BACKGROUND M290SAP, a murine CD103 antibody conjugated with the immunotoxin saporin, has been found to induce the indefinite acceptance of transplanted pancreatic islets in mice. We sought to understand the underlying mechanism of this alloacceptance, particularly with respect to the CD4 CD25 T regulatory phenotype. METHODS In this study, we established the kinetics of M290SAP and evaluated the requirement of alloantigen for the induction and maintenance of CD4 CD25 T regulatory cells (Tregs). Naive C57BL/6 mice were treated with several doses of M290SAP with and without donor-specific blood or splenocytes. Blood and spleens were collected at specific time points and underwent FACS analysis. RESULTS M290SAP significantly depleted CD103 cells and induced the up-regulation of CD4 CD25 T regulatory population in spleen cell preparations. The combination of alloantigen in the form of donor-specific blood or splenocytes, with M290SAP, further induced the up-regulation of CD4 CD25 Tregs in the spleen compared with either M290SAP alone or alloantigen alone. The generation of CD4 CD25 cells and the depletion of CD103 cells reached a maximum at 7 d and by 3 wk CD103 and CD4 CD25 T regulatory cell populations returned to baseline. When multiple antigenic challenges were administered, the splenic CD4 CD25 cell population was again up-regulated and persisted for 3 wk. CONCLUSIONS Our data confirm that M290SAP induces the generation of the CD4 CD25 T regulatory phenotype in spleens of naïve mice. Alloantigen further enhances and rejuvenates the CD4 CD25 cell population in mice treated with M290SAP.