Isolating, immunophenotyping and ex vivo stimulation of CD4+ and CD8+ gastric lymphocytes during murine Helicobacter pylori infection
Isolating, immunophenotyping and ex vivo stimulation of CD4+ and CD8+ gastric lymphocytes during murine Helicobacter pylori infection
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DOI：
10.1016/j.jim.2012.07.002
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发表时间：
2012-10-31
影响因子：
2.2
通讯作者：
Moss, Steven F.
中科院分区：
文献类型：
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作者：
Ruiz, Victoria E.;Sachdev, Monisha;Moss, Steven F.
幽门螺杆菌感染与严重的慢性炎症有关，但宿主的免疫反应很少能够清除细菌。已知T辅助细胞1、T辅助细胞17和调节性T细胞等Th细胞源性淋巴细胞在H.幽门螺杆菌感染以及促进进行性胃病理学。这些免疫细胞群在H.幽门感染小鼠感染模型提供了详细研究这些相互作用的机会。流式细胞仪分析提供了极好的淋巴细胞特性，由于其高特异性，灵敏度和潜力，执行多个同时测量。然而，这需要在充分的组织解离后活的富集的单细胞悬浮液，这由于胃组织的异质性而带来挑战。我们已经评估了几种分离技术，并优化了一个协议，以分离和富集淋巴细胞从H。幽门感染的小鼠胃。EDTA/UIT随后胶原酶IV消化成功解离平均每只小鼠1 x 107个细胞。使用Lympholyte M梯度进一步富集，每个胃平均产生4 x 10（6）个CD 45（+）淋巴细胞。分离后，我们比较了CD 3/CD 28、佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）和离子霉素或H. pylori裂解物，并确定CD 3/CD 28有效地诱导IFN γ和IL 17 A的刺激，但损害Foxp 3表达。使用优化的方案，我们观察到在H期间特异性定位于胃室的表达CD 8（+）IFN γ的淋巴细胞增加2倍。幽门感染对H. pylori免疫发病机制仍然被认为是谜，因此这种优化的方案可以帮助描绘进一步的新的免疫细胞靶介导H.幽门螺杆菌诱导的病理学，并确定疫苗开发的免疫相关性。出版社：Elsevier B. V.
Helicobacter pylori infection is associated with severe chronic inflammation, yet the host immune response is rarely able to clear the bacterium. Thymus derived lymphocyte populations such as T helper 1,T helper 17, and T regulatory cells are known to play important roles in the chronicity of H. pylori infection as well as contributing to ongoing gastric pathology. It is yet to be established how these immune cell populations interact in the gastric environment during H. pylori infection. Mouse models of infection offer an opportunity to investigate these interactions in detail. Flow cytometric analysis provides excellent lymphocyte characterization due to its high specificity, sensitivity and potential to perform multiple simultaneous measurements. However, this requires a viable enriched single cell suspension after adequate tissue dissociation, which poses a challenge due to the heterogeneity of gastric tissue. We have evaluated several isolation techniques and have optimized a protocol to isolate and enrich lymphocytes from the H. pylori-infected murine stomach. EDTA/UIT followed by Collagenase IV digestion successfully dissociates an average of 1 x 10(7) cells per mouse. Further enrichment using Lympholyte M gradient yields on average 4 x 10(6) CD45(+) lymphocytes per stomach. Following isolation we compared lymphocyte stimulation by CD3/CD28, phorbol 12-myristate 13-acetate (PMA) and ionomycin or H. pylori lysate and determined that CD3/CD28 effectively induces stimulation of IFN gamma and IL 17A, but impairs Foxp3 expression. Using an optimized protocol we observed a 2-fold increase of CD8(+) IFN gamma-expressing lymphocytes localized specifically to the gastric compartment during H. pylori infection. The mechanisms of H. pylori immunopathogenesis are still considered enigmatic, therefore this optimized protocol can help delineate further novel immune cell targets that mediate H. pylori-induced pathology and identify the correlates of immunity for vaccine development. Published by Elsevier B.V.