Walravens K, Wellemans V, Weynants V, Boelaert F, deBergeyck V, Letesson J-J, Huygen K, Godfroid J
Veterinary and Agrochemical Research Center, Groeselenberg 99, B-1180, Brussels, Belgium.
Vet Immunol Immunopathol. 2002 Jan 1;84(1-2):29-41. doi: 10.1016/s0165-2427(01)00398-1.
Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG. In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.
选用3头10月龄的牛，通过气管内途径用10(6)cfu牛分枝杆菌的一株田间菌株进行感染。定期采集血样，用于抗原刺激后体外干扰素-γ产生情况的检测。感染动物的外周血细胞在感染后3 - 4周对牛分枝杆菌粗抗原（活的和热灭活的卡介苗以及蛋白纯化衍生物（PPD））产生干扰素-γ。对牛PPD与禽PPD的反应比值表明对牛分枝杆菌抗原有特异性致敏。感染后3个月（PI），宰杀动物，从结核病变中培养出牛分枝杆菌。在不同时间点，通过流式细胞术检测外周血中产生特异性牛分枝杆菌干扰素-γ的CD4 +、CD8 +和WC1 + T细胞的频率。细胞内干扰素-γ和牛细胞表面分子的双色免疫荧光染色显示，在用PPD、活的或灭活的卡介苗刺激后，CD4 +和CD8 + T细胞而非WC1 + T细胞产生干扰素-γ。在分析的2只动物中，感染后第5周和第9周之间，循环中产生干扰素-γ的CD8 +细胞的相对百分比下降。分泌干扰素-γ的CD4 +细胞未观察到相同的变化。对感染动物的T细胞进行磁性阳性分选显示，CD4 + T细胞仅在存在抗原呈递细胞（APC）时产生特异性干扰素-γ。阳性分选的CD8 + T细胞仅在存在重组人白细胞介素-2和APC时分泌干扰素-γ。体外去除CD4 + T细胞而非CD8 +或WC1 + T细胞导致特异性干扰素-γ产生的消除，表明该细胞群体在特异性干扰素-γ产生中起关键作用。
