The elusive nature of assessing immunological processes in situ in organ transplantation is one of the major impediments to improve diagnostics and treatment. Here, we present a proof-of-concept study using multiplexed in situ hybridization (ISH) (RNAscope) to detect low-abundance cytokines in formalin-fixed paraffin-embedded (FFPE) human transplant kidney biopsies in combination with immunofluorescence (IF) for cell phenotyping. We show that a multiplex IF and ISH (mIFISH) assay is feasible to identify the cellular source of cytokines and chemokines (tumor necrosis factor-α, interferon-γ, and CXCL9) in FFPE transplant kidney biopsies and that quantification of the mRNA and protein signal is also possible at single-cell resolution in the context of tissue complexity. Furthermore, the mIFISH assay allows precise quantitative assessment of tubulitis, one of the key morphological correlates of alloimmune injury. Simultaneous in situ identification and quantification of multiple cellular phenotypes and mRNA expression of proinflammatory cytokines in FFPE tissues offer a novel insight into the biology of alloimmune injury in kidney transplantation and may contribute to improved diagnostic accuracy and patient care.

评估器官移植原位免疫过程的难以捉摸的性质是改善诊断和治疗的主要障碍之一。在这里，我们提出了使用多重原位杂交（ISH）（RNAscope）的概念验证研究，以检测福尔马林固定石蜡包埋（FFPE）人移植肾活检组织中的低丰度细胞因子，并结合免疫荧光（IF）细胞表型。我们显示了IF和ISH（mIFISH）多重检测法在确定FFPE移植肾活检组织中细胞因子和趋化因子（肿瘤坏死因子-α，干扰素-γ和CXCL9）的细胞来源以及mRNA和蛋白质的定量分析方面是可行的在组织复杂的情况下，信号在单细胞分辨率下也是可能的。此外，mIFISH分析可精确定量评估肾小管炎，同种免疫损伤的关键形态学相关因素之一。FFPE组织中多种细胞表型和促炎细胞因子的mRNA表达的同时原位鉴定和定量分析为肾脏移植中同种免疫损伤的生物学提供了新的见解，并可能有助于提高诊断准确性和患者护理。