Estimate the utilization of V β –J β and D β

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Transcript Estimate the utilization of V β –J β and D β

Overlap of the human
+
CD8
T cell receptor repertoire
Harlan S. Robins1,2, SK Srivastava1, P Campregher1, CJ Turtle1, J Andriesen2, SR Riddell1, CS Carlson1,2, EH Warren1
Fred Hutchinson Cancer Research Center, Seattle, WA1, Adaptive TCR Technologies, Seattle, WA2
The antigenic specificity of T lymphocytes is in large part
determined by the amino acid sequence in the
hypervariable complementarity-determining region 3
(CDR3) of the α and β chains of the T cell receptor (TCR).
The nucleotide sequences that encode the CDR3 regions
are generated by somatic rearrangement between
variable (V), diversity (D), and joining (J) region gene
segments.
Diversity generated by the somatic
rearrangement of germline genomic DNA is augmented by
the deletion of nucleotides adjacent to the recombinational
signal sequences (RSSs) of the V, D, J segments and
template-independent insertion of nucleotides at the VβDβ, Dβ-Jβ, and Vβ-Jβ junctions. Together, these processes
allow for tremendous variation within the CDR3 region.
High-throughput sequencing on the immunoSEQ system
enables us to globally capture the TCRB CDR3
repertoires of multiple individuals.
We examine the
repertoire of TCRB CDR3 sequences expressed in the
naïve and memory CD8+ T cells from the peripheral blood
of seven healthy adults, and assess the extant of overlap
between any two individuals TCRB CDR3 repertoires.
With this information we:
1. Estimate the utilization of Vβ–Jβ and Dβ-Jβ gene
segment pairs in CD8+ T cells across seven healthy
donors.
2. Estimate the extent to which CD8+ T cell TCRB
CDR3 repertoire overlaps between any two
individuals.
3. Screen for characteristics that distinguish shared
and nonshared TCRB CDR3 sequences.
 Millions of unique TCRB CDR3 sequences
were identified (Table 2).
Results
 Utilization of Dβ-Jβ gene segment pairs is
non-random. The Dβ2-Jβ1 gene segment
pair is observed less often relative to other
Dβ-Jβ gene segment pairs (Fig. 3)
 Unexpectedly high TCRB CDR3 amino-acid
sequence overlap in both the CD8+ naïve
and memory compartments (Fig. 5a).
Fig. 3. Relative utilization of specific Dβ-Jβ gene
segment pairs in naïve and memory CD8+ T cells. The
proportion of all in-frame, TCRB CDR3 sequences
obtained from (A) naïve or (B) memory CD 8+ T cells that
utilize each Dβ-Jβ pairing. Gene pairings include a D
segment, Dβ1 or Dβ2, and Jβ family, Jβ1 or Jβ2.
Fig. 5. Characteristics of public CD8+ TCRB CDR3
amino-acid sequences.
(A) Number of shared
sequences in the naive and memory CD8+ CDR3
repertoires of every possible pair of individuals. A colored
shape represents the relationship of a pair of individuals:
HLA identical siblings (blue circle), mother daughter pair
(red square), and unrelated individuals (black triangle). (
B and C) Frequency distribution of shared (red) and
nonshared (blue) CDR3 sequences observed in the (B)
naïve and memory (C) CD8+ compartments of every
possible pair of individuals as a function of the total
number of nucleotide insertions at the Vβ-Dβ and Dβ-Jβ
junctions.
A
 Utilization of specific Vβ and Jβ segments is
variable within an individual, but relatively
consistent between individuals (Fig. 2).
Fig. 2. Non-uniform utilization of Vβ–Jβ gene
combinations in CD8+ T cells of seven healthy adults.
Mean utilization frequency of specific Vβ–Jβ gene segment
combinations in TCRB CDR3 sequences expressed in (A)
naïve and (B) memory CD8+ T cells.
B
A
A
 The actual TCRB CDR3 repertoire overlap
between any two individuals is tens of
thousands of amino-acid sequences (Fig.
4).
 PBMC collected from seven HLA typed donors
(Table 1).
Fig 4. Calculation of the total number of TCRB CDR3
amino acid sequence overlaps between the naïve
CD8+ T cell compartments of two donors. The colored
points indicate the observed number of naïve and
memory CD8+ TCRB CDR3 amino-acid sequences
between donor 3 and the other donors. The solid lines
indicate the estimated total number of CD8+ TCRB CDR3
amino-acid sequences donor 3 shares with the other
donors.
Table 1: Donor characteristics
B
Number of shared CDR3 sequences in CD8+ memory
C
B
Total number of nucleotide insertions
 Genomic DNA was extracted.
 TCRB sequences were amplified and sequenced
using the immunoSEQ assay (Fig. 1)
Fig. 1:
 Shared naïve and memory CD8+ T cells
have fewer non-template insertions than
nonshared CD8+ T cells (Fig. 5b, 5c).
Table 2. Summary of the TCRB CDR3 sequence data
Materials and Methods
 PBMC were FACS-sorted to isolate and collect
memory and naïve CD8+ αβ T cells.
Results
Number of shared CDR3 sequences in CD8+ naive
Results
Probability of insertion
Introduction
Conclusions
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,
September 2010, Vol 2, Issue 47.
Adaptive TCR Technologies
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307 Westlake Ave N
Seattle, WA 98109
 We sequenced, from the peripheral blood
CD8+ T cell compartment of seven healthy
adults, ~40 million rearranged TCRB CDR3
sequences. Approximately 3 million of
these sequences were unique.
 The realized CDR3 sequence repertoire
within each individual is strongly biased
toward specific Vβ–Jβ pair utilization, and
dominated by sequences containing fewer
than 8 non-template nucleotide insertions.
 The overlap in the naïve CD8+ CDR3
sequence repertoires of any two of the
individuals is ~7000 fold larger than
predicted.