Reprogramming systemic and local immune function to empower immunotherapy against glioblastoma

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Standard-of-care (surgery, radiotherapy, temozolomide) rarely prevents recurrence due to GBM’s invasive heterogeneity and the blood–brain barrier. While immunotherapy has transformed care in other cancers, GBM is sparsely infiltrated by effector T cells, limiting responses. Prior efforts have focused on reversing local tumor microenvironment (TME) immunosuppression, yet GBM patients also exhibit profound systemic immunosuppression and lymphopenia, with CD4 counts approaching those seen in AIDS. Clinical treatments (radiation/TMZ) and dexamethasone further exacerbate lymphopenia. The research question is whether simultaneously correcting systemic immunosuppression (to increase circulating, functional T cells) and local immunosuppression (to enable their tumor entry and activity) can empower T cell–based immunotherapy in GBM. The authors propose Nano-reshaper, a nanoparticle co-delivering a CBD prodrug to alleviate lymphopenia and a LIGHT-encoding plasmid to remodel TME and recruit lymphocytes.

GBM is considered immunologically cold with abundant immunosuppressive cells (TAMs, MDSCs, Tregs) and factors (IL-10, TGF-β) that impede T-cell recruitment and function. Strategies to modulate the TME—reducing MDSCs, repolarizing M2 TAMs, delivering T-cell chemokines, and normalizing vasculature—have shown preclinical promise but limited clinical success, suggesting local modulation alone is insufficient. Emerging clinical observations highlight profound systemic immunosuppression in GBM, including bone marrow sequestration of naïve T cells via S1PR1 loss and serum factors inhibiting T-cell proliferation. Age-related immunosenescence, treatment-related lymphopenia (radiation/TMZ), and dexamethasone-induced T-cell depletion further reduce systemic T cells. Cytokine therapies (e.g., IL-2, IL-7, IL-12) can boost T cells but suffer from toxicity and pharmacologic limitations. LIGHT has been shown to normalize vasculature, induce HEVs, and enhance lymphocyte recruitment. CAR-T therapy passively increases T-cell numbers but faces antigen heterogeneity and trafficking barriers in GBM. These data support a dual approach addressing both systemic and local immunosuppression.

Design and formulation: The authors developed Nano-reshaper to co-deliver a cannabidiol (CBD) prodrug (CP) and a plasmid encoding LIGHT (PLIGHT) using lipid-coated calcium phosphate (CaP) nanoparticles. CBD was phosphorylated to CP to enable loading via Ca2+-phosphate precipitation; ApoE-PEG2000-DSPE was conjugated and incorporated at 5 mol% to enhance BBB crossing via ApoE receptors. Preparation used reversed-phase microemulsion to form CP/PLIGHT CaP cores followed by lipid film hydration with DOTAP, cholesterol, MPEG-DSPE, and ApoE-PEG-DSPE. Characterization: Particle size (DLS/TEM), zeta potential, encapsulation efficiency (EE), loading efficiency (LE), pH-dependent release (PBS pH 7.4/6.5/5.0), cellular colocalization of carrier (DiD) and plasmid (YOYO-1), CP-to-CBD intracellular conversion (HPLC), and in vitro transfection (EGFP reporter) were assessed. In vivo LIGHT expression in orthotopic GL261 tumors was quantified by His-tag ELISA and EGFP imaging. BBB targeting and biodistribution: An in vitro BBB transwell model (bEnd.3) measured nanoparticle transcytosis with or without ApoE competition. In vivo brain accumulation was quantified by DiR imaging in healthy and GL261-bearing mice. Cellular distribution to T cells, macrophages, DCs, and tumor cells in brain tumors, spleen, lymph nodes, and thymus was analyzed by flow cytometry. Systemic immunity assays: The effect of CBD/CP and Nano-reshaper on systemic immunity was tested in GBM-bearing mice by thymus and spleen indices, total and subset T-cell counts (blood, spleen, DLNs), activation markers (IFN-γ, granzyme B), and tumor-specific CD8+ T cells (H-2Kb Trp-2 tetramer). In vitro CFSE proliferation assays assessed T-cell proliferation in the presence of GBM serum and CBD. APC function: Phagocytosis was assessed using coumarin-6 labeled MPEG-PLA nanoparticles in RAW264.7, BV2, and DC2.4 cells; endocytosis inhibitors probed mechanisms (macropinocytosis). Intracellular Ca2+ (Fluo-3 AM) was measured after CBD exposure. DC maturation (CD80/CD86) and cross-presentation (SIINFEKL–MHC-I) were evaluated in BMDCs with OVA antigen. TME remodeling: Tumor vasculature structure (CD31) and adhesion molecules (ICAM-1, VCAM-1) were assessed by IHC/IF. HEVs were identified via MECA79 staining. Lymphocyte recruitment chemokines (CCL21, CXCL13) and immunosuppressive cytokines (TGF-β, IL-10) in tumors were quantified by ELISA. Tumor-infiltrating immune cells (CD8+, CD4+, Tregs, MDSCs, TAM phenotypes, CD103+ DCs, activated DCs) and functional status of CD8+ T cells (IFN-γ, GzmB, PD-1, TIM-3) were assessed by flow cytometry; absolute T-cell numbers per gram tumor were calculated. Therapeutic studies: Orthotopic GL261-luc and GL261/G422 models in male mice were used. Treatments included 5% glucose control, ApoE-CaCP (CP 10 mg/kg i.v.), ApoE-PLIGHT@CaP (PLIGHT 1.5 mg/kg i.v.), Nano-reshaper (CP 10 mg/kg + PLIGHT 1.5 mg/kg i.v.), temozolomide (20 mg/kg i.p.), anti-PD-1 (5 mg/kg i.p.). Tumor burden was monitored by bioluminescence imaging. Survival analyses (Kaplan–Meier) assessed efficacy. CD8+ or CD4+ T-cell depletion (10 mg/kg i.p.) and FTY720 (1.5 mg/kg i.p.) experiments probed mechanism (CD8 dependence and peripheral T-cell trafficking). Rechallenge: Long-term survivors from Nano-reshaper + anti–PD-1 were re-implanted contralaterally with GL261 without further treatment to assess immunological memory; memory T-cell subsets (TEM, TCM) in blood were analyzed. Safety: Hematology, serum biochemistry, and histology of major organs were evaluated in healthy mice after treatments.

• Nano-reshaper formulation: Size ~35 nm by DLS (~20 nm TEM); zeta potential ~+13 mV. Encapsulation efficiency: PLIGHT 51.7 ± 4.8%, CP 53.4 ± 5.5%; loading efficiency: PLIGHT 0.21 ± 0.02%, CP 1.45 ± 0.18%. pH-dependent release accelerated at pH 5.0. High colocalization of carrier and plasmid in tumor-site cells (88.6 ± 3.3%). In vitro transfection efficiency comparable to commercial liposomal reagent; in vivo, higher EGFP and LIGHT expression with Nano-reshaper vs PLIGHT@CaP.
• Targeting/BBB: In vitro BBB model showed 1.6-fold higher transcytosis vs PLIGHT@CaCP; competition with free ApoE reduced transport. In vivo, increased brain and tumor accumulation vs PLIGHT@CaCP, with higher uptake in T cells, macrophages, DCs, and tumor cells.
• Systemic immunity: ApoE-CaCP (CP alone) increased thymus/spleen indices and T-cell numbers (thymus T cells up to 6.6-fold at 10 mg/kg; peripheral CD8+/CD4+ counts approximated sham), alleviating lymphopenia, but did not increase tumor T-cell infiltration. Nano-reshaper significantly increased percentages and counts of CD8+ and CD4+ T cells in spleen, blood, and DLNs, elevated CD8+ IFN-γ and GzmB, and increased CD80+CD86+ DCs in DLNs. Tumor-specific Trp-2+ CD8+ T cells in blood increased. Synergy of CP and PLIGHT surpassed either component alone.
• APC activation: CBD enhanced phagocytosis in RAW264.7, BV2, DC2.4; inhibition profiles indicated macropinocytosis mediation; CBD increased intracellular Ca2+ in DC2.4 (TRPV2 agonism implicated). CBD promoted DC maturation (↑CD86) and cross-presentation (↑SIINFEKL–MHC-I). In BMDCs, Nano-reshaper increased CD80, CD86, and SIINFEKL–MHC-I vs controls. TAM polarization shifted toward M1 (↓CD206, ↑CD80) under ApoE-CaCP, ApoE-PLIGHT@CaP, and Nano-reshaper.
• TME remodeling: Nano-reshaper normalized vasculature (more homogeneous, reduced mean vessel length), increased endothelial ICAM-1 and VCAM-1 coverage. HEVs (MECA79+) induced in ApoE-PLIGHT@CaP and Nano-reshaper groups. Chemokines CCL21 and CXCL13 were upregulated; T and B cell infiltration increased. Cross-presenting CD11c+CD103+ DCs and activated DCs (CD80+CD86+) increased in tumors. Immunosuppressive cells decreased: MDSCs, Tregs, and M2 TAMs; M1/M2 and CD8+/Treg ratios increased. Immunosuppressive cytokines TGF-β and IL-10 were reduced. CD8+ T-cell function improved (↑IFN-γ+, ↑GzmB+; reduced exhaustion markers).
• T-cell infiltration: Absolute intratumoral T cells increased (e.g., CD8+ T cells per gram tumor significantly higher vs controls; exact values shown in Fig. 6j). Trp-2–specific CD8+ T cells increased in tumors.
• Antitumor efficacy: In GL261-luc model, Nano-reshaper reduced tumor bioluminescence vs control and monotherapies. Median survival: control 18.5 d, ApoE-CaCP 19 d, ApoE-PLIGHT@CaP 23 d, Nano-reshaper 31 d. CD8 depletion abrogated efficacy (median 18 d vs 27 d with IgG), while CD4 depletion had little effect (19 d). Blocking peripheral T-cell trafficking with FTY720 abrogated benefit (Nano-reshaper median 34 d vs 18.5 d with FTY720 co-treatment).
• Combination therapy: Nano-reshaper + anti–PD-1 provided superior tumor control and survival; in GL261, 83.3% (5/6) achieved long-term survival without recurrence; similar synergy observed in G422 model. Long-term survivors resisted contralateral rechallenge with 100% second long-term survival and exhibited increased CD8+ TEM and TCM.
• Safety: Hematology/biochemistry largely within normal ranges; modest increases in WBC subsets; no histopathological damage in major organs, including brain and thymus.

The study addresses the central hurdle of insufficient effector T cells in GBM by demonstrating that overcoming both systemic and local immunosuppression is essential. CBD restored systemic immune competence by alleviating lymphopenia, expanding thymic and splenic T-cell compartments, and enhancing APC phagocytosis and antigen cross-presentation. LIGHT expression remodeled the TME, normalizing vasculature, inducing HEVs, and upregulating lymphoid chemokines to recruit lymphocytes while reducing suppressive cells and cytokines. Together, these effects substantially increased intratumoral, tumor-specific, functional CD8+ T cells, which were shown to be indispensable for tumor control. The necessity of continuous peripheral T-cell influx was confirmed by the FTY720 experiment. Importantly, Nano-reshaper sensitized GBM to PD-1 blockade, yielding high long-term survival rates and durable immunological memory against rechallenge, highlighting the translational relevance of dual reprogramming to amplify T cell–based immunotherapies in GBM and potentially other cold tumors.

The authors developed Nano-reshaper, a BBB-targeted nanoplatform co-delivering a CBD prodrug and a LIGHT plasmid to simultaneously reprogram systemic and local immunity in GBM. This approach increased systemic T-cell output, enhanced APC function, normalized tumor vasculature, induced HEVs, elevated lymphocyte-recruiting chemokines, and reduced immunosuppression, collectively driving robust CD8+ T-cell infiltration and function. Nano-reshaper inhibited GBM growth, prolonged survival, and synergized with PD-1 blockade to achieve 83.3% long-term survival and durable protection against rechallenge in mouse models. This platform may generalize to other immunologically cold tumors and synergize with vaccines, oncolytic viruses, and checkpoint blockade. Future work should validate efficacy in clinical settings, elucidate CBD’s precise immunomodulatory mechanisms, and assess effects on broader immune cell populations.

• Preclinical models (GL261, G422) may not fully recapitulate human GBM, including lower tumor mutational burden in patients; clinical confirmation is needed.
• CBD increased lymphocytes in tumor-free mice, indicating systemic immune effects independent of tumor; the precise mechanisms underlying CBD’s immunomodulatory actions in vitro and in vivo remain to be defined.
• The study focused on T cells and APCs; other immune cell types may be affected by Nano-reshaper and warrant investigation.
• Translation requires assessment of dosing, pharmacokinetics, and long-term safety in humans, as well as potential variability due to steroid use and standard-of-care treatments in patients.