The Vα14i-TCR knock-in mouse produces large numbers of correctly selected, bona fide NKT cells.

<p>(A) Schematic representation of the knock-in transgene. The <i>Vα14</i> promoter, <i>loxP</i> (triangle)-flanked STOP cassette, and pre-rearranged <i>Vα14i</i> (Vα14-Jα18, red square) sequences were inserted 3′ of <i>Jα1</i> and 5′ of the first <i>Cα</i> exon (coding exons are highlighted in blue); 4pA = 4 SV40 polyadenylation sites. AH, arms of homology. <i>Eα</i>, enhancer (black oval). (B) Representative proportions of NKT cells and conventional T cells of total lymphocytes in thymus and spleen. Numbers indicate mean percentages ± SD of at least seven age-matched mice per genotype. (C) Representative proportions of splenic CD4+, CD8+, and DN (CD4− CD8−) NKT cells. Numbers indicate mean percentages ± SD of seven mice per genotype. (D) The Vβ repertoires of splenic NKT cells of the indicated genotypes. Bars indicate means and error bars SD of three independent experiments. (E) Representative flow cytometric analysis of the indicated cell-surface proteins on conventional CD4+ T cells and NKT cells. (F) Intracellular flow cytometric staining of PLZF, GATA-3, ROR-γt, and Th-POK in the depicted NKT cells. Numbers indicate means of the median fluorescence intensities (MFIs), normalized to CD4+ tetramer− T cells of CTR animals, or percentage of ROR-γt+ cells among DN NKT cells; calculated from three animals per genotype. Histograms are representative of three independent experiments with eight mice in total. Throughout the figure, NKT cells were gated as tetramer+ TCRβ+, conventional (conv) T cells as tetramer− TCRβ+; CTR, <i>CD4-Cre</i> or <i>Vα14i^StopF/wt</i>.</p

TCR switch on mature conventional T cells.

<p>(A) Genetic set-up of the TCR switch experiment. In <i>Mx-Cre Cα^F/Vα14i^StopF</i> mice, the endogenous TCRα-chains (Vα(x)Jα(y)) are exclusively expressed from the <i>Cα^F</i> allele. Cre-mediated recombination leads to termination of expression from the <i>Cα^F</i> allele, and simultaneous start of expression of the Vα14i-TCRα-chain from the <i>Vα14i^StopF</i> allele. (B) T-cell-deficient mice were reconstituted with NKT cell-depleted splenocytes of the indicated genotypes. After 2 wk, the TCR switch was induced by poly(I:C) injection. Eight weeks later, percentages of tetramer+ and tetramer− T cells (TCRβ+) were analyzed in spleen and liver. Black numbers indicate percentages of total lymphocytes, red numbers absolute cell number calculated from 9–17 animals. (C) Bars indicate means and SD (error bars) of CD4+, CD8+, or DN (CD4− CD8−) cells among tetramer− and tetramer+ T cells, calculated from at least nine mice per genotype. (D, E) The Vβ repertoires of the depicted splenic CD4+ (D) or CD8+ (E) T cell subsets isolated from T-cell-deficient animals that received NKT cell-depleted <i>Mx-Cre Cα^F/Vα14i^StopF</i> splenocytes. Some of these mice were injected with poly(I:C) 2 wk later to induce the TCR switch. Eight weeks after poly(I:C) injection, the Vβ repertoires were analyzed. Data represent means and SD (error bars) of two independent experiments with a total of three mice (tetramer− without poly(I:C) injection) or eight mice (poly(I:C) injected) per T cell population. Vβs typical for glycolipid selection of NKT cells are highlighted in red.</p

TCR signaling is not required for the steady state homeostasis of mature NKT cells.

<p>(A) Percentages of TCRβ− cells of the depicted T cell subsets 2 wk after poly(I:C) injection into <i>Mx-Cre Cα^F/F</i> mice. Bars show means and SD (error bars) of 3–5 mice. (B) Surface TCRβ expression of splenic CD4+ NKT cells (NK1.1+ CD5+ CD62L^low) 2 wk after poly(I:C) injection. Numbers indicate means ± SD of three independent experiments with altogether five mice per genotype. (C, D) Total cell counts of splenic naïve conventional CD4+ T cells (CD5+ CD44^low NK1.1−; C) or of memory/effector-like CD4+ T cells (CD5+ CD44^high NK1.1−; D) from 26 control <i>Cα^F/F</i> (CTR, TCR+) animals as well as from 24 <i>Mx-Cre Cα^F/F</i> animals, all after poly(I:C) injection (TCR+, TCR−). (E) Splenic CD4+ NKT cell numbers from in total 32 control <i>Cα^F/F</i> animals (CTR, TCR+) as well as TCRβ+ and TCRβ− CD4+ NKT cell numbers from in total 27 <i>Mx-Cre Cα^F/F</i> animals, at the indicated time after poly(I:C) injection. (C–E) Half-lives were calculated with GraphPad Prism software using nonlinear regression, one-phase decay analysis. (F) BrdU was administered for 4 wk via the drinking water, starting 2 wk after poly(I:C) injection. Directly afterwards, animals were sacrificed and BrdU incorporation was measured by flow cytometry. Representative blots of 2 <i>Cα^F/F</i> and 4 <i>Mx-Cre Cα^F/F</i> mice are shown. (G) Bar chart showing proportion of cells that incorporated BrdU of the indicated T cell subtypes. Bars show means calculated from 2 <i>Cα^F/F</i> and means and SD (error bars) 4 <i>Mx-Cre Cα^F/F</i> mice. *** <i>p</i><0.001; * <i>p</i><0.05; ns, not significant; one-way ANOVA.</p

TCR-signals are not required for the innate activation of NKT cells.

<p>(A) Intracellular Egr2 expression of the depicted splenic T cells, 90 min after PBS or αGalCer injection, or 6 h after LPS injection. Plots are representative for at least three independent experiments. (B) Intracellular IFN-γ expression of the depicted cells stained directly ex vivo without addition of brefeldin or monensin. Splenic cells were isolated 90 min after PBS or αGalCer injection, or 6 h after LPS injection. Plots are representative for at least three independent experiments.</p

Premature Vα14i-TCR expression impairs NKT and conventional T cell development.

<p>(A–E) Absolute cell numbers in thymus and spleen of 7–13 mice of the indicated genotypes: NKT cells (A), CD4+ NKT cells (B), DN NKT cells (C), total cells (D), and DN tetramer− T cells (E). Bars indicate medians. *** <i>p</i><0.001; ** <i>p</i><0.01; * <i>p</i><0.05; ns, not significant; one-way ANOVA. (A) Mean cell numbers are depicted below the scatter blot. (F) The Vβ repertoires of splenic NKT cells of the depicted animals. Data for <i>CD4-Cre Vα14i^StopF/wt</i> are the same as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001589#pbio-1001589-g001" target="_blank">Figure 1D</a>. Bars indicate means and error bars SD of 3–4 mice per genotype of 3–4 independent experiments. Throughout the figure, NKT cells were gated as tetramer+ TCRβ+, conventional (conv) T cells as tetramer− TCRβ+; CTR, <i>CD4-Cre</i> or <i>Vα14i^StopF/wt</i>.</p

The maintenance of NKT lineage identity does not depend on TCR-signals.

<p><i>Cα^F/F</i> and <i>Mx-Cre Cα^F/F</i> mice were injected with poly(I:C) and analyzed 6 wk later. (A, B) Intracellular expression of Egr2 (A) and PLZF (B) in T cells from the depicted mice. Plots are representative for at least three independent experiments. (C) Flow cytometric analysis of NK1.1 expression on splenic naïve (CD62L^high CD5+), memory/effector-like (CD62L^low CD5+) CD4+ T cells, and CD4+ NKT cells (NK1.1+ CD5+ CD62L^low), with or without TCR expression. Median fluorescence intensity, normalized to NK1.1 expression of NK cells (NK1.1+ TCRβ− CD5−). Bars indicate medians. *** <i>p</i><0.001; ** <i>p</i><0.01; * <i>p</i><0.05; ns, not significant; one-way ANOVA. (D) Flow cytometric expression analysis of extra- and intracellular markers of splenic T cells. Median fluorescence intensities of at least four mice per analyzed protein were normalized to the expression on/in conventional CD4+ T cells (tetramer− TCRβ+) to account for interexperimental variations. Expression of NK1.1, CD122, FasL, and T-bet were normalized to NK cells (NK1.1+ TCRβ− CD5−) and then set to 1 for naïve T cells. Data are shown as heatmap, calculated by Perseus software. Blue letters, significantly reduced on splenic TCR− CD4+ NKT cells in comparison to TCR+ CD4+ NKT cells from <i>Cα^F/F</i> and <i>Mx-Cre Cα^F/F</i> mice; analyzed by one-way ANOVA.</p

NKT cell overproduction affects their maturation and NK cell homeostasis.

<p>(A) Intracellular IL-4, IL-13, IFN-γ, and TNF expression of splenic CD4+ NKT cells isolated from the depicted animals 90 min after αGalCer injection. Cells were stained directly ex vivo without addition of brefeldin or monensin. Black numbers indicate mean percentages ± SD, and red numbers indicate mean total NKT cell counts expressing the respective cytokine. Data are from three animals per genotype; FSC, forward scatter. (B) Representative proportions of stage 1 (CD44^low NK1.1^low), stage 2 (CD44^high NK1.1^low), and stage 3 (CD44^high NK1.1^high) thymic and splenic NKT cells. Numbers indicate mean percentages ± SD of 10 mice per genotype. (C) Flow cytometric analysis of the depicted markers on thymic and splenic, transgenic, and control NKT cells. Bars indicate means and error bars SD calculated from 4–7 mice. (D) Extracellular and intracellular flow cytometric stainings of CD69 and T-bet in the depicted NKT cell subpopulations. Numbers in representative histogram indicate percentage of CD69^high or T-bet+ cells among the indicated NKT cells calculated from eight animals per genotype (CD69) or three animals per genotype (T-bet). Histograms are representative of at least three independent experiments with each at least seven mice in total. (E) Absolute splenic NK cell numbers (NK1.1+ TCRβ− tetramer–) of age-matched 6–12-wk-old animals (7–16 per genotype). Bars indicate medians. *** <i>p</i><0.001; ns, not significant; one-way ANOVA. Throughout the figure, NKT cells were gated as tetramer+ TCRβ+, conventional (conv) T cells as tetramer− TCRβ+; CTR, <i>CD4-Cre</i> or <i>Vα14i^StopF/wt</i>.</p

Signs of sterile inflammation in mice harboring TCR-switched T cells.

<p>T-cell-deficient mice were reconstituted with NKT-cell-depleted splenocytes of the indicated genotypes. Spleen weight (A), absolute splenic cell numbers (B–E, G, H), and serum TNF levels (F) of 3–28 mice per genotype were determined 8 wk after poly(I:C) administration where indicated. Bars indicate medians. Red points show six animals with near absence of B cells and dendritic cells. (B) Total splenocytes; (C) Macrophages/monocytes (Mac1+ Gr1^int SiglecF−); (D) Neutrophils (Mac1+ Gr1^high SiglecF−); (E) Erythroblasts (Ter119+); (G) B cells (B220+ TCRβ−); (H) Dendritic cells (CD11c+). *** <i>p</i><0.001; ** <i>p</i><0.01; * <i>p</i><0.05, one-way ANOVA.</p