Human cytomegalovirus (CMV) is a common immune-evasive herpes family virus leading to lifelong asymptomatic infection in 50 to 80% of humans. Current research evaluating the use of TCR sequencing to predict response to immunotherapy has focused on measurements of T cell clonal expansion and TCR convergence (2,3,4) as potential predictive biomarkers for response. Given that CMV infection has been reported to elicit large clonal proliferations of CMV reactive T cells (1), and is a source of chronic antigen stimulation, we hypothesized that CMV infection might alter T cell repertoire features in a manner relevant to the potential biomarker use of TCR sequencing. Here we sought to identify features of CMV infection using TCRB profiling of peripheral blood (PBL) total RNA. We identify reduced T cell evenness and elevated TCR convergence as features of chronic CMV infection.

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ROC for Prediction of CMV Status by TCR Evenness and Convergence
Specificity
Sensitivity
1.0 0.8 0.6 0.4 0.2 0.0
0.0
0.2
0.4
0.6
0.8
1.0
Model Features
Convergence, AUC = .68
Evenness, AUC = .86
Convergence + Evenness, AUC = .93
T Looney1, J Zheng1 , G Lowman1, D Topacio-Hall1, F Hyland1. (1) Thermo Fisher Scientific
TCRB chain convergence in chronic cytomegalovirus infection and cancer:
insights from a novel potential immune repertoire biomarker
METHODS – Study Overview
Figure 5. TCR evenness is reduced in CMV+ donors.
Evenness values were lower in CMV+ subjects profiled
using the TCRB-LR and SR assays, suggesting that
CMV infection elicits T cell clonal expansion in
asymptomatic subjects (one-sided student’s t-test).
ABSTRACT
Human cytomegalovirus (CMV) is a common
immune-evasive herpes family virus leading to
lifelong asymptomatic infection in 50 to 80% of
humans. Current research evaluating the use of
TCR sequencing to predict response to
immunotherapy has focused on measurements of
T cell clonal expansion and TCR convergence
(2,3,4) as potential predictive biomarkers for
response. Given that CMV infection has been
reported to elicit large clonal proliferations of CMV
reactive T cells (1), and is a source of chronic
antigen stimulation, we hypothesized that CMV
infection might alter T cell repertoire features in a
manner relevant to the potential biomarker use of
TCR sequencing. Here we sought to identify
features of CMV infection using TCRB profiling of
peripheral blood (PBL) total RNA. We identify
reduced T cell evenness and elevated TCR
convergence as features of chronic CMV infection.
INTRODUCTION -- Definition of TCR
Convergence and Evenness
TCR Evenness is a measure of the similarity of
clone frequencies in a TCR repertoire. It is also
referred to as the normalized Shannon Entropy and
is equivalent to 1 - “clonality”. Evenness nearing 1
indicates that all clones are found at similar
frequencies in a sample.
Convergent TCRs are identical in amino acid
space but different in nucleotide space. They
represent instances where T cells independently
underwent VDJ recombination and proliferated in
response to a common antigen. In the context of
cancer, TCR convergence has been proposed to
serve as an indicator of the immunogenicity of a
tumor and thus its sensitivity to checkpoint blockade
therapy (2,3,4 and Table 2). Importantly, TCR
convergence has been proposed to arise in
response to a broad range of tumor associated
antigens (Table 1), including those derived from
viral infection.
CONCLUSIONS
• We identify reduced T cell evenness and elevated
TCR convergence as features of chronic CMV
infection.
• CMV infection appears to significantly alter the T
cell repertoire, suggesting that CMV status may be
required for proper interpretation of T cell clonal
expansion and TCR convergence in the context of
immunotherapy for cancer.
REFERENCES
1. Khan et al. Cytomegalovirus seropositivity drives the
CD8+ T cell repertoire toward greater clonality in elderly
individuals. J Immunol 2002; 169:1984
2. Zhang et al. Peripheral blood TCRB repertoire
convergence and evenness predict response to CTLA-4
monotherapy for cancer. SITC 2018.
3. Storkus et al. Peripheral blood TCRB chain convergence
predicts response to dendritic cell-based immunotherapy
in advanced-stage melanoma patients. SITC 2018.
4. Looney et al. Evidence for antigen-driven TCRB chain
convergence in the melanoma infiltrating T cell repertoire.
AACR 2018
Figure 7. TCR convergence and evenness are
uncorrelated. Correlation was evaluated using
Spearman test (TCRB-SR data shown). The lack of
correlation suggests these features may be combined
to improve the prediction of CMV status.
Ion Reporter Analysis of
TCR Repertoire
Features
Sequencing on Ion Gene
Studio S5, targeting
1.5M reads depth per
library
FR1-C or FR3-J
multiplex PCR from 25ng
PBL cDNA
(17 CMV-, 18 CMV+)
Study Workflow
RESULTS – Elevated TCR Clonal
Expansion and Convergence in CMV+
Donors
RESULTS – Combination of TCR
Convergence and Evenness Improves
Prediction of CMV Status
SensitivityTable 1. Types of antigens measured by tumor
mutation burden and TCR convergence.
Antigen Source
Tumor
Mutation
Burden
TCR
Convergence
Non-Synonymous Mutations ✔ ✔
Aberrant Post-Translational
Modifications ✖ ✔
Ectopic Gene Expression ✖ ✔
Splicing Defects ✖ ✔
Autoantigens ✖ ✔
Virus-Derived Antigens ✖ ✔
Figure 2. Experimental Study.
Total RNA from PBL was
obtained from 35 healthy blood
donors of known CMV status,
then used for TCRB sequencing
via the Oncomine TCRB-LR
assay (amplicon spanning
CDR1, 2 and 3) and the
Oncomine TCRB-SR assay
(CDR3 only). Libraries were
sequenced using the Ion Gene
Studio S5, then analyzed via
Ion Reporter. Data were used to
identify TCRB repertoire
features correlated with CMV
status and compare repertoire
features across the two assays.
Figure 3. Assay Design. The Oncomine TCRB-LR
assay utilizes AmpliSeq multiplex framework 1 (FR1)
and constant gene primer to amplify all three CDR
domains of the rearranged TCRB chain (~330bp
amplicon) from non-FFPE RNA. The Oncomine TCRB-
SR assay utilizes framework 3 (FR3) and joining gene
(J) primers to amplify the CDR3 region of the TCRB
chain (~80bp amplicon) from fresh or FFPE-preserved
RNA or DNA input.
4 For Research Use Only. Not for use in diagnostic procedures.
Oncomine TCR Beta Sequencing | Enabling TCR Profiling from RNA and DNA Input
V D J C
FR1 FR2 FR3
CDR1 CDR2 CDR3
325-400bp amplicon64 FR1
V-region
primers
2 C- region
primers
V D J C
FR1 FR2 FR3
CDR1 CDR2 CDR3
80bp amplicon59 FR3
V-region
primers
14 FR3 J
region
primers
Oncomine TCR Beta-LR* Assay
Oncomine TCR Beta-SR** Assay
α β
T Cell
FFPE
Blood
*LR = Long Read
**SR = Short Read
Figure 6. TCR convergence is elevated in CMV+
donors. The aggregate frequency of convergent TCRs
was higher in CMV+ subjects profiled using the TCRB-
LR and SR assays, suggesting that T cell responses to
chronic CMV infection give rise to convergent TCR
responses (one-sided student’s t-test).
Figure 8. ROC curve for the prediction of CMV
status using TCR convergence and evenness.
ROC curves are presented for each feature alone,
and for a logistic regression classifier (R caret
package) trained using TCR evenness and
convergence as features to predict response to
immunotherapy. The combination of features
improves the prediction of CMV status (AUC = .93)
0.4
0.5
0.6
0.7
0.8
0.9
TCR Evenness Across Datasets
TCREvenness(1-clonality)
CMV Negative
CMV Positive
0.00
0.02
0.04
0.06
0.08
0.10
Frequency of Convergent Rearrangements Across Datasets
ConvergentTCRFrequency
CMV Negative
CMV Positive
0.02 0.04 0.06 0.08
0.4
0.5
0.6
0.7
0.8
0.9
cmv$SR_convergent_TCR_frequency
cmv$SR_Evenness
CMV Negative
CMV Positive
TCREvenness(1-clonality)ConvergentTCRFrequency
TCREvenness
TCR Convergence
Specificity
p = 7.1E-4 p = 1.4E-4
p = .03 p = .05
TCRB-SR TCRB-LR
TCRB-SR TCRB-LR
cor = -.03
INTRODUCTION – Recent Reports
Correlating TCR Convergence with
Immunotherapy Response
Study Lead and
Publication
Cancer and
Therapy
Correlation
between TCR
Convergence
and Response
Walt Storkus
UPMC
SITC 2018 (2)
Dendritic cell
vaccine for
advanced
melanoma
++
Larry Fong
UCSF
SITC 2018 (3)
Anti CTLA-4 for
multiple cancers +
Philip Jermann
Univ of Basel
EACR 2019
Anti PD-1 for
NSCLC +
+ Indicates significant positive correlation between
pretreatment TCR convergence and response