Malignant gliomas are heavily infiltrated by immature myeloid cells that mediate immunosuppression. Agonistic CD40 monoclonal antibody (mAb) has been shown to activate myeloid cells and promote antitumor immunity. Our previous study has also demonstrated blockade of cyclooxygenase-2 (COX-2) reduces immunosuppressive myeloid cells, thereby suppressing glioma development in mice. We therefore hypothesized that a combinatory strategy to modulate myeloid cells via two distinct pathways, i.e., CD40/CD40L stimulation and COX-2 blockade, would enhance anti-glioma immunity. We used three different mouse glioma models to evaluate therapeutic effects and underlying mechanisms of a combination regimen with an agonist CD40 mAb and the COX-2 inhibitor celecoxib. Treatment of glioma-bearing mice with the combination therapy significantly prolonged survival compared with either anti-CD40 mAb or celecoxib alone. The combination regimen promoted maturation of CD11b(+) cells in both spleen and brain, and enhanced Cxcl10 while suppressing Arg1 in CD11b(+)Gr-1(+) cells in the brain. Anti-glioma activity of the combination regimen was T-cell dependent because depletion of CD4(+) and CD8(+) cells in vivo abrogated the anti-glioma effects. Furthermore, the combination therapy significantly increased the frequency of CD8(+) T-cells, enhanced IFN-γ-production and reduced CD4(+)CD25(+)Foxp3(+) T regulatory cells in the brain, and induced tumor-antigen-specific T-cell responses in lymph nodes. Our findings suggest that the combination therapy of anti-CD40 mAb with celecoxib enhances anti-glioma activities via promotion of type-1 immunity both in myeloid cells and T-cells.

MicroRNAs (miRs) play important roles in regulation of a variety of cell functions, including immune responses. We have previously demonstrated that miR-17-92 expression in T-cells enhances Th1 phenotype and provides a long-term protection against glioblastoma when co-expressed as a transgene in T-cells along with a chimeric antigen receptor. To further elucidate the function of miR-17-92 in tumor antigen-specific CD8(+) T-cells, we generated transgenic (Tg) mice in which CD8(+) T-cells overexpress transgene-derived miR-17-92 under the lck promoter as well as T-cell receptor specific for human gp10025-33 (Pmel-1) (miR-17-92/Pmel-Tg). CD8(+) T-cells from miR-17-92/Pmel-Tg mice demonstrated enhanced interferon (IFN)-γ production and cytotoxicity in response to the cognate antigen compared with those from control Pmel-Tg mice without the transgene for miR-17-92. In addition, miR-17-92/Pmel-Tg mouse-derived CD8(+)CD44(+) T-cells demonstrated increased frequencies of cells with memory phenotypes and IFN-γ production. We also found that miR-17-92/Pmel-Tg-derived CD8(+) T-cells expressed decreased levels of transforming growth factor (TGF)-β type II receptor (TGFBR2) on their surface, thereby resisting against suppressive effects of TGF-β1. Our findings suggest that engineering of tumor antigen-specific CD8(+) T-cells to express miR-17-92 may improve the potency of cancer immunotherapy.

Background

While adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based immunotherapy for central nervous system (CNS) tumors, such as glioblastoma (GBM), still needs to overcome multiple challenges, including effective homing and persistence of T-cells. Based on previous observations that interleukin (IL)-17-producing T-cells can traffic to the CNS in autoimmune conditions, we evaluated CD8⁺ T-cells that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells in a preclinical GBM model.

Methods

We differentiated Pmel-1 CD8⁺ T-cells into Tc17-1 cells and compared their phenotypic and functional characteristics with those of IFN-γ-producing CD8⁺ T (Tc1) and IL-17-producing CD8⁺ T (Tc17) cells. We also evaluated the therapeutic efficacy, persistence, and tumor-homing of Tc17-1 cells in comparison to Tc1 cells using a mouse GL261 glioma model.

Results

In vitro, Tc17-1 cells demonstrated profiles of both Tc1 and Tc17 cells, including production of both IFN-γ and IL-17, although Tc17-1 cells demonstrated lesser degrees of antigen-specific cytotoxic activity compared with Tc1 cells. In mice-bearing intracranial GL261-Quad tumor and treated with temozolomide, Tc1 cells, but not Tc17-1, showed a significant prolongation of survival. However, when the T-cell transfer was combined with poly-ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17-1 cells exhibited significantly prolonged survival associated with upregulation of very late activation antigen-4 on Tc17-1 cells in vivo. Glioma cells that recurred following the therapy lost the susceptibility to Pmel-1-derived cytotoxic T-cells, indicating that immuno-editing was a mechanism of the acquired resistance.

Conclusions

Tc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.

Background

As therapies for systemic cancer improve and patients survive longer, the risk for brain metastases increases. We evaluated whether immune mechanisms are involved in the development of brain metastasis.

Methods

We conducted our studies using BALB/c mice bearing syngeneic 4T1 mammary adenocarcinoma cells in the mammary gland.

Results

The brains of mice bearing 4T1 tumors at day 14 had no detectable metastatic tumor cells but presented with marked accumulation of bone marrow-derived CD11b(+)Gr1(+) myeloid cells, which express high levels of inflammatory chemokines S100A8 and S100A9. In vitro, S100A9 attracts 4T1 cells through Toll-like receptor 4 and CD11b(+)Gr1(+) myeloid cells through Toll-like receptor 4 and the receptor for advanced glycation end-products. Systemic treatment of 4T1-bearing mice with anti-Gr1 (RB6-8C5) monoclonal antibody reduces accumulation of CD11b(+)Gr1(+) myeloid cells in the day-14 premetastatic brain as well as subsequent brain metastasis of 4T1 cells detected on day 30. Furthermore, treatment of 4T1 tumor-bearing mice with the cyclooxygenase-2 inhibitor celecoxib or genetic disruption of cyclooxygenase-2 in 4T1 cells inhibits the inflammatory chemokines and infiltration of CD11b(+)Gr1(+) myeloid cells in the premetastatic brain and subsequent formation of brain metastasis.

Conclusions

Our results suggest that the primary tumor induces accumulation of CD11b(+)Gr1(+) myeloid cells in the brain to form "premetastatic soil" and inflammation mediators, such as S100A9, that attract additional myeloid cells as well as metastatic tumor cells. Celecoxib and anti-Gr1 treatment may be useful for blockade of these processes, thereby preventing brain metastasis in patients with breast cancer.

We recently reported that STING contributes to antiglioma immunity by triggering type I IFN induction in glioma microenvironment. Moreover, intratumoral administration of STING agonist improved the efficacy of peptide vaccination in a mouse glioma model, suggesting the rational use of STING agonists in the immunotherapy of brain tumor.

Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b(+) brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting(Gt) (/Gt) mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting(Gt) (/Gt) mice showed increased CD11b(+) Gr-1(+) immature myeloid suppressor and CD25(+) Foxp3(+) regulatory T cells (Treg) and decreased IFNγ-producing CD8(+) T cells. CD4(+) and CD8(+) T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).