Journal: Frontiers in Immunology

Article Title: Differential effects of immobilized CCL21 and ICAM1 on TILs with distinct expansion properties

doi: 10.3389/fimmu.2025.1625118

Figure Lengend Snippet: Molecular and morphometric phenotyping of pre-REP TILs enables differential prediction of their expansion potency. Flow cytometry-based phenotyping of pre-REP samples (n=10). Gating strategy is shown in Supplementary Figure 2 . Representative FSC (A) and SSC (a’) profiles of high- and low-expanding pre-REP TILs (T6 and T93, respectively). (B , b’ ) Bar graphs showing the median fluorescence intensity (MFI) of FSC and SSC, respectively, obtained for 10 independent TILs. Data are shown as mean ± SEM of three independent experiments. Calculated p-values (using standard t-test) are as indicated in the figure. (C) Fold expansion as a function of mean frequencies of CD4 + , CD8 + and CD4 - CD8 - T cells within the CD3 + lymphocyte population in pre-REP TILs. Calculated p-values (using standard t-test) are as indicated in the figure. Data shown here are representatives of three independent experiments. (D) CD8/CD4 ratio in pre-REP TILs. Data are shown as mean ± SEM of three independent experiments. Calculated and p-values (using standard t-test) are as indicated in the figure. (E) Distribution of subsets of differentiation markers in pre-REP TILs. The percentages (mean ± SEM) of naïve (TN, CD45RO + CCR7 + ), central memory (TCM, CD45RO − CCR7 + ), effector memory (TEM, CD45RO − CCR7 − ) and effector (TEMRA, CD45RO + CCR7 − ) T cells in CD3 population are shown. Data presented here are representatives of three independent experiments.

Article Snippet: Following the establishment of TIL cultures, a standard 14-days REP was initiated by stimulating 10,000 pre-REP TILs with anti-CD3 (OKT-3) antibody (30 ng/ml, MACS GMP CD3 pure; Miltenyi Biotec, Bergisch Gladbach, Germany), 3,000 IU/ml IL-2 (Akron Biotech), and a mixture of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) feeder cells, obtained from two non-related donors at a 200:1 ratio of feeder cells to T cells.

Techniques: Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Differential effects of immobilized CCL21 and ICAM1 on TILs with distinct expansion properties

doi: 10.3389/fimmu.2025.1625118

Figure Lengend Snippet: Molecular phenotyping of SIN-stimulated TILs at the end of the REP process (day 14). Low- and high-expanding TILs (upper panel (A–D) and lower panel (E–H), respectively) were harvested on day 14, at the end of the REP process and subjected to spectral flow cytometry using selected phenotypic markers. The low-expanding TILs underwent simultaneous treatment with feeder cells and SIN for 14 days, whereas the high-expanding TILs were treated for 7 days with feeder cells only, followed by SIN treatment, without feeder cells for an additional period of 7 days. Data are shown as mean ± SEM of three independent experiments. (A, E) Bar graphs illustrating the percentage of CD8 + and CD4 + T cells among the CD3 + T cells as determined by flow cytometry analysis. (B, C, F, G) Distribution of surface markers among CD8 + (B, F) and CD4 + T cells (C, G) , that are related to T-cell activation (CD25, CD69), exhaustion (LAG-3, PD-1), and cytotoxic potency (GranzB, perforin) in low- (B, C) and high-expanding TILs (F, G) , in the presence or absence of SIN stimulation. (D, H) Distribution of differentiation subsets markers in low- (D) and high (H) -expanding TILs on day 14. The percentages (mean ± SEM) of naïve (TN, CD45RO + CCR7 + ), central memory (TCM, CD45RO − CCR7 + ), effector memory (TEM, CD45RO − CCR7 − ) and effector (TEMRA, CD45RO + CCR7 − ) T cells in CD3 population are represented. The data shown here are representatives of three independent experiments. Calculated p-values (using standard t-test) between the proportion of effector memory T cell subset in SIN-treated cells compared to untreated cells are as indicated in the figure.

Article Snippet: Following the establishment of TIL cultures, a standard 14-days REP was initiated by stimulating 10,000 pre-REP TILs with anti-CD3 (OKT-3) antibody (30 ng/ml, MACS GMP CD3 pure; Miltenyi Biotec, Bergisch Gladbach, Germany), 3,000 IU/ml IL-2 (Akron Biotech), and a mixture of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) feeder cells, obtained from two non-related donors at a 200:1 ratio of feeder cells to T cells.

Techniques: Flow Cytometry, Activation Assay