Results: We identified that Th SD4 subset appeared to be primary target of CTLA-4 therapy. The effect consisted of increased ratio of Th/CD8 and skew of Th cells from IFNg-secreting towards proliferating cells within the tumor. Analysis of major lymphocyte subsets from LN revealed an increased percentage of activated CD69+/ CD25+ cells from Th, CD8+ and B cells and IFNg+ secretion by Th. We found that the number of TCR clusters significantly increased in Th after therapy. This indicates proliferation of certain Th clones, yet with unknown specificities. To identify the antigen specificity of these clones we immunized mice with melanoma peptide neoantigens and harvested T-cells from LN. Then T-cells were cocultured with APCs loaded with different melanoma peptides. We identified up to 75% CD69+/CD25+ activated T-cells to the specific peptides. We detected clones of TCR, which were found with a high frequency in cells reactivated to specific peptides and were not found in the control. Now the data set is in progress. Future experiments will be directed to compare B16F0-specific TCRs with proliferative clones after anti-CTLA4 blockade.Conclusions: We found that anti-CTLA-4 immunotherapy leads to activation and clonal expansion of lymphocytes with similar TCRs. For detailed analysis, we developed the approach to identify the TCR specificity to specific B16F0 peptides.
The purpose of the present study was to investigate how the effects of high-fat diet feeding on the skeletal muscle persisted during aging using mice. Post-weaned male mice were fed a high-fat diet between 1-and 3-mo-old followed by return to supply a normal diet until 13-mo-old. Monthly physical tests demonstrated that age-related glucose intolerance that was generally developed after 10-mo-old in the control mice was significantly improved in mice fed a high-fat diet. Interestingly, mRNA expressions of Pdk4, Ucp3, and Zmynd17 were up-regulated by high-fat feeding and persisted in the tibialis anterior muscle until 13-mo-old. At Pdk4 and Ucp3 loci, enhanced distributions of active histone modifications were noted in the high-fat-fed mice at 13-mo-old. In contrast, age-related accumulation of histone variant H3.3 at these loci was suppressed. These results indicated that epigenetic modifications caused by early nutrition mediated the changes in skeletal muscle gene expression during aging.
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