Immune profiling of γδ T cells after human intestinal transplantation

项目摘要 γδ T细胞可以识别不同的抗原并发挥不同的功能。先天和适应性特征 人γδ T细胞的分化可能是由不同的γδ T细胞受体（TCR）库驱动的，其可以通过以下方式形成： 组织区室化、年龄和抗原暴露史。尽管占很大比例， 包括肠道和肝脏在内的许多器官中的常驻T细胞，γδ T细胞及其在移植中的可能作用 结果在很大程度上是研究不足的。我们的首要目标是阐明 人小肠移植后γδ T细胞参与同种异体排斥反应和宿主防御的机制 移植（ITx）。我们发现，ITx后的临床结局主要取决于 移植物抗宿主（GvH）和宿主抗移植物（HvG）同种异体反应性之间的关系， 基于TCRβ CDR 3高的Tx前混合淋巴细胞反应后的同种异体反应性T细胞克隆 通量测序（TCRβ-seq）。我们发现，GvH反应性αβ T细胞，从预先存在的 供体驻留记忆T细胞（TRM）在遇到快速再增殖的受体抗原呈递细胞后 在移植粘膜中，获得效应T细胞（Teff）功能并迁移到循环和骨髓（BM）中， 其中它们减弱HvG应答，促进供体细胞移植和控制排斥。采用单细胞 通过RNA-seq，我们发现BM浸润的供体γδ T细胞，由“公共”Vδ2克隆型主导， 细胞毒性Teff表型与CD 8 αβ T细胞相似，表明它们也有减弱HvG反应的潜力 并为供体造血干细胞和祖细胞移植腾出空间。我们发现受体γδ T 细胞逐渐填充同种异体移植物，经历从Teff到TRM的表型变化，主要是“私有” Vδ1克隆型。我们假设γδ T细胞不仅参与宿主对病原体的防御， 还具有调节人ITx后的双向同种异体反应的潜力。我们现在建议研究 ITx后局部和全身人γδ T细胞的表型和克隆追踪， 它们对同种异体反应性的调节作用以及与移植结果的关联。我们建议进行三次 具体目的：确定供体（目的1）和受体（目的2）的嵌合体、表型和克隆型 通过流式细胞术、质谱细胞术和TCRγδ-seq.散装 Tx前未刺激和离体刺激的γδ T细胞库和Tx后γ δ T细胞库的TCRγδ-seq ITx患者的循环、肠移植物和BM中的谱系将与单个细胞连接， 使用iRepertoire和10 x Genomics平台对γδ Τ细胞的转录谱进行了分析（Aim 3）。我们提出的 研究将提供更深入的了解γδ T细胞嵌合体背后的机制， TRM特征及其对局部和全身同种异体反应的调节作用，促进了 新的策略来调节γδ T细胞，以克服排斥反应，感染和增加ITx的利用， 拯救生命，提高生活质量的手术

PROJECT SUMMARY γδ T cells can recognize diverse antigens and exert disparate functions. The innate- and adaptive-like features of human γδ T cells may be driven by differential γδ T cell receptor (TCR) repertoires, which can be shaped by tissue compartmentalization, age and history of antigen exposure. Despite comprising a significant proportion of resident T cells in many organs, including gut and liver, γδ T cells and their possible role in transplantation outcomes are largely under‐researched. Our overarching goals are to elucidate the fundamental mechanisms of how γδ T cells participate in allograft rejection and host defense after human intestinal transplantation (ITx). We revealed that clinical outcomes after ITx are largely determined by the balance between graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivities, using an approach that identifies alloreactive T cell clones following pre-Tx mixed lymphocyte reactions on the basis of TCRβ CDR3 high throughput sequencing (TCRβ-seq). We showed that GvH-reactive αβ T cells, expanded from preexisting donor resident memory T cells (TRM) after encountering rapidly-repopulating recipient antigen-presenting cells in the graft mucosa, acquire effector T cell (Teff) functions and migrate into circulation and bone marrow (BM), where they attenuate HvG responses, facilitate donor cell engraftment and control rejection. Using single cell RNA-seq, we found that BM-infiltrating donor γδ T cells, dominated by “public” Vδ2 clonotypes, showed cytotoxic Teff phenotypes similar to CD8 αβ T cells, suggesting their potential to also attenuate HvG reactions and make space for donor hematopoietic stem and progenitor cell engraftment. We found that recipient γδ T cells gradually populated the allograft, undergoing phenotypic changes from Teff to TRM, mainly with “private” Vδ1 clonotypes. We hypothesize that γδ T cells not only participate in host defense against pathogens, but also have the potential to modulate two-way alloresponses after human ITx. We now propose a study of phenotypic and clonal tracking of human γδ T cells locally and systemically after ITx and further investigation of their regulatory roles on alloreactivity and association with graft outcomes. We propose to pursue three Specific Aims: To determine the chimerism, phenotype and clonotype of donor- (Aim 1) and recipient- (Aim 2) derived γδ T cells in graft mucosa, circulation and BM by flow cytometry, mass cytometry and TCRγδ-seq. Bulk TCRγδ-seq of pre-Tx unstimulated and ex vivo-stimulated γδ T cell repertoires and post-Tx γδ T cell repertoires in the circulation, intestinal allograft and BM of ITx patients will be linked with single cell transcriptional profiles of γδ T cell using the iRepertoire and 10x Genomics platforms (Aim 3). Our proposed research will provide a deeper understanding of the mechanisms behind γδ T cell chimerism, maturation of TRM features, and their modulatory roles on local and systemic alloresponses, facilitating the development of novel strategies to regulate γδ T cells to overcome rejection, infection and increase the utilization of ITx as a life-saving, quality of life-improving procedure.