Systematic identification of minor histocompatibility antigens to address GVHD

摘要 异基因造血干细胞移植(allo-HSCT)有可能治愈许多血液疾病。 但通常会并发移植物抗宿主病(GvHD)。尽管治疗策略取得了重大进展， 与GVHD相关的发病率和死亡率仍然很高，迫切需要更好的工具来预测GVHD风险。 移植物抗宿主病的免疫学基础是供者T细胞识别次要组织相容性抗原(MHAgs)， 由供受者之间的非同义单核苷酸多态(SNP)差异引起。 然而，到目前为止，只有大约50 mHAgs被报道。近些年来国内外研究的新进展 通过下一代测序，编码外显子体内的生殖系和体细胞事件，与 人类白细胞抗原I类和II类表位预测的改进，为系统地 在个性化的基础上确定候选的mHAg。我们假设对组织特异性mHAgs的鉴定， 通过对患者和捐赠者DNA的基线分析预测，可以实现个性化的基因组学信息 GvHD的风险评估，有可能帮助完善当前的捐赠者选择算法，并使 移植后免疫抑制的个性化定制。因此，我们的目标是：(1)构建表位 系统识别mHAgs的预测流水线，结合最先进的SNP发现工具 从WES出发，通过对单细胞RNA-SEQ和蛋白质组的综合分析，设计了表达过滤器 一组共同靶向的GvHD组织(即皮肤、肝脏、结肠和肺)的简档，炎症特征， 和Y染色体特异性表达(在女性男性移植的情况下)。然后，这将与 人类白细胞抗原I、II类预测。通过直接检测与人类白细胞抗原结合的抗原表位预测将得到证实 通过基于免疫肽组学的GvHD受影响的靶组织评估的多肽。(2)定义和跟踪T 移植物抗宿主病患者对预测mHAgs的细胞反应。将MHAG目标的预测与泛函联系起来 T细胞反应，我们将通过测试mhag特异性T细胞反应来建立概念验证 临床相关环境：(I)经历器官特异性移植物抗宿主病的患者，免疫蛋白质组学特征为 目的1；(Ii)接受来自2个不同的不同的HLA相合的捐赠者的序贯allo-HSCT的患者 一个移植后而不是另一个移植后发生GVHD；以及(Iii)接受移植后治疗的患者 作为GvHD预防措施的一部分，匹配亲属供者(MRD)HSCT后的环磷酰胺。(3)测试 MHAG预测产生预后GvHD风险评分的可行性。以确定单个mhag是否 负担与临床观察到的GvHD有关，我们将重点关注髓系肿瘤患者，并进行WES 从DFCI治疗的患者的~200对MRD受者-供者对中发现一组DNA，然后 在来自URD HSCT的~200对的扩展集上，关于GvHD发生的信息是 可用。通过对来自CIBMTR的约600对其他配对(也包括髓系肿瘤)的计划验证分析，我们 将评估这种方法是否适用于跨机构和跨种族的患者。

Summary Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially curative for many blood disorders but is often complicated by graft-versus-host disease (GvHD). Despite major advances in therapeutic strategies, GvHD-related morbidity and mortality remain high, and better tools to predict the GvHD risk are urgently needed. The immunologic basis of GvHD is the recognition by donor T cells of minor histocompatibility antigens (mHAgs), arising from nonsynonymous single nucleotide polymorphism (SNP) differences between donor and recipient. However, only some 50 mHAgs have been reported thus far. Current advances in the characterization of germline and somatic events within coding exomes through next-generation sequencing, coupled with improvements in HLA class I and II epitope prediction, offer an unprecedented opportunity to systematically identify candidate mHAgs on an individualized basis. We hypothesize that identification of tissue-specific mHAgs, predicted through baseline analysis of patient and donor DNA, can enable a personalized genomics-informed risk assessment of GvHD, with the potential to help refine current donor selection algorithms and to enable personalized tailoring of post-transplant immunosuppression. Our aims are thus to: (1) Build an epitope prediction pipeline to systematically identify mHAgs, incorporating state-of-the-art tools for SNP discovery from WES, expression filters designed through comprehensive analysis of single-cell RNA-seq and proteomic profiling of a set of commonly targeted GvHD tissues (i.e. skin, liver, colon and lung), inflammation signatures, and Y-chromosome specific expression (in the case of female male transplants). This will be then coupled with HLA class I and II prediction. Epitope predictions will be confirmed through direct detection of HLA-bound peptides by immunopeptidome-based evaluations of GvHD-affected target tissues. (2) Define and track the T cell responses to predicted mHAgs in patients with GvHD. To link prediction of mHAg targets with functional T cell responses, we will establish proof-of-concept by testing for mHAg-specific T cell responses across three clinically relevant settings: (i) patients experiencing organ-specific GvHD, immunoproteomically characterized in Aim 1; (ii) patients who received sequential allo-HSCT from 2 distinct HLA-identical donors and experienced GvHD following one but not the other transplant; and (iii) patients who received post-transplant cyclophosphamide after matched related donor (MRD) HSCT as part of GvHD prophylaxis. (3) Test the feasibility of mHAg prediction to generate a prognostic GvHD risk score. To determine if individual mHAg burden relates to clinically observed GvHD, we will focus on patients with myeloid neoplasia, and perform WES analysis on a discovery set of DNA from ~200 MRD recipient-donor pairs from patients treated at DFCI, and then on an extension set of ~200 pairs from URD HSCT, from whom information regarding GvHD occurrence is available. Through a planned validation analysis of ~600 other pairs (also myeloid neoplasia) from CIBMTR, we will assess if this approach is applicable cross-institutionally, and for patients across ethnicities.