Transcript Title: adaptive TCR Stuff name, name, institutions

Immune profiling with high-throughput sequencing
Harlan Robins1,2 Cindy Desmarais2, Chris Carlson1,2
Fred Hutchinson Cancer Research Center, Seattle, WA1, Adaptive TCR Technologies, Seattle, WA2
Introduction
Results
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.
Because of the potential diversity of receptors, for
example a healthy adult has approximately 10 million
different TCRB chains contained within their 1012
circulating T cells, it is improbable to randomly converge
on the same CDR3 sequence, effectively making each
CDR3 sequence a unique nucleotide tag. This property
suggests that unique TCRB chains can be used to monitor
blood cancers, like T-cell lymphoma and leukemia.
Adaptive TCR has developed a novel method to amplify
and sequence rearranged TCRB CDR3 sequences (Fig.
1). We present the results of a test of the precision,
accuracy, sensitivity, and repeatability of this reaction and
results of a potential application, identifying Minimal
Residual Disease in T cell Acute Lymphoblastic Leukemia
(T-ALL) patients.
1. Test the precision, accuracy, and sensitivity of our
assay using clone spike-in experiments (6 clones,
spiked in at 10-1,000,000 cells).
2. Assess the repeatability of A) PCR and B)
Sequencing through replication (Fig. 3A).
3. Use our assay to detect MRD and compare to
current method, flow sorting
 Assay is accurate across five-orders of
magnitude: observed clone frequencies are
within 3-fold range of expected clone
frequencies.
 Assay is precise across five-orders of
magnitude: each clone’s observed
frequency is internally consistent.
 Assay is sensitive: clones spiked-in at 1 in
100,000 background cells are detectable.
Fig. 2: Expected versus observed
frequencies for 6 spiked-in clones in multiple
mixes. Four CD4+ clones (A,B,C,D) and two
CD8+ clones (E and F) and were spiked into
either complex PBMC or sorted CD4+ T cell
populations at concentrations spanning five
orders of magnitude.
Results
Results
 Sequencing reaction highly reproducible:
>97% of TCRB chains detected in Sample
A1a are also detected in Sample A1b and
vis versa (Fig. 3B)
 PCR reaction reproduces frequent clones:
>54% of TCRB chains detected in Sample A
also detected in Sample B, and vis versa
(Fig.3C and D)
 TCRB sequencing detected cancer clones
in 21 of 28 samples, whereas Flow sorting
detected cancer clones in 28 of 28
samples.
 TCRB sequencing detected MRD in all
cases in which flow sorting detected MRD.
 TCRB sequencing detected MRD in an
additional 7 cases for which flow sorting
did not detect MRD.
Fig. 3: Experimental design and results of
repeatability experiment. A) Schematic of
experimental design. B) Sequence copy counts of
TCRB clones obtained by sequencing the same
PCR library on two different lanes of a flow cell.
Each point represents a unique clone in this loglog scatter plot. The points in red are found in
both repetitions, while those in blue are only
found in one repetition. C) Sequence copy counts
obtained by sequencing two separate PCR
reactions performed using template from the
same DNA extraction.
Fig. 4: Log transformed frequencies of Cancer clones
in 28 T-ALL patients pre-treatment (A) and 29 days
following treatment (B). Black circles represent the
frequency of cancer clones detected using immunerepertoire sequencing, and red circles represent the
frequency of cancer clones detected using flow sorting.
Day 29 TCRB sequencing clone frequencies were not
included for the 7 samples for which MRD was not
detected at Day 0.
Materials and Methods
Samples:
1)Spike-in Experiment: 6 clones (A-F) independently
spiked into a diverse background at 3 concentrations (AD) or 4 concentrations (E-F).
2) Repeatability Experiment: PBMC isolated from one
healthy adult. DNA extraction, PCR reaction, and
Sequencing reaction repeated twice.
3) MRD: DNA extracted from T cells isolated form 28 TALL patients pre-treatment and 29 days after treatment.
PCR and Sequencing:
TCRB chains from all samples were sequenced using
Adaptive TCRs TCRB immunoSEQ assay (Fig. 1).
Analysis:
1)Spike-in Experiment: Expected and observed
frequencies were calculated for each clone at each spikein concentration.
2) Repeatability Experiment: Overlap between repeated
samples.
3) MRD: Frequency of cancer clone calculated pre and
post-treatment using TCRB SEQ and flow sorting.
Fig. 1:
Conclusions
 TCRB repertoire sequencing is accurate
across five orders of magnitude.
Assay
 TCRB sequencing is sensitive and can
detect rare clones, down to 1:100.000 of
total repertoire.
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.
Some of this work is published in Journal of Immunological
Methods, September 2011.
Adaptive TCR Technologies
Suite 300
307 Westlake Ave N
Seattle, WA 98109
 TCRB repertoire sequencing can detect
most cancer clones.
 TCRB repertoire sequencing detects MRD
at levels un-detectable by Flow sorting.