Wyckoff J H, Howland J L, Confer A W
Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078-0353.
Vet Immunol Immunopathol. 1993 Feb;36(1):45-64. doi: 10.1016/0165-2427(93)90005-o.
Three Brucella abortus antigen preparations were tested for stimulatory activity with immune bovine T-lymphocyte cell lines in vitro. A total of 32 polyclonal T-lymphocyte cell lines were derived from two steers each from four immunization groups: (1) B. abortus Strain 19 (S19) alone, (2) heat-killed B. abortus whole bacterial cells (HKC) alone, (3) S19 with recombinant human interleukin 2 (rHuIL-2), (4) HKC with rHuIL-2. Peripheral blood mononuclear cells were isolated at 2 and 9 weeks post immunization and cultured in vitro with either HKC antigen or B. abortus soluble antigen (BASA) with recombinant bovine interleukin 2 (rBoIL-2) to initiate four cell lines per steer. Sixteen of the resulting T-lymphocyte cell lines (from the S19 and S19+IL-2 groups) were tested through indirect immunofluorescence for expression of cell surface markers CD2, CD4, CD6, CD8, major histocompatibility complex (MHC) Class II molecules and a marker expressed on a subset of helper T-lymphocytes (Th) as well as sIgM, CD1 and a MHC Class II+ monocyte/macrophage marker. The T-lymphocyte cell lines were used to evaluate antigen-induced lymphoproliferative (LP) responses in a titration assay with HKC, BASA and gamma-irradiated B. abortus (gamma BA) antigens. The results indicate that most of the cells in many of the cell lines were typical activated T-lymphocytes as determined by surface marker expression and included cells positive for all T-lymphocyte markers tested. The cell lines contained no B-lymphocytes or mononuclear phagocytes. However, two cell lines contained significant populations (> 80%) of CD2-, CD4-, CD6-, CD8- cells that were both responsive to exogenous rBoIL-2 and were capable of exhibiting antigen-induced LP responses. In 22 of the 32 cell lines tested, gamma BA was superior to HKC at nearly every concentration tested in stimulating LP responses. This observation was independent of the immunization used to prime the T-lymphocytes in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins with relative molecular masses common to all three antigen preparations as well as significant (P < 0.05) quantitative and qualitative differences in individual proteins between HKC and gamma BA. Together, the results suggest gamma BA may provide an in vitro antigenic stimulus which is deficient in HKC.
对三种流产布鲁氏菌抗原制剂进行了体外刺激免疫牛T淋巴细胞系活性的测试。总共32个多克隆T淋巴细胞系来自四个免疫组，每组两头公牛：（1）单独的流产布鲁氏菌19株（S19）；（2）单独的热灭活流产布鲁氏菌全菌细胞（HKC）；（3）S19与重组人白细胞介素2（rHuIL-2）；（4）HKC与rHuIL-2。在免疫后2周和9周分离外周血单核细胞，并在体外与HKC抗原或流产布鲁氏菌可溶性抗原（BASA）及重组牛白细胞介素2（rBoIL-2）一起培养，以启动每个公牛的四个细胞系。通过间接免疫荧光对产生的16个T淋巴细胞系（来自S19和S19+IL-2组）进行测试，检测细胞表面标志物CD2、CD4、CD6、CD8、主要组织相容性复合体（MHC）II类分子以及辅助性T淋巴细胞（Th）亚群上表达的一种标志物以及sIgM、CD1和MHC II+单核细胞/巨噬细胞标志物的表达情况。这些T淋巴细胞系用于在HKC、BASA和γ射线照射的流产布鲁氏菌（γBA）抗原的滴定试验中评估抗原诱导的淋巴细胞增殖（LP）反应。结果表明，通过表面标志物表达确定，许多细胞系中的大多数细胞是典型的活化T淋巴细胞，包括所有测试的T淋巴细胞标志物呈阳性的细胞。这些细胞系中不含B淋巴细胞或单核吞噬细胞。然而，两个细胞系含有大量（>80%）的CD2-、CD4-、CD6-、CD8-细胞，这些细胞对外源rBoIL-2有反应，并且能够表现出抗原诱导的LP反应。在测试的32个细胞系中的22个中，在几乎每个测试浓度下，γBA在刺激LP反应方面都优于HKC。这一观察结果与体内启动T淋巴细胞所用的免疫方式无关。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，所有三种抗原制剂都有相对分子质量相同的蛋白质，并且HKC和γBA之间的个别蛋白质在定量和定性上存在显著（P<0.05）差异。综合来看，结果表明γBA可能提供了一种HKC中缺乏的体外抗原刺激。
