1. Supplementary figure 1. Gating strategy used for FACS sorting of TIL subsets (CD3+, CD3+CD8+/-, CD3+CD8+PD-1+/-). Two representative fresh tumor samples are shown (3903 and 3998).
3903
3998
Supplementary Fig.1

2. Supplementary Fig.2 A B C D E Shannon entropy
Supplementary figure 2. Comparison of Shannon entropy in TIL subsets. (A) Shannon entropy (a measure of clonotypic diversity) is plotted for melanoma bulk TIL and sorted TIL subsets. Entropy is calculated by summing the frequency of each clone times the log (base 2) of the same frequency over all productive reads in the sample. Samples with higher entropy have a greater diversity of clonotypes while samples with lower entropy have less clonotypic diversity. Wilcoxon matched-pairs signed rank test was applied (n=10). For samples 1913 and 3922 the CD8- subset was not available. The number of non-synonymous mutations is plotted in relationship with the percentage of CD8+PD-1+ TIL (B), Shannon entropy of TIL CD8+PD-1+ (C) , Shannon entropy of bulk TIL (D) and Shannon entropy of TIL CD8+ (E) (n=9). For samples 1913 and 3922 the CD8- subset was not available and for sample 3977 the number of non-synonymous mutations was not available.
Shannon entropy
p=0.002
Supplementary Fig.2
Number of non-synonymous mutations
% CD8+PD-1+ TIL
Shannon entropy bulk TIL
Shannon entropy TIL CD8+
Shannon entropy TIL CD8+PD-1+
r2=0.1037
p=0.3980
r2=0.0145
p=0.7577
r2=0.0009
p=0.9366
r2=0.0575
p=0.5342 A B C D E

3. Supplementary Fig.3 A B Frequency TCRB clonotype
Supplementary figure 3. PD-1 separates TIL into 2 separate subsets with different TCR repertoires. (A) The frequency of the most dominant CD8+PD-1+ TCRB clonotype is represented in the CD8+PD-1- and CD8+PD-1+ TIL compartments respectively. (n=12) (B) The frequency of the most dominant CD8+PD-1+ TCRB clonotype is represented in the CD8+ and CD8+PD-1+ TIL compartments respectively (n=11, for sample 3922 the CD8+ subset was not available). Wilcoxon matched-pairs signed rank test was applied.
Supplementary Fig.3 A B
p=0.0003
p=0.001
Frequency TCRB clonotype
Frequency TCRB clonotype

4. 3998 Supplementary Fig.4 A C B TCR pair 3998-7
Supplementary figure 4. CD137 up-regulation by CD8+mTCRB+ cells from patient PBL transduced with reconstructed TCR pairs , , A1, , , and were tested against autologous B-cells transfected with tandem minigenes (TMG) encoding shared melanoma/melanocyte and cancer-germline antigens. One representative experiment is shown (A). PBL transduced with reconstructed TCR pair was tested against autologous B-cells transfected with tandem minigenes (TMG) encoding NY-ESO antigen. One representative experiment is shown (B). Murine TCRB expression and CD137 up-regulation are shown for reconstructed TCR pair after co-culture with, unpulsed autologous B-cells, autologous B-cells pulsed with 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml of the mutated MAGEA6 peptide (KVDPIGHVY) and wild type MAGEA6 peptide (EVDPIGHVY) respectively (C).
Tumor exomic sequencing was performed by Axeq, Rockville MD. 345 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 115 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3998
Supplementary Fig.4
B-cells only
OKT-3
CD137
mTCRB
100 ng/ml
10 ng/ml
1 ng/ml
0.1 ng/ml
MAGEA6mut peptide
MAGEA6wt peptide
1 μg/ml A C B
TCR pair

5. Supplementary figure 5. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs , , , , , A1, A2 and , were tested against COS-A2 cells transfected with the wt form of the HLA-11 molecule pulsed with 1 µg/ml of irrelevant peptide or CDKN2Amut peptide; COS-A2 cells transfected with mutant form of the HLA-11 molecule40; autologous tumor cell line (TC 1913). One representative experiment is shown. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32). Inhibition was 46% and 39% for reconstructed TCR pairs and , respectively.
Tumor exomic sequencing was performed by the Broad Institute, Cambridge MA putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against two of these mutations (HLA-A11mut and CDKN2Amut) and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
1913 A B
Supplementary Fig.5

6. Supplementary figure 6. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs , were tested against the autologous tumor cell line (TC 2650) and a mismatched tumor cell line. One representative experiment is shown. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32).
Tumor cell line exomic sequencing was performed by the NIH Intramural Sequencing Center (NISC). 431 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against the tumor cell line and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
2650 A B
Supplementary Fig.6

7. Supplementary figure 7. CD137 up-regulation by CD8+mTCRB+ cells from patient PBL transduced with reconstructed TCR pairs , , and , were tested against autologous B-cells transfected with tandem minigenes (TMG) encoding for tumor neo-antigens or pulsed with 1 µg/ml of mutated and wild type form of peptides FBXO21 and UGGT2. One representative experiment is shown.
Tumor exomic sequencing was performed by Axeq, Rockville MD. 504 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 157 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3678
Supplementary Fig.7

8. Supplementary Fig.8 3713 A B C D
Supplementary figure 8. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) T-cell clones (3713-2, , , , and ) were tested against the autologous tumor cell line (TC 3713) and a mismatched tumor cell line. One representative experiment is shown. (B) T-cell clones (3713-2, , , and ) were tested against autologous B-cells pulsed with 0.1 mM of mutated and wt form of peptides WDR, SRPX, HELZ2; tumor cell line (TC 3713). One representative experiment is shown. (C) PBL transduced with reconstructed TCR pairs A1, A2, were tested against the autologous tumor cell line (TC 3713) and a mismatched tumor cell line. One representative experiment is shown.
Tumor exomic sequencing was performed by Personal Genomics Diagnostic (PGDX), Baltimore MD putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 11 of these mutations (WDRmut, SRPXmut, AFMIDmut, HELZ2mut, GCN1L1mut, PLSCR4mut, CENPLmut, AHNAKmut, TPX2mut, SECO2mut, PRPX3mut) and (D) for reactivity against t MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3. A B C D
3713
Supplementary Fig.8

9. Supplementary figure 9. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs , , A1, A2, , and were tested against autologous B-cells transfected with tandem minigenes (TMG) encoding for tumor neo-antigens; autologous tumor cell line (TC 3759) and a mismatched tumor cell line. One representative experiment is shown. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32).
Tumor exomic sequencing was performed by the Surgery Branch, NIH, Bethesda MD putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 70 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3759 A B
Supplementary Fig.9

10. Supplementary figure 10. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs , , , , , , , A1 and A2 were tested against autologous B-cells transfected with tandem minigenes (TMG) encoding for tumor neo-antigens and the autologous tumor cell line (TC 3784). One representative experiment is shown. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32).
Tumor exomic sequencing was performed by the Broad Institute, Cambridge MA. 662 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 140 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3784 A B
Supplementary Fig.10

11. Supplementary figure 11. CD137 up-regulation by CD8+mTCRB+ cells from patient PBL transduced with reconstructed TCR pairs , , A1, A2, , , , , A1 and A2 were tested against autologous B-cells pulsed with 1 µg/ml of mutated and wt Kiaa peptide and the autologous tumor cell line (TC 3903). One representative experiment is shown.
Tumor exomic sequencing was performed by Personal Genomics Diagnostic (PGDX), Baltimore MD. 385 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against two of these mutations (Kiaa mut and VPS13B mut) and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3903
Supplementary Fig.11

12. Supplementary figure 12. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs , , , and were tested against autologous B-cells transfected with tandem minigenes (TMG) encoding for tumor neo-antigens and the autologous tumor cell line (TC 3922). One representative experiment is shown.
Tumor exomic sequencing was performed by Personal Genomics Diagnostic (PGDX), Baltimore MD. 449 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 141 of these mutations and for (B) reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3. A B
3922
Supplementary Fig.12

13. Supplementary figure 13. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs A1, A2, , , A1, A2 and were tested against the autologous tumor cell line (TC 3926) and a mismatched tumor cell line. One representative experiment is shown. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32).
Tumor exomic sequencing was performed by Broad Institute, Cambridge MA. 340 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 197 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3926 A B
Supplementary Fig.13

14. Supplementary figure 14. CD137 up-regulation by CD8+mTCRB+ cells from patient (A) PBL transduced with reconstructed TCR pairs A1, A2, A1, A2, , , and were tested against the autologous tumor cell line (TC 3977) and a mismatched tumor cell line. One representative experiment is shown. The reconstructed TCR pairs were tested also for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3. (B) CD137 up-regulation inhibition by pan MHC-I blocking antibody (clone W6-32).
3977 A B
Supplementary Fig.14

15. Supplementary figure 15. CD137 up-regulation by CD8+mTCRB+ cells from patient PBL transduced with reconstructed TCR pairs , , , and were tested against the autologous tumor cell line (TC 3992) and a mismatched tumor cell line. One representative experiment is shown.
Tumor exomic sequencing was performed by Axeq, Rockville MD. 159 putative exomic mutations were found; in the present study the reconstructed TCR pairs were tested for reactivity against 70 of these mutations and for reactivity against MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3.
3992
Supplementary Fig.15

16. Supplementary Table 1. Primers used for the single cell RT-PCR

17. Supplementary Table 2. Frequency of the top 10 CD8+PD-1+ TCRB in bulk unsorted TIL and sorted TIL subsets
N/A not available
nd not detected

18. Supplementary Table 3. Retrospective analysis of pairing the most frequent TCRB in the CD8+PD-1+ TIL subset with the most frequent TCRA from the same subset
* Pairs found by single cell RT-PCR and pairSEQ.
♯ Tumor reactivity was evaluated after co-culture with the autologous TC.

19. Supplementary Table 4. Method of identification of TCR pairs.
** For this TCRB 2 TCRA were identified and the expression of each pair is reported.
P = these TCRA-TCRB pairs were identified by pairSEQ
SC = these TCRA-TCRB pairs were identified by single cell RT-PCR
C = These TCRA-TCRB pairs were tested as T-cell clones and the sequences were confirmed by 5’ RACE

20. Supplementary Table 5. TCR expression on genetically engineered PBL *
* The frequency of CD3+CD8+mTCR+ cells among genetically engineered PBL is reported for every reconstructed TCRA-TCRB pair.
** For this TCRB 2 TCRA were identified and the expression of each pair is reported.
N/A these TCRA-TCRB pairs were not identified.
¶ These TCRA-TCRB pairs were tested as T-cell clones and the sequences were confirmed by 5’ RACE.