Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

Frequencies and Characterization of HBV-specific Cytotoxic T Lymphocytes in Self-limited and Chronic Hepatitis B Viral Infection in China

Hepatitis B virus （HBV）-specific cytotoxic T lymphocytes （CTLs） are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A＊0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells （PBMCs） from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8＋T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8＋T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8^＋ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8^＋T response but also improving its function.

Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

Objective: To investigate potential human leucocyte antigen(HLA)-A2-restricted epitope peptides of glypican-3(GPC3) and determine the cytotoxicity of peptidespecific cytotoxic T lymphocytes(CTLs) against hepatocellular carcinoma(HCC) cells.Methods: The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3+SMMC 7721 and Hep G2 cells was detected using IFN-g based enzymelinked immunospot and lactate dehydrogenase release assays in vitro.Results: A total of six peptides were identified for bindings to HAL-A2 and the GPC3522–530 and GPC3 229–237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522–530 or positive control GPC3 144–152 peptide responded to the peptide by producing IFN-g, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522–530-specific CTLs responded to and killed SMMC 7721 and Hep G2 cells, instead of GPC3-silenced SMMC7721 or Hep G2 cells. GPC3 522–530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.Conclusions: The GPC3 522–530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.

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.

Association of Graves’ disease and Graves’ ophthalmopathy with the polymorphisms in promoter and exon 1 of cytotoxic T lymphocyte associated antigen-4 gene

Objective: To investigate the association of Graves’ disease and Graves’ ophthalmopathy with the C/T transition polymorphism at position –318 of promoter and the A/G transition polymorphism at position 49 of exon 1 within cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene. Methods: Thirty-three patients with ophthalmopathy of Graves’ disease, fifty-six Graves’ patients without ophthalmopathy and sixty normal subjects as control were involved in the present case-control study. The polymorphisms were evaluated by polymerase chain reaction fragment length polymorphism (PCR-RFLP). Com-parisons were made of gene frequencies and allele frequencies between the groups. Results: The gene frequencies of CT and allele frequencies of T were much higher in Graves’ patients with ophthalmopathy than that in the group without ophthalmopathy (P=0.020, P=0.019). The gene frequencies of GG and allele frequencies of G in patients with Graves’ disease were significantly increased as compared with control group (P=0.008, P=0.007). The data suggest that smokers with Graves’ disease seemed to be more predisposed to ophthalmopathy than non-smokers (P=0.018). Conclusion: Our results suggest that an allele of T at position –318 of promoter is associated with genetic susceptibility to Graves’ ophthalmopathy while an allele of G at position 49 of exon 1 is associated with genetic susceptibility to Graves’ disease instead. Smoking is believed to be a major risk factor for ophthalmo-pathy.

Effect of cytotoxic T-lymphocyte antigen-4,TNF-alpha polymorphisms on osteosarcoma: evidences from a meta-analysis

Objective： Previous studies have investigated the role of cytotoxic T-lymphocyte antigen-4 （CTLA-4） and tumor necrosis factor-alpha （TNF-a） in carcinogenesis of osteosarcoma, but their results were inconsistent. We aimed to clarify the associations between CTLA-4, TNF-a polymorphism and osteosarcoma risk by using meta-analysis. Methods： We searched relevant studies without language restriction in PubMed, EMbase, Cochrane Library, Google Scholar databases, Chinese National Knowledge Infrastructure （CNKI） and conference literature in humans published prior to March 2013. The strengths of the associations between genetic variants and osteosarcoma risk were estimated by odds ratio （OR） with 95% confidence interval （95% CI）. Results： A total of seven studies with 1,198 osteosarcoma patients and 1,493 controls were selected. Four studies were eligible for CTLA-4 （1,003 osteosarcoma and 1,162 controls）, and three studies for TNF-a （195 osteosarcoma and 331 controls）. Pooled results showed that rs231775 polymorphism of CTLA-4 was associated with osteosarcoma risk （GG vs. AA： OR=1.63, 95% CI=1.24-2.13; GG ＋ GA vs. AA： OR=1.56, 95% CI=1.21-2.01; AA ＋ GA vs. GG： OR=0.83, 95% CI=0.71-0.97; G vs. A： OR=1.21, 95% CI=1.08-1.36）. No significant heterogeneity was observed across the studies. No significant associations were found between rs5742909 polymorphism of CTLA-4 or rs1800629 polymorphism of TNF-a and osteosarcoma risk. Conclusions： These results suggest that the rs231775 polymorphism of CTLA-4 may play an important role in carcinogenesis of osteosarcoma.

INVESTIGATION OF INDUCING EFFECT OF SPECIFIC CYTOTOXICITY OF CTLS BY ANTIGEN PEPTIDES FROM T LYMPHOCYTIC LEUKEMIA CELLS

Objective: To investigate the characteristics of specific antitumor immunity induced by antigen peptides mixture from T lymphocytic leukemia cells. Method: Antigen peptides mixtures were prepared from different leukemia cell lines and then bound with Hsp70 in vitro. Human peripheral blood mononuclear cells (PBMC) were cultured in vitro, and activated with Hsp70-antigen peptides. The activated PBMC was cultured continuously in vitro, and used as effector cells in vitro test of cytotoxicity to different target cells. Results: The antigen peptides from different leukemia cell lines were peptides mixture and could activate PBMC effectively if they were presented by Hsp70. The activated PBMC could proliferate in the presence of IL-2 and Hsp70-antigen peptides. The proliferative PBMC had specific cytotoxicity to leukemia cells corresponding to the antigen peptides. PBMC activated by antigen peptides from T lymphocytic leukemia cell lines could effectively kill T lymphocytic leukemia cells, and the cytotoxicity of these PBMC to T lymphocytic leukemia cells was significantly stronger than that of PBMC activated by antigen peptides from other leukemia cells (P < 0.05). PBMC activated by either Hut78-peptides or Molt 4-peptides could effectively kill Jurkat cells. And the cytotoxicity of PBMC activated by Hut78/Molt-4-peptides to Jurkat cells was significantly stronger than that of PBMC activated by either Hut78-peptides or Molt-4-peptides alone (P<0.05). Conclusion: Antigen peptides mixture from T lymphocytic leukemia cell lines can induce specific cytotoxic effect to T lymphocytic leukemia cells. There exists cross-reactivity among antigen peptides mixture from different T lymphocytic leukemia cell lines. The cross-reactivity could be amplified by blending of different antigen peptides from different T lymphocytic leukemia cell lines, suggesting that it is possible to prepare broad-spectrum antigen peptide vaccine against T lymphocytic leukemia by using multiple leukemia cell lines.

Effect of IFNα-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B

Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

Relationship between serum HBV DNA level and HBV-specific, nonspecific cytotoxic T lymphocytes and natural killer cells in patients with chronic hepatitis B

Background The response of patients with chronic hepatitis B （CHB） to antiviral therapy against hepatitis B virus （HBV） is related to the base line level of HBV DNA, but the mechanism is not clear. The present study aimed to understand the possible relationship between the level of HBV DNA and HBV-specific, nonspecific cytotoxic T lymphocytes （CTL） and natural killer （NK） cells of CHB patients and the mechanism how the HBV DNA level influences the antiviral therapeutic effect. Methods Totally 100 adult patients with CHB who were positive for HBV DNA, HBeAg and （HLA）-A2 were enrolled into this study. HBV DNA was tested by real time fluorescence quantitative polymerase chain reaction （PCR）. HBV specific and nonspecific CTL and NK cells were tested by flowcytometry. Serum alanine aminotransferase （ALT） and total bilirubin （TBil） were determined for each patient using routine biochemical tests. The 100 cases were assigned to two groups based on their HBV DNA level： group A had 48 cases, their HBV DNA level was 104-105 copies/ml, group B had 52 cases their HBV DNA level was 106-107 copies/ml. HBV specific CTL, nonspecific CTL, NK cells, ALT and TBil of the two groups were compared. Results HBV DNA level of groups A and B was （4.81±0.39） log10 copies/ml and （6.81±0.40） log10 copies/ml, respectively （t=25.32, P 〈0.001）. HBV specific CTL and NK cells of group A were significantly higher than those of group B （P 〈0.001 for both）. Nonspecific CTL of group A was significantly lower than that of group B （P 〈0.01）. ALT and TBil of group A were significantly lower than those of group B （P 〈0.01 and P 〈0.05, respectively）. Conclusions Serum HBV DNA level of patients with CHB is related to HBV specific CTL, nonspecific CTL and NK cells, which might result in inflammatory reaction of liver and cause more damage to liver function. Mechanism of HBV DNA level affecting the efficacy of anti-viral treatment may be related to the levels of HBV specific CTL and NK cells.

Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4^＋CD25^＋ T ceils in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen

Background Epstein-Barr virus （EBV） is a herpesvirus commonly associated with several malignant diseases including nasopharyngeal carcinoma （NPC）, which is a common cancer in Southeastern Asia. Previous studies showed that plasma levels of EBV-DNA might be a sensitive and reliable biomarker for the diagnosis, staging and evaluating of therapy for NPC. There are a few analyses of the levels of EBV-latent membrane protein 2 （LMP2）-specific cytotoxic T-lymphocytes （CTLs） in patients with NPC. This study was conducted to investigate the levels of EBV-LMP2-specific CTLs, EBV-DNA load and the level of CD4^＋CD25^＋T cells in such patients. Methods From February 2006 to April 2006, 62 patients with NPC, 40 healthy virus carriers positive for EBV viral capsid antigen （EBV-IgA-VCA） and 40 controls were enrolled in the study. We used a highly sensitive ELISPOT assay, real-time polymerase chain reaction （PCR） and flow cytometry to measure the EBV-LMP2-specific CTL response, the EBV DNA load and the level of CD4^＋CD25^＋T cells, respectively. Results The EBV-LMP2-specific CTL responses of the samples from the control, healthy virus carriers and patients with NPC were significantly different from the LMP2 epitopes, with the control and healthy virus carrier samples displaying a stronger response in three cases. There were significant differences in EBV DNA load in serum between NPC and the healthy groups; patients with NPC at stages Ⅲ or Ⅳ had significantly higher viral loads compared with those at stages Ⅰ or Ⅱ. A significantly higher percentage of CD4^＋CD25^＋ T lymphocytes were detected in the patients, compared with healthy virus carriers and healthy controls. Moreover, patients with advanced stages of NPC （Ⅲ and Ⅳ） had significantly higher percentages than the patients with early stages （Ⅰ and Ⅱ）. Conclusions Patients with NPC are frequently unable to establish or maintain sufficient immunosurveillance to control proliferating B cells harboring EBV and to destroy the tumor cells that express immunodominant LMP2 proteins. Controlling the activity of CD4^＋CD25^＋T cells and elevating CD8^＋ cells specific for LMP2 epitopes could be an effective immunotherapy for patients with NPC.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy

Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte （CTL） numbers,immunophenotype,tumor-specificity,and in vivo residence time,migration,and distribution.Therefore,tracing in vivo persistence,migration,and distribution of CTLs is important for cancer immunotherapy.Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 （CCK-8） assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester （CFSE）-labeled melanoma antigen-specific cytotoxic T lymphocytes （CFSE-CTLs） for malignant melanoma cells in vitro were compared.Additionally,CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma,and their residence time,migration,and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue （％FI/g） in various tissues and analyzing tumor/non-tumor （T/NT） values.Anti-tumor effects of transferred CTLs and correlation between ％FI/g and D-value of tumor size were analyzed.Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity.No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing （P=0.849） or interleukin-2 （P=0.318） and interferon-y （P=0.201）levels.Distribution of CTLs in vivo varied with time.A negative correlation between ％FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed.CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion.CTLs were observed up to three weeks later in the tumor,liver,kidneys,and spleen; this was related to the abundant blood supply or the nature of immune organs.Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations.Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors.CFSE fluorescent markers can trace in vivo CTL persistence,migration,and distribution because of its stability,long half-life,and low toxicity.

Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

Background Immunoglobulin heavy chain variable region （IgHV） is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte （CTL） responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions （FR） shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia （B-ALL）. Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A^＊0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay. Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A^＊0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten （83%） of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 （3-11） shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A^＊0201＋ healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family. Conclusion Anti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 （PGE2） and recruit myeloid-derived suppressor cells （MDSCs）. Activated cytotoxic T lymphocytes （CTLs） induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8^＋ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.

GPC3刺激对DC-CTL靶向杀伤肝细胞癌的影响

目的本研究将磷脂酰肌醇蛋白聚糖3(GPC3)作为肝细胞癌(LIHC)的靶分子,研究其在激活树突状细胞(DC)-细胞毒性T淋巴细胞(CTL)和增强肿瘤杀伤能力中的作用。方法为更系统、更全面地鉴定GPC3在多种恶性肿瘤中的意义,对GPC3在泛癌中的基因表达水平进行评估,并从LIHC与临床特征的相关性角度进一步研究GPC3。分别通过生存预后分析(Kaplan-Meier图)和受试者工作特征(ROC)曲线分析评估GPC3在LIHC中的预后价值和诊断价值。重组GPC3蛋白用于刺激DC,然后与CTL细胞共培养,经酶联免疫吸附试验(ELISA)分析以检测DC-CTL细胞中细胞因子的表达。通过CCK8测定法测定在Huh7细胞上用GPC3培养刺激的DC-CTL细胞的细胞毒性。结果GPC3的基因表达水平在大多数癌症中显著不同,并且在LIHC中与正常组织相比更高。GPC3在血清中与年龄和甲胎蛋白(AFP)水平相关(均P<0.001),而与LIHC的TNM分期、病理分期和组织学分级无关。ROC曲线分析表明,GPC3可用于准确预测LIHC。GPC3的表达水平与LIHC的总生存期无关。联合治疗显著上调了IFN-γ和TNF-α的分泌。用150 ng/ml和200 ng/ml GPC3刺激的DC-CTL细胞杀死Huh7细胞的能力强于不使用GPC3刺激的DC-CTL细胞(均P<0.05)。结论用GPC3刺激DC-CTL细胞通过促进细胞毒性在体外抑制肿瘤生长。GPC3可能是一种潜在的肿瘤标志物,可用于提高DC-CTL细胞的治疗效率。

Effect of oxymatrine on specific cytotoxic T lymphocyte surface programmed death receptor-1 expression in patients with chronic hepatitis B

Background Oxymatrine has certain antiviral effects in the treatment of chronic hepatitis B （CHB）, but its exact mechanism is unclear. The objective of the present study was to explore oxymatrine＇s antiviral mechanism by studying its effect on the hepatitis B virus （HBV） specific cytotoxic T lymphocyte （CTL） surface programmed death receptor-1 （PD-1） expression in CHB patients. Methods Sixty-five CHB patients who had HBV DNA≥104 copies/mL positive HBeAg, positive human leukocyte antigen （HLA）-A2, alanine aminotransferase （ALT） 〉2xupper limit of normal value （ULN） were randomly divided into two groups： treatment group （n=33）, treated with an intravenous infusion of 600 mg oxymatrine in glucose solution once a day for a month, then with a 200 mg oxymatrine oral capsule three times a day, and a 200 mg silibin meglumine tablet three times a day; control group （n=32） patients were treated only with silibin meglumine tablet, method and dosage were the same as those of treatment group. Three months later, peripheral blood HBV-specific CTL surface PD-1 expression, HBV-specific CTL level, HBV DNA, HBeAg, and results of liver function tests were analyzed and compared. Results Three months post-treatment, in the treatment group, peripheral blood HBV-specific CTL surface PD-1 expression （（19.42±15.94）%） decreased significantly compared to the pretreatment level （（31.30±24.06）%; P 〈0.05）, and decreased significantly compared to that of control group three months after treatment （（29.45±21.62）%; P 〈0.05）. HBV-specific CTL level （（0.42±0.07）%） significantly increased compared with the pretreatment （（0.29±0.15）%; P 〈0.01）, and the control group posttreatment level was （0.31±0.15）% （P 〈0.05）. HBV DNA level in 11 cases became negative （HBV DNA〈500 copies/ml, 33.33%）, which was higher than that of the control group after treatment （two cases, 6.25%; X2=7.45, P 〈0.01）, HBeAg of nine cases turned negative （27.27%）, which was higher than that of the control group after treatment （one case, 3.13%; X2=7.27, P 〈0.01）. Conclusion Oxymatrine could downregulate peripheral blood HBV-specific CTL surface PD-1 expression in CHB patients, increase HBV-specific CTL level, which may be one of the possible mechanisms by which oxymatrine clears or inhibits HBV in CHB Datients.

Demethylating agent decitabine induces autologous cancer testis antigen specific cytotoxic T lymphocytes in vivo

Background Cancer testis antigens （CTAs） are a novel group of tumor associated antigens.Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism,thus enhance the immunogenicity of leukemia cells.However,few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes （CTLs） in vivo,and if so,whether this effect contributes to disease control.In this study,we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.Methods Several mouse CTAs were screened by RT-PCR.CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry.The activity of specific CTLs was measured by real time RT-PCR.Results We firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs.Then we measured the CTLs＇ activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment.Finally,we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.Conclusions Our study showed the autologous immune response induced by decitabine in vivo.And more importantly,we firstly proved that this response may contribute to disease control.We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine,and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.

初诊SLE患者抗CTLA-4抗体水平及其临床意义

目的探讨初诊系统性红斑狼疮(SLE)患者血清中抗CTLA-4抗体水平及其临床意义。方法选取2022年1月至2023年6月在苏州大学附属第一医院风湿免疫科就诊的初诊SLE患者56例作为病例组,同时选取我院体检中心52例体检者作为对照组。采用酶联免疫吸附试验(ELISA)检测两组血清中IgG和IgM型抗CTLA-4抗体。比较不同临床特征(包括皮疹、口腔溃疡、浆膜炎等等)初诊SLE患者组间抗CTLA-4抗体水平差异。采用Pearson相关分析评估初诊SLE患者抗CTLA-4抗体与疾病活动指标[包括SLE疾病活动指数2000版(SLEDAI-2K)、抗ds-DNA以及C反应蛋白(CRP)]的关系。结果病例组IgG和IgM型抗CTLA-4水平均高于对照组(t=4.657、5.014,P均<0.05);IgG和IgM型抗CTLA-4抗体水平在SLE合并浆膜炎的患者中高于未合并浆膜炎患者(t=1.849、2.013;P=0.046、0.032);Pearson相关分析显示,IgM型抗CTLA-4抗体与SLEDAI-2K(r=0.425,P=0.029)及补体C3(r=-0.494,P=0.029)呈正相关;IgG和IgM型抗CTLA-4抗体与CRP(r=0.301、0.404;P=0.041、0.022)和抗ds-DNA抗体(r=0.644、0.363;P=0.002、0.036)呈正相关。结论初诊SLE患者血清抗CTLA-4抗体呈高表达,且该抗体与浆膜炎、疾病活动度相关。

Clinical evaluation of sintilimab in conjunction with bevacizumab for advanced colorectal cancer with microsatellite stable-type after failure of first-line therapy

BACKGROUND At present,immune checkpoint inhibitors(ICIs)remain the 1st-line therapy me-thod for patients suffering from high microsatellite instability/deficient misma-tch repair metastatic colorectal cancer(mCRC).However,ICI treatments demon-strate minimal therapeutic efficacy against microsatellite stable(MSS)/proficient mismatch repair(pMMR)CRC.This is mainly because this type of tumor is a“cold tumor”with almost no lymphocyte infiltration.Anti-angiogenic drugs have been found to improve the immune microenvironment by promoting many immune cells to enter the immune microenvironment,thereby exerting anti-tumor effects.AIM To investigate the effects of ICIs combined with bevacizumab monoclonal anti-body on tumor immune cells in MSS/pMMR advanced CRC patients with first-line treatment failure.METHODS A total of 110 MSS/pMMR patients with advanced CRC after first-line treatment failure in the Affiliated Hospital of Qinghai University were enrolled for a ran-domized controlled trial.In short,patients in the experimental group(n=60)were given sintilimab plus bevacizumab for 4 cycles,and those in the control group(n=50)patients were treated with FOLFIRI combined with bevacizumab for 4 cycles.The expression levels of cluster of differentiation(CD)8(+)T cells,tumor-associated macrophages(TAMs),and cancer-associated fibroblasts(CAFs)were comprehensively evaluated to assess the effects of sintilimab combined with bevacizumab on MSS/pMMR advanced CRC sufferers following failure of 1st-line therapy.RESULTS The positive expression rates of CD8(+)T lymphocytes(30%vs 50%),TAMs(23.30%vs 60%),and CAFs(23.30%vs 50%)before and after treatment in both groups exhibited statistical significance(P<0.05).Additionally,the therapeutic effects of both groups(partial remission:26.67%vs 10%;objective response rate:26.70%vs 10%)were significantly different(P<0.05).Although the experimental group showed a higher progression-free survival,median progression-free survival,and disease control rate than the control group,the difference was not statist-ically significant.Moreover,no significant difference in the occurrence rate of drug-related adverse reactions after treatment between the two groups was found(P>0.05).CONCLUSION ICIs in combination with bevacizumab can not only improve the patient’s prognosis but also yield safe and controllable adverse drug reactions in patients suffering from MSS/pMMR advanced CRC after failure to a 1st-line therapy.

基于BCMA突变体构建BCMA CAR-T细胞体外杀伤功能评价模型

目的:为解决野生型B细胞成熟抗原(BCMA)被γ分泌酶切割导致表达不稳定的问题,构建抵抗γ分泌酶切割的BCMA突变体并构建靶细胞,用于评价BCMA CAR-T细胞的杀伤功能。方法:将野生型BCMA的穿膜域替换为人CD8α穿膜域,构建抵抗γ分泌酶切割的BCMA突变体(BCMA-CD8αTM),构建过表达该突变体的U266(U266^(BCMA Mut))、K562(K562^(BCMA Mut))、SKOV3(SKOV3^(BCMA Mut))和CHO(CHO^(BCMA Mut))细胞;构建装载NFAT-EGFP报告基因的BCMA CAR Jurkat细胞(BCMA-CAR-Jurkat-Reporter)与U266^(BCMA Mut)细胞共培养,采用FCM检测该细胞中EGFP表达水平以指示NFAT激活水平,荧光素酶法检测BCMA CAR-T细胞对Luciferase标记的K562^(BCMA Mut)细胞的杀伤作用,实时无标记动态细胞分析技术(RTCA)检测BCMA CAR-T细胞对SKOV3^(BCMA Mut)和CHO^(BCMA Mut)细胞的杀伤作用。结果:应用γ分泌酶抑制剂LY411575抑制γ分泌酶活性,显著增强野生型U266细胞表面BCMA表达水平,平均荧光强度上调10倍以上;但撤除抑制剂后BCMA表达水平逐渐降低(P<0.01);BCMA-CD8αTM突变体可抵抗γ分泌酶的切割作用,在U266细胞表面稳定表达(P>0.05);U266细胞及过表达BCMA-CD8αTM的U266细胞与BCMA-CAR-Jurkat-Reporter细胞共培养后都可激活Reporter系统、增强EGFP表达,但该效应在BCMA-CD8αTM过表达的U266细胞中更显著(P<0.01);BCMA-CD8αTM在BCMA表达阴性的K562、SKOV3和CHO 3种靶细胞中成功过表达,且在LY411575处理下该突变体的表达水平仅有小幅度升高;荧光素酶法检测结果显示,不同效靶比下,BCMA CAR-T细胞均可特异、高效杀伤过表达BCMA-CD8αTM的K562细胞;RTCA结果显示,不同效靶比下,BCMA CAR-T细胞均可有效识别、杀伤过表达BCMACD8αTM的SKOV3和CHO细胞,但同等效靶比下的Mock-T细胞无此效应。结论:本实验构建的BCMA-CD8αTM突变体能够抵抗γ分泌酶的切割,在多种靶细胞表面稳定表达,为评价BCMA CAR-T细胞体外杀伤的有效性和特异性提供多种检测手段。