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.

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.

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.

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。

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。

基于LCK募集和活化的嵌合抗原受体T细胞的结构优化及其功能研究

目的 通过对二代嵌合抗原受体(CAR)进行淋巴细胞特异蛋白酪氨酸激酶(LCK)结构改造,探讨能否增强二代CD137 CAR-T细胞的活化水平及杀伤肿瘤细胞效率,为制备新型二代CAR-T细胞奠定基础。方法 利用基因工程技术,构建靶向人表皮生长因子受体-2(HER2)的CD137-LCK2 CAR和CD137-PYAP2 CAR的慢病毒载体,经慢病毒包装、感染活化T细胞后制备出CD137-LCK2和CD137-PYAP2 CAR-T细胞。采用流式细胞术、酶联免疫吸附实验、细胞毒性实验分别检测新构建的CAR-T细胞在活化分子CD137的表达水平、表型分布、分泌的细胞因子水平及对表达HER2的肿瘤细胞的杀伤效率。结果相较于二代CD137 CAR-T细胞,经过结构改造的靶向HER2的CD137-LCK2和CD137-PYAP2 CAR-T细胞能够上调活化分子CD137的表达、增加细胞因子IFN-γ的分泌、增强对靶细胞的杀伤能力,且均有统计学差异;同时能够维持在记忆状态、受抗原刺激后可迅速活化增殖分化。结论 靶向HER2的CD137-LCK2和CD137-PYAP2 CAR-T细胞有望比二代CD137 CAR-T细胞能更有效活化并发挥抗肿瘤效应。

猪源病毒细胞毒性T淋巴细胞表位研究进展

主要组织相容性复合体(major histocompatibility complex,MHC)Ⅰ类分子,也称为猪白细胞抗原(swine leukocyte antigen,SLA)Ⅰ类分子,在机体抗原递呈、器官移植、细胞免疫应答和调控等方面起重要作用。细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位是抗原经抗原递呈细胞加工后,与SLA-Ⅰ类分子结合并递呈的线性肽段,具有高度特异性,在机体抗原识别递呈和细胞免疫抵抗病毒感染方面发挥着关键作用。CTL表位能够刺激CTLs发挥细胞杀伤作用,主要表现为杀伤病毒。作者将对口蹄疫病毒、非洲猪瘟病毒和猪繁殖与呼吸综合征病毒等几种重要的猪源病毒的CTL表位研究现状进行综述,并利用生物信息学手段分析SLA-Ⅰ与CTL表位结合基序特征,以期为今后相关猪源病毒CTL表位的研究和多表位疫苗的设计提供参考。

恶性肿瘤中免疫检查点抑制剂与甲状腺功能异常风险的Meta分析

目的 Meta分析免疫检查点抑制剂(immune checkpoint inhibitors,ICIs)治疗引起恶性肿瘤患者发生甲状腺功能异常的风险。方法 检索从建库至2022年10月15日Pub Med、Embase、Cochrane Library、中国知网及万方医学网中关于恶性肿瘤ICIs治疗的相关不良反应研究。根据纳入与排除标准筛选文献,提取研究数据,采用Stata 12.0软件进行Meta分析。结果 共纳入32项随机对照试验,其中使用程序性细胞死亡蛋白1(programmed cell death protein-1,PD-1)、程序性细胞死亡配体1(programmed cell death ligand-1,PD-L1)、细胞毒性T淋巴细胞相关蛋白4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)抑制剂单药治疗分别为17篇、6篇、3篇,两种ICIs联合使用为8篇。Meta分析显示,ICIs单药治疗引起恶性肿瘤患者发生甲状腺功能减退(RR=9.04,95%CI为7.21~11.33)、甲状腺功能亢进(RR=10.83,95%CI为6.96~16.90)、甲状腺炎(RR=7.61,95%CI为2.99~19.37)的风险明显高于对照组。PD-1(RR=8.61,95%CI为6.47~11.46)、PD-L1(RR=6.75,95%CI为4.33~10.52)、CTLA-4(RR=7.12,95%CI为3.16~16.08)和联合应用(RR=52.56,95%CI为13.15~210.12)引起的甲状腺功能减退的风险比对照组均升高。与单一ICIs比较,ICIs联合使用引起甲状腺功能减退(RR=1.77,95%CI为1.41~2.22)及甲状腺功能亢进(RR=3.79,95%CI为2.42~5.96)的风险进一步升高。结论 ICIs治疗引起恶性肿瘤患者甲状腺功能异常的风险明显升高,以发生甲状腺功能减退的风险最高,其次是甲状腺功能亢进和甲状腺炎。与ICIs单药比较,ICIs联合使用可进一步升高风险。

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.

MUC1 Antigen-Specific CD8 T Lymphocytes Targeting MCF7 and MDA-MB-231 Human Breast Adenocarcinoma Cell Lines

MUC1 is an antigen that is overexpressed on the cell surface of many human breast adenocarcinomas and other types of cancer. The cancer immunity cycle has seven steps, starting with release of cancer cell antigen and following with cancer antigen presentation. Priming, activation and trafficking of T cells to tumors are the next steps and the infiltration of T cells into tumors, the recognition of cancer cells by T cells and killing of cancer cells are the final steps. We have tested a synthetic peptide for the MUC1 antigen and generated dendritic cells (DCs) that were pulsed with the specific peptide. Mature DCs were used to activate naive T cells to differentiate into antigen-specific CTLs. CTLs were tested for proliferation, cytokine release (IFNγ), activation markers and cytotoxicity against human breast adenocarcinoma cell lines. The cytotoxic effect of those CTLs was higher against MCF7 human cell line than against MDA-MB-231 human cell line.

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。

隔药饼灸对环磷酰胺诱导免疫抑制兔共刺激分子CTLA-4、4-1BB、CD28的影响

目的探讨隔药饼灸对环磷酰胺诱导免疫抑制兔共刺激分子细胞毒性T淋巴细胞相关抗原4(CTLA-4)、4-1BB、CD28的影响。方法选择大耳白兔32只,随机分为空白组、模型组、艾条灸组和隔药饼灸组,每组8只。模型组、艾条灸组和隔药饼灸组予环磷酰胺诱导免疫抑制模型。模型制备成功次日,艾条灸组和隔药饼灸组分别予艾条灸和隔药饼灸,隔日1次,共10次。空白组和模型组不予艾灸。艾条灸组和隔药饼灸组末次干预次日,空白组和模型组同时间,采集腹腔静脉血,离心留取血清,采用ELISA法检测血清CTLA-4、4-1BB、CD28;腹腔静脉取血后,摘取脾脏和肝脏,免疫组化法检测脾脏和肝脏组织CTLA-4、4-1BB蛋白表达。结果模型组血清CTLA-4、CD28水平均高于空白组,血清4-1BB水平低于空白组(P均<0.05);与模型组比较,艾条灸组和隔药饼灸组血清CTLA-4、CD28水平均降低,血清4-1BB水平均升高(P均<0.05);与艾条灸组比较,隔药饼灸组血清CTLA-4、CD28水平均降低,血清4-1BB水平升高(P均<0.05)。模型组脾脏和肝脏组织CTLA-4阳性表达均高于空白组,4-1BB阳性表达均低于空白组(P均<0.05)。与模型组比较,艾条灸组脾脏和肝脏组织CTLA-4阳性表达均降低(P均<0.05),肝脏组织4-1BB阳性表达升高(P<0.05),而脾脏组织4-1BB阳性表达升高不明显(P>0.05);隔药饼灸组脾脏和肝脏组织CTLA-4阳性表达均降低(P均<0.05),4-1BB阳性表达均升高(P均<0.05)。艾条灸组与隔药饼灸组脾脏和肝脏组织CTLA-4、4-1BB阳性表达比较差异均无统计学意义(P均>0.05)。结论隔药饼灸能够通过调节共刺激分子CTLA-4、4-1BB、CD28而改善环磷酰胺诱导免疫抑制兔的免疫功能,其效果优于艾条灸。

CTLA-4与PD-1/PD-L1免疫检查点抑制剂治疗子宫内膜癌新进展

免疫检查点抑制剂(ICIs)是可调节免疫反应。细胞毒性T淋巴细胞相关抗原4 (CTLA-4)和程序性死亡受体1 (PD-1)/程序性死亡受体配体1 (PD-L1)对应的免疫检查点抑制剂已在临床抗肿瘤中展现出极大优势。免疫检查点抑制剂通过阻断免疫检查点通路,激活免疫细胞识别和杀伤的功能治疗子宫内膜癌,从而达到抗肿瘤效果,应用前景广阔。

Magnetic Field Emulations of Small Inhibitor RNA: Effects on Implanted GL261 Tumors in C57BL/6 Immune Competent Mice

EMulate Therapeutics has developed a system for emulating the effects of solvated molecules via their magnetic field recordings. Recordings of magnetic field emissions of select small inhibitor RNAs (siRNAs;murine targeting CTLA-4 and murine targeting PD-1) were tested on C57Bl/6 mice implanted subcutaneously with the GL261 murine tumor cell line. A signal composed of concatenated recordings of siRNA molecules targeting the murine CTLA-4 and PD-1 receptors (labeled A2) was used in immune competent C57Bl/6 mice. The mice were flank implanted with the murine glioblastoma cell line GL261. Mice were exposed to the signal continuously (24 hours a day) until tumor volumes reached the designated volume limit. Tumors were excised and analyzed via PAGE/Western blot for the expression of CTLA-4, PD-1, Ki67, Caspase 3, CD4 and CD8. Terminal blood draws were used for CBCs. We report the down regulation of the checkpoint inhibitors CTLA-4 in the exposed mice. Significant tumor volume reduction was observed in mice exposed to the siRNA signal compared to control mice;no adverse events were recorded. Cell blood counts (CBC) and protein expression patterns were observed to correlate with the expected function of protein expression inhibition of the targets.

外周血CTLA4、TIM3对重症肺炎合并呼吸衰竭患儿疾病转归的预测价值

目的探讨外周血细胞毒性T淋巴细胞相关抗原(CTLA4)、T细胞免疫球蛋白黏蛋白分子3(TIM3)对重症肺炎合并呼吸衰竭患儿疾病转归的预测价值。方法选取2021年5月至2022年8月该院收治的108例重症肺炎合并呼吸衰竭患儿作为研究组。另选取同期来该院体检的120例健康儿童作为健康组。根据研究组30 d转归情况不同分为存活组(n=77)和死亡组(n=31)两个亚组。采用流式细胞仪检测外周血T细胞上CTLA4、TIM3表达水平。采用受试者工作特征(ROC)曲线评估CTLA4、TIM3对重症肺炎合并呼吸衰竭患儿预后的评估价值,多因素Logistic回归分析探讨重症肺炎合并呼吸衰竭患儿死亡的影响因素。结果研究组外周血T细胞上CTLA4、TIM3水平高于健康组,差异有统计学意义(P<0.05)。死亡组外周血T细胞上CTLA4、TIM3水平高于存活组,差异有统计学意义(P<0.05)。血清CTLA4、TIM3预测重症肺炎合并呼吸衰竭患儿预后的曲线下面积(95%CI)分别为0.779(95%CI:0.728~0.828)、0.842(95%CI:0.791~0.893),最佳截断值分别为9.67%、49.22%,特异度分别为57.14%、62.34%,灵敏度分别为90.32%、90.32%,二者联合检测的曲线下面积(95%CI)为0.929(0.888~0.970),特异度为85.71%,灵敏度为87.10%。存活组年龄、出生体质量、急性生理学与慢性健康状况评分系统Ⅱ(APACHEⅡ)评分、合并低氧血症、合并高碳酸血症、白细胞计数(WBC)、C反应蛋白(CRP)与死亡组比较差异有统计学意义(P<0.05)。多因素Logistic回归分析显示,APACHEⅡ评分≥18分(OR=2.143,95%CI:1.503~3.055)、CTLA4≥9.67%(OR=2.497,95%CI:1.658~3.761)、TIM3≥49.22%(OR=3.025,95%CI:1.943~4.711)是重症肺炎合并呼吸衰竭患儿死亡的危险因素(P<0.05)。结论重症肺炎合并呼吸衰竭患儿外周血T细胞上CTLA4、TIM3水平明显升高,且与疾病转归密切相关,有望作为评估患儿病情变化的生物学指标。