Self-Engagement Of An NKT Cell Inhibitory Receptor During Development Alters Receptor Expression and Cellular Function

Invariant NKT (iNKT) cells have been shown to be pro-inflammatory or anti-inflammatory in many pathologic conditions including infections, cancer, autoimmune diseases and allergy. Additionally, early expansion of iNKT cells correlates with successful engraftment and low incidence of graft-versus host disease suggesting a role for iNKT cells in tolerance to allogeneic transplantation. iNKT cells recognize glycolipids bound to CD1d via their T cell receptor (TCR), but also express the inhibitory receptors for MHC class I ligands (MHC I.) Little is known regarding the importance of iNKT cell self-recognition of MHC I during development and the occurrence of autoimmunity, allergy or tolerance to in utero hematopoietic cellular transplantation (IUHCT). Given the importance of NK cell self-recognition during development, we hypothesized that, like NK cells, self-engagement of MHC I inhibitory receptors during development alters the phenotype and function of self-responsive iNKT cells.

Prenatal allogeneic chimeras were established by in utero transplantation of Balb/c fetal liver light density cells at E14 into age-matched C57BL/6 fetal recipients. A kinetic evaluation of iNKT cell receptor expression and function was performed after birth. iNKT cells were evaluated for expression of activating (Ly49D) and inhibitory (Ly49A, F, G) receptors specific for the donor MHC I (H-2^d.) The functional response of iNKT cell subsets was measured following in vivo activation with KRN7000, the ligand for iNKT cells. Intracellular cytokine production (IFN-gamma and IL-4) production was assessed in the alloresponsive Ly49A+ iNKT cell subset and compared to naïve controls and B10.D2 (H2^d) mice.

We found that a negligible fraction of iNKT cells expressed the activating Ly49D receptor in either chimera or controls (<1%.) In chimeric mice, the frequency of iNKT cells expressing Ly49A, F, or G did not change significantly. However, the levels of Ly49A expression were significantly reduced on iNKT cells from all organs tested suggesting developmental recognition of donor class I ligands had occurred. These reduced receptor levels did not appear to be due to sequestration as acid stripping did not alter the phenotype. Lastly, Ly49A+ but not Ly49A- iNKT cells in chimeric mice exhibited significantly increased production of IL-4 in response to TCR stimulation suggesting a shift toward a regulatory phenotype. Consistent with this finding, IFN-gamma levels in chimeras were minimally changed compared to controls.

These findings indicate that inhibitory receptor self-engagement during development leads to: 1) alterations in receptor expression; 2) shift toward a tolerogenic phenotype in self-responsive iNKT cell phenotypes; and 3) no change in the receptor expression or function of non-responsive iNKT cell phenotypes. Further studies will directly evaluate the impact of self-recognition of iNKT cells in the pathogenesis of autoimmunity and allergy or tolerance following IUHCT.