Prediction of graft-versus-host disease in humans by donor gene expression profiling. Homo sapiens

Graft-versus-host disease (GVHD) results from recognition of host antigens by donor T cells following allogeneic hematopoietic cell transplantion (AHCT). Notably, histoincompatibility between donor and recipient is necessary but not sufficient to elicit GVHD. Therefore, we tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we analyzed the gene expression profile of CD4 and CD8 T cells from 50 AHCT donors. We found that pre-AHCT gene expression profiling segregates donors whose recipient suffered from GVHD or not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate diverse cell functions including TGF-β signaling and cell proliferation. We also performed gene expression profiling in T cells harvested from 40 AHCT recipients on day 365 post-AHCT. The donor gene profile defined on day 0 showed exceedingly strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. These data suggest that donor gene profiles linked with GVHD were largely imprinted at the hematopoietic stem cell level. The ability to discriminate strong and weak alloresponders using gene expression profiling could pave the way to personalized transplantation medicine. Keywords: comparative gene profile, cell type comparaison, GVHD+, GVHD- Overall design: Peripheral blood was obtained from 50 AHCT donors pre-transplant (referred to as day 0) and from 40 recipients on day 365. Donors and recipients were HLA-identical siblings. Recipients were regarded as negative for acute GVHD (aGVHD) when they lived at least 100 days without presenting GVHD. Recipients were considered negative for chronic GVHD (cGVHD) when they remained cGVHD-free for 365 days post-AHCT. CD4 and CD8 T-cell subsets were purified with microbeads. Total RNA was purified, amplified, reverse transcribed and hybridized on microarrays developed by The Microarray Centre of The Toronto University Health Network (http://www.microarrays.ca/). RNA from donor and recipient T cells was hybridized on the human H19K array (19,008 ESTs), and donor T-cell RNA was also hybridized on the ImmunArray (3,411 ESTs from immune related genes).

移植物抗宿主病（Graft-versus-host disease, GVHD）是异基因造血干细胞移植（allogeneic hematopoietic cell transplantation, AHCT）后，供者T细胞识别宿主抗原所引发的疾病。尤为关键的是，供者与受者之间的组织不相容性虽是引发GVHD的必要条件，但并非充分条件。为此，本研究验证了如下假说：部分供者可能较其他供者具有更强的同种免疫应答能力，因而更易诱发GVHD。
为验证该假说，我们对50名AHCT供者的CD4阳性与CD8阳性T细胞的基因表达谱进行了分析。研究结果显示，移植前的基因表达谱可有效区分受者发生与未发生GVHD的供者。“高危供者”表型（对应受者发生GVHD）受多基因调控，其形成受调控多种细胞功能的基因活性影响，涵盖转化生长因子-β（TGF-β）信号通路及细胞增殖相关基因。
此外，我们还对40名AHCT受者在移植后365天采集的T细胞开展了基因表达谱分析。移植当日（第0天）确立的供者基因谱，与受者移植后一年采集的CD4阳性及CD8阳性T细胞基因谱呈现极强的相关性。上述数据表明，与GVHD相关的供者基因谱在造血干细胞层面即已基本定型。借助基因表达谱区分强、弱同种免疫应答供者的能力，可为个性化移植医学的发展铺平道路。
关键词：比较基因谱分析，细胞类型比较，GVHD+，GVHD-
整体实验设计：采集50名AHCT供者移植前（记为第0天）以及40名受者移植后365天的外周血样本。供者与受者为人类白细胞抗原（Human Leukocyte Antigen, HLA）相合同胞。若受者在移植后至少存活100天且未出现急性移植物抗宿主病（acute GVHD, aGVHD），则判定为aGVHD阴性；若受者在移植后365天内未发生慢性移植物抗宿主病（chronic GVHD, cGVHD），则判定为cGVHD阴性。采用磁珠纯化CD4阳性与CD8阳性T细胞亚群。提取总RNA后进行扩增、反转录，并在多伦多大学健康网络微阵列中心（http://www.microarrays.ca/）制备的微阵列芯片上进行杂交。供者与受者T细胞的RNA均在人类H19K芯片（包含19008条表达序列标签（Expressed Sequence Tag, EST））上杂交，同时供者T细胞RNA还在免疫芯片（包含3411条免疫相关基因的EST）上进行了杂交。