CD4+ CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+ KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.

Cancer/testis (CT) antigens are expressed in normal germ line tissues and various cancers. They are considered promising target molecules for immunotherapy for patients with various cancers. To identify CT antigens, we performed serological identification of antigens by recombinant expression cloning. The humoral immune response of cancer patients against a newly defined antigen was analyzed. A testicular cDNA library was immunoscreened with serum obtained from a gastric adenocarcinoma patient whose primary cancer had regressed once and most liver metastasies had disappeared transiently. We isolated 55 positive cDNA clones comprising 23 different genes. They included 4 genes with testis-specific expression profiles in the Unigene database, including coiled-coil domain containing 62 (CCDC62). RT-PCR analysis showed that the expression of 2 splice variants of CCDC62 was restricted to the testis in normal adult tissues. In malignant tissues, CCDC62 variant 2 (CCDC62-2) was aberrantly expressed in a variety of cancers, including stomach cancer. A serological survey of 191 cancer patients with a range of different cancers by ELISA revealed antibodies to CCDC62-2 in 13 patients, including stomach cancer. None of the 41 healthy donor serum samples were reactive in the same test. The serum reaction against CCDC62-2 was confirmed by western blot. CCDC62-2 is a CT antigen that is immunogenic in cancer patients.

BackgroundIdentification of new cancer antigens is necessary for the efficient diagnosis and immunotherapy. A variety of tumor antigens have been identified by several methodologies. Among those antigens, cancer/testis (CT) antigens have became promising targets.MethodsThe serological identification of antigens by the recombinant expression cloning (SEREX) methodology has been successfully used for the identification of cancer/testis (CT) antigens. We performed the SEREX analysis of colon cancer.ResultsWe isolated a total of 60 positive cDNA clones comprising 38 different genes. They included 2 genes with testis-specific expression profiles in the UniGene database, such as TEKT5 and a CT-like gene, A kinase anchoring protein 3 (AKAP3). Quantitative real-time RT-PCR analysis showed that the expression of TEKT5 was restricted to the testis in normal adult tissues. In malignant tissues, TEKT5 was aberrantly expressed in a variety of cancers, including colon cancer. A serological survey of 101 cancer patients with different cancers by ELISA revealed antibodies to TEKT5 in 13 patients, including colon cancer. None of the 16 healthy donor serum samples were reactive in the same test.ConclusionWe identified candidate new CT antigen of colon cancer, TEKT5. The findings indicate that TEKT5 is immunogenic in humans, and suggest its potential use as diagnostic as well as an immunotherapeutic reagent for cancer patients.

KEYWORDSCancer/testis antigen; SEREX; Antibody response; Squamous cell carcinoma of the head and neck Summary Numerous tumor antigens have so far been identified from various tumors using the serological identification of antigens by recombinant expression cloning (SEREX) method. Among them, cancer/testis (CT) antigens are considered promising target molecules for immunotherapy for patients with various cancers. We performed several SEREX analyses of various cancers to identify CT antigens, including gastric adenocarcinoma, lung adenocarcinoma, and colon cancer, and consequently identified additional CT antigens, such as XAGE-1, CCDC62-2, GKAP1, and TEKT5. However, although SEREX analysis of squamous cell carcinoma of the head and neck (HNSCC) has been performed several times, only a few CT or HNSCC specific antigens have yet been isolated. Compared with other tumors, a small number of studies have been reported on the antigen proteins specific to HNSCC. We here reported the expression of selected CT antigens and their immunogenicity in patients with HNSCC. The results obtained suggested that CCDC62-2, GKAP1, and TEKT5 are immunogenic in HNSCC and also demonstrated their potencies as diagnostic markers for patients with HNSCC in combination with other CT antigens such as NY-ESO-1, MAGE-A3, and MAGE-A4.

Purpose Sonic Hedgehog (SHH) is a regulatory protein involved in bone fracture healing. Orthognathic surgery involves surgical osteotomy of the mandible or maxilla to restore the proper anatomic and functional position in patients with dentofacial deformity. The purpose of this study was to analyze SHH local blood serum concentrations after osteotomy to gain further understanding of the molecular regulation of the initial stage of osteotomy healing. Methods Serum samples (local drainage and peripheral venous) of 34 patients (24 females and 10 males, mean age was 23.4 (16–42) years) who underwent orthognathic surgery were isolated from patients at different time points during the perioperative period. The levels of SHH, soluble receptor activator of nuclear factor-κB ligand (sRANKL) and osteoprotegerin (OPG) were measured using ELISA. Results SHH was detected in the local drainage immediately after osteotomy (309.5 ± 58.2 pg/ml), and decreased for 2 days after the operation (197.5 ± 43.6 pg/ml). The sRANKL local serum concentrations were at the maximum level immediately after the operation (141.4 ± 22.6 pg/ml) and decreased for 2 days (110.1 ± 23.4 pg/ml). On the other hand, the OPG concentration in the local serum was at a minimum after osteotomy (59.4 ± 4.6 pg/ml) and reached its maximum (181.5 ± 17.8 pg/ml, P < 0.01) at 2 days after osteotomy. SHH and OPG local serum levels on day 2 were associated with the amount of bleeding during the operation. The local drainage serum level of SHH of maxillary/mandibular osteotomy had a tendency to be higher than that of mandible-only osteotomy at 2 days after operation. Conclusions Elevated levels of SHH in local serum after osteotomy, especially during the initial stage of healing, indicates its importance in osteotomy healing. Highlights