The beneficial effects of a gas-permeable flask for expansion of Tumor-Infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity

Abstract

Adoptive transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) is a highly successful cell therapy approach in the treatment of late-stage melanoma. Notwithstanding the success of this therapy, only very few centers worldwide can provide it. To make this therapy broadly available, one of the major obstacles to overcome is the complexity of culturing the TIL. Recently, major efforts have been deployed to resolve this issue. The use of the Gas-permeable flask (G-Rex) during the REP has been one application that has facilitated this process. Here we show that the use of this new device is able to rescue poor TIL growth and maintain clonal diversity while supporting an improved mitochondrial function.

Keywords: TIL ACT; gas-permeable membrane; mitochondrial function; oxidative phenotype; tumor-infiltrating lymphocytes.

Figure 1.

Assessment of TIL growth after rapid expansion in the traditional flask and bag vs. Gas-permeable flask (G-Rex) reveals a trend toward improved TIL expansion when using the G-Rex flask. (A) Fold expansion of post-REP TIL lines expanded shows a trend toward a better expansion when using the G-Rex. N = 10. (B) The G-Rex flask facilitates the expansion of TIL lines from which the growth is impaired in the REP using the traditional flask and bag devices N = 4. Statistical significance was determined by using a paired t-test.

Figure 2.

Rapid expansion of TIL in the Gas-permeable flask (G-Rex) does not favor selection and expansion of specific T cell clones. Clonal diversity was evaluated by measurement of the expression of major TCR α and β chain gene by the NanoString nCounter® technology. The analysis of the level of expression of 45 Vα and 46 Vβ genes in RNA isolated from the pre-REP TIL lines in comparison with the different expansion devices demonstrated no difference in the clonal diversity obtained post REP. Two out of 4 TIL lines from 4 melanoma patients are shown. Statistical analysis using a Spearman correlation comparing cells grown in traditional flask and bag (red) vs. Gas-permeable flask (blue) is shown for both Vα and Vβ genes.

Figure 3.

Bioenergetic analysis of melanoma TIL lines to evaluate mitochondrial function. (A–D) OCR of 4 TIL lines on day 7 of the REP (A and B) or sorted, pre-REP CD8⁺BTLA⁺ and CD8⁺BTLA⁻ TIL line from 3 patients (C and D) were determined using a Seahorse XP96 Bioanalyzer. OCR were calculated after 3 min of mix time and 4 min of measurement time. OCR[Mito] is the total mitochondrial OCR, the value just prior to the Oligomycin (OLG) injection minus the non-mitochonddrial OCR component determine by Antimycin (AA) treatment. OCR[OLG] is the component of the OCR that is sensitive to Oligomycin treatment, the rate used by ATP-syntase. The OCR on a per cell basis was determined by dividing the OCR by the seeded TIL count of 250000. (E) OCR/ECAR ratio is the OCR[OLG] divided by the ECAR basal which is reflextive of a cell dependence on glycolysis. (F) pre-REP TIL were stained using a MitoTracker dye. The MFI of the MitoTracker staining of the CD8⁺BTLA⁺ and CD8⁺BTLA⁻ subset from 3 TIL lines is shown. The histogram shows staining of a representative TIL line. Statistical significance was determined by using a paired t-test.