Platt Ratree, Sponseller Brett A, Chiang Yu-Wei, Roth James A
Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, United States.
Vet Immunol Immunopathol. 2010 Jun 15;135(3-4):275-81. doi: 10.1016/j.vetimm.2009.12.010. Epub 2010 Jan 7.
The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both tests were averaged before statistical analysis. Following EHV-1 stimulation, the MHC II expression index (EI) increased significantly in CD2+CD4+CD8- and CD2+CD4-CD8+ subsets of the infected group. At 4 days after incubation, the non-antigen stimulated CD2+CD4-CD8- subset of the infected group expressed a high percentage (61.1%) of MHC II. When stimulated with EHV-1, the MHC II expression declined significantly but remained at a relatively high percentage (34.4%). The IFN-gamma EI was significantly higher in infected foals in all major T cell subsets (CD2+) while only the CD2+CD4+CD8- subset showed a significant increase in intracellular IL-4 EI. The FCM results showed strong specific CMI responses to EHV-1 by all three tested parameters compared to the control group (p<0.01). The high MHC II expression in the CD2+CD4-CD8- subset suggests that this T cell subset may represent a gammadelta TCR repertoire and thereby plays an important role as antigen presenting cells in the horse, as reported in other species. Being able to simultaneously quantify the frequency of specific lymphocyte subsets and the expression of cytokines that characterize activation of lymphocytes and protective CMI by multi-parameter FCM enables evaluation of subset-specific CMI responses to EHV-1 infection. This system can be applied to measure CMI responses to other equine vaccines and pathogens.
通过多参数流式细胞术（FCM）评估马驹对强毒马疱疹病毒1型（EHV-1）感染的细胞介导免疫（CMI）反应。10匹7至8月龄的EHV-1血清阴性马驹经鼻内感染强毒EHV-1，10匹未感染的马驹作为对照。在感染后6周和7周采集血样，检测对活的异源EHV-1回忆抗原的特异性CMI反应。激活标志物包括主要组织相容性复合体II类（MHC II）、细胞内干扰素γ（IFN-γ）和白细胞介素4（IL-4）。在进行统计分析之前，将两项检测结果进行平均。EHV-1刺激后，感染组的CD2+CD4+CD8-和CD2+CD4-CD8+亚群中MHC II表达指数（EI）显著升高。孵育4天后，感染组未受抗原刺激的CD2+CD4-CD8-亚群表达高百分比（61.1%）的MHC II。用EHV-1刺激时，MHC II表达显著下降，但仍保持在相对较高的百分比（34.4%）。在所有主要T细胞亚群（CD2+）中，感染马驹的IFN-γ EI显著更高，而只有CD2+CD4+CD8-亚群的细胞内IL-4 EI显著增加。FCM结果显示，与对照组相比，所有三个检测参数均显示出对EHV-1的强烈特异性CMI反应（p<0.01）。CD2+CD4-CD8-亚群中高MHC II表达表明，该T细胞亚群可能代表γδTCR库，从而在马中作为抗原呈递细胞发挥重要作用，正如在其他物种中所报道的那样。通过多参数FCM能够同时定量特定淋巴细胞亚群的频率以及表征淋巴细胞激活和保护性CMI的细胞因子表达，从而能够评估对EHV-1感染的亚群特异性CMI反应。该系统可用于测量对其他马用疫苗和病原体的CMI反应。
