Abstract
Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.
Electronic supplementary material
The online version of this article (10.1007/s00262-018-2152-x) contains supplementary material, which is available to authorized users.
Keywords: Therapeutic vaccine, Cancer/testis antigen, CTL, TCR, Next-generation sequencing
Introduction
Therapeutic immunization with epitope peptides derived from tumor-associated antigens has shown meaningful efficiency (prolongation of survival) without remarkable adverse reactions in clinical trials; nonetheless, this approach failed to demonstrate adequate clinical efficacy required for approval [1–3]. However, vaccination with neoantigen is a promising new strategy to induce and activate cytotoxic T-lymphocytes (CTLs) that target tumor-specific mutant antigens [4–8]. For the appropriate clinical application of therapeutic cancer vaccines, surrogate and predictive biomarkers and/or reliable monitoring methods with which to examine CTLs during treatment are required [1–3, 8]. To this end, characterization of the T-cell receptor (TCR) repertoire by next-generation sequencing (NGS) is an attractive candidate approach to assess anti-tumor immune responses during immunotherapy [9–12]. This analysis has provided substantial information regarding the therapeutic mechanisms of immune checkpoint blockade [13–15]. Similarly, it would be of interest to examine the TCR repertoire and function of tumor-specific CTLs over the course of long-term immune therapy. However, this analysis requires the collection of a substantial number of peripheral blood mononuclear cells (PBMCs) or tumor-infiltrating lymphocytes (TILs) throughout the course of treatment. We have previously conducted phase I clinical trial using multiple therapeutic vaccines consisting of the cancer/testis antigens [upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)] and vascular endothelial growth factor receptor (VEGFR) 1 and 2. This therapeutic vaccination protocol proved not only safe, but also suggested the induction of CTLs against multiple antigens in some patients with advanced, recurrent non-small cell lung cancer [16]. We kept a number of frozen aliquots of PBMCs stocks from two patients who showed CTL induction against multiple antigens (patients #10 and #12) of the fifteen patients enrolled in this phase I clinical trial (Supplementary table 1). Here, we examine the frequency of CTLs expressing TCRs specific for URLC10 or CDCA1 in these PBMCs by NGS and also show the results of functional analysis of vaccine-induced CTL clones to explore CTL induction, maintenance and function during long-term therapeutic cancer vaccination.
Materials and methods
Patients
We selected two patients, patient #10 and #12, with advanced, recurrent non-small cell lung cancer from the patients enrolled in the phase I clinical trial at the Fukushima Medical University (Supplementary Table 1) [16]. This phase I clinical trial was approved by the ethical committee of Fukushima Medical University (approval number: 810) and was registered with ClinicalTrials.gov (NCT00874588). Written, informed consent was obtained from all individual patients included in this study. The trials were carried out in accordance with the Helsinki declaration on experimentation on human subjects.
Peptides
URLC10-derived HLA-A24 (A*24:02)-restricted peptide, URLC10-177 (RYCNLEGPPI), CDCA1-derived HLA-A24 (A*24:02)-restricted peptide, CDCA1-64 (VYGIRLEHF), HIV-derived HLA-A24 (A*24:02)-restricted peptide, HIV epitope peptide (RYLRDQQLL), and CMV-derived HLA-A24 (A*24:02)-restricted peptide, CMV pp65 peptide (QYDPVAALF) were synthesized as GMP grade by the American Peptide Company (Sunnyvale, CA, USA), as previously described [16–19].
Lymphocyte preparation for immunomonitoring
Immunological assays were periodically standardized and validated using the Clinical Laboratory Improvements Amendments (CLIAs) and the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) guidelines. PBMCs were obtained from patients before the treatment and after every 4th vaccination [at the end of every treatment course (TC)] as described previously [16–19].
Measurement of the peptide-specific IFN-γ response
To evaluate the peptide-specific CTL response, enzyme-linked immunospot (ELISPOT) assay was performed following ex vivo culture, as described previously [16–19]. Briefly, frozen PBMCs derived from the same patient were thawed at the same time and the viability was confirmed to be > 90%. PBMCs (5 × 105 /ml) were cultured with 10 µg/ml of the respective peptide and 100 IU/ml of interleukin-2 (Novartis, Emeryville, CA) at 37 °C for 2 weeks. The respective peptide was added into the culture on Day 0 and 7. Following CD4+ cell depletion using the Dynal CD4 Positive Isolation kit (Invitrogen, Carlsbad, CA, USA), IFN-γ ELISPOT assay was performed using the Human IFN-γ ELISPOT assay Plus kit (MabTech AB, Nacka Strand, Sweden) according to the manufacturer’s instructions. Briefly, HLA-A24:02-positive B-lymphoblast TISI cells (IHWG Cell and Gene Bank, Seattle, WA, USA) were incubated with 20 µg/ml of vaccinated peptides overnight, then the residual peptide in the media was washed out to prepare peptide-pulsed TISI cells as the stimulator cells. Prepared CD4− cells were cultured with peptide-pulsed TISI cells (2 × 104 cells/well) at 1, 0.5, 0.25 and 0.13 responder to stimulator ratio on a 96-well plate (Millipore, Bedford, MA) at 37 °C overnight. HIV epitope peptide-pulsed TISI cells were used as negative control stimulator cells. To confirm IFN-γ productivity, responder cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (66 ng/ml) and ionomycin (3 µg/ml) overnight, and then applied to the IFN-γ ELISPOT assay (2.5 × 103 cells/well) without stimulator cells. All ELISPOT assays were performed using triplicate wells. The plates were analyzed by an automated ELISPOT reader, ImmunoSpot S4 (Cellular Technology Ltd., Shaker Heights, OH) and ImmunoSpot Professional Software version 5.0 (Cellular Technology Ltd.). The number of peptide-specific spots was calculated by subtracting the spot number in the control well from the spot number in the well with the peptide-pulsed TISI cells. The sensitivity of our ELISPOT assay was estimated to be approximately average by the ELISPOT panel of the Cancer Immunotherapy Consortium [20].
Flow cytometry
Expression of peptide-specific TCR was examined on FACS-Canto II (BD Biosciences, San Jose, CA) using the URLC10-177/HLA-A*24:02 tetramer-PE (URLC10/MHC-multimer) (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), CDCA1-64/HLA-A*24:02 pentamer-PE (CDCA1/MHC-multimer) (ProImmune Ltd., Oxford, UK), or HIV epitope peptide/HLA-A*24:02-tetramer-PE or pentamer-PE (HIV/MHC-multimer) (Medical & Biological Laboratories Co., Ltd.), according to the manufacturer’s instructions as described previously [16–19].
For detection of phosphorylation of CD3 ζ CTL clones were stimulated with URLC10-177-pulsed or non-pulsed TISI cells for 45 min, and were then stained with FITC-conjugated anti-human CD8 mAb (SK1) and PE-Cy7-conjugated anti-human CD4 mAb (SK3), fixed and permeabilized with Fixation and Permeabilization Solution (BD Bioscience), blocked in 5% casein/50 µg/ml mouse IgG in PBS, and stained with PE-conjugated anti-CD247 (phosphorylated CD3 ζ chain) (pY142) mAb (K25-407.69) for 30 min. Relative mean fluorescence intensity (MFI) was calculated according to the following formula: relative MFI = MFI when stimulated URLC10-177 pre-pulsed TISI cells/ MFI when stimulated with HIV pre-pulsed TISI cells. All data were analyzed with FlowJo software (Tree Star, Inc, Ashland, OR, USA). All antibodies and 7-AAD were purchased from BD Biosciences.
IFN-γ enzyme-linked immunosorbent assay (ELISA)
CTLs were stimulated with respective peptide-pulsed cells (1 × 104 cells/well) at several responder/stimulator ratios in 200 µl of AIM-V/5% FCS on 96 well round-bottom plates (Corning Inc.) as described previously [21, 22]. After 24 h of incubation, cell-free supernatants were harvested and the IFN-γ production was examined using an IFN-γ ELISA kit (BD Biosciences) according to the manufacturer’s instructions.
CTL expansion by ex vivo stimulation and establishment of CTL clones
Peptide-specific CTLs were harvested from ELISPOT-positive wells and expanded as described previously [21, 22]. On day 14, CTLs were harvested and the CTL activity was examined by IFN-γ ELISA. Then, CTL clones were established by the limiting dilution method as described previously [21, 22].
Calcium mobilization
CTL clones were labeled with Fluo-4 AM (Molecular Probes, Eugene, OR), then stimulated with URLC10-177-pulsed or non-pulsed TISI cells. We monitored MFI as a measure of cytosolic Ca2+ concentrations for 120 s using FACS-Canto II (BD Bioscience) and analyzed the data using FlowJo software (BD Bioscience) [23].
Cytotoxicity assay
The cytotoxic activity of the established CTL clones was examined using a 4 h 51Cr release assay as described previously [24]. To demonstrate HLA-A24 (A*24:02)-restricted URLC10-specific cytotoxicity, the following cells were used as target cells: HLA-A24-positive TISI cells with or without URLC10-177 pre-pulse, URLC10-expressing HLA-A24-positive TE1 cells, URLC10-expressing HLA-A24-positive TE11 cells, and URLC10-expressing HLA-A24 negative TE14 cells obtained from the Japanese Collection of Research Biosources (JCRB) [21]. Data are presented as the means ± SD of triplicate samples.
TCR α and TCR β diversity analysis by deep sequencing
Total RNA was extracted from CTL clones, freshly thawed frozen-stock PBMCs, PBMCs cultured with appropriate peptides and IL-2 (100 IU/ml) for 7 and 14 days, and purified CD8 T cells cultured with the appropriate peptide and IL-2 for 14 days. TCRα and TCRβ chain sequencing was performed at Repertoire Genesis Incorporation using the unbiased gene amplification method with Adaptor-Ligation PCR. Bioinformatic analysis was performed on the sequencing data using the repertoire analysis software, Repertoire Genesis (Repertoire Genesis Inc., Osaka, Japan) [25].
Statistical analyses
Statistical analysis was performed using unpaired, two-tailed Student’s t test for the ELISA. P values less than 0.05 were considered as significant. All statistical analyses were conducted using the SSPS statistics software v. Twenty-one (IBM).
Results
Establishment of peptide-specific CTL clones from PBMCs of patient #10
To explore changes in cancer peptide vaccine-induced CTLs over time, we conducted CTL cloning and functional analysis coupled with NGS analysis of the TCR repertoire in bulk PBMCs. We first examined PBMCs of patient #10 who had received nine treatment courses (TCs) (Supplementary table 1). URLC10-specific and CDCA1-specific CTLs were demonstrated from post TC2 to post TC8 and from post TC4 to post TC8, respectively, by ELISPOT assay (Supplementary Fig. 1). While URLC10/MHC-multimer positive CD8+ T cells were detected consistently, CDCA1/MHC-multimer positive CD8+ T cells were not detected (Supplementary Fig. 2). We established URLC10-specific CTL clones from PBMCs of post TC2 and TC8 (Table 1). Two URLC10-specific CTL clones (1-2-U#1 and #5) established from the post TC2 PBMCs expressed the same 1-U-C1 TCR that bound the URLC10/MHC-multimer. At post TC8, five URLC10-specific CTL clones (1-5-U#1–5) were established, and these clones expressed two different TCRs (1-U-C2 and 1-U-C3) that did not bind the URLC10/MHC-multimer (Table 1). Concerning CDCA1, we established specific CTL clones from PBMCs of post TC5 and TC8 (Table 1). All ten CTL clones (1-5-C#1–10) established from the post TC5 PBMCs expressed the same 1-C-C1 TCR, which did not bind the CDCA1/MHC-multimer. The original ex vivo stimulated cells used for CTL cloning were not stained with the CDCA1/MHC-multimer at post TC8 (Supplementary Fig. 2); however, we successfully established CDCA1-specific CTL clones (1-8-C #1–5) all expressing the same 1-C-C2 TCR that bound the CDCA1/MHC-multimer (Table 1).
Table 1.
Characterization of CTL clones established from PBMCs of patient # 10
| Antigen peptide | Time point | CTL clone | IFN-γ (pg/mL) R/S ratio = 5 | Peptide/Multimer + in CD3 + CD4-CD8+ (%) | TRA | TRB | TCR sequence name | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| V | J | CDR3 | V | J | CDR3 | ||||||
| URLC10 | Post TC2 | 1-2-U#1 | 1351.5 ± 94.6 | 98.3 | TRAV19 | TRAJ49 | CALSEPHKAGNQFYF | TRBV9 | TRBJ2-7 | CASSVGENEQYF | 1-U-C1 |
| 1-2-U#5 | 261.7 ± 114.5 | 96.9 | |||||||||
| Post TC8 | 1-8-U#1 | 1560.2 ± 169.3 | 0.00 | TRAV19 | TRAJ4 | CALSGGYNKLIF | TRBV9 | TRBJ2-2 | CASSVDGRTGGMTGELFF | 1-U-C2 | |
| 1-8-U#2 | 1428.6 ± 135.2 | 0.00 | |||||||||
| 1-8-U#3 | 595.9 ± 198.1 | 0.00 | TRAV13-2 | TRAJ16 | CADPDGQKLLF | TRBV7-9 | TRBJ1-2 | CASSHGVEYGYTF | 1-U-C3 | ||
| 1-8-U#4 | 906.4 ± 276.5 | 0.00 | |||||||||
| 1-8-U#5 | 477.2 ± 249.5 | 0.00 | |||||||||
| CDCA1 | Post TC5 | 1-5-C#1 | 858.8 ± 139.1 | 0.20 | TRAV8-6 | TRAJ43 | CAVSYNNNDMRF | TRBV5-6 | TRBJ2-7 | CASSSYYEQYF | 1-C-C1 |
| 1-5-C#2 | 1092.0 ± 122.1 | 0.60 | |||||||||
| 1-5-C#3 | 1260.4 ± 131.2 | 0.10 | |||||||||
| 1-5-C#4 | 1309.2 ± 121.1 | 2.10 | |||||||||
| 1-5-C#5 | 735.5 ± 227.9 | 1.00 | |||||||||
| 1-5-C#6 | 1512.0 ± 59.1 | 0.20 | |||||||||
| 1-5-C#7 | 803.9 ± 56.3 | 0.70 | |||||||||
| 1-5-C#8 | 593.1 ± 56.6 | 0.20 | |||||||||
| 1-5-C#9 | 386.5 ± 57.9 | 0.30 | |||||||||
| 1-5-C#10 | 776.3 ± 78.7 | 0.30 | |||||||||
| Post TC8 | 1-8-C#1 | 7419.6 ± 287.1 | 9.80 | TRAV8-6 | TRAJ11 | CASGYSTLTF | TRBV7-9 | TRBJ2-5 | CASSSAGAQETQYF | 1-C-C2 | |
| 1-8-C#2 | 3652.5 ± 121.6 | 81.4 | |||||||||
| 1-8-C#3 | 6757.5 ± 161.3 | 29.2 | |||||||||
| 1-8-C#4 | 3409.2 ± 53.5 | 85.0 | |||||||||
| 1-8-C#5 | 2070.4 ± 97.6 | 88.3 | |||||||||
| CMV | Post TC9 | 1-9-CMV#11 | 1951.6 ± 62.8 | NT | TRAV24 | TRAJ40 | CALGTYKYIF | TRBV7-3 | TRBJ2-2 | CASSLIQGGAGELFF | 1-CMV-C1 |
| 1-9-CMV#13 | 826.4 ± 84.5 | NT | |||||||||
When CTL clones were stimulate with peptide-pulsed target cells, URLC10-specific CTL clones (1-8-U#1 and 1-8-U#2) expressing the same 1-U-C2 TCR that do not bind the URLC10/MHC-multimer produced comparable amounts of IFN-γ as the 1-2-U#1 CTL clone expressing the 1-U-C1 TCR that substantially bound the URLC10/MHC-multimer (Table 1). Furthermore, the 1-2-U#1 CTL clone produced approximately five times the amount of IFN-γ as the 1-2-U#5 CTL clone which expresses the same 1-U-C1 TCR (Table 1). We also established two CMV-specific CTL clones expressing identical TCRs from the post TC9 PBMCs and found that these two CTL clones produced significantly different amounts of IFN-γ (Table 1). Thus, the difference in IFN-γ productivity was not due to TCR binding activity to URLC10/MHC-multimer alone and this phenomenon was not specific for tumor vaccine-induced CTL clones.
Taken together, these results suggest that vaccination induces peptide-specific CTLs expressing various TCRs at different time points.
Direct TCR sequencing of PBMCs obtained from patient #10
We next utilized NGS to analyze the TCR repertoire in PBMCs taken from patient #10 at pre-treatment and post TC2, 8, and 9. Analysis of the top 20 TCR sequences revealed that some TCR sequences were present prior to treatment and persisted over the course of treatment (Supplementary table 2). However, these TCRs were different from the TCRs expressed on URLC10, CDCA1, and CMV-specific CTL clones. In read-out ranging in number from 2,456 to 10,792, we never identified the same TCR α or β sequences expressed on URLC10, CDCA1 or CMV-specific CTL clones, even at the same time points that CTLs were demonstrated by ELISPOT assay and/or peptide/MHC-multimer staining or that the CTL clones were established. These results suggest that CTL clones induced by vaccination were quite rare in the PBMCs of patient #10.
ELISPOT assay and URLC10/MHC-multimer analysis of CTLs in PBMCs of patient #12
We further examined CTLs in PBMCs from patient #12 who received 19 TCs and survived more than 2,958 days (Supplementary table 1). In patient #12, the number of both URLC10- and CDCA1-specific IFN-γ spots was significantly increased over background in ELISPOT assays from post TC2 to TC19. However, the number of IFN-γ specific spots varied significantly over the course of treatment. URLC10-specific spots decreased from post TC9 to TC15 before increasing again at post TC16 (Supplementary Fig. 3). This fluctuation was consistent with URLC10/MHC-multimer staining (Supplementary Fig. 4a). On the other hand, the population of CDCA1/MHC-multimer positive CD8+ T cells ranged from, at most, 2.3% to less than 1% even when the number of CDCA1-specific spots was found to be substantially increased in the ELISPOT assay (Supplementary Fig. 3 and Supplementary Fig. 4b). These results suggest that URLC10 induces CTLs expressing TCRs that bind the URLC10/MHC-multimer strongly, whereas CDCA1 induces CTLs expressing TCRs that weakly bind the CDCA1/MHC-multimer.
Establishment of peptide-specific CTL clones from PBMCs of patient #12
We established URLC10-specific CTL clones from PBMCs taken at post TC1, 11, 14 and 16, and CDCA1-specific CTL clones from the PBMCs taken at post TC2, 12 and 18 (Table 2). All URLC10-specific CTL clones established from the post TC1 PBMCs expressed an identical TCR (3-U-CS1) with high binding activity to the URLC10/MHC-multimer. On the other hand, all CTL clones established from the post TC11 or TC14 PBMCs were not stained with the URLC10/MHC-multimer. Interestingly, three out of four CTL clones established from the post TC16 PBMCs expressed different TCRs with high binding activity to the URLC10/MHC-multimer. Therefore, induction of CTLs expressing TCRs with high binding activity to the URLC10/MHC-multimer did not always precede that of CTLs expressing TCRs with low binding activity to the URLC10/MHC-multimer. Notably, the 3-U-CS1 TCR expressed on two CTL clones established from the post TC16 PBMCs (3-16-U#7 and 9) was the same as that expressed on CTL clones established from the post TC1 PBMCs (3-1-U#1, 2, 3, 5 and 6), suggesting that CTL clones expressing the 3-U-CS1 TCR were maintained from post TC1 to post TC16 (15 months).
Table 2.
Characterization of CTL clones established from PBMCs of patient #12
| Antigen peptide | Time point | CTL clone | IFN-γ (pg/mL) R/S ratio = 5 | Peptide/multimer + in CD3 + CD4-CD8+ (%) | TRA sequence | TRB sequence | TCR sequence name | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| V | J | CDR3 | V | J | CDR3 | ||||||
| URLC10 | Post TC1 | 3-1-U#1 | 4626.5 ± 138.6 | 98.9 | TRAV17 | TRAJ39 | CATDDSAGNMLTF | TRBV9 | TRBJ2-1 | CASSLNNNEQFF | 3-U-CS1 |
| 3-1-U#2 | 5382.3 ± 152.8 | 99.2 | |||||||||
| 3-1-U#3 | 4632.2 ± 131.7 | 99.0 | |||||||||
| 3-1-U#5 | 4751.0 ± 149.5 | 99.7 | |||||||||
| 3-1-U#6 | 3982.0 ± 43.6 | 99.8 | |||||||||
| Post TC11 | 3-11-U#1 | 287.8 ± 72.2 | 0.08 | TRAV8-4 | TRAJ45 | CAVSDPGADGLTF | TRBV5-5 | TRBJ1-1 | CASSPLRVMNTEAFF | 3-U-CS2 | |
| 3-11-U#5 | 2868.0 ± 12.4 | 0.03 | |||||||||
| 3-11-U#6 | 7082.2 ± 206.6 | 0.03 | |||||||||
| 3-11-U#8 | 1033.5 ± 165.1 | 0.04 | |||||||||
| 3-11-U#9 | 3030.0 ± 31.7 | 0.00 | |||||||||
| 3-11-U#10 | 290.0 ± 98.5 | 0.03 | |||||||||
| 3-11-U#11 | 948.7 ± 156.4 | 0.07 | |||||||||
| Post TC14 | 3-14-U#1 | 1030.1 ± 45.9 | 0.17 | TRAV14 | TRAJ28 | CAMRDPWAGSYQLTF | TRBV5-1 | TRBJ2-3 | CASSPRDTTTDTQYF | 3-U-CS3 | |
| 3-14-U#2 | 1379.5 ± 209.9 | 0.11 | |||||||||
| 3-14-U#4 | 603.1 ± 50.6 | 0.14 | |||||||||
| 3-14-U#5 | 751.4 ± 74.3 | 0.03 | |||||||||
| 3-14-U#6 | 6119.1 ± 134.1 | 0.02 | |||||||||
| 3-14-U#7 | 2648.3 ± 97.1 | 0.02 | |||||||||
| 3-14-U#8 | 3136.1 ± 270.0 | 0.02 | |||||||||
| 3-14-U#9 | 1626.3 ± 22.6 | 0.02 | |||||||||
| Post TC16 | 3-16-U#1 | 234.5 ± 91.6 | 94.7 | TRAV4 | TRAJ33 | CLVGVWLPSNYQLIW | TRBV24-1 | TRBJ2-3 | CATSDPGESEVHTQYF | 3-U-CS4 | |
| 3-16-U#2 | 21058.2 ± 162.1 | 97.6 | TRAV12-2 | TRAJ13 | CAVNMWNSGGYQKVTF | TRBV12-5 | TRBJ2-7 | CASGFGGSYEQYF | 3-U-CS5 | ||
| 3-16-U#4 | 11112.8 ± 63.9 | 99.9 | |||||||||
| 3-16-U#5 | 4684.4 ± 41.0 | 0.02 | TRAV6 | TRAJ42 | CALRFYGGSQGNLIF | TRBV25-1 | TRBJ1-2 | CASSADSYTF | 3-U-CS6 | ||
| 3-16-U#7 | 3346.2 ± 83.4 | 79.9 | TRAV17 | TRAJ39 | CATDDSAGNMLTF | TRBV9 | TRBJ2-1 | CASSLNNNEQFF | 3-U-CS1 | ||
| 3-16-U#9 | 17344.5 ± 60.4 | 99.3 | |||||||||
| CDCA1 | Post TC2 | 3-2-C#2 | 1380.7 ± 196.5 | 0.13 | TRAV24 | TRAJ28 | CAYTSGAGSYQLTF | TRBV9 | TRBJ1-2 | CASSESRDRIHYGYTF | 3-C-CS1 |
| 3-2-C#3 | 145.3 ± 43.2 | 0.09 | TRAV24 | TRAJ10 | CAAGNILTGGGNKLTF | TRBV27 | TRBJ1-6 | CASSLGRGNSPLHF | 3-C-CS2 | ||
| 3-2-C#4 | 1154.7 ± 74.1 | 0.61 | |||||||||
| 3-2-C#8 | 195.3 ± 51.2 | 1.19 | |||||||||
| 3-2-C#11 | 105.8 ± 61.7 | 0.21 | |||||||||
| 3-2-C#5 | 788.8 ± 72.8 | 0.53 | TRAV8-6 | TRAJ26 | CAVSDPRNYGQNFVF | TRBV6-3 | TRBJ2-7 | CATRGQSYEQYF | 3-C-CS3 | ||
| 3-2-C#6 | 216.2 ± 43.1 | 0.29 | |||||||||
| 3-2-C#7 | 522.9 ± 50.6 | 0.71 | TRAV23 | TRAJ22 | CAAPLSAASGSARQLTF | TRBV24-1 | TRBJ1-5 | CATSDPRDWTGEANQPQHF | 3-C-CS4 | ||
| 3-2-C#10 | 240.3 ± 41.9 | 1.90 | |||||||||
| Post TC12 | 3-12-C#1 | 2435.1 ± 129.8 | 0.02 | TRAV21 | TRAJ30 | CAVGGLRDDKIIF | TRBV27 | TRBJ1-2 | CASSSGQTNYGYTF | 3-C-CS5 | |
| 3-12-C#2 | 3458.6 ± 198.1 | 0.17 | |||||||||
| 3-12-C#3 | 1119.2 ± 42.3 | 0.22 | |||||||||
| 3-12-C#4 | 968.8 ± 57.0 | 0.97 | |||||||||
| Post TC18 | 3-18-C#17 | 3128.1 ± 156.6 | 0.03 | TRAV12-1 | TRAJ37 | CVVNRASNTGKLIF | TRBV27 | TRBJ2-7 | CASSLYRGLYEQYF | 3-C-CS6 | |
| 3-18-C#20 | 1170.6 ± 88.3 | 0.06 | TRAV21 | TRAJ30 | CAVGGLRDDKIIF | TRBV27 | TRBJ1-2 | CASSSGQTNYGYTF | 3-C-CS5 | ||
| 3-18-C#29 | 934.4 ± 89.0 | 0.01 | |||||||||
| 3-18-C#31 | 1035.9 ± 118.1 | 0.01 | |||||||||
Concerning CDCA1-specific CTLs, none of the CTL clones established from patient #12 were stained with the CDCA1/MHC-multimer (Table 2). This is consistent with the fact that the cells used for ELISPOT assay, which were used for CTL cloning, failed to show a substantial population expressing a TCR with high binding activity to the CDCA1/MHC-multimer (Supplementary Fig. 4b). CTL clones established from PBMCs at post TC2 expressed four different TCRs. Moreover, all CTL clones established from the post TC12 PBMCs expressed the same TCR (3-C-CS5), which was expressed on three out of four CTL clones established from the post TC18 PBMCs, suggesting that the CTL clone expressing the 3-U-CS1 TCR was maintained from post TC12 to post TC18 (6 months).
Taken together, there was a large variation in TCRs expressed on vaccination-induced CTLs between treatment courses. However, some CTL clones, regardless of the activity of their TCR to bind the peptide/MHC-multimer, were maintained in PBMCs throughout the treatment course.
TCR-mediated signaling and Ca2+ mobilization in URLC10/MHC multimer-positive and negative CTL clones
We next examined differences in the intracellular signaling downstream of the TCR between URLC10/MHC-multimer positive and negative clones. Phosphorylation of CD3 ζ (pCD3 ζ; pY-142), which is a read-out of an early step in TCR signaling, was observed in the URLC10/MHC-multimer positive clone 3-16-U#9 (Fig. 1a). Although all URLC10/MHC-multimer negative clones tested produced a substantial amount of IFN-γ, significant phosphorylation of CD3 ζ was not observed in these clones. On the other hand, in an intracellular Ca2+ mobilization analysis, we observed substantial Ca2+ influx not only in URLC10/MHC-multimer positive clones, but also in all URLC10/MHC-multimer negative clones tested (Fig. 1b). However, Ca2+ influx did not appear to correlate with IFN-γ productivity among URLC10/MHC-multimer negative clones, since clone 3-14-U#5 produced only a miniscule amount of IFN-γ. In a cytotoxic activity assay, both URLC10/MHC-multimer positive and negative clones exerted marginal, but significant, HLA-A24-restricted URLC10-specific cytotoxicity (Fig. 1c). These results suggest that CTL functions (IFN-γ productivity and cytotoxic activity) do not simply correlate with the phosphorylation level of the CD3 ζ chain or Ca2+ influx, and that some CTLs expressing TCRs with low binding activity to the URLC10/MHC-multimer exert a similar level of effector functions as those with TCRs that exhibit high binding activity to the URLC10/MHC-multimer.
Fig. 1.
Phosphorylation of CD3 ζ and Ca2+ mobilization in URLC10/MHC multimer-positive and negative CTL clones. CTL clones positive or negative for URLC10/MHC-multimer staining were stimulated with HIV epitope peptide pre-pulsed TISI cells (gray filled) or URLC10-177 pre-pulsed TISI cells (bold line). Phosphorylation of the CD3 ζ chain (pCD3 ζ; pY-142) was then examined by flow cytometry using anti-pCD3 ζ mAb (a). Relative mean fluorescence intensity (MFI) is indicated in each panel. Ca2+ influx was also examined using flow cytometry following the stimulation with URLC10-177 pre-pulsed TISI cells (b). Arrow indicates the time of stimulation with URLC10-177 pre-pulsed TISI cells. c Specific cytotoxic activity of URLC10/MHC-multimer positive or negative CTL clones. Specific cytotoxic activity of URLC10/MHC-multimer positive CTL clone 3-16-U#9 and URLC10/MHC-multimer negative CTL clones 3-14-U#7 and 3-14-U#9 was examined by 51Cr release assay. HLA-A24-positive TISI cells with (open circle) or without (open square) URLC10-177 pre-pulse (in upper panels) as well as URLC10-expressing TE1 cells (HLA-A24-positive) (closed square), URLC10-expressing TE11 cells (HLA-A24-positive) (closed circle) and URLC10-expressing TE14 cells (HLA-A24 negative)(closed triangle) (in lower panels) were used as target cells. *P < 0.05 compared with the non-pulsed TISI cells (upper panels) or TE14 cells (lower panels) at the same effector/ target ratio. Data are representative of two independent experiments
Direct TCR sequencing of PBMCs obtained from patient #12
We next analyzed the TCR repertoire in PBMCs of patient #12 by NGS.
Among the top 20 TCR sequences, several TCR sequences appeared over the course of treatment, and the frequency of some increased following vaccine treatments (Supplementary table 3). However, all of the top 20 TCRs identified in PBMCs differed from those expressed on established URLC10- and CDCA1-specific CTL clones.
The URLC10-specific 3-U-CS3 TCR α chain and CDCA1-specific 3-C-CS2 TCR α chain were detected in PBMCs at the same time points as used for CTL cloning (Table 3). Moreover, three TCR sequences of CTL clones were found in PBMCs at time points different from those used to establish CTL clones expressing these TCRs: 3-C-CS2 TCR α chain at post TC17, 3-C-CS2 TCR β chain at post TC1 and TC16, and 3-C-CS3 TCR β chain at post TC12. Other URLC10- or CDCA1-specific TCR sequences were not detected in PBMCs by direct NGS even at the same time point as that used for CTL cloning. These results suggest that these vaccine-induced CTL clones were maintained at a level of 0.003–0.089% in PBMCs.
Table 3.
The number of TCR sequences expressed on established CTLs in all TCR sequence read-outs of PBMCs from Patient #12
| Antigen peptide | Time point when CTL clone was established | TCR sequence name of CTL clone | Pre | Post TC1 | Post TC2 | Post TC3 | Post TC7 | Post TC10 | Post TC12 | Post TC14 | Post TC16 | Post TC17 | Post TC18 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TRA | |||||||||||||
| URLC10 | Post TC1 and 16 | 3-U-CS1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Post TC11 | 3-U-CS2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC14 | 3-U-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | |
| Post TC16 | 3-U-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-U-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| 3-U-CS6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| CDCA1 | Post TC2 | 3-C-CS1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3-C-CS2 | 0 | 0 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | ||
| 3-C-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| 3-C-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Post TC12 and 18 | 3-C-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC18 | 3-C-CS6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total number of TRA read sequences | 171,464 | 15,006 | 8981 | 8970 | 12,653 | 10,524 | 37,870 | 107,386 | 93,409 | 8302 | 100,438 | ||
| Clone % | 0.000 | 0.000 | 0.089 | 0.000 | 0.000 | 0.000 | 0.000 | 0.003 | 0.000 | 0.024 | 0.000 | ||
| TRB | |||||||||||||
| URLC10 | Post TC1 and 16 | 3-U-CS1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Post TC11 | 3-U-CS2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC14 | 3-U-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC16 | 3-U-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-U-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| 3-U-CS6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| CDCA1 | Post TC2 | 3-C-CS1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3-C-CS2 | 0 | 39 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 0 | 0 | ||
| 3-C-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 0 | 0 | 0 | 0 | ||
| 3-C-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Post TC12 and 18 | 3-C-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC18 | 3-C-CS6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total number of TRB read sequences | 95,914 | 92,441 | 4742 | 5222 | 5205 | 5384 | 98,166 | 116,575 | 94,742 | 4578 | 85,551 | ||
| Clone % | 0.000 | 0.042 | 0.000 | 0.000 | 0.000 | 0.000 | 0.008 | 0.000 | 0.006 | 0.000 | 0.000 | ||
Positive results are indicated in bold
TCR sequencing analysis following ex vivo stimulation of PBMCs obtained from patient #12
We next used the portion of the PBMC aliquots remaining after the above-mentioned analyses to examine the efficacy of CTL expansion by ex vivo stimulation (Table 4). Following ex vivo stimulation of the post TC16 PBMCs with URLC10-177 for 14 days, we detected not only the 3-U-CS4 and 3-U-CS5 TCR α chains, both of which were expressed on CTL clones established at post TC16, but also the 3-U-CS2 and 3-U-CS3 TCR α chains expressed on CTL clones established at post TC11 and TC14 respectively, but not found at post TC16. Moreover, the 3-U-CS5 and 3-U-CS6 TCR α chains, that were expressed on CTL clones established at post TC16 but not post TC14, were detected in the post TC14 PBMCs after 14 days of ex vivo stimulation with URLC10-177.
Table 4.
Effect of ex vivo stimulation on NGS analysis
| Antigen peptide | Time point when CTL clone was established | TCR sequence name of CTL clone | Ex vivo stimulation with URLC10 | |||||
|---|---|---|---|---|---|---|---|---|
| Post TC14 | Post TC16 | |||||||
| Day7 | Day14 | Day7 | Day14 | Day14CD8 | ||||
| URLC10 | Post TC1 and 16 | 3-U-CS1 | 0 | 0 | 0 | 0 | 0 | |
| Post TC11 | 3-U-CS2 | 0 | 0 | 0 | 1 | 0 | ||
| Post TC14 | 3-U-CS3 | 0 | 0 | 0 | 1 | 0 | ||
| Post TC16 | 3-U-CS4 | 0 | 0 | 0 | 9 | 84 | ||
| 3-U-CS5 | 0 | 3 | 4 | 7 | 125 | |||
| 3-U-CS6 | 0 | 1 | 0 | 0 | 0 | |||
| CDCA1 | Post TC2 | 3-C-CS1 | 0 | 0 | 0 | 0 | 0 | |
| 3-C-CS2 | 0 | 0 | 0 | 0 | 0 | |||
| 3-C-CS3 | 0 | 0 | 0 | 0 | 0 | |||
| 3-C-CS4 | 0 | 0 | 0 | 0 | 0 | |||
| Post TC12 and 18 | 3-C-CS5 | 0 | 0 | 0 | 0 | 0 | ||
| Post TC18 | 3-C-CS6 | 0 | 0 | 0 | 0 | 0 | ||
| Total TRA sequence number | 10,638 | 10,079 | 12,106 | 7460 | 6175 | |||
| Clone % | 0.000 | 0.040 | 0.033 | 0.241 | 3.384 | |||
| Antigen peptide | Time point when CTL clone was established | TCR sequence name of CTL clone | Ex vivo stimulation with CDCA1 | |||||
|---|---|---|---|---|---|---|---|---|
| Post TC2 | Post TC 18 | |||||||
| Day7 | Day14 | Day14CD8 | Day7 | Day14 | Day14CD8 | |||
| URLC10 | Post TC1 and 16 | 3-U-CS1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Post TC11 | 3-U-CS2 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC14 | 3-U-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC16 | 3-U-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-U-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| 3-U-CS6 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| CDCA1 | Post TC2 | 3-C-CS1 | 0 | 0 | 24 | 0 | 0 | 0 |
| 3-C-CS2 | 0 | 2 | 39 | 0 | 0 | 0 | ||
| 3-C-CS3 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| 3-C-CS4 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Post TC12 and 18 | 3-C-CS5 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Post TC18 | 3-C-CS6 | 11 | 26 | 327 | 0 | 1 | 0 | |
| Total TRB sequence number | 5527 | 5141 | 5542 | 7451 | 7537 | 5546 | ||
| Clone % | 0.199 | 0.545 | 7.037 | 0.000 | 0.013 | 0.000 | ||
Positive results are indicated in bold
When post TC2 PBMCs were stimulated with CDCA1-64, not only 3-C-CS1 and 3-C-CS2 TCR β chains, which were expressed on CTL clones established at post TC2, but also the 3-C-CS6 TCR β chain, which was expressed on CTL clones established at post TC18, were detected. Thus, CTLs expressing the 3-C-CS6 TCR β chain were induced at post TC2, although this TCR was not found on established CTL clones at post TC2 or by direct NGS sequencing of PBMCs.
URLC10-specific 3-U-CS5 and 3-U-CS4 TCR α chains ranked in the top 20 sequences from CD8+ T cells after 14 days ex vivo stimulation of the post TC16 PBMCs (Supplementary table 4a). The 3-C-CS6 and 3-C-CS2 TCR β chains also ranked in the top 20 sequences from CD8+ T cells after 14 days ex vivo stimulation of the post TC2 PBMCs (Supplementary table 4b). No CDCA1-specific TCR α chains were found to be present after ex vivo stimulation with URLC10. Likewise, the URLC10-specific TCR β chain was not detected after ex vivo stimulation with CDCA1. These results suggest that the majority of vaccine-induced specific CTL clones were not detectable by direct NGS of PBMCs; however, these rare CTL clones were efficiently expanded and became readily detectable following ex vivo stimulation with a specific peptide.
Maintenance of CTLs expressing TCRs with low binding activity to the CDCA1/MHC-multimer in patient #12
A summary of the CTL analyses in PBMCs from patient #12 is shown in Table 5. The URLC10-specific CTL clone expressing the 3-U-CS1 TCR, which was induced at post TC1 and maintained until post TC16, is presumably responsible for the URLC10-specific CTL response until post TC11 when CTL clones expressing the 3-U-CS2 TCR, which did not bind the URLC10/MHC-multimer, replaced it as the dominant CTL clone in PBMCs. After this point, CTLs expressing various URLC10-specific TCRs are responsible for the URLC10-specific CTL response seen at post TC14 and 16. Concerning CDCA1, CTL clones that express five different CDCA1-specific TCRs were induced by post TC2, and three of these were maintained after post TC11. Notably, all of CDCA1-specific CTL clones were not stained with the CDCA1/MHC-multimer. These results suggest that therapeutic vaccination induces antigen-specific CTLs expressing various TCRs over the course of the long-term vaccination. Moreover, the induction timing and the fate of CTLs vary and are not restricted by the binding activity of the TCR to a peptide/MHC-multimer.
Table 5.
Summary of CTL analyses in the PBMCs of patient #12
| Binding activity to peptide/MHC multimer | Pre | Post TC1 | Post TC2 | Post TC3 | Post TC7 | Post TC10 | Post TC11 | Post TC12 | Post TC14 | Post TC16 | Post TC17 | Post TC18 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| URLC10 | 3-U-CS1 | High | – | C | – | – | – | – | – | – | – | C | – | – |
| 3-U-CS2 | Low | – | – | – | – | – | – | C | – | – | SS | – | – | |
| 3-U-CS3 | Low | – | – | – | – | – | – | – | – | C S | SS | – | – | |
| 3-U-CS4 | High | – | – | – | – | – | – | – | – | – | C SS | – | – | |
| 3-U-CS5 | High | – | – | – | – | – | – | – | – | SS | C SS | – | – | |
| 3-U-CS6 | Low | – | – | – | – | – | – | – | – | SS | C | – | – | |
| Performed analyses | S | C S | S | S | S | S | C | S | C S SS | C S SS | S | S | ||
| ELISPOT assay | – | +++ | +++ | +++ | +++ | +++ | +++ | ++ | +++ | +++ | +++ | +++ | ||
| % of peptide/multimer positive cells | 0 | 2.2 | 16.4 | 17.2 | 11.6 | 3.3 | 0.6 | 0.4 | 0.4 | 4.7 | 0.6 | 0.5 | ||
| CDCA1 | 3-C-CS1 | Low | – | – | C SS | – | – | – | NT | – | – | – | – | – |
| 3-C-CS2 | Low | – | S | C S SS | – | – | – | NT | – | – | S | S | – | |
| 3-C-CS3 | Low | – | – | C | – | – | – | NT | S | – | – | – | – | |
| 3-C-CS4 | Low | – | – | C | – | – | – | NT | – | – | – | – | – | |
| 3-C-CS5 | Low | – | – | – | – | – | – | NT | C | – | – | – | C | |
| 3-C-CS6 | Low | – | – | SS | – | – | – | NT | – | – | – | – | C SS | |
| Performed analyses | S | S | C S SS | S | S | S | NT | C S | S | S | S | C S SS | ||
| ELISPOT assay | – | – | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | ||
| % of peptide/multimer positive cells | NT | NT | NT | NT | 0.6 | 0 | 0 | 1.6 | 0.4 | 0.1 | 0.2 | 0.1 | ||
Positive results are indicated in bold
C CTL cloning, S direct sequencing of PBMCs, SS sequencing after ex vivo stimulation, NT not tested
The results of the ELIPOT assays were classified into four groups (−, +, ++ or +++) according to a previously reported protocol [19]
Discussion
Quantitative NGS of TCRs in PBMCs and/or TILs is an attractive and novel method to examine tumor-specific immune responses in cancer patients. NGS has revealed the complexity and intratumor heterogeneity of TILs in renal cell carcinoma [26] and homogeneous T-cell infiltration in ovarian cancer [27]. Moreover, there are some reports suggesting that TCR clonal expansion (reduced TCR diversity) in TILs correlates with the therapeutic effects of immunochemotherapy and neoadjuvant chemotherapy [28, 29]. The TCR repertoire in PBMCs is reportedly distinct from that of TILs [26–30]; however, there are few reports that have analyzed the TCR repertoire in PBMCs and/or TILs in concert with the analysis of tumor-specific CTLs. Here, we analyzed vaccine-induced CTLs in PBMCs by NGS with respect to their TCR and conducted functional analysis of CTL clones using frozen PBMCs aliquot stocks from two non-small cell lung cancer patients who had been enrolled in our previously reported phase I trials [16]. In the analysis of patient #10, direct TCR sequencing did not demonstrate any TCR sequences identical to those of specific CTL clones generated from PBMCs, including the TCR expressed on CMV-specific CTL clones. In the analysis of patient #12, CDCA1-specific 3-C-CS2 TCR was detected in the post TC1 PBMCs (39 in 92,441; 0.042%), even when the ELISPOT assay did not demonstrate a CTL response. However, TCRs expressed on the specific CTL clones were not always identified in PBMCs by NGS, even after ex vivo stimulation, at the same time point as CTL clones were established. It is possible that frozen PBMCs yield less RNA of a lower quantity than that prepared from fresh PBMCs. Thus, preparation of cDNAs from RNA of fresh PBMCs might result in more frequent detection of vaccine peptide-specific TCRs by NGS. However, TCR sequences of the CTL clones were not observed in PBMCs even when approximately 100,000 sequences were read at the same time point as the CTL clones were established (e.g., post TC14 and 16 for patient #12). This suggests that CTLs exist in PBMCs near the minimum threshold required for detection by direct NGS. A previous report using NGS demonstrated that clonal expansion of CTLs in PBMCs precedes the advanced effect of ipilimumab (anti-CTLA4 mAb) [31] and unique and productive TCR V-β CDR3 sequences closely correlate with toxicity of CTLA4 blockade by tremelimumab [32]. These reports suggest that extended expansion of CTL clones in PBMCs to levels detectable by NGS would correlate with advanced effects. The demonstration of a limited number of CTL clones in PBMCs by NGS would be consistent with the fact that therapeutic vaccines do not induce remarkable adverse reactions in clinical trials.
In this study, the high-frequency, top 20 TCR read-outs from PBMCs were never the same as those expressed on established CTL clones, even when these sequences appeared to increase in PBMCs following treatment. It should be noted that we did not exclude the possibility that these CTLs are specific for VEGFR1 or VEGFR2, which were also immunized in conjunction with URLC10 and CDCA1 in our phase I trials [16]. However, the frequency of CTLs against VEGFR1 or VEGFR2 would be much less than that of URLC10 or CDCA1, since VEGFR1 and VEGFR2 are likely expressed on normal endothelium during physiological angiogenesis. Further, it is possible that the CTLs frequently detected in PBMCs might be specific for other things, such as infectious pathogens present in the lungs of the individual patient. At several time points, the ELISPOT assay and peptide/MHC-multimer staining demonstrated the presence of specific CTLs more efficiently than direct NGS. These results indicate that combining biological analysis with TCR sequencing using NGS as well as specific T-cell cloning would be required for appropriate CTL analysis in the PBMCs of cancer patients treated with immunotherapies, and especially in those treated with vaccine therapies.
Notably, although CDCA1-specific 3-C-CS2, 3-C-CS3, or 3-C-CS6 TCR-expressing CTLs were not stained with the CDCA1/MHC-multimer, they were maintained through the long-term treatment course in patient #12. It has been reported that CTLs effective for tumor elimination are required to express TCRs with high-binding activity to the targeting peptide/MHC on tumor cells [33, 34]. However, the peptide/MHC-multimer occasionally fails to stain CTLs that specifically and effectively respond to the same peptide/MHC on target cells [35–39]. Moreover, self-antigen specific CD4+ T cells that are not stained by peptide/MHC-multimer are functionally competent and pathogenic in autoimmune diseases [40, 41]. Thus, it is possible that these CDCA-1/MHC-multimer negative TCR-expressing CTL clones could work as effector CTLs in patient #12, and these TCRs would provide appropriate signals resulting in increased survival without the induction of exhaustion during long-term vaccine therapies. Furthermore, concerning CTL clones expressing the URLC10/MHC-multimer positive 3-U-CS1 TCR, IFN-γ production was comparable among the five clones established at post TC1 (3-1-U#1–6), whereas there was a greater than fivefold difference between two clones established at post TC16 (3-16-U#7 and 9). These results might suggest that 3-U-CS1 TCR provided relatively strong signals resulting in induction of an exhausted phenotype in some CTLs following repeated vaccinations.
To our best knowledge, this is the first report to analyze the TCR repertoire of vaccine-induced tumor-specific CTLs in PBMCs over the long-term course of a vaccine treatment. While future studies will be required to examine the responses of vaccine-induced CTLs to patient’s tumor cells, and thus determine the contribution of vaccine-induced CTLs to anti-tumor effects, the results presented suggest that long-term vaccination induces antigen-specific CTLs expressing various TCRs and that some CTLs are maintained throughout treatment. Thus, therapeutic tumor antigen vaccination combined with the use of immune checkpoint blockade (anti-CTLA4 and anti-PD-1/PD-L1 mAb) might be an attractive and rational strategy for comprehensive cancer immune therapy, which would result in the induction of CTLs, facilitation of CTL infiltration, and withdrawal of immune suppression in tumor tissue [42–45].
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We are grateful to OncoTherapy Science, Inc. (Kanagawa, Japan) for their technical support.
Abbreviations
- CDCA1
Cell division associated 1
- CDCA1-64
CDCA1-derived HLA-A24 (A*24:02)-restricted peptide
- CDCA1/MHC-multimer
CDCA1-64/ HLA-A*24:02 pentamer-PE
- CMV pp65 peptide
CMV-derived HLA-A24 (A*24:02)-restricted peptide
- HIV epitope peptide
HIV-derived HLA-A24 (A*24:02)-restricted peptide
- NGS
Next-generation sequencing
- TC
Treatment course
- URLC10
Upregulated lung cancer 10
- URLC10-177
URLC10-derived HLA-A24 (A*24:02)-restricted peptide
- URLC10/MHC-multimer
URLC10-177/ HLA-A*24:02 tetramer-PE
- VEGFR
Vascular endothelial growth factor receptor
Author contributions
Kazuyoshi Takeda designed this study, interpreted the data, and wrote the manuscript. Kazutaka Kitaura and Ryuji Suzuki carried out next-generation sequencing. Yuki Owada, Satoshi Muto, Naoyuki Okabe, Takeo Hasegawa, Jun Osugi, and Mika Hoshino carried out biological analysis. Takuya Tsunoda, Ko Okumura and Hiroyuki Suzuki revised the manuscript. All authors had final approval of the submitted and published versions.
Funding
This work was supported by the Ministry of Education, Science, and Culture, Japan (15K14410) to K. Takeda.
Compliance with ethical standards
Conflict of interest
K. Kitaura and R. Suzuki are currently employed by Repertoire Genesis, Inc. The other authors declare that they have no conflicts of interest.
Ethical approval and ethical standards
This study was approved by the ethical committee of Fukushima Medical University (approval number: 810) and was registered with ClinicalTrials.gov (NCT00874588). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any animal studies performed by any of the authors.
Informed consent
Informed consent was obtained from all individual participants included in the study.
References
- 1.Schlom J. Therapeutic cancer vaccines: current status and moving forward. J Natl Cancer Inst. 2012;104(8):599–613. doi: 10.1093/jnci/djs033. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Melero I, Gaudernack G, Gerritsen W, Huber C, Parmiani G, Scholl S, Thatcher N, Wagstaff J, Zielinski C, Faulkner I, Mellstedt H. Therapeutic vaccines for cancer: an overview of clinical trials. Nat Rev Clin Oncol. 2014;11(9):509–524. doi: 10.1038/nrclinonc.2014.111. [DOI] [PubMed] [Google Scholar]
- 3.van der Burg SH, Arens R, Ossendorp F, van Hall T, Melief CJ. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat Rev Cancer. 2016;16(4):219–233. doi: 10.1038/nrc.2016.16. [DOI] [PubMed] [Google Scholar]
- 4.Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, Franci C, Cheung TK, Fritsche J, Weinschenk T, Modrusan Z, Mellman I, Lill JR, Delamarre L. Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature. 2014;515(7528):572–576. doi: 10.1038/nature14001. [DOI] [PubMed] [Google Scholar]
- 5.Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014;515(7528):577–581. doi: 10.1038/nature13988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015;348(6230):69–74. doi: 10.1126/science.aaa4971. [DOI] [PubMed] [Google Scholar]
- 7.Carreno BM, Magrini V, Becker-Hapak M, Kaabinejadian S, Hundal J, Petti AA, Ly A, Lie WR, Hildebrand WH, Mardis ER, Linette GP. Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science. 2015;348(6236):803–808. doi: 10.1126/science.aaa3828. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Yarchoan M, Johnson BA, Lutz ER, Laheru DA, Jaffee EM. Targeting neoantigens to augment antitumour immunity. Nat Rev Cancer. 2017;17(4):209–222. doi: 10.1038/nrc.2016.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Calis JJ, Rosenberg BR. Characterizing immune repertoires by high throughput sequencing: strategies and applications. Trends Immunol. 2014;35(12):581–590. doi: 10.1016/j.it.2014.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Linnemann C, Heemskerk B, Kvistborg P, Kluin RJ, Bolotin DA, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu CJ, Mamedov IZ, Velds A, Blank CU, Haanen JB, Turchaninova MA, Kerkhoven RM, Spits H, Hadrup SR, Heemskerk MH, Blankenstein T, Chudakov DM, Bendle GM, Schumacher TN. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013;19(11):1534–1541. doi: 10.1038/nm.3359. [DOI] [PubMed] [Google Scholar]
- 11.Kobayashi E, Mizukoshi E, Kishi H, Ozawa T, Hamana H, Nagai T, Nakagawa H, Jin A, Kaneko S, Muraguchi A. A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days. Nat Med. 2013;19(11):1542–1546. doi: 10.1038/nm.3358. [DOI] [PubMed] [Google Scholar]
- 12.Fang H, Yamaguchi R, Liu X, Daigo Y, Yew PY, Tanikawa C, Matsuda K, Imoto S, Miyano S, Nakamura Y. Quantitative T cell repertoire analysis by deep cDNA sequencing of T cell receptor a and b chains using next-generation sequencing (NGS) Oncoimmunology. 2014;3(12):e968467. doi: 10.4161/21624011.2014.968467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West AN, Carmona M, Kivork C, Seja E, Cherry G, Gutierrez AJ, Grogan TR, Mateus C, Tomasic G, Glaspy JA, Emerson RO, Robins H, Pierce RH, Elashoff DA, Robert C, Ribas A. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515(7528):568–571. doi: 10.1038/nature13954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Twyman-Saint Victor C, Rech AJ, Maity A, Rengan R, Pauken KE, Stelekati E, Benci JL, Xu B, Dada H, Odorizzi PM, Herati RS, Mansfield KD, Patsch D, Amaravadi RK, Schuchter LM, Ishwaran H, Mick R, Pryma DA, Xu X, Feldman MD, Gangadhar TC, Hahn SM, Wherry EJ, Vonderheide RH, Minn AJ. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015;520(7547):373–377. doi: 10.1038/nature14292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Inoue H, Park JH, Kiyotani K, Zewde M, Miyashita A, Jinnin M, Kiniwa Y, Okuyama R, Tanaka R, Fujisawa Y, Kato H, Morita A, Asai J, Katoh N, Yokota K, Akiyama M, Ihn H, Fukushima S, Nakamura Y. Intratumoral expression levels of PD-L1, GZMA, and HLA-A along with oligoclonal T cell expansion associate with response to nivolumab in metastatic melanoma. Oncoimmunology. 2016;5(9):e1204507. doi: 10.1080/2162402X.2016.1204507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Suzuki H, Fukuhara M, Yamaura T, Mutoh S, Okabe N, Yaginuma H, Hasegawa T, Yonechi A, Osugi J, Hoshino M, Kimura T, Higuchi M, Shio Y, Ise K, Takeda K, Gotoh M. Multiple therapeutic peptide vaccines consisting of combined novel cancer testis antigens and anti-angiogenic peptides for patients with non-small cell lung cancer. J Transl Med. 2013;11:97. doi: 10.1186/1479-5876-11-97. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Yoshitake Y, Fukuma D, Yuno A, Hirayama M, Nakayama H, Tanaka T, Nagata M, Takamune Y, Kawahara K, Nakagawa Y, Yoshida R, Hirosue A, Ogi H, Hiraki A, Jono H, Hamada A, Yoshida K, Nishimura Y, Nakamura Y, Shinohara M. Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS. Clin Cancer Res. 2015;21(2):312–321. doi: 10.1158/1078-0432.CCR-14-0202. [DOI] [PubMed] [Google Scholar]
- 18.Okuyama R, Aruga A, Hatori T, Takeda K, Yamamoto M. Immunological responses to a multi-peptide vaccine targeting cancer-testis antigens and VEGFRs in advanced pancreatic cancer patients. Oncoimmunology. 2013;2(11):e27010. doi: 10.4161/onci.27010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kono K, Iinuma H, Akutsu Y, Tanaka H, Hayashi N, Uchikado Y, Noguchi T, Fujii H, Okinaka K, Fukushima R, Matsubara H, Ohira M, Baba H, Natsugoe S, Kitano S, Takeda K, Yoshida K, Tsunoda T, Nakamura Y. Multicenter, phase II clinical trial of cancer vaccination for advanced esophageal cancer with three peptides derived from novel cancer-testis antigens. J Transl Med. 2012;10:141. doi: 10.1186/1479-5876-10-141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, Kalos M, Maecker HT, Romero P, Yuan J, Kast WM, Hoos A, Elispot Proficiency Panel of the CVCIAWG (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother. 57(3): 303–315. 10.1007/s00262-007-0380-6 [DOI] [PMC free article] [PubMed]
- 21.Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H. Identification of human leukocyte antigen-A24-restricted epitope peptides derived from gene products upregulated in lung and esophageal cancers as novel targets for immunotherapy. Cancer Sci. 2007;98(11):1803–1808. doi: 10.1111/j.1349-7006.2007.00603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Yoshimura S, Tsunoda T, Osawa R, Harada M, Watanabe T, Hikichi T, Katsuda M, Miyazawa M, Tani M, Iwahashi M, Takeda K, Katagiri T, Nakamura Y, Yamaue H. Identification of an HLA-A2-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2) PLoS One. 2014;9(1):e85267. doi: 10.1371/journal.pone.0085267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Bernhagen J, Krohn R, Lue H, Gregory JL, Zernecke A, Koenen RR, Dewor M, Georgiev I, Schober A, Leng L, Kooistra T, Fingerle-Rowson G, Ghezzi P, Kleemann R, McColl SR, Bucala R, Hickey MJ, Weber C. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med. 2007;13(5):587–596. doi: 10.1038/nm1567. [DOI] [PubMed] [Google Scholar]
- 24.Takeda K, Yamaguchi N, Akiba H, Kojima Y, Hayakawa Y, Tanner JE, Sayers TJ, Seki N, Okumura K, Yagita H, Smyth MJ. Induction of tumor-specific T cell immunity by anti-DR5 antibody therapy. J Exp Med. 2004;199(4):437–448. doi: 10.1084/jem.20031457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kitaura K, Shini T, Matsutani T, Suzuki R. A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) a and b repertoires and identifying potential new invariant TCR a chains. BMC Immunol. 2016;17(1):38. doi: 10.1186/s12865-016-0177-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Gerlinger M, Quezada SA, Peggs KS, Furness AJ, Fisher R, Marafioti T, Shende VH, McGranahan N, Rowan AJ, Hazell S, Hamm D, Robins HS, Pickering L, Gore M, Nicol DL, Larkin J, Swanton C. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas. J Pathol. 2013;231(4):424–432. doi: 10.1002/path.4284. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Emerson RO, Sherwood AM, Rieder MJ, Guenthoer J, Williamson DW, Carlson CS, Drescher CW, Tewari M, Bielas JH, Robins HS. High-throughput sequencing of T-cell receptors reveals a homogeneous repertoire of tumour-infiltrating lymphocytes in ovarian cancer. J Pathol. 2013;231(4):433–440. doi: 10.1002/path.4260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Tamura K, Hazama S, Yamaguchi R, Imoto S, Takenouchi H, Inoue Y, Kanekiyo S, Shindo Y, Miyano S, Nakamura Y, Kiyotani K. Characterization of the T cell repertoire by deep T cell receptor sequencing in tissues and blood from patients with advanced colorectal cancer. Oncol Lett. 2016;11(6):3643–3649. doi: 10.3892/ol.2016.4465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Park JH, Jang M, Tarhan YE, Katagiri T, Sasa M, Miyoshi Y, Kalari KR, Suman VJ, Weinshilboum R, Wang L, Boughey JC, Goetz MP, Nakamura Y. Clonal expansion of antitumor T cells in breast cancer correlates with response to neoadjuvant chemotherapy. Int J Oncol. 2016;49(2):471–478. doi: 10.3892/ijo.2016.3540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Jang M, Yew PY, Hasegawa K, Ikeda Y, Fujiwara K, Fleming GF, Nakamura Y, Park JH. Characterization of T cell repertoire of blood, tumor, and ascites in ovarian cancer patients using next generation sequencing. Oncoimmunology. 2015;4(11):e1030561. doi: 10.1080/2162402X.2015.1030561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Subudhi SK, Aparicio A, Gao J, Zurita AJ, Araujo JC, Logothetis CJ, Tahir SA, Korivi BR, Slack RS, Vence L, Emerson RO, Yusko E, Vignali M, Robins HS, Sun J, Allison JP, Sharma P. Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities. Proc Natl Acad Sci U S A. 2016;113(42):11919–11924. doi: 10.1073/pnas.1611421113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Robert L, Tsoi J, Wang X, Emerson R, Homet B, Chodon T, Mok S, Huang RR, Cochran AJ, Comin-Anduix B, Koya RC, Graeber TG, Robins H, Ribas A. CTLA4 blockade broadens the peripheral T-cell receptor repertoire. Clin Cancer Res. 2014;20(9):2424–2432. doi: 10.1158/1078-0432.CCR-13-2648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Leisegang M, Engels B, Schreiber K, Yew PY, Kiyotani K, Idel C, Arina A, Duraiswamy J, Weichselbaum RR, Uckert W, Nakamura Y, Schreiber H. Eradication of large solid tumors by gene therapy with a T-cell receptor targeting a single cancer-specific point mutation. Clin Cancer Res. 2016;22(11):2734–2743. doi: 10.1158/1078-0432.CCR-15-2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Engels B, Engelhard VH, Sidney J, Sette A, Binder DC, Liu RB, Kranz DM, Meredith SC, Rowley DA, Schreiber H. Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity. Cancer Cell. 2013;23(4):516–526. doi: 10.1016/j.ccr.2013.03.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Hernández J, Lee PP, Davis MM, Sherman LA. The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire. J Immunol. 2000;164(2):596–602. doi: 10.4049/jimmunol.164.2.596. [DOI] [PubMed] [Google Scholar]
- 36.Burrows SR, Kienzle N, Winterhalter A, Bharadwaj M, Altman JD, Brooks A. Peptide-MHC class I tetrameric complexes display exquisite ligand specificity. J Immunol. 2000;165(11):6229–6234. doi: 10.4049/jimmunol.165.11.6229. [DOI] [PubMed] [Google Scholar]
- 37.Buslepp J, Zhao R, Donnini D, Loftus D, Saad M, Appella E, Collins EJ. T cell activity correlates with oligomeric peptide-major histocompatibility complex binding on T cell surface. J Biol Chem. 2001;276(50):47320–47328. doi: 10.1074/jbc.M109231200. [DOI] [PubMed] [Google Scholar]
- 38.Laugel B, van den Berg HA, Gostick E, Cole DK, Wooldridge L, Boulter J, Milicic A, Price DA, Sewell AK. Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties. J Biol Chem. 2007;282(33):23799–23810. doi: 10.1074/jbc.M700976200. [DOI] [PubMed] [Google Scholar]
- 39.Stone JD, Artyomov MN, Chervin AS, Chakraborty AK, Eisen HN, Kranz DM. Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs. J Immunol. 2011;187(12):6281–6290. doi: 10.4049/jimmunol.1101734. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Sabatino JJ, Jr, Huang J, Zhu C, Evavold BD. High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses. J Exp Med. 2011;208(1):81–90. doi: 10.1084/jem.20101574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Schubert DA, Gordo S, Sabatino JJ, Jr, Vardhana S, Gagnon E, Sethi DK, Seth NP, Choudhuri K, Reijonen H, Nepom GT, Evavold BD, Dustin ML, Wucherpfennig KW. Self-reactive human CD4 T cell clones form unusual immunological synapses. J Exp Med. 2012;209(2):335–352. doi: 10.1084/jem.20111485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348(6230):56–61. doi: 10.1126/science.aaa8172. [DOI] [PubMed] [Google Scholar]
- 43.Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, Araki K, Ahmed R. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017;355(6332):1423–1427. doi: 10.1126/science.aaf0683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, Sasmal DK, Huang J, Kim JM, Mellman I, Vale RD. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017;355(6332):1428–1433. doi: 10.1126/science.aaf1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Takeda K, Kojima Y, Uno T, Hayakawa Y, Teng MW, Yoshizawa H, Yagita H, Gejyo F, Okumura K, Smyth MJ. Combination therapy of established tumors by antibodies targeting immune activating and suppressing molecules. J Immunol. 2010;184(10):5493–5501. doi: 10.4049/jimmunol.0903033. [DOI] [PubMed] [Google Scholar]
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