Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.comFor Research Use Only. Not for use in diagnostic procedures. The content provided herein may relate to products that have not been officially released and is subject to change without notice.
G Lowman1, T Looney2, E Linch1, L Miller1, D Topacio-Hall1, A Pankov2, J Zheng2, R Hartberg3, H Almåsbak3, T E Stav-Noraas3, A Kullmann3, F Hyland2, M Andersen1
(1) Thermo Fisher Scientific, Carlsbad, CA, USA (2) Thermo Fisher Scientific, South San Francisco, CA, USA (3) Thermo Fisher Scientific, Oslo, Norway
Insights into the tumor microenvironment and therapeutic T cell manufacture revealed by long amplicon immune repertoire sequencing
ABSTRACTTCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy1-4. Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells.
CONCLUSIONSThese results demonstrate: (1) The accuracy and versatility of immune repertoire sequencing using the Ion AmpliSeqTM
Immune Repertoire Assay Plus – TCRβ, (2) The benefit of combining targeted gene expression and repertoire profiling for studies of the tumor microenvironment, (3) The utility of repertoire sequencing covering all CDR regions in monitoring the manufacture of therapeutic T cells.
REFERENCES1. Robins, H. S. et al. Blood 114:19 (2009)2. Carlson, C. S. et al. Nat. Commun. 4, 2680 (2013)3. Li, B. et al. Nature Genetics 48, 725–732 (2016)4. Sheikh, N. et al. Cancer Res. 76:13 (2016)5. Sandberg et al. Leukemia 21:2 (2007) 6. Liu, X. et al. PLOS One 11:3 (2016)7. Thompson, J.R. et al. Nucleic Acids Res. 30:9 (2002)8. Qiu, X. et al. App. and Env. Microbiology 67:2 (2001)9. Wang, G. et al. App. and Env. Microbiology 63:12 (1997)
0.2
0.4
0.6
0.8
1.0
1
T ce
ll ev
enne
ss
1 = Most even sizes0 = Least even sizes
19 NSCLC biopsies
Number of input T cells (log10)
Nu
mb
er
of u
niq
ue
re
arr
an
ge
me
nts
(lo
g1
0)
3 4 5
3
4
5
1,000 10,000 100,0001,000
10,000
100,000
Number of Input T cells
Clo
nes
Det
ecte
d
Sequencing of Counted T cells
Repressive Tumor Microenvironment
Permissive Tumor Microenvironment
Inhibits T cell responses to tumorPermits T cell expansion and
anti-tumor activity
High T cell evenness Low T cell evenness
0.6
0.7
0.8
0.9
1.0
T cell clone evenness during in vitro expansionvia anti-CD3/CD28 beads
Clo
ne E
venn
ess
Donor 1Donor 2
Day
0 P
BM
C p
re-is
olat
ion
Day
0 P
BM
C p
ost-i
sola
tion
Day
3 p
re-b
ead
rem
oval
Day
3 p
ost-b
ead
rem
oval
Day
10
0.00001
0.0001
0.001
0.01
0.1
1
0.00001 0.0001 0.001 0.01 0.1 1
57 copies
566 copies
5,655 copies
56,552 copies
Obs
erve
d Pl
asm
id F
requ
ency
30plasmidspike-in
Detection of reference rearrangement spike-ins
Plasmid Input (pg)
Figure 1. Ion AmpliSeq Immune Repertoire Assay Plus – TCRβMultiplex AmpliSeq primers target the framework region 1 (FR1) and constant (C) regions of the TCRβ producing a ~330bp amplicon which covers the entire variable gene and the CDR3 region. The assay utilizes RNA input from blood leukocytes, fresh-frozen tissue, or sorted T cells and has a flexible input range between 10ng and 1μg.
INTRODUCTIONTo evaluate assay accuracy we sequenced libraries derived from 30 well-studied T cell lymphoma rearrangements5,6, then compared our results with those reported by another commercially available immune repertoire sequencing technology. We next used the assay to profile tumor infiltrating T cell repertoires for a cohort of 19 individuals with non-small cell lung cancer. We correlate repertoire features with gene expression profiling data. We then harnessed the long read capability of the assay to profile T cells at various stages of the therapeutic T cell manufacturing process.
ASSAY ACCURACYLong read TCRβ sequencing of 30 reference T cell lymphoma rearrangements cloned into plasmids in a background of PBL yielded strong linearity in detection of clonal frequencies in reference spike-in experiments. We further demonstrate the quantitative nature of the assay by studying populations of counted T cells.
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
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
FR1 FR2 Diversity(D) Joining (J)
Constant
Variable gene (V)
CDR3
FR3
~330bp Amplicon
CDR1 CDR2
Figure 2. Detection of reference rearrangements Libraries were prepared using pools of 30 known lymphoma rearrangements at known input concentrations (calculated to equal ~50,000 to ~5 copies of RNA) in a background of 100ng of leukocyte RNA. We observe strong linearity across five orders of magnitude of control input concentration.
Figure 3. Detection of counted T cells Libraries were prepared using RNA extracted from counted populations of T cells (1,000 – 100,000). The detected clone frequency is linear and in agreement with known T cell input. Importantly, there is not evidence of a large false positive rate for clone detection.
To test the reproducibility of the assay between library replicates, we constructed 16 libraries from the peripheral blood leukocyte sample. Correlations between variable gene usage and Top 50 clone detection frequency yield minimum correlation values of r=0.97 for variable gene usage and r=0.96 for Top 50 clone detection frequency.
Tumor biopsy revealed 589 unique TCR. • Oligoclonal repertoire with a small
number of dominating clones; • Shannon diversity: 6.78
PBL revealed 45305 unique TCR.• Diverse, polyclonal repertoire with few
highly expanded T cells • Shannon diversity: 13.95
RESULTSThe Ion AmpliSeq Immune Repertoire Assay Plus TCRβ kit was applied to study the overlap between between circulating and tumor-infiltrating T cells. 100ng of total RNA derived from PBL and tumor biopsy from an individual with Stage 1B squamous cell carcinoma of lung was used as template.
Sample Types:• Blood • Fresh Frozen Tissue • Sorted T cells
Sequencing of matched TIL and peripheral blood exhibited differing repertoire features between samples. The tumor biopsy showed an oligoclonal repertoire with a small number of dominating clones and a Shannon diversity value of 6.78. The peripheral blood samples exhibited a diverse, polyclonal repertoire with few highly expanded T cells and a Shannon diversity value of 13.95. By correlating the repertoires, we observe 219 clones that are shared between samples. There are 370 clones that are unique to the tumor sample, with a subset of these clones which are highly expanded. This highly expanded set of clones unique to the tumor could potentially point to T cells responding to tumor-specific antigen.
To further study the ability of immune repertoire sequencing to probe the tumor microenvironment (TME), we sought to correlate repertoire features with gene expression profiles derived from the OncomineTM Immune Response Research Assay (OIRRA). In a repressive TME the T cell repertoire may exhibit high evenness (with few expanded clones) due to inhibition of T cell response. In a permissive TME the repertoire may exhibit low evenness (with T cell expansion) due to T cell response to the tumor. We sought to correlate these metrics with several immune response gene expression categories.
Clone overlap for PBL and Tumor
Log10 frequency in PBL
Log1
0 fre
quen
cy in
tum
or-8
0-2
-4-6
-8 -6 -4 -2 0
45086 clones unique to PBL
219 shared clones
370 clones unique to tumor
Figure 6. Correlation of T cell evenness and gene expression profile (A) Schematic illustrating T cell response in repressive/permissive tumor microenvironments. (B-C) Libraries were prepared from 19 NSCLC biopsies using the Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ and the OncomineTM Immune Response Research Assay (OIRRA). The majority of this cohort exhibited high T cell evenness – suggesting repressive tumor microenvironments. Using a largest principle component analysis of each gene category within the OIRRA panel, we see correlation between evenness values and markers for myeloid response.
(A)
(B)Proliferation
Drug_target
Checkpoint_pathway
NK_cell_marker
T_cell_differentiation
Housekeeping
Leukocyte_inhibition
Antigen_processing
Macrophage
Type_I_interferon_signaling
Neutrophil
Lymphocyte_infiltrate
NK_activation
Dendridic_cell
Interferon_signaling
Tumor_marker,stemness
Antigen_presentation
Cytokine_signaling
Lymphocyte_development
T_cell_regulation
Chemokine_signaling
Tumor_antigen
Type_II_interferon_signaling
TCR_coexpression
Helper_T_cells
Tumor_marker
PD-1_signaling,tumor_marker
Myeloid_marker,MDSC
Leukocyte_migration
Lymphocyte_activation
Adhesion,migration
Dendridic_cell,macrophage
Apoptosis
Myeloid_marker
PD-1_signaling
T_cell_receptor_signaling
T_cell_regulation,trafficking
B_cell_marker
Innate_immune_response
B_cell_receptor_signaling
Myeloid_marker,stem_cell
Gene function correlation with clone evenness
Correlation
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Correlation with Oncomine™ Immune Response Research Assay
Gene categories
(C)
Figure 7. T cell evenness measurement during therapeutic T cell manufacturing process. T cell clone evenness is tracked for two separate donors from Day 0 (PBMC pre- & post-isolation), Day 3 (pre- and post-bead removal), and after Day 10 in culture. Importantly, T cell evenness increases over time, showing that there is no bias for expansion in particular T cell populations in culture using CTSTM DynabeadsTM anti-CD3/CD28 beads and CTSTM OpTimizerTM serum-free media.
PBMC-derived T cells were subjected to in vitro expansion via CTSTM DynabeadsTM anti-CD3/CD28 beads and CTSTM
OpTimizerTM serum-free media as part of a therapeutic T cell research study. T cell expansion was measured using T cell clone evenness output by Ion ReporterTM analysis of data generated by the Ion AmpliSeq Immune Repertoire Assay Plus – TCRβ panel. There is a consistent increase in evenness with cell culture time, suggesting that anti-CD3/CD28 beads and CTS™ OpTmizer™ media promote polyclonal (unbiased) T cell expansion.
Table 2. Steps in the manufacture of therapeutic T cells. Explanation of the role of T cell repertoire sequencing in the steps involved in carrying T cells from isolation, through expansion, transduction, and introduction
Figure 5. Overlap of clones identified in matched TIL and PBL samples Plot showing clones identified and unique to peripheral blood (45,086 - along x-axis), unique to TIL (370 - along y-axis), and found in both samples (219 - middle of plot).
Figure 4. Correlation of variable gene usage and Top 50 clone frequency across 16 libraries. Plot showing correlation between 16 replicate libraries for (A) variable gene usage and (B) Top 50 clone detection frequency.
Top Related