McAb4F8,McAb1F11与囊尾蚴抗原作用免疫印渍分析

将猪囊尾蚴囊液抗原（ＣＣＦＡ）和脑囊虫病病人脑脊液（ＰＣＳＦ）进行ＳＤＳ－ＰＡＧＥ电泳分析，发现在ＣＣＦＡ中有１４条蛋白带，范围在２８～７６ｋＤａ。脑囊虫病病人ＣＳＦ中有６条蛋白整，范围在３３～８３ｋＤａ。用ＣＣＦＡ和ＰＣＳＦ与ＭｃＡｂ４Ｆ     

SDS－PAGE酶联免疫印渍实验对人、猪蛔虫抗原特性的分析

以ＳＤＳ－ＰＡＧＥ方法对人蛔虫和猪蛔虫成虫可溶性抗原分析发现，在分子量１４ｋＤ～２３０ｋＤ之间两种抗原有许多相同蛋白带，未见明显的特异性蛋白带存在。应用ＳＤＳ－ＰＡＧＥ酶联免疫印渍方法发现以该两种抗原制备的兔抗血清均能从两种抗原中辨认出多个蛋白带。猪蛔虫成虫可溶性抗原中分子量２１．７ｋＤ和３４ｋＤ蛋白带能被部分兔抗血清所识别，而人蛔虫成虫可溶性抗原未出现这２条反应带。以人蛔虫感染者血清进行酶联免疫印渍也发现人蛔虫、猪蛔虫成虫可溶性抗原相似。两抗原中许多蛋白带可被病人血清所识别，其主要蛋白带为６６ｋＤ，提示蛔虫病人血清中含有大量抗体。另外，其中一份病人血清能从猪蛔虫成虫可溶性抗原中识别出４８．５ｋＤ蛋白带，而在人蛔虫抗原中未发现有该蛋白带。     

细胞毒性T淋巴细胞相关抗原4基因微卫星多态性与炎症性肠病的关系

目的探讨细胞毒性T淋巴细胞相关抗原4（CTLA-4）基因微卫星多态性与浙江省炎症性肠病（IBD）患者的相关性。方法对118例无血缘关系的IBD患者（99例溃疡性结肠炎，19例克罗恩病）以及140例正常对照者，采用特异性等位基因PCR方法，检测CTLA-4外显子4的3′非翻译区包含（AT）。重复序列的等位基因。扩增产物用12％非变性聚丙烯酰胺凝胶电泳，硝酸银染色。结果CTLA-4微卫星共有20种等位基因。与正常对照组比较，122bp等位基因频率在溃疡性结肠炎患者（P=0.0001／Pc=0．0025，OR=11.393，95％CI：2．574～50．429）和克罗恩病患者（P=0．0003／Pc=0.0050，OR=21.061，95％CI：3，927～112．94）中均显著增高。结论CTLA-4基因微卫星多态性与浙江省IBD患者显著相关。     

Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti–PD-1 therapy may primarily act at the tumor site.

Immune checkpoint–targeting therapies, in particular those targeting the programmed cell death protein 1/ligand 1 (PD-1/PD-L1) axis, now form the standard of care for advanced melanoma (1) and a number of other solid cancers including non–small cell lung cancer (NSCLC) (2), renal cell carcinoma (3), and urothelial carcinoma (4). Despite the widespread clinical use of PD-1/PD-L1 blocking agents, the mechanism by which these therapies enhance immune-mediated tumor control remains incompletely understood. Early work addressing the mechanism of action of PD-1 blockade showed increased numbers of intratumoral proliferating (Ki-67⁺) CD8 T cells and T cell receptor (TCR) clones after treatment (5). These findings provide evidence for a boosting effect on tumor-infiltrating CD8 T cells. In line with these findings, PD-L1 expression on tumor cells has been shown to have predictive value for therapy outcome (5–7) and influence the activity of PD-1 blockade in at least some mouse models (8, 9). Other studies, however, have suggested that PD-1 blockade may also exert its effect through activation of circulating tumor-reactive CD8 T cell responses. First, studies in mouse models of chronic viral infection have shown the recruitment of CXCR5⁺ Tim-3⁻ CD8 T cells from the white pulp of the spleen upon PD-1 blockade (10, 11). Second, data obtained using mouse tumor models demonstrated that the proliferative response to anti–PD-1 therapy is dependent on CD28-mediated costimulation (12), and these findings are in line with a mechanistic study showing that PD-1 signaling inhibits T cell functionality through attenuation of CD28 costimulation (13). Collectively, the latter findings have been interpreted as PD-1 blockade potentially having a role in inducing proliferation of the tumor-reactive CD8 T cell pool. Recent data from clinical studies in patients provide evidence supporting such a hypothesis. First, NSCLC and melanoma patients treated with PD-1 blockade showed an increase in proliferating (Ki-67⁺) CD8 T cell subsets (14–17). Second, PD-1 blockade was shown to result in clonal replacement of tumor-infiltrating CD8 T cells in patients with squamous cell carcinoma (18). In contrast, however, an analysis of peripheral blood from melanoma patients showed no consistent increase in TCR diversity after treatment (19). Although the current knowledge suggests that PD-1 blockade may alter the circulating tumor-reactive CD8 T cell compartment, direct evidence for such a hypothesis is currently lacking.Importantly, most studies that have addressed the impact of PD-1 blockade on CD8 T cells focused the analyses on bulk CD8 T cells without assessing the T cell specificity. However, work from Schreiber and colleagues has shown that “bystander” CD8 T cells respond differentially to checkpoint-targeting therapies compared to tumor-reactive CD8 T cells in their mouse model (20). These findings demonstrate the importance of dissecting the mechanism of action of checkpoint-targeting therapies on the tumor-reactive CD8 T cell response rather than the bulk CD8 T cell compartment. In this study, we assessed whether PD-1 blockade can increase the magnitude of preexisting melanoma-reactive CD8 T cells (boosting) and/or lead to newly detectable tumor-reactive CD8 T cell responses (broadening) in peripheral blood of melanoma patients (Fig. 1A).Open in a separate windowFig. 1.Hypothesis and kinetics of melanoma-reactive CD8 T cell responses during anti–PD-1 therapy. (A) Potential mechanisms of anti–PD-1 therapy include expansion of preexisting tumor-reactive CD8 T cells (boosting) and induction of novel, tumor-reactive CD8 T cell responses (broadening). (B) Overview of the HLA-A*02:01–restricted epitope panel. A total of 71 shared melanoma-associated epitopes were included to analyze the tumor-reactive CD8 T cell responses. Viral epitopes served as positive control for the generation of pMHC multimers. Detailed information about the epitopes is provided in SI Appendix, Table S1. (C) Representative flow cytometry plots of melanoma-reactive CD8 T cell responses (blue, located in the diagonal of the plot because of the dual coding strategy) before and during anti–PD-1 therapy. The magnitude of melanoma-reactive CD8 T cell responses (blue, upper right corner) represents the percentage of total CD8 T cells (gray). A representative example of the full gating strategy is provided in SI Appendix, Fig. S1. (D) Kinetics of melanoma-reactive CD8 T cell responses detected in melanoma patients (n = 5) following anti–PD-1 therapy. A ≥twofold increase (on-therapy versus pretherapy) in magnitude is indicated (*).     

Detection and quantification of HIV-1 specific CD4 helper and CD8 cytotoxic cells: their role in HIV-1-infected individuals and vaccine recipients

There is a strong link between virus specific CD8 T-cell function and the efficiency of regulatory CD4 helper T cells. Controlling viraemia in HIV-1-infected individuals requires the maintenance of strong CD4 and CD8 T-cell responses. These responses should be elicited by prophylactic vaccination and by postexposure immunotherapy. This review will examine the methods that are available for the detection and quantification of HIV-1 specific CD4 and CD8 T-cell responses. We will also discuss the methods that should be used to identify these responses in HIV-1-infected individuals, seropositive recipients of immunotherapy and seronegative vaccinees. Finally, we will give examples of how responses observed in vitro relate to those known to occur in vivo .     

Relationship between the ratios of CD4/CD8 T-lymphocytes in the bronchoalveolar lavage fluid and lymph nodes in patients with sarcoidosis

BackgroundEvaluating the ratio of CD4/CD8 T-lymphocytes in the bronchoalveolar lavage fluid (BALF) is important for understanding the clinical and pathological conditions of patients with sarcoidosis. However, few studies have thus far demonstrated the usefulness of evaluating the relationship between the ratios of CD4/CD8 T-lymphocytes in the mediastinal lymph nodes and BALF. This study aimed to investigate and identify the relationships between CD4/CD8 T-lymphocyte ratio in the mediastinal lymph nodes and BALF in patients with sarcoidosis.MethodsThirty-three consecutive patients with sarcoidosis with enlarged mediastinal and/or hilar lymphadenopathy were enrolled in the study, and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and bronchoalveolar lavage (BAL) were simultaneously performed. The CD4/CD8 T-lymphocyte ratios in the mediastinal lymph nodes and BALF were evaluated using immunohistochemistry and flow cytometry, respectively.ResultsThe interobserver variability in the CD4/CD8 ratio in the mediastinal lymph nodes as determined by immunostaining was low, and the pathological and cytological profiles of T-lymphocytes in the mediastinal and/or hilar lymph nodes and BALF were correlated in patients with sarcoidosis. Additionally, the CD4/CD8 T-lymphocyte ratios in BALF were significantly higher than those in the mediastinal lymph nodes. Importantly, non-caseating granulomas were detected at a high rate by using EBUS-TBNA.ConclusionsPerforming EBUS-TBNA in patients with sarcoidosis allows correct diagnosis as well as the estimation of the ratio of CD4/CD8 T-lymphocytes in BALF.     

肺结核患者外周血CD_4~+、CD_8~+T细胞Blimp-1表达下降

目的研究活动性肺结核患者外周血单个核细胞(PBMCs)Blimp-1的表达及临床意义。方法采集31例活动期肺结核患者和45位健康对照组外周血,纯化PBMCs,用结核分枝杆菌ESAT-6和CFP-10混合性抗原肽库刺激,通过细胞表面标记和细胞内细胞因子染色技术,采用流式技术检测CD+4、CD+8T细胞Blimp-1的表达。结果与对照组比较,肺结核患者PBMCs中的CD+4、CD+8T细胞亚群分布出现显著性下降,且肺结核患者CD+4T细胞中Blimp-1的表达比例(%)下降(肺结核组89.5%(83.8%,95.7%),对照组94.5%(89.8%,98.7%),P0.05),且CD+4、CD+8T细胞中Blimp-1的表达量(平均荧光强度)也显著性下降(CD+4T细胞:肺结核组9.28(7.5,18.9),对照组15.4(11,25.4),P0.05);CD+8T细胞:肺结核组9.01(6.08,14.7),对照组14.2(9.53,23.1),P0.05)。结论活动期肺结核CD+4、CD+8T细胞群内Blimp-1的表达下降可能会使效应性和调节性T细胞的分化出现异常。Blimp-1可能参与结核病的疾病进程,这为研究结核病的诊断和治疗提供了线索。     

1型糖尿病患者外周血中CD4^＋CD25^＋与CD8^＋CD28^-调节性T细胞水平的研究

流式细胞仪检测1型糖尿病（T1DM）患者外周血CD4^＋CD25^＋与CD8^＋CD28^-调节性T细胞的水平,发现其外周血CD4^＋CD25^＋T淋巴细胞水平[（2．02±0．43）％]显著低于2型糖尿病（T2DM）组[（6．79±1．75）％]和健康对照（NC）组[（7．84±1．45）％],而CD8^＋CD28^-调节性T细胞水平三组间无差异。     

Effects of low-dosage simvastatin on rheumatoid arthritis through reduction of Th1/Th2 and CD4/CD8 ratios

The objective of this study was to assess both the anti-inflammatory and immunomodulatory effects of low-dosage simvastatin on rheumatoid arthritis (RA). In each patient, simvastatin at 10 mg/day was administered for 12 weeks. The other treatments were unchanged at least 3 months before simvastatin administration to the end of the study. Patients were assessed for the improvement in clinical, laboratory, and immunological parameters of RA and for adverse events. Twenty-four patients with RA were enrolled. Clinical symptoms, including patient's assessment of pain and disease activity on visual analog scale (VAS), the swollen joint and tender joint counts, and handgrip strength significantly improved. Physician's assessment of disease activity on VAS, a period of morning stiffness and modified health assessment questionnaire showed a tendency of improving after administration of low-dosage of simvastatin. Of special interest was that the median levels of erythrocytes sedimentation rate, C-reactive protein, and rheumatoid factor were significantly decreased from 54.0 mm/h to 45.5 mm/h, from 1.50 mg/dl to 0.85 mg/dl, and from 57.0 IU/ml to 28.0 IU/ml, respectively, after administration of simvastatin. ACR20 and ACR50 responses were achieved in 62% and 38%, respectively, of simvastatin-treated patients for 12 weeks. Immunological assessment in peripheral blood revealed that the Th1/Th2 and CD4/CD8 ratios were significantly reduced by simvastatin. No adverse events were reported during simvastatin treatment. Immunomodulation through the alteration of Th1/Th2 and CD4/CD8 ratios may be a pharmacological mechanism in the anti-rheumatic effect of low-dosage simvastatin. Although it is necessary to evaluate the long-term effects of statins, low-dosage statins appear to be good as additional therapeutic agents.