Abstract
Abstract: :
Purpose:Previously we reported that ACAID is dependent on invariant (i)NKT cells for the generation of antigen specific efferent CD8+ T regulatory cells (Tr). Here we show that the subpopulation of CD4+ iNKT cells that is responsible for tolerance induction expresses Ly49 C/I. Another purpose of this study was to determine if there were a role for the Ly49 C/I molecule in the generation of ACAID. Materials and Methods: ACAID was induced in vivo by inoculation of antigen into the anterior chamber of mice that were treated with or without antibodies specific for the inhibitory Ly49 C/I or Ly49 G2 molecules. The development of ACAID was assessed by the ability of the experimental group to show suppressed DTH response. In vitro ACAID was performed with TGFß2 treated, antigen pulsed, APCs which were cultured with whole spleen cells with or without the addition of F(ab’)2 Ly49 C/I antibodies. NKT cells were enriched from the non–adherent cell population and IL–10 mRNA was assessed by RT–PCR. Flow cytometry was used to verify that Ly49 C/I was expressed by CD4+ iNKT cells in ACAID. Results:In vivo, Ly49 C/I (1F8 and 5E6 F(ab’)2), and not by Ly49 G2 (4D11) antibodies blocked the induction of ACAID in C57 B1/6 mice. Moreover, the addition of F(ab’)2 Ly49 C/I antibodies to in vitro cultures of ACAID conferring APCs and splenocytes interfered with the production of IL–10 by NKT cells. Flow cytometry showed that CD4+ iNKT cells increased in ACAID and that they expressed Ly49 C/I. Conclusions:The expression of Ly49 C/I on iNKT cells is required for the development of Tr cells and ACAID. Previously, we showed that interaction of the iTCR on the NKT cell with CD1d is required for the production of NKT cell derived IL–10. Data here suggest that Ly49 C/I may serve as a second signal for the production of NKT cell derived IL–10 during ACAID. Thus, while ligation of the Ly49 inhibitory molecule with its MHC class I ligand is known to inhibit NK cell mediated lysis and IFN gamma production, this report suggests that the ligation of the Ly49 inhibitory molecule during immunosuppressive responses induces the production of immunosuppressive cytokines.
Keywords: ACAID • immunomodulation/immunoregulation • inhibitory receptors