Natural killer cell licensing in mice with inducible expression of MHC class I

Abstract

Mouse natural killer (NK) cells acquire effector function by an education process termed "licensing" mediated by inhibitory Ly49 receptors which recognize self-MHC class I. Ly49 receptors can bind to MHC class I on targets (in trans) and also to MHC class I on the NK-cell surface (in cis). Which of these interactions regulates NK-cell licensing is not yet clear. Moreover, there are no clear phenotypic differences between licensed and unlicensed NK cells, perhaps because of the previously limited ability to study NK cells with synchronized licensing. Here, we produced MHC class I-deficient mice with inducible MHC class I consisting of a single-chain trimer (SCT), ovalbumin peptide-β2 microgloblin-H2K(b) (SCT-K(b)). Only NK cells with a Ly49 receptor with specificity for SCT-K(b) were licensed after MHC class I induction. NK cells were localized consistently in red pulp of the spleen during induced NK-cell licensing, and there were no differences in maturation or activation markers on recently licensed NK cells. Although MHC class I-deficient NK cells were licensed in hosts following SCT-K(b) induction, NK cells were not licensed after induced SCT-K(b) expression on NK cells themselves in MHC class I-deficient hosts. Furthermore, hematopoietic cells with induced SCT-K(b) licensed NK cells more efficiently than stromal cells. These data indicate that trans interaction with MHC class I on hematopoietic cells regulates NK-cell licensing, which is not associated with other obvious phenotypic changes.

Keywords: immunity; lymphocytes; tolerance.

Fig. 1.

Inducible SCT-K^b mice. (A) A schematic figure of inducible SCT-K^b Tg mice. (B) At day 1 after i.p. injection of doxycycline or PBS, SCT-K^b expression on splenocyte subsets was assessed by flow cytometry in ROSA-rtTA β2m^−/− mice treated with doxycycline (thin line), TRE-SCT Tg β2m^−/− mice treated with doxycycline (dotted line), and (TRE-SCT Tg × ROSA-rtTA) β2m^−/− mice treated with PBS (gray shading) and doxycycline (thick line). (C) Immunohistochemistry of spleen for SCT-K^b and VCAM-1 expression as indicated 1 d after i.p. injection of doxycycline. VCAM-1 is a marker of splenic stromal cells.

Fig. 2.

SCT-K^b induction specifically licensed Ly49C⁺ NK cells but not Ly49A⁺ NK cells. Doxycycline or PBS was i.p. injected into the indicated mice on days 1 and 3. Splenocytes were stimulated with plate-bound anti-NK1.1 antibody and analyzed for IFN-γ production by CD3⁻/CD19⁻/NKp46⁺ cells. (A) Representative dot plots demonstrating IFN-γ production by Ly49C⁺ or Ly49C⁻ NK cells. Data are representative of three independent experiments. (B and C) Frequency of IFN-γ–producing NK cells in the Ly49C⁺ and Ly49C⁻ populations (B, Left) and Ly49A⁺ and Ly49A⁻ populations (C, Left) and the ratio of IFN-γ–producing Ly49C⁺ NK cells/Ly49C⁻ NK cells (B, Right) and IFN-γ–producing Ly49A⁺ NK cells/Ly49A⁻ NK cells (C, Right). (D) The ratio of IFN-γ–producing Ly49C⁺ NK cells to Ly49C⁻ NK cells on days 1 and 3 of doxycycline treatment. Dox, doxycycline. Data in B–D are shown as mean ± SEM (n = 3). *P < 0.01 by unpaired Student t test.

Fig. 3.

NK-cell maturation is not enhanced after doxycycline treatment or SCT-K^b induction. The mice were i.p. injected with doxycycline on days 1 and 3. Markers of NK cell maturation were assessed by flow cytometry. (A) Representative dot plots of CD27 and CD11b expression on CD3⁻/CD19⁻/NK1.1⁺ cells. (B) Representative histograms depicting markers of NK-cell maturation. PBS treatment (red line) and doxycycline exposure on day 1 (green line) and day 3 (blue line) are shown. Data are representative of three independent experiments.

Fig. 4.

Cis interaction is dispensable for NK-cell licensing. (A and B) ROSA-rtTA β2m^−/− (non-Tg) NK cells or (TRE-SCT Tg × ROSA-rtTA) β2m^−/− (Tg) NK cells were labeled with CFSE and adoptively transferred into Tg or non-Tg mice. At days 7 and 9 posttransfer, doxycycline was injected i.p.. At day 10, CSFE-labeled (donor) and unlabeled (host) CD3⁻/CD19⁻/NK1.1⁺ cells were assessed for SCT-K^b expression and IFN-γ production by plate-bound anti-NK1.1 stimulation. (A) Histograms depicting expression of SCT-K^b by donor and host NK cells. Data are representative of three independent experiments. (B) Frequency of IFN-γ–producing NK cells in the Ly49C⁺ or Ly49C⁻ population (Left) and the ratio of IFN-γ–producing Ly49C⁺ NK cells/Ly49C⁻ NK cells (Right) for donor and host NK cells, as indicated. Data in B are shown as mean ± SEM (n = 2 samples measured in duplicate). *P < 0.01 by unpaired Student t test.

Fig. 5.

NK cells were licensed through trans interaction with hematopoietic cells. Ly5.1/β2m^−/− (Ly5.1/non-Tg) and Ly5.2/(TRE-SCT Tg × ROSA-rtTA)β2m^−/− (Ly5.2/Tg) BM cells were i.v. injected into irradiated (9.5 Gy) Ly5.2/Tg mice and Ly5.1/non-Tg mice, respectively. At 6 wk after transfer, the mice were treated with doxycycline on days 1 and 3. Then splenocytes were stimulated with plate-bound anti-NK1.1 to analyze the frequency of IFN-γ–producing NK cells among the Ly49C⁺ or Ly49C⁻ population. (A) SCT-K^b expression of donor and host CD3⁻/CD19⁻/NK1.1⁺ cells in the BM chimeras. (B) Frequency of IFN-γ–producing NK cells in the Ly49C⁺ or Ly49C⁻ population (Left) and the ratio of IFN-γ–producing Ly49C⁺ NK cells/Ly49C⁻ NK cells (Right) for donor and host NK cells, as indicated. Data in B are shown as mean ± SEM (n = 2 samples measured in duplicate). *P < 0.01 by unpaired Student t test.