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