Role of the Cytokine Profiles Produced by Invariant Natural Killer T Cells in the Initial Phase of Cyclophosphamide-Induced Tolerance

Abstract

Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance induction protocol. Recently, we reported that invariant natural killer T (iNKT) cells were essential for the tolerance induction in this system. In this study, we evaluated the roles of the cytokines produced by iNKT cells. DBA/2 (H-2d) mice and BALB/c (H-2d) wild-type (WT) or iNKT knock out (KO) mice were used as donors and recipients. WT recipients received three doses (days -7, -4, -1 or 35, 38, 41) or a single dose (day -1 or 0) of alpha-galactosylceramide (GC) in conjunction with our conditioning regimen that consisted of 10(8) donor spleen cells on day 0 and 200 mg/kg CP on day 2. To investigate the iNKT cell function, iNKT KO recipients were reconstituted with cytokine (interferon-gamma, interleukin [IL]-4, or IL-10) KO iNKT cells and received donor spleen cells and CP. Mixed chimerism was observed in WT recipients, but was reduced in iNKT KO recipients. However, mixed chimerism was absent in WT recipients given GC on days -7, -4, -1, but not in WT recipients given GC on day 35, 38, 41. Donor skin grafts were chronically rejected when mixed chimerism was diminished. Skin grafts were accepted in iNKT KO recipients reconstituted with iNKT cells from interferon-gamma, IL-4, or IL-10 KO mice and receiving our conditioning regimen. Invariant NKT cells were required in the initial phase of the induction of chimerism. Our results indicated that known major cytokines produced by iNKT cells were dispensable for the regulatory function of iNKT cells.