NIK-dependent RelB activation defines a unique signaling pathway for the development of V alpha 14i NKT cells - PubMed (original) (raw)

. 2003 Jun 16;197(12):1623-33.

doi: 10.1084/jem.20030141.

Raziya B Shaikh, Kirsten J L Hammond, Hilde De Winter, Andrew J Leishman, Stephane Sidobre, Olga Turovskaya, Theodore I Prigozy, Lisa Ma, Theresa A Banks, David Lo, Carl F Ware, Hilde Cheroutre, Mitchell Kronenberg

Affiliations

NIK-dependent RelB activation defines a unique signaling pathway for the development of V alpha 14i NKT cells

Dirk Elewaut et al. J Exp Med. 2003.

Abstract

A defect in RelB, a member of the Rel/nuclear factor (NF)-kappa B family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-kappa B-inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/- x aly/+ compound heterozygous mice. After stimulation with alpha-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/- or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

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Figures

Figure 1.

Figure 1.

Vα14_i_ NKT cell deficiency in RelB− / − mice. (A) Representative dot plots showing TCRβ versus α-GalCer/CD1d tetramer binding in the thymus and liver of RelB+/+, RelB+/−, or RelB−/− mice. The average percentage of Vα14_i_ NKT lymphocytes is indicated. Numbers are mean ± SEM of 4 to 17 mice analyzed in each group. (B) Total number of Vα14_i_ NKT cells. Thymus, liver, and spleen mononuclear cells of the indicated mice were labeled with mAbs against TCRβ and α-GalCer/CD1d tetramers. Using the total cell count obtained from each organ, absolute numbers of NKT cells (gated as shown in A) were determined. Numbers are mean ± SEM of 4 to 17 mice analyzed in each group. *Significantly different from RelB+/+ (P < 0.05, Kruskal-Wallis; Dunn's post-hoc test), †RelB+/− versus RelB−/− (P < 0.05, Kruskal-Wallis; Dunn's post-hoc test). (C) Measurement of serum IL-4 upon in vivo administration of α-GalCer. RelB+/+ (n = 7), RelB+/− (n = 3), and RelB−/− (_n_=2) mice on the C57BL/6 background were immunized with α-GalCer (2 μg/mouse) and analyzed 4 h after immunization for serum levels of IL-4 as determined by ELISA. (D) α-GalCer presentation by splenic DCs. (Top panel) Purified DCs from the indicated mice were isolated as described and stained with anti-CD1d mAb or isotype control and analyzed by flow cytometry. Representative histograms for CD1d (black) or controls (open lines) are shown. (Bottom panel) α-GalCer-pulsed DCs were seeded at 6 × 104 cells/well with responder spleen cells from C57BL/6 mice at 2.5 × 105 cells/well. After 3 d of culture, IFN-γ levels were assayed by ELISA. Data represent mean ± SEM of triplicate cultures. One representative experiment of three is shown.

Figure 2.

Figure 2.

Development of CD1d-dependent Vα14_i_ NKT cells is restored in bone marrow chimeras. (A) Representative staining with α-GalCer/CD1d tetramers. Bone marrow chimeric mice were made as described in Materials and Methods, and liver mononuclear cells of the indicated recipient mice were stained with mAb against TCRβ and with α-GalCer/CD1d tetramers. The fraction of total intrahepatic lymphocytes staining with α-GalCer/CD1d tetramers in RAG2−/− recipients reconstituted with either RelB + / + or RelB − / − bone marrow is indicated. The numbers represent mean ± SEM of at least six individual mice in each group. (B) Mononuclear cells of liver, spleen, and thymus were stained with anti-TCRβ and α-GalCer/CD1d tetramers, and the fraction of CD1d tetramer+ cells among the gated, TCRβ+ lymphocytes was determined. Numbers are the mean ± SEM of at least six mice analyzed in each group.

Figure 3.

Figure 3.

The RelB expressing cell required for Vα14_i_ NKT cell development does not need to express β2m. Liver mononuclear cells of the indicated recipient mice were stained with mAb against TCRβ and with α-GalCer/CD1d tetramers. The fraction of TCRβ+ cells staining with α-GalCer/CD1d tetramers in β2m−/− recipients reconstituted with either RelB + / + or RelB − / − bone marrow was determined. Numbers represent mean ± SEM of two to three individual mice analyzed in each group.

Figure 4.

Figure 4.

Vα14_i_ NKT cell deficiency in aly/aly mice. (A) Representative dot plots showing TCRβ versus α-GalCer/CD1d tetramer staining in thymus and liver from aly/+ and aly/aly mice. Percentage of Vα14_i NKT lymphocytes is indicated. Numbers are mean ± SEM of four mice analyzed in each group. (B) Total number of Vα14_i NKT cells. Thymus, liver, and spleen mononuclear cells of the indicated mice were labeled with mAbs against TCRβ and α-GalCer/CD1d tetramers. Using the total cell count obtained from each organ, absolute numbers of Vα14_i_ NKT lymphocytes (gated as shown in A) were determined. Numbers are mean ± SEM of four mice analyzed in each group. *P < 0.05 Mann Whitney U (rank sum) test. This data is representative of two separate experiments in which four mice of each strain were analyzed. (C) Measurement of IFN-γ and IL-4 release upon in vivo administration of α-GalCer. _aly/_+ or aly/aly mice were immunized with α-GalCer (2 μg/mouse) and analyzed four (IL-4) and 16 h (IFN-γ) after immunization. Serum levels of IL-4 and IFN-γ were tested by ELISA. Numbers are the mean ± SEM of four mice analyzed in each group.

Figure 5.

Figure 5.

LTβR mediated activation of RelB through NIK. (A) ICAM-1 induction by LTβR stimulation. Fibroblasts from the kidneys of RelB + / + or RelB − / − mice were stimulated with an agonistic α-LTβR mAb (2 μg/ml) for 24 h. Cell surface levels of ICAM-1 were determined by flow cytometry. Histograms representing ICAM-1 levels before (thin line) and after stimulation (bold line) are shown. One representative example of three independent experiments is shown. (B) NF-κB/Rel binding activities in wild-type and aly/aly mice after LTβR ligation. MEFs from C57BL/6 and aly/aly mice were stimulated with an α-LTβR mAb (2 μg/ml) for 8 h. Nuclear extracts were prepared from unstimulated and LTβR triggered cells. Extracts were incubated with a palindromic κB-binding site as described in Materials and Methods. The results from addition of specific anti-sera against RelA (p65) and RelB are indicated at the bottom. One representative experiment of three is shown.

Figure 6.

Figure 6.

In vivo requirement of NIK for RelB activation. (A) The number of Peyer's patches depends upon RelB and NIK. Peyer's patches were counted in RelB − / −, aly/aly mice, and RelB/aly compound heterozygotes. Each dot represents the number of Peyer's patches in an individual mouse and the horizontal bar indicates the average value. *RelB + / − × _aly/_+ versus _aly/_+ and RelB + / − mice (P < 0.05, Student's t test). (B) Development of normal sized Peyer's patches is RelB dependent. Shown are representative Peyer's patches in adult RelB − / −, RelB + / −, and RelB + / + mice. (C) Measurement of IFN-γ release upon in vivo administration of α-GalCer. RelB + / −, RelB + / − × _aly/_+ compound heterozygotes and _aly/_+ mice were immunized with α-GalCer (2 μg/mouse) and analyzed 16 h after immunization. Serum levels of IFN-γ were tested by ELISA. Numbers represent mean ± SEM of four to six individual mice analyzed in each group. *RelB + / − × _aly/_+ versus _aly/_+ and RelB + / − mice (P < 0.05, Student's t test).

Figure 7.

Figure 7.

Vα14_i_ NKT cell differentiation, not homeostasis, is affected by disrupting NIK signaling. (A) Homeostatic proliferation of α-GalCer reactive Vα14_i_ NKT cells. CD45.1+, CD8 depleted thymocytes were labeled with CFSE and adoptively transferred to CD45.2+ _aly/_+ or aly/aly recipients as described. 7 d after transfer the number of cell divisions of CD45.1+ α-GalCer/CD1d-tetramer+ NKT cells in the liver of the recipient mice was analyzed by flow cytometry. Histograms representing CFSE staining in gated tetramer+ cells in aly/+ or aly/aly recipients are shown. One representative example of four independent experiments is shown. (B) Maturity of thymic NKT cells. Thymocytes of the indicated mice were stained with mAbs against TCRβ, CD44, NK1.1, and with α-GalCer/CD1d tetramers. Vα14_i NKT cells were gated as shown in Fig. 1 A and Fig. 3 A and analyzed for the expression of CD44 and NK1.1. The average percentage of CD44+NK1.1+ Vα14_i NKT cells is indicated. Numbers are mean ± SEM of four (aly/aly) or five (C57BL/6) mice analyzed in each group.

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