In vivo microarray expression data from WT, Irf4-/- and checkpoint blockade treated Irf4-/- TEa CD4+ T cells. In vivo microarray expression data from WT, Irf4-/- and checkpoint blockade treated Irf4-/- TEa CD4+ T cells

Previously we found that Irf4-deficient T cells were dysfunctional and unable to reject heart allografts and checkpoint blockade treatment could reinvigorate dysfunctional Irf4-deficient T cells and enable them reject heart allografts. But shortly after the reinvigoration those T cells went back to dysfunctional state. TCR-transgenic TEa CD4+ T cells (B6 background) recognize a Balb/c I-Eα allopeptide presented by B6 antigen presenting cells, and were used to assess the effects of immune checkpoint blockades on Irf4-deficient alloreactive T cells. WT, Irf4-/- and checkpoint blockade treated Irf4-/- TEa CD4+ T cells were isolated from Balb/c heart transplanted B6 mice, followed by microarray analysis. To define un-restored gene expressions in Irf4-deficient alloreactive T cells upon immune checkpoint blockades, we compared genes expression level among three groups. We revealed that checkpoint blockade restored the expression levels of the majority of wild-type T cell-expressed genes in Irf4-/- T cells, indicating the reinvigoration of Irf4-/- T cells. The remaining un-restored genes following checkpoint blockade, though minimal in number, may be responsible for the reinvigorated Irf4-/- T cells to become re-dysfunction. Overall design: TCR(Vα2+Vβ6+)CD45.2+CD4+ TEa cells were isolated from splenocytes of WT TEa or Irf4−/− TEa mice by a FACSAria flow cytometer (BD Biosciences). B6.SJL CD45.1+ congenic mice were adoptively transferred with either 5 x 10e6 CD45.2+ WT TEa or 5 x 10e6 CD45.2+ Irf4‒/‒ TEa cells on day -1, and transplanted with Balb/c hearts on day 0. CD45.1+ mice adoptively transferred with either CD45.2+ Irf4‒/‒ TEa cells were i.p. injected with 400 μg Rat IgG, or 200 μg anti-PD-L1 plus 200 μg anti-CTLA-4 (9D9) mAbs on days 0, 3, 5. On day 6, adoptively transferred CD45.2+ TEa cells were sorted from splenocytes of transplant recipients by the FACSAria flow cytometer. Total RNA was extracted from sorted cells with RNeasy mini kit (Qiagen), followed by microarray analysis. Two independent experiments for each group were performed.

此前我们发现，Irf4缺陷型T细胞功能失调，无法排斥心脏同种移植物；而免疫检查点阻断（checkpoint blockade）治疗可重振功能失调的Irf4缺陷型T细胞，使其获得心脏同种移植物排斥能力。但在重振后不久，这些T细胞会再次回归功能失调状态。TCR转基因TEa CD4+ T细胞（B6背景）可识别由B6抗原呈递细胞（antigen presenting cells）呈递的Balb/c来源I-Eα同种变肽，该细胞系被用于评估免疫检查点阻断对Irf4缺陷同种反应性T细胞的作用。我们从接受Balb/c心脏移植的B6小鼠体内分离得到野生型（wild-type，WT）、Irf4缺陷型（Irf4-/-）以及经免疫检查点阻断处理的Irf4缺陷型TEa CD4+ T细胞，随后进行基因芯片分析（microarray analysis）。为明确免疫检查点阻断处理后Irf4缺陷同种反应性T细胞中未被恢复的基因表达情况，我们对三组样本的基因表达水平进行了比较。我们发现，免疫检查点阻断可恢复Irf4缺陷T细胞中绝大多数野生型T细胞表达的基因的表达水平，这表明Irf4缺陷T细胞的功能得到了重振。尽管经免疫检查点阻断处理后剩余的未恢复基因数量极少，但它们可能是重振后的Irf4缺陷T细胞再次出现功能失调的原因。实验整体设计：通过FACSAria流式细胞仪（BD Biosciences）从野生型TEa或Irf4缺陷型TEa小鼠的脾脏细胞中分离得到TCR(Vα2+Vβ6+)CD45.2+CD4+ TEa细胞。在第-1天，向B6.SJL CD45.1+同源基因小鼠过继转移（adoptively transferred）5×10^6个CD45.2+野生型TEa细胞，或5×10^6个CD45.2+ Irf4缺陷型TEa细胞；并于第0天为这些小鼠移植Balb/c来源的心脏。对于过继转移了CD45.2+ Irf4缺陷型TEa细胞的CD45.1+小鼠，分别于第0、3、5天腹腔注射（intraperitoneal，简称i.p.）400 μg大鼠IgG，或200 μg抗PD-L1抗体联合200 μg抗CTLA-4（9D9）单克隆抗体（monoclonal antibodies，mAbs）。在第6天，通过FACSAria流式细胞仪从移植受体小鼠的脾脏细胞中分选得到过继转移的CD45.2+ TEa细胞。使用RNeasy迷你试剂盒（Qiagen）从分选得到的细胞中提取总RNA，随后进行基因芯片分析。每组均设置两次独立重复实验。