T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors (TCRs) having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we use the Ion AmpliSeq™ Immune Repertoire Assay Plus – TCRβ to evaluate evidence for T cell convergence within melanoma tumor biopsy research samples from a set of 63 subjects plus peripheral blood leukocytes (PBL) from four healthy subjects. We find that the melanoma TME is highly enriched for convergent TCRs compared to healthy donor peripheral blood. We discuss the potential use of TCR convergence as a liquid biopsy compatible predictive biomarker for immunotherapy response.

1. For Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
T Looney1, S Pabla2, S Glenn2, G Lowman1, L Miller1, D Topacio-Hall1, E Linch1, A Pankov1, J Zheng1, J Conroy2, C Morrison2, F Hyland1.
(1) Thermo Fisher Scientific (2) OmniSeq, Inc., Buffalo, NY
Evidence for antigen-driven TCRβ chain convergence in the melanoma-infiltrating T cell repertoire
Abstract
T cell convergence refers to the phenomenon whereby
antigen-driven selection enriches for T cell receptors (TCRs)
having a shared antigen specificity but different amino acid or
nucleotide sequence. T cell recruitment and expansion within
the tumor microenvironment (TME) may be directed by
responses to tumor neoantigen, suggesting that elevated T
cell convergence could be a general feature of the tumor
infiltrating T cell repertoire. Here we use the Ion AmpliSeq™
Immune Repertoire Assay Plus – TCRβ to evaluate evidence
for T cell convergence within melanoma tumor biopsy
research samples from a set of 63 subjects plus peripheral
blood leukocytes (PBL) from four healthy subjects. We find
that the melanoma TME is highly enriched for convergent
TCRs compared to healthy donor peripheral blood. We
discuss the potential use of TCR convergence as a liquid-
biopsy compatible predictive biomarker for immunotherapy
response.
For Research Use Only. Not for use in diagnostic
procedures.
© 2018 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific
and its subsidiaries unless otherwise specified.
Conclusions
1.Ruggiero, E et al. Nature Communications (2015). 8081
2.Emerson, RO et al. Nature Genetics (2017). 49(5):659-665
The Ion Ampliseq Immune Repertoire Assay Plus – TCRβ is
available via www.thermofisher.com Catalog Number: A35386
Methods
References
Introduction
Results
Figure 4. A) Convergent clone score for healthy PBL (N=4) and melanoma
biopsies having greater than 100 detected clones (N=63). Convergence
scores are elevated in tumor compared to PBL (p<1E-7). Convergence
score is calculated as the number of clone pairs that are identical in amino
acid space divided by the total number of clone pairs, calculated as nCr2,
where n is the number of clones detected in a sample. B) Frequency of
convergent TCRs, calculated as the sum of the frequency of all convergent
clones detected in a sample. Tumors have more frequent convergent
clones than PBL (p<1E-4).
Here we report elevated TCRβ chain amino acid
convergence in melanoma biopsy T cell repertoires compared to
peripheral blood T cell repertoires derived from healthy donors.
Convergent TCR groups are typically not shared across subjects,
consistent with T cell responses to tumor-specific (i.e. private) neo-
epitopes.
Convergent TCRs are the result of T cell responses to antigen,
and thus may serve as a metric for quantifying tumor
immunogenicity. Importantly, we propose that TCR convergence is
a T cell repertoire feature which preferentially arises following
chronic antigen stimulation rather than the acute but transient
antigen challenges elicited by infectious disease. Given that tumor
antigens may prime T cells over months or years time,
measurements of TCR convergence may provide an accurate
estimate of tumor immunogenicity. To the extent that tumor
immunogenicity is a predictor of anti-tumor T cell responses during
immunotherapy, T cell convergence may hold particular utility as a
liquid biopsy compatible predictive biomarker for immunotherapy
response, potentially more so than metrics like tumor mutation
burden which indirectly assess tumor immunogenicity.
The extent to which elevated TCR convergence is a feature of
other cancer types, and is quantifiable in the peripheral blood of
individuals with cancer, will be addressed by ongoing studies.
Owing to tumor heterogeneity and greater variation in tumor
biopsy sample quality, one might predict PBL to be a preferred
input material for measurement of TCR convergence.
In reviewing commercially-derived and academic TCRβ
sequencing datasets generated using the Illumina platform (1,2),
we note that baseline convergent TCR frequencies in healthy
donors are far higher than those reported here (average 15% vs
.2%, 75-fold difference). Given that artifacts resembling
convergent TCRs may arise owing to base substitution sequencing
errors, these data suggest that TCR convergence may have been
overlooked as a potential predictive biomarker for clinical response
to immunotherapy owing to obfuscating platform-specific noise.
Accepting that members of a convergent TCR group share antigen
specificity, and given that the unsequenced TCRα chains of group
members are likely to differ, one can infer that convergent TCRs
are likely beta chain dominant. In such case, the full length TCRβ
chains provided by this assay could be paired with generic TCRα
chains to rapidly generate tumor antigen specific TCRs for future
therapeutic T cell applications.
Healthy PBL Tumor
-9
-8
-7
-6
-5
Convergence Scores
for PBL and Tumor
Log10ConvergenceScore
Healthy PBL Tumor
0.00
0.01
0.02
0.03
0.04
0.05
0.06
Convergent TCR Frequency
for PBL and Tumor
ConvergentTCRFrequency
Number of individuals
sharing convergent TCRB group
0
200
400
600
800
1000
1200
1400 1
2
3
4
5
6
7
8
10
CDR
loops
T Cell
Receptor
HLA
presenting
peptide
Variable Diversity Joining Constant
N1 N2
A. Adult IGH orTCRBeta chain rearrangement
In adult B andT cells, the process ofVDJ rearrangement very often involves
exonucleotide chewback ofVDJ genes and the addition of
non-templated bases, forming N1 and N2 regions in the B cell receptor
heavy chain CDR3 and theT cell receptor Beta chain CDR3.
These processes vastly increase IGH andTCRB CDR3 diversity.
Variable Diversity Joining Constant
B. Fetal IGH orTCRBeta chain rearrangement
In the fetus, the process ofVDJ rearrangement often occurs without
exonucleotide chewback ofVDJ genes and addition of non-templated
bases, resulting in a restricted IGH andTCRB CDR3 repertoire that is
distinct from the adult repertoire.
These structural differences can be used to distinguish fetal B andT cell
CDR3 receptors from maternal B andT cell CDR3 receptors in cell free
DNA present in maternal peripheral blood. In this way, fetal B andT
cell health and development may be monitored in a non-invasive
manner.
Figure 1. Structural differences between fetal and adult B andT cell receptors
~330bp amplicon
1B
Sequencing in multiplex on
S5 via 530 chip
FR1-C multiplex PCR (
AmpliSeq)
Ion Reporter analysis of
repertoire features and
polymorphism
Workflow
Figure 2. The assay utilizes AmpliSeq multiplex PCR primers to target
the TCRβ Framework 1 and Constant regions to enable both clonotyping
and detection of TRBV gene polymorphism linked to adverse events
during immunotherapy. AmpliSeq™ technology allows for use of a large
primer set enabling comprehensive coverage of potential TCRβ
rearrangements. TCRβ libraries are sequenced via the S5 and analyzed
on Ion Reporter server or cloud.
Tumor Antigen
T cells
Variable CDR3 AA CDR3 NT Freq
TRBV7-8 ASSLGQAYEQY GCCAGCAGCTTAGGTCAGGCATACGAGCAGTAC 1.8E-3
TRBV7-8 ASSLGQAYEQY GCCAGCAGCTTGGGACAGGCCTACGAGCAGTAC 4.8E-4
TRBV7-8 ASSLGQAYEQY GCCAGCAGCTTAGGGCAGGCCTACGAGCAGTAC 1E-4
●
0
20
40
60
80
TCRB V−gene usage and number of clones for TCR−005−RS−02738603_RNA_OIRRA−7_RNA_v1_20170517212600606
CDR3length(nt)
1
23
46
Number of Clones
201100 readsClone Shannon Diversity: 6.6392
Clone Evenness: 0.6278 1525 clones
Mean CDR3 NT length: 35.4133 +/− 5.3053
V−gene Shannon Diversity: 3.9098
V−CDR3 Frequency
0.10
0.05
●0.01
●0.005
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TRBV1
TRBV2
TRBV3−1
TRBV4−1
TRBV5−1
TRBV6−1
TRBV7−1
TRBV4−2
TRBV6−2
TRBV3−2
TRBV4−3
TRBV6−3
TRBV7−2
TRBV6−4
TRBV7−3
TRBV5−3
TRBV9
TRBV10−1
TRBV11−1
TRBV12−1
TRBV10−2
TRBV11−2
TRBV12−2
TRBV6−5
TRBV7−4
TRBV5−4
TRBV6−6
TRBV5−5
TRBV6−7
TRBV7−6
TRBV5−6
TRBV6−8
TRBV7−7
TRBV5−7
TRBV6−9
TRBV7−8
TRBV5−8
TRBV7−9
TRBV13
TRBV10−3
TRBV11−3
TRBV12−3
TRBV12−4
TRBV12−5
TRBV14
TRBV15
TRBV16
TRBV17
TRBV18
TRBV19
TRBV20−1
TRBV21−1
TRBV23−1
TRBV24−1
TRBV25−1
TRBV26
TRBV27
TRBV28
TRBV29−1
TRBV30
Figure 3. Spectratyping plot highlighting clones detected in a melanoma
biopsy. VDJ rearrangements are arranged along the X-axis according to
the variable gene of the rearrangement, and along the Y- axis according
to the CDR3 length in nucleotides (NT). This repertoire includes
dominating clones typical of T cell clonal expansion following tumor
antigen stimulation.
Table 1. Example of a convergent TCR group detected in a melanoma
biopsy. This group contains three TCRβ clones that are identical in TCRβ
amino acid space but have distinct CDR3 NT junctions owing to
differences in non-templated bases at the V-D-J junction. Blue indicates
bases contributed by the variable gene while yellow indicates bases
contributed by the joining gene. Black bases are those arising from
exonucleotide chewback and N-additions by the enzyme TdT. Red arrows
indicate positions where clones differ. Illumina-style base substitution
sequencing errors and PCR errors can create artifacts that resemble
convergent TCRs.
Metric Average (Range)
Total Clones Detected 5262 (113 - 44610)
Convergence Score 5.17E-6 (0 - 4.19E-5)
Convergent TCR Frequency 0.024 (0 - .231)
Convergent TCR Groups 22 (0 - 183)
Convergent TCR Clones 47 (0 - 399)
4A 4B
Table 5. Summary repertoire metrics
from TCR sequencing of melanoma
biopsies from 63 subjects. Convergent
TCR groups indicates the number of
TCRβ AA sequences found in more than
one clone.
Figure 5 (right). Sharing of convergent
TCR groups across subjects. Convergent
TCR groups are typically found in a single
donor.
Figure 6 (below). Heatmap of distribution
of convergent TCR groups across sample
set. Columns indicate different
convergent TCR groups detected in the
dataset (1341 total), while rows indicate
different donors. Red color indicates TCR
group is abundant in a subject.
Tumor neoantigens may stimulate the proliferation of T cells
possessing a stereotyped TCRβ amino acid sequence. Due to
the degeneracy of the amino acid code, T cell clones having
the same amino acid sequence may have different nucleotide
sequences (“convergent” TCRs). Measuring the features of
convergent TCRs, rather than all detected TCRs, allows one
to eliminate noise from TCRs that are not involved in tumor
antigen specific responses.
Figure 1. Convergence is a Hallmark of Chronic Antigen Stimulation
Priming and proliferation
of tumor antigen specific
T cells.
T cells do not destroy
tumor.
Tumor antigen continues
to prime naive T cells
with shared antigen
specificity.
This distinguishes T cell
responses to cancer
from those typical of
acute responses to
infectious disease..
Over time, convergent
TCR groups become
detectable in sample.
NumberofconvergentTCRBgroups
5
Number of samples
having TCR group
Convergence Scores For
PBL and Tumor
Convergent TCR Frequency for
PBL and Tumor