The significance of T-BET-positive CD8 T-cells with diminished CD5 expression in Kikuchi-Fujimoto disease.

Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare condition characterized by benign localized lymphadenopathy and clinical symptoms such as fever, sore throat, odynophagia, and leukopenia. Though the etiology of KFD is unknown, this condition is similar to viral infection, including increased infiltration of activated plasmacytoid dendritic cells. KFD exhibits three histological phases that reflect its progression status: proliferative, necrotic, and xanthomatous lesions. The expression loss of pan T-cell markers, such as CD2, CD5, and CD7, of infiltrating T-cells is observed in KFD cases, complicating the distinction from T-cell lymphoma. However, reports on the loss of their expression in KFD have been limited. Furthermore, the precise population of the T-cell subset in KFD is still unclear. Here, we focused on surface markers and transcription factors for T-cell differentiation and analyzed them immunohistochemically in 46 KFD cases. We observed diminished CD5 expression of CD8-positive (CD5dim CD8+) T-cells in the proliferative lesion of KFD cases. Furthermore, these CD5dim CD8+ T-cells expressed T-BET, a master regulator of type 1 helper T-cells. The upregulation of T-BET and downregulation of CD5 in CD8+ T-cells causes dysregulated activation and proliferation of CD8+ T-cells, potentially contributing to the unique histopathological features of KFD. Recognizing the frequent infiltration of T-BET-positive CD5dim CD8+ T-cells in KFD is important for distinguishing it from mature T-cell lymphoma. Our findings suggest that the immune response in KFD shares similarities with viral infections and highlight the importance of characterizing T-BET-positive CD5dim CD8+ T-cell populations for understanding KFD pathogenesis.

A Rare Case of Leukemoid Reaction During Mechanical Circulatory Support in a Patient With Severe Heart Failure: An Autopsy Study.

The leukemoid reaction (LR) is reported to be caused by severe stress conditions such as infection, malignancies, intoxication, severe hemorrhage, or acute hemolysis; this condition is attributed to a very severe prognosis. Some reports have suggested that the LR was associated with a systemic stress response. A 36-year-old man who required mechanical circulatory support (MCS), including veno-arterial extracorporeal membrane oxygenation and Impella 5.5 due to severe heart failure, was transferred to our hospital. He showed a markedly elevated WBC count and died of multiple organ failure. The autopsy revealed the possibility that leukocytosis might have been due to an LR; however, the cause of the cardiac failure was unknown. To the best of our knowledge, this study is the first to report a rare case of LR in a patient with severe heart failure requiring MCS.

An infantile case of hereditary folate malabsorption with sudden development of pulmonary hemorrhage: a case report.

BACKGROUND: Hereditary folate malabsorption-a rare disorder caused by impairment of the folate transporter-can develop into severe folate deficiency manifesting as megaloblastic anemia and occasionally thrombocytopenia. Reportedly, megaloblastic anemia can manifest with hemorrhagic episodes, possibly due to ineffective platelet production and platelet dysfunction. However, life-threatening hemorrhage events in hereditary folate malabsorption have not been well investigated. CASE PRESENTATION: A 3-month-old Japanese boy was transferred to our hospital due to thrombocytopenia and severe megaloblastic anemia. During a thorough examination of hematopoietic abnormalities, the patient suddenly went into cardiac arrest due to pulmonary hemorrhage. Although intravenous folate supplementation was started soon after the identification of folate deficiency, the patient died of circulatory defect and multiple organ failure. The cause of pulmonary hemorrhage, such as respiratory infection, could not be confirmed. Genetic investigation revealed a mutation in the SLC46A1 gene to be the cause of the hereditary folate malabsorption. CONCLUSION: We report an infantile case of hereditary folate malabsorption that progressed to lethal pulmonary hemorrhage before folate deficiency was identified. Clinicians should consider that megaloblastic anemia could lead to severe bleeding without warning, and that nutrient supplementation should be initiated as soon as possible.

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.

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.

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.