ZBTB7B (Th-POK) regulates the development of IL-17-producing CD1d-restricted mouse NKT cells

Abstract

CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor γt positive, and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.

Fig 1. CD8+ NKT-cells predominate in a Zbtb7b mutant mouse strain

A: Homozygotes for the helpless mutation (hpls/hpls) have reduced percentage of CD4⁺ T-cells in the peripheral blood and spleen and a block in thymic T-cell development at the CD4⁺ CD8^dim stage. B: T480G mutation in Zbtb7b exon 1, altering codon 102 from leucine to arginine within the BTB-POZ domain. The bottom panel shows an alignment of parts of the BTB-POZ domains from the indicated proteins, with the mutated residue highlighted in red. C: Flow cytometric analysis of spleen cells stained with α-GalCer-loaded CD1d tetramers and antibodies to TCR, CD4, and CD8. Numbers in the upper panel show percentage of spleen cells within the gate, and lower panels show the percentage of these gated NKT-cells that are CD4⁺, CD8⁺ or negative for both. D: Percentage of NKT-cells in thymus, blood, spleen, lymph node, bone marrow (left axis) and liver (right axis) of Zbtb7b^hpls/hpls, Zbtb7b^hpls/+ and Zbtb7b^+/+ mice. Each datapoint represents a different mouse and the bars represent the mean. The data for the percentages and absolute cell numbers of NKT-cells in thymus, spleen and liver are pooled from at 2 (liver and bone marrow) or more different experiments with some animals for the liver data on a mixed CBAxC57BL/6 background. Mixed background mice are depicted with hexagons, pure B6 mice are depicted with triangles. E: Absolute cell number of NKT-cells in thymus, spleen, bone marrow (left axis) and lymph node (LN) and liver (right axis) of Zbtb7b^hpls/hpls, Zbtb7b^hpls/+ and Zbtb7b^+/+ mice. Each datapoint represents a different mouse and the bars represent the mean. The percentage of CD4 and CD8-defined NKT-cellsubsets within thymus, spleen and liver. Each datapoint represents a different mouse. F: Expression of CD8α and CD8β chains on CD4-negative NKT-cells from wild-type and mutant mice in thymus, spleen and liver. Except for liver data for hpls/+ mice all flow cytometric data representative of at least 3 different experiments with at least 2 animals per genotype and experiment. Statistics were calculated using the Kruskal-Wallis test with * = p < 0.05, ** = p < 0.005 and *** = p < 0.0005.

Figure 2. Divergent NKT-cell development in the thymus of Zbtb7b mutant mice

A: Thymic NKT-cells, gated on α-GalCer-CD1d tetramer⁺ and TCRβ⁺ cells, showing subsets resolved by CD44 and NK1.1 expression. The bottom panels are further gated on CD44⁺ NK1.1⁺ mature NKT-cells, showing CD4 and CD8 expression. B: The percentage of CD8⁺ NKT-cells within each stage of hpls/hpls NKT-cell maturation in the thymus, as shown in A (second row). Each symbol represents an individual mouse, data is from at four independent experiments with 2-4 mice per group. C: Mixed bone marrow chimera results showing relative percentage of CD4/CD8 defined NKT-cell subsets in thymus. Each symbol represents a different recipient mouse in a single experiment. Equal mixtures of CD45.1 marked +/+ bone marrow and either CD45.2 hpls/hpls bone marrow (filled circles) or CD45.2 +/+ bone marrow (open circles) were used to reconstitute irradiated CD45.1 recipients. After hemopoietic reconstitution, thymocytes of individual animals were analysed by flow cytometry to identify NKT-cells subsets from either wt or hpls bone marrow origin. D: Relative contribution of hpls/hpls and wild-type cells to the stages of NKT-cell maturation in mixed bone marrow chimeras, generated as described in C and the ratio of CD45.2+ cells to CD45.1+ cells within each subset calculated. To account for small inter-individual differences in overall hemopoietic reconstitution, the CD45.2:CD45.1 cell ratios in NKT-cell subsets of each animal were normalized by dividing by the ratio in DP thymocytes in the same mouse. E: Percentage of NKT-cells staining for the cell cycle marker Ki67 in individual mixed bone marrow chimeras, gated on CD45.2+ hpls/hpls cells (filled circles) or CD45.1+ wild-type cells in the same thymus (open circles). F: Flow cytometric histograms of Ki67 staining in all thymic NKT-cells (top panel) or in the indicated NKT-cell subsets (bottom), showing concatenated data for hpls/hpls and wild-type cells in the thymus from all 4 mice shown in Figure E. G: Percentage of NKT cells in the indicated organs in mixed bone marrow chimeras analysed separately for CD45.1⁺ +/+ and CD45.2⁺ hpls/hpls cells. Statistics were calculated using the Mann-Whitney test, with * = p < 0.05 and ** = p < 0.005.

Figure 3. Altered expression of surface markers on NKT-cells of Zbtb7b^hpls/hpls mice

A: Expression of the indicated surface markers and inhibitory and activating NK receptors on NKT-cells in the thymus, spleen and liver was measured by flow cytometry. Histograms show concatenated samples from all mice shown in panel B. B: Each dot represents an individual mouse, the bar graph shows the mean of all samples. T, S and L denotes Thymus, spleen and liver respectively.

Figure 4. Altered cytokine production by thymic Zbtb7b mutant NKT-cells

A Thymocytes from Zbtb7b mutant and wild-type mice were activated for 2.5h with PMA and Ionomycin and stained for surface markers followed by intracellular staining with antibodies to IL17, IFNγ and TNF or with isotype control antibodies. All plots are gated on NKT-cells (TCRβ⁺, αGalCer-CD1d-tetramer⁺). Gates for cytokine-producing cells were set on isotype control stains, and the numbers show the percentage of NKT-cells within these gates. Data for IL17 is from 3 different experiments on a pure C57BL/6 background. Data for IFNγ production is from two independent experiments, with mice used in experiment 1 from a pure C57BL/6 background, while mice for experiment 2 were on a mixed CBAxC57BL/6 background. B Sorted thymic NKT-cells were stimulated with 10 μg/ml anti-CD3 and anti-CD28, plate-bound CD1d and αGalCer or sorted splenic dendritic cells and αGalCer. After 24 hours the supernatant was assayed for the concentration of the indicated cytokines, determined by a cytometric bead array. Concentrations are in pg/ml, represented on a logarithmic scale. Values below 1pg/ml were given a baseline value of 1. The results are derived from four to ten separate cultures collected over two to three independent experiments. Bars depict mean of all samples collected.

Figure 5. RORγt expression in Zbtb7b mutant NKT-cells

A: Expression of Zbtb7b, RORγt, IL23R, Runx1, Runx3 and Tbet mRNA in sorted thymic NKT-cells from Zbtb7b mutant, heterozygous and wild-type mice was measured by RT-PCR. B and C: Thymocytes and splenocytes from Zbtb7b mutant and wild-type mice were activated for 3 hours in the presence of GolgiStop (BD Pharmingen) with PMA and Ionomycin and stained for surface markers followed by intracellular staining with antibodies recognising IL-17 and the transcription factor RORγt. All plots are gated on NKT-cells (TCRβ⁺ or CD3⁺, PBS57-CD1d-tetramer+). B shows representative FACS plots, C shows the percentages of RORγt⁺ NKT-cells in thymus, spleen, liver and lymph node. Data are representative of two (thymus, liver and spleen) or one (lymph node) experiments with 2-5 mice (C57BL/6 background) per group. Data for heterozygous mice for spleen and liver are from a single experiment. D shows the percentage of IL-17⁺ cells out of all RORγt⁺ NKT-cells in the thymus. E shows the relative expression of RORγt⁺ and T-Bet in individual NKT-cells.