Polymorphism within the TCRB variable gene (TRBV) has been linked to chronic autoimmune diseases such as Type 1 Diabetes, Rheumatoid Arthritis, Psoriatic Arthritis, Multiple Sclerosis and Asthma (1-8), and may also be mechanistically linked to immune mediated adverse events (IMAEs) during immunotherapy (9-11). Here we use the Ion-AmpliSeq™ Immune Repertoire Plus TCRB assay to evaluate TRBV gene polymorphism in a group of 85 Caucasians with melanoma. The assay provides coverage of all three CDR domains to enable detection of TRBV polymorphism. We find evidence of extensive genetic diversity within the TRBV gene, including 15 nonsynonymous variants that are absent from the IMGT database (12). TRBV gene allele typing may provide rich biomarker information for the prediction of IMAEs and chronic autoimmune disease.

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T Looney1, A Glavin1, S Pabla2, S Glenn2, L Miller1, D Topacio-Hall1, E Linch1, A Pankov1, J Zheng1, J Conroy2, C Morrison2,
G Lowman1, M Andersen1, F Hyland1. (1) Thermo Fisher Scientific (2) OmniSeq, Inc., Buffalo, NY
Widespread human T cell receptor beta variable gene polymorphism:
implications for the prediction of IMAEs and immunotherapy outcome
METHODS – Informatics Workflow
Figure 4. V-gene usage plot highlighting detected IMGT
alleles. Reads mapping to each variable gene within the
repertoire are color-coded by the best matching IMGT allele.
Allele designations are also indicated on the X-axis label.
Heterozygous loci manifest as multi-colored bars. In some
cases, the best-matching IMGT allele imperfectly matches a
sample variable gene sequence. This may indicate the
presence of an allele that is absent from the IMGT database.
Figure 7. Example of a non-synonymous IMGT variant.
IgBLAST (14) alignment of an allele having two amino acid
substitutions compared to the best matching IMGT allele. This
particular allele was detected in our sample cohort and the
Lym1K database derived from 1000 genomes data.
Figure 5. Clones detected per subject
from a combination of tumor biopsy
and peripheral blood TCRB profiling.
Fresh frozen RNA from melanoma biopsy
from 85 Caucasians was used for TCRB
sequencing. When available, PBL RNA
was also used for TCRB sequencing. We
typically detected between 1000 and
10000 clones per individual, which were
analyzed for the presence of non-IMGT
TRBV gene alleles. Subjects having
fewer than 100 detected clones were
excluded from downstream analysis.
ABSTRACT
Polymorphism within the TCRB variable gene (TRBV)
has been linked to chronic autoimmune diseases such
as Type 1 Diabetes, Rheumatoid Arthritis, Psoriatic
Arthritis, Multiple Sclerosis and Asthma (1-8), and may
also be mechanistically linked to immune mediated
adverse events (IMAEs) during immunotherapy (9-11).
Here we use the Ion-AmpliSeq™ Immune Repertoire
Plus TCRB assay to evaluate TRBV gene
polymorphism in a group of 85 Caucasians with
melanoma. The assay provides coverage of all three
CDR domains to enable detection of TRBV
polymorphism. We find evidence of extensive genetic
diversity within the TRBV gene, including 15 non-
synonymous variants that are absent from the IMGT
database (12). TRBV gene allele typing may provide
rich biomarker information for the prediction of IMAEs
and chronic autoimmune disease.
INTRODUCTION
The antigen specificity of the T cell receptor is determined in
part by the sequence of the CDR and Framework regions
encoded by the TRBV gene (1A). Polymorphism within the
TRBV CDR1 and 2 regions can modulate TCR interaction with
HLA, potentially increasing the likelihood of auto-antigen
recognition. (1B) The assay utilizes AmpliSeq multiplex PCR
primers to target the TCRB Framework 1 and Constant regions
to enable detection of TRBV gene polymorphism. AmpliSeq
technology allows for use of a large primer set enabling
comprehensive coverage of potential TCRB rearrangements.
FR1 CDR3 Constant
CONCLUSIONS
We find evidence for extensive TRBV gene allelic
diversity beyond what is represented in the IMGT
database. The results are particularly striking given
that Caucasians are a well-studied ethnic group. The
significance of TRBV gene polymorphism in the
context of checkpoint blockade is not yet known,
though we note evidence suggesting that uncommon
non-synonymous variants may be disfavored during T
cell development, potentially due to auto-reactivity.
TRBV allele typing can be performed using as little as
10ng PBL RNA, facilitating retrospective analyses.
Finally, we note that the variable gene allele is
germline encoded, giving it the potential to act as a
true predictive biomarker for IMAEs and chronic
autoimmune disorders.
REFERENCES
1. Concannon et al. J Exp Med (1987) 165:1130
2. Subrahmanyan et al. Am J Hum Genet (2001) 69:381
3. Pierce et al. J Diabetes Res (2013) 737485
4. McDermott et al. Arth Rheum 38:1 (1995)
5. Maksmoyowych et al. Immunogenetics (1992) 35:257
6. Uebe et al. BMC Medical Genetics (2017) 18:92
7. Hockertz et al. Am J Hum Genet (1998) 62:373
8. Gras et al. J Ex Med (2010) 207:7
9. Hughes et al. Diabetes Care (2015) 38:e55
10. Okamoto et al. J Diabetes Investig (2016) 7:915
11. Gaudi et al. Diabetes Care (2015) 38:e182
12. Lefranc et al. Nucleic Acids Res (2015). 43:D413
13. Yu et al. J Immunol (2017) 198:2202
14. Ye et al. Nucleic Acids Res (2013) 41:W34Thymic Selection
Severe IMAE
Auto-reactive CDR3
Allele name
Location of
AA variant
Individuals
having allele
In Lym1k?
In NCBI NR
database?
TRBV11-2*32k
FR3
1
No
No
TRBV11-3*1k
FR2
17
No
Yes
TRBV12-4*46k
FR2
1
No
No
TRBV12-5*4k
FR2
1
No
No
TRBV19*17k
FR2
1
No
No
TRBV23-1*2k
FR3
1
No
No
TRBV24-1*1k
FR2
39
No
Yes
TRBV5-3*1k
FR2
1
No
No
TRBV5-8*1k
FR1
17
No
No
TRBV6-2*156k
FR1, CDR1,
FR2, CDR2,
FR3
1
No
No
TRBV6-5*106k
CDR2
1
No
No
TRBV11-1*11p
CDR1, FR2/
CDR2
1
Yes
No
TRBV30*6p
FR3
1
Yes
No
TRBV5-5*9p
FR3
2
Yes
No
TRBV5-6*11p
FR3
3
Yes
No
Non-synonymous variant
Synonymous variant
Key
12345
1
100000
10000
1000
100
10
Synonymous Non-synonymous
0.0
0.2
0.4
0.6
0.8
1.0
fractionofclonespossessingvariant
Figure 6. Distribution of synonymous and non-synonymous
variants in sample cohort. 74 subjects having more than 100
detected clones were evaluated for the presence of synonymous
or non-synonymous variants of IMGT alleles. 16 of 74 (22%)
subjects possess an uncommon, non-synonymous variant.
Figure 8. Fraction of clones
possessing variant allele. For
each instance where a novel allele
was detected, we determined the
fraction of clones having that TRBV
gene which also possessed the
novel allele. Under neutral selection,
approximately 50% of the clones
should possess a novel allele in a
heterozygous carrier. Non-
synonymous variants tend to be
found at lower frequency than
synonymous variants, suggesting
that they may be disfavored during
T cell maturation, potentially due to
auto-reactivity.
Auto-reactive TRBV
Thymic Selection
Healthy
Checkpoint blockade
Figure 2. Model for the emergence of IMAEs in a carrier
of an auto-reactive TRBV allele (2A) T cells possessing
auto-reactive TCRs are eliminated by thymic negative
selection, preventing autoimmune disease in a carrier of an
auto-reactive TRBV allele. (2B) Checkpoint inhibition reduces
thymic negative selection, leading to the emergence of auto-
reactive T cells that may cause IMAEs.
Identify mismatches to
IMGT
Evidence-based filtering
Identify clones
FR1-C multiplex PCR
Report novel alleles
Compare to 1000
genomes and NCBI NR
Ion Reporter WorkflowTCRB sequences are amplified using
non-FFPE RNA from tumor biopsy,
peripheral blood or sorted cells,
followed by multiplex sequencing via
the Ion S5 530 chip (15-20M reads).
PCR and sequencing errors are
eliminated before clone reporting. In
some cases, an individual will
possess a plurality of clones that do
not match any IMGT variable gene
allele; this may indicate presence of a
novel allele. If sufficient clone support
exists, Ion Reporter classifies the
sequence as a putative novel variant.
As a last step, putative variants are
compared to those found in the
Lym1k database (13) derived from
1000 genomes data and the NCBI NR
database.
Figure 3. Spectratyping plot highlighting clones detected
in a peripheral blood sample. Clones detected from
sequencing of 50ng peripheral blood leukocyte (PBL) RNA.
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). The
ordering of variable genes respects the genomic position of
the variable genes within the TRB locus. Size of the circle
indicates the frequency of a particular V-gene + CDR3 NT
length combination, while color indicates the number of clones
having each V-gene + CDR3 NT combination. Key repertoire
metrics are displayed along bottom of plot. Carriers of non-
IMGT TRBV gene alleles will present with a plurality of clones
sharing a mismatch to the IMGT reference over the variable
gene of the rearrangement. Plot is taken from Ion Reporter.
RESULTS – TCRB sequencing of
85 Caucasians with melanoma
Attributes of non-synonymous TRBV
alleles detected in 85 Caucasians
●
0
20
40
60
80
TCRB V−gene usage and number of clones for B501175_−_PBMC_Leukocyte_−_50ng_RNA_v1_20170811155825372
CDR3length(nt)
1
787
1574
Number of Clones
2112118 readsClone Shannon Diversity: 11.7485
Clone Evenness: 0.742 58431 clones
Mean CDR3 NT length: 37.4392 +/− 5.311
V−gene Shannon Diversity: 4.3052
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
Clonespersubject
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
1A
1B
2A 2B
TRBV1
TRBV2*01/02
TRBV3−1*01
TRBV4−1*01
TRBV5−1*01
TRBV6−1*01
TRBV7−1
TRBV4−2*01
TRBV6−2*01
TRBV3−2
TRBV4−3*01
TRBV6−3
TRBV7−2*01/02
TRBV6−4*01/02
TRBV7−3*01/03
TRBV5−3*01/02
TRBV9*01/02
TRBV10−1*02
TRBV11−1*01
TRBV12−1
TRBV10−2*01
TRBV11−2*03
TRBV12−2
TRBV6−5*01
TRBV7−4*01
TRBV5−4*01
TRBV6−6*02/01
TRBV5−5*01/02
TRBV6−7*01
TRBV7−6*01
TRBV5−6*01
TRBV6−8*01
TRBV7−7*01
TRBV5−7*01
TRBV6−9*01
TRBV7−8*01
TRBV5−8*01
TRBV7−9*01
TRBV13*01
TRBV10−3*02/03/01
TRBV11−3*01
TRBV12−3*01
TRBV12−4*01
TRBV12−5*01
TRBV14*01/02
TRBV15*02
TRBV16*01
TRBV17
TRBV18*01
TRBV19*01
TRBV20−1*01/05/02
TRBV21−1
TRBV23−1*01
TRBV24−1*01
TRBV25−1*01
TRBV26
TRBV27*01
TRBV28*01
TRBV29−1*01
TRBV30
V−gene usage, colored by allele for B501175_−_PBMC_Leukocyte_−_50ng_RNA_v1_20170811155825372
readshavingv−gene(thousands)
0
100
200
300
400
500