Transplant tolerance to cardiac allografts induced in neonatal mice with allogeneic neonatal liver and thymic cells

Introduction: Medawar showed that donor-specific tolerance induced in neonatal mice by injection of adult-derived semi-allogeneic bone marrow and spleen cells resulted in long-term acceptance of skin allografts; we subsequently demonstrated that this protocol also induced tolerance to cardiac allografts. In human infants, transplants (Tx) require allogeneic donors of comparable age given the size and functional constraints of the transplanted organ. Hematopoietic stem/progenitor cells and T cells play important roles in tolerance, and in neonatal mice reside in liver and thymus respectively. We now tested neonatal liver (NLC) and thymus (NTC) cells from fully allogeneic donors together with anti-CD8a (T cell-depleting) and anti-CD154 (co-stimulation blockade) antibodies (mAbs) to induce tolerance in neonatal mice.  
Methods: C3H/He (C3H; H-2^k) neonates were injected i.v. with neonatal GFP+ B6 (H-2^b) NLC/NTC 1:1 mixture (~15x10⁶ cells/mouse) + CD8a + CD154 mAbs. Trafficking of injected cells was monitored by whole body/organ imaging; donor/host cells were identified by immunostaining with high resolution microscopy. Hearts from B6 donors were transplanted at age 6 wks to test tolerance induction in neonatally-treated (n=9) or untreated mice (n=3).
Results: Six days after allo-NLC/NTC + CD8a + CD154 antibody injection, whole organ imaging detected donor cell chimerism in spleen, liver, lymph node, lung, and bone. Donor GFP+ cells in spleen white pulp regions included both T (CD3) and B (B220) cells and in red pulp regions monocytes (CD11b, infrequent) and megakaryocytes (CD41). Host CD4 Foxp3 Tregs were observed interacting with donor single-positive CD4 T cells expressing little if any Foxp3. At 100d post-injection persistent chimerism in spleen, thymus, liver, lymph node, and bone was verified by immunostaining with anti-GFP antibody to include donor macrophages (F4/80), B cells (B220) and few T cells (thymus). GVHD was not observed. At 100d post-Tx, allografts were beating in all treated recipients (score range 2-4; median beat score = 4); all control animals rejected grafts by day 11. H&E-stained cryosections of a B6 heart Tx in a neonatally-treated C3H mouse (post-tx d100) showed normal cardiomyocyte structure with sparse if any cellular infiltration (myocardium); CD8 T cells were present primarily in the pericardium and CD4 Tregs were present without damage, suggesting they play active roles in the tolerance process. In contrast, a rejected allograft (post-Tx d20) in an untreated mouse showed extensive cardiomyocyte damage and considerable cellular infiltration that included many CD8 T cells.
Conclusion: Neonatal injection of allo-NLC/NTC + CD8a + CD154 mAbs resulted in long-term chimerism without GVHD and 100% heart allograft survival at 100 days, demonstrating that neonatal tolerance can be induced with fully allogeneic cells from age-matched neonatal donors. Studies are underway to further analyze the nature of the induced tolerance.