Quantification of newly developed T cells in mice by real-time quantitative PCR of T-cell receptor rearrangement excision circles.
OBJECTIVE
Thymic output of newly developed ab T cells in humans can be measured via signal joint T-cell receptor rearrangement excision circles (sjTRECs). Deletion of the TCRD locus via dRec to psiJa recombination during TCRA rearrangement results in the production of such sjTRECs. The deleting elements dRec and psiJa are highly conserved between humans and mice and used in a comparable manner. We developed and evaluated a real-time quantitative PCR (RQ-PCR) to detect and quantify dRec-psiJa sjTRECs in murine peripheral blood leukocytes for estimation of thymic output of newly developed ab T cells in mice.


METHODS
The threshold cycle (Ct) of the sjTREC RQ-PCR was related to the Ct value of an endogenous reference gene. The difference in Ct value (DCt) was correlated to the absolute numbers of CD45+ and CD3+ cells per mL of blood, as obtained by a single platform flow cytometric assay, resulting in the frequency of sjTRECs in CD45+ and CD3+ cells.


RESULTS
The RQ-PCR proved to be sensitive with a detection level of approximately one sjTREC copy in 100 ng of DNA. SjTRECs could not be detected in peripheral blood leukocytes of RAG-1(-/-) mice, demonstrating the specificity of the assay. As in humans and primates, sjTREC levels declined in aging and thymectomized mice. Remarkably, significant mouse strain-dependent differences in sjTREC levels were observed. 129Sv and C57BL/6 mice had significantly lower sjTREC levels in blood than Balb/c and DBA2 mice.


CONCLUSIONS
Quantification of murine sjTRECs by RQ-PCR may allow for accurate assessment of thymic output in mice.
Department of Hematology, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands.
Annoek E C
AE
Broers
Jules P P
JP
Meijerink
Jacques J M
JJ
van Dongen
Sandra J
SJ
Posthumus
Bob
B
Löwenberg
Eric
E
Braakman
Jan J
JJ
Cornelissen
D000375:Aging / Q000276:immunology; D000818:Animals; D001483:Base Sequence; D017252:CD3 Complex / Q000097:blood; D003199:Computer Systems; D004270:DNA, Circular / Q000097:blood*; D005434:Flow Cytometry; D015329:Gene Rearrangement, T-Lymphocyte; D017493:Leukocyte Common Antigens / Q000097:blood; D018655:Lymphocyte Count; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013047:Specific Pathogen-Free Organisms; D013934:Thymectomy; D013950:Thymus Gland / Q000166:cytology*
D017252:CD3 Complex; D004270:DNA, Circular; D016693:Receptors, Antigen, T-Cell, alpha-beta; D017493:Leukocyte Common Antigens