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transcript
Supplementary Materials for
Direct measurement of T cell receptor affinity and sequence from naïve
antiviral T cells
Shu-Qi Zhang, Patricia Parker, Ke-Yue Ma, Chenfeng He, Qian Shi, Zhonghao Cui,
Chad M. Williams, Ben S. Wendel, Amanda I. Meriwether, Mary Alice Salazar,
Ning Jiang*
*Corresponding author. Email: jiang@austin.utexas.edu
Published 1 June 2016, Sci. Transl. Med. 8, 341ra77 (2016)
DOI: 10.1126/scitranslmed.aaf1278
The PDF file includes:
Materials and Methods
Fig. S1. Nonspecific adhesion on primary CD8+ T cells is negligible and not
correlated with pMHC site density.
Fig. S2. Multivalency of pMHC does not affect 2D affinity measurement.
Fig. S3. 2D affinity kinetic curves for three additional CD8+ T cell clones with
varying affinities.
Fig. S4. Streptamer staining is fully reversible, and streptamers stain with the
same intensity as conventional tetramers.
Fig. S5. Estimating the error of single-cell 2D affinity measurements.
Fig. S6. Peptide titration curves of 15 HCV-specific CD8+ T cell clones.
Fig. S7. Conventional 2D affinity measurement on HCV-specific CD8+ T cell
clones shows similar correlations as single-cell 2D affinity.
Fig. S8. Gating strategy for sorting HCV-streptamer+ T cells.
Fig. S9. Ratio of high-affinity/low-affinity HCV-specific CD8+ T cells based on
functional potential.
Fig. S10. Amplification efficiency of affinity-tested cells.
Fig. S11. TRAV and TRBV usage of affinity-tested primary HCV-specific CD8+
T cells by donor.
Table S1. Comparison of single-cell 2D affinity derived from a primary T cell and
conventional 2D affinity derived from the T cell’s in vitro expanded clone.
Table S2. Single-cell 2D affinity and SPR 3D affinity for the native and peptide
variants of NY-ESO-1 against 1G4 TCR.
www.sciencetranslationalmedicine.org/cgi/content/full/8/341/341ra77/DC1
Table S3. Median 2D affinity and ratio of high/low 2D affinity from primary
HCV-specific CD8+ T cells.
Table S4. Single-cell 2D affinity and correlated TCR and TCR CDR3
sequences.
Reference (29)
Supplemental Materials and Methods
Micropipette adhesion frequency assay:
The TCR-pMHC affinity is measured using a micropipette adhesion frequency assay that was
previously described (5, 15).
For CD8+ T cell clones, an adhesion curve can be determined by obtaining the adhesion
probability at multiple contact times. This is fit with a known model for reversible bimolecular
interactions at two-dimensional surfaces (15):
]}[exp{1 offaclra tkKAmmP (eq. S1)
Pa = Adhesion frequency
mr = TCR site density
ml = pMHC site density
Ac = contact area between RBC and T cell
AcKa = 2D Affinity
koff = 2D dissociation rate constant
t = contact time
Ac, 3 μm2, is kept constant for all measurements, and we estimate it to be within several percent
of 3 µm2 based on the length-calibrated images from the microscope. However, since the actual contact
area cannot be known, the true 2D affinity, Ka, cannot be measured. As a surrogate, AcKa is used as the
effective 2D affinity in all 2D affinity publications (5,15), with a unit of µm4. Similarly, we use AcKa as
the 2D affinity in this study.
The site density of T cell receptor and pMHC is assessed by flow cytometry. For site density,
~105 T cells or RBCs are incubated with 5 μl of PE labeled TCRαβ antibody (Biolegend) or 5 μl PE
labeled HLA-A2 antibody (Biolegend) for 1 hour at 4C in staining buffer, respectively. Measurements
are performed in BD Accuri, and total TCR or pMHC expression level is quantified using BD
Quantibrite beads according to manufacturer instructions. Site density of TCR or pMHC is acquired by
dividing average T cell or RBC surface area respectively.
For single-cell 2D affinity, TCR site density is derived from the bulk CD8+ T cell population
from the same patient, and the measurement variance was estimated using the parameters from
supplementary fig 6. For bulk 2D affinity on CD8+ T cell clones, TCR site density can be directly
measured using a separate aliquot of the clone; the measurement variance is determined by averaging
the adhesion frequencies from multiple T cell-RBC measurements.
Estimating the error of single-cell 2D affinity measurements
To measure the error in the 2D affinity calculation of primary T cells, we considered all possible sources
of error to the measured adhesion frequency, Pa, where σ refers to the standard deviation:
2
RBC-RBC
2
aceTcell_surf
2
Tcell-Tcell
2
binom
2 (eq. S2)
σbinom: Bernoulli process variation
σTcell-Tcell : Variation in TCR site density between T cells
σTcell_surface: Variation in TCR site density across the surface of one T cell
σRBC-RBC : Variation in pMHC site density between red blood cells (RBC).
From Eq. 1, TCR expression level differences will be a function of the variation in adhesion
frequency Pa given a constant 2D affinity and pMHC site density. Measuring TCR expression level
differences using pMHC ligand cannot be done on primary CD8+ T cells because each TCR-pMHC
interaction will have a different 2D affinity due to the polyclonality of TCRs. As such, RBCs coated
with biotinylated TCR antibody is used as a surrogate. TCR antibody interaction with TCR fits the
model for a biomolecular interaction just like TCR-pMHC interaction (Fig. S5 and Eq. S1), and hence
can be used to measure TCR expression variability.
The Bernoulli process variation σbinom is a function of the adhesion probability (Pa) and the
number of contacts between RBC and T cell. We isolated σTcell-Tcell variation by using one RBC coated
with biotinylated TCR antibody and measured its adhesion frequency against multiple primary CD8+ T
cells (factor i); this case contains the largest source of variation. σTcell_surface was isolated by measuring
the adhesion frequency of one RBC coated with biotinylated TCR antibody against one primary CD8+ T
cell at multiple areas of the cell, performed by releasing and re-aspirating the T cell at a different
location (case iia,b); we tested this using two T cells and the adhesion frequencies for the both cells have
similar variances but different mean values, emphasizing that each T cell has its own unique adhesion
frequency which can be attributed to differences in TCR expression. Lastly, σRBC-RBC was isolated by
measuring the adhesion frequency of one HCV-specific CD8+ T cell clone against multiple RBCs coated
with the cognate pMHC (case iii).
LDH Cytotoxicity Assay:
JY cells were cultured with varying concentration of HCVns3:1406-1415 peptide for 3 hours.
HCV-specific T cell clones and JY cells were washed 3 times with CTL media before using. For lysis
capacity, 104 JY cells and 105 T Cells were put into contact by centrifugation and then cultured for 4
hours at 37ᵒC. For peptide sensitivity, 6x103 JY cells and 6x104 T Cells are used. Individual conditions
were performed in triplicates. The Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific) was
used according to manufacturer’s instructions. Lysis capacity, defined as percent specific lysis, was
calculated according to the following formula:
specific control
max spontaneous
% Specific Lysis
Where α is the difference in absorbance between 490nm and 680nm, performed in triplicates.
αspecific refers to T cells incubated with HCV-pulsed JY cells. αcontrol refers to T cells incubated with non
peptide-pulsed JY cells. αmax refers to JY cells lysed according to Pierce instructions. αspontaneous refers to
JY cells incubating by themselves.
Single Cell TCR Amplification:
Single cell TCR amplification and sequencing was done following published protocol (13) with
minor modifications. Briefly, single CD8+ T cells were directly picked into lysis buffer and reverse
transcription was done either right away or after thawing the frozen lysate. After PCR, multiple cells
were pooled, purified by electrophoresis and gel extraction, and sequenced using Ilumina Mi-seq V2 kit.
TCR sequence analysis
Raw reads were first filtered and separated into α and β chain groups based on constant
sequences. For both α and β chain groups, reads were further separated according to cell barcodes.
Within each cell barcode, reads were clustered based on sequence similarity. From each cluster, one
sequence was generated based on the consensus of nucleotides weighted by the quality score at that
position. Following this method, TCR α and β chain sequences for each single cells can be generated. It
is possible for individual T cells to have a second α and/or β chain. Given the read distribution of
consensus sequences for each cell, the second most abundant consensus is determined to be a second
chain if the read number is at least 10x higher than the third most abundant consensus and has a read
number greater than 3. MIGEC was used for V/J assignment and CDR3 annotation (29).
Supplementary Figure 1. Nonspecific adhesion on primary CD8+ T cells is negligible and not
correlated with pMHC site density.
Red blood cells bearing HLA-A2-CD8mut/HCV of varying site densities were used to interact with
freshly purified CD8+ T cells from human PBMCs to assess the background adhesion level. Adhesion
frequency is measured by the average of 30 contacts with 4 seconds at each contact. pMHC site densities
were measured using HLA-A2 antibody.
0%
20%
40%
60%
80%
100%
7 38 107 478 1865A
dhesio
n F
requency, P
aHLA-A2-CD8mut/HCV site density (sites/μm2)
A B
Supplementary Figure 2. Multivalency of pMHC does not affect 2D affinity measurement.
To exclude the possibility of multivalent TCR-pMHC interactions introduced by immobilizing multiple
pMHCs onto one streptavidin molecule, we compare the affinity measurement obtained from one HCV-
specific T cell clone using either red blood cells conjugated with pMHC captured by wild type
tetravalent streptavidin (SA) or mutant divalent streptavidin. Divalent streptavidin would represent a
biomolecular interaction, as one binding pocket is occupied to the biotin on the red blood cell, and the
other one by the pMHC. (A, B) Adhesion frequency (A) and conventional 2D affinity (B) of wildtype
SA and divalent SA as described previously (5) (two-tailed t test).
0%
20%
40%
60%
80%
100%
Wildtype SA Divalent SA
Adhesio
n F
requency, Pa
9.5 sites/μm213.3 sites/μm2
p = n.s.
Wildtype SA Divalent SA
Conventional 2D
Aff
inity
(μm4)
1.6x10-3
1.2x10-3
8.0x10-4
4.0x10-4
0
p = n.s.
A B
C
Supplementary Figure 3. 2D affinity kinetic curves for three additional CD8+ T cell clones with
varying affinities.
Adhesion probability (Pa) was plotted as a function of contact time (s). 2D TCR-pMHC affinity, AcKa,
for each clone is indicated. Solid line is the model fit (Eq. 1). Error bars denote standard deviation.
0%
20%
40%
60%
80%
100%
0 2 4 6 8 10Adhesio
n F
requency, P
a
Contact Time (sec)
AcKa = 2.3x10-4 ± 2.8x10-5μm4
mTCR = 26 μm-2
mpMHC = 85 μm-2
0%
20%
40%
60%
80%
100%
0 5 10Adhesio
n F
requency, P
a
Contact Time (sec)
AcKa = 1.2x10-3 ± 1.4x10-4 μm4
mTCR = 11 μm-2
mpMHC = 104 μm-2
0%
20%
40%
60%
80%
100%
0 2 4 6 8 10
Adhesio
n F
requency,
Pa
Contact Time (sec)
AcKa = 3.1x10-4 ± 4.5x10-5 μm4
mTCR = 74 μm-2
mpMHC = 57 μm-2
A B
Supplementary Figure 4. Streptamer staining is fully reversible, and streptamers stain with the
same intensity as conventional tetramers.
(A) Streptamers can be completely dissociated off of the surface of antigen-specific T cells (14). An
HCV-specific CD8+ T cell clone is stained with PE-labeled streptamer bearing HLA-A2/HCV at 4°C in
staining buffer. Cells were then dissociated of streptamer by biotin competition. (B) An HCV-specific
CD8+ T cell clone is stained with either streptamer HLA-A2/HCV or conventional streptavidin-based
tetramer HLA-A2/HCV.
Streptamer Fluorescence Intensity (a.u)
Tetramer Fluorescence Intensity (a.u)
A
B Contributions to Variance in Pa (σ2)
Factor Total variance (σ2) Binomial T cell to T cell TCR surface uniformity RBC to RBC
i 1.5x10-2 3.9x10-3 1.1x10-2 ~5.8x10-4 (factor ii mean) 0
iia 5.0x10-3 4.8x10-3 0 2.2x10-4 0
iib 4.5x10-3 3.6x10-3 0 9.4x10-4 0
iii 7.0x10-3 4.0x10-3 0 0 3.0x10-3
Supplementary Figure 5. Estimating the error of single-cell 2D affinity measurements.
(A) Adhesion probability (Pa) as a function of contact time (s) for primary CD8+ T cells contacted with
a RBCs bearing TCR antibody.
(B) Calculate contributions of variance from individual variables based on measurements from Fig. 2D
and Eq. 3
0%
20%
40%
60%
80%
100%
0 10 20
Adhesio
n F
requency, Pa
Contact Time (sec)
Clone name Single-cell 2D affinity (μm4)
Peptide potency
([M])
Tetramer
MFI
661-3 B9 2.1E-04 2.0E-07 954
661-3 C5 1.5E-03 1.6E-07 23096
661-3 C8 7.3E-05 2.7E-05 182
661-3 D10 1.6E-04 3.7E-08 147
2B2 1.4E-04 9.0E-07 225
4A1 4.2E-05 8.9E-06 267
4D1 2.6E-04 1.2E-07 1863
B8 Smer 1.3E-03 1.5E-07 1227
B4 1.6E-04 2.0E-07 130
C9 3.8E-04 4.5E-06 129
D2 6.2E-04 1.5E-06 340
D9 3.9E-05 6.4E-05 135
E8 1.6E-05 1.5E-05 151
G9 2.2E-03 2.0E-08 740
G11 4.7E-05 1.5E-05 616
Supplementary Figure 6. Peptide titration curves of 15 HCV-specific CD8+ T cell clones.
Peptide titration curves of 15 HCV-specific CD8+ T cell clones against JY cells pulsed with HCV
peptide. Here, we define peptide potency as the peptide concentration required to induce 10% specific
cell lysis.
-10%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Lys
is C
apacity
Peptide Concentration ([M])
661-3 B9 661-3 C5661-3 C8 661-3 D102B2 4A14D1 B8 SmerB4 C9D2 D9E8 G9G11
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
A B
C
Supplementary Figure 7. Conventional 2D affinity measurement on HCV-specific CD8+ T cell
clones shows similar correlations as single-cell 2D affinity.
Bulk 2D affinity versus (A) 3D affinity by SPR (similar to Fig. 3B), (B) Lysis capacity (similar to Fig.
4A, and (C) Peptide Potency (similar to Fig. 4B. Conventional 2D affinity is measured by taking the
average single-cell 2D affinities from at least four cells per clone.
3D
Affin
ity (µM
-1)
Bulk 2D Affinity (µm4)
10-1
10-2
10-3 10-5 10-4 10-3
r = 0.83
p < 0.02
Lysis
Ca
pa
city
Bulk 2D Affinity (µm4)
r = 0.76
p < 10-5
100%
70%
40%
10%
10-5 10-4 10-3
Pe
ptid
e P
ote
ncy [
M]
Bulk 2D Affinity (µm4)
10-5 10-4 10-3
10-4
10-5
10-6
10-7
10-8
r = -0.77
p < 0.001
Conventional 2D Affinity (μm4) Conventional 2D Affinity (μm4)
Conventional 2D Affinity (μm4)
A B C
D E F
G H
Supplementary Figure 8. Gating strategy for sorting HCV-streptamer+ T cells.
Magnetic beads enriched antigen-specific CD8+ T cells, as well as the flow-through fraction were used
to setup various gates for antigen-specific T cell sorting. (A,B) Starting from the enriched fraction, gates
were first set for single cell lymphocytes. Counting beads were added prior to analysis to allow counting
of the antigen-specific T cell frequency. Although the enriched fraction is a subset of a CD8+ T cell
enriched sample, a minority of other cell populations still exists. (C) As such, CD8+ T cells were chosen
using negative selection using markers for macrophages, neutrophils, natural killer cells, CD4+ T cells,
and B cells. CD8 antibody was not added due to its tendency to activate T cells and alter the T cell
receptor expression level. In the majority of these experiments, we also observed an enrichment of dead
cells using the magnetic enrichment procedure, and hence 7-aminoactinomycin D is always used to
discern dead populations. (D) Afterwards, the antigen-specific T cells were gated. (E) The gating
HC
V-s
pecific
CD
8+
T c
ells
P
er
Tota
l C
D8
+T
cells 10-5
10-6
threshold was set by using the flow-through from the same sample, which has been stained with the
same panel as the enriched fraction but should not contain streptamer labeled cells. (F, G) naïve
antigen-specific T cells were isolated based on positive expression of CCR7, CD45RA, and CD27.
(H) The frequency of HCV-specific CD8+ T cells from the four HCV-seronegative human
samples are calculated as follows:
𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 =# 𝑠𝑡𝑟𝑒𝑝𝑡𝑎𝑚𝑒𝑟+ 𝑐𝑒𝑙𝑙𝑠 ∗
# 𝑏𝑒𝑎𝑑𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 # 𝑏𝑒𝑎𝑑𝑠 𝑎𝑑𝑑𝑒𝑑
𝑡𝑜𝑡𝑎𝑙 𝐶𝐷8+ 𝑇 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡
Total CD8+ T cell count was determined by measuring the fraction of CD8+ T cells in the flowthrough
using CD8 and TCR antibodies, and multiplying that by the total live cell count of flowthrough by
Cellometer.
Supplementary Figure 9. Ratio of high-affinity/low-affinity HCV-specific CD8+ T cells based on
functional potential.
From Fig. 4A, a single-cell 2D affinity of ~2x10-5 μm4 represents a sharp cutoff between in vitro
functional and non-functional T cells. Using this threshold to define high and low affinity T cells,
the ratio of high/low affinity CD8+ T cells was plotted from the primary T cell affinity data of 9 unique
donors from Fig. 5A (two-tailed Mann-Whitney U Test).
0
0.5
1
1.5
2
2.5
3
3.5
4R
atio H
igh
/Lo
w A
ffin
ity T
ce
lls
Age
≤33 ≥49
p < 0.005
A
Amplification efficiency
Sample 4B Sample 5A Sample 6 Sample 7
TCRα 22/44 (50%) 9/16 (56%) 13/25 (52%) 9/21 (43%)
TCRβ 32/44 (73%) 12/16 (75%) 11/25 (44%) 11/21 (52%)
TCRαβ 18/44 (41%) 8/16 (50%) 7/25 (28%) 4/21 (19%)
B
Supplementary Figure 10. Amplification efficiency of affinity-tested cells.
(A) Amplification efficiency of productive TCRα and TCRβ chains from donor 4B, 5A, 6, and 7. (B,
left) In another experiment, the T cell transfer pipette was first dipped into the chamber to simulate cell
picking and then dipped into lysis media to simulate transfer of zero cells. Amplification of TCR
amplicons, which are 300 base pair in length, was not seen. (B, right) Two T cells were aspirated by
transfer pipette, injected into one tube, and then the pipette was immediately dipped into a second tube
to test possible carry-over contamination. TCR amplicons were seen in the transfer to the first tube but
not the second, indicating that both T cells were deposited into the first tube and no carry over
contamination exists between samples.
Supplementary Figure 11. TRAV and TRBV usage of affinity-tested primary HCV-specific CD8+
T cells by donor.
0
51015
202530354045
5 8-1 8-3 8-4 12-3 14 19 22 26-1 26-2 41 38-2
Cou
nt
TRAV
4B5A67
0
2
4
6
8
10
12
14
16
2
4-1
4-3
5-1
6-2
/3
6-5
6-6
7-8
7-9 9
10-3
11-2
12-3
/4 13
14
15
18
19
20-1
25-1 27
28
30
Co
un
t
TRBV
4B
5A
6
7
Supplementary Table 1. Comparison of single-cell 2D affinity derived from a primary T cell and
conventional 2D affinity derived from the T cell’s in vitro expanded clone.
Multiple data points for conventional 2D affinity reflects the single-cell 2D affinity value from each cell
of the CD8+ T cell clone measured.
Single-cell 2D affinity
(x10-3μm4)
Average 2D affinity ± SD
(x10-3 μm4)
Primary (single cell) 1.9 1.9 ± 0.79*
Clone (conventional)
3.6
1.8 ± 0.94
1.7
0.66
1.5
1.4
2.4
1.3
*standard deviation is estimated based on model from fig. S5
Supplementary Table 2. Single-cell 2D affinity and SPR 3D affinity for the native and peptide
variants of NY-ESO-1 against 1G4 TCR.
Table of the native and peptide variants of NY-ESO-1, the measured 2D affinities, and the published 3D
affinities (3). Affinity listed as mean ± standard error of the mean (SEM) for 3D is from (3) and SEM
for 2D affinity is estimated using model from fig. S5)
Peptide name Sequence
Single-cell 2D affinity
(x10-5 μm4) ± SEM 3D affinity, Ka
(mM-1) ± SEM
ESO-3M SLMMWITQV 49 ± 18 109 ± 3
ESO-7H SLLMWIHQV 1.5 ± 0.4 10 ± 0.8
ESO-9C
(native) SLLMWITQC 12 ± 2.5 69 ± 3
ESO-3A SLAMWITQV 53 ± 32 152 ± 12
ESO-9L SLLMWITQL 5.5 ± 0.73 18 ± 2
ESO-4D SLLDWITQV 4.5 ± 2.3 4.0 ± 0.2
ESO-9V SLLMWITQV 53 ± 15 139 ± 10
Supplementary Table 3. Median 2D affinity and ratio of high/low 2D affinity from primary HCV-
specific CD8+ T cells.
Primary HCV-specific CD8+ T cells from Fig. 5A are defined as high and low affinity T cells using a 2D
affinity threshold of 2x10-5 μm4. Multiple blood draws from the same donor are aggregated together
prior to calculation.
Donor
Median Single-
cell 2D affinity
Ratio high/low
single-cell 2D
affinity T cell
1 6.3E-05 2.1
2 8.7E-05 2.4
3 6.7E-05 3.5
4 5.9E-05 1.9
5 1.6E-05 0.7
6 2.0E-05 1.0
7 8.0E-06 0.4
8 1.9E-05 1.0
9 6.8E-06 0.2
Supplementary Table 4. Single-cell 2D affinity and correlated TCRα and TCRβ CDR3 sequences.
TCRα and TCRβ CDR3 amino acid sequences from primary HCV-specific CD8+ T cells that have been
affinity-tested. Blank cells indicate no TCR sequence detected. Red highlight denotes two pairs of T
cells observed to have the same TCRα chain but different TCRβ chain.
TCRα TCRβ
Donor Affinity V-gene CDR3 AA V-gene CDR3 AA
5A 5.6E-06 5*01 CAAVHDYKLSF
5A 8.5E-06 38-2/DV8*01 CASYAGGTSYGKLTF 4-1*01 CASSPAPSASSYEQYF
5A 7.2E-06 38-2/DV8*01 CATHTGKLIF 19*01 CASSWSASYEQYF
5A 5.5E-05 8-3*01 CAVGSNSGYALNF 9*01 CASSSSWADTQYF
5A 4.2E-06 38-2/DV8*01 CAYGDDKIIF 25-1*01 CASGQGQETQYF
5A 6.1E-05 38-2/DV8*01 CAYLGGADGLTF 19*01 CASSMGANEQFF
5A 3.0E-06 38-2/DV8*01 CAYLVYDMRF 27*01 CASSLAQGQPQHF
5A 2.2E-04 38-2/DV8*01 CAYTEDKIIF 6-2/3*01 CASSYWQGELFF
5A 1.2E-04 38-2/DV8*01 CAYYGGSQGNLIF 13*01 CASTNRQEGQETQYF
5A 1.2E-05 9*01 CASSPGGELFF
5A 6.6E-07 10-3*01 CAISGQAVSTDTQYF
5A 2.8E-05 11-2*01 CASSPYPRGGTGELFF
5A 7.0E-06 5-1*01 CASSPADPFSNTQYF
4B 1.5E-06 5*01 CAEIGTDKLIF 20-1*01 CSANSRTGLGYTF
4B 4.0E-04 38-2/DV8*01 CAHNTGNQFYF 6-6*01 CASSYGSYEQYF
4B 1.1E-05 38-2/DV8*01 CAKTGANNLFF
4B 4.7E-04 38-2/DV8*01 CALSGHDKVIF 25-1*01 CASSWGGGEQYF
4B 4.8E-05 38-2/DV8*01 CARDAGNMLTF 7-9*01 CASSLEGSYEQYF
4B 2.3E-04 38-2/DV8*01 CATSGTYKYIF 20-1*01 CSAIDFPMGNEQFF
4B 1.1E-05 38-2/DV8*01 CAYDDMRF 11-2*01 CASSSRHEGEDTEAFF
4B 1.6E-04 38-2/DV8*01 CAYFGGGADGLTF 2*01 CASREDGASGSPDTQYF
4B 2.2E-04 38-2/DV8*01 CAYGDDKIIF 25-1*01 CASKMGAEAFF
4B 4.8E-05 38-2/DV8*01 CAYGEDNDMRF
4B 1.1E-04 38-2/DV8*01 CAYHGGGATNKLIF
4B 4.1E-04 38-2/DV8*01 CAYKDTASKLTF 12-3/4*01 CASSLGGDIQYF
4B 1.3E-04 38-2/DV8*01 CAYKPDTPLVF 25-1*01 CASTGGLGYTF
4B 1.5E-05 38-2/DV8*01 CAYNAGNMLTF 13*01 CASSYQSGNTEAFF
4B 4.5E-06 38-2/DV8*01 CAYPGGGADGLTF 25-1*01 CASNRGDGYTF
4B 8.0E-04 38-2/DV8*01 CAYRDDKIIF 19*01 CASSIALSDNYGYTF
4B 1.1E-05 38-2/DV8*01 CAYREGAQKLVF 30*01 CAWSINSAEAFF
4B 9.0E-04 38-2/DV8*01 CAYREVNDMRF 25-1*01 CASSDSYGYTF
4B 1.1E-05 38-2/DV8*01 CAYSGGGADGLTF 13*01 CASSLEREGRGEQFF
4B 4.2E-05 38-2/DV8*01 CAYVQDDKIIF 19*01 CASSQGSYEQYF
4B 6.7E-04 38-2/DV8*01 CAYYGGNQFYF 25-1*01 CQAGDTEAFF
4B 2.7E-05 38-2/DV8*01 CVLMDSSYKLIF
4B 3.3E-04 4-1*01 CASTQSPLAGNEQYF
4B 8.0E-05 4-3*01 CASSGYTGELFF
4B 1.7E-05 25-1*01 CASTNSGNTIYF
4B 3.6E-04 25-1*01 CASSSGATEAFF
4B 1.3E-06 14*01 CASSQVGQNLYEQYF
4B 5.9E-05 9*01 CASSVEGGGAKETQYF
4B 8.7E-06 19*01 CASSIGHNTGELFF
4B 5.5E-05 6-5*01 CASTQGSTDTQYF
4B 1.9E-05 19*01 CASSVAIWRGSYNEQFF
4B 5.3E-05 6-5*01 CASSSPGASTYEQYF
4B 7.4E-06 6-2/3*01 CASSYNIAGELFF
4B 1.0E-03 19*01 CASSIAGSAYEQYF
4B 2.0E-06 19*01 CASSIRRIDPYNEQFF
4B 3.0E-05 7-8*01 CASSVELAGTYEQYF
6 1.8E-04 22*01 CADYNDYKLSF
6 1.0E-04 5*01 CAESEGKLIF
6 4.3E-06 38-2/DV8*01 CALTGYSTLTF
6 1.0E-05 14/DV4*01 CAMRGGSYIPTF
6 2.5E-05 38-2/DV8*01 CATYSGGGADGLTF 6-6*01 CASSYTGELFF
6 1.0E-04 8-4*01 CAVSDLEPNSSASKIIF 25-1*01 CASSFGADTQYF
6 1.5E-05 38-2/DV8*01 CAYKDNDMRF 25-1*01 CASSAETQYF
6 2.2E-05 38-2/DV8*01 CAYNDGNQFYF 18*01 CASSSPHRDSYSPLHF
6 1.5E-05 38-2/DV8*01 CAYNDMRF 25-1*01 CASSQETQYF
6 6.2E-06 38-2/DV8*01 CAYRDDYKLSF 19*01 CASSIGQYEQYF
6 2.2E-05 38-2/DV8*01 CAYRTDNDMRF
6 1.5E-05 38-2/DV8*01 CAYRTISELTF
6 1.4E-04 26-1*01 CIPEGRKLTF 28*01 CASSYRGSGNTIYF
6 1.7E-05 6-6*01 CASSYAQGNEQFF
6 4.9E-06 25-1*01 CASSLGRGQFF
6 1.4E-04 25-1*01 CASSRGDTEAFF
6 7.4E-06 27*01 CASSLWSAGVHNEQFF
7 1.1E-05 41*01 CADLNARLMF
7 1.5E-05 5*01 CAEGGGSYIPTF 25-1*01 CASSEGDTEAFF
7 3.5E-06 38-2/DV8*01 CAHDTGNQFYF
7 5.0E-06 12-3*01 CALLSSNTGKLIF 9*01 CASSVEGIDEKLFF
7 2.7E-04 19*01 CALSEPYSGAGSYQLTF 15*01 CATSSGDLSSGANVLTF
7 4.4E-06 38-2/DV8*01 CAYSGGGADGLTF 15*01 CATSIDRGREKLFF
7 4.5E-04 38-2/DV8*01 CAYWEGQKLLF
7 6.2E-06 26-2*01 CILDDNNDMRF
7 1.6E-06 TRAV8-1*01 CAVFMDSNYQLIW
7 4.4E-06 19*01 CASSMGGNPEQYF
7 3.1E-06 2*01 CASSDDRGPYEQYF
7 1.2E-04 5-1*01 CASSQVRLNTEAFF
7 7.2E-05 19*01 CASSRSLNVNSNQPQHF
7 5.0E-06 7-9*01 CASSLGEKLFF
7 3.7E-05 25-1*01 CASDSNTEAFF
7 1.3E-05 19*01 CASSMGGEQYF