Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination
Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination
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DOI：
10.1093/jnci/92.16.1336
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发表时间：
2000-08-16
期刊：
影响因子：
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通讯作者：
Rosenberg, SA
中科院分区：
文献类型：
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作者：
Kammula, US;Marincola, FM;Rosenberg, SA
背景：监测癌症患者对表位特异性疫苗接种的免疫应答对于疫苗开发非常重要。传统的方法是比较疫苗接种前后外周血单个核细胞（PBMC）对抗原表位的体外致敏性，这种方法耗时长，并且只能定性评估应答。我们使用快速、定量、实时聚合酶链反应（PCR）测定来直接测量患者PBMC对疫苗抗原表位的免疫反应性。在用g209- 2 M（衍生自黑素瘤蛋白gp 100的肽）（n = 24）或ESg 209 - 2 M（该肽的修饰形式）（n = 20）进行两轮疫苗接种之前和之后，从黑素瘤患者获得PBMC，通过测定干扰素γ检测PBMC的免疫反应性用表位体外致敏后的IFN γ蛋白释放或通过实时PCR用于IFN γ信使RNA表达，认为接种后PBMC中IFN γ蛋白释放增加2倍或以上或IFN γ转录物积累增加1.5倍或以上是特异性应答的证据。将结果分为阳性和阴性后，采用斯皮尔曼相关系数检验两种方法之间的相关性。所有统计学检验均为双侧检验。结果：两种方法获得的结果强相关（斯皮尔曼rho = 0.72; P = 0.0006）。在体外致敏后，g209- 2 M和Esg 209 - 2 M肽在PBMC中产生相似百分比的疫苗特异性反应性（分别为63%和65%的患者; Fisher精确检验P = 0.6用于两组的比较）。PCR方法可以在一部分患者中检测到疫苗特异性反应（分别为38%和35%的患者; Fisher精确检验P = 0.7用于两组的比较）。结论：疫苗接种诱导循环抗肿瘤淋巴细胞，虽然在低频率，能够直接与肿瘤抗原反应。接种疫苗的个体的PBMC可以在不需要长时间体外操作的直接测定中对疫苗特异性刺激作出应答。
Background: Monitoring the immune response to epitope-specific vaccination in cancer patients is important for vaccine development. The traditional method, in which the in vitro sensitization of peripheral blood mononuclear cells (PBMCs) with epitope is compared before and after vaccination, is time-consuming and allows only a qualitative assessment of the response, We used a rapid, quantitative, realtime polymerase chain reaction (PCR) assay to directly measure the immune reactivity of patients' PBMCs to the vaccine epitope, Methods: PBMCs were obtained from melanoma patients before and after two rounds of vaccination with either g209-2M, a peptide derived from melanoma protein gp100 (n = 24), or ESg209-2M, a modified version of this peptide (n = 20), PBMCs were tested for immune reactivity by assaying interferon gamma (IFN gamma) protein release after in vitro sensitization with the epitope or for IFN gamma messenger RNA expression by real-time PCR, A twofold or more increase in IFN gamma protein release or a 1.5-fold or more increase in IFN gamma transcript accumulation in PBMCs after vaccination was considered to be evidence of a specific response. Correlation between the two methods was tested by use of the Spearman correlation coefficient after the results were ranked as positive or negative. All statistical tests were two-sided. Results: The results obtained with the two methods were strongly correlated (Spearman's rho = 0.72; P = .0006). The g209-2M and Esg209-2M peptides resulted in similar percentages of vaccine-specific reactivity in PBMCs after in vitro sensitization (63% and 65% of patients, respectively; Fisher's exact test P = .6 for comparison of the two groups). The PCR method could detect vaccine-specific reactivity in a subset of patients (38% and 35% of patients, respectively; Fisher's exact test P = .7 for comparison of the two groups). Conclusion: Vaccination induces circulating antitumor lymphocytes, albeit in low frequencies, capable of directly reacting with tumor antigen. PBMCs of vaccinated individuals can respond to a vaccine-specific stimulus in a direct assay that does not require prolonged irt vitro manipulations.