Article Figures & Data
Figures
- FIGURE 1.
Comparison of TRBV sequencing results with FACS analysis. The percentages of individual rearranged TRBV sequences obtained using 5′ RACE analysis were compared with the results of FACS analysis conducted with specific TRBV Abs. Samples were only analyzed if the appropriate TRBV Ab was available, and TRBV products that were represented at levels below 5% as determined either by TRBV sequence analysis or by Ab staining were excluded from the analysis. The results obtained using these methods were highly correlated using linear regression analysis (R = 0.8975, p < 0.001).
- FIGURE 2.
Comparisons of the persistence of individual T cell clonotypes in responders and nonresponders. The percentages of all persistent T cell clonotypes that were detected in samples of PBMC obtained between 23 and 63 days following adoptive transfer, as determined using TRBV sequence analysis, were plotted for the responding and nonresponding patients. Each point represents a distinct T cell clonotype that was identified in an individual patient sample.
Tables
Total Number of TRBV Sequences Analyzed Number of Distinct Clonal Sequencesa Number of Clonotypes Represented in TIL at: <5% 5–9% 10–20% >20% Responder 6 118 41 37 1 3 0 9 128 10 4 2 2 2 10 92 20 17 0 0 3 16 64 20 16 2 0 2 17 81 24 21 0 2 1 19 78 38 32 4 2 0 21 91 37 32 2 3 0 25 81 39 35 3 1 0 26 84 37 32 3 1 1 28 78 15 12 1 0 2 30 81 37 33 0 3 1 31 75 18 13 4 0 1 34 80 21 19 1 0 1 Nonresponder 7 80 34 31 2 0 1 11 82 4 3 0 0 1 12 89 78 78 0 0 0 13 70 22 18 1 2 1 14 85 3 1 0 1 1 15 67 14 10 2 1 1 18 61 22 17 3 1 1 20 86 34 32 0 1 1 22 81 15 10 1 2 2 24 85 23 19 1 1 2 27 73 26 23 1 1 1 29 78 21 17 1 1 2 -
a Clonotypes present within TIL samples were identified by comparison of the TRBV, D, and J region sequences of cDNA clones that were derived from individual TRBV genes.
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PBMC Sample Daya Lymphocyte Number Per mm3 (days 5–15) Persistence Total (%)b Lymphocyte Number Per mm3 (days 23–63) Persistence Total (%)b Responder 6(2)c 9, 55 2,085 49 1,646 24 9 9, 29 21,436 96 7,086 96 10 7, 23 12,150 94 3,410 84 16 7, 61 389 36 973 14 17(2)c 8, 28 555 43 1,328 10 19 5, 33 1,956 57 2,740 12 21 6, 27 1,407 45 1,079 15 25 7, 31 168 56 455 8 26 8, 34 711 44 483 2 28 9, 63 576 74 1,382 7 30(2)c 5, 26 111 61 283 11 31 7, 26 739 36 643 4 34 9, 37 82 62 639 51 Mean 3,258 58 1,704 26 SE 1,756 5 515 9 Nonresponder 7 15, 37 NDd 9 1,893 <1 11 8, 35 810 4 760 <1 12 8, 35 791 5 864 9 13 8, 29 1,073 36 1,027 <1 14 8, 29 532 <1 1,792 <1 15 7, 34 760 60 871 5 18 13, 29 287 2 385 <1 20 8, 25 548 18 592 2 22 8, 34 305 41 307 1 24 7, 29 2,282 87 535 41 27 7, 24 2,496 47 200 1 29 8, 32 164 25 186 <1 Mean 913 28 784 5 SE 225 8 163 3 p2 = 0.663e p2 = 0.008 p2 = 0.115 p2 = 0.001 -
a The days given are relative to the date of TIL infusion.
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b The values indicate the sum of the percentages of individual clonotypes detected in PBMC samples that were also present in the administered TIL.
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c Patients 6, 17, and 30 had received a single prior ablation followed by an adoptive transfer of autologous TIL.
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d A PBL count was not carried out for patient no. 7 at this time point.
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e The lymphocyte counts and the percentage of persistent clonotypes present in responders and nonresponders were compared using the Wilcoxon rank sum test.
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Patient No. Ag Reactivity of TILa Reactivity of Most Prevalent Persistent Clonotypeb Most Prevalent Persistent Clonotype (%) Responder 6 Autol. Autol. 16 9 MART-1 MART-1 83 10 MART-1 MART-1 77 16 Autol. Autol. 12 17 Autol. Autol. 8 19 Autol. N/Tc 5 21 Autol. Autol. 8 25 Autol. N/T 7 26 MART-1 - 1 28 MART-1 MART-1 7 30 gp100:209–217 Autol. 7 31 MART-1 - 2 34 Autol. N/T 43 Nonresponder 7 MART-1 - <1 11 MART-1 - <1 12 Autol. - 4 13 MART-1 - 1 14 Autol. - <1 15 MART-1 - 4 18 gp100:209–217 - <1 20 MART-1 - 2 22 Autol. - 3 24 Autol. N/T 17 27 MART-1 - 1 29 MART-1 - <1 -
a Ag reactivity was evaluated by determining the ability of TIL to release IFN-γ in response to autologous (Autol.) or HLA-matched allogeneic tumors, or to T2 cells that were pulsed with either the MART-1:26–35 or gp100:209–217 HLA-A2-restricted peptides.
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b The Ag reactivity of the most persistent clonotype detected in PBMC 23–63 days after transfer is indicated. Isolated T cell clones corresponding to the dominant persistent clonotypes from TIL from patients 9 and 10 released IFN-γ in response to T2 cells that were pulsed with the MART-1:27–35 peptide, and clones corresponding to the dominant persistent clonotypes within TIL from patients 6, 16, and 22, released IFN-γ in response to autologous tumor cells. The dominant persistent clonotype within the TIL from patient 28 was identified by staining with anti-TRBV Abs and a MART-1:26–35(2L) tetramer. Tumor reactivity of the dominant persistent clonotypes from patients 17 and 30 was determined by measuring the ability of these T cells, which were identified within the TIL using specific TRBV Abs, to specifically up-regulate expression of cell surface CD107a expression following stimulation with autologous tumor cells. A dash indicates either that no persistent clonotype was detected or that the most persistent clonotype was present at a frequency of between 1 and 5% and was not evaluated due to the limited number of cells available for analysis.
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c For samples designated N/T (not tested), the ability of the persistent clonotype to recognize autologous tumor has not been verified.
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