MHC class II-expressing thymocytes suppress invariant NKT cell development

Abstract

Natural killer T (NKT) cells are positively selected on cortical thymocytes expressing the non-classical major histocompatibility complex (MHC) class I CD1d molecules. However, it is less clear how NKT cells are negatively selected in the thymus. In this study, we investigated the role of MHC class II expression in NKT cell development. Transgenic mice expressing MHC class II on thymocytes and peripheral T cells had a marked reduction in invariant NKT (iNKT) cells. Reduced numbers of iNKT cells correlated with the absence of in vivo production of cytokines in response to the iNKT cell agonist alpha-galactosylceramide. Using mixed bone marrow chimeras, we found that MHC class II-expressing thymocytes suppressed the development of iNKT cells in trans in a CD4-dependent manner. Our observations have significant implications for human iNKT cell development as human thymocytes express MHC class II, which can lead to an inefficient selection of iNKT cells.

Figure 1

Reduction of iNKT cells in CIITA^Tg mice. Expression of TCRβ and NK1.1 markers (a) or TCRβ and tetramer (b) on indicated cell types from WT (CIITA^Tg littermates), CIITA^Tg, and CD1d^−/− mice. The number in the dot plots represents the percentage of NK1.1⁺TCRβ⁺ NKT cells (a) or iNKT cells (b). Mice used were 10–11 weeks old and similar results were obtained from at least four mice per group. (c) Absolute numbers of iNKT cells. Values are mean ± SD of iNKT cells present in thymi (n=10), spleens (n=6), and livers (n=4). Mice were 2–3 months old. (d) Representative iNKT cell staining of 3 pairs of WT (CIITA^Tg littermates) and CIITA^Tg on the H-2^d background. (e) Analysis of NK1.1 and CD44 expression on thymic iNKT cells. Tetramer⁺HSA^lo cells were gated and percentages of cells in each quadrant are shown. Results are representative of four pairs of WT and CIITA^Tg littermates. Mice were 10 weeks old.

Figure 2

MHC class II inhibits iNKT cell development. (a) MHC class II expression in thymocytes is necessary for the inhibition of iNKT cell generation. Total thymocytes from the indicated mice were analyzed for TCRβ and tetramer reactivity. WT and CIITA^Tg mice on the Abb^+/− and Abb^−/− background were littermates. Results are representative of at least 4 experiments. (b) Relative percentages of thymic iNKT cells in at least 8 mice of Abb^+/−, CIITA^Tg/Abb^+/−, CIITA^Tg /Abb^−/−, or Abb^−/−. The value of the percentage of iNKT cells in WT mice was set at 100%. Mice were 2–3 months old. (c) Suppression of iNKT cell development by MHC class II-expressing thymoyctes in trans. BM cells from WT (CD45.2) and WT (CIITA^Tg littermates; CD45.1/CD45.2) or WT (CD45.2) together with CIITA^Tg (CD45.1/CD45.2) were co-injected into B6.SJL (CD45.1) recipients. Shown at the top is the composition of total thymocytes in reconstituted mice. TCRβ versus tetramer profiles of thymocytes are shown at the bottom. Chimeras were analyzed 10–12 weeks post BM transplantation. (d) MHC class II and CD4 expression is required for the disappearance of iNKT cells in mixed BM chimeric mice as indicated. BM were prepared from WT (CD45.1/2), Abb^−/− (CD45.2), CIITA^Tg/Abb^−/− (CD45.2), CIITA^Tg/CD1d^−/− (CD45.2), CIITA^Tg (CD45.1/2), and CD4^−/− (CD45.2) mice and transferred to B6.SJL mice. The number in the dot plots represents the percentage of iNKT cells developed from WT BM cells except the bottom group that shows CD4^−/− iNKT cells. Chimeras were analyzed 2–3 months post BM transplantation.