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IFN-g (pg/ml) C-REP T-REP MART-1 peptide (mM) C-REP T-REP 18 6 2 0.67 0.22 E:T ratio A B IFN-g (pg/ml) 0.5 0 0.1 1 TGF-b (ng/ml) C Sup. Fig. 2

1.8 4.2 Mart-1 CD8 CD107a 44.2 37.8 Mart-1 CD8 IFNg D 0.1 nM 10 nM 1 mM 16 0 63 0.2 18 28 C-REP 11 0.2 21 0.6 0.2 9 60 13 34 T-REP 0.8 16 19 Sup. Fig. 2

Supplementary Figure 2.TIL expanded under T-REP and C-REP conditions demonstrated equivalent MART-1-specific antitumor function on per-cell basis.TIL2935 were rapidly expanded using T-REP or C-REP for 14 days, and expanded CD8+ T cells were isolated by negative selection. Normalized numbers of MART-1 tetramer-reactive T cells were co-incubated with (A) Melanoma tumor cell lines Mel888 or HLA-A2-transduced Mel888A2 for 18h to assess IFN-g secretion, or (B) 51Cr labeled Mel888 (dotted lines) or Mel624 (solid lines) at indicated ratios for 4 hours to assess cytotoxicity by chromium release, or (C) T2 cells loaded with titrated amounts of MART-1 peptide for 18hrs to assess IFN-g release. (D) Post-expansion TIL were incubated with MART-1 peptide-pulsed T2 cells for 4 hours, and surface CD107a and intracellular IFN-g expression of gated MART-1 tetramer-reactive T cells was evaluated by flow cytometry.

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