Stratification of the immunotypes of tongue squamous cell carcinoma to improve prognosis and the response to immune checkpoint inhibitors

An understanding of the tumor immune microenvironment is required to improve treatment, especially the selection of immune checkpoint inhibitors (ICIs). In this study, we stratified the immunotypes of tongue squamous cell carcinoma (TSCC) based on the results of comprehensive immune profiling. We enrolled 87 therapy-naïve TSCC and 17 ICI-treated TSCC patients who underwent glossectomy without any other prior therapy. Comprehensive immune profile analyses employed multiplex immunofluorescence and tissue imaging. Based on the hierarchies of 58 immune parameters and the spatial distances between cytotoxic T lymphocytes (CTL) and tumor cells, we stratified five immunotypes: Immunoactive type I, border type II, immunosuppressed type III, immunoisolating type IV, and immunodesert type V. The type I frequency was only 16%. Most TSCCs (~ 70%) were of types III-V. The CTL density (CTL-D) was closely correlated with the PD-L1+ pan-macrophages (panM)-D, and the panM-D closely correlated with the PD-1+ CTL-D. This indicated that PD-1 and PD-L1 expression required macrophages and CTL recruitment in the tumor microenvironment. No ICI-treated TSCC patients, all of whom were recurrent/metastatic cases, were of the type I immunotype, and almost half (47.0%) were of the immunodesert type V. Most cases exhibited an imbalance between T-cell PD-1 and macrophage PD-L1 expression. We defined five TSCC-specific immunotypes based on the results of comprehensive immune profiling analyses. Immunoactive type, which would be sensitive to ICI monotherapy, was rare, and most TSCC cases exhibited immune-regulated immunotypes. Immunotype-based personalized treatments are required to improve clinical outcomes.


Automatic detection of spongiosis associated with oral lichenoid lesions by machine learning

Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology


Personal immune profiles: Diversity and prognostic value for oral tongue squamous cell carcinoma evaluated by comprehensive immune parameter analyses with multiplex immunofluorescence

Understanding the tumor immune microenvironment is becoming increasingly necessary for risk prediction and treatment selection. In particular, oral cancer has various immunosuppressive characteristics in the tumor microenvironment. Therefore, we comprehensively assessed the immune profiles of oral tongue squamous cell carcinoma (OTSCC). Multiplex immunofluorescence and tissue imaging analyses were performed to evaluate immune profiles at the invasive tumor front of 60 OTSCC surgical specimens. We analyzed 58 immune parameters including the density and proportion (%) of total leukocytes (Leu) and T cells, six subsets of T and myeloid cells, and the expression of programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1). The density, proportion, and location of CD45+ Leu, three T cell subsets (CD8+, Foxp3-CD4+ conventional, and Foxp3+CD4+ regulatory T cells), CD163-CD68+ M1 and CD163+CD68+ M2 macrophages, and neutrophils were highly variable at the individual level. The density and proportion of M2 macrophages were significantly lower in the T1 stage group. Risk prediction analyses for recurrence and/or metastasis (R/M) showed that R/M (+) T1 cases had significantly higher M2 density and percentages. The immune profiles of OTSCC patients are diverse and cannot be predicted from clinicopathological information alone. The M2 macrophage abundance is a potential candidate biomarker for R/M in the early stage of OTSCC. Personal immune profiling may provide beneficial information for risk prediction and treatment selection.


High expression of Sam68 contributes to metastasis by regulating vimentin expression and a motile phenotype in oral squamous cell carcinoma

The present study aimed to investigate the clinical and biological significance of Src-associated in mitosis 68 kDa (Sam68) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis was performed on tissue samples obtained from 77 patients with OSCC. Univariate analysis revealed that the high expression of Sam68 was significantly correlated with advanced pathological T stage (P=0.01), positive lymphovascular invasion (P=0.01), and pathological cervical lymph node metastasis (P<0.01). Moreover, multivariate analysis demonstrated that the high expression of Sam68 was an independent predictive factor for cervical lymph node metastasis (odds ratio, 4.39; 95% confidence interval, 1.49-14.23; P<0.01). These results indicated that high Sam68 expression contributed to tumor progression, especially cervical lymph node metastasis, in OSCC. mRNA sequencing was also performed to assess the changes in the transcriptome between OSCC cells with Sam68 knockdown and control cells with the aim of elucidating the biological roles of Sam68. Gene Ontology enrichment analysis revealed that downregulated differentially expressed genes (DEGs) were concentrated in some biological processes related to epithelial-mesenchymal transition. Among these DEGs, it was established that vimentin was particularly downregulated in these cells. It was also confirmed that Sam68 knockdown reduced the motility of OSCC cells. Furthermore, the immunohistochemical study of vimentin identified the association between vimentin expression and Sam68 expression as well as cervical lymph node metastasis. In conclusion, the present study suggested that the high expression of Sam68 may contribute to metastasis by regulating vimentin expression and a motile mesenchymal phenotype in OSCC.


Oral lichenoid contact lesions related to dental metal allergy may resolve after allergen removal


Mandibular Reconstruction with Scapular Systems: A Single-Center Case Series Involving 208 Flaps

The scapular flap is the most versatile composite flap used for mandibular reconstruction. The purpose of this study was to review and summarize findings of cases of mandibular reconstruction with a scapular flap and describe associated outcomes and complications. A total of 208 microvascular scapular free flaps were performed for mandibular reconstruction in a total of 205 patients from 2003 to 2018. This study involved a retrospective review of all eligible patients' medical records. There were seven cases (3.4 percent) of microvascular thrombosis. Postoperative bone union was achieved by 201 patients, except for five with total flap necrosis and two with partial flap necrosis. There were four cases (1.9 percent) of mandibular condyle dislocation. Two major types of complications were observed at the donor site, including four cases of infection and six cases of scapular body fracture. Postoperative denture prosthesis was introduced to 97 patients (47.3 percent). Implant treatment was performed in 10 patients (4.9 percent). Functional and aesthetic outcomes were good to excellent. The scapular composite free flap for mandibular reconstruction was associated with favorable outcomes and demonstrated satisfactory results. Although scapular bone fracture is rare, patients who have undergone mandibular reconstruction using a scapular flap should be monitored for its presence. . Therapeutic, IV.


Retrospective analysis of schwannoma in the oral and maxillofacial region: clinicopathological characteristics and specific pathology of ancient change

Schwannomas are benign tumours that originate from the Schwann cells of the nerve. Despite the frequency of their occurrence in the head and neck, data relating to their clinicopathological features in the region are limited. This study reviews the clinicopathological characteristics and specific pathology of ancient (degenerative) change in 40 cases in the oral and maxillofacial region. Medical records were reviewed of the 40 cases treated at Tokyo Medical and Dental University Hospital Faculty of Dentistry between 2000 and 2020. The most frequently involved site was the tongue, and the average tumour size was 13.2 mm. Degenerative changes were observed in eight cases. All tumours were completely excised through biopsy or local excision, and no recurrence was observed on clinical follow up. Statistical analyses revealed significant associations (p < 0.05) between ancient change and tumour size, and between ancient change and the apparent diffusion coefficient (ADC) value derived from diffusion-weighted magnetic resonance imaging (MRI). Analysis suggests that schwannoma progresses over a long period and subsequently undergoes secondary ancient changes. Pathological events such as cystic formation and haemorrhage that are associated with ancient changes, cause hypocellularity and lead to high ADC values. These values may indicate ancient change and should be considered when distinguishing schwannoma from other lesions.

The British Journal of Oral & Maxillofacial Surgery


Malignant transformation of oral lichen planus: a retrospective study of 565 Japanese patients

Oral lichen planus (OLP) is a chronic inflammatory oral mucosa disease that is recognized as an oral potentially malignant disorder. However, the potentially malignant nature of OLP remains unclear. We designed this study to examine the demographic and clinical characteristics of patients with OLP and evaluate the associated malignant transformation rate. A total of 565 patients with a clinical and histopathological diagnosis of OLP who presented at our department between 2001 and 2017 were retrospectively studied. Patients who had clinical and histopathological features of oral lichenoid lesions (OLLs) classified as oral lichenoid contact lesions, oral lichenoid drug reactions and oral lichenoid lesions of graft-versus-host disease were excluded. The study population included 123 men and 442 women aged 21-93 years (mean ± standard deviation, 60.5 ± 11.8). The 565 patients were followed up for a duration of 55.9 ± 45.3 months, during which 4 (0.7%) patients developed squamous cell carcinoma (SCC). In three of these 4 patients who developed SCC, the clinical type of OLP was the red type. Our results suggested that OLP was associated with a low risk of malignant transformation. We recommend regular follow-up for OLP patients and clear differentiation of oral epithelial dysplasia and OLLs to enable early detection of malignant transformation. Further investigation of the clinical risk factors associated with malignant transformation is necessary.


Management of retropharyngeal lymph node metastasis in oral cancer

Retropharyngeal lymph node (RPLN) metastasis is extremely rare, and prognosis is significantly poor in oral cancer. We retrospectively examined the management of RPLN metastases in oral cancer. A total of 1247 patients with oral cancer were treated at our department from January 2002 and December 2016. Among these patients, 374 (30%) had histologically positive lymph node metastases. Of these, 15 patients (1.2%) were diagnosed with RPLN metastases. We evaluated the diagnostic period, size, recurrence pattern, laterality, treatment, and therapeutic outcomes. The Kaplan-Meier method was used to determine overall survival (OS) among the RPLN metastasis group, cervical lymph node (CLN) metastases group, and treatment methods group for RPLN metastases. One patient had RPLN involvement at the initial treatment, and RPLN involvement in other patients was found subsequently. The mean duration in confirming RPLN metastases was 228 days (range, 50-867 days). Surgical therapy was performed in 5 patients, chemoradiotherapy in 7 patients, and best supported care (BSC) in 3 patients. The cumulative 5-year OS rate for the RPLN metastasis group (n = 15) was 38.1%, compared with the rate of 71.3% for the CLN group (n = 359). Regarding the therapeutic approach for RPLN metastases, OS rates were 80.0% (n = 5) in the surgical therapy group, 28.6% (n = 7) in the chemoradiotherapy group, and 0% (n = 3) in the BSC group. Early detection and surgical treatment of RPLN metastases are associated with increased survival rate in oral cancer.


Detection of extraoral primary cancers by positron emission tomography/computed tomography in patients with oral squamous cell carcinoma

Extraoral primary cancers (EPCs) can appear in patients with oral cancer. This study investigated the use of positron emission tomography/computed tomography (PET/CT) to detect EPCs and to identify characteristics of these cancers. The disease-specific survival rate and the overall survival rate were calculated. We studied records for 891 patients with oral cancer who underwent PET/CT between January 2006 and December 2016 for the detection of EPCs. A total of 35 EPCs were detected by PET/CT in 34 patients with primary oral carcinoma (3.8%). Of this total, 33 patients had 1 EPC and 1 patient had 2 EPCs. The extraoral lesions were found in the upper gastrointestinal tract (11), colon (6), lung (5), kidney (3), liver (2), prostate (2), breast (2), bladder (1), thyroid (1), pancreas (1), and hypopharynx (1). The disease-specific survival rate was 84.8% and the overall survival rate was 64.6%. PET/CT was useful for early detection of EPCs. The use of PET/CT for the early detection and treatment of EPCs with no symptoms can improve treatment outcomes.

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology


Clinicopathological Study of 24 Cases of Pemphigoid

類天疱瘡24例に関する臨床病理学的検討

Pemphigoid is an autoimmune bullous disease characterized by a subepithelial blister. We report 24 cases of pemphigoid who visited our department from 2007 through 2017. Twenty-one cases were classified as mucous membrane pemphigoid and 3 cases as bullous pemphigoid. They consisted of 5 males and 19 females, 44 to 91 years of age (median: 65.5 years). Lesions in the oral cavity were most frequently located in the gingiva (19 cases), followed by the buccal mucosa (7 cases) and the palate (6 cases). Eighteen of the 24 cases showed only oral manifestations. Treatment with systemic steroid was given to 12 cases, and topical steroid to 12 cases. In 8 of the 12 cases with topical steroid, we co-administered tetracycline, diaminodiphenyl and/or nicotinamide. The lesions of all patients were improved, and we have continued to treat them with attention to relapse. It is considered that a prompt diagnosis should be made and appropriate treatment for pemphigoid, which often shows only oral manifestations, should be provided in cooperation with dermatologists.

類天疱瘡は，上皮下水疱を呈する自己免疫性水疱症である．2007年から2017年の間に類天疱瘡と診断された24例について臨床病理学的検討を行ったので報告する．内訳は，粘膜類天疱瘡が21例，水疱性類天疱瘡が3例であった．性別は男性5例，女性19例，年齢は44歳から91歳(中央値65.5歳)であった．口腔内での発生部位は歯肉が19例と最も多く，頬粘膜が7例，口蓋が6例であった．口腔内病変のみ認めたのは18例であった．治療法は，ステロイドの全身投与が12例，局所投与が12例であった．局所投与の12例中8例は，ミノサイクリン塩酸塩，ジアフェニルスルフォンおよびニコチン酸アミドが単独または併用で投与されていた．全例の病状は改善し，現在も再燃に注意しながら経過観察を行っている．類天疱瘡は，口腔内のみに病変を認める症例も多く，皮膚科と連携し早期に診断し適切な治療を行う必要があると思われた．

Journal of Japanese Society of Oral Medicine


Quantitation and distribution of metallic elements in sequestra of medication-related osteonecrosis of jaw (MRONJ) using inductively coupled plasma atomic emission spectroscopy and synchrotron radiation X-ray fluorescence analysis

Medication-related osteonecrosis of the jaw (MRONJ) is a serious adverse effect of antiresorptive agents like bisphosphonates. Abnormal concentrations of various trace metallic elements contained in bone minerals have been associated with MRONJ. In this study, we focused on trace metallic elements contained in the MRONJ sequestrum; their content and distribution were compared to those in osteomyelitis and non-inflammatory bones using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and synchrotron radiation X-ray fluorescence analysis (SR-XRF). On ICP-AES analyses, various trace elements (Co, Cr, Cu, Fe, K, Mg, Ni, Sb, Ti, V, Pb) were significantly more in MRONJ sequestra than non-inflammatory bones. The Cu content was significantly higher in MRONJ sequestra than osteomyelitis and non-inflammatory bones. The Cu content in MRONJ sequestra was high even after decalcification. Additionally, Cu was distributed along the trabecular structures in decalcified MRONJ specimens, as observed using SR-XRF analysis. Therefore, this study was indicative of the characteristic behavior of Cu in MRONJ.


Quantification of tissue dimensional changes during preparation of histopathological specimen

病理組織標本作製過程における組織の寸法変化に関する検討

It has been reported that histological specimens shrink and/or expand during the formalin fixation process and preparation of histopathological specimens. In this study, to quantify the dimensional changes of histological specimens, we investigated the size of hard and soft tissues during the following processes: formalin fixation (study ), and from post-trimming to completion of the histopathological specimen (study ).
Study consisted of soft tissue samples from 15 tongue resected patients, and hard tissue samples from 15 segmental mandibular resected patients. The dimensions were measured before and after formalin fixation. The mean shrinkage in soft tissues and hard tissues were -0.40% and 2.79%, respectively. The larger the surgical soft tissue specimen, the significantly smaller was its mean shrinkage.
Study consisted of the soft tissue samples from 67 tongue resected patients, and hard tissue samples from 19 segmental mandibular resected patients. The dimensions were measured at the time of post-trimming and after completing the histopathological specimens. The mean shrinkage in soft tissues was 6.81%, compared to 9.35% in hard tissues. The mean shrinkage in hard tissues was significantly higher than that in soft tissues. In contrast to study , study showed that as the size of soft tissue specimen increased, its mean shrinkage also increased significantly.
These results provide useful feedback from histopathological diagnosis to clinical diagnosis, and help to choose more appropriate diagnosis and treatments.

Journal of The Japanese Stomatological Society


Clinical Course of Oral Squamous Cell Carcinoma in Patients on Immunosuppressant and Glucocorticoid Therapy

Depressed immune function is a serious adverse effect of long-term immunosuppressant or steroid administration at doses that exceed the physiologically required amount. The purpose of this study was to determine the effects of immunosuppression on carcinogenesis, particularly malignant tumor development, in patients with oral squamous cell carcinoma who had immunosuppression because of immunosuppressant therapy with or without steroid therapy administered for different underlying diseases. In this retrospective chart review, 886 patients with oral squamous cell carcinoma who received treatment at the authors' department from April 2001 through December 2011 were included. Their clinical characteristics; tumor, node, and metastasis (TNM) stage; initial treatment for the primary cancer; mode of cervical lymph node metastasis; incidence rate of distant metastases; white blood cell, neutrophil, and lymphocyte counts on initial examination; and therapeutic outcomes were evaluated and compared between patients on and those not on immunosuppressant therapy with or without steroid therapy. Survival rates were calculated using the Kaplan-Meier method. Fourteen eligible patients (5 men, 9 women; mean age, 65.2 yr) were identified who were on immunosuppressant therapy with or without steroid therapy. They exhibited considerably more metastases, extracapsular spread, and distant metastases, and the number of metastases and extracapsular spread were statistically significant (P = .0213, P = .042, respectively). In 9 patients, total lymphocyte count in the peripheral blood was no higher than 1,500/μL, indicating the lower limit of the normal range. One patient died of recurrence of the primary tumor. Another patient died of cervical lymph node recurrence. Distant metastases occurred in 2 patients. The cumulative disease-specific 5-year survival rate of patients receiving immunosuppressive therapy was 62.3% and that of patients with cervical lymph node metastasis was 25%. The results of this study suggest that patients with oral squamous cell carcinoma on immunosuppression therapy show progression of cervical lymph node metastasis and extracapsular spread and are at high risk of developing distant metastases.

Journal of Oral and Maxillofacial Surgery


Clinical evaluation of coverage of open wounds: Polyglycolic acid sheet with fibrin glue spray vs split thickness skin

This study aimed to evaluate the coverage of oral wounds using either a polyglycolic acid (PGA) sheet or split-thickness skin grafting (STSG). A total of 119 cases of wound coverage using a PGA sheet and fibrin glue spray as well as 132 cases of wound coverage cases using STSG were reviewed retrospectively. The site of the excision area, perioperative conditions, and postoperative functional problems were evaluated. The PGA group had significantly shorter operation time, earlier start of oral intake, and shorter hospitalization than the STSG group. If the PGA sheet over the wound with exposed bone could be protected by a surgical sprint, oral food intake could be started on the day after surgery at the earliest. When the size of the wound in the buccal excisional area was classified into two groups (<6 or ≥6 cm2), mouth opening in the STSG group was significantly larger at 3 months postoperatively. When the size of the wound in the tongue and floor of mouth was classified into two groups (<12 or ≥12 cm2), the STSG group had a significantly higher score in postoperative speech intelligibility. Selection of a PGA sheet or STSG based on the consideration of defect size, tumor location, patients' local and general condition and tolerance for surgery could reduce the patients' postsurgical dysfunctional problems.


Long-term outcome of non-surgical treatment in patients with oral leukoplakia

The standard treatments for oral leukoplakia range from careful observation to complete resection. No surgical intervention is chosen for several supposable reasons. Surgical treatment and no surgical treatment for oral leukoplakia have no defined basis for comparisons, and few studies have reported on the long-term outcomes of oral leukoplakia without surgery. This study aimed to identify the important factors using a long-term wait-and-see policy in patients with oral leukoplakia. In total, 237 lesions from 218 patients selected for non-surgical therapy between 2001 and 2010 were analyzed. On the basis of long-term follow-up data, lesions were classified as unchanged, reduced, disappeared, expanded, and malignantly transformed. In total, 135 (57.0%) lesions remained unchanged, 30 (12.7%) lesions were characterized by a reduction in size or clinical severity, and 44 (18.6%) lesions had disappeared. Another 17 (7.2%) lesions resulted in spread or clinical deterioration, and 11 (4.6%) lesions developed oral squamous cell carcinoma. We demonstrated a cumulative malignant transformation rate of 11.6% in 10years without resection. The lesions that were nonhomogeneous, and higher degree of epithelial dysplasia, located on the tongue were likely to progress into cancer. In addition, 32.5% of lesions without surgical treatment were reduced or disappeared. There is a possibility that removal of considerable irritation for a long time contributes to the treatment of this disease. The development of appropriate treatments for oral leukoplakia is required, which will enable successful differentiation between surgical and observation cases.


Temporomandibular joint intermittent closed lock: Clinic and magnetic resonance imaging findings

This study was carried out to describe the clinical and magnetic resonance imaging (MRI) findings of patients with intermittent closed lock (ICL) of the temporomandibular joint (TMJ). This retrospective study included 58 joints with ICL and 526 joints without ICL as controls. We compared the MRI findings between the patients with and without ICL and investigated clinical and MRI finding of ICL patients. We divided ICL patients into 2 groups based on the treatment efficacy: effective and ineffective. There were significant differences in the prevalence of disk deformity between the joints with and without ICL. Masticatory muscle pain was observed in 41.7% of the effective group and in 80% of the ineffective group, respectively. These results suggest that there is a relationship among the onset of ICL and disk deformation. Masticatory musclepain was significantly observed in the ineffective group.

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology


Clinical evaluation of malignant transformation in oral leukoplakia

口腔白板症の癌化に関する臨床的検討

The purpose of this study was to examine clinical risk factors of oral leukoplakia that were associated with malignant transformation. A total of 512 patients (554 lesions) with clinical and histopathological diagnoses of oral leukoplakia who presented at our department from 2001 through 2010 were retrospectively studied. They consisted of 278 males and 234 females 11 to 84 years of age (median, 61 years). The mean follow-up was 36 months. The sites of oral leukoplakia included the tongue (176 lesions), gingiva (219 lesions), buccal mucosa (76 lesions), hard palate (50 lesions), soft palate (21 lesions), lips (7 lesions), and floor of the mouth (5 lesions). Squamous cell carcinoma developed in 13 of the 512 patients during follow-up. The development occurred after a mean period of 39 months (range, 9 to 91 months). The sites of malignant transformation included the ventral aspect of the tongue (10 lesions), gingiva (1 lesion), buccal mucosa (1 lesion), and soft palate (1 lesion). In 9 of these 13 malignant lesions, the clinical type of oral leukoplakia was non-homogeneous. The degree of epithelial dysplasia was none (2 lesions), mild (5 lesions), or moderate (6 lesions). On univariate analysis, site, clinical type, and treatment significantly correlated with malignant transformation. On multivariate analysis, these 3 factors retained their statistical significance. The cumulative malignant transformation rate was 5.6% at 5 years and 8.7% at 10 years.

Japanese Journal of Oral & Maxillofacial Surgery


Recurrence patterns of oral leukoplakia after curative surgical resection: Important factors that predict the risk of recurrence and malignancy

Oral leukoplakia can be treated using a variety of treatment procedures; however, the lesions recur in many cases irrespective of the treatment procedure used. The rate of recurrence was from 7.7% to 38.1%. This study aims to identify the important factors that can lower the risk of recurrence of oral leukoplakia treated by curative surgical resection. The clinical records of 52 patients with oral leukoplakia (53 lesions) who underwent curative surgical resection between 2004 and 2009 were retrospectively analyzed for the rate of recurrence, clinical outcome, epithelial dysplasia, lesion location, and resection margins. The recurrence rate following curative surgical resection was 15.1%, with the most common site being the gingiva. Malignant transformation occurred in a single patient (1.9%). Minimal resection margins (<3 mm) were observed in many patients with recurrent disease, and recurrence was more likely in cases with positive margins (epithelial abnormalities at the resection margins) than in those with negative margins. There was no significant association between recurrence and the degree of epithelial dysplasia. Our data suggest that surgical resection of oral leukoplakia is curative only if all areas of epithelial abnormalities are identified and resected. Moreover, an adequate resection margin may reduce the risk of recurrence.


B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti-B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were re-exposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance.


Keratinocyte-associated B7-H1 directly regulates cutaneous effector CD8+ T cell responses

Keratinocytes (KCs) may play important roles for maintenance of peripheral tolerance in the upper layers of the skin. Coinhibitory signals mediated via the programmed death (PD)-1 and its ligand B7-H1 (PD-L1/CD274) are crucial for the downregulation of T cell immune responses and for the maintenance of peripheral tolerance. In this study, to investigate the role of KC-expressed B7-H1 in the regulation of T cell immune responses, we generated transgenic (tg) mice overexpressing B7-H1 under the control of keratin 14 (K14) promoter (K14-B7-H1 tg). K14-B7-H1 tg mice displayed impaired contact hypersensitivity (CH) responses to primary and secondary hapten challenges. The K14-B7-H1 tg mice did not exhibit substantial impairment of cutaneous dendritic cell migration after sensitization and of hapten-specific proliferation and IFN-gamma production of CD4(+) and CD8(+) T cells in the draining lymph nodes, suggesting that overexpression of B7-H1 on KCs did not affect the induction phase of the CH response. The systemic or s.c. injection of hapten-sensitized T cells into the K14-B7-H1 tg mice did not efficiently induce the CH response. IFN-gamma expression and apoptosis of KCs in the challenged ears were impaired in K14-B7-H1 tg mice. IFN-gamma production by presensitized CD8(+) T cells stimulated with hapten-pulsed KCs was markedly impaired for the KCs obtained from the K14-B7-H1 tg mice but was restored by the addition of an anti-B7-H1 mAb. These results suggest that KC-associated B7-H1 directly downregulates the effector function of CD8(+) T cells by associating with PD-1 at local inflammatory sites and that it plays a role in peripheral T cell tolerance against exogenous Ags.


Leucocyte-associated immunoglobulin-like receptor-1 is an inhibitory regulator of contact hypersensitivity

Leucocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a membrane receptor of the immunoglobulin (Ig) superfamily that is expressed on most types of haematopoietic cells, and delivers inhibitory signals through interacting with collagens. In order to elucidate the immunological functions of LAIR-1 in vivo, we established transgenic mice expressing a chimeric protein composed of the extracellular domain of LAIR-1 fused with an Ig tag (LAIR-1-Ig), which acts as a decoy by competing with endogenous LAIR-1. The transgenic mice showed an increased susceptibility for development of contact hypersensitivity (CHS), an experimental model of allergic contact dermatitis, in association with enhanced hapten-specific T-cell responses. When T cells from the hapten-sensitized donor mice were transferred into non-sensitized recipients, treatment of either donor mice or recipient mice with LAIR-1-Ig protein accelerated CHS, suggesting a potentially negative role of LAIR-1 in both the sensitization and the elicitation of hapten-reactive T cells. In vitro assays revealed that LAIR-1 decreased the production of interleukin-6 and interleukin-12 in dendritic cells, and inhibited the proliferation and cytokine production of naïve and memory T cells along with G(0)/G(1) cell cycle arrest. Collectively, our findings suggest that LAIR-1 plays a crucial inhibitory role in CHS by regulating antigen-presenting cell and T-cell functions.


Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy

Although self-reactive T-cell precursors can be eliminated upon recognition of self-antigen presented in the thymus, this central tolerance process is often incomplete, and additional mechanisms are required to prevent autoimmunity. Recent studies indicates that the interaction between B7-H1 and its receptor PD-1 on activated T cells plays an important role in the inhibition of T-cell responses in peripheral organs. Here, we show that, before their exit to the periphery, T cells in lymphoid organs rapidly up-regulate PD-1 upon tolerogen recognition. Ablation of the B7-H1 and PD-1 interaction when T cells are still in lymphoid organs prevents anergy. Furthermore, blockade of B7-H1 and PD-1 interaction could render anergic T cells responsive to antigen. Our results thus reveal previously unappreciated roles of B7-H1 and PD-1 interaction in the control of initiation and reversion of T-cell anergy.


Expression and regulation of human CD275 on endothelial cells in healthy and inflamed mucosal tissues

The CD275-CD278 costimulatory pathway is a new pathway for CD28-B7 family molecules involved in the effector phase of T-cell-mediated immune responses. Expression of CD275 in oral mucosa at healthy and disease states has not been examined. We generated monoclonal antibodies against human CD275 and investigated its expression and regulation in cultured tissue cell lines and oral mucosal tissues. CD275 on monocytes was efficiently upregulated by interleukin-4, while interferon-gamma abrogated this effect. CD275 on cultured endothelial cells (EC) was rapidly enhanced by tumour necrosis factor-alpha. In healthy oral mucosa, CD275 was not detected on keratinocytes, Langerhans cells or intraepithelial lymphocytes within the epithelium or on interstitial dendritic cells or lymphocytes in the sub-epithelium. Constitutive expression of CD275 on EC in the connective tissues was observed in healthy mucosa, but CD275 expression on EC in oral lichen planus was either upregulated or down regulated. Approximately 20% of the T cells found within infiltrating mononuclear cells in the sub-epithelium expressed high levels of the CD278 receptor. CD275 on lymphoid and nonlymphoid cells is positively or negatively regulated by various cytokines. Our results suggest that CD275 on EC is involved in the recruitment or extravasation of receptor-positive effector T cells into inflamed tissues.


Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma

We examined cell surface expression of five B7 costimulatory molecules (B7-H1, B7-DC, B7h, CD80 and CD86) in human oral squamous cell carcinoma (SCC) lines. Most human SCC cell lines expressed various levels of B7-H1 and B7-DC. Their expression was further upregulated by interferon (IFN)-gamma stimulation. Immunohistochemical staining revealed substantial and predominant expression of B7-H1 on human primary oral SCC. A murine SCC line, NR-S1, neither expressed B7-H1 nor B7-DC, but induced B7-H1 by IFN-gamma stimulation in culture and the inoculation in vivo. Although NR-S1 tumors grew progressively in immunocompetent syngeneic mice, the administration of blocking anti-B7-Hl or anti-PD-1 mAb significantly inhibited the tumor growth, suggesting the negative regulation of host immune responses by the PD-1:B7-H1 pathway. Our results demonstrate that B7-H1 is predominantly induced on oral SCC within the B7 family molecules. A successful inhibition of tumor growth by blockade of the PD-1:B7-H1 pathway may implicate a new approach for immunotherapy of oral SCC.


Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer

The negative regulatory programmed death-1/programmed death-1 ligand (PD-1/PD-L) pathway in T-cell activation has been suggested to play an important role in tumor evasion from host immunity. In this study, we investigated the expression of PD-L1 and PD-L2 in human esophageal cancer to define their clinical significance in patients' prognosis after surgery. PD-L1 and PD-L2 gene expression was evaluated in 41 esophagectomy patients by real-time quantitative PCR. The protein expression was also evaluated with newly generated monoclonal antibodies that recognize human PD-L1 (MIH1) and PD-L2 (MIH18). The protein and the mRNA levels of determination by immunohistochemistry and real-time quantitative PCR were closely correlated. PD-L-positive patients had a significantly poorer prognosis than the negative patients. This was more pronounced in the advanced stage of tumor than in the early stage. Furthermore, multivariate analysis indicated that PD-L status was an independent prognostic factor. Although there was no significant correlation between PD-L1 expression and tumor-infiltrating T lymphocytes, PD-L2 expression was inversely correlated with tumor-infiltrating CD8(+) T cells. These data suggest that PD-L1 and PD-L2 status may be a new predictor of prognosis for patients with esophageal cancer and provide the rationale for developing novel immunotherapy of targeting PD-1/PD-L pathway.


Clinical observation of mouth-opening exercise and self-reduction manipulation on the habitual luxation of the temporomandibular joint

習慣性顎関節脱臼に対する開口訓練と自己整復操作についての臨床的観察

The objective of this study was to evaluate the effect of mouth opening exercise and self-reduction manipulation on the habitual luxation of the temporomandibular joint (TMJ), which is extremely difficult or impossible to reduce by oneself. Ten patients with habitual luxation of TMJ were enrolled and underwent these procedures. One month later, in all cases there was no recurrence of habitual luxation and the maximum mouth opening without pain to the predicted mouth opening increased significantly compared with that at the first visit. After self-reduction manipulation, the patients had no fear of the luxation at all, and we did not identify TMJ sound by palpation under observation. We suggest that mouth-opening exercise and self-reduction manipulation might be indicated as initial measures for patients with habitual luxation of TMJ.

習慣性顎関節脱臼とは, 下顎頭が下顎窩から逸脱し自力での復帰がきわめて困難かあるいはできなくなった状態が習慣性に生じるものである．非観血的療法として過開口制限指導などがあるが, 十分な効果が得られないことがある．本症患者には日常生活における脱臼発生の恐怖感が強く, そのため正常な顎運動を自制して咀嚼筋がこわばっているものが多いことに著者らは注目し, 過開口制限とは逆の開口訓練と自己整復操作を指導した．2002年2月から2003年10月に当科を受診した習慣性顎関節脱臼患者のうち, 触診にて関節雑音を認めなかった10例について検討した．年齢18∼72歳 (平均34歳), 男性2例女性8例, 病悩期間10日∼8年 (平均3.7年) で, 脱臼の発生頻度はさまざまであった．初診時, 自己整復操作指導後, 治療開始1か月後および最終観察時で, 脱臼の発生の有無・塚原らが報告した予測開口域に対する無痛自力最大開口域の割合・脱臼に対する恐怖感の有無・関節雑音の発生の有無の4項目を検討した．平均観察期間15.8か月 (8か月∼2年4か月) であった．治療開始1か月以降には全症例において脱臼の発生を認めず, 予測開口域に対する無痛自力最大開口域の割合は初診時に比べて有意に増大した．初診時全症例で認められた脱臼に対する恐怖感は, 自己整復操作指導後にすべての症例で消失した．また観察期間中にすべての症例で, 関節雑音の発生は認められなかった．

Journal of the Japanese Society for the Temporomandibular Joint


Blockade of the interaction between PD-1 and PD-L1 accelerates graft arterial disease in cardiac allografts

Programmed death-1 (PD-1), a member of the CD28 family, has been identified. PD-1 is involved in the negative regulation of some immune responses. We evaluated the role of PD-ligand 1 (PD-L1) in graft arterial disease (GAD) of cardiac allografts and in smooth muscle cells (SMCs). C57BL/6 murine hearts were transplanted into B6.C-H2<bm12>KhEg mice for examination of GAD. PD-L1 was expressed in SMCs of the thickened intima in the graft coronary arteries, and administration of anti-PD-L1 monoclonal antibody (mAb) enhanced the progression of GAD (luminal occlusion: 55+/-5.0% versus 9.8+/-4.3%, P<0.05). The expressions of interferon gamma (IFN-gamma) and tumor necrosis factor alpha of cardiac allografts were upregulated in response to anti-PD-L1 mAb treatment. In vitro, PD-L1 expression was induced in SMCs in response to IFN-gamma stimulation. Sensitized splenocytes increased SMC proliferation, and anti-PD-L1 mAb in combination with IFN-gamma stimulation increased this proliferation. The PD-L1 pathway regulates both the proliferation of SMCs and GAD. Thus, control of this interaction is a promising strategy for suppression of GAD.

Arteriosclerosis, Thrombosis, and Vascular Biology


The expression of B7-H1 on keratinocytes in chronic inflammatory mucocutaneous disease and its regulatory role

PD-1 and its ligands, B7-H1/PD-L1 and B7-DC/PD-L2, have been identified recently as CD28-B7 family molecules that are implicated in immune regulation. Lichen planus (LP) is a T cell-mediated chronic inflammatory mucocutaneous disease. We investigated the expression and function of PD-1 and its two ligands in LP. Immunohistochemical examination revealed the abundant expression of PD-1 and B7-H1 in infiltrating T cells and macrophages, and lower-level expression of B7-DC on macrophages in the subepithelium. Interestingly, substantial expression of B7-H1 on keratinocytes (KCs) was found close to the numerous T cell infiltrates in the subepithelium. Unstimulated cultured KCs expressed both B7-H1 and B7-DC, and their expression was upregulated by proinflammatory cytokines, particularly IFN-gamma. The T-cell proliferative responses and IFN-gamma production that were induced by IFN-gamma-treated KCs were augmented preferentially by anti-B7-H1 mAb, but not by anti-B7-DC mAb. These results indicate the regulatory role of B7-H1 on KCs in the interactions with T cells. Our results suggest that the induction of B7-H1 on KCs may play an important role in tolerance induction in the inflamed oral mucosa and skin.


The Expression and Function of Costimulatory Molecules B7h and B7-H1 on Colonic Epithelial Cells

Previous studies have suggested that intestinal epithelial cells (IECs) may function as antigen-presenting cells for CD4+ and CD8+ T cells. However, these cells fail to express conventional costimulatory molecules (CD80, CD86), leading to the possibility that antigen presented by normal IECs could result in anergy. Other members of the B7 family have recently been identified. B7h interacts with inducible costimulator (ICOS) on T cells and provides a positive signal, whereas B7-H1 and B7-DC interact with PD-1 and transmit an inhibitory signal. Our aim was to determine whether IECs express novel B7 family members and whether these molecules play a role in IEC:T-cell interactions. B7h and B7-H1 expression was assessed in isolated IECs and IEC lines. The functional role of B7h and B7-H1 in the interaction between IECs and T cells was assessed in coculture experiments using purified anti-B7h or B7-H1 monoclonal antibodies (mAbs), B7h immunoglobulin (Ig), or B7-H1 fusion proteins. B7h and B7-H1 messenger RNA was detected in IEC lines and IECs from healthy controls and patients with inflammatory bowel disease (IBD). IECs from patients with IBD but not healthy controls expressed B7h and B7-H1 protein on their surface. Proliferation of IEC-stimulated T cells was inhibited only by B7h immunoglobulin treatment, whereas interferon gamma secretion in these cocultures was inhibited by both anti-B7h mAb and B7h Ig. No difference was seen between IBD or normal IEC populations. These data suggest that the B7h-ICOS costimulatory pathway may be important in IEC:T-cell interactions.


B7-DC regulates asthmatic response by an IFN-gamma-dependent mechanism.

B7-H1 (PD-L1) and B7-DC (PD-L2) are the ligands for programmed death-1 (PD-1), which is a member of the CD28/CTLA-4 family and has been implicated in peripheral tolerance. We investigated the roles of B7-H1 and B7-DC in a murine OVA-induced allergic asthma model. B7-H1 was constitutively expressed on dendritic cells, macrophages, B cells, and T cells in the lungs of naive mice, and its expression could be dramatically increased after allergen challenge. In contrast, B7-DC expression was scarcely expressed on dendritic cells in naive mice, but was up-regulated after allergen challenge, although the up-regulation of B7-DC expression on macrophages was minimal. Treatment of mice with anti-B7-DC mAb at the time of allergen challenge, but not at the time of sensitization, significantly increased their airway hyper-reactivity and eosinophilia. Such treatment also resulted in the increased production of IL-5 and IL-13, and decreased IFN-gamma production in the lungs and draining lymph node cells. These changes were diminished when mice were depleted of IFN-gamma by anti-IFN-gamma mAb pretreatment. Interestingly, treatment with anti-B7-H1 or anti-PD-1 mAb did not significantly affect the asthmatic response. These results suggest a unique role for B7-DC in the regulation of asthmatic response through an IFN-gamma-dependent, but PD-1-independent, mechanism.


Preferential contribution of B7-H1 to programmed death-1-mediated regulation of hapten-specific allergic inflammatory responses

Programmed death-1 (PD-1) and its ligands, B7-H1/PD-L1 and B7-DC/PD-L2, are new CD28-B7 family members that may be involved in the regulation of immune responses. We examined the roles of these molecules in mouse hapten-induced contact hypersensitivity (CH). Administration of anti-PD-1 mAb at sensitization significantly enhanced and prolonged ear swelling. Treatment with anti-B7-H1 mAb, but not anti-B7-DC mAb, also enhanced CH reactions. The anti-PD-1 mAb treatment at sensitization significantly increased the T cell number of draining lymph nodes (DLN). B7-H1 was induced on activated T cells and antigen-presenting cells (APC) in the skin and the DLN, whereas B7-DC expression was restricted to dendritic cells (DC) in the dermis and the DLN. A particular subset of DC, B7-H1(+)B7-DC(-)CD86(low), was found in sensitized DLN. The blockade of B7-H1, but not B7-DC, dramatically enhanced the initial T cell proliferative responses against hapten-pulsed DLN APC, suggesting the preferential contribution of B7-H1 to the T cell-APC interaction. Our results demonstrate the regulatory role of PD-1 and the differential roles of B7-H1 and B7-DC in hapten-induced immune responses. The PD-1-B7-H1 pathway may play a unique role in regulating inflammatory responses.


Involvement of inducible costimulator-B7 homologous protein costimulatory pathway in murine lupus nephritis

Inducible costimulator (ICOS)-B7 homologous protein (B7h) is a new member of the CD28-B7 family of costimulatory molecules that regulates T cell-dependent humoral immune responses. In this study, we examined the involvement of this costimulatory pathway in the development and progression of lupus in NZB/W F(1) mice. Expression of ICOS on T cells was enhanced with disease progression, whereas B7h expression on B cells was down-regulated. Administration of anti-B7h mAb before the onset of renal disease significantly delayed the onset of proteinuria and prolonged survival. Blockade of B7h effectively inhibited all subclasses of IgG autoantibody production and accumulation of both Th1 and Th2 cells. Hypercellularity and deposition of IgG and C3 in glomeruli were significantly reduced. B7h blockade after the onset of proteinuria prevented the disease progression and improved the renal pathology. Our results demonstrated the involvement of the ICOS-B7h costimulatory pathway in the pathogenesis of lupus nephritis, and the blockade of this pathway may be beneficial for the treatment of human systemic lupus erythematosus.


Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon gamma-producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.


Clinical Study on the Effect of Fluocinonide and Clobetasol Propionate Oral Ointments in the Treatment of Oral Vesiculoerosive Diseases

口腔粘膜疾患に対するフルオシノニドならびにプロピオン酸クロベタゾール口腔用軟膏の治療効果に関する臨床的検討

Journal of Japanese Society for Oral Mucous Membrane