Development of a TCR beta repertoire assay for profiling liquid biopsies from NSCLC donors

Isolation of PBL RNA from NSCLC donors

Whole blood was collected from 5 donors in Streck BCT®. Specimens were shipped at ambient temperature to our centralized clinical testing laboratory and processed within 4 days after blood was drawn. Whole blood was centrifuged at 800 ×g for 20 min, and buffy coat cells were recovered from the plasma/red blood cell (RBC) interface and purified using RBC Lysis Buffer (ammonium chloride-based lysing reagent), according to the manufacturer’s technical data sheet (Tonbo Biosciences, CA). RNA was extracted from purified buffy coat cells using the RNeasy® FFPE Kit, including a DNase treatment step (Qiagen, CA). The specimens used in our study were Institutional Review Board waived as they were remnant, de-identified specimens.

T-cell expansion with CMV model antigen stimulation

Cryopreserved PBMC, initially recovered by leukapheresis using Ficol, were thawed in X-VIVO^TM 15 (Lonza, Basel, Switzerland) medium. We chose 4 different normal healthy, CMV-seropositive, and HLA-A*0201-positive donors for this study. Thawed cells were collected at baseline or cultured for 6 days in the presence of 1 µg/mL CMV whole antigen (partially purified lysate from CMV-infected human fibroblast cells) or 1 µg/mL HLA-A*0201^-restricted CMV immunodominant peptide (pp65_495-503, NLVPMVATV). IL-2 (10 U/mL) was added one day after the start of the culture.

For sequencing, cells were lysed using Buffer RLT Plus (Qiagen, CA) supplemented with β-mercaptoethanol and frozen at -80°C until RNA extraction. RNA was isolated according to the manufacturer’s instructions using the RNeasy-Plus® Mini Kit (Qiagen, CA).

PBMC were prepared for flow cytometry using HLA-A*0201-CMVpp65495-503 tetramer-PE reagent (MBL International, MA). Cells were incubated with HLA-A*0201-CMVpp65_495-503 tetramer-PE reagent (MBL International, Woburn, MA) for 15 min at room temperature, followed by incubation with anti-human CD8a-FITC antibody (clone RPB-T8, BioLegend, San Diego, CA) at 4 °C for 30 min. 7-AAD was included in the final buffer to exclude dead cells. Data were acquired in a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using BD Cell Quest software. Analyses were done using FCS Express version 4 (De Novo Software, Pasadena, CA). Percent HLA-A*0201/CMVpp65_(495-503) tetramer-positive population was quantified from live (7-AAD^-) CD8⁺ cells.

TCRβ sequencing with Ion Torrent

The Qubit^TM RNA HS assay (ThermoFisher Scientific, MA) was used to quantify RNA. Agilent RNA 6000 Nano Assay was used to determine RNA integrity number (RIN) and DV200 scores. cDNA was generated using the SuperScript^TM IV VILO^TM Kit (ThermoFisher Scientific, MA). Following cDNA synthesis, the input for library preparation was determined based on a functional CD3 RNA qualification assay, which measures the CD3 fraction of mixed cell populations and cDNA amplifiability^[6].

The Oncomine TCR Beta-SR Assay (ThermoFisher Scientific, MA) was used to sequence CDR2, CDR3, and joining regions of the TCR Beta locus, as illustrated in Figure 1 (used with permissions from ThermoFisher Scientific). Sequencing was performed on the Ion GeneStudio^TM S5 Plus. Twelve specimens were multiplexed per Ion 530^TM Chip prior to sequencing using the Ion Chef^TM instrument.

Figure 1. The Oncomine TCR Beta-SR Assay targets the complementarity-determining region 3 (CDR3) within the TCR beta chain locus. The assay consists of multiplexed primers that bind to framework region 3 (FR3) and joining regions of the locus, producing amplicons approximately 80 bp in length for sequencing. TCR: T-Cell receptor

TCR beta sequencing in NSCLC donor specimens collected in Streck BCT

To assess TCR repertoire features in clinical specimens, whole blood was collected from 5 different NSCLC donors in Streck RNA BCT and shipped to the centralized laboratory. RNA was isolated for TCR sequencing from the buffy coat layer, which contains a large fraction of PBLs. DV200 score, which was developed to measure the quality of highly degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples for RNA-seq, was used to measure RNA quality. This method is suitable for the assessment of PBL RNA from Streck BCT, which tends to be fragmented following shipment at ambient temperature.

The input quantity used for library preparation was based on a functional CD3 RNA qualification assay, which measures both the T-cell RNA content and the amplifiable quality of the sample. As shown in Table 1, the extracted RNA for each sample varied in quality and T-cell content. DV200 values ranged from 52%-77%, and RNA input ranged from 34-400 ng. For donors 1-4, these highly variable inputs were successfully normalized by the qualification assay and resulted in consistent library metrics (1.55-2.13 million raw reads, 73%-83% usable reads, 44,299-61,936 clones). However, for donor 5, the number of raw and usable reads (1.17 million, and 63%, respectively) was relatively low and the assay detected many fewer clones compared to the other samples (n = 10,731), potentially due to lower input quantity.

For the Oncomine TCR Beta-SR assay, T-cell repertoire metrics are reported using several precalculated outputs, including TCR Convergence and Evenness scores. For this study, Clonality was determined from the Evenness score and was defined as 1- Evenness. Scores ranged 0.005-0.033 for TCR Convergence and 0.09-0.48 for Clonality across the 5 donors [Table 1]. Donor 5 had the highest clonality score, and the expansion of 2 different clones was visible in the Spectratyping plot [Figure 2E, as compared to Figure 2A-D], which is a standard output of the Ion Reporter software.

Figure 2. Spectratyping plots for the five NSCLC specimens. Donor 1-5 correspond to A-E, respectively. Circles are bins containing all clones with a particular V gene-CDR3 nucleotide (nt) length combination. Circle size indicates the frequency of all clones contained in the bin, and color indicates the frequency of the most prevalent clone present in the bin (darker orange with higher frequency). NSCLC: non-small cell lung cancer

T-cell expansion with CMV model antigen stimulation

To study the TCR repertoire changes due to antigen stimulation, T-cell populations from four CMV-seropositive donors were analyzed after a 6-day in vitro stimulation with CMV whole antigen (“Lysate”) or a HLA-A*0201^-restricted CMV immunodominant peptide (“Peptide”).

RNA was isolated from PBMC prior to antigen challenge (“Baseline”) and after stimulation and used to prepare libraries for TCR sequencing following the same workflow used for the NSCLC specimens. As shown in Table 2, a lower input of RNA (25-107 ng) was required due to the high quality of the RNA (RIN Scores 9.9-10) and resulted in similar sequencing metrics to what was seen with relatively fragmented RNA from Streck BCT (1.43-2.16 million raw reads, 81%-85% usable reads).

TCR Convergence scores varied across donors at baseline and in response to antigen challenge, as shown in Figure 3A. The highest TCR Convergence score was observed in peptide-stimulated cells from Donor 3 and was driven by two sets of TCR convergent clones [Figure 3B].

Figure 3. TCRβ convergence scores for the CMV model antigen stimulation study. Results are shown for two replicate Ion 530 chips (A); Two examples of convergent T-cell clones are shown for cells stimulated with pp65 peptide from Donor 3 (B). TCRβ: T-Cell receptor beta; CMV: cytomegalovirus

Clonality scores increased in the CMV lysate condition relative to baseline for all 4 donors, and the increase was most dramatic for Donor 4 [Figure 4A]. This clonal expansion could be clearly observed in the Spectratyping plots, as shown in Figures 4B and 4C. The most prevalent clone and driver of the high clonality score was observed at 76% frequency within the dataset and is represented by the prominent white circle [Figure 4B, red box]. This clone was present in the corresponding baseline sample at 6.5% frequency [Figure 4C, yellow box].

Figure 4. Clonality scores for the CMV model antigen study (A); Spectratyping plots for Donor 4 showing clonal expansion in the lysate condition (B) relative to baseline (C). Color indicates evenness and clonality, where darkest blue is highest evenness and lowest clonality. Size indicates Frequency for each bin containing clones of a particular V-gene/CD3 nt length combination, where larger indicates an increasing frequency. TCRβ: T-Cell receptor beta; CMV: cytomegalovirus

Expansion of CMVpp65_(495-503)-specific T-cells post-antigen challenge was also analyzed by flow cytometry using HLA-A*0201/CMVpp65_(495-503) tetramer reagent. Figure 5 shows CD8⁺ T-cell populations from each of the 4 donors prior to in vitro stimulation and after stimulation with CMV lysate or CMVpp65_495-503 peptide. In the T-cell populations from donors 1-3, a very low abundance of tetramer⁺ CD8⁺ T-cells was observed prior to stimulation, while undetectable or marginal increase in tetramer⁺ CD8⁺ T-cells was observed after stimulation with CMV lysate. However, stimulation of these 3 donors’ T-cells with CMVpp65_(493-503) peptide resulted in a significant increase in tetramer⁺ T-cells [Figure 5]. Analysis of the T-cell population from donor 4 showed that 13.72% of the CD8⁺ T cells bound the CMVpp65 tetramer prior to in vitro stimulation. The tetramer-positive population expanded to 26.24% after stimulation with CMV whole antigen but decreased to 8.29% after stimulation with CMVpp65_495-503 peptide.

Figure 5. pp65 tetramer assay results for the CMV model antigen study. Positive FITC and PE staining indicates the presence of HLA-A*0201-restricted, pp65(495-503)-responsive CD8+ T-cells. The % positive cells are shown. CMV: cytomegalovirus; FITC: fluorescein isothiocyanate; PE: phycoerythrin

As shown in Table 3, a single family of clones common to all donors (TRBV12-3 TRBJ1-2 ASSSAxYxYT) expanded in response to stimulation with both pp65 peptide and whole-cell lysate. In the CMV lysate challenge, this expansion observed by sequencing correlated strongly with pp65 tetramer staining [Figure 6]. We compared the fold change in clone frequency for the ASSSAxYxYT family of clones in response to CMV lysate challenge to the % pp65-responsive CD8+ cells in response to CMV lysate challenge for all four donors. The coefficient of determination (R²) for the correlation analysis was 0.9376. A similar result was observed in the case of the pp65 peptide stimulation for 3 out of the 4 donors (R² = 0.9454) (data not shown).

Figure 6. T-cell clone dynamics as measured by the TCR Beta assay correlate with changes in the pp65-responsive CD8+ T-cell population as measured by flow cytometry. The fold change in clone frequency for the ASSSAxYxYT family of clones in response to CMV lysate challenge is plotted relative to the fold increase in % pp65-responsive CD8+ cells in response to CMV lysate challenge for all four donors. Blue dotted line: linear regression analysis. The coefficient of determination (R²) for the correlation is shown. TCR: T-Cell receptor; CMV: cytomegalovirus