Antibody-mediated T-cell reduction or increased levels of chimerism overcome resistance to cyclophosphamide-induced tolerance in NKT-deficient mice

We reported that invariant NKT-cell knockout (iNKT KO) mice are resistant to the induction of intrathymic chimerism and clonal deletion in the cyclophosphamide (CP)-induced tolerance system (CPS). However, another report shows that clonal deletion with chimerism may be intact in iNKT KO recipients in a bone marrow transplantation model. We also reported that pretreatment with anti-Thy1.2 mAb, which reduces the number of T cells and iNKT cells, promotes allograft tolerance across H-2 barriers in the CPS. In this study, we evaluated the efficacy of T-cell depletion in the CPS, and the relationship between the role played by iNKT cells in central tolerance and mixed chimerism. BALB/c (H-2^d) wild-type, or iNKT KO (Jα18^-/-) mice were pretreated with 20-100 μg of anti-Thy1.2 mAb and given 10⁸ donor DBA/2 (H-2^d) spleen cells on Day 0, and 200 mg/kg CP on Day 2. Pretreatment with T-cell depletion resulted in higher levels of mixed chimerism, increased intrathymic clonal deletion of donor-reactive cells, and the induction of skin graft tolerance in iNKT KO recipients in CPS. This suggests that the high levels of mixed chimerism overcame the resistance to CP-induced tolerance in iNKT KO mice. Consistently, the enhancement of mixed chimerism by injection of tolerant donor spleen cells (SC) rendered iNKT KO recipients susceptible to CP-induced tolerance. These results suggest that iNKT-cell-mediated immunoregulation of central tolerance is evident at low levels of peripheral mixed chimerism in the CPS.