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. Author manuscript; available in PMC: 2020 Sep 15.

Published in final edited form as: Clin Cancer Res. 2019 Dec 23;26(6):1349–1358. doi: 10.1158/1078-0432.CCR-19-1068

Figure 1: — Activated, non-exhausted TIL express high levels of 4-1BB in GBM and are subject to improved function by 4-1BB agonism. A. Frequency of 4-1BB expression on CD8⁺ T cells, p values as calculated by unpaired t-test. B. Representative histogram of 4-1BB expression on control PBMC, GBM PBMC and GBM TIL. Plots are gated on singlets, live cells, and CD3+CD8⁺. C. Frequency of CD8⁺ 4-1BB+ TIL expressing PD-1 alone or PD-1, TIM-3, and LAG-3 (Triple+). ***p<0.001 by paired t-test. D. Median fluorescent intensity (MFI) of 4-1BB levels on PD-1+ or PD-1+TIM-3+LAG-3+ (Triple+) TIL isolated from human GBM. E. Frequency of IFN-γ+ TIL among PD-1 negative, PD-1 single positive, or PD-1/TIM-3/LAG-3 triple-positive CD8 TIL. **p<0.01 by paired t-test. F. Representative histogram showing TIL samples expressing either low (black) or high (red) levels of 4-1BB. Gated on singlets, live cells, lymphocytes, CD3+CD8⁺. G. Representative contour plot of IFN-γ following in vitro stimulation with a 4-1BB agonist antibody in patients’ CD8 TILs expressing either high or low levels of 4-1BB. H. IFN-γ production among CD8⁺ TIL from patients with either high or low levels of 4-1BB. Median Fluorescent Intensity (MFI) is depicted. *p<0.05 by unpaired t-test. I. Linear regression of IFN-γ production and expression of 4-1BB. p<0.0702. J. Frequency of 4-1BB expression on CD8 T cells in the blood of control mice vs. various compartments in tumor-bearing (TB) mice. CLN = ipsilateral tumor-draining cervical lymph nodes. ***p<0.001 by One-way ANOVA followed by Bonferroni’s Multiple Comparison Test. K. Frequency of CD8⁺ 4-1BB+ TIL expressing PD-1 alone or PD-1, TIM-3, and LAG-3. **p<0.01 by paired t-test. L. MFI of 4-1BB on PD-1+ or PD-1+TIM-3+LAG-3+ (Triple+) T cells isolated from CT2A TIL. M. Frequency of IFN-γ on cells expressing PD-1 and 4-1BB; 4-1BB, PD-1, TIM-3, and LAG-3; and PD-1, TIM-3, and LAG-3 but not 4-1BB as determined by Boolean gating of CT2A TIL re-stimulated in vitro with PMA/Ionomycin.

Figure 1: — Activated, non-exhausted TIL express high levels of 4-1BB in GBM and are subject to improved function by 4-1BB agonism. A. Frequency of 4-1BB expression on CD8⁺ T cells, p values as calculated by unpaired t-test. B. Representative histogram of 4-1BB expression on control PBMC, GBM PBMC and GBM TIL. Plots are gated on singlets, live cells, and CD3+CD8⁺. C. Frequency of CD8⁺ 4-1BB+ TIL expressing PD-1 alone or PD-1, TIM-3, and LAG-3 (Triple+). ***p<0.001 by paired t-test. D. Median fluorescent intensity (MFI) of 4-1BB levels on PD-1+ or PD-1+TIM-3+LAG-3+ (Triple+) TIL isolated from human GBM. E. Frequency of IFN-γ+ TIL among PD-1 negative, PD-1 single positive, or PD-1/TIM-3/LAG-3 triple-positive CD8 TIL. **p<0.01 by paired t-test. F. Representative histogram showing TIL samples expressing either low (black) or high (red) levels of 4-1BB. Gated on singlets, live cells, lymphocytes, CD3+CD8⁺. G. Representative contour plot of IFN-γ following in vitro stimulation with a 4-1BB agonist antibody in patients’ CD8 TILs expressing either high or low levels of 4-1BB. H. IFN-γ production among CD8⁺ TIL from patients with either high or low levels of 4-1BB. Median Fluorescent Intensity (MFI) is depicted. *p<0.05 by unpaired t-test. I. Linear regression of IFN-γ production and expression of 4-1BB. p<0.0702. J. Frequency of 4-1BB expression on CD8 T cells in the blood of control mice vs. various compartments in tumor-bearing (TB) mice. CLN = ipsilateral tumor-draining cervical lymph nodes. ***p<0.001 by One-way ANOVA followed by Bonferroni’s Multiple Comparison Test. K. Frequency of CD8⁺ 4-1BB+ TIL expressing PD-1 alone or PD-1, TIM-3, and LAG-3. **p<0.01 by paired t-test. L. MFI of 4-1BB on PD-1+ or PD-1+TIM-3+LAG-3+ (Triple+) T cells isolated from CT2A TIL. M. Frequency of IFN-γ on cells expressing PD-1 and 4-1BB; 4-1BB, PD-1, TIM-3, and LAG-3; and PD-1, TIM-3, and LAG-3 but not 4-1BB as determined by Boolean gating of CT2A TIL re-stimulated in vitro with PMA/Ionomycin.