Biology subjects

Chuntova, P.

Publications and source records attributed to Chuntova, P..

6 recordsLinked to original sources

bioRxiv2026

Ultrasound-mediated blood-brain barrier modulation enhances T-cell access but requires immune activation for effective CNS immunity

Immunotherapy shows limited efficacy in brain tumours, where restricted immune access, antigenic heterogeneity and local immunosuppression constrain durable responses. Low-intensity pulsed ultrasound with microbubbles (LIPU+MB) transiently modulates the blood-brain barrier (BBB) and is widely assumed to enhance immunotherapy by facilitating drug and immune cell penetration into the central nervous system (CNS). However, whether increased anatomical access alone is sufficient to generate effective CNS immunity remains unclear. Here, using a transgenic mouse model with astrocyte-restricted antigen expression, we showed that BBB modulation alone is insufficient to generate functional T-cell immunity in the CNS. Although LIPU+MB enabled rapid T-cell entry, accumulation required prior T-cell activation and integrin-dependent mechanisms, indicating that entry remains governed by canonical immune processes. Moreover, T-cells failed to persist owing to insufficient activation of antigen-presenting cells (APCs) within the CNS. Systemic immune adjuvants (poly-ICLC and IL-2; PI) induced APC activation, promoted tissue-resident-memory-like differentiation and supported durable T-cell responses. LIPU+MB further enhanced these responses by increasing T-cell recruitment, resulting in greater accumulation than with PI alone. Mechanistically, antigen presentation by bone marrow-derived APCs was more critical than that by microglia for the accumulation and persistence of antigen-specifc T-cells in the CNS. In antigenically heterogeneous glioma models resistant to CAR T-cell therapy, combining PI with BBB modulation enhanced the efficacy of immunotherapy, which was mirrored by prolonged survival and endogenous tumour-specific T-cell responses, consistent with epitope spreading. Together, these findings define key limitations of LIPU+MB in enabling effective T-cell therapy and establish that BBB modulation must be coupled to systemic immune activation to support T-cell-mediated antitumour immunity in the CNS.

immunology↗

bioRxiv2024

Replicating retroviral delivery of an IL-15 superagonist improves antitumor immunity and long-term survival in poorly immunogenic glioblastoma models

Glioblastoma (GBM) is the most lethal primary brain neoplasm due to its highly immunosuppressive microenvironment and resistance to conventional therapies. To overcome this challenge, we engineered a replicating retrovirus (RRV) to deliver a superagonist interleukin-15 receptor-linked fusion protein (RLI) directly to tumor cells, engineering them into local immunotherapy biofactories. This strategy leverages the tumor-selective replication of RRV to achieve localized and sustained RLI expression within the tumor microenvironment. In two orthotopic poorly immunogenic GBM mouse models, intratumoral administration of RRV RLI significantly reduced tumor growth and prolonged survival compared to controls, with some mice achieving long-term remission and demonstrating immunologic memory upon rechallenge. Transcriptomic and flow cytometric analyses revealed that RRV RLI treatment enhanced infiltration and activation of CD8 T cells, NK cells, and upregulated antigen presentation pathways within the tumor microenvironment. Depletion studies indicated that the therapeutic efficacy of RRV RLI is dependent on both CD4 and CD8 T cells. Notably, combining RRV RLI with the GBM standard of care chemotherapeutic agent temozolomide (TMZ) synergistically improved survival outcomes. Subsequent single-cell RNA and T cell receptor sequencing identified enhanced effector cell activation, antigen presentation, and clonal T cell expansion in the combination therapy group. Further T cell receptor analysis and clustering implied a tumor-specific immune response rather than one targeting the viral delivery vehicle, suggesting that this therapeutic approach could be reapplied without eliciting anti-vector immunity. Our findings suggest that RRV-mediated delivery of RLI effectively transforms GBM tumors into immunostimulatory hubs, eliciting a potent anti-tumor immune response. This novel viral immunotherapy holds significant promise for clinical translation in the treatment of GBM and other difficult-to-treat solid tumors.

cancer biology↗

bioRxiv2024

IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma

BackgroundGlioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. MethodsEffects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. ResultsMice with RRV-IRF8 pre-transduced intracerebral tumors had significantly longer survival and slower tumor growth compared to controls. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naive T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. ConclusionsOur results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control. Key pointsO_LIGBM intra-tumoral myeloid cells are proliferative and targets for RRV therapy. C_LIO_LIExpression of IRF8 significantly improves survival and slows tumor growth in murine GBM. C_LIO_LIIRF8 expression in MDSCs reduces immunosuppression and enriches cDC1s in vivo. C_LI Importance of the studyRecent publications have presented conflicting studies regarding the role of IRF8 in GBM. While some studies showed IRF8 as a negative prognostic factor, others demonstrated the conversion of tumor cells into DCs using IRF8. Here, we show that RRV-mediated delivery of IRF8, a clinically relevant modality, allows for transduction of both tumor and immune cells in vivo. We show that a significant survival effect relies heavily on the infection and modulation of both populations, and that even a modest number of reprogrammed intra-tumoral MDSCs can have a substantial impact on the immunological milieu, significantly enriching and activating cytotoxic T-cells. Further, this work reveals intra-tumoral myeloid cells as a target for other RRV-based gene therapies.

immunology↗

bioRxiv2023

Enhancing CAR-T Cell Metabolism to Overcome Hypoxic Conditions in the Brain Tumor Microenvironment

The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1 (PGC-1) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

immunology↗

bioRxiv2021

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

BackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART)-cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T-cells and evaluated the function of the CART-cells both in vitro and in vivo. In order to inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). ResultsBoth RV- and Tg-CART-cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART-cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies. Importance of studyThe majority of preclinical studies evaluating CART therapy for GBM have utilized xenografts implanted into immunocompromised mice. Because the successful development of these strategies will depend on the understanding of critical interactions between therapeutic cells and the endogenous immune environment, it is essential to develop a novel immunocompetent system which allows us to study these interactions in a robust and reproducible manner. To this end, we created a Tg mouse strain in which all T-cells express a murinized EGFRvIII-CAR. T-cells derived from these mice demonstrated consistent CAR expression and EGFRvIII-specific cytotoxicity while traditional transduction with a CAR vector showed batch-to-batch variability. The syngeneic system also gave us the opportunity to evaluate a combination regimen with blockade of myeloid-derived suppressor cells. The Tg-CART mice represent a novel system for robust, and reproducible preclinical investigations.

immunology↗

bioRxiv2020

Deep immune profiling reveals targetable mechanisms of immune evasion in checkpoint blockade-refractory glioblastoma

BackgroundGlioblastoma (GBM) is refractory to checkpoint blockade immunotherapy (CPI). We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed. MethodsWe used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally CPI-refractory (GBM and sarcoma) or CPI-responsive (renal cell carcinoma), as well as mouse models of GBM that are CPI-responsive (GL261) or CPI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations. ResultsCPI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells, and fewer myeloid cells, in particular PD-L1+ tumor associated macrophages. The SB28 mouse model of GBM responded to CPI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying CPI resistance in GBM. The response to CPI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded dendritic cells, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation. ConclusionsOur data suggest that a major obstacle for effective immunotherapy of GBM is the low antigenicity of the tumor cells coupled with poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified dendritic cells and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/404939v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@bdd0e4org.highwire.dtl.DTLVardef@1dd335forg.highwire.dtl.DTLVardef@157bfa6org.highwire.dtl.DTLVardef@1e76151_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefIn mice and humans, tumors that were sensitive to checkpoint blockade had consistent immunological features. A mouse model of glioma that is refractory to checkpoint blockade was sensitized by increasing antigen presentation through a variety of approaches.

cancer biology↗