Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells

Background. Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natura l killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neo. natal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell re constitution, with less reliable tolerance induction. For a number of reaso ns, it would be advantageous to use neonatal rather than fetal pigs as dono rs. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved o utcomes to be achieved. Materials and methods. Multiple or single fragments of neonatal porcine thy mus tissue were grafted with a splenic fragment to different sites (mediast inum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK- cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitonea l injection containing 1 x 10(7) donor splenocytes. Donor-specific skin gra ft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xen oantibodies were followed by flow cytometry. Results. Peripheral repopulation of CD4(+) cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tis sue in either the mediastinum or the mesentery, but not in mice grafted und er both kidney capsules with the same amount of tissue. The level of CD4 re constitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five per. cent of mice with >20% of CD4 (+) cells among peripheral blood lymphocytes (PBL) by 13 weeks posttranspla ntation accepted donor porcine skin, while rejecting either non-donor neona tal porcine or mouse BALB/c skin allografts. In contrast, only 29% of graft ed mice with <20% CD4(+) cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either lo w or high levels of anti-pig xenoantibodies of the IgM, IgG(1), and IgG(2a) isotypes. Grafted mice with >20% CD4(+) cells all had low levels of anti-p ig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MIR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. Conclusion. Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 co mpartment than does grafting under the kidney capsule. Good CD4 reconstitut ion was associated with optimal donor-specific skin graft tolerance and avo idance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.