Brauer Patrick M, Pessach Itai M, Clarke Erik, Rowe Jared H, Ott de Bruin Lisa, Lee Yu Nee, Dominguez-Brauer Carmen, Comeau Anne M, Awong Geneve, Felgentreff Kerstin, Zhang Yuhang H, Bredemeyer Andrea, Al-Herz Waleed, Du Likun, Ververs Francesca, Kennedy Marion, Giliani Silvia, Keller Gordon, Sleckman Barry P, Schatz David G, Bushman Frederic D, Notarangelo Luigi D, Zúñiga-Pflücker Juan Carlos
Sunnybrook Research Institute, and Department of Immunology, University of Toronto, Toronto, ON, Canada;
Division of Immunology, Boston Children's Hospital, Boston, MA; Department of Pediatric Critical Care, Sheba Medical Center, Tel-Aviv University, Tel-Aviv, Israel;
Blood. 2016 Aug 11;128(6):783-93. doi: 10.1182/blood-2015-10-676304. Epub 2016 Jun 14.
Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.
原发性免疫缺陷病由一组影响免疫系统发育和/或功能的异质性基因缺陷组成。在此，我们利用从患有不同重组激活基因1（RAG1）突变的患者产生的人诱导多能干细胞（iPSC）进行体外分化，以评估T细胞发育和T细胞受体（TCR）V（D）J重组。来自严重联合免疫缺陷（SCID）患者细胞的RAG1突变体显示，无法维持T细胞发育超越CD3（--）CD4（-）CD8（-）CD7（+）CD5（+）CD38（-）CD31（- / lo）CD45RA（+）阶段，从而无法达到CD3（- / +）CD4（+）CD8（+）CD7（+）CD5（+）CD38（+）CD31（+）CD45RA（-）阶段。尽管来自奥门综合征（OS）患者的RAG1突变体具有残余的重组活性，但由于可能由N端截短的RAG1和催化失活的RAG1组成的异二聚体导致单链DNA断裂增加，仍观察到类似的T细胞分化受损。此外，对CD3（-）CD4（+）CD8（-）未成熟单阳性和CD3（+）CD4（+）CD8（+）双阳性细胞的TCR-β（TRB）和TCR-α（TRA）重排进行深度测序分析显示，谱系多样性严重受限，少数可变、多样和连接基因被优先使用，并且来自SCID和OS iPSC衍生细胞的TRB和TRA互补决定区3序列的长度分布存在偏差，而对照iPSC产生的T细胞祖细胞具有广泛多样的谱系。最后，在SCID和OS衍生的T系细胞中未检测到TRA/δ切除环（TREC），这是TRA/δ基因座重排的标志物，与T细胞发育中的前TCR阻滞一致。本研究比较了SCID和OS患者的人类T细胞发育，并阐明了重要差异有助于解释同一基因内不同突变导致的不同患者广泛的免疫表型。
