Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method.
OBJECTIVE
Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a host's immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples.


METHODS
The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility.


RESULTS
Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells.


CONCLUSIONS
We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.
Mariani
Sara
S
Divisione di Ematologia dell'Università di Torino, Torino, Italy.
Hwang
Seung Yae
SY
Foglietta
Myriam
M
Bonello
Lisa
L
Vitale
Candida
C
Coscia
Marta
M
Fiore
Francesca
F
Bruno
Benedetto
B
Massaia
Massimo
M
D000940:Antigenic Variation / Q000235:genetics; D002460:Cell Line; D022801:Complementarity Determining Regions / Q000235:genetics; D006801:Humans; D008722:Methods; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D013601:T-Lymphocytes; D050378:T-Lymphocytes, Regulatory
D022801:Complementarity Determining Regions; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta