Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient''s human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8⁺ and CD4⁺ T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8⁺ response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4⁺ response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8⁺ and CD4⁺ T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.     

肾移植后外周血CD4~+及CD8~+T细胞亚群计数的临床意义

背景:临床上常以流式细胞检测受者外周血CD4、CD8细胞比值来揭示与排斥或感染相关的关系。目的:探讨肾移植后排斥或感染时外周血CD4+及CD8+T细胞(简称CD4和CD8细胞)亚群计数的变化和意义。方法:应用流式细胞仪检测肾移植121例受者CD4、CD8细胞数进行检测。根据入院病情将患者分为移植后正常组、急性排斥组、肺部感染组进行观察。结果与结论:移植后正常患者和急性排斥患者相比,CD4、CD8细胞数差异均无显著性意义(P>0.05)。肾移植后肺部感染患者CD4、CD8细胞数则均显著低于移植后正常组(P<0.001)。当感染控制、症状改善时,CD4、CD8细胞数显著升高(P<0.001)。说明肾移植后CD4和CD8细胞计数可以作为免疫状态的参考,其对于感染的参考价值大于排斥,动态观察分析有助于指导治疗。     

Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum—a possible indicator of immune response—increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.     

血清OPN、血CD4~+、CD8~+水平变化与肾癌患者临床分期的关联性分析

目的研究血清骨桥蛋白(OPN)、血CD4~+、CD8~+水平变化与肾癌患者临床分期的关联性。方法选取2017年9月～2018年9月我院收治的肾癌患者180例为观察组,经病理切片证实,临床分期:Ⅰ期44例、Ⅱ期70例、Ⅲ期39例、Ⅳ期27例,另选同期健康体检者90例为对照组。均检测血清OPN、血CD4~+、CD8~+水平,对比不同受检对象间上述物质水平,分析其水平与肾癌临床分期的相关性。结果观察组血清OPN、血CD8~+水平高于对照组,血CD4~+水平低于对照组(P0.05);经单因素方差分析,不同临床分期间血清OPN、血CD4~+水平对比,差异有统计学意义(P0.05);不同临床分期间血清OPN、血CD4~+水平两两对比,Ⅳ期血清OPN水平高于Ⅲ期,血CD4~+水平低于Ⅲ期,Ⅲ期血清OPN水平高于Ⅱ期,血CD4~+水平低于Ⅱ期,Ⅱ期血清OPN水平高于Ⅰ期,血CD4~+水平低于Ⅰ期(P0.05);不同临床分期间血CD8~+水平对比,差异无统计学意义(P0.05);经Spearman分析,血清OPN水平与肾癌临床分期呈正相关(P0.05);血CD4~+水平与肾癌临床分期呈负相关(P0.05)。结论肾癌患者血清OPN、血CD4~+、CD8~+水平变化显著,且血清OPN、血CD4~+水平与临床分期关系密切,通过检测上述指标,可为临床疗效及预后评估提供数据支持。     

SARS患者外周血CD4+CD8+T淋巴细胞的变化

目的 观察严重急性呼吸综合征(severe acute respiratory syndrome，SARS)患者外周血CD4^ 和CD8^ T淋巴细胞的变化，探讨SARS患者机体的免疫状况。方法 10位健康人(对照组)和13例确诊为SARS患者于发病第1、2、3、4周采静脉血，用流式细胞仪检测CD4^ 、CD8^ T淋巴细胞。结果 与对照组比较，SARS患者从发病1至4周外周血CD4^ 、CD8^ T淋巴细胞百分比均有不同程度的降低，以病程发展的2周左右为最明显。结论 SARS患者外周血CD4^ 、CD8^ T淋巴细胞有不同程度的降低，机体呈异常的免疫反应。     

Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.     

大鼠实验性变态反应性脑脊髓炎CD4+,CD8+的改变及意义

目的:观察实验性变态反应性脑脊髓炎模型大鼠外周血CD4 ,CD8 的改变及免疫调节剂左旋咪唑的作用。方法:实验于2005-05/07在遵义医学院附属医院神经科实验室和贵州省细胞工程重点实验室完成。取Wistar大鼠23只单纯随机分为4组:①正常组(n=5):不干预。②模型组(n=8):每只大鼠一次性注入0.5mL豚鼠脊髓匀浆(相当髓鞘碱性蛋白3.5mg)和完全福氏佐剂混合乳剂,其中0.4mL注入双后足掌皮下,0.1mL注入尾近端。在注入抗原乳剂后即刻、24,48h共3次腹腔注射左旋咪唑10mg/kg。③完全福氏佐剂组(n=5):注射无豚鼠脊髓匀浆生理盐水 完全福氏佐剂混合液0.5mL,用法同模型组,不注射左旋咪唑。④左旋咪唑组(n=5):每间隔24h腹腔注射左旋咪唑,用法同模型组。将注射日规定为第0天,每日观察2次记录大鼠神经体征变化,所有大鼠于注射第16天取材,流式细胞仪检测外周血CD4 ,CD8 的变化,苏木精-伊红染色观察病理变化,Loyez氏髓鞘染色法观察髓鞘改变。结果:23只大鼠全部进入结果分析。①神经体征变化:正常组、完全福氏佐剂组和左旋咪唑组均未出现症状,模型组8只大鼠中有7只于第14天出现症状,发病率为87.5%。②CD8 的变化:左旋咪唑组和模型组低于正常组犤(31.24±7.01)%,(20.64±5.52)%,(41.73±7.28)%,P<0.05,0.01犦,且模型组低于左旋咪唑组(P<0.05)。③CD4 的变化:左旋咪唑组和模型组高于正常组犤(45.21±5.35)%,(43.30±5.47)%,(34.44±7.01)%,P<0.05犦,但前2组比较差异不显著。④CD4 /CD8 :左旋咪唑组和模型组高于正常组(1.53±0.49,2.33±1.04,0.86±0.32,P<0.05,0.01)。⑤模型组苏木精-伊红染色见大脑白质及脊髓血管周围有大量炎性细胞浸润;Loyez氏染色见部分脱髓鞘改变。结论:①大鼠实验性变态反应性脑脊髓炎是通过降低CD8 、增高CD4 及CD4 /CD8 介导的免疫性疾病。②左旋咪唑破坏了机体的免疫平衡,是诱导实验性变态反应性脑脊髓炎发生的重要免疫调节剂。     

NOD鼠体内CD_4~+NKG2D~+T细胞与CD_8~+T细胞间关系

目的探讨NOD鼠体内CD_4~+NKG2D~+T细胞与CD_8~+T细胞间关系。方法动态监测NOD鼠外周血中CD_4~+NKG2D~+T及CD8+NKG2D~+T细胞在不同周龄频率。利用IGRP206-214特异性CTL清除的NOD鼠,对其外周血中CD_4~+NKG2D+/CD_4~+T及CD8+NKG2D+/CD_8~+T细胞频率进行分析。将磁珠分选所得CD_4~+NKG2D~+T细胞分别与经CFSE标记的纯CD_4~+NKG2D-T细胞、CD_8~+T细胞共培养4 d,检测体系中CD_4~+NKG2D-T细胞、CD_8~+T细胞CFSE的荧光强度变化。结果 NOD鼠外周血CD_4~+NKG2D+/CD_4~+T及CD8+NKG2D+/CD_8~+T细胞频率均随其1型糖尿病疾病进程发展逐渐升高,且二者之间呈正相关。IGRP206-214特异性CTL清除的NOD鼠,外周血中CD_4~+NKG2D+/CD_4~+T细胞频率随CD8+NKG2D+/CD_8~+T细胞频率降低显著下降。与单独培养的CD_4~+NKG2D-T细胞相比,加入CD_4~+NKG2D~+T细胞的培养体系中,CD_4~+NKG2D-T细胞增殖加快。但CD_4~+NKG2D~+T细胞对CD_8~+T增殖作用不明显。结论 NOD鼠体内存在着一群与1型糖尿病疾病进程正相关的CD_4~+NKG2D~+T细胞,且该群细胞极有可能是通过直接作用于CD_4~+NKG2D-T细胞而间接对CD_8~+T细胞产生作用。     

视神经脊髓炎的CD4+和CD8+T细胞内细胞因子的分析

目的：探讨视神经脊髓炎（NMO）的免疫反应类型以及在疾病的复发期和缓解期其免疫反应类型的变化情况。方法：利用流式细胞仪并采用细胞内抗原染色的方法对22名临床诊断为NMO的患者（复发期患者8名;缓解期患者14名）进行CD4＋和CD8＋T细胞内细胞因子白介素（IL）-13I、L-5测定并加以分析。20名健康人为对照组。结果：与健康对照组比较,NMO组的CD4＋IL-13＋T细胞和CD8＋IL-13＋T细胞显著增加（P〈0.05）,但将复发期和缓解期分开来分析时发现仅仅在复发期显著增加,在缓解期无明显变化（P〉0.05）。CD4＋IL-5＋T细胞和CD8＋IL-5＋T细胞在复发期和缓解期均无显著变化（P〉0.05）。结论：NMO的发病及发病阶段与II型辅助性T细胞（Th2）和II型细胞毒性T细胞（Tc2）所分泌的IL-13有关,而与IL-5无关。同时也说明对T细胞内细胞因子进行测定在分析神经系统自身免疫性疾病的发病过程有一定的意义。     

淋巴细胞CD3~+、CD4~+、CD8~+亚群检测在早发型新生儿败血症诊断中的价值

目的探讨早发型新生儿败血症诊断中,淋巴细胞CD3~+、CD4~+、CD8~+百分比检测的价值。方法将2014年1月至2015年6月出生,7d内发病,疑似感染的患儿纳入本研究,入院24h内采集静脉血,检测CD3~+、CD4~+、CD8~+淋巴细胞百分比,C反应蛋白(CRP)及血常规,在抗菌药物使用前做血培养。将其中血培养证实为早发型新生儿败血症的足月新生儿作为败血症组,血培养阴性的作为局部感染组。同时选择同期因高胆红素血症(排除感染等因素所致)而住院的足月新生儿作为非感染组。采用流式细胞仪对上述患儿标本进行检测,观察并比较3组间淋巴细胞亚群CD3~+、CD4~+、CD8~+百分比。结果败血症组CD3~+[(40.3±10.6)%],CD4~+[(28.6±11.2)%],CD8~+[(10.8±2.6)%]低于局部感染组的CD3~+[(64.8±9.8)%],CD4~+[(48.9±10.2)%],CD8~+[(17.6±5.6)%]和非感染组的CD3~+[(62.6±11.6)%]、CD4~+[(46.4±13.6)%]、CD8~+[(16.5±7.3)%],差异有统计学意义(P0.05),局部感染组的CD3~+、CD4~+、CD8~+与非感染组之间比较,差异无统计学意义(P0.05)。结论CD3~+、CD4~+、CD8~+百分比可以作为诊断早发型新生儿败血症的指标。     

活动性肺结核与潜在感染者CD8+、CD4+记忆T细胞表达水平的比较

[目的]探讨CD8+、CD4+记忆T细胞表达水平在活动性肺结核与潜伏感染者中的表达差异及其意义.[方法]本院确诊的25例活动性肺结核患者(A组)、25例肺结核潜伏感染者(B组)、30例健康人群作为健康对照组(C组),检测各组外周血CD4+记忆T细胞、CD8+记忆T细胞的表达水平,并探讨其与高分辨率CT评分(HRCT)的相关性.[结果]B组的CD4+ Tcm、CD8+ Tcm水平显著高于A组、C组(P＜0.05),A组的CD4+ Tcm、CD8+ Tcm水平显著的高于C组(P＜0.05).B组、A组的CD4+Tem、CD8+Tem水平显著的低于C组(P＜0.05),B组、A组的CD4+ Tem、CD8+ Tem水平相比差异无显著性(P＞0.05).A组患者CD4+ Tcm、CD8+ Tcm水平与HRCT评分呈显著的负相关关系(P＜0.05);A组患者CD4+Tem、CD8+ Tem水平与HRCT评分无显著的相关系(P＞0.05).[结论]CD4+、CD8+中心记忆性T细胞在活动性肺结核与潜伏感染者中水平差异显著,并且与肺结核患者病例损伤程度有关.     

Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro

Using a novel system that supports positive selection in vitro, we have investigated the cellular requirements for this process by testing the ability of individual thymic and nonthymic stromal cell types to support the maturation of CD4+CD8+ thymocytes into CD4+ or CD8+ T cells. We show that thymic cortical epithelial cells are unique in their ability to mediate this maturation, and suggest that in addition to TCR ligation, these cells supply specific signals for positive selection. Moreover, by demonstrating positive selection on ECDI (1- ethyl-3-[3'dimethyl-aminopropyl]-carbodiimide)-fixed epithelial cells in this system, we provide direct evidence that the provision of these signals involves interactions with epithelial cell surface molecules rather than the release of soluble factors.     

CD4^＋CD25^＋调节性T细胞在ITP患者发病机制中的作用

目的研究慢性特发性血小板减少性紫癜（CITP）患者外周血CD4^＋CD25^＋调节性T细胞数量及Foxp3基因的表达水平,探讨它们在CITP发病机制中的作用。方法分别收集40例CITP患者及健康对照组外周抗凝静脉血,分离纯化T淋巴细胞。利用PE标记的抗-CD4单抗,FITC标记的抗-CD25单抗,作双色流式细胞术,分析CITP患者外周血CD4^＋CD25^＋调节性T细胞百分率,采用RT-PCR法检测T细胞Foxp3 mRNA表达水平。结果CITP患者外周血CD4^＋T、CD4^＋CD25^＋T细胞百分率及T细胞Foxp3 mRNA水平均低于对照组（P〈0.01）,且CD4^＋CD25^＋T细胞百分率与Foxp3 mRNA水平呈正相关。结论CITP患者细胞免疫功能失调,CD4^＋CD25^＋调节性T细胞的数量与功能发生改变,T淋巴细胞耐受机制的破坏可能与CITP的发病机制有关。     

CD8^＋CD28^-T细胞在原发性肝癌患者外周血的表达及意义

目的探讨原发性肝癌（PHc）患者外周血中CD8^＋CD28^-T细胞在PHC细胞免疫中所发挥的作用。方法采用流式细胞技术对38例PHC患者及40例健康对照人群外周血中的CD8^＋T细胞表面CD28分子的表达进行检测。结果38例PHC患者与40例正常对照组外周血测定结果比较发现：PHC患者外周血中CD8^＋测定值较正常对照组显著增高;CD8^＋CD28^-较正常对照组明显增高;而PHC患者外周血中CD8^＋CD28^＋与正常组比较明显下降,统计学差异均具有显著性。结论CD8^＋CD28^-T细胞在PHC患者外周血中增生活跃,提示高表达的CD8^＋CD28^-T细胞在肝癌免疫应答中具有重要意义。     

RA患者外周血CD4/CD8 T细胞比值及CD4+ CD25+ T细胞的检测及其临床意义

目的 观察活动期类风湿性关节炎（RA）患者外周血T细胞CD4／CD8比值及CD4^＋ CD25^＋T细胞（Tn细胞）的数量,探讨其在RA诊治中的价值。方法 用流式细胞术检测CD4^＋ CD25^＋T细胞及TR细胞的数量。结果 49例急性期RA患者的CD4／CD8比值和TR细胞数量显著高于正常人群组。28例RA患者治疗后的TR细胞数量显著高于治疗前,而CD4／CD8比值的变化无统计学意义。结论 RA治疗后症状改善患者,TR细胞数量明显上升,RA的发病和发展与该细胞数量密切相关。     

Quantitative analysis of CD6, CD4 and CD8 cell surface molecules compared to the absolute numbers of CD6+, CD4+ and CD8+ T-cells in peripheral blood in patients with HIV-infection.

T lymphocyte subsets were determined on blood samples from 16 HIV-seropositive patients with manifest AIDS (CDC IV), 24 HIV-seropositive patients with lymphadenopathy syndrome (LAS, CDC III), 16 HIV-seropositive clinical healthy persons (CDC II) and 11 HIV-seronegative homosexuals as control group. Absolute numbers of T-cells (CD6+), T-helper/inducer-cells (CD4+) and T-suppressor/cytotoxic-cells (CD8+), obtained by immunofluorescence staining were compared with the absolute amount of subset specific surface molecules, obtained by a T-cell-ELISA. With both, indirect immunofluorescence technique and ELISA technique a highly significant decrease of the absolute numbers of CD4+ cells and the absolute amount of CD4 surface molecules, respectively, was found in asymptomatic HIV-infection, LAS and in manifest AIDS. In all HIV-seropositive groups the relative decrease of CD4 surface molecules was significantly greater than the decline of CD4+ cells. This phenomenon however was not seen in HIV-seronegative homosexuals. The absolute number of CD6+ cells and the amount of CD6 surface molecules were found significantly lowered in AIDS compared to HIV-seronegative homosexuals. No significant changes were found for CD8+ cell numbers and CD8 surface molecule in the progression of the HIV-infection.     

CD3~+、CD4~+、CD8~+T细胞水平在胃癌患者外周血中的变化

目的探讨T淋巴细胞亚群CD3+、CD4+、CD8+和CD4+/CD8+比值水平在胃癌患者外周血中的表达及意义。方法采用血液分析仪技术,检测40例胃癌手术前患者(按临床分期为Ⅰ～Ⅱ级组18例,Ⅲ～Ⅳ级组22例)及25名正常对照组外周血CD3+、CD4+、CD8+细胞和CD4+/CD8+比值T细胞水平。结果胃癌患者外周血CD3+、CD4+细胞、CD4+/CD8+比值分别为0.78%±0.35%、0.37%±0.21%、0.61%±0.42%,明显低于正常对照组(P<0.05);胃癌Ⅲ～Ⅳ级组CD3+、CD4+细胞CD4+/CD8+比值分别为0.42%±0.27%、0.21%±0.17%、0.29%±0.25%,明显低于Ⅰ～Ⅱ级组(0.95%±0.35%、0.45%±0.19%、0.76%±0.30%,P<0.05)。结论检测胃癌患者手术前外周血CD3+、CD4+、CD8+细胞和CD4+/CD8+比值T细胞水平,可以初步评估胃癌患者机体免疫状态,也为肿瘤的免疫增强治疗提供了理论依据。