Wataru Kumamaru

T-cell receptor Vβ gene usage by T cells reactive with the tumor-rejection antigen SART-1 in oral squamous cell carcinoma

We recently described that the SART‐1690–698 peptide could induce HLA‐A24‐restricted cytotoxic T lymphocytes (CTLs), which recognize the SART‐1  259+ tumor cells from peripheral blood mononuclear cells (PBMCs) of HLA‐A24+ cancer patients. In our study, in 5 of 14 HLA‐A24+ patients with oral squamous cell carcinomas (SCCs), CTLs could be induced with the SART‐1690–698 peptide from the PBMCs. In 2 of the patients from whom the highest CTL activities were induced, the T‐cell receptor (TCR) Vβ repertoire expressed by the SART‐1690–698‐specific CTLs was found to be restricted and multiple Vβ families were predominantly expressed in each patient. Although the predominant Vβ families were different between the 2 patients, Vβ7 was highly and commonly predominant. The same predominant Vβ families were also detected in the tumor‐infiltrating lymphocytes (TILs) from each patient, and each Vβ family contained one or more unique T‐cell clonotypes. The unique T‐cell clonotypes were found to be common between the TILs and SART‐1690–698‐specific CTLs from each patient, and especially 2 T‐cell clonotypes with Vβ7 were identical even in the 2 patients. One of the 2 T‐cell clonotypes with Vβ7 was detected in the TILs from 11 of 14 HLA‐A24+ patients and another was found in those from 8 of HLA‐A24+ patients, while none of 10 HLA‐A24− patients demonstrated both T‐cell clonotypes. These results strongly suggest that the T‐cell clonotypes with Vβ7 are major TCR Vβ genes expressed by SART‐1690–698‐specific CTLs. Furthermore, autologous tumor cells from one of the HLA‐A24+ patients stimulated the PBMCs and regional lymph node cells (LNCs) to expand the same T‐cell clonotypes as those in the SART‐1690–698‐specific CTLs. These results strongly suggest that the SART‐1690–698‐specific CTLs clearly accumulate in vivo, especially in the TILs, as a consequence of in situ antigenic stimulation by autologous tumor cells. The identification of the unique TCR Vβ genes used by SART‐1259‐specific CTLs should help to improve the diagnosis of the specific immune response in patients with SART‐1  259+ cancers, especially during anticancer immunotherapy.

Possible involvement of cytokines, chemokines and chemokine receptors in the initiation and progression of chronic GVHD

Chronic GVHD (cGVHD) after allogeneic hematopoietic SCT (HSCT) is characterized by an infiltration of T cells into target organs including the oral mucosa and salivary glands. This study was designed to clarify the molecular mechanism of the local accumulation of pathogenic T cells in cGVHD. The expression of cytokines, chemokines and chemokine receptors in the buccal mucosa (BM), labial salivary glands (LSG) and PBMC from 16 patients with cGVHD after allogeneic HSCT was examined. The mRNA expression of T helper 1 (Th1) and Th2 cytokines, and several chemokines and chemokine receptors was significantly increased in the BM and LSG from cGVHD patients, in comparison with both those in the BM and LSG from controls, respectively, and also with those in the PBMC from cGVHD patients. Furthermore, the mRNA expression of Th2 cytokines, macrophage-derived chemokine and CC chemokine receptor 4 was closely associated with a strong T-cell infiltration in the BM and LSG from cGVHD patients. These results suggest that cGVHD might be initiated and/or maintained by Th1/Th0 cells and thereafter progresses in association with Th2 cell accumulation via the interaction of particular chemokine and chemokine receptors.

Multiple schwannomas in the oral floor: case report.

We present a case of multiple schwannomas of the oral floor in a 62-year-old man, which met the diagnostic criteria of schwannomatosis.

A novel measurement method for the morphology of the mandibular ramus using homologous modelling.

OBJECTIVES It is important to assess the mandibular morphology when orthognathic surgery, especially mandibular ramus osteotomy, is performed. Several studies on three-dimensional (3D) facial asymmetry have reported differences in linear and angle measurements between the deviated and contralateral sides in asymmetric mandibles. However, methods used in these studies cannot analyse the 3D morphology of the ramus. In this study, we aimed to evaluate the differences in mandibular ramus between the deviated and contralateral sides in asymmetric mandibles using traditional measurements as well as 3D shape analysis. METHODS 15 Japanese females with jaw deformities treated by orthodontic surgery were enrolled. 3D CT images were reconstructed, and 14 landmarks were identified on the model surface. Ten linear and four angle measurements were calculated using these landmarks. Homologous ramus models were constructed for each sample, and after converting all homologous models to the right side, 30 homologous models of the ramus were analysed using principal component analysis. RESULTS Firstly, eight principal components explained >80% of the total variance. Differences between the deviated and contralateral sides in measurements and scores of the eight principal components were tested. Significant difference at the 5% level between the deviated and contralateral sides was observed in five linear measurements, three angle measurements and the third principal component. The variance of the deviated side was significantly larger in the diameter between the mandibular notch and coronoid process, horizontal dilated angle of the mandibular ramus and vertical dilated angle of the mandibular ramus. The variance of the contralateral side was significantly larger in the height of mandibular ramus, height of posterior of mandibular ramus, condylar width, height of condylar head and mandibular angle. The squared multiple correlation coefficient adjusted for the degrees of freedom was 0.815. The third principal component showed the difference between the deviated and contralateral sides. Shape variation represented by the third principal component visually indicated that the contralateral side was larger and had inwardly directed coronoid process and the deviated side had a mandibular angle that was turned inwards to a greater extent. CONCLUSIONS In conclusion, we successfully created a homologous model of the mandibular ramus and demonstrated the effectiveness of this model in the 3D comparison of the ramus morphology between the contralateral and deviated sides in asymmetric mandibles.

In vitro induction of specific CD8+ T lymphocytes by tumor-associated antigenic peptides in patients with oral squamous cell carcinoma

The aim of this study was to clarify candidate peptides for peptide-based specific immunotherapy of patients with oral squamous cell carcinoma (SCC). Thirteen peptides were examined for in vitro induction of peptide-specific CD8(+) T lymphocyte (CD8(+)TL) activity in peripheral blood mononuclear cells from 35 patients with oral SCC. A correlation between the induction ability of CD8(+)TL and in vivo immune response of host was carried out immunohistochemically in 23 patients. Peptide-specific activities of CD8(+)TL for at least one peptide were detectable in 21/35 patients (60.0%). The potent peptides were SART-1(690) in 9/35 (25.7%), SART-2(93), and ART4(75) in 7/35 (20.0%), respectively. In the 9 patients with SART-1(690)-specific activity, the whole of activities was significantly inducible for more number of other peptides compared to that in 26 patients without the activity (P=0.035). Cellular responses in 7 patients with SART-1(690)-specific activity were significantly stronger than those in 16 patients without the activity (P=0.027). Furthermore, the number of CD3(+) T cells around the SCC was also significantly different between the 2 groups of patients (P=0.041). In conclusion, SART-1(690), SART-2(93), and ART4(75) could be applicable as peptide-based specific immunotherapies for the majority of patients with oral SCC.

Efficient regulation of branching morphogenesis via fibroblast growth factor receptor 2c in early-stage embryonic mouse salivary glands.

Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.

Involvement of the T-box transcription factor Brachyury in early-stage embryonic mouse salivary gland

The mouse submandibular gland (SMG) is important organ for embryonic development, and branching morphogenesis is regulated by many molecules containing transcription factors. Real-time reverse transcriptase polymerase chain reaction revealed that the expression of Brachyury increased in the SMG and peaked between E12.5-E13.5, concomitant with the early stage of branching morphogenesis. The expression of Brachyury in SMG rudiments between E12.5-E13.5 was confirmed by western blotting. In addition, fibronectin and Btbd7 (regulated by fibronectin), which are both essential for cleft formation, were expressed strongly during the same period. The Sox2 and Wnt3a, which regulate cell growth, were also expressed strongly during E12.5-E13.5. On the other hand, cleft formation and branching morphogenesis was suppressed by knockdown of Brachyury gene, suggesting that Brachyury plays a central role in regulating cell growth and cleft formation in early-stage embryonic mouse salivary gland development.

Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva.

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.