Masaki Terabe

Background: Glioblastoma is a lethal brain tumor notable for its limited spontaneous induction of CD8 ⁺ T cell responses. Vaccination may overcome this limitation by generating tumor-specific CD8 ⁺ T cells capable of mediating anti-tumor immunity in the brain. However, preclinical vaccine development is constrained by a lack of immunologically stringent glioblastoma models with defined endogenous antigens. We therefore sought to identify endogenous, vaccine-targetable antigens expressed by the immune checkpoint-refractory SB28 syngeneic mouse glioblastoma model and to evaluate their targetability using a heterologous prime-boost regimen with the chimpanzee adenovirus ChAdOx1 and the modified vaccinia Ankara (MVA) viral vector-based vaccines. Methods: RNA sequencing and whole-exome sequencing were used to identify candidate tumor-associated antigens (TAAs) and neoantigens, and tumor-specific expression was validated by RT-qPCR. ChAdOx1 and MVA vectors encoding candidate antigens were generated and evaluated for immunogenicity and anti-tumor efficacy. Antigen-specific CD8 ⁺ T cell responses and minimal epitopes were characterized by ex vivo restimulation, MHC-I binding assays, tetramer staining, and tumor cytotoxicity assays. Results: We identified five candidate TAAs ( Gpr149, Cdh6, Fbn2, Igf2bp1, and Shox2) but no suitable neoantigen targets in SB28. Vaccination induced CD8 ⁺ T cell responses against the candidate TAAs, and immunogenic minimal epitopes were identified for each. Among the candidates, Gpr149 elicited the strongest vaccine-induced CD8 ⁺ T cell responses. In mice challenged intracranially with SB28, Gpr149-targeted ChAdOx1/MVA vaccination combined with immune checkpoint blockade significantly prolonged median survival and resulted in long-term survival in 50% of mice, whereas vaccination targeting the other TAAs did not improve survival. CD8 ⁺ T cells recognizing the immunodominant Gpr149 _128-135 epitope (VCYNFYTM) specifically recognized and lysed wild-type SB28 cells, but not Gpr149-knockout SB28 cells, confirming endogenous processing and presentation of the targeted epitope. Conclusions: Gpr149 is an endogenous SB28 tumor antigen that can be effectively targeted by ChAdOx1/MVA vaccination to generate protective anti-tumor immunity against intracranial glioblastoma. Protection was associated with a dominant CD8 ⁺ T cell response to the Gpr149 _128-135 epitope VCYNFYTM. These findings establish endogenously expressed Gpr149 in SB28 as a physiologically relevant model for evaluating antigen-targeted immunotherapies and provide a systematic approach for identifying endogenous vaccine targets in other preclinical models. Key Messages: What is already known on this topic – The immune checkpoint-refractory SB28 glioblastoma model provides a stringent setting for preclinical immunotherapy studies, but endogenous tumor antigens suitable for antigen-targeted vaccination have not been defined, requiring studies of antigen-specific immunity to rely largely on transgenic foreign antigens. What this study adds – We identify Gpr149 as an endogenous SB28 tumor antigen and show that ChAdOx1/MVA vaccination generates functional Gpr149-specific CD8+ T cells that confer protective immunity against intracranial tumor challenge. How this study might affect research, practice or policy – Gpr149 provides an endogenous-antigen system for more physiologically relevant testing of antigen-targeted immunotherapies in immunologically cold tumors, while the antigen-discovery approach and ChAdOx1/MVA platform provide a framework for evaluating clinically relevant glioblastoma antigens in future therapeutic studies.

The P2Y receptor (P2YR) family comprises eight members that signal through either Gi (P2Y12, P2Y13, and P2Y14) or Gq/G11 (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) proteins1,2. Among these, the ADP-activated ...

Background: Triple-negative breast cancer requires new treatment strategies due to poor responses to current therapies. While myeloid SIRPα mediates immunosuppression, its cancer intrinsic role remains poorly understood. Methods: Human breast cancer scRNAseq profiles were used to examine SIRPα expression across different cell populations and subtypes. TNBC brain-tropic cells were injected into the mouse mammary fat pad for the orthotopic tumor model and intracardiac-injected for brain metastasis models. Bulk RNA sequencing was used to determine SIRPα-regulated pathway. Stably SIRPα overexpressed and knockout TNBC cell lines were established to determine SIRPα intracellular regulation. Digital spatial profiling was utilized to investigate the orthotopic and brain metastasis tumor immune microenvironment. Results: Human single-cell data showed that SIRPα levels increased in malignant TNBC epithelial cells. We observed that SIRPα is upregulated in patient breast-to-brain metastatic lesions. SIRPα is overexpressed in TNBC brain-tropic cells compared to parental cells. Bulk RNA-Seq showed that targeting SIRPα affects genes involved in mitochondrial dynamics, and that SIRPα upregulates mitochondrial fission and induces metastasis through the SHP2/Erk/Drp1 signaling pathway. In vivo, overexpression of SIRPα in cancer cells significantly increases TNBC systemic metastasis. Next, spatial proteomics revealed changes in the immune microenvironment associated with the SIRPα-regulated ECM protein fibronectin. Fibronectin induces microglial tolerance by impairing inflammatory signaling and metabolic reprogramming, allowing cancer to escape microglial immunosurveillance. Most importantly, SIRPα inhibition reduced TNBC brain metastatic lesions in mouse metastasis models. Conclusion: Cancer-intrinsic SIRPα promotes TNBC brain metastasis through increased mitochondria fission and triggering microglia tolerance, and targeting SIRPα reduces brain metastasis.

Glioblastoma is a lethal brain tumor that is unresponsive to current cancer immunotherapeutic approaches, including immune checkpoint blockade (ICB). This suggests that initial priming of T cells, rather than their expansion and licensing as effectors, is a restricting feature in this tumor setting. To overcome the limited initiation of CD8 ⁺ T cell responses, we employed a strong heterologous prime-boost vaccination with the simian adenovirus ChAdOx1 and poxvirus modified vaccinia Ankara (MVA). Vaccination conferred therapeutic efficacy against orthotopic, immune checkpoint-blockade (ICB)-refractory SB28 murine glioblastoma. Vaccination was effective against both the murine tumor antigen, P1A, and a newly identified glioblastoma-associated antigen, Gpr149. Additional treatment with ICB provided no additional benefit. Systemic ChAdOx1/MVA vaccination induced robust infiltration of antigen-specific T cells in tumor-challenged brains, the majority of which exhibited a CD103 ⁺ CD69 ⁺ CD8 ⁺ tissue-resident memory (TRM)-like phenotype. These cells were polyfunctional, durable in brains with sustained tumor control, and mediated tissue-specific immunological memory. Moreover, intracranial adoptive transfer of glioblastoma-derived antigen-specific TRM-like cells was sufficient to protect naïve recipients from subsequent orthotopic tumor challenge. Together, these findings establish that viral vector vaccination can generate tumor-specific TRM-like cells that mediate effective anti-glioblastoma immunity, providing a rationale for clinical evaluation of ChAdOx1/MVA-based strategies in glioblastoma.

Background: Immunotherapy, while effective for many extra-cranial solid tumors, has not shown benefits against glioblastoma. Limited T cell infiltration and an immunosuppressive tumor microenvironment (TME) are among the main barriers to successful immunotherapy. We hypothesized that blocking the chemokine CXCL12 would modulate the TME composition and reverse GBM resistance to immunotherapy. Methods: We used a syngeneic murine SB28 GBM model resistant to immune checkpoint inhibitor (ICI) and compared orthotopic and subcutaneous tumors to differentiate tumor-intrinsic factors from CNS-related barriers. We treated SB28 tumor-bearing mice with vehicle, ICI, CXCL12 inhibitor (NOX-A12), or a combination of NOX-A12 with ICI. We assessed treatment effects on immune cell populations in the blood and the TME, and on tumor growth and mouse survival. Results: ICI alone increased effector CD8+T cells in subcutaneous tumors but did not alter immune subsets in intracranial tumors. The combination of NOX-A12 and ICI increased effector CD8+ and CD4+ T cells in both models. In subcutaneous tumors, combination treatment also reduced MHC-IIlow tumor-associated macrophages (TAM) and slowed tumor growth. However, in orthotopic tumors, TAM populations remained unaffected, and survival was not extended, despite similar T-cell modulation, supporting the role of TAM in mediating GBM resistance to ICI. Conclusion: CXCL12 inhibition demonstrates therapeutic potential in facilitating anti-tumor immune response by dual mechanism, including T cell expansion and TAM reduction. The inability to deplete TAMs and improve survival in intracranial GBM underscores the need to address brain-specific mechanisms underlying TAM persistence to advance immunotherapy in GBM.

Background: To develop effective therapies for glioblastoma (GBM), a deeper understanding of its underlying immunoregulatory mechanisms is needed. Invariant natural killer T (iNKT) cells are unconventional T cells that recognize lipid antigens and are known to regulate tumor immunity in other cancer types. Given the lipid-rich nature of the brain and the unique metabolic activity of GBM cells, we hypothesized that GBM-enriched lipids could direct iNKT cells to contribute to the immunosuppressive nature of the disease. Methods: Lipid levels of multiple human GBM stem-like cell (GSC) lines, low grade-glioma lines, and normal human astrocytes were determined using liquid-chromatography mass spectrometry. GBM stem-like cell-enriched lipids were tested in iNKT stimulation assays, with either human iNKT cell lines or PBMC samples from both healthy donors and GBM patients, to determine antigenicity and characterize the nature of iNKT activation. Results: Multiple lipid species were found to be uniquely enriched in GSCs. Many of these lipids, called sulfatides, were recognized by and activated iNKT cells in a dose-dependent manner when presented by CD1d. Pharmaceutical and genetic targeting of the sulfatide synthetic pathway within GSCs resulted in an altered ability to activate iNKT cells. However, one lipid, lyso-sulfatide, inhibited the activation of iNKT cells and suppressed activation induced by a cognate antigen, α-galactosylceramide. Conclusions: The modulation of iNKT cell functions by GSC-enriched glycosphingolipids may contribute to the immunosuppression of GBM and highlights sulfatide production as a potential therapeutic target for GBM treatment.

Background: Glioblastoma (GBM) prognosis remains poor, and although immune checkpoint inhibitors (ICI) have transformed the treatment of many tumors, they are ineffective in GBM. However, response to ICIs occurs in high-tumor-mutational-burden (TMB) GBMs. To address the immunological impact of high TMB in GBM, we created a high TMB syngeneic mouse model from the low TMB SB28 GBM cell line. Methods: We used CRISPR-Cas9 to target murine Msh2, Mlh1, CXCL10, and CCL5. Single-cell-sorted clones were characterized by whole exome, bulk RNA-sequencing, and neoantigen prediction. Clones were injected subcutaneously or intracranially with or without anti-PD-1/anti-CTLA4 and dexamethasone. Results: Loss of mismatch repair (MMR) proteins Msh2 or Mlh1 increased nonsynonymous mutations. A fraction of mice with intracranial Msh2KO but not Mlh1KO SB28 showed long-term survival with anti-PD-1/anti-CTLA4 treatment plus dexamethasone. Long-term surviving mice from Msh2 KO SB28 rejected rechallenged subcutaneous tumors. Subcutaneous tumors from clones with increased TMB grew more slowly. This was fully abrogated in Rag1 null mice for Msh2KO but only partially for Mlh1KO SB28. Hypermutant Msh2KO clones spontaneously secreted CXCL10, CCL5, and increased pro-inflammatory chemokines after IFN-γ stimulation. Knockout of CXCL10 or CCL5 in the highest TMB Msh2KO clone restored flank tumor growth, indicating loss of immune response despite elevated TMB. Conclusion: Mismatch repair-deficient SB28 tumors were more immunogenic, but this was not completely correlated with TMB. Rather, rejection depended on increased secretion of pro-inflammatory chemokines. Msh2 and Mlh1 loss was not equivalent, suggesting that additional studies are needed to elucidate germline and somatic mismatch repair gene-specific immune alterations.

Background: Glioblastoma (GBM) is the most aggressive primary brain cancer in adults and remains incurable. Mucosal-associated invariant T (MAIT) cells are unconventional T cells with a semi-invariant T cell receptor and have been shown to regulate immune responses. However, the role of MAIT cells in glioblastoma (GBM) has not been well characterized. Methods: We used flow cytometry, bulk RNA-seq and scRNA-seq, and multiplexed tissue imaging to investigate the role of MAIT cells in GBM. Results: Flow cytometry analysis of peripheral blood samples of GBM patients showed a significant reduction of MAIT cell frequency and the ability to produce Th1 cytokines. In bulk RNA sequencing data analysis of GBM tissues, the MAIT cell gene signature significantly correlated with poor patient survival. A scRNA-seq of CD45+ cells from 20 GBM tissue samples showed 12 (60%) were positive for MAIT cells and the enrichment of RORC-expressing MAIT17. The MAIT cell signature significantly correlated with tumor-associated neutrophil (TAN) activities. Multiple immune suppressive genes known to be used by TANs were upregulated in MAIT-positive tumors. Spatial imaging analysis of GBM tissues showed that all specimens were positive for both MAIT cells and TANs, and localized enrichment of TANs. Conclusion: These findings revealed an immunosuppressive role of MAIT cells in GBM and highlighted the MAIT-TAN axis as a potential novel therapeutic target to modulate GBM’s immunosuppressive tumor microenvironment.

Background: While chimeric antigen receptor (CAR) T-cells are promising, there is a rapidly growing interest in developing other CAR-expressing immune cells. However, to date, no reported studies evaluated these cells side-by-side in immune-competent glioma models. Methods: We developed a novel C57BL/6-background transgenic mouse strain with all hematopoietic cells carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Crossing with mice transgenic for Vav-Cre allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK-cells, and CAR-macrophages in a syngeneic mouse SB28EGFRVIII glioma model. Results: CAR-NK and CAR-NKT-cells demonstrated anti-tumor effects comparable to CAR-T cells in vitro. A single intratumoral administration of CAR-T and CAR-NKT cells in combination mediated superior therapeutic efficacy compared to CAR-T cells or CAR-NKT-cells alone. A single intravenous infusion of CAR-NK cells following lymphodepletion failed to mediate significant anti-glioma effects. Additionally, intratumoral injection of CAR-NK cells did not confer therapeutic benefit. Contrary to previous reports using human macrophages, CAR-macrophages did not demonstrate enhanced antigen-presentation activity against glioma cells compared to non-CAR macrophages. Intratumorally administered CAR-macrophages failed to demonstrate local persistence or anti-tumor effects in vivo. Conclusions: These data provide a valuable basis as to which immune cells can mediate effective anti-glioma response in an immuno-competent glioma environment. Our data also suggest that a combination of CAR-T and CAR-NKT-cells may represent a promising therapeutic strategy.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow–derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding β-galactosylceramide (βGalCer) without sulfate. C24:2 induced IFN-γ–dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell–stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid’s function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.