The NFκB-inducing kinase is essential for the developmental programming of skin-resident and IL-17-producing γδ T cells
- University of Zurich, Switzerland
- Kantonsspital St. Gallen, Switzerland
- Johannes-Gutenberg University of Mainz, Germany
Figures
Figure 1 with 1 supplement
In the absence of NIK, the development of DETCs is blocked in the embryonic thymus.
(A) Lymphocytes isolated from the epidermis of adult heterozygous control (left panel) and Map3k14-/- animals were analysed for the presence of Vγ5+ DETCs. Pregating is on live singlets and CD45+ CD11b- cells. (B) Analysis of the epidermal γδ T cell compartment of heterozygous control (upper panel) and Map3k14-/- embryos (day 19 post conception) after pregating on live singlets and CD45+ CD11b- cells. (C) Summary of the frequency of total γδ T cells as well as Vγ5+ cells within the γδ T cell gate. Data are mean +/- SD and are representative of two similar experiments. (D) Analysis of developing Vγ5+γδ thymocytes in the thymi of E19 embryos. Flow Plots have been pregated on live singlets and CD45+ CD4- cells. Lower panel depicts the summary of the frequency of total γδ thymocytes as well as Vγ5+ cells within the γδ T cell gate in d19 embryonic thymi, and the median fluorescence intensity of the indicated markers. Data are mean +/- SD and representative of two similar experiments. (E) Analysis of the expression level of CD45RB, CD122, CD24 and CD62L on developing Vγ5+γδ thymocytes isolated from E19 embryonic thymi. Grey shaded histograms depict heterozygous controls, red histograms Map3k14-/- cells. Lower panel shows the summary for the frequency of positive cells for CD45RB and CD122 and the median fluorescence intensity of CD24 and CD62L, respectively. Data are mean +/- SD and are representative of two similar experiments.
Figure 1—figure supplement 1
DETC development in NIK-deficient thymi at embryonic day 17.
(A) Analysis of developing Vγ5+ γδ thymocytes in E17 thymi. Flow Plots have been pregated on live singlets and CD45+ CD4- cells. Right panels depict the median fluorescence intensity of CD3 and Vγ5 expression. Data are mean +/- SD and representative of two similar experiments. (B) Analysis of the expression level of CD45RB on developing Vγ5+ γδ thymocytes isolated from E17 embryonic thymi. Grey shaded histograms depict heterozygous controls, red histograms Map3k14-/- cells. Right panel shows the summary for the frequency of CD45RB positive cells. Data are mean +/- SD and are representative of two similar experiments.
Figure 2 with 1 supplement
In NIK-deficient mice, γδ T cells selectively lose their ability for production of IL-17.
(A) Flow cytometric analysis of the splenic γδ T cell compartment of heterozygous control (upper panel) and Map3k14-/- animals after pregating on live singlets and CD45+ CD11b- B220- cells. The distribution of Vγ1.1+, Vγ4+, and CD27- Vγ4+ γδ T cells is shown. (B) Summary of two independent experiments. Data represent mean +/- SD of the indicated cell subsets. (C) Analysis of the lung-resident γδ T cell compartment of heterozygous control (upper panel) and Map3k14-/- animals after pregating on live singlets and CD45+ CD11b- B220- cells. The distribution of Vγ1.1+, Vγ6+, and CD27- Vγ6+ γδ T cells is shown. (D) Flow cytometric analysis of IFN-γ, IL-17 and TNF-α expression by peripheral γδ T cells after PMA/Ionomycin stimulation, isolated from the spleen of heterozygous control (upper panel) and Map3k14-/- animals (lower panel). Left plots have been pregated on CD45+ CD11b- CD4- live singlets. (E) Summary of the frequency of cytokine producing cells within the γδ T cell compartment. Data represent mean +/- SD. (F) Flow cytometric analysis of IFN-γ and IL-17 expression by lung-resident γδ T cells isolated from the lung of heterozygous control (upper panel) and Map3k14-/- animals (lower panel). Left plots have been pregated on CD45+ CD11b- CD4- live singlets. (G) Summary of the frequency of cytokine producing cells within the lung-resident γδ T cell compartment. Data represent mean +/- SD. (H) Flow cytometric analysis of IFN-γ and IL-17 expression by dermal γδ T cells isolated from the skin of heterozygous control and Map3k14-/- animals. Right panel depicts the frequency of cytokine producing cells within the dermal γδ T cell compartment. Data show mean +/- SD and are representative of at least three independent experiments.
Figure 2—figure supplement 1
Ratio of Vγ4+ and Vγ6+ cells in the lung-resident IL-17-producing γδ T cell compartment.
(A) Flow cytometric analysis of Vγ4+ and Vγ6+ cells within IFN-γ and IL-17 expressing γδ T cells isolated from the lung of heterozygous control (upper panel) and Map3k14-/- animals (lower panel). Left plots have been pregated on CD45+ CD11b- CD3+ TCRγδ+ live singlets. (B) Summary of the frequency of Vγ4+ and Vγ6+ cells within the lung-resident γδ T cell compartment. Data represent mean +/- SD.
Figure 3
NIK-deficient CD27- γδ T cells show reduced expression of Rorc and and Sox13.
(A) Gating strategy used for sorting of CD27+ and CD27-γδ T cells. Right plot depicts post-sort reanalysis, which routinely yielded purities >95%. (B) Analysis of the expression level of Rorc, Il23r and Tbet mRNA in sorted CD27+ and CD27- γδ T cells isolated from the spleen of the indicated genotypes. Data represent mean +/- SD. (C) Analysis of the expression level of Sox13 and Egr3 in sorted CD27+ and CD27- splenic γδ T cells isolated from the indicated genotypes. (D) Backskin thickness after Aldara treatment of heterozygous control (open triangles) and Map3k14-/- animals (closed squares). Data are representative of two independent experiments. (E) IL17-A and IL17-F expression of dermal γδ T cell on day 6 after Aldara treatment.
Figure 4 with 1 supplement
Conditional deletion of NIK in mTECs causes impaired DETC development.
(A) Map3k14flox/wt mice have been crossed to Deleter Cre animals for deletion of the Loxp flanked gene locus. After subsequent breeding to homozygosity, Map3k14del/del mice were analyzed for the presence of lymph nodes. Upper panel depicts heterozygous controls. (B) Splenic γδ T cells from control, Map3k14-/- and Map3k14del/del mice were assessed for production of IL-17 (left panel) and IFN-γ (right panel) by intracellular cytokine staining. (C) The epidermal γδ T cell compartment of control, Map3k14-/- and Map3k14del/del animals was assessed for the frequency of Vγ5+ DETCs. (D) Lymphocytes isolated from the epidermis of adult control (upper panel), Map3k14-/- and Ccl19-Cre+ Map3k14flox/flox animals were analysed for the presence of Vγ5+ DETCs. Pregating is on live singlets and CD45+ cells. Right panel depicts the summary of the frequency of total γδ T cells as well as Vγ5+ γδ T cells in the epidermis of Control, Map3k14-/- and Ccl19-Cre+ Map3k14flox/flox animals. Data represent mean +/- SD. (E) Frequency of total γδ T cells in the skin of 12-–13 day old control littermates and Foxn1-Cre+ Map3k14flox/flox animals. Data represent mean +/- SD and is pooled from several independent experiments.
Figure 4—figure supplement 1
Targeting strategy used for generation of the conditional Map3k14flox/wt strain.
(A) Schematic representation of the targeted Map3k14 gene locus showing the position of the Loxp Sites flanking exon 4-–6. Abbreviations are denoted in the figure. (B) Schematic representation of the Map3k14 locus after Cre-mediated recombination, leading to a frameshift and a stop codon in exon 7.
Figure 5
Conditional deletion of NIK in TECs causes not only a loss of DETCs, but also IL-17-producing γδ T cells.
(A) γδ T cells were isolated from the lung of Rorc-Cre+ R26-Stop-flox YFP-mice and assessed for the frequency of YFP+ cells within the CD27-γδ T cell compartment. (B) Lung-resident γδ T cells from adult control, Map3k14-/- and Rorc-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 (left panel) and IFN-γ (right panel) by intracellular cytokine staining. (C) Lung-resident γδ T cells from adult control and CD4-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 (left panel) and IFN-γ (right panel). (D) Lung-resident as well as splenic γδ T cells from adult control and Ccl19-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 and IFN-γ. Data is representative of three independent experiments. (E) Frequency of IL-17-producing γδ T cells in the lung of 12-–13 day old control littermates and Foxn1-Cre+ Map3k14flox/flox animals, plots have been pregated on live CD45+ singlets, CD3+ TCRγδ+ cells. Right panels depict the summary of several independent experiments for cytokine production in the lung (left) and spleen (right). Data are mean +/- SD.
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The NFκB-inducing kinase is essential for the developmental programming of skin-resident and IL-17-producing γδ T cells
eLife 4:e10087.
https://doi.org/10.7554/eLife.10087
