Fig 2: miR-21 Promotes Effector Cell Differentiation through AP-1 Activation(A) Naive CD4+ T cells were transfected with either scrambled control or miR-21-blocking locked nucleic acid (LNA21). After 48 hr, PDCD4 and b-actin expression were assessed by western blot. Representative blots and mean normalized intensities from four experiments are shown (mean ± SEM, two-tailed paired t test).(B) Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control or anti-miR-21. The representative histogram shows phosphorylated JNK in GFP+ cells on day 3. The filled gray histogram represents unstimulated naive CD4+ T cells. Results from 7 experiments are expressed relative to the geometric mean fluorescence intensity (MFI) of controls (mean ± SEM, two-tailed paired t test).(C) Naive CD4+ T cells were activated and transduced as described in (B) and co-transfected with the AP-1 luciferase reporter plasmid and the Renilla luciferase control construct. On day 3, the activity of AP-1 firefly luciferase was measured and normalized to that of Renilla luciferase (n = 4, mean ± SEM, two-tailed paired t test).(D–F) Naive CD4+ T cells isolated from 20- to 35-year-old and 65- to 85-year-old individuals were activated with beads coated with anti-CD3 and anti-CD28 antibodies.(D) PDCD4 expression was quantified by RT-PCR on day 3 and day 5. Results are normalized to ACTB and presented relative to those of cells on day 3 from young individuals. The horizontal lines represent mean values (n = 12–19, two-tailed unpaired t test).(E and F) Representative histograms (E) and geometric MFI of phosphorylated JNK on day 3 and day 4 from 10 individuals (F). The filled gray histograms represent unstimulated naive CD4+ T cells. The horizontal lines represent mean values, and significance was calculated by two-tailed unpaired t test.(G) Naive CD4+ T cells from older adults were activated with anti-CD3 and anti-CD28 beads in the presence of either DMSO or the AP-1 inhibitor SR11302 for 5 days. Surface expression of IL7Ra, CCR7, IL2Ra, and CD39 and intracellular production of IL-2, TNF-a, and granzyme B after re-stimulation with PMA and ionomycin were assessed by flow cytometry (n = 12, two-tailed paired t test).(H) Activated naive CD4+ T cells were transfected with scramble control (si-ctrl) or c-FOS-specific siRNA (si-FOS) on day 2. IL-2, TNF-a, and granzyme B production were assessed on day 5 (n = 4, two-tailed paired t test).(I) Naive CD4+ T cells were activated as described in (G). miR-21 expression was measured on day 3 by qRT-PCR. Results are normalized to the expression of RNU48 and presented relative to those of unstimulated naive CD4+ T cells (n = 6, two-tailed paired t test).(J) Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-miR-21. After 36 hr, activated cells were transfected with si-ctrl or PDCD4-specific siRNA (si-PDCD4). The graphs show marker expression in GFP+ cells on day 5 (n = 8, one-way ANOVA followed by Tukey’s multiple comparison test).*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Tumor-infiltrating Tpex cells from human RCC have the superior proliferative capacity in response to ex vivo IL-15 treatment compared with the Ttex subset of CD8 TILs. (A) Representative flow plots of PD-1 expression on CD8 TILs (left) and the frequency of TCF1+Tim-3- Tpex and TCF1-Tim-3+ Ttex cells among PD-1+ CD8 TILs (right) in human RCC tumors. (B) Frequency of PD-1+ cells among total CD8 TILs. The graph shows the mean and SEM. (C) Frequency of Tpex and Ttex cells among PD-1+ CD8 TILs. (D, E) Representative flow plots (D) and summary graphs (E) showing the expression of PD-1, CD39, Ki-67, and CD28 on Tpex and Ttex cells. Data were combined from three independent experiments with samples from 8 patients. *P < 0.05; **P < 0.01 (Student’s t-test, paired). (F, G) Expression of CD122 (F) and CD132 (G) on Tpex and Ttex cells among PD-1+ CD8 TILs. Data were combined from two independent experiments with samples from 6 patients. No statistical significance was determined by one-way ANOVA. (H-J) RCC TILs were cultured with recombinant human IL-15 (10 ng/ml) in the presence of irradiated autologous PBMCs for 3 days. (H, I) Frequency of Ki-67+ cells in the Tpex and Ttex subsets pre- and post-treatment. (J) The expansion ratio from pre- to post-treatment in each subset. Data were combined from two independent experiments with samples from 7 patients. **P < 0.01 (Student’s t-test, paired).

Fig 4: Phenotypic diversity of expanded TILs across tumor types. (A) Expanded tumor-infiltrating lymphocytes (TILs) showed high purity, and that of CD45+CD3+ cells consistently exceeded 93.0%. The proportion of CD8+ cells among CD3+ T cells varied across tumor types. (B) Frequencies of exhaustion markers within CD3+ T cells: CD39 and lymphocyte activation gene 3 (LAG-3) showed variable positivity, whereas programmed cell death protein 1 (PD-1) remained negligible in all groups. (C, D) Memory subset distribution. Both CD4+ (C) and CD8+ (D) TILs were predominantly effector memory T-cells (TEMs), whereas central memory T-cells (TCMs) were present at low frequencies. Each dot represents one patient sample (melanoma, n = 5; pancreatic cancer, n = 3; gastric cancer, n = 3; cervical cancer, n = 5; and colorectal cancer, n = 5). Bars indicate mean ± SD. Colors represent tumor types: orange, melanoma (MM); blue, pancreatic cancer (PC); purple, gastric cancer (GC); brown, cervical cancer (CxCa); and black, colorectal cancer (CRC). Dashed lines indicate the 50% reference threshold.