Kovacs Birgit, Maus Marcela V, Riley James L, Derimanov Geo S, Koretzky Gary A, June Carl H, Finkel Terri H
Division of Rheumatology, Department of Pediatrics, Children's Hospital of Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):15006-11. doi: 10.1073/pnas.232058599. Epub 2002 Nov 5.
Lipid rafts are important signaling platforms in T cells. Little is known about their properties in human CD8(+) T cells. We studied polarization of lipid rafts by digital immunofluorescence microscopy in primary human T cells, using beads coated with anti-CD3 and anti-CD28 mAbs (CD3/28 beads). Unlike CD4(+) T cells, CD8(+) T cells did not polarize lipid rafts when stimulated with CD3/28 beads, when the anti-CD28 antibody was substituted with B7.2Ig, or if an anti-CD8 antibody was added to the CD3/28 beads. This phenomenon was also observed in human antigen-specific CD8(+) T cells. On stimulation with CD3/28 beads, the T cell antigen receptor clustered at the cell/bead contact area in both CD4(+) and CD8(+) T cells. Examination of lipid rafts isolated by sucrose density gradient centrifugation revealed the constitutive expression of p(56)Lck in the raft fractions of unstimulated CD8(+) T cells, whereas p(56)Lck was recruited to the raft fraction of CD4(+) T cells only after stimulation with CD3/28 beads. Stimulation with CD3/28 beads induced marked calcium flux, recruitment of PKC-theta and F-actin to the cell/bead contact site, and similar proliferation patterns in CD4(+) and CD8(+) T cells. Thus, polarization of lipid rafts is not essential for early signal transduction events or proliferation of human CD8(+) lymphocytes. It is possible that the lower stringency of CD8(+) T cell activation obviates a requirement for raft polarization.
脂筏是T细胞中重要的信号转导平台。关于它们在人CD8(+) T细胞中的特性，人们了解甚少。我们使用包被抗CD3和抗CD28单克隆抗体的珠子（CD3/28珠子），通过数字免疫荧光显微镜研究了原代人T细胞中脂筏的极化情况。与CD4(+) T细胞不同，当用CD3/28珠子刺激时，当抗CD28抗体被B7.2Ig替代时，或者如果向CD3/28珠子中添加抗CD8抗体时，CD8(+) T细胞不会使脂筏极化。在人抗原特异性CD8(+) T细胞中也观察到了这种现象。在用CD3/28珠子刺激时，T细胞抗原受体在CD4(+)和CD8(+) T细胞的细胞/珠子接触区域聚集。对通过蔗糖密度梯度离心分离的脂筏进行检查发现，未刺激的CD8(+) T细胞的脂筏部分中组成性表达p(56)Lck，而p(56)Lck仅在CD3/28珠子刺激后才被募集到CD4(+) T细胞的脂筏部分。用CD3/28珠子刺激可诱导明显的钙流、PKC-θ和F-肌动蛋白募集到细胞/珠子接触部位，并且在CD4(+)和CD8(+) T细胞中具有相似的增殖模式。因此，脂筏极化对于人CD8(+)淋巴细胞的早期信号转导事件或增殖并非必不可少。有可能CD8(+) T细胞激活的较低严格性消除了对脂筏极化的需求。
