Extended Data Fig. 1. Establishment of patient derived co-culture model.

a, Approach for imaging-based quantification of TIL-mediated killing of melanoma target cells. Plates were imaged at 0, 24, 48 and 72 hours, and viable cell counts were normalized to starting counts to quantify outgrowth of target cells. b-d, Sorting and gating strategy to isolate and expand TIL cultures prior to co-culture. b. TILs grown in IL2 or OKT3-stimulated for 72 hours and analyzed after 4 hours of PMA-I-stimulation. FMO, fluorescent-minus-one control b. TILs from 2686 retain ability to induce IFNγ and TNFa, and OKT-3 reactivation leads to an increase in Granzyme-B production compared to TILs grown in IL2 alone. c, MaMel-134 TILs produce IFNγ, TNFa, and Granzyme-B. d, MaMel-80 TILs produce IFNγ, TNFa, and Granzyme-B. e-h. Impact of time, dose, IFNγ pre-treatment, MHC-I blocking, and OKT3 on TIL-mediated killing in the co-culture system from patient 2686. Ratio of viable cancer cells (y axis, relative to t0) in co-cultures: (e) after different time points of co-culture at increasing TIL:cancer cell ratios (x axis), where TILs were restimulated with immobilized OKT3 for 72 h prior to co-culture; f, after 48 h of co-culture, where cancer cells were pre-treated with 1 ng/ml IFNγ for 16 hours (without prior OKT3-reactivation); g, after 48 hours of co-culture as in (f) but using OKT3-reactivated TILs. h, Specificity of IFNγ pre-treatment approach. Ratio of viable allogenic cancer cells (y axis, relative to t0) in different culture conditions with or without IFNγ pre-treatment (x axis) grown from 0 to 72 hours (color bars) with 2686 TILs with or without prior reactivation with OKT3. For e-h, we performed a one-way ANOVA with Tukey post hoc test. Error bars: Mean±SD. All experiments were performed in triplicates in each of at least two independent experiments.