Background & Aims

Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4⁺ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.

Methods

We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13−30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4⁺ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4⁺ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.

Results

Circulating and gut-resident CD4⁺ T cells from controls responded to bacteria at frequencies of 40−4000 per million for each bacterial species tested. Microbiota-reactive CD4⁺ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4⁺ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.

Conclusions

In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4⁺ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

中文翻译：

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对肠道微生物群有反应性的循环和组织驻留 CD4+ T 细胞在健康个体中含量丰富，并且在炎症期间功能会发生改变

背景与目标

共生微生物与免疫系统之间的相互作用受到严格调节并维持肠道稳态，但对人类中的这些相互作用知之甚少。我们研究了人类 CD4 ⁺ T 细胞对肠道微生物群的反应。我们测量了循环和肠道组织中对肠道微生物有反应的 T 细胞的丰度，并确定了它们的克隆多样性。我们还评估了它们的功能表型和对肠道常驻细胞群的影响，并研究了慢性肠道炎症患者微生物反应性 T 细胞的变化。

方法

我们收集了来自健康个体（对照组，n = 13-30）和炎症性肠病患者（n = 119；59 例溃疡性结肠炎和 60 例克罗恩病）的外周血单个核细胞和肠道组织样本。我们使用了 2 种独立的检测（CD154 检测和羧基荧光素琥珀酰亚胺酯稀释检测）和 9 种肠道细菌（大肠杆菌、嗜酸乳杆菌、动物双歧杆菌乳亚种、普氏粪杆菌、普通拟杆菌、Roseburia gutis、Ruminococcus obeum、鼠伤寒沙门氏菌和艰难梭菌）来量化、扩展和表征微生物反应性 CD4 ⁺T细胞。我们对扩增的微生物反应性 T 细胞系中的 T 细胞受体 Vβ 基因进行了测序，以确定它们的克隆多样性。我们检查了微生物反应性 CD4 ⁺ T 细胞对肠基质和上皮细胞系的影响。通过流式细胞术和定量聚合酶链反应测量细胞因子、趋化因子和基因表达模式。

结果

对于每个测试的细菌种类，来自对照的循环和肠道驻留 CD4 ⁺ T 细胞对细菌的反应频率为 40-4000/百万。微生物群反应性 CD4 ⁺ T 细胞主要具有记忆表型，存在于外周血单核细胞和肠道组织中，并具有多种 T 细胞受体 Vβ 库。这些细胞功能异质，产生屏障保护细胞因子，并通过白细胞介素 17A、干扰素 γ 和肿瘤坏死因子刺激肠基质和上皮细胞。在炎症性肠病患者中，微生物群反应性 CD4 ⁺与肠道相比，血液中的 T 细胞减少；与来自对照血液或肠道组织的 T 细胞的反应相比，我们检测到的 T 细胞反应具有更高的白细胞介素 17A 产生频率。

结论

在对炎症性肠病患者与对照组的外周血单个核细胞和肠道组织进行分析时，我们发现对肠道细菌的反应是人类 CD4 ⁺ T 细胞库的正常特性，并不一定表明免疫系统之间的相互作用被破坏。细胞和共生微生物群。T 细胞对共生体的反应可能通过产生屏障保护细胞因子和提供大量对病原体作出反应的 T 细胞来支持肠道内稳态。