Immunoactive Tripeptide RMK Alters T-Cell Subset Proportions and Remodels the Gut Microbiome–Metabolite Axis in S180 Tumor-Bearing Mice

Background: Immunoactive peptides are emerging as safe oral immunomodulators, but whether a single tripeptide can alter T-cell subsets and the gut microbiome–metabolite axis is unclear. Methods: The synthetic tripeptide RMK (Arg–Met–Lys; HPLC purity > 98%) was given orally once daily to S180 sarcoma-bearing Kunming mice from 7 days before to 14 days after tumor inoculation, a host-preconditioning plus continuous-exposure regimen. Results: RMK reduced tumor growth at both doses (inhibitory rates 40.03% and 48.58%), attenuated thymic atrophy and splenomegaly, and was accompanied by higher proportions of thymic CD4+CD8+ double-positive cells, splenic CD4+ and CD8+ T cells and tumor-infiltrating CD8+ T cells. Tumor-derived suspensions showed mitochondrial depolarization and ROS accumulation, readouts of mitochondrial and redox status that do not identify a cell-death pathway. 16S rRNA sequencing showed higher relative abundances of Lachnospiraceae and Ruminococcaceae, although community structure did not differ significantly. Untargeted metabolomics showed altered bile acid, sphingolipid and steroid-hormone metabolites, with lower aldosterone and cortisol than in tumor-bearing controls. Molecular docking, as an exploratory prediction only, gave the most favorable poses for MHC-I/II (H-2Kb, ΔG = −7.09 kcal/mol) and TCR-proximal kinases (AKT1, ZAP-70, Lck). Conclusions: These findings identify RMK as a candidate oral immunomodulatory peptide whose site of action remains to be defined.