Expression of soluble CD27 in extranodal natural killer/T-cell lymphoma, nasal type: potential as a biomarker for diagnosis and CD27/CD70-targeted therapy.

The engagement of CD27 on lymphocytes with its ligand, CD70, on tumors is believed to mediate tumor immune evasion and the elevation of serum soluble CD27 (sCD27) levels in patients with CD70-positive malignancies. We previously showed that CD70 is expressed in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), an Epstein-Barr virus (EBV)-related malignancy. However, little is known about serum sCD27 expression and its association with the clinical characteristics of, and the CD27/CD70 interaction in, ENKL. In the present study, we show that serum sCD27 is significantly elevated in the sera of patients with ENKL. The levels of serum sCD27 provided excellent diagnostic accuracy for discriminating patients with ENKL from healthy subjects, correlated positively with the levels of other diagnostic markers (lactate dehydrogenase, soluble interleukin-2 receptor, and EBV-DNA), and decreased significantly following treatment. Elevated serum sCD27 levels also correlated significantly with advanced clinical stage and tended to correspond with shorter survival, in patients with ENKL. Immunohistochemistry indicated that CD27-positive tumor-infiltrating immune cells exist adjacent to CD70-positive lymphoma cells. In addition, serum sCD27 levels in patients with CD70-positive ENKL were significantly higher than those in patients with CD70-negative ENKL, suggesting that the intra-tumoral CD27/CD70 interaction boosts the release of sCD27 in serum. Furthermore, the EBV-encoded oncoprotein latent membrane protein 1 upregulated CD70 expression in ENKL cells. Our results suggest that sCD27 may serve as a novel diagnostic biomarker and also may serve as a tool for evaluating the applicability of CD27/CD70-targeted therapies by predicting intra-tumoral CD70 expression and CD27/CD70 interaction in ENKL.

Manipulation of diacylglycerol and ERK-mediated signaling differentially controls CD8+ T cell responses during chronic viral infection.

Introduction: Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen exposure is less understood. Here, we investigated the role of diacylglycerol (DAG)-mediated signaling downstream of the TCR during chronic lymphocytic choriomeningitis virus clone 13 (LCMV CL13) infection by blocking DAG kinase zeta (DGKζ), a negative regulator of DAG. Methods: We examined the activation, survival, expansion, and phenotype of virus-specific T cell in the acute and chronic phases of LCMV CL13-infected in mice after DGKζ blockade or selective activation of ERK. Results: Upon LCMV CL13 infection, DGKζ deficiency promoted early short-lived effector cell (SLEC) differentiation of LCMV-specific CD8+ T cells, but this was followed by abrupt cell death. Short-term inhibition of DGKζ with ASP1570, a DGKζ-selective pharmacological inhibitor, augmented CD8+ T cell activation without causing cell death, which reduced virus titers both in the acute and chronic phases of LCMV CL13 infection. Unexpectedly, the selective enhancement of ERK, one key signaling pathway downstream of DAG, lowered viral titers and promoted expansion, survival, and a memory phenotype of LCMV-specific CD8+ T cells in the acute phase with fewer exhausted T cells in the chronic phase. The difference seen between DGKζ deficiency and selective ERK enhancement could be potentially explained by the activation of the AKT/mTOR pathway by DGKζ deficiency, since the mTOR inhibitor rapamycin rescued the abrupt cell death seen in virus-specific DGKζ KO CD8+ T cells. Discussion: Thus, while ERK is downstream of DAG signaling, the two pathways lead to distinct outcomes in the context of chronic CD8+ T cell activation, whereby DAG promotes SLEC differentiation and ERK promotes a memory phenotype.

CD47 blockade enhances the efficacy of intratumoral STING-targeting therapy by activating phagocytes.

Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.

A stealth antigen SPESP1, which is epigenetically silenced in tumors, is a suitable target for cancer immunotherapy.

Recent studies have revealed that tumor cells decrease their immunogenicity by epigenetically repressing the expression of highly immunogenic antigens to survive in immunocompetent hosts. We hypothesized that these epigenetically hidden "stealth" antigens should be favorable targets for cancer immunotherapy due to their high immunogenicity. To identify these stealth antigens, we treated human lung cell line A549 with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza) and its prodrug guadecitabine for 3 d in vitro and screened it using cDNA microarray analysis. We found that the gene encoding sperm equatorial segment protein 1 (SPESP1) was re-expressed in cell lines including solid tumors and leukemias treated with 5Aza, although SPESP1 was not detected in untreated tumor cell lines. Using normal human tissue cDNA panels, we demonstrated that SPESP1 was not detected in normal human tissue except for testis and placenta. Moreover, we found using immunohistochemistry SPESP1 re-expression in xenografts in BALB/c-nu/nu mice that received 5Aza treatment. To assess the antigenicity of SPESP1, we stimulated human CD4+ T-cells with a SPESP1-derived peptide designed using a computer algorithm. After repetitive stimulation, SPESP1-specific helper T-cells were obtained; these cells produced interferon-γ against HLA-matched tumor cell lines treated with 5Aza. We also detected SPESP1 expression in freshly collected tumor cells derived from patients with acute myeloid leukemia or lung cancer. In conclusion, SPESP1 can be classified as a stealth antigen, a molecule encoded by a gene that is epigenetically silenced in tumor cells but serves as a highly immunogenic antigen suitable for cancer immunotherapy.

A critical role of STING-triggered tumor-migrating neutrophils for anti-tumor effect of intratumoral cGAMP treatment.

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-ß1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

Phosphorylated vimentin as an immunotherapeutic target against metastatic colorectal cancer.

Colorectal cancer (CRC) patients with metastatic lesions have low 5-year survival rates. During metastasis, cancer cells often obtain unique characteristics such as epithelial-mesenchymal transition (EMT). Vimentin a biomarker contributes to EMT by changing cell shape and motility. Since abnormal phosphorylation is a hallmark of malignancy, targeting phosphorylated vimentin is a feasible approach for the treatment of metastatic tumors while sparing non-tumor cells. Recent evidence has revealed that both CD8 cytotoxic T lymphocytes (CTLs) and also CD4 helper T lymphocytes (HTLs) can distinguish post-translationally modified antigens from normal antigens. Here, we showed that the expression of phosphorylated vimentin was upregulated in metastatic sites of CRC. We also showed that a chemotherapeutic reagent augmented the expression of phosphorylated vimentin. The novel phosphorylated helper peptide epitopes from vimentin could elicit a sufficient T cell response. Notably, precursor lymphocytes that specifically reacted to these phosphorylated vimentin-derived peptides were detected in CRC patients. These results suggest that immunotherapy targeting phosphorylated vimentin could be promising for metastatic CRC patients.

Expression of placenta-specific 1 and its potential for eliciting anti-tumor helper T-cell responses in head and neck squamous cell carcinoma.

Placenta-specific 1 (PLAC1) is expressed primarily in placental trophoblasts but not in normal tissues and is a targetable candidate for cancer immunotherapy because it is a cancer testis antigen known to be up-regulated in various tumors. Although peptide epitopes capable of stimulating CD8 T cells have been previously described, there have been no reports of PLAC1 CD4 helper T lymphocyte (HTL) epitopes and the expression of this antigen in head and neck squamous cell carcinoma (HNSCC). Here, we show that PLAC1 is highly expressed in 74.5% of oropharyngeal and 51.9% of oral cavity tumors from HNSCC patients and in several HNSCC established cell lines. We also identified an HTL peptide epitope (PLAC131-50) capable of eliciting effective antigen-specific and tumor-reactive T cell responses. Notably, this peptide behaves as a promiscuous epitope capable of stimulating T cells in the context of more than one human leukocyte antigen (HLA)-DR allele and induces PLAC1-specific CD4 T cells that kill PLAC1-positive HNSCC cell lines in an HLA-DR-restricted manner. Furthermore, T-cells reactive to PLAC131-50 peptide were detected in the peripheral blood of HNSCC patients. These findings suggest that PLAC1 represents a potential target antigen for HTL based immunotherapy in HNSCC.

Intratumoral STING activations overcome negative impact of cisplatin on antitumor immunity by inflaming tumor microenvironment in squamous cell carcinoma.

Although cisplatin (CDDP) has been used as a major chemotherapeutic drug for head and neck squamous cell carcinoma (HNSCC), its impact on T-cell functions is controversial. Therefore, we investigated the immunologic effects of CDDP and antitumor effects by combination therapy of CDDP with a ligand for stimulator of interferon genes, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Direct impacts of CDDP on T-cell functions were addressed by comparing T-cell functions between human subjects treated and untreated with CDDP. The immune responses and the efficacy of combination therapy using CDDP and cGAMP were assessed using BALB/c mice inoculated with mouse squamous cell carcinoma (SCC) cell lines. CDDP inhibited T-cell proliferation in a dose-dependent manner. T-cell functions of CDDP-treated HNSCC patients were comparable to those of healthy donors and CDDP-untreated HNSCC patients. In the mice bearing SCC cell lines, combination therapy using CDDP and cGAMP enhanced the gene expressions of CXCL9 and CXCL10 in the tumor tissues and inhibited tumor growth. The antitumor effect was cancelled by anti-CXCR3 monoclonal antibody. These findings suggest that the combination therapy using CDDP and an immunomodulating drug like cGAMP would be a rational cancer immunotherapy for patients with HNSCC.

Lung Adenocarcinoma with Cheek Dysesthesia as an Initial Symptom: A Case Report and Literature Review.

Metastasis from lung carcinoma to the sphenoid bone is rare. Patients with symptoms related to sphenoid bone metastasis as the initial presentation of carcinoma are thus also rare. Herein, we report the case of a patient presenting with only cheek dysesthesia as the first sign of lung adenocarcinoma. The 74-year-old woman presented with a 2-month history of left cheek dysesthesia. CT showed a tumor around 2.5 cm in diameter with heterogeneous enhancement of the central focus at the left foramen rotundum in the sphenoid bone. We endoscopically biopsied the tumor through the left sphenoid sinus. Results of histologic examination were consistent with lung adenocarcinoma. FDG-PET/CT analysis demonstrated lung carcinoma that had already metastasized to mediastinal lymph nodes and multiple bones, such as the ribs and lumbar vertebras, in addition to the sphenoid bone. As EGFR gene mutation (p.L858R) was identified, the patient was treated with oral gefinitib. This treatment proved quite effective, and the patient remains alive without tumor growth as of 18 months.

PD-L1-specific helper T-cells exhibit effective antitumor responses: new strategy of cancer immunotherapy targeting PD-L1 in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) originates from squamous epithelium of the upper aerodigestive tract and is the most common malignancy in the head and neck region. Among HNSCCs, oropharynx squamous cell carcinoma (OSCC) has a unique profile and is associated with human papillomavirus infection. Recently, anti-programmed cell death-1 monoclonal antibody has yielded good clinical responses in recurrent and/or metastatic HNSCC patients. Therefore, programmed death-ligand 1 (PD-L1) may be a favorable target molecule for cancer immunotherapy. Although PD-L1-expressing malignant cells could be targeted by PD-L1-specific CD8+ cytotoxic T lymphocytes, it remains unclear whether CD4+ helper T lymphocytes (HTLs) recognize and kill tumor cells in a PD-L1-specific manner. METHODS: The expression levels of PD-L1 and HLA-DR were evaluated using immunohistochemical analyses. MHC class II-binding peptides for PD-L1 were designed based on computer algorithm analyses and added into in vitro culture of HTLs with antigen-presenting cells to evaluate their stimulatory activity. RESULTS: We found that seven of 24 cases of OSCC showed positive for both PD-L1 and HLA-DR and that PD-L1241-265 peptide efficiently activates HTLs, which showed not only cytokine production but also cytotoxicity against tumor cells in a PD-L1-dependent manner. Also, an adoptive transfer of the PD-L1-specific HTLs significantly inhibited growth of PD-L1-expressing human tumor cell lines in an immunodeficient mouse model. Importantly, T cell responses specific for the PD-L1241-265 peptide were detected in the HNSCC patients. CONCLUSIONS: The cancer immunotherapy targeting PD-L1 as a helper T-cell antigen would be a rational strategy for HNSCC patients.

Cyclin-dependent kinase 1 and survivin as potential therapeutic targets against nasal natural killer/T-cell lymphoma.

Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression of lesions in the affected organs. Recent data have shown that NNKTL is associated with the aberrant expression of cyclin-dependent kinase 1 (CDK1) and its downstream target survivin, but little is known about the functional roles of CDK1 and survivin in NNKTL. In the current study, we show that knockdown of the EBV-encoded oncoprotein latent membrane protein 1 (LMP1) induces downregulation of CDK1 and survivin in NNKTL cells. Immunohistochemistry detected CDK1 and survivin expression in LMP1-positive cells of NNKTL biopsy specimens. Inhibition of CDK1 and survivin in NNKTL cells with several inhibitors led to a dose-dependent decrease in cell proliferation. In addition, the Sp1 inhibitor mithramycin, which can downregulate both CDK1 and survivin, significantly suppressed the growth of established NNKTL in a murine xenograft model. Our results suggest that LMP1 upregulation of CDK1 and survivin may be essential for NNKTL progression. Furthermore, targeting CDK1 and survivin with Sp1 inhibitors such as mithramycin may be an effective approach to treat NNKTL, which is considered to be a treatment-refractory lymphoma.

Targeting phosphorylated p53 to elicit tumor-reactive T helper responses against head and neck squamous cell carcinoma.

The human T cell receptor is capable of distinguishing between normal and post-translationally modified peptides. Because aberrant phosphorylation of cellular proteins is a hallmark of malignant transformation, the expression of the phosphorylated epitope could be an ideal antigen to combat cancer without damaging normal tissues. p53 activates transcription factors to suppress tumors by upregulating growth arrest and apoptosis-related genes. In response to DNA damage, p53 is phosphorylated at multiple sites including Ser33 and Ser37. Here, we identified phosphorylated peptide epitopes from p53 that could elicit effective T helper responses. These epitope peptides, p5322-41/Phospho-S33 and p5322-41/Phospho-S37, induced T helper responses against tumor cells expressing the phosphorylated p53 protein. Moreover, chemotherapeutic agents augmented the responses of such CD4 T cells via upregulation of phosphorylated p53. The upregulation of phosphorylated p53 expression by chemotherapy was confirmed in in vitro and xenograft models. We evaluated phosphorylated p53 expression in the clinical samples of oropharyngeal squamous cell carcinoma and revealed that 13/24 cases (54%) were positive for phosphorylated p53. Importantly, the lymphocytes specific for the phosphorylated p53 peptide epitopes were observed in the head and neck squamous cell cancer (HNSCC) patients. These results reveal that a combination of phosphorylated p53 peptides and chemotherapy could be a novel immunologic approach to treat HNSCC patients.

ANCA-Negative Granulomatosis with Polyangiitis Presenting with Hypertrophic Cranial Pachymeningitis, Abducens Nerve Palsy, and Stenosis of the Internal Carotid Artery.

We report a rare case of granulomatosis with polyangiitis (GPA) presenting with hypertrophic cranial pachymeningitis (HCP), abducens nerve palsy, and stenosis of the internal carotid artery (ICA). A 59-year-old Japanese man presented with a year history of nasal obstruction and a 2-month history of slight headache. Histopathological examination of the granulomatous mucosa in the ethmoid sinuses resected by endoscopic sinus surgery revealed necrotizing vasculitis with multinucleated giant cells. The patient was diagnosed with the limited form of GPA as a result of the systemic examination. He declined immunosuppressive treatment. Eighteen months after the diagnosis of GPA, he presented with diplopia and severe headache. Though nasal findings indicating GPA were not observed in the nasal cavity, CT scan revealed a lesion of the right sphenoid sinus eroding the bone of the clivus. Gadolinium-enhanced MRI of the brain showed thickening of the dura mater around the right cavernous sinus and clivus. Magnetic resonance angiography and cerebral angiography revealed narrowing at the C5 portion of the ICA. Intravenous methylprednisolone pulse therapy followed by oral prednisolone and cyclophosphamide resolved headache and dramatically improved HCP and stenosis of the ICA.