Transcriptional signatures of Itk-deficiency using CD3+, CD4+ and CD8+ T-cells. Mus musculus

The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk. Overall design: CD3+ CD4+ and CD8+ T-cells from pooled suspensions of spleen and lymph nodes of Wt and Itk knockout mice on C57BL/6 background were isolated after negative depletion. Unstimulated as well as stimulated T-cells were studied. Stimulations were done with anti-CD3 (1 mg/ml) for 24 hrs. For the CD4+ T-cells we collected triplicates from the Itk knockout mice and duplicates from the Wt group. For the CD8+ T-cells, we got duplicates from Itk knockout , while we obtained a single sample from Wt owing to the low cell yield for resting Wt CD8+ T-cells. After CD3-stimulation we got a single sample from the CD8+ subset of both Wt and Itk knockout, while for the CD4+ subsets we collected duplicates.

Tec家族激酶Itk（Tec-family kinase Itk）在T细胞活化与功能调控过程中发挥关键作用，同时亦可调控常规T细胞与先天样T细胞的发育分化。本研究利用Affymetrix基因芯片（Affymetrix microarrays）对Itk缺陷型CD3阳性（CD3+）T细胞（包含CD4阳性（CD4+）与CD8阳性（CD8+）亚群）的转录组进行了系统表征。相较于野生型（Wt）CD3+ T细胞，Itk基因敲除（Itk-/-）CD3+ T细胞的最大转录差异出现在未刺激状态下，例如杀伤细胞凝集素样受体相关基因的表达变化。与单纯抗CD3刺激相比，抗CD3/CD28共刺激处理显著降低了差异表达转录本的数量，提示CD28共刺激通路的激活主要不依赖于Itk。CD4+与CD8+ T细胞亚群的转录特征显示，其差异表达基因的数量多于总CD3+ T细胞群体。环孢素（Cyclosporin, CsA）处理对转录调控的影响强于Itk缺陷，这表明T细胞受体（TCR, T cell receptor）介导的钙调神经磷酸酶/NFAT（calcineurin/NFAT）激活通路中，仅有部分环节依赖于Itk。对同时受Itk缺陷与CsA处理共同调控的转录本集合进行NFAT结合位点的生物信息学分析，并结合染色质免疫沉淀（chromatin-immunoprecipitation）实验，结果证实NFATc1可结合Bub1、IL7R、Ctla2a、Ctla2b及Schlafen1基因的调控区域。最后，为全面鉴定Tec家族激酶共同调控的转录本，本研究将Itk缺陷型T细胞的表达谱与Btk缺陷型B细胞的表达谱进行比对，筛选得到二者共有的差异表达转录本集合。综上，本研究系统阐明了Itk缺失条件下全球转录组的整体变化特征。

实验设计概况：从C57BL/6背景的野生型（Wt）与Itk基因敲除小鼠的脾脏及淋巴结混合悬液中，通过阴性分选法分离得到CD3+ CD4+与CD8+ T细胞。本研究同时分析了未刺激与经抗CD3刺激处理的T细胞，刺激处理采用1 mg/ml抗CD3抗体孵育24小时。对于CD4+ T细胞亚群，Itk基因敲除组设置3次生物学重复，野生型组设置2次生物学重复；对于CD8+ T细胞亚群，Itk基因敲除组获取2次生物学重复，而野生型组因静息态CD8+ T细胞的细胞得率较低，仅获取1次单样本。经抗CD3刺激后，野生型与Itk基因敲除小鼠的CD8+ T细胞亚群均仅获取1次单样本，而CD4+ T细胞亚群则设置2次生物学重复。