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Bone Marrow Failure
Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia
Valentina Giudice,1 Xingmin Feng,1 Zenghua Lin,1,2 Wei Hu,3 Fanmao Zhang,3 Wangmin Qiao,3 Maria del Pilar Fernandez Ibanez,1 Olga Rios,1
and Neal S. Young1
1Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA; 2Department of Hematology, Affiliated Hospital of Nantong University, Jiangsu, China and 3BGI Genomics, BGI-Shenzhen, China
ABSTRACT
Oligoclonal expansion of CD8+CD28– lymphocytes has been con- sidered indirect evidence for a pathogenic immune response in
+–
acquired aplastic anemia. A subset of CD8 CD28 cells with
CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8+CD28–CD57+ cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8+CD57+ cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodomi- nant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8+ cells from aplastic anemia patients with CD8+CD57+ cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8+CD57+ cells, which also showed decreased diversity compared to total CD4+ and CD8+ cell pools. From analysis of complementarity-deter- mining region 3 sequences in the CD8+ cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8+ T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diag- nosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064)
Introduction
Acquired aplastic anemia (AA) is a bone marrow (BM) failure syndrome charac- terized by peripheral blood (PB) pancytopenia and BM hypocellularity due to hematopoietic stem and progenitor cell destruction.1-7 There is indirect evidence to support the hypothesis of autologous immune attack: clinical response to immunosuppressive therapies (IST),1,2,8-10 the predominant role of activated cyto- toxic T cells (CTLs) in BM growth inhibition,1,10-12 identification of putative autoantigens,13,14 and oligoclonal expansion of CD8+ lymphocytes.8,14-17
Oligoclonality of T-cell populations has been defined by flow cytometry and deep sequencing of the T-cell receptor (TCR) Vβ repertoire and by spectratyping of complementarity-determining region 3 (CDR3) length skewing.18-21 The TCR, an aβ or γd heterodimer, is responsible for antigen recognition and T-cell activation.22-
Ferrata Storti Foundation
Haematologica 2018 Volume 103(5):759-769
Correspondence:
fengx2@nhlbi.nih.gov
Received: July 21, 2017. Accepted: December 30, 2017. Pre-published: February 1, 2018.
doi:10.3324/haematol.2017.176701
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/5/759
©2018 Ferrata Storti Foundation
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Haematologica | 2018; 103(5)
759
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