor immunosuppression through the down-regulation of immunosuppressive molecules is one of the novel antitumor mechanisms of traditional Chinese medicines. The clinical use of compounded prescriptions of ART combined with AMB and LHC should be considered to avoid the reduced treatment efficiency caused by tumor im- munosuppression.

Dendritic cells engineered to secrete anti-Dc R3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro

AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

Liver tumor infiltrating lymphocytes: Comparison of hepatocellular and cholangiolar carcinoma

AIM: To investigate the role of tumor inf iltrating lym-phocytes (TIL) in primary hepatocellular and cholangio-lar carcinomas of the liver.METHODS: Immunohistochemical analysis was per-formed including antibodies to CD3, CD4, CD8, CD20, CD56 and TIA-1 in formalin-f ixed and paraff in-embed-ded tissue of 35 liver resection specimens of hepatocel-lular or cholangiocellular carcinomas. Semiquantitative evaluation was performed with emphasis on the area of the tumor itself and of the tumor/liver interface.RESULTS: All hepatocellular carcinomas showed in-filtration of lymphocytes predominantly around the tumor in the tumor/liver interface consisting mainly of CD3+ CD4+ T lymphocytes [164.3/10 high power f ields (HPF)] and in the tumor itself of CD8+ cells (54.9/10 HPF). Cholangiocarcinomas contained a heterogeneous amount of TIL, composed mainly of CD3+ T cells with a predominance of CD8+ cells in the tumor tissue (52.6/10 HPF) and of CD4+ cells in the interface region (223.1/10 HPF). CD56+ cells of the innate immune system were scarce. There was no significant difference between hepatocellular or cholangiolar carcinoma. No correlation with the clinicopathological data was seen. CONCLUSION: Liver TIL consists of intratumoral CD8+ T cells and peritumoral CD4+ T cells indepen-dent of histogenetic origin. Different functions of lym-phocytes in these regions seem possible.

Induction of cytotoxic T lymphocyte respones in vivo after immunotherapy with dendritic cells in patients with nasopharyngeal carcinoma

The aim of the present study was to determine the efficacy of immunotherapy with dendritic cells to elicit EBV-specific CTL-immunity in advanced cases of EBV-positive patients with nasopharyngeal carcinoma （NPC） and to determine the safety and toxicity of this preparation. Nine cases of histologically confirmed patients with NPC undergoing treatment with radiological therapy were enrolled in this study. Dendritic cells, generated in vitro from blood monocytes of patients were cultured and matured with cytokines and then infected with recombinant adenovims vaccine containing EBV-latent membrane protein-2 （Ad-LMP2）. On 9 days＇ cultivation of cells, the matured DCs were harvested, irradiated with 60^Co and then injected intradermally to patients with NPC. The injections were performed 3 times totally. After immunization, the CTL responses were assayed by means of cytotoxicity and epitope-specific IFN-γ production. The results of this trial showed that all patients could tolerate this kind of treatment without any side effect, during which marked increase of LMP2-specific CIL-responses could be demonstrated in 5 patients of this group. And the level of IgA/VCA antibody decreased in 8 of 9 patients, thus accounting for a better prognosis for these patients. All patients will be followed up for another one year. At least, the present work shows that intradermal vaccination with autologons DCs infected with recombinant Ad-LMP2 adenovirus is a safe procedure in NPC patients, in which this procedure can enhance the LMP2-specific CTL responses in patients. These data are encouraging to develop more effective vaccine strategies for the treatment of nasopharyngeal carcinoma.

ESTABLISHMENT OF A HUMAN T－LYMPHOMA CELL LINE（H－TL90） AND ANALYSIS OF ITS BIOLOGICAL CHARACTERISTICS

We established a human T-lymphoma cell line from the cancerous ascrtes of a male patient with prostate cancer which was named H-TL90. This cell line was characterized by its histological features, and by chromosomal and immunological analysis. Immunophenotypic analysis revealed that the cells expressed surface antigen CD3- CD4- CD7+ CD8-. Biological analysis revealed that the cell can promote lymphoryte proliferation. This suggested that the cell line has an autosecretion function. Cytogenetic analysis revealed that H-TL90 was a hyperdiploid with 47 chromosomes and had characteristic transloration between chromosome 3 and 11, and the deletion of the long arm of chromosome 6. These resuhs demonstrated the H-TL90 cell line can be a useful model for the study of human T-lymphoma.

肠病相关性T细胞淋巴瘤3例

1资料与方法1.1临床资料例1男性,31岁,因右下腹疼痛2d入院,既往有长期腹泻史。行剖腹探查术,术中见回盲部明显增厚、水肿,并穿孔,行回盲部切除术。例2男性,24岁,腹痛、腹胀伴大便习惯改变2个月,腹部可触及一个肿物,直径约4cm,有轻压痛,行部分小肠切除术。例3男性,59岁,因左下腹疼痛,伴恶心、呕吐,左下腹压痛、反跳痛阳性。既往有长期腹部不适和腹泻史。行剖腹探查、部分回肠切除术。