supplementary materials forthe includes: fig. s1. validation study design and analysis details of...

www.sciencetranslationalmedicine.org/cgi/content/full/7/272/272ra10/DC1

Supplementary Materials for

Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients

Heather Morris, Susan DeWolf, Harlan Robins, Ben Sprangers, Samuel A. LoCascio,

Brittany A. Shonts, Tatsuo Kawai, Waichi Wong, Suxiao Yang, Julien Zuber, Yufeng Shen,* Megan Sykes*

*Corresponding author. E-mail: [email protected] (M.S.); [email protected] (Y.S.)

Published 28 January 2015, Sci. Transl. Med. 7, 272ra10 (2015) DOI: 10.1126/scitranslmed.3010760

The PDF file includes:

Fig. S1. Validation study design and analysis details of the tracking of alloreactive clones in healthy controls. Fig. S2. Anti-donor clonal analysis at increasing fold expansion criteria. Fig. S3. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in CKBMT recipients (CD4). Fig. S4. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in the CKBMT recipients (CD8). Fig. S5. LDA (CTLp and HTL) for subjects 1, 2, 4, and 5. Fig. S6. Comparison of pre- and posttransplant anti-donor MLR in subject 4. Fig. S7. Frequencies of donor-reactive clones before and after transplant in the conventional kidney transplant recipients. Fig. S8. Detection of third party–reactive TCRs before and after transplant. Table S1. Cell counts, entropy, and clonality for healthy control experiments. Table S2. Sum frequency of alloreactive clones in healthy controls (%). Table S3. Cell numbers, number of unique clones, and total number of reads for each patient sample. Table S4. Tabulated data from clonal analysis in subjects 1, 2, 4, 5, IS#1, and IS#2 (Figs. 3B and 4). Table S5. Tabulated data from clonal analysis in healthy controls (Fig. 4B). Table S6. Sum frequency of alloreactive clones in subject 4 (%). Table S7. Relative turnover analysis in tolerant subjects (Fig. 5B). Table S8. Cell numbers, number of unique clones, and total number of reads for anti–third party responses.

Table S9. Relative turnover analysis at increasing fold expansion criteria: Subject 1 (Fig. 5C).

Supplemental Figure 1. Validation study design and analysis details of the tracking of alloreactive clones in healthy controls. A. CFSE staining in representative unstimulated control sample. B. Healthy control (HC) #1 experimental design. C. HC #2, HC #3 experimental design. PBMCs obtained from healthy controls at different time points separated by two week (HC#1) or one year (HC#2, HC#3) intervals were used in parallel MLRs with the same responder-stimulator pairs for each time point. We then performed deep sequencing on the divided CFSE-low T cells in the MLR as well as on unstimulated T cells. D. Clonal frequencies in the stimulated populations (T1) are plotted against frequencies in the unstimulated population (T1) for HC#2 and HC#3.

Supplemental Figure 2. Anti-donor clonal analysis at increasing fold expansion criteria. Odds ratio of detecting donor-reactive clones post-transplant versus pre-transplant when donor reactivity is defined by various fold expansion criteria (frequency in pre-transplant anti-donor MLR/frequency in unstimulated pre-transplant sample > 5, 7, or 10). Red points are statistically significant (P < 0.05). B. Individual P-values for results plotted in A.

Supplemental Figure 3. Frequencies of donor-reactive clones in unstimulated PMBCs before and after transplant in CKBMT recipients (CD4). Scatter plots showing frequency of individual donor-reactive T cell clones in pre-transplant stimulated samples versus unstimulated samples at each indicated time point. Donor-reactive clones (defined in Fig. 3 legend) are pink. The blue dotted line denotes the frequency threshold of detection.

Supplemental Figure 4. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in the CKBMT recipients (CD8). Scatter plots showing frequency of individual donor-reactive T cell clones in pre-transplant stimulated samples versus unstimulated samples at each indicated time point. Donor-reactive clones (defined in Fig. 3 legend) are pink. The blue dotted line denoting the frequency threshold of detection for the unstimulated sample.

Supplemental Figure 5. LDA (CTLp and HTL) for subjects 1, 2, 4, and 5. A. CTLp for subjects 1, 2, 4, and 5. Frequencies calculated according to the single hit Poisson model using L-calc software are shown. All dilution curves fit the assumptions for the model except where marked with #. CTLp data have been previously summarized in Kawai et al.(3). B. HTLfor subjects 1, 2, 4, and 5. Frequencies calculated according to the single hit Poisson model are shown. For some analyses, responses did not decline with decreasing responder numbers at the highest responder concentrations. In these instances, the frequency was calculated on the diluting portion of the curve. At some time points (marked with #), the dilution curve did not fit the single hit Poisson model.

Supplemental Figure 6. Comparison of pre- and posttransplant anti-donor MLR in subject 4. A. Pie charts showing relative overlap of the donor-reactive repertoire (“fingerprint” as defined in Figure 1) defined in pre-transplant as compared to one year post-transplant MLR in subject 4. Each circle represents the sum frequency of all donor-reactive clones identified in the sample; the red segment shows the percentage of that total frequency arising from donor-reactive clones identified in both pre-transplant (dark blue) and 12 months post-transplant (light blue) MLRs (tabulated data in Supplemental Table 6). B. Scatter plots showing frequency of individual T cell clones in 12 month post-transplant stimulated samples versus unstimulated samples at each indicated time point. Post-transplant donor-reactive clones (clones with frequencies ≥10-4 in the 12 month post-transplant MLR expanded at least 5-fold compared to the unstimulated 12 month post-transplant sample) are shown in pink and the blue dotted line denotes the frequency threshold of detection in the unstimulated sample.

Supplemental Figure 7. Frequencies of donor-reactive clones before and after transplant in the conventional kidney transplant recipients. Scatter plots showing frequency of individual T cell clones in pre-transplant stimulated samples versus unstimulated samples at each indicated time point. Donor-reactive clones (defined in Fig. 4 legend) are shown in pink and the blue dotted line denotes the frequency threshold of detection for the unstimulated sample.

Supplemental Figure 8. Detection of third party–reactive TCRs before and after transplant. Anti-third party clones defined as clones (defined by amino acid CDR3 sequence + V family & J gene in subjects 1, 2, 5, and nucleotide CDR3 sequence in subject 4) in pre-transplant MLR with ≥10-4 frequency that were expanded at least 5-fold relative to their frequency in the pre-transplant unstimulated sample. Frequency defining detection in unstimulated populations was 10-5 in subject 1, subject 2, subject 4, and 5x10-5 in subject 5. * P < 0.05 (significant decrease); individual P-values shown in table. The number of cells, unique clones, and total reads for divided CD4 and CD8 populations responding in each anti-third party MLR are summarized in Supplemental Table 8.

Sample

# productive reads

(millions)

# of cells (millions)

# unique productive

CDR3s Entropy Clonality

CD4

HC #1 T1 unstim 1.1 3.7 84,091 15.2 0.072

HC #1 T1 stim 3.3 6.3 33,568 13.1 0.13

HC #1 T2 stim 2.1 4.4 14,824 12.4 0.11

HC #2 T1 unstim 1.8 3.1 128,678 16.0 0.06

HC #2 T1 stim 1.8 1.1 12,806 11.0 0.19

HC #2 T2 unstim 1.9 3.9 136,186 16.2 0.05

HC #2 T2 stim 1.7 0.5 12,041 11.2 0.17

HC #3 T1 unstim 1.8 2.4 102,269 15.3 0.08

HC #3 T1 stim 1.8 1.9 18,566 11.5 0.19

HC #3 T2 unstim 2.1 2.9 121,093 15.9 0.06

HC #3 T2 stim 1.9 1.4 11,798 10.7 0.21

CD8

HC #1 T1 unstim 1.5 1 52,941 13.8 0.12

HC #1 T1 stim 2.2 1.6 8,989 9.1 0.3

HC #1 T2 stim 3.1 1.2 6,482 8.7 0.3

HC #2 T1 unstim 2.0 2 112,235 13.3 0.2

HC #2 T1 stim 1.9 2 14,646 10.7 0.23

HC #2 T2 unstim 1.9 1.9 96,613 14.0 0.15

HC #2 T2 stim 1.9 2 14,624 11.6 0.17

HC #3 T1 unstim 2.0 1 67,493 11.9 0.26

HC #3 T1 stim 2.0 1.2 7,958 10.2 0.22

HC #3 T2 unstim 1.8 1.9 94,052 13.7 0.17

HC #3 T2 stim 2.2 0.7 6,592 9.9 0.22

Supplemental Table 1. Cell counts, entropy, and clonality for healthy control experiments. Summary of CD4 and CD8 T cell numbers sorted for DNA sequencing, number of productive reads, unique reads, entropy, and clonality from the healthy control experiments. The data are corrected for erroneous reads, for which the average error rate is about 0.25% in typical Illumina HiSeq runs.

Stimulated CD4

Unstimulated CD4

Stimulated CD8

Unstimulated CD8

Overlapping CD4

Overlapping CD8

HC#1 T1 47.91 0.50 67.74 0.34 24.71 17.30

HC#1 T2 62.92 0.48 64.01 0.43 26.28 19.39

HC#2 T1 76.61 0.72 66.63 0.74 27.44 25.42

HC#2 T2 80.36 0.34 73.46 0.51 14.97 15.58

HC#3 T1 66.00 1.06 79.30 2.30 42.40 12.09

HC#3 T2 79.63 0.65 80.03 0.27 48.37 14.93

Supplemental Table 2: Sum frequency of alloreactive clones in healthy controls (%).

Patient Time point Cell Number Unique Clones Total Reads

CD4 CD8 CD4 CD8 CD4 CD8

Subject 1

Pre-tx stim 2.12x10^6 645,000 13,185 8,494 4,146,788 2,198,370

Pre-tx unstim 3.88x10^6 1.07x10^6 108,699 79,501 3,212,755 5,000,388

6 months post-tx 1.31 x10^6 664,000 55,172 40,056 4,565,095 1,178,654

18 months post-tx 291,000 441,000 49,996 24,747 3,153,867 3,211,016

Subject 2

Pre-tx stim 5,380 16,200 731 882 477,330 2,152,104

Pre-tx unstim 1.90 x10^6

595,000 91,510 59,217 1,492,110 2,106,439

6 months post-tx 96,300 178,000 12,066 4,871 2,193,860 2,666,969

12 months post-tx 390,000 488,000 28,250 12,767 2,278,401 2,440,492

24 months post-tx 500,000 608,000 59,776 21,494 2,341,825 2,441,681

Subject 4

Pre-tx stim 380,000 730,000 33,430 17,630 3,862,177 4,442,167

12 months post-tx stim 222,000 245,000 17,775 12,704 3,618,166 4,508,554

Pre-tx unstim 3.7 x10^6 3.1 x10^6 199,315 132,651 3,115,662 2,403,665

6 months post-tx 1.4 x10^6 2.95 x10^6 137,614 44,774 2,552,126 4,397,347

12 months post-tx 1.8 x10^6 1.6 x10^6 195,797 102,398 3,688,291 3,679,239

24 months post-tx 4.7 x10^6 3.9 x10^6 176,347 109,110 3,207,898 4,137,164

Subject 5

Pre-tx stim 106,000 654,000

Pre-tx unstim 6.43 x10^6 1.87 x10^6 109,369 74,792 1,088,847 832,182

6 months post-tx 3.12 x10^6 996,000 48,030 33,566 2,110,247 3,190,886

14 months post-tx 148,000 62,500 10,510 2,384 1,171,575 2,246,579

IS#1 Pre-tx stim 927,000 124,000 28,180 4,405 4,656,330 5,002,719

Pre-tx unstim 2.95 x10^6 587,000 137,377 30,187 6,224,428 5,701,089

10 months post-tx 564,000 800,000 48,845 10,587 4,828,252 7,364,468

17 months post-tx 555,000 511,000 66,266 15,895 6,378,135 8,496,445

IS#2

Pre-tx stim 244,000 741,000 15,870 13,207 5,131,739 2,340,113

Pre-tx unstim 431,000 242,000 112,383 61,629 6,613,583 4,137,730

2.5 months post-tx 1.90 x10^6 1.41 x10^6 98,163 90,666 6,968,221 4,443,222

12 months post-tx 82,600 171,000 13,000 9,452 2,983,646 4,884,200

Supplemental Table 3. Cell numbers, number of unique clones, and total number of reads for each patient sample. “Stim” denotes sorted CD4 and CD8 populations from pre-transplant MLRs. The remaining samples were sorted CD4 and CD8 populations from unstimulated samples obtained at the indicated times, including pre-transplant (pre-tx unstim) and post-transplant (post-tx) time points. Host-vs-Graft HLA mismatches (HLA-A,B,DQ,DR): subject 1: 2/4 Class I, 2/4 Class II; subject 2: 1/4 Class I, 0/4 Class II; subject 4: 2/4 Class 1, 0/4 class II; subject 5: 2/4 Class I; 0/4 Class II, IS#1: 2/4 Class I, 2/4 Class II; IS#2: 2/4 Class I, 4/4 Class II.

Subject 1 Donor-reactive clones detected

Donor-reactive clones undetected OR P-value

Pre-transplant (CD4) 89 2111

6 months post-transplant (CD4) 60 2140 0.665 0.0193

18 months post-transplant (CD4) 34 2166 0.372 5.22E-07








12 months post-transplant (CD4) 20 414 0.49 0.0146 24 months post-transplant (CD4) 17 417 0.413 0.00338


6 months post-transplant (CD8) 0 279 0 0.000883



















IS #1 Donor-reactive clones detected




17months post-transplant (CD4) 403 1794 2.008 3.53E-15



IS #2 Donor-reactive clones detected



2.5 months post-transplant (CD4) 28 2072 4.039 0.000483 12 months post-transplant (CD4) 58 2042 8.489 3.13E-11


2.5 months post-transplant (CD8) 3 2428 1 1 12 months post-transplant (CD8) 7 2424 2.337 0.343

Supplemental Table 4. Tabulated data from clonal analysis in subject 1, 2, 4, 5, IS#1, and IS#2 (Figs. 3B and 4).

Healthy Controls

Donor-reactive clones

detected (T1)


undetected (T1)


detected (T2)


undetected (T2)

OR P-value

Healthy Control #2 CD4 (n = 1693 ) 177 1516 178 1515 1.006 1

Healthy Control #2 CD8 (n = 1949) 60 1889 70 1879 1.173 0.422



Supplemental Table 5. Tabulated data from clonal analysis in healthy controls (Fig. 4B). Change over one year in detection in unstimulated T cell populations of alloreactive CD4 and CD8 clones for HC#2 and HC#3. Alloreactive clones are defined as clones expanded at least 5-fold in the T1 MLR relative to frequencies in T1 unstimulated populations and present at ≥10-4

frequency in the MLR. Minimum frequency of 10-5 defined as detectable in unstimulated sample at T2.

Stimulated CD4 Stimulated CD8 Overlapping CD4 Overlapping CD8

pre-transplant 63.51 76.67 4.50 1.52

post-transplant 71.81 72.84 4.49 1.96

Supplemental Table 6. Sum frequency of alloreactive clones in subject 4 (%).

Pre-transplant

clones detected

Pre-transplant

clones undetected


detected


undetected Odds Ratio P-value

Subject 1

CD4: 6 months 3464 84459 74 2126 0.85 0.18

CD4: 18 months 1720 86203 36 2164 0.83 0.31

CD8: 6 months 1413 56672 29 1163 1 1

CD8: 18 months 1210 56875 23 1169 0.92 0.84

Subject 2

CD4: 6 months 1476 75604 13 421 1.58 0.11

CD4: 12 months 1843 75237 23 411 2.28 0.00045

CD4: 24 months 2228 74852 21 413 1.71 0.021

CD8: 6 months 1053 48409 0 279 0 0.0053

CD8: 12 months 1121 48341 2 277 0.31 0.1

CD8: 24 months 1302 48160 2 277 0.27 0.038

Subject 4

CD4: 6 months 5285 141444 82 1654 1.33 0.016

CD4: 12 months 3523 143206 45 1691 1.08 0.58

CD4: 24 months 3361 143368 32 1704 0.8 0.26

CD8: 6 months 4255 103810 34 1981 0.42 1.40E-08

CD8: 12 months 4169 103896 26 1989 0.33 1.30E-11

CD8: 24 months 3898 104167 24 1991 0.32 5.30E-11

Supplemental Table 7. Relative turnover analysis in tolerant subjects (Fig. 5B).

Anti-Third Party

Subject 1 Anti-Third Party



Subject 5

CD4: Cell number 301,000 946,000 4,353,000 606,000

CD4: Unique clones 28,672 34,872 86,654 38,963

CD4: Total reads 3,495,497 2,954,100 2,325,923 4,684,892

CD8: Cell number 437,000 395,000 2,611,000 790,000

CD8: Unique clones 25,422 18,981 63,459 30,633

CD8: Total reads 5,261,157 2,109,027 4,315,089 5,442,251

Supplemental Table 8. Cell numbers, number of unique clones, and total number of reads for anti–third party responses.

Subject 1 Fold Expansion

Pre-transplant

clones detected

Pre-transplant

clones undetected


detected


undetected OR P-

value

6 months post-

transplant (CD4)

5 3464 84459 74 2126 0.85 0.18 7 3464 84459 63 2121 0.72 0.01 10 3464 84459 57 2110 0.66 0.0013

18 months post-

transplant (CD4)

5 1720 86203 36 2164 0.83 0.31 7 1720 86203 32 2152 0.75 0.12 10 1720 86203 28 2139 0.66 0.027

6 months post-

transplant (CD8)

5 1413 56672 29 1163 1 1 7 1413 56672 25 1156 0.87 0.57 10 1413 56672 16 1153 0.56 0.016

18 months post-

transplant (CD8)

5 1210 56875 23 1169 0.92 0.84 7 1210 56875 17 1164 0.69 0.15 10 1210 56875 10 1159 0.41 0.0017

Supplemental Table 9. Relative turnover analysis at increasing fold expansion criteria: Subject 1 (Fig. 5C).

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