GITRL COEXPRESSION IN ANTI-GD2 CAR-T CELLS ENHANCES RESISTANCE TO TREG-MEDIATED IMMUNOSUPPRESSION, MITIGATES EXHAUSTION, AND BOOSTS CYTOTOXICITY AGAINST GLIOBLASTOMA SPHEROIDS AND ORGANOTYPIC TUMOR SLICES
- 1Centro de Terapia Celular (CTC), Hemocentro de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil
- 2Mie University, Tsu, Japan
Abstract
The CAR-T cell therapies have revolutionized hematological malignancies treatment. However, their efficacy against solid tumors in early-phase trials showed modest responses. This is largely due to the heterogeneous and immunosuppressive tumor microenvironment (mediated predominantly by regulatory T cells), which impairs CAR-T cell trafficking, persistence and function. Furthermore, the lack of reliable and predictive preclinical platforms to elucidate CAR-T cell behavior within tumor tissues poses a significant obstacle for clinical translation. Notably, conventional two-dimensional assays fail to recapitulate the complexity of the tumor microenvironment, lacking essential immunosuppressive cell populations as well as the physiological gradients of oxygen and nutrients. Adopting more reliable and predictive preclinical platforms, and based on previous reports, we hypothesized that the glucocorticoid-induced TNFR-related protein ligand (GITRL), through engagement of its receptor GITR, could potentiate T cell activity in CAR-T cells and overcome Treg-mediated suppression in 3D glioblastoma cocultures containing regulatory T cells, as well in organotypic tumor slices. To this purpose, we generated by lentiviral transduction human anti-GD2 CAR-T cells coexpressing GITRL (CAR.GD2/GITRL) also CAR-T cells expressing the GD2-specific CAR alone (CAR.GD2). Firstly, to assess their in vitro functional activity we tested both CAR-T cells against T98G glioblastoma cells. By incucyte analysis, CAR.GD2/GITRL showed superior lysis capacity against tumor cells in all effector:target ratios (E:T). Phenotypic analysis of CAR-T cells recovered from cocultures revealed reduced expression of exhaustion markers, such as PD-1 and LAG-3, in CAR.GD2/GITRL cells. To reliably test our CAR-T cells, we established subcutaneous xenografts in NSG mice using luciferase-expressing T98G cells. Ex vivo 400µm agarose-embedded tumor slices were cocultured with CAR-T cells for 5 days. Both CAR-T demonstrated bioluminescence reduction and pro-inflammatory cytokine secretion. In order to reproduce the tumor microenvironment and predict the immunosuppressed effect, we generated iTregs by inducing the transcription factor Foxp3 superexpression in CD4+ T lymphocytes. These cells were evaluated in a multicellular system comprising tumor spheroids and CAR-T cells. Importantly, we confirmed that iTregs did not demonstrated cytotoxic effect against the spheroids at the same time that suppressed the CAR-T proliferation. Indeed, CAR.GD2/GITRL cells demonstrated increased capacity to diminished the spheroids compared to CAR.GD2, confirming the less susceptibility of the immunosuppression driven by iTregs. These findings indicate that GITRL coexpression increases CAR-T cell resistance to exhaustion and immunosuppressive signals. Furthermore, we validate tumor slice and spheroid coculture models with iTregs as highly predictive platforms to assess CAR-T efficacy in solid tumor microenvironments.
Related
- Lipid Nanoparticle Delivery of CAR276 mRNA Enables Antigen-Specific Cytotoxicity in T Cells Against Glioblastoma Cells
- Combinatorial treatment with formononetin and magnolol induces synergistic cytotoxicity and suppresses migration in glioblastoma cells
- Rigorous nested cross-validation and external generalization-gap analysis of a machine-learning classifier for U-251 glioblastoma cytotoxicity
- 241 Engineering CAR-T cell resistance to CAF-derived paracrine cAMP-mediated immunosuppression
- Stress-adapted cancer-associated fibroblasts as mediators of immunosuppression and therapy resistance
BibTeX
@article{furtado2026gitrl,
title = {{GITRL COEXPRESSION IN ANTI-GD2 CAR-T CELLS ENHANCES RESISTANCE TO TREG-MEDIATED IMMUNOSUPPRESSION, MITIGATES EXHAUSTION, AND BOOSTS CYTOTOXICITY AGAINST GLIOBLASTOMA SPHEROIDS AND ORGANOTYPIC TUMOR SLICES}},
author = {Izadora Peter FURTADO and Millena BRANDãO and Raíssa Raminelli REZENDE and Karolina Santos ESTEVES and Hiroshi SHIKU and Diego Villa CLé and Dimas Tadeu COVAS and Lucas Eduardo Botelho SOUZA},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.106874},
url = {https://doi.org/10.1016/j.htct.2026.106874},
note = {CC BY},
}
RIS
TY - JOUR
TI - GITRL COEXPRESSION IN ANTI-GD2 CAR-T CELLS ENHANCES RESISTANCE TO TREG-MEDIATED IMMUNOSUPPRESSION, MITIGATES EXHAUSTION, AND BOOSTS CYTOTOXICITY AGAINST GLIOBLASTOMA SPHEROIDS AND ORGANOTYPIC TUMOR SLICES
AU - Izadora Peter FURTADO
AU - Millena BRANDãO
AU - Raíssa Raminelli REZENDE
AU - Karolina Santos ESTEVES
AU - Hiroshi SHIKU
AU - Diego Villa CLé
AU - Dimas Tadeu COVAS
AU - Lucas Eduardo Botelho SOUZA
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.106874
UR - https://doi.org/10.1016/j.htct.2026.106874
AB - The CAR-T cell therapies have revolutionized hematological malignancies treatment. However, their efficacy against solid tumors in early-phase trials showed modest responses. This is largely due to the heterogeneous and immunosuppressive tumor microenvironment (mediated predominantly by regulatory T cells), which impairs CAR-T cell trafficking, persistence and function. Furthermore, the lack of reliable and predictive preclinical platforms to elucidate CAR-T cell behavior within tumor tissues poses a significant obstacle for clinical translation. Notably, conventional two-dimensional assays fail to recapitulate the complexity of the tumor microenvironment, lacking essential immunosuppressive cell populations as well as the physiological gradients of oxygen and nutrients. Adopting more reliable and predictive preclinical platforms, and based on previous reports, we hypothesized that the glucocorticoid-induced TNFR-related protein ligand (GITRL), through engagement of its receptor GITR, could potentiate T cell activity in CAR-T cells and overcome Treg-mediated suppression in 3D glioblastoma cocultures containing regulatory T cells, as well in organotypic tumor slices. To this purpose, we generated by lentiviral transduction human anti-GD2 CAR-T cells coexpressing GITRL (CAR.GD2/GITRL) also CAR-T cells expressing the GD2-specific CAR alone (CAR.GD2). Firstly, to assess their in vitro functional activity we tested both CAR-T cells against T98G glioblastoma cells. By incucyte analysis, CAR.GD2/GITRL showed superior lysis capacity against tumor cells in all effector:target ratios (E:T). Phenotypic analysis of CAR-T cells recovered from cocultures revealed reduced expression of exhaustion markers, such as PD-1 and LAG-3, in CAR.GD2/GITRL cells. To reliably test our CAR-T cells, we established subcutaneous xenografts in NSG mice using luciferase-expressing T98G cells. Ex vivo 400µm agarose-embedded tumor slices were cocultured with CAR-T cells for 5 days. Both CAR-T demonstrated bioluminescence reduction and pro-inflammatory cytokine secretion. In order to reproduce the tumor microenvironment and predict the immunosuppressed effect, we generated iTregs by inducing the transcription factor Foxp3 superexpression in CD4+ T lymphocytes. These cells were evaluated in a multicellular system comprising tumor spheroids and CAR-T cells. Importantly, we confirmed that iTregs did not demonstrated cytotoxic effect against the spheroids at the same time that suppressed the CAR-T proliferation. Indeed, CAR.GD2/GITRL cells demonstrated increased capacity to diminished the spheroids compared to CAR.GD2, confirming the less susceptibility of the immunosuppression driven by iTregs. These findings indicate that GITRL coexpression increases CAR-T cell resistance to exhaustion and immunosuppressive signals. Furthermore, we validate tumor slice and spheroid coculture models with iTregs as highly predictive platforms to assess CAR-T efficacy in solid tumor microenvironments.
SN - 2531-1379
SN - 2531-1387
ER -