Supplementary Materials, Methods, Tables and Figures:

The following supplementary information is included in this section.

1. Detailed Materials and Methods

2. Supplementary Tables

a. Supplementary Table 1: TCRβ Primer Sequences used in single-cell RT-PCR.

b. Supplementary Table 2: Identified peptide epitopes.

c. Supplementary Table 3: Recurrent tetramer-associated TCRβ clonotypes.

d. Supplementary Table 4: CDR3 amino acid length distribution.

3. Supplementary Figures

a. Supplementary Figure 1: Generation of an oligoclonal population of UNC-CDK4-1 specific CD8+ T-cells

b. Supplementary Figure 2: Measurement of tetramer “specific” T-cell responses in post-SCT samples.

Detailed Materials and Methods

Identification of HLA-A0201 restricted peptides by HPLC-MS

The mouse hybridoma line HB-82, source of the anti-HLA-A0201 antibody BB7.2, and the U937 leukemia cell line stably transfected with HLA-A0201 (U937.A2) were grown in a medium consisting of RPMI 1640 supplemented with 10% heat inactivated (56 °C for 30 minutes) fetal bovine serum (FBS), 1% penicillin streptomycin, and 1% L-glutamine. 3×1012 HB-82 cells were pelleted, and the 3L of resulting supernatant containing the secreted BB7.2 IgG2b antibody were collected, concentrated and affinity purified. Approximately 6×109 U937 cells were lysed in 20 mM Tris-HCl, pH 8, 150 mM NaCl, 1 % (mass:volume) 3- [(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS), 1 mM (phenylmethylsulfonyl fluoride), 10 μg/mL pepstatin, 5 μg/mL aprotinin, and 10 μg/mL leupeptin. The cell lysate was subjected to ultra-centrifugation at 100,000 G for 1 hr. Using an AKTAprime FPLC, a 5 mL GE Healthcare HiTRAP recombinant protein A column was loaded with 1 mg/mL of purified BB7.2 antibody. The cell lysate supernatant was then loaded onto the column. The BB7.2/HLA/peptide complexes were eluted with 0.2 M aqueous acetic acid solution and the concentration of acetic acid in pooled fractions was adjusted to 10% to separate the peptides from the HLA molecules and β2-microglobulin (β2m). The eluate was then passed through Microcon 3 K filters (14,000 rpm for 40 minutes) and subjected to liquid chromatography (LC) tandem mass spectrometry (MS/MS).

An Hitachi NanoFrontier Nano LC / Linear ion trap time-of-flight mass spectrometer was used for online LC-MS/MS experiments. A MonoCap monolithic trap column was used prior to the analytical column. Separation was achieved on a nano LC (75 µm I.D. x 150 mm) packed with Vydac C18 (5 µm particles) at a flow rate of 200 nL/min and a 60 minute gradient of 5% to 70% solvent B. Mobile phase A was composed of 98% H2O, 2% acetonitrile and 0.1% formic acid. Mobile phase B consisted of 98% acetonitrile, 2% H2O and 0.1% formic acid. 1 μL of the purified peptide was injected and subjected to data dependent acquisition using collision induced dissociation (CID) for peptide ion activation. MS/MS ion search was performed using the Mascot search engine with the no enzyme option and non-identical protein database (NCBInr).

Peptide-specific CTL lines generated using peptide pulsed T2 cells as stimulators

PBMCs were isolated from HLA-A*02:01 positive buffy coats (Gulf Coast Regional Blood Center, Houston, TX) using Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI) and cryopreserved in FBS (Gemini Bioproducts, West Sacramento, CA) with 5% DMSO. CTL cultures were initiated on day 0. T2 cells were washed in serum-free RPMI 1640 medium (Life Technologies, Inc., Carlsbad, CA), resuspended at a concentration of 2.5×106 cells/mL in serum-free RPMI 1640, and incubated with the UNC-CDK4-1 peptide (ALTPVVVTL, Biosynthesis, Lewisville, TX) at 20 µg/ml for 90 minutes at 37C. Peptide-pulsed T2 cells were irradiated with 7000 cGy in an RS 2000 X-Ray irradiator (Rad Source Technologies, Inc., Suwanee, GA), pelleted, and resuspended in complete culture medium (CM; RPMI 1640 medium supplemented with 2 mM L-glutamine and penicillin-streptomycin (all from Life Technologies) and 10% Human AB serum (Gemini Bioproducts)). Frozen PBMCs were thawed and washed twice in cold CM. CTL cultures were initiated at a ratio of 2 PBMCs to 1 live T2 cell, at a concentration of 2×106 PBMCs and 1×106 T2 cells per mL. Half of the CM was replaced as needed without disturbing the cell layer at the bottom of the flask. The CTL cultures were restimulated with peptide-pulsed, irradiated T2 cells at a 2:1 PBMC to T2 ratio on days 7, 14 and 21. On days 8, 15 and 22, 20 IU/mL of recombinant human interleukin-2 (IL-2; R&D Systems, Minneapolis, MN) was added to the cultures. If medium was replaced within 72 hours of restimulation, IL-2 was added to the CM. Cytotoxicity assays and tetramer staining were performed 4 days after the final restimulation (Day 25).

Cytotoxicity Assay

CD8+ effector cells were isolated from CTL cultures by indirect magnetic labeling using the CD8+ T cell Isolation Kit (Miltenyi Biotec, Auburn, CA) and resuspended at a concentration of 1×106 cells/mL in CM. 1×106 U937 or U937.A2 target cells were incubated in 200 L of 0.6 M Cell Trace Far Red DDAO-SE (Life Technologies) in Dulbecco’s PBS (DPBS) for 15 minutes at 37oC, washed once in CM, and resuspended at 1×106 cells/ml in CM. 100,000 effector cells (100 L) and 100,000 target cells (100 L) were coincubated in a 96-well V-bottom plate for 5 ½ hours at 37oC. Target cells were incubated in the absence of effector cells to control for non-specific cell death. After incubation, the cells were washed twice in cold DPBS and incubated for 5 minutes at 4oC in 100 L buffer (10 mM HEPES pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) with 2 L Live/Dead Fixable Violet Stain (Life Technologies). Cells were washed once in DPBS, fixed in 250 L DPBS with 1% paraformaldehyde, and analyzed on a MACSQuant flow cytometer (Miltenyi Biotec).

Peptide-specific CTL lines generated using peptide pulsed autologous dendritic cells (DCs) as stimulators

Antigen-specific T-cell expansion from naïve CD8+ T-cells was performed based upon the method of Wölfl and Greenberg (1). PBMCs were isolated from HLA-A*02:01 positive buffy coats using Ficoll-Paque PLUS and cryopreserved in FBS with 5% DMSO. The purified PBMCs were adhered to plastic for 3 hours. Non-adherent cells were collected and cryopreserved as a source of T-cells. The adherent cells were resuspended at 10×106 cells/mL in AIM V 10% media, and 2 mL were placed into a 6 well plate. The cells were maintained with IL-4 10 ng/mL and GM-CSF 800 IU at 37 °C. On the fifth day, the generated immature DCs were replated and treated with 10 ng/mL LPS, 100 IU/mL IFN-γ, 10 ng/mL IL-4 and 800 IU GM-CSF. After 16 hours the matured DCs were pulsed with 20 μg/mL UNC-CDK4-1 peptide and irradiated at 30 Gy. During DC maturation, the non-adherent cell fraction was thawed, and CD8+ cells were isolated by negative selection using Miltenyi MACS beads. Following re-suspension the CD8+ cells were incubated with 10 μL anti-CD57 and 10 μL anti-CD45RO per 1×107 CD8+ cells. The flow through was collected and considered a naïve CD8+ fraction. The naïve CD8+ cells were co-incubated with the peptide pulsed DCs at a ratio of 4 T-cells:1 DC in AIM V 5% media with IL-21 at 30 ng/mL. On day 3 of co-culture IL-15 at 5 ng/mL and IL-7 at 5 ng/mL. The cultures were continued for 11 days of stimulation and then analyzed.

CD107 / IFN-γ T-cell activation assay.

Antigen-specific T-cell stimulation and analysis of cultured T-cells was performed based upon the method of Betts et al (2). Autologous DCs were used as the targets for the T-cell cultures generated above. The DCs were either pulsed with 20 μg/mL of PR1 peptide, 20 μg/mL of UNC-CDK4-1 peptide or unpulsed. After pulsing the DCs were washed, and then suspended at 1×106 cells/mL. T-cell cultures were pelleted and resuspended at 1×106 cells/mL. For each experiment 5×105 T-cells were mixed with DCs at a 1:1 ratio to give a final volume of 1 mL. An additional control using phytohemagglutinin (PHA) was made by adding 8 μL PHA to 5×105 T-cells. After combining the cells, 60 μL PE- labeled anti-CD107a, 60 μL PE-labelled anti-CD107b and 60 μL anti-CD28/49d were added. The cells were incubated for 1 hr and then treated with 5 μL Brefeldin A and 5 μL monensin. The cells remained in the incubator for another 5 hours. The cells were subsequently washed (PBS, 10% BSA, 10% sodium azide), lysed (1× FACS lyse solution, 0.05% Tween) and permeabilized (1× FACS Perm, 0.05% Tween). The permeabilized cells were washed a second time (PBS, 10% BSA, 10% sodium azide, 0.05% Tween) and fixed with paraformaldehyde (1%). After fixing the cells were resuspended in 300 μL of FACS wash buffer and blocked with IgG. After blocking the cells were treated with PerCP-labelled anti-CD8 and FITC-labelled anti-IFNγ. After 30 minutes the cells were washed twice and analyzed by flow cytometry.

Western Blot Analysis

Three human AML PBMC lysates, a healthy donor PBMC lysate and a Jurkat cell lysate were prepared using RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 1% Triton-X and SigmaFast Protease Inhibitor (Sigma-Aldrich, St.Louis, MO)), and the protein concentrations were measured using the Micro BCA protein assay (Thermo Fisher Scientific, Waltham, MA). 20 μg of each lysate were incubated with loading dye (2% SDS, 10% glycerol, 62.5 mM Tris, pH 6.8, 5% beta-mercaptoethanol and 0.002% bromophenol blue) in a total volume of 15 L for 5 min at 98C. Samples were loaded onto a 4-12% NuPAGE gradient gel (Life Technologies, Inc., Carlsbad, CA) and electrophoresed at 150 V. The proteins were transferred to a PVDF membrane using a Mini-PROTEAN Trans-Blot Module (Bio-Rad Laboratories, Hercules, CA) at 250 mA for 70 min. CDK4 was detected with a primary antibody from Abcam (ab75511; Cambridge, MA) followed by an HRP-conjugated anti-mouse antibody (GE Healthcare, Waukesha, WI). Bands were visualized using Amersham ECL Western blotting detection reagents (GE Healthcare). The membrane was reprobed with a monoclonal anti-GAPDH antibody (Sigma-Aldrich).

Tetramer Flow Cytometry

Frozen post-transplant PBMC samples from AML patients were thawed and washed twice in CM. Depending on the number of cells recovered, 5×105 to 1×106 cells were incubated in 100 μL DPBS with Pacific Blue-conjugated CD4 (4 μL; 558116; BD Biosciences, San Jose, CA), CD14 (2 μL; 558121; BD Biosciences), CD16 (2 μL; 558122; BD Biosciences) and CD19 (1 μL; 302224; Biolegend, San Diego, CA) antibodies (‘dump’ channel), 2 μL FITC-conjugated CD8 antibody (MHCD0801; Life Technologies, Carlsbad, CA), and 5 L PE-conjugated tetramer (Beckman Coulter, Brea, CA) at 4°C for 25 minutes. Two μL Live/Dead Fixable Far Red Stain (Life Technologies, Inc.) was added and cells were incubated for 5 minutes at 4oC. Samples were washed once in DPBS after incubation and analyzed immediately on a MACSQuant flow cytometer (Miltenyi Biotec, Inc., Auburn, CA). Compensation was performed using single stained controls with the Anti-mouse Ig, Compensation Particles Set (BD Biosciences) for Pacific Blue, FITC and PE (a PE-conjugated CD8 antibody was used in place of the tetramer for compensation) and the ArC Amine Reactive Compensation Bead Kit (Life Technologies) was used for the Live/Dead Far Red Stain. Tetramer positive cells were enumerated in the live (far red stain negative), lymphocyte (FSC/SSC), Pacific Blue ‘dump’ negative, CD8 (FITC) positive gate.

iTopia Affinity and Off-rate Assays

The affinity of the CDK4 peptide (ALTPVVVTL) for HLA-A0201 and the relative stability of the peptide-HLA complexes were measured using the iTopia Epitope Discovery System (Beckman Coulter, Brea, CA), following the manufacturer’s protocol. Both assays use a FITC-conjugated anti-HLA antibody that binds to the correctly folded HLA-peptide complex and a positive control peptide (FLPSDFFPSV) for comparison with the test peptide. For the affinity assays, peptides were incubated in HLA-A0201 coated wells at concentrations ranging from 10-4 to 10-8 M at 21oC overnight, in the presence of the anti-HLA antibody. After washing the wells to remove unbound antibody and peptide, fluorescence was read on a Synergy 2 microplate reader (BioTek, Winooski, VT) with the excitation set at 485 nm and emission detected at 528 nm. Results were graphed relative to the binding of the positive control peptide at 10-4 M, and the ED50 was determined using GraphPad Prism’s nonlinear regression ‘log (agonist) versus response – variable slope (four parameter)’ curve. For the off-rate assay, peptides were incubated in HLA-A0201 coated wells at a concentration of 11 M at 21oC overnight, then washed and incubated at 37oC to allow the peptides to dissociate. Wells were washed again at the times indicated on the graph to remove dissociated peptide and antibody, and fluorescence was read on the microplate reader. Results were graphed relative to the positive control peptide as 100% binding at each time point (the control peptide does not significantly dissociate over the eight hour time course). The t1/2 was calculated using GraphPad Prism’s nonlinear regression, ‘dissociation – one phase exponential decay’ curve.

FACS Tetramer analysis and single-cell sorting

Peptide-HLA-A0201 iTAg tetramer assembled with CMV pp65 (NLVPMVATV) and UNC-CDK4-1 (ALTPVVVTL) peptides were purchased from Beckman-Coulter and used per manufacturer’s recommendations. The HLA-A0201 iTAg negative tetramer (also from Beckman Coulter) was used as a negative control. PBMCs were isolated by Ficoll density-gradient centrifugation from a single patient three months following SCT. Cells were stained with CD8-Pacific Orange (MHCD0830; Life Technologies) along with PE-HLA-A0201 iTAg tetramers generated with CMV-pp65, UNC-CDK4-1, and negative control peptides (Beckman-Coulter). For single-cell sorting, sort gates were determined by setting the CD8+ Tetramer+ gate such that it included no cells in the negative tetramer sample. Tetramer+CD8+ T cells were sorted by an iCyt Reflection high-speed sorter at 1 cell/well into a 96-well PCR plate (USA Scientific, Ocala, FL), each well containing 4 μL buffer (0.5× PBS, 10 mM DTT, and 8 U RNaseOUT RNase inhibitor (Invitrogen)). Plates were kept frozen at −80°C prior to RT-PCR analysis.

Single cell PCR and sequencing

RT-PCR amplification and sequencing of TCRβ clonotypes was performed as previously described (3), using a multiplex primer set including primers specific for all TCRβ variable region genes and the beta-chain constant region (Supplementary Table 1). RT-PCR amplicons were used as templates for a second round of PCR amplification using a panel of nested TCRβ-specific primers. PCR products were treated with Exonuclease I (New England Biolabs, Ipswich, MA) and shrimp alkaline phosphatase (Roche, Basel, Switzerland) and sequenced by the University of North Carolina Genome Analysis Facility (Chapel Hill, NC). TCRβ sequence gene-usage identifications were performed using the SoDA software tool (4). Sequences were defined as identical if they shared the same Vβ and Jβ gene usage along with identical β-CDR3 regions. As the iCyt Reflection saves the well location information indexed to the analyzed parameters for sorted cells, sequences derived from sorted cells can be compared for their associations with signals in each fluorescence channel. Tetramer-PE fluorescence vs. CD8-PacificOrange fluorescence values were plotted for each T-cell’s TCRβ clonotype using an R script (Figure 3A-B). Tetramer-PE fluorescence was used as an index of TCR binding avidity and was plotted for each sequence using Prism (GraphPad) (Figure 3C-D).

Bulk TCRβ repertoire sequencing

PBMCs were isolated from a second sample taken 6 months post-SCT from the same patient whose earlier sample was used for the single-cell sorting experiments. Bulk CD8+ T cells were sorted (without tetramer staining) on an iCyt Reflection hi-speed sorter. Three million CD8+ T cells were sorted in total. From this bulk-sorted population, RNA was extracted using the RNEasy kit protocol (Qiagen). The TCRβ repertoire was amplified using a RT-PCR protocol and multiplex primer set designed to minimize amplification bias and produce amplicon libraries that can be run on the Illumina HiSeq sequencing platform (iRepertoire, Huntsville AL) (5). The sequencing library was run using 2x100 paired-end chemistry on an Illumina HiSeq 2000 sequencer in the UNC High Throughput Sequencing Facility.

High-throughput sequencing data analysis

Sequence data were processed using Python and R scripts developed in the lab. Paired-end reads were joined across regions of overlap to produce a single joined contig corresponding to each read. These were analyzed for the presence of the conserved invariant cysteine and FGXG motifs that define the CDR3 region in one reading frame. Sequences that did not exhibit this motif, had stop codons in the motif reading frame, or were present at less than three copies in the dataset were excluded from further analysis. Vβ and Jβ gene usage identifications were made as follows: nucleotides upstream of the invariant cysteine position were analyzed for exact identity with germline V gene sequences. For reads whose Vβ gene was not identified by exact matching, identification was determined by finding the highest-scoring Smith-Waterman alignment between the nucleotides upstream of the invariant cysteine codon and the germline Vβ sequences (6). A similar procedure was used to identify Jβ usage based on the nucleotides downstream of the invariant phenylalanine. All germline reference gene sequences were downloaded from IMGT (http://www.imgt.org/IMGTrepertoire/) (7).

A heatmap of Vβ and Jβ usage was generated using the R function heatmap.2 in the gplots package (version 2.10.1) (8).

Supplementary Table 1: TCRβ Primer Sequences used in single-cell RT-PCR.

External Primer set

hb2E

TCACACAGATGGGACAGGAA

hb3E

AGCTGTTTCCCAGACTCCAA

hb4E

GGGAATGACAAATAAGAAGT

hb5E

CAAAACGAGAGGACAGCA

hb6E

AATGCTGGTGTCACTCAGA

hb6-4E

GGATATGAGACATAATGCCATG

hb7E

CTCAGGTGTGATCCAAT

hb7-4E

CTCAGGTGTGATTCAAT

hb9E

TCTGGAGACCTCTCTGTG

hb10E

AATCACCCAGAGCCCAAGA

hb11E

GATTCACAGTTGCCTAAGGAT

hb12E

GACAGAGATGGGACAAGAA

hb13E

GAGTCATCCAGTCCCCAAGA

hb14E

CCAATTTCTGGACATGATAATCT

hb15E

GGTTACCCAGTTTGGAAAGC

hb16E

TGAAGAAGTCGCCCAGA

hb18E

TCATGCAGAACCCAAGACAC

hb19E

GTACCTGTTCAGAAAGGAAGGA

hb20E

TGCTGTCGTCTCTCAACA

hb24E

AAGAGGATTATGCTGGAATGTT

hb25E

AAGCTGACATCTACCAGACC

hb27E

CCAAGATACCTCATCACAGT

hb28E

ATATGTTCTGGTATCGACAAGAC

hb29E

AGTGTCAAGTCGATAGCC

hb30E

ACTGTGGAGGGAACATCAA

hbCe

CTTGACAGCGGAAGTGGTT

Internal Primer set

hb2i

CTGTGTCCCCATCTCTAATC

hb3i

TGGGAAACGACAAGTC

hb4i

AGCTCATGTTTGTCTACA

hb5i

AGCTGAATGTGAACGCC

hb5-1i

GCCTTCAGTTCCTCTTTG

hb6i

CGACAAGACCCAGGCATG

hb6-4i

GCTAAGGCTCATCCATTATTCAA

hb7-2/3i

ATCCAATTTCAGGTCATACT

hb7-8/9i

TTCCAGAATGAAGCTCAACT

hb7-4/6/7i

CCCAGAGTTTCTGACTTAC

hb9i

CCGCACAACAGTTCCCTG

hb10i

GACATGGGCTGAGGCT

hb11i

GTAGACTCCACTCTCAAGAT

hb12i

GTACAGACAGACCATGATGC

hb13i

GGAAACAGCCACTCTGAAA

hb14i

TATTGGTATCGACGTGTTATG

hb15i

GCCAGTGACCCTGAGTTGTT

hb16i

ACTCCAAAACATCTTGTCAGAG

hb18i

GATGCAGCCCAATGAAAGG

hb19i

GAATTTGAACCACGATGCCA

hb20i

TTATCTGTAAGAGTGGAACCTC

hb24i

GCCTACGGTTGATCTATTACTC

hb25i

CACCTCATCCACTATTCCTATG

hb27i

AATATGAACCATGAGTATATGTCC

hb28i

CTACGGCTGATCTATTTCTC

hb29i

TCAGCCGCCCAAACCTAA

hb30i

CAGAATCTCTCAGCCTCCA

hbCi

CACCTCCTTCCCATTCAC

All primers are listed in the 5’ 3’ direction.

Peptide

Protein

E-value

IC50 (ANN)

ENVNTYFVL

dnaJ homolog subfamily C member 5

0.0003

24,302.04

PAASDCRAAE

AT-hook DNA-binding motif-containing protein 1

0.0003

36,500.44

ALTGWLPEV

Calpain-7-like protein

0.0007

7.79

MELSVLLFL

Cytochrome P450 2B6

0.0007

3,283.68

DYDIALLEL

Suppressor of tumorigenicity 14 protein

0.0007

22,841.13

ALPEIFTEL

Eukaryotic translation initiation factor 2, subunit 3

0.001

18.13

SLQEEHVAV

Plectin-1

0.002

31.66

AAFVRLNEA

Anion exchange protein 3

0.003

8,783.20

SLLPAIVEL

Protein phosphatase 2, regulatory subunit

0.004

10.29

LLIAVNSPV

Schlafen family member 14

0.004

10.70

LLIENVASL  

Glutathione peroxidase 1

0.004

13.50

FLLPTGAEA

Cathepsin G

0.004

20.07

LSLPSPVEL

Ras and Rab interactor

0.004

5,953.69

LEPWRASL

Separin

0.004

N/A *

ALFPGVALL

PDIA3 protein disulfide isomerase family A, member 3

0.005

12.48

LLIERGASL

Ankyrin repeat domain 17

0.005

29.19

LLDVPTAAV

Interferon gamma inducible protein 30

0.005

43.78

ALTPVVVTL

Cyclin dependent kinase 4

0.005

46.88

AVAVITVQL

Amphiregulin preproprotein

0.005

1,211.34

EDAPVGSLL

Protocadherin-8 precursor

0.005

24,752.51

ALSEKIVSV

Coiled-coil domain containing protein 93

0.008

10.15

KLLVTVTAV

Synaptotagmin-14

0.008

15.08

ILLAAVPTA

Major facilitator superfamily domain containing 2B

0.008

19.74

LLIPGLATA

NADH dehydrogenase 1 alpha subcomplex subunit 1

0.008

34.55

AFQHAVKI

Glycerophophodiester phosphodiesterase domain-containing protein 1

0.012

N/A *

SLAGGIIGV

Heterogeneous nuclear ribonucleoprotein K

0.015

9.42

FLISLVFLL

MAL-like protein

0.022

6.67

DQNKVVSV

Olfactory receptor 5P2

0.025

N/A *

APDVFVL

Predicted ankyrin repeat domain-containing protein 60

0.16

N/A *

LFTHLGV

ER mannosyl-oligosaccharide 1,2-alpha-mannosidase

0.16

N/A *

GARVGLLARVQA

GAS2-like protein 1 isoform a

2.7 §

≥ 8,313.74

IRAVGGRAV

Domain-containing protein 4B

2.9 §

22,923.82

AGPVREAEA

Disintegrin and metalloproteinase domain-containing protein 12

2.9 §

24,050.92

LIINTALA

Catenin alpha-3

6.7 §

N/A *

Supplementary Table 2: Identified peptide epitopes

§ Peptides with an E-blast score > 1 did not completely align with known human peptide sequences.

* Peptide sequences < 9 amino acids long cannot bind to HLA-A0201.

The MHC-I binding predictions were made using the IEDB analysis resource ANN aka NetMHC (ver. 3.4) tool (9, 10).

Supplementary Table 3: Recurrent tetramer-associated TCRβ clonotypes.

pp65NLV

N=63

UNC-CDK4-1

N=25

Sequence

V

J

n

Sequence

V

J

n

ASRPFVNTEA

V6-5

J1-1

6

ASRMNTEA

V19-1

J1-1

2

ASSFSLPYEQY

V12-3

J2-7

3

ASSAQGFGEL

V9-01

J2-2

4

ASSSEFGDTAYNEQ

V7-3

J2-1

24

ASSEGRETQY

V25-1

J2-5

2

ASSSVNEQ

V12-3

J2-1

3

ASSIGQGPYEQY

V19-1

J2-7

2

ASTIVSGYT

V12-3

J1-2

21

ASSLGGRRYEQY

V11-2

J2-7

2

ASSLGQKEQY

V11-2

J2-7

4

The recurrent TCRβ clonotypes are shown for both pp65NLV and UNC-CDK4-1 associated T-cell responses. Each sequence contains a Vβ-CDR3-Jβ component. The Vβ contribution to the clonotype is noted by the first few amino acids up to the true CDR3 region that is denoted in bold type. The Jβ contribution to the clonotype sequence is denoted by the regular type face amino acids following the CDR3 amino acids. The Vβ and Jβ families for each clonotype are also shown as are the number of times each clonotype was identified in the single-cell sorting experiment.

Supplementary Table 4: CDR3 amino acid length distribution.

CDR3 amino acid length

4

5

6

7

8

9

10

11

# of reads in repertoire

4

435

17

1,299

5.840×106

5.081×106

1.574×107

5.953×107

Percentage of repertoire

< 4×10-6

4×10-6

1.8×10-5

1.364×10-3

6.130

5.334

16.52

62.47

CDR3 amino acid length

12

13

14

15

16

17

18

# of reads in repertoire

2.666×106

6.116×106

1.930×105

8.293×104

3,454

5,990

5,885

Percentage of repertoire

2.798

6.420

0.203

0.087

3.626×10-3

6.288×10-3

6.178×10-3

The number of TCRβ reads that yielded a functional CDR3 clonotype are shown listed by predicted CDR3 length. Predicted CDR3 length contains the amino acid sequence for the CDR3 region starting immediately after the N-terminal invariant cysteine residue and ends immediately before the C-terminal invariant phenylalanine residue. The percentage of the TCRβ repertoire supported by the various CDR3 lengths is also given. There was a marked skewing of the CDR3 length distribution, with 78.99% of the repertoire being supported by TCRβ sequences with CDR3 regions of either 10 or 11 amino acids.

Supplementary Figure 1.

Supplementary Figure 1: Measurement of UNC-CDK4-1 specific T-cell responses in post-SCT samples. The gating strategy used for tetramer enumeration is shown at the top of the figure. The 6 post-SCT samples shown in Figure 2 are included in this figure with the additional fluorescence minus one (FMO) and negative tetramer (in 3 of the 6 samples where it was used) added to show gating and the low amount of non-specific tetramer reactivity. Each row represents 1 patient sample denoted by its label in Figure 2. For comparison the pp65NLV tetramer FACS plot for patient 5 (2E) is also shown.

Supplementary Figure 2.

Supplementary Figure 2: Generation of UNC-CDK4-1 specific T-cells using UNC-CDK4-1 pulsed T2 cells as stimulators. Five HLA-A*02:01 expressing buffy coats were tested to generated UNC-CDK4-1 specific T-cells. All 5 buffy coats were stimulated with peptide pulsed irradiated T2 cells in IL-2 for 3 weeks. (A) Of the 5 cultures, 1 yielded an expanded tetramer+ T-cell population on 0.2% of CD8+ cells. The other 4 cultures yielded tetramer+ populations of 0.01%, which was considered non-specific binding by the tetramer (data not shown). (B) The T-cell culture shown in (A) was able to selectively kill the U937.A2 cell line that had been the source of the UNC-CDK4-1 peptide (the U937 control cell line does not express an HLA predicted to bind the UNC-CDK4-1 peptide with high affinity).

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8.Gregory R. Warnes. Includes R source code and/or documentation contributed by: Ben Bolker LB, Robert Gentleman, Wolfgang Huber Andy Liaw, Thomas Lumley, Martin Maechler, Arni Magnusson, Steffen Moeller, Marc Schwartz and Bill Venables (2011). gplots: Various R programming tools for plotting data. R package version 2.10.1. http://CRAN.R-project.org/package=gplots. Heatmap R code.

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0

10

20

30

40U937U937-A2

CD8-FITC

Tetr

amer

-PE

0.20 P = 0.0327

Perc

ent C

ytot

oxic

ity

A. B.

0

10

20

30

40

U937

U937-A2

CD8-FITC

T

e

t

r

a

m

e

r

-

P

E

0.20

P = 0.0327

P

e

r

c

e

n

t

C

y

t

o

t

o

x

i

c

i

t

y

A. B.

CD8-FITC

UN

C-C

DK

4-1

tetr

amer

-PE

FMO UNC-CDK4-1 Neg. Tetramer

2A

2B

2C

2D

2E

2F

0.00 0.17

0.00 0.08

0.00 0.01 0.00

0.00 0.01 0.00

0.00 0.04 0.01

0.00 0.00

Far Red Viability

SSC

SSC

FSC CD8-FITC

Dum

p-Pa

cBlu

e

live cells87.36

lymphocytes70.92

56.26 3.18

3.9437.24

0.07

pp65

tetr

amer

-PE

CD8-Pac Orange

CD8-FITC

U

N

C

-

C

D

K

4

-

1

t

e

t

r

a

m

e

r

-

P

E

FMOUNC-CDK4-1 Neg. Tetramer

2A

2B

2C

2D

2E

2F

0.00 0.17

0.00 0.08

0.00 0.01 0.00

0.00 0.01 0.00

0.00 0.04 0.01

0.00 0.00

Far Red Viability

S

S

C

S

S

C

FSC CD8-FITC

D

u

m

p

-

P

a

c

B

l

u

e

live cells

87.36

lymphocytes

70.92

56.263.18

3.94

37.24

0.07

p

p

6

5

t

e

t

r

a

m

e

r

-

P

E

CD8-Pac Orange

0

10

20

30

40U937U937-A2

CD8-FITC

Tetr

amer

-PE

0.20 P = 0.0327

Perc

ent C

ytot

oxic

ity

A. B.

0

10

20

30

40

U937

U937-A2

CD8-FITC

T

e

t

r

a

m

e

r

-

P

E

0.20

P = 0.0327

P

e

r

c

e

n

t

C

y

t

o

t

o

x

i

c

i

t

y

A. B.