Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

CD1d expression in glioblastoma is a promising target for NKT cell-based cancer immunotherapy.

Glioblastoma is the most common and aggressive type of brain tumor with high recurrence and fatality rates. Although various therapeutic strategies have been explored, there is currently no effective treatment for glioblastoma. Recently, the number of immunotherapeutic strategies has been tested for malignant brain tumors. Invariant natural killer T (iNKT) cells play an important role in anti-tumor immunity. To address if iNKT cells can target glioblastoma to exert anti-tumor activity, we assessed the expression of CD1d, an antigen-presenting molecule for iNKT cells, on glioblastoma cells. Glioblastoma cells from 10 of 15 patients expressed CD1d, and CD1d-positive glioblastoma cells pulsed with glycolipid ligand induced iNKT cell-mediated cytotoxicity in vitro. Although CD1d expression was low on glioblastoma stem-like cells, retinoic acid, which is the most common differentiating agent, upregulated CD1d expression in these cells and induced iNKT cell-mediated cytotoxicity. Moreover, intracranial administration of human iNKT cells induced tumor regression of CD1d-positive glioblastoma in orthotopic xenografts in NOD/Shi-scid IL-2RγKO (NOG) mice. Thus, CD1d expression represents a novel target for NKT cell-based immunotherapy for glioblastoma patients.

Immunological features of a lung cancer patient achieving an objective response with anti-programmed death-1 blockade therapy.

The role of immune checkpoint inhibitors in metastatic lung cancer has been established in recent years and the pretherapeutic profiles of the tumor microenvironment in responders have been increasingly reported. The role of salvage surgery and the immune profiles of the posttherapeutic specimens in patients achieving an objective response have rarely been studied. We report a case of metastatic lung cancer treated by anti-programmed death-1 Ab followed by surgical resection. The immune status of the tumor was assessed, showing germinal center formation, memory B cell infiltration, and a high frequency of interferon gamma -secreting T cells.

Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation.

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line-derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL-12 and tumor necrosis factor (TNF)-α production from mature DC, while no effect of SH-SY5Y cell supernatant was noted. In addition, IL-6 and IL-10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α-Galactosylceramide-pulsed DC co-cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)-γ after stimulation with neuroblastoma cell line supernatant-cultured DC was reversed by addition of IL-12. CD40 expression and IL-12 production in NLF-sup-treated DC were increased by addition of exogenous IFN-γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer-pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN-γ production.

Regulatory T cells induce CD4- NKT cell anergy and suppress NKT cell cytotoxic function.

BACKGROUND: Due to the strong tumoricidal activities of activated natural killer T (NKT) cells, invariant NKT cell-based immunotherapy has shown promising clinical efficacy. However, suppressive factors, such as regulatory T cells (Tregs), may be obstacles in the use of NKT cell-based cancer immunotherapy for advanced cancer patients. Here, we investigated the suppressive effects of Tregs on NKT cells and the underlying mechanisms with the aim to improve the antitumor activities of NKT cells. METHODS: Peripheral blood samples were obtained from healthy donors, patients with benign tumors, and patients with head and neck squamous cell carcinoma (HNSCC). NKT cells, induced with α-galactosylceramide (α-GalCer), and monocyte-derived dendritic cells (DCs) were co-cultured with naïve CD4+ T cell-derived Tregs to investigate the mechanism of the Treg suppressive effect on NKT cell cytotoxic function. The functions and phenotypes of NKT cells were evaluated with flow cytometry and cytometric bead array. RESULTS: Treg suppression on NKT cell function required cell-to-cell contact and was mediated via impaired DC maturation. NKT cells cultured under Treg-enriched conditions showed a decrease in CD4- NKT cell frequency, which exert strong tumoricidal responsiveness upon α-GalCer stimulation. The same results were observed in HNSCC patients with significantly increased effector Tregs. CONCLUSION: Tregs exert suppressive effects on NKT cell tumoricidal function by inducing more CD4- NKT cell anergy and less CD4+ NKT cell anergy. Both Treg depletion and NKT cell recovery from the anergy state may be important for improving the clinical efficacy of NKT cell-based immunotherapy in patients with advanced cancers.

Sublingual administration of liposomes enclosing alpha-galactosylceramide as an effective adjuvant of allergen immunotherapy in a murine model of allergic rhinitis.

BACKGROUND: Sublingual immunotherapy (SLIT) is an established efficacious approach for the treatment of allergic rhinitis (AR). However, SLIT requires a long administration period to establish stable and adequate responses. This study investigated the efficacy of the sublingual administration of an allergen with liposomes enclosing α-GalCer (α-GC-liposome) as a potential adjuvant in mice with AR. METHODS: Mice with AR induced by OVA received the sublingual administration of OVA, α-GC-liposomes, or OVA plus α-GC-liposomes for 7 days. After nasal re-challenge with OVA, nasal symptoms were evaluated. The serum levels of OVA-specific Ig, the cytokine production of CD4+ T cells in the cultures of cervical lymph node (CLN) cells, and the gene expression of CLNs were analyzed. RESULTS: Although IL-4, IL-5 and IL-13 production from CD4+ T cells in CLN cells was significantly inhibited by the sublingual administration of OVA alone in mice with AR induced by OVA, their nasal symptoms were not significantly diminished. However, the combined sublingual administration of α-GC-liposomes and OVA completely suppressed nasal symptoms, downregulated Th2 and Th17 type cytokine production in CD4+ T cells as well as Th2 and Th17 gene expressions, and upregulated Th1 type cytokine production as well as Th1 gene expressions in CLN cells. Additionally, the serum levels of specific IgG2a were promoted, and specific IgE and IgG1 were inhibited. CONCLUSIONS: Our findings suggest that the sublingual administration of an allergen with α-GC-liposomes as an adjuvant might increase the therapeutic efficacy and effectiveness of this treatment method.

Activated iNKT cells enhance the anti-tumor effect of antigen specific CD8 T cells on mesothelin-expressing salivary gland cancer.

BACKGROUND: Salivary gland cancers are not sensitive to conventional radiotherapy or chemotherapy regimens. Therefore, the development of a new treatment strategy is of critical importance for improving the prognosis. We examined the expression of mesothelin molecules in salivary gland cancers and the efficacy of adoptive cell therapy based on mesothelin-specific chimeric antigen receptor transduced T cells. METHODS: The expression of mesothelin molecule was studied in salivary gland cancer samples obtained from 16 patients as well as a salivary gland cancer cell line (A-253) and five other cell lines. The activation of mesothelin-specific chimeric antigen receptor-expressing CD8 T cells after stimulation with mesothelin and the effects of invariant natural killer T cells on this activation were evaluated. RESULTS: Mesothelin was detected in the A-253 cells and the surgical specimens except for the case of squamous cell carcinoma to various degrees. Following stimulation with mesothelin expressing cancer cells, chimeric antigen receptor T cells were dose-dependently activated; this activation was enhanced by co-culture with invariant natural killer T cells and subsequently abrogated by treatment with anti-interferon-γ antibodies. Furthermore, the cytotoxicity of chimeric antigen receptor T cells against various cancer cells was further augmented by invariant natural killer T cells. CONCLUSIONS: The use of adoptive transfer with mesothelin-specific chimeric antigen receptor-expressing CD8 T cells against salivary gland cancers is an effective therapy and invariant natural killer T cells are expected to be used in adjuvant treatment for T cell-based immunotherapy.

Frequency and proliferative response of circulating invariant natural killer T cells in pediatric patients with malignant solid tumors.

BACKGROUND: Invariant natural killer T (iNKT) cells play an important role in tumor immunity, enhancing both innate and acquired immunity. We have previously shown the enhancement of antibody-dependent cellular cytotoxicity against neuroblastoma by activated iNKT cells. As a first step towards clinical application, we studied the frequency and proliferative response of circulating iNKT cells in children with and without cancer. METHODS: Blood samples were collected from 10 patients with pediatric malignant solid tumors and 11 patients with non-neoplastic diseases (control). The frequency of circulating iNKT cells was quantified by flow cytometry. Whole peripheral blood mononuclear cells were then stimulated with α-galactosylceramide (α-GalCer) for 7 days, and the expansion rate of the iNKT-cell fraction was assessed. RESULTS: The frequency of iNKT cells in the patients of the cancer and control group did not differ to a statistically significant extent. The iNKT-cell population increased after α-GalCer stimulation in all cases. The iNKT cells of patients who had undergone intensive chemotherapy also had the potential to expand in vitro. CONCLUSIONS: Unlike adult cancer patients, the numbers of circulating iNKT cells were not decreased in pediatric cancer patients. α-GalCer stimulation induced a proliferative response in all of the patients.

Invariant natural killer T infiltration in neuroblastoma with favorable outcome.

BACKGROUND: Tumor immunity has been suggested to play a key role in clinical and biological behavior of neuroblastomas. Given that CD1-restricted invariant natural killer T (iNKT) cells enhance both innate and acquired tumor immunity, we investigated the expression of the iNKT-cell-specific T-cell receptor Vα24-Jα18 in neuroblastoma tissues and its correlation with clinical and biological characteristics. METHODS: Using real- time quantitative PCR, we quantified the expression of Vα24-Jα18 in untreated tumor samples from 107 neuroblastoma cases followed in our institution and analyzed the correlation between the presence of infiltrated iNKT cells and clinical characteristics or patients' outcome. RESULTS: Vα24-Jα18 receptor was detected in 62 untreated cases (57.9%). The expression was significantly higher in stages 1, 2, 3, or 4S (P = 0.0099), in tumors with low or intermediate risk (P = 0.0050), with high TrkA expression (P = 0.0229), with favorable histology (P = 0.0026), with aneuploidy (P = 0.0348), and in younger patients (P = 0.0036). The overall survival rate was significantly higher in patients with iNKT-cell infiltration (log-rank; P = 0.0089). CONCLUSIONS: Since tumor-infiltrating iNKT cells were predominantly observed in neuroblastomas undergoing spontaneous differentiation and/or regression, we suggest that iNKT cells might play a key role in these processes.

CD45RA-Foxp3high regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma.

BACKGROUND: Although regulatory T cells (Tregs) are thought to play an important role in immune suppression, their clinical significance in head and neck squamous cell carcinoma (HNSCC) is unclear. A recent study reported Tregs could be divided into functional subsets based on the expression of CD45RA and Foxp3. METHOD: The frequency of circulating Treg subsets was analyzed in patients with HNSCC and compared with the frequency in patients with benign tumors. The association of Treg subsets with the frequency of lymphocyte subsets, status of progression, clinical course, and prognosis were also examined. RESULTS: The frequency of CD4+Foxp3+ Tregs was comparable between HNSCC patients and age-matched benign tumor patients; however, CD45RA-Foxp3high Tregs were significantly increased in HNSCC patients, in particular those with advanced stage tumors. The high frequency of CD45RA-Foxp3high Tregs correlated with a poor prognosis and the low frequency of CD45RA-Foxp3high Tregs before treatment showed a better clinical outcome, even in patients with advanced stage tumors. CD45RA-Foxp3high Treg numbers were decreased after intensive treatments; however, Treg numbers recovered in the early stages of recurrent cases, even before the clinical manifestation. CONCLUSION: CD45RA-Foxp3high Tregs are associated with the clinical course of HNSCC and might be a new target for treatment and an early marker of tumor recurrence in HNSCC patients.

Invariant NKT cell-based immunotherapy for lung cancer and head and neck cancer.

CD1d-restricted invariant natural killer T (iNKI) cells are the unique lymphocyte subpopu- lation that interacts with glycolipid via the invariant T cell receptor. Ligand activated iNKT cells produce a variety of cytokines, and thus bridging the innate and adaptive immune sys- tems. Since recent studies have highlighted iNKT cells to have potent anti-tumor effects, greater attention are being given to the development of iNKT cell-based immunotherapy for advanced solid tumors. This article summarizes the progress of our recent works on active immunotherapies aiming at the augmentation of iNKT cell function in vivo in patients with non-small cell lung cancer, and discusses the role of iNKT cell-induced immune responses in cancer immunotherapy.

Invariant NKT cells are resistant to circulating CD15+ myeloid-derived suppressor cells in patients with head and neck cancer.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti-tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15+ granulocytic MDSC (G-MDSC) and CD14+ monocytic MDSC (M-MDSC) was demonstrated in HNSCC patients. The percentage of G-MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G-MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15+ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α-galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15+ G-MDSC. These results indicate that increased G-MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G-MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G-MDSC.

Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells.

The role of invariant natural killer T (iNKT) cells in antitumor immunity has been studied extensively, and clinical trials in patients with advanced cancer have revealed a prolonged survival in some cases. In recent years, humanized blocking antibodies against co-stimulatory molecules such as PD-1 have been developed. The enhancement of T cell function is reported to improve antitumor immunity, leading to positive clinical effects. However, there are limited data on the role of PD-1/programmed death ligand (PDL) molecules in human iNKT cells. In this study, we investigated the interaction between PD-1 on iNKT cells and PDL on antigen-presenting cells (APCs) in the context of iNKT cell stimulation. The blockade of PDL1 at the time of stimulation resulted in increased release of helper T cell (Th) 1 cytokines from iNKT cells, leading to the activation of NK cells. The direct antitumor function of iNKT cells was also enhanced after stimulation with anti-PDL1 antibody-treated APCs. According to these results, we conclude that the co-administration of anti-PDL1 antibody and alpha-galactosylceramide (αGalCer)-pulsed APCs enhances iNKT cell-mediated antitumor immunity.

[A Clinical Study of a Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma of Salivary Glands].

Malignant lymphoma involving the salivary glands is a rare entity that accounts for only 1 to 4% of all salivary gland tumors. We have analyzed the clinical features of MALT lymphoma of the salivary glands in 7 patients whom we have treated in the past 10 years, including data from 43 patients in the literature. The most common symptom was persistent or progressive swelling of the salivary glands without pain or facial palsy. The affected glands were the unilateral parotid in 62%, the unilateral submandibular gland in 6.8%, and the bilateral parotid or submandibular gland in 6.8%. The salivary gland swelling as evaluated by MRI or ultrasonography consisted of a localized solitary tumor in 68.9%, multiple tumors in 10.3%, and diffuse swelling of the gland in 26.1%. In 24 out of 50 patients, MALT lymphoma developed on the basis of Sjögren's syndrome. As for diagnosis, malignant lymphoma was suspected only in 4 cases out of 11 (36.7%) based on the fine needle aspiration cytology. In all patients, a definitive diagnosis was obtained based on histopathological study of the localized tumor or the biopsy specimens. When patients with Sjögren's syndrome have developed persistent swelling of the salivary glands, it is necessary to rule out malignant lymphomas, especially MALT lymphoma, by combination of ultrasonography, MRI, FNA, and biopsy or open surgery depending on image findings. The treatment of MALT lymphoma has not yet been standardized because MALT lymphoma is a rare entity. The choice of initial treatment is important because it is related to its prognosis. If MALT lymphoma remains in a local site, we should undertake an operation or radiotherapy. If MALT lymphoma has dissemineated systemically, we should choose chemotherapy.

[Experience of two cases of tetanus without a clear history of trauma].

Tetanus develops following inoculation of damaged human tissue with Clostridium tetani which transforms into a vegetative rod-shaped bacterium and produces the tetanospasmin. Usually we make a diagnosis of tetanus based on typical symptoms and history of trauma. But, when patients have no noticeable history of trauma, we have to diagnose tetanus on the basis of the clinical course and symptoms. We report herein on two cases of tetanus without a clear history of trauma. The first patient visited us with the chief complaints of pain in the neck and shoulder, and difficulty in opening the mouth. Based on these symptoms, we diagnosed tetani in the first stage and we started treatment, consisting mainly of human anti-tetanus immunoglobulin on the first day. The second patient also had typical symptoms of tetanus. However, he rejected the use of human anti-tetanus immunoglobulin on the first day. Because his symptoms worsened on the 2nd day, we insisted that he used it. On the 5th hospital day he developed partial opisthotonus of neck. However he recovered without tracheotomy or intratracheal intubation.

[The efficacy of superselective intra-arterial chemo-radiotherapy for cervical neck lymph node metastases].

We report herein on 32 cases of head and neck carcinoma with cervical lymph node metastases treated by radiotherapy and concomitant intraarterial cisplatin (RADPLAT) from April 2009 to May 2013. N3 cases revealed residual disease of the cervical lymph nodes in 7/9 cases. Among the 22 patients excluding N1 and N3 cases, the pathological CR rate was 63.6%. Among the 13 patients in whom the anticancer drug was directly infused into the cervical lymph nodes, the pathological CR rate was 76.9%, whereas in the 9 patients without direct infusion of the cervical lymph nodes, the pathological CR rate was 44.4%. Therefore, we recommend the direct infusion into cervical lymph node metastases for not only N3 cases but also N2 cases if a feeding artery is identified easily. When clinical examination after RADPLAT leads to suspected residual disease, neck dissection should be adapted. If the clinical examination leads to a diagnosis of CR, we recommend a biopsy of the original cervical lymph nodes because the cases which we diagnosed as CR revealed residual disease of the cervical lymph nodes in 4/16.

Histopathological study of lymph node lesions in the acute phase of Kawasaki disease.

AIMS: To review the histopathological features of cervical LNs, and to clarify the changes in extracervical LNs, in acute Kawasaki disease (KD). METHODS AND RESULTS: The samples were obtained from 33 patients with acute-phase KD. We divided the LNs into those in the neck (n = 23) and those located elsewhere (n = 26), and investigated them histologically. Changes occur not only in the cervical region, but also in LNs throughout the body. Most lymphadenopathy is non-specific, caused by sinus expansion and paracortical zone enlargement, but there are also necrotic lesions of various sizes that can be surmised to result from ischaemic changes in some LNs. Necrotic foci start to develop immediately below the capsule, and are accompanied by fibrin thrombi in the small vessels and perivascular nuclear debris. Especially in the case of cervical LNs with necrosis, a high degree of non-purulent inflammation develops in the LN capsule and surrounding connective tissue. CONCLUSIONS: In addition to lymphadenopathy with necrosis, KD should be suspected if there is non-purulent inflammation of the LN capsule and/or surrounding connective tissue featuring mainly monocytes/macrophages.

[A clinical study on 8 cases of sarcoidosis with a cervical mass as a symptom].

Sarcoidosis is a multiorgan granulomatous disease of unknown origin, which frequently involves the lung and the eyes. It is rare that sarcoidosis causes cervical lymphadenopathy and a chronic continuous salivary gland swelling. In the present study, we examined how to diagnose sarcoidosis in patients complaining of a cervical mass and its clinical presentation according to 8 cases which we experienced. We undertook biopsy of the cervical lymph nodes in 7 cases, and biopsy of the parotid gland in 1 case. In cases non-caseating epithelioid cell granuloma was proved, diagnosed as sarcoidosis histopathlogically. In 6 patients, the sarcoidal lesion was in other organs, whereas in 2 patients the sarcoidal lesion was confined in the head and neck. Bilateral hilar lymphadenopathy and an elevated level of angiotensin-converting enzyme which are typical in sarcoidosis were confirmed in only one of our patients. Therefore, the diagnostic value of these tests is low for the diagnosis of sarcoidosis in the patients whose chief complaint is a cervical mass. Because 4 out of 7 cases were positive for the tuberculin test, it is imperative to distinguish between sarcoidosis and cervical tuberculous lymphadenitis by combining the culture of acid-bacilli and TB-PCR with histopathological evaluation of the biopsy specimen. We experienced one patient in whom the sarcoidal lesion developed in the skin and the eyes 20 years after the onset of cervical lymphadenopathy. It is important to follow the patients carefully, even if we diagnose the disease as sarcoidosis confined in the head and neck.

Primary cardiac synovial sarcoma: a case report and literature review.

Primary cardiac synovial sarcoma is a rare disease. A 51-year-old man visited our hospital with the chief complaint of palpitations and shortness of breath while exercising. Copious bloody pericardial effusion and a multicystic intrapericardial tumor were detected. A primary cardiac malignant tumor was suspected, an open-chest tumor resection was performed with the objectives of diagnosis and treatment. Histologically, the tumor cells were uniformly spindle-shaped with an ovoid or oval nucleus, they had proliferated in fascicular fashion. In addition myxoid degeneration, a hemangiopericytomatous vascular pattern and pseudorosette formation were seen in some areas of the tumor. Based on the histopathological and immunohistochemical findings and reverse transcription polymerase chain reaction detection of SS18-SSX1 fusion transcripts, a monophasic fibrous type synovial sarcoma was diagnosed. Postoperative radiation therapy was administered and there had been no recurrence 9 months after the surgery.