急性移植物抗宿主病（GvHD）仍然是同种异基因造血干细胞移植（allo-HSCT）后的最大临床挑战和决定预后的并发症。供体T细胞被认为是对宿主组织（尤其是肠道）的异反应性的关键介体。与先前的研究支持一致，我们发现，在全MHC-I不匹配的allo-HSCT过程中，肠道上皮内淋巴细胞（IEL）组分在动态调节。然而，肠上皮细胞（IEC）损伤危及肠道屏障的完整性，是肠GvHD的核心特征，但IEL在IEC调控中的贡献程度尚不清楚。为了研究淋巴上皮细胞的相互作用，我们使用了一种新型的外体T细胞/器官样体共培养模型系统。在这里，异基因肠道上皮T细胞在诱导IEC死亡方面强于同基因IE L和异基因非IEL T细胞。诱导IEC死亡的能力主要限于TCRβ+ T细胞，并且大部分是通过IFNγ依赖性执行的。异源性反应性需要多样的T细胞受体（TCR）亚型，因为经基因改造以表达仅限于单个非内源性抗原的TCR的IEL无法介导IEC病理学。有趣的是，微小的组织相容性抗原（miHA）不匹配就足以引发IEL驱动的IEC损伤。最后，先进的活细胞成像分析揭示，与同基因对照组相比，异基因IEC中的异反应性IEL在肠道样体内巡视了较小的区域，表明它们在同基因IEC内具有独特的迁移特性。因此，我们在这里提供了实验证据，证明了外体协同培养系统在模拟IEL与IEC之间的细胞和分子特征的异基因环境中的相互作用方面的实用性。鉴于失调的免疫上皮稳态作为肠GvHD的核心方面的新概念，该方法代表了一种新的实验体系，例如筛选治疗策略，以评估其规范化T细胞/IEC相互作用的潜力。因此，在临床前的体内同种异基因HSCT模型系统中进行的分析可能仅限于此处所选择的积极策略。版权所有© 2023 Matthe，Dinkel，Schmid，Vogler，Neurath，Poeck，Neufert，Büttner-Herold和Hildner。

Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.Copyright © 2023 Matthe, Dinkel, Schmid, Vogler, Neurath, Poeck, Neufert, Büttner-Herold and Hildner.