Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8 + tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8 + TILs capable of producing multiple cytokines were mainly PD-1 − Tim-3 + , an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8 + TIL multifunctionality. The adoptive transfer of antigen-specific CD8 + T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8 + T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.immune exhaustion | CD8T cells | antitumor immunity | tumor microenvironment | multifunctionality

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.

CD11b + myeloid subpopulations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), play crucial roles in the suppression of T-cell-mediated anti-tumor immunity. Regulation of these cell types is a primary goal for achieving efficient cancer immunotherapy. We found that metformin (Met) induces CD11b + -cell-mediated growth inhibition of a K7M2neo osteosarcoma independent of T cells, as growth inhibition of K7M2neo was still observed in wild-type (WT) mice depleted of T cells by antibodies and in SCID; this contrasted with the effect of Met on Meth A fibrosarcoma, which was entirely T-cell-dependent. Moreover, the inhibitory effect seen in SCID was abrogated by anti-CD11b antibody injection. PMN-MDSCs were significantly reduced in both spleens and tumors following Met treatment. In TAMs, production of IL-12 and TNF-α, but not IL-10, became apparent, and elevation of MHC class II with reduction of CD206 was observed, indicating a shift from an M2- to M1-like phenotype via Met administration. Metabolically, Met treatment decreased basal respiration and the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio of CD11b + cells in tumors, but not in the spleen. In addition, decreased reactive oxygen species (ROS) production and proton leakage in MDSCs and TAMs were consistently observed in tumors. Uptake of both 2-deoxy-2- d -glucose (2-NBDG) and BODIPY® decreased in MDSCs, but only BODIPY® incorporation was decreased in TAMs. Overall, our results suggest that Met redirects the metabolism of CD11b + cells to lower oxidative phosphorylation (OXPHOS) while elevating glycolysis, thereby pushing the microenvironment to a state that inhibits the growth of certain tumors.

The spontaneous immune responses against XAGE-1b (GAGED2a) were analyzed in non-small cell lung cancer (NSCLC) patients. An antibody response against XAGE-1b (GAGED2a) was observed in 10% (20/200) of NSCLC patients and in 19% (13/69) of stage IIIB/IV lung adenocarcinoma patients. A CD4 T-cell response was detected in 88% (14/16) and a CD8 T-cell response in 67% (6/9) in the XAGE-1b (GAGED2a) antibody-positive patients examined. Frequent antibody responses and CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients indicate the strong immunogenicity of the XAGE-1b (GAGED2a) antigen in NSCLC patients. We established T-cell clones from PBMCs of antibody-positive patients and determined the DRB1*04:05-restricted XAGE-1b (GAGED2a) 18-31 peptide (14-mer) as a CD4 T cell epitope and the A*02:06-restricted XAGE-1b (GAGED2a) 21-29 peptide (9-mer) as a CD8 T cell epitope. As for peptide recognition, CD4 and CD8 T-cell clones responded to naturally processed antigen. The CD4 T-cell clone recognized DCs pulsed with the synthetic protein or a lysate from XAGE-1b-transfected 293T cells. The CD8 T-cell clone showed cytotoxicity against a tumor expressing XAGE-1b (GAGED2a) and the appropriate HLA class I allele. These findings establish XAGE-1b (GAGED2a) as a promising target for a lung cancer vaccine.More than 70 cancer/testis (CT) antigen gene families have been identified by immunological or genetic approaches. 1-3Several CT antigens such as the NY-ESO-1 antigen etc. have been shown to elicit humoral and cellular immune responses in cancer patients. 4,5 Because of their restricted expression in normal tissues and high immunogenicity, CT antigens are considered attractive targets for cancer vaccines. 6-9XAGE-1 was originally identified by the search for PAGE/ GAGE-related genes using an expression sequence tag database 10 and was shown to exhibit CT antigen characteristics. 11,12 Five identical genes XAGE1A to E have now been identified, located in dispersed fashion in different orientations in a region of approximately 350 kilobases on chromosome Xp11.22.13 They belong to X antigen family genes. The associated protein is designated as G antigen family D member 2 (GAGED2), and GAGED2a and d isoforms have been identified. 10,13 Four transcript variants XAGE-1a, b, c and d have been extensively studied and were shown to be expressed in metastatic melanoma, Ewing sarcoma, and various epithelial tumors such as breast, lung and prostate cancers.14-17 In a serologic search for antigens using recombinant expression cloning (SEREX), we identified XAGE-1b as a dominant antigen recognized by serum from a lung adenocarcinoma patient using an autologous tumor cell line established from malignant pleural effusion as a source of the cDNA library.18 From the analysis with transfected 293T cells using a USO 9-13 mAb specific for XAGE-1b (GAGED2a) protein, we showed that the XAGE-1a and b transcripts code for the 81 amino acid XAGE-1b (GAGED2a) protein. 19 The XAGE-1c transcript codes for 9-and 17-a.a. peptides from an ...

The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.