Polyclonal expansion of functional tumor-reactive lymphocytes infiltrating glioblastoma for personalized cell therapy

Abstract

Tumor-infiltrating lymphocyte (TIL)-therapy has received FDA approval for the treatment of advanced melanoma and shows potential for broader applications in solid tumors, including glioblastoma. In this study, tumor-reactive TILs (tr-TILs) are isolated and enriched for CD137 expression from cavitron ultrasonic aspirator (CUSA) emulsions of 161 adult patients diagnosed with diffuse gliomas. Tr-TILs are successfully expanded in 87 out of the 161 patients, reflecting an expansion rate of 54%. Notably, the presence of IDH1 mutation and the cumulative dose of steroids are identified as significant negative predictors of expansion efficacy. The expanded tr-TILs exhibit distinct phenotypic and molecular dysfunctional features yet show upregulated expression of progenitor/memory-like markers and polyclonal T-cell receptors. Importantly, these tr-TILs demonstrate specific antitumor reactivity against autologous tumor cells in both in vitro and in vivo xenograft models. These findings provide a compelling background for a personalized immunotherapeutic approach while tackling one of the most significant challenges in oncology.

Fig. 1. CD137+ tr-TILs are efficiently enriched from CUSA material.

a–f Scatter plots with bars showing (a) the frequency of CD137 on CD8⁺ and CD4⁺ T cells after debris removal, and (b) on CD3⁺ T cells compared with matched PBMCs (n = 161 vs. n = 77), before and after CD137 enrichment; c the number of cells, and the percentage of (d) CD45⁺, (e, f) CD8⁺ and CD4⁺ T cells before and after CD137 enrichment (n = 161). Data are presented as mean ± SD and statistical significance was assessed using a two-tailed unpaired t test. g ELISA quantification of IFNγ secreted by CD137⁺ tr-TILs and CD137- TILs co-cultured with matched GB-NS at 24 h (n = 12, with 2 technical replicates averaged prior to statistical analysis using a two-tailed unpaired t test). h Bar histograms showing CD137 MFI of macrophages, monocytes, neutrophils and tr-TILs before (pre) and after (post) enrichment (n = 5) and (i) of GB-NS belonging to proneural, classical and mesenchymal molecular subtype (n = 12). Data are presented as mean ± SD and statistical significance was assessed using a two-tailed unpaired t-test. j–m CD137 mRNA expression levels in (j) glioblastoma samples compared with tumor-free brain samples (n = 160); k CD137 expression across glioblastoma molecular subtypes: proneural, classical, and mesenchymal (n = 156); l, m CD137 expression in glioblastoma stratified by (l) MGMT promoter methylation status (n = 122) and (m) IDH1 mutation status (n = 150). Box plots show median (center line), 25th and 75th percentiles (bounds of box), and minimum and maximum values (whiskers). Statistical differences were analyzed using Tukey’s Honest Significant Difference (HSD) test. n–p Kaplan Meier curves of glioblastoma patients based on (n) the expression of TNFRSF9 (High TNFRSF9 [78 patients] vs. Low TNFRSF9 [77 patients]), o in MGMT met patients (High [28 patients] vs. Low [28 patients]), and (p) in MGMT unmet patients (High [33 patients] vs. Low [33 patients]); GlioVis dataportal; statistical significance between survival curves was assessed using the Log-rank (Mantel–Cox) test. Full numerical data and comprehensive details of statistical analyses are available in the corresponding source data tables for each figure. Source Data are provided as a Source Data file.

Fig. 2. tr-TILs retain memory status and express exhaustion markers.

a–c Representative proliferation kinetics of (a) expanded tr-TILs divided into high (n = 3), and (b) low-moderate recovery (n = 3), and (c) not expanded (n = 3). d, e Scatter plot with bar graphs showing duration (d) and dosage (e) of steroid treatment with dexamethasone (dex) between expanded tr-TILs-patients (n = 62) vs. not expanded tr-TILs-patients (n = 53). Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed Mann–Whitney test. f, g Scatter plots with bars showing (f) central (Tcm) and effector (Tem) memory status of CD8⁺ and CD4⁺ TILs of expanded (n = 71) and not expanded (n = 74) samples at the end of expansion process (statistical significance was assessed using a two-tailed unpaired t test); (g) and exhaustion immunophenotype on CD8⁺, CD4⁺ tr-TILs for both expanded (n = 72) and not expanded (n = 75) tr-TILs at the end of expansion process. Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed Mann–Whitney test. h–j Real-time PCR relative expression of genes related to (h) progenitor and (i) intermediate exhaustion status from Day 7 to Day 18 in expanded tr-TIL samples (n = 5 and 7 respectively, in blue in the supplementary tables), or related to (j) effector status in not expanded samples from Day 7 to Day 14 (n = 6, marked with an * in the supplementary tables). Data are presented as mean ± SD, with 2 technical replicates averaged prior to statistical analysis assessed using a two-tailed paired t-test. Full numerical data and comprehensive details of statistical analyses are available in the corresponding source data tables for each figure. Source Data are provided as a Source Data file.

Fig. 3. Expanded tr-TILs preserve tumor-specific T cell clonotypes and maintain progenitor status.

a Entire tissue sections (upper panel- scale bar, 5 mm) and selected region (lower panel- scale bar, 900 μm) of a representative glioblastoma stained for CD8 (red) and CD137 (brown). b, c Two distinct areas showing CD8⁺ TILs co-expressing CD137 (scale bars, 200 μm and 50 μm), and (d, e) coexistence of CD8⁺ CD137⁺ TILs and CD8+ TILs lacking CD137 (scale bar, 50 μm). f, g Representative immunofluorescence images showing DAPI (blue), CD8 (red) and TCF1 (green) staining and CD8/TCF1 colocalization (merge) of (f) TIL 390 (scale bar, 50 μm) and (g) TIL 431 (scale bar, 50 μm). Immunohistochemistry and immunofluorescence staining were performed on a sample size of n = 10. h Distribution of TCRβ clonotypes by tr-TIL samples from 10 glioma patients. TCRβ chain frequencies are expressed as a percentage, with 2% as the lower cut-off value, and are shown in a Stacked Barplot generated using R 4.2.3 (URL: https://www.R-project.org/). The absolute count of TCR clonotypes is indicated above each barplot.

Fig. 4. Tumor-reactive TILs exhibit specific antitumor activity against autologous tumor cells.

a–h tr-TILs co-cultured with matched GB-NS. a–c Scatters plots showing the residual number of tumor cells (count) from different GB molecular subtypes when in co-culture with autologous tr-TILs or PBMCs (ptPBMC), and healthy donor PBMCs (dPBMC) at (a) 24, (b) 48, and (c) 72 h, at E: T ratio = 3:1. d Quantification of IFNγ (pg/ml) secreted by tr-TILs co-cultured with autologous tumor cells from different GB molecular subtypes (24 h); as controls dPBMC and ptPBMCs were used. Data were derived from 12 cases, 4 with mesenchymal, 4 with proneural and 4 with classical subtype. Data are presented as mean ± SD, with 2 technical replicates averaged prior to statistical analysis assessed using a two-tailed unpaired t test. e Representative flow cytometry histograms showing PD-L1 expression levels of GB-NS in co-culture with tr-TILs (orange: TILs + GB-NS Day 3; red: TILs + GB-NS Day 5) compared to FMO (fluorescence minus one) control (black) and GB-NS alone (blue). f Bar histograms showing PD-L1 MFI of GB-NS alone and in co-culture with tr-TILs (n = 6). Data are presented as mean ± SD and statistical significance was assessed using a two-tailed unpaired t test. g Scatter plot showing residual tumor cells (count) in co-culture with tr-TILs at Day 3 and Day 5 (n = 6). Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed unpaired t test. h ELISA quantification of IFNγ secreted by tr-TILs co-cultured with GB-NS at Day 3 and Day 5 (n = 5). Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed unpaired t test. (i–k) PD-L1 blocking in tr-TILs co-cultured with GB-NS. i Representative flow cytometry plot of tr-TILs co-cultured with GB-NS in the presence of αPD-L1 antibody and in the presence of the isotype control (IgG2a/eBM2a). j Scatter plot showing the residual number of tumor cells (count) in co-culture with tr-TILs in the presence of αPD-L1 antibody and isotype control (IgG2a/eBM2a) at 24 and 48 h (n = 6). k ELISA quantification of IFNγ secreted by tr-TILs co-cultured with GB-NS in the presence of αPD-L1 antibody and isotype control at 24 and 48 h (n = 5). Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed unpaired t test. Full numerical data and comprehensive details of statistical analyses are available in the corresponding source data tables for each figure. Source Data are provided as a Source Data file.

Fig. 5. Tumor-reactive TILs control tumor growth in xenograft models.

a–d Frequency of CD4⁺ and CD8⁺ T cells of 4 tr-TILs samples during in vitro expansion. e Experimental schema of in vivo studies. f–i Kaplan Meier curves showing survival of nude mice treated with a dose escalation of tr-TILs. Four different TILs were tested (TIL 240, TIL 247, TIL 254, TIL 273). Control mice were treated with PBMCs or left untreated (control TILs - cTs). Comparisons between groups were performed using the log-rank (Mantel–Cox) test. j–l Expression of the activation markers CD69 and CD137 on tr-TILs isolated and analyzed 3, 6, and 24 h after intracranial injection of 0.75 × 10⁶ (j), 1.5 × 10⁶ (k), and 5 × 10⁶ (l) in xenograft models. Data are presented as mean ± SD from 5 mice per group (n = 5), and statistical significance was assessed using a two-tailed paired t test. m Expression of the activation markers CD69 and CD137 on tr-TILs (n = 4) during in vitro co-cultures at 24, 48, and 72 h. Data are presented as mean ± SD, and statistical significance was assessed using a two-tailed paired t test. n, o Representative MRI performed with T2-weighted images (T2-wi) and T1-weighted images (T1-wi) with contrast agent (ca) (n) on control and (o) TIL-treated xenograft mice; a total of 12 mice/condition were studied. p Bar graph showing the tumor volume measured using MRI and expressed as mm³ (controls vs TIL-treated mice). Data are presented as mean ± SD from 7 mice per group (n = 7) and statistical significance was assessed using a two-tailed unpaired t test. Full numerical data and comprehensive details of statistical analyses are available in the corresponding source data tables for each figure. Source Data are provided as a Source Data file.

Fig. 6. Persistence of CD8⁺ TILs and PD-L1 overexpression on tumor cells are found in xenograft gliomas.

a, b Representative Hematoxylin&Eosin staining images showing the morphological characteristics of (a) tumors from control (n = 7) and (b) TIL-treated (n = 7) mice 60 days after tumor cell implantation (Scale bar, 500 and 400 μm); c, d anti-human CD3 staining performed on tumors revealed the presence of TILs in treated but not control mice (Scale bar, 300 and 100 μm, respectively); (d inset) TILs persisting within the tumor mass were positive for CD8 (Scale bar, 100 μm). e, f PD-L1 staining of tumors (e) from control and (f) TIL-treated mice 60 days after tumor cell implantation (Scale bar, 100 and 500–300 μm, respectively). g, h Anti-human GFAP staining showing (h) a barrier surrounding tumor masses in treated mice (Scale bar 300 and 200 μm).