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Deep sequencing of the human TCRγ and TCRβ
repertoires provides evidence that TCRβ rearranges
after αβ, γδ T cell commitment
C.S. Carlson1, A. Sherwood2, C. Desmarais2, R.J. Livingston2, J. Andriesen2, M. Haussler3, H. Robins1
1Fred
Hutchinson Cancer Research Center, 2Adaptive TCR Corporation 3 University of Manchester
Results
Introduction
The ability of T lymphocytes to mount an
immune response against a diverse array of
pathogens is primarily conveyed by the aminoacid
sequence
of
the
hypervariable
complementary determining region 3 (CDR3)
regions of the T cell receptor (TCR). The
genes that encode the two primary types of
TCRs, αβ and γδ, undergo somatic
rearrangement during T cell development.
TCRβ and TCRδ genes are assembled via
recombination of Variable (V), Diversity (D),
and Joining (J) gene segments (VDJ
recombination) and similarly, the TCRα and
TCRγ genes by recombination of Variable and
Joining gene segments (VJ recombination) to
form productive αβ and γδ Y-like surface
receptors.
During development, the TCR variable regions
do not rearrange simultaneously in the multipotent precursor T cell; TCRδ rearranges first,
followed by TCRγ and TCRβ. The TCRα locus
rearranges last, after the surface expression of
both pre-TCRα and TCRβ chains(18). Both the
order and the effect of TCR rearrangement and
expression on γδ and αβ T-cell lineage
commitment remains controversial (1, 19-23).
The canonical model proposes that TCRγ,
TCRβ, and TCRδ rearrange prior to T cell
lineage commitment. Adaptive TCR has
developed a method to deeply sequence both
TCRB and TCRG CDR3 chains. By
sequencing the TCRB and TCRG repertoire of
both types of T cells, we will be able to
estimate:
1. Abundance of rearranged
TCRγ CDR3 chains in αβ T
cells.
2. Abundance of rearranged
TCRβ CDR3 chains in γδ T
cells
3. Clonality of the TCRγ and
TCRβ repertoire.
4. Overlap of γδ T cell TCRG
CDR3 repertoire between any
two individuals.
 Both αβ and γδ T cells carry
rearranged TCRγ CDR3 chains
(Table 1).
Table 1. Summary of total and unique
TCRγ Sequences
Results
 Utilization of Vγ-Jγ gene segment
pairs is non-random. The Vγ9-JPγ1
gene segment pair is observed
much more often relative to other
Vγ-Jγ gene segment pairs (Fig. 3A).
 Utilization of specific Vβ and Jβ
segments is variable within an
individual, but relatively
consistent between individuals
(Fig. 3C).
 γδ T cells carry few to no
rearranged TCRβ CDR3 chains
(Table 2).
Table 2: Summary of total and unique
TCRβ Sequences
Fig. 3: Average V-J gene utilization of
sequenced TCRγ and TCRβ
sequences across three samples:
Average V-J utilization of gene
segments in TCRγ CDR3 sequences
amplified from γδ T cells (3A), TCRγ
CDR3 sequences amplified from αβ T
cells (3B), and TCRβ sequences
amplified from αβ T cells (3C).
Fig. 4: Shared nucleotide identical
TCRγ CDR3 sequences: Nine
nucleotide identical TCRγ CDR3
sequences amplified from γδ T cells are
shared by all three individuals. For each
shared sequence, the copy count
detected for each individual is indicated
on the Y-axis.
and TCRβ sequences
amplified from αβ T cells (3C).
 For all three individuals, the γδ T
cell TCRγ repertoire is dominated
by one or two clones (>50% of
total repertoire) (Fig. 2).
Fig. 2: Frequency of the 25 most
common TCR sequences: For each
sample we plot the proportion of
productive sequences accounted for by
the 25 most numerous productive TCR
sequences. (2A) TCRγ chains amplified
from γδ T cells and αβ T cells and (2B)
TCRβ chains amplified from αβ T cells.
Conclusions
1. The TCRγ CDR3 region
rearranges prior to T cell
differentiation (Table 1, Fig.
5).
2. The TCRβ CDR3 region
rearranges
after
T
cell
commitment (Table 2, Fig. 5).
3. The TCRγ CDR3 repertoire is
clonal (Fig. 2A), and >70% of
chains carried by γδ T cells
use Vγ9-JγP1 gene segments
(Fig. 3A).
4. The highest frequency TCRγ
CDR3 sequence in each
individual is public and
shared by all 3 subjects (Fig.
4).
A
 40 ml of whole blood collected from
three healthy adult donors.
 PBMC isolated with Ficoll gradient and
bead-sorted using Miltenyi kits to isolate
and collect αβ and γδ T cells.
 Genomic DNA extracted from sorted
cells with Qiagen DNAeasy macro-kit
Fig. 1: TCRB
 The
most
frequent
TCRG
nucleotide
clone
in
each
individuals is public and shared
by all three healthy individuals
(Fig. 4).
3B),
Materials and Methods
 TCRβ and TCRγ sequences amplified
and sequenced from both αβ and γδ T
cells using the immunoSEQ assay (Fig.
1)
Results
B
C
Assay
For additional information about immunoSEQ assays and
the immunoSEQ Analyzer suite of bioninformatics
applications at Adaptive TCR Technologies, visit our booth
or contact us on the web at www.adaptivetcr.com and
www.immunoseq.com.
This work is published in Science Translational Medicine,
July 2011, Vol. 3, Issue 90.
Adaptive TCR Technologies
Suite 300
307 Westlake Ave N
Seattle, WA 98109
Fig 5: TCR CDR3 rearrangement
schema