Biomarkers of TIL
Current immunotherapies may be ineffective because immune cells in tumors (tumor infiltrating lymphocytes A.K.A. TILs) do not harbor the metabolic fitness to mount effective responses. We have found that mitochondrial Reactive Oxygen Species (MtROS) in TILs are a negative biomarker of TIL metabolic fitness in tumors. This assay may be useful on fresh patient biopsies to predict responses to immunotherapy and determine secondary interventions needed to invigorate TILs.
Figure 3. Mitochondrial reactive oxygen species signify mitochondrial exhaustion. Representative FACS plots of (A) Naïve WT OT-1+ CD8+ T cells activated and expanded with cognate peptide or (B) human PBMC expanded in high dose IL-2 (3000U/mL) and CD8+ T cells FACS stained at indicated time points. FACS gates are set from fluorescence minus one controls. Data points represent quantification of five individual mice or human samples and are represented as SEM, students t test performed for each time point versus T0 control. Experiments repeated twice. (C) Representative FACS plot with gating from FACS sorts of mtROS/ CD8+ T cells. Lowest 25% - and highest 25% +[A1] mtROS populations were collected and (D) Representative oxygen consumption rate (OCR) trace and quantification of spare respiratory capacity (SRC) from sorted populations at indicated time points measured via Seahorse Bioanalysis. Spare respiratory capacity (SRC) calculated as the difference between initial OCR rate and the maximal OCR rates achieved after FCCP uncoupling. Data are quantified and represented as SEM, students t test performed for each condition versus control T cells. (E) IFN-g production from sorted subsets at indicated time points. Post-sort purity was >97%. (F) Representative FACS plot and quantification of mtROS-Annexin co-staining on day 7 WT OT-1+ T cells. FACS gates are set from fluorescence minus one controls. Data are quantified and represented as SEM, students t test. Individual experiments repeated three times. * p<0.05, ****p<0.0001.