Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.
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.
Sunnybrook Research Institute, and Department of Immunology, University of Toronto, Toronto, ON, Canada;
Patrick M
0000-0001-7788-5204
PM
Brauer
Division of Immunology, Boston Children's Hospital, Boston, MA; Department of Pediatric Critical Care, Sheba Medical Center, Tel-Aviv University, Tel-Aviv, Israel;
Itai M
IM
Pessach
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;
Erik
E
Clarke
Division of Immunology, Boston Children's Hospital, Boston, MA;
Jared H
JH
Rowe
Division of Immunology, Boston Children's Hospital, Boston, MA;
Lisa
L
Ott de Bruin
Division of Immunology, Boston Children's Hospital, Boston, MA;
Yu Nee
YN
Lee
Ontario Cancer Institute, University Health Network, Toronto, ON, Canada;
Carmen
C
Dominguez-Brauer
Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA;
Anne M
AM
Comeau
Sunnybrook Research Institute, and Department of Immunology, University of Toronto, Toronto, ON, Canada; McEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, Canada;
Geneve
G
Awong
Division of Immunology, Boston Children's Hospital, Boston, MA;
Kerstin
K
Felgentreff
Departments of Immunobiology and Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT;
Yuhang H
YH
Zhang
Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO;
Andrea
A
Bredemeyer
Department of Pediatrics, Faculty of Medicine, Kuwait University, Kuwait City, Kuwait;
Waleed
W
Al-Herz
Division of Immunology, Boston Children's Hospital, Boston, MA;
Likun
L
Du
Division of Immunology, Boston Children's Hospital, Boston, MA;
Francesca
F
Ververs
McEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, Canada;
Marion
M
Kennedy
"Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia, Brescia, Italy;
Silvia
S
Giliani
McEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, Canada;
Gordon
G
Keller
Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO;
Barry P
BP
Sleckman
Departments of Immunobiology and Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT; Howard Hughes Medical Institute, New Haven, CT; and.
David G
DG
Schatz
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;
Frederic D
FD
Bushman
Division of Immunology, Boston Children's Hospital, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA.
Luigi D
0000-0002-8335-0262
LD
Notarangelo
Sunnybrook Research Institute, and Department of Immunology, University of Toronto, Toronto, ON, Canada;
Juan Carlos
JC
Zúñiga-Pflücker
D002478:Cells, Cultured; D053960:DNA Breaks; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000473:pathology*; D007223:Infant; D009154:Mutation; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D016511:Severe Combined Immunodeficiency / Q000235:genetics* / Q000473:pathology*; D013601:T-Lymphocytes / Q000473:pathology*; D060152:V(D)J Recombination
D018398:Homeodomain Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; C064658:RAG-1 protein