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BackgroundWhile 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.MethodsWe 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.ResultsIn 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.ConclusionsTc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.

Simple Summary: Intratumoral hypoxia is associated with poor prognosis and therapeutic resistance by modifying the tumor microenvironment in several cancers. MTH1, a member of the Nudix family, maintains the genomic integrity and viability of tumor cells under hypoxic conditions. This study aimed to investigate whether the antitumor activity of immune cells is effective under hypoxia and whether hypoxia-induced MTH1 could be a target for immunotherapy. We found that MTH1 expression is elevated in hypoxic head and neck cancer cell lines and tissues. A novel MTH1 epitope peptide activates CD4+ helper T cells with cytotoxic activity, and is effective even under hypoxic conditions. Combining MTH1-targeted immunotherapy with PD-1 blockade enhances cytotoxicity. These results suggest that MTH1-targeted immunotherapy combined with checkpoint blockade could effectively treat hypoxic tumors by maintaining T-cell activity and increasing cytotoxicity under hypoxic conditions. Intratumoral hypoxia is associated with tumor progression, aggressiveness, and therapeutic resistance in several cancers. Hypoxia causes cancer cells to experience replication stress, thereby activating DNA damage and repair pathways. MutT homologue-1 (MTH1, also known as NUDT1), a member of the Nudix family, maintains the genomic integrity and viability of tumor cells in the hypoxic tumor microenvironment. Although hypoxia is associated with poor prognosis and can cause therapeutic resistance by regulating the microenvironment, it has not been considered a treatable target in cancer. This study aimed to investigate whether hypoxia-induced MTH1 is a useful target for immunotherapy and whether hypoxic conditions influence the antitumor activity of immune cells. Our results showed that MTH1 expression was elevated under hypoxic conditions in head and neck cancer cell lines. Furthermore, we identified a novel MTH1-targeting epitope peptide that can activate peptide-specific CD4+ helper T cells with cytotoxic activity. The proliferation and cytotoxic activity of T cells were maintained under hypoxic conditions, and PD-1 blockade further augmented the cytotoxicity. These results indicate that MTH1-targeted immunotherapy combined with checkpoint blockade can be an effective strategy for the treatment of hypoxic tumors. [ABSTRACT FROM AUTHOR]

Background: The identification of epitope peptides from tumor‐associated antigens (TAAs) is informative for developing tumor‐specific immunotherapy. However, only a few epitopes have been detected in mouse TAAs of head and neck cancer (HNSCC). Methods: Novel mouse c‐Met‐derived T‐cell epitopes were predicted by computer‐based algorithms. Mouse HNSCC cell line‐bearing mice were treated with a c‐Met peptide vaccine. The effects of CD8 and/or CD4 T‐cell depletion, and vaccine combination with immune checkpoint inhibitors (ICIs) were evaluated. Tumor re‐inoculation was performed to assess T‐cell memory. Results: We identified c‐Met‐derived short and long epitopes that elicited c‐Met‐reactive antitumor CD8 and/or CD4 T‐cell responses. Vaccination using these peptides showed remarkable antitumor responses via T cells in which ICIs were not required. The c‐Met peptide‐vaccinated mice rejected the re‐inoculated tumors. Conclusions: We demonstrated that novel c‐Met peptide vaccines can induce antitumor T‐cell response, and could be a potent immunotherapy in a syngeneic mouse HNSCC model. [ABSTRACT FROM AUTHOR]

The recognition by cytotoxic T cells (CTLs) is essential for the clearance of SARS-CoV-2 virus-infected cells. Several viral proteins have been described to be recognized by CTLs. Among them, the spike (S) protein is one of the immunogenic proteins. The S protein acts as a ligand for its receptors, and several mutants with different affinities for its cognate receptors have been reported, and certain mutations in the S protein, such as L452R and Y453F, have been found to inhibit the HLA-A24-restricted CTL response. In this study, we conducted a screening of candidate peptides derived from the S protein, specifically targeting those carrying the HLA-A24 binding motif. Among these peptides, we discovered that NF9 (NYNYLYRLF) represents an immunogenic epitope. CTL clones specific to the NF9 peptide were successfully established. These CTL clones exhibited the ability to recognize endogenously expressed NF9 peptide. Interestingly, the CTL clone demonstrated cross-reactivity with the Y453F peptide (NYNYLFRLF) but not with the L452R peptide (NYNYRYRLF). The CTL clone was able to identify the endogenously expressed Y453F mutant peptide. These findings imply that the NF9-specific CTL clone possesses the capability to recognize and respond to the Y453F mutant peptide. [ABSTRACT FROM AUTHOR]

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.

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.

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).

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.

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.

In CD70‐expressing tumors, the interaction of CD70 on tumor cells with its lymphocyte receptor, CD27, is thought to play a role in immunosuppression in the tumor microenvironment and elevated serum levels of soluble CD27 (sCD27). Previous studies showed that CD70 is expressed in nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)‐related malignancy. However, the association between intratumoral CD70/CD27 expression and serum levels of sCD27 in NPC remains unclear. In the present study, we show that CD70 is primarily expressed by tumor cells in NPC and that CD27‐positive lymphocytes infiltrate around tumor cells. NPC patients with CD27‐positive lymphocytes had significantly better prognosis than patients lacking these cells. In addition, high CD70 expression by tumor cells tended to be correlated with shorter survival in NPC patients with CD27‐positive lymphocytes. Serum sCD27 levels were significantly increased in patients with NPC and provided good diagnostic accuracy for discriminating patients from healthy individuals. The concentration of serum sCD27 in patients with CD70‐positive NPC with CD27‐positive lymphocytes was significantly higher than in patients with tumors negative for CD70 and/or CD27, indicating that the intratumoral CD70/CD27 interaction boosts the release of sCD27. Furthermore, positive expression of CD70 by NPC cells was significantly correlated with EBV infection. Our results suggest that CD70/CD27‐targeted immunotherapies may be promising treatment options and that sCD27 may become an essential tool for evaluating the applicability of these therapies by predicting the intratumoral CD70/CD27 interaction in NPC. [ABSTRACT FROM AUTHOR]

Recent studies have shown that activation of the cGAS‐STING pathway is a key process in antitumor immune responses and various kinds of STING agonists have been developed for cancer immunotherapy. Despite promising preclinical studies, preliminary clinical results have shown only a modest effect of STING agonists. There is therefore a need to develop more effective treatment strategies. Based on previous observations that COX‐2 is frequently overexpressed not only in a variety of cancers but also in tumor myeloid cells and that it suppresses antitumor immunity and promotes tumor survival by producing PGE2, we investigated the antitumor effects of combination therapy with a STING agonist cGAMP and the selective COX‐2 inhibitor celecoxib in mouse models. Combination treatment with cGAMP and celecoxib inhibited tumor growth compared with either monotherapy, and the combination therapy induced both local and systemic antitumor immunity. cGAMP treatment decreased PD‐1 expression on tumor‐infiltrating T‐cells and enhanced T‐cell activation in tumor‐draining lymph nodes regardless of the presence of celecoxib. Meanwhile, although celecoxib treatment did not alter the frequency of CD4+CD25+Foxp3+ regulatory T‐cells, it enhanced the expression of costimulatory molecules and glycolysis‐associated genes in tumor‐infiltrating CD11b+Ly6G+ cells. Moreover, we also found that celecoxib decreased lactate efflux and increased the frequency of IFN‐γ‐ and TNF‐α‐producing CD8+ T‐cells in the tumor microenvironment. Taken together, our findings suggest that combined treatment with celecoxib may be an effective strategy to improve the antitumor efficacy of STING agonists. [ABSTRACT FROM AUTHOR]

Homeobox B7 (HOXB7) is a master regulatory gene that regulates cell proliferation and activates oncogenic pathways. Overexpression of HOXB7 correlates with aggressive behavior and poor prognosis in patients with cancer. However, the expression and role of HOXB7 in head and neck squamous cell carcinoma (HNSCC) remain unclear. In this study, we observed that most samples from patients with oropharyngeal cancer and HNSCC expressed HOXB7. As no direct inhibitor has been reported, we identified a potent peptide epitope to target HOXB7‐expressing tumors through immune cells. A novel HOXB7‐derived peptide epitope (HOXB78–25) elicited antigen‐specific and tumor‐reactive promiscuous CD4+ T cell responses. These CD4+ T cells produced γ‐interferon (IFN‐γ) and had the direct ability to kill tumors through granzyme B. Notably, downregulation of HOXB7 using siRNA enhanced human leukocyte antigen class II expression on tumor cells by decreasing the phosphorylation of MAPK. Mitogen‐activated protein kinase inhibition augmented IFN‐γ production by HOXB7‐reactive CD4+ T cell responses without decreasing the expression of HOXB7. These results suggest that combining HOXB7 peptide‐based vaccine with MAPK inhibitors could be an effective immunological strategy for cancer treatment. [ABSTRACT FROM AUTHOR]