Type 1 diabetes (T1D) arises secondary to immune-driven destruction of pancreatic b- cells and manifests as insulin-deficient hyperglycemia. We showed that oral vaccination with live attenuated Salmonella, which simultaneously delivers autoantigens and a TGFb expression vector to immune cells in the gut mucosa, provides protection against the progression of T1D in non-obese diabetic (NOD) mice. In this study we employed the Sleeping Beauty (SB) transposon system that is composed of a transposase and transposon encoding the td-Tomato to express red fluorescent protein (RFP) to permanently mark the cells that take up the Salmonella vaccine. After animal vaccination, the transposon labeled-dendritic cells (DCs) with red fluorescence appeared throughout the secondary lymphoid tissues. Furthermore, Sleeping Beauty containing tgfb1 gene (SB-tgfb1) co-expressed TGFb and RFP. The labeled DCs were detected predominantly in Peyer’s patches (PP) and mesenteric lymph nodes (MLN) and expressed CD103 surface marker. CD103C DCs induced tolerogenic effects and gut homing. TGFb significantly increased programmed death-ligand-1 (PDL-1 or CD274) expression in the DCs in the MLN and PP of treated mice. Also, TGFb increased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) levels in CD4C cells in MLN and PP. Interestingly, DCs increased in all lymphatic organs of mice vaccinated with oral live Salmonella-based vaccine expressing preproinsulin (PPI), in combination with TGFb, IL10, and subtherapeutic-doses of anti-CD3 mAb compared with vehicletreated mice. These DCs are mostly tolerogenic in MLN and PP. Furthermore the DCs obtained from vaccine-treated but not vehicle-treated mice suppressed in vitro T cell proliferation. These data suggest that the MLN and the PP are a central hub for the beneficial anti-diabetic effects of an oral Salmonella-based vaccine prevention of diabetes in rodents. Keywords: Salmonella-based