Figure 1.

Effective isolation and expansion of TILs from NSCLC-derived lesions. Single cell suspensions were obtained from distal lung tissue and tumor sections of 23 donors directly after resection. (a) Numbers of life cells/mg tissue. Dead cells were excluded by trypan blue. Each dot represents one donor, box and whisker plots depict the mean (dotted line), minimum and maximum values (whiskers) and 25^th pct and 75^th pct (box). (b) Gating strategy of CD56⁻/CD3⁺ T cells and lymphocytes, and (c) percentage of CD3⁺ cells within the lymphocyte population in tumor and lung, to calculate (d) the numbers of T cells/mg tissue by flow cytometry, depicted as in (A). (e) Of 9 normal lung tissues (left panel), and of 17 tumor tissues (right panel), single cell suspensions were cultured for 10–13 days with 6000IU/ml IL-2 (pre-REP), followed by 2 weeks of culture with 30ng/ml anti-CD3, feeder mix, and 3000IU/ml IL-2 (REP). The fold change of total cell count was determined from the number of cells used as input. (f, g) The ratio of CD8⁺ over CD4⁺ T cells of the CD3⁺ T cells was determined by flow cytometry in tumor digests directly ex vivo, and after pre-REP and REP cultures. (a, c, d, e and g) Significance was calculated with paired student’s T test; * p < .05; *** p < .001. If no indication, p ≥ 0.05. (h) T cell clonality was determined by CDR3 sequencing of the TCRb chain of FACS-sorted CD4⁺ and CD8⁺ T cells from tumor digest and from corresponding expanded TILs (CD25^hiCD127^hi CD4⁺ T cells containing the Treg population were excluded from this analysis). Relative individual T cell clone frequency ordered from the largest (bottom) to the smallest (top). Color-coded pairs represent shared TCR sequences, light-grey bars depict non-shared TCR sequence.