Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.
Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.
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DOI：
10.1172/jci170191
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发表时间：
2023-07-17
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对肾同种异体移植物活检 (rBx) 进行批量分析，鉴定出与急性细胞排斥 (ACR) 相关的 RNA 转录物；然而，这些细胞缺乏对理解免疫抑制（IS）下排斥如何发生的机制至关重要的细胞背景。我们对使用不同 IS 药物（他克莫司、艾斯卡利单抗和贝拉西普）的 ACR 患者的 rBx 进行了组合单细胞 RNA 转录组和 TCR-α/β 测序。我们发现不同的 CD8+ T 细胞表型（例如，效应细胞、记忆细胞、耗尽细胞）取决于 IS 类型，特别是在扩展的 CD8+ T 细胞克隆型 (CD8EXP) 中。 CD8EXP 的基因表达确定了受 IS 类型影响的治疗靶点。 TCR 分析显示 CD8EXP 的数量受到高度限制，与 HLA 不匹配或 IS 类型无关。将 CD8EXP 中的 TCR-α/β cDNA 亚克隆到 Jurkat 76 细胞 (TCR–/–) 中，通过混合淋巴细胞反应赋予同种异体反应性。对连续 rBx 样本的分析显示，在成功的抗排斥治疗后，CD8EXP 持续存在，减少但并未消除。相比之下，CD8EXP 在治疗难治性排斥反应中得以维持。最后，大多数 rBx 衍生的 CD8EXP 也在匹配的尿液样本中观察到，为使用尿液衍生的 CD8EXP 作为排斥同种异体移植物中发现的 CD8EXP 的替代品提供了先例。总体而言，我们的数据定义了克隆 CD8+ T 细胞对 ACR 的反应，为改进排斥反应的检测、评估和治疗铺平了下一步。
Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR–/–) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.