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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:W34

Thymic 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

12

34

5

1

100000

10000

1000

100

10

Synonymous Non-synonymous

0.0

0.2

0.4

0.6

0.8

1.0

fract

ion

of c

lone

s po

sses

sing

var

iant

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 Workflow TCRB 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

CD

R3

leng

th (n

t)

1

787

1574

Number of Clones

2112118 readsClone Shannon Diversity: 11.7485Clone Evenness: 0.742 58431 clones

Mean CDR3 NT length: 37.4392 +/− 5.311V−gene Shannon Diversity: 4.3052

V−CDR3 Frequency

0.10

0.05

●0.01

●0.005

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●

● ●

TRBV

1TR

BV2

TRBV

3−1

TRBV

4−1

TRBV

5−1

TRBV

6−1

TRBV

7−1

TRBV

4−2

TRBV

6−2

TRBV

3−2

TRBV

4−3

TRBV

6−3

TRBV

7−2

TRBV

6−4

TRBV

7−3

TRBV

5−3

TRBV

9TR

BV10−1

TRBV

11−1

TRBV

12−1

TRBV

10−2

TRBV

11−2

TRBV

12−2

TRBV

6−5

TRBV

7−4

TRBV

5−4

TRBV

6−6

TRBV

5−5

TRBV

6−7

TRBV

7−6

TRBV

5−6

TRBV

6−8

TRBV

7−7

TRBV

5−7

TRBV

6−9

TRBV

7−8

TRBV

5−8

TRBV

7−9

TRBV

13TR

BV10−3

TRBV

11−3

TRBV

12−3

TRBV

12−4

TRBV

12−5

TRBV

14TR

BV15

TRBV

16TR

BV17

TRBV

18TR

BV19

TRBV

20−1

TRBV

21−1

TRBV

23−1

TRBV

24−1

TRBV

25−1

TRBV

26TR

BV27

TRBV

28TR

BV29−1

TRBV

30

Clo

nes

per s

ubje

ct

CDR loops

T Cell Receptor

HLA presenting peptide

Variable Diversity Joining Constant

N1 N2

A. Adult IGH or TCRBeta chain rearrangement

In adult B and T cells, the process of VDJ rearrangement very often involves exonucleotide chewback of VDJ genes and the addition of non-templated bases, forming N1 and N2 regions in the B cell receptorheavy chain CDR3 and the T cell receptor Beta chain CDR3. These processes vastly increase IGH and TCRB CDR3 diversity.

Variable Diversity Joining Constant

B. Fetal IGH or TCRBeta chain rearrangement

In the fetus, the process of VDJ rearrangement often occurs withoutexonucleotide chewback of VDJ genes and addition of non-templated bases, resulting in a restricted IGH and TCRB CDR3 repertoire that is distinct from the adult repertoire.

These structural differences can be used to distinguish fetal B and T cellCDR3 receptors from maternal B and T cell CDR3 receptors in cell freeDNA present in maternal peripheral blood. In this way, fetal B and Tcell health and development may be monitored in a non-invasivemanner.

Figure 1. Structural differences between fetal and adult B and T cell receptors

~330bp amplicon

1A

1B

2A 2B

TRBV

1TR

BV2*

01/0

2TR

BV3−

1*01

TRBV

4−1*

01TR

BV5−

1*01

TRBV

6−1*

01TR

BV7−

1TR

BV4−

2*01

TRBV

6−2*

01TR

BV3−

2TR

BV4−

3*01

TRBV

6−3

TRBV

7−2*

01/0

2TR

BV6−

4*01

/02

TRBV

7−3*

01/0

3TR

BV5−

3*01

/02

TRBV

9*01

/02

TRBV

10−1

*02

TRBV

11−1

*01

TRBV

12−1

TRBV

10−2

*01

TRBV

11−2

*03

TRBV

12−2

TRBV

6−5*

01TR

BV7−

4*01

TRBV

5−4*

01TR

BV6−

6*02

/01

TRBV

5−5*

01/0

2TR

BV6−

7*01

TRBV

7−6*

01TR

BV5−

6*01

TRBV

6−8*

01TR

BV7−

7*01

TRBV

5−7*

01TR

BV6−

9*01

TRBV

7−8*

01TR

BV5−

8*01

TRBV

7−9*

01TR

BV13

*01

TRBV

10−3

*02/

03/0

1TR

BV11−3

*01

TRBV

12−3

*01

TRBV

12−4

*01

TRBV

12−5

*01

TRBV

14*0

1/02

TRBV

15*0

2TR

BV16

*01

TRBV

17TR

BV18

*01

TRBV

19*0

1TR

BV20−1

*01/

05/0

2TR

BV21−1

TRBV

23−1

*01

TRBV

24−1

*01

TRBV

25−1

*01

TRBV

26TR

BV27

*01

TRBV

28*0

1TR

BV29−1

*01

TRBV

30

V−gene usage, colored by allele for B501175_−_PBMC_Leukocyte_−_50ng_RNA_v1_20170811155825372

read

s ha

ving

v−g

ene

(thou

sand

s)

0

100

200

300

400

500