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, b and gd, 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 TCR and
TCRg genes by recombination of Variable and
Joining gene segments (VJ recombination) to
form productive b and gd Y-like surface
receptors.
During development, the TCR variable regions
do not rearrange simultaneously in the multipotent precursor T cell; TCRd rearranges first,
followed by TCRg and TCRb. The TCR locus
rearranges last, after the surface expression of
both pre-TCR and TCRb chains(18). Both the
order and the effect of TCR rearrangement and
expression on gd and b T-cell lineage
commitment remains controversial (1, 19-23).
The canonical model proposes that TCRg,
TCRb, and TCRd 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:
Results
 Both b and gd T cells carry
rearranged TCRg CDR3 chains
(Table 1).
 Utilization of Vg-Jg gene segment
pairs is non-random. The Vg9-JPg1
gene segment pair is observed
much more often relative to other
Vg-Jg gene segment pairs (Fig. 3A).
Table 1. Summary of total and unique
TCRg Sequences
 Utilization of specific Vβ and Jβ
segments is variable within an
individual, but relatively
consistent between individuals
(Fig. 3C).
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).
 gd T cells carry few to no
rearranged TCRb CDR3 chains
(Table 2).
Table 2: Summary of total and unique
TCRb Sequences
 For all three individuals, the gd T
cell TCRg 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.
A
Conclusions
1. The TCRg CDR3 region
rearranges prior to T cell
differentiation (Table 1, Fig.
5).
2. The TCRb CDR3 region
rearranges
after
T
cell
commitment (Table 2, Fig. 5).
3. The TCRg CDR3 repertoire is
clonal (Fig. 2A), and >70% of
chains carried by gd T cells
use Vg9-JgP1 gene segments
(Fig. 3A).
4. The highest frequency TCRg
CDR3 sequence in each
individual is public and
shared by all 3 subjects (Fig.
4).
0.70
Proportio of t otal TCRg sequences
0.60
Assay
gd T cells Sample B
gd T cells Sample C
ab T cells Sample A
0.30
ab T cells Sample B
ab T cells Sample C
0.20
0.00
5
10
15
20
25
n
25 most common TCRg CDR3 Sequences
B
0.70
C
0.60
Proportio of t otal TCRb sequences
Fig. 1: TCRB
gd T cells Sample A
0.40
0.10
 Genomic DNA extracted from sorted
cells with Qiagen DNAeasy macro-kit
 TCRb and TCRg sequences amplified
and sequenced from both b and gd T
cells using the immunoSEQ assay (Fig.
1)
0.50
0.50
0.40
Fig 5: TCR CDR3 rearrangement
schema
Stochastic & Signal Strength Model
ab T cells Sample A
ab T cells Sample B
ab T cells Sample C
n
 PBMC isolated with Ficoll gradient and
bead-sorted using Miltenyi kits to isolate
and collect αβ and gd T cells.
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).
A
 40 ml of whole blood collected from
three healthy adult donors.
 The
most
frequent
TCRG
nucleotide
clone
in
each
individuals is public and shared
by all three healthy individuals
(Fig. 4).
3B),
1. Abundance of rearranged
TCRg CDR3 chains in b T
cells.
2. Abundance of rearranged
TCRb CDR3 chains in gd T
cells
3. Clonality of the TCRg and
TCRb repertoire.
4. Overlap of gd T cell TCRG
CDR3 repertoire between any
two individuals.
Materials and Methods
Results
0.30
0.20
TCRB
TCRG
ab T cell
TCRB
TCRG
gd T cell
TCRB
TCRG
ab T cell
TCRG
gd T cell
TCRB
TCRG
0.10
0.00
5
10
15
20
25
25 most common TCRb CDR3 Sequences
Sequential TCRRearrangement
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
TCRG
uncommitted T cell
committed T cell