Contrasting roles for IKK-regulated inflammatory signalling pathways for development and maintenance of type 1 and adaptive γδ T cells
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, United Kingdom
Figures
Figure 1 with 2 supplements
Development of type 1 and persistence of adaptive and type 17 γδ T cells depends on IKK expression.
Thymi, lymph nodes, and spleen from IKK∆TCD2 mice (n = 14) and Cre −ve littermates (n = 7) were enumerated and analysed by flow. (A) Representative flow plots are of thymocytes with the indicated gates, showing gates used to identify progenitor, adaptive, and type 1 subsets intrathymically (see Materials and methods for details). (B) Scatter plots are of total cell numbers of the indicated subset from the thymus of IKK∆TCD2 mice or Cre −ve littermates. Horizontal bars are mean. (C) Representative flow plots illustrate the gating strategy to identify adaptive, type 1, and type 17 subsets in lymph nodes and spleen (see Materials and methods for details). (D) Scatter plots are of total cell numbers recovered from lymph nodes (L), spleen (S), and both spleen and lymph nodes combined (T) of the indicated subset from IKK∆TCD2 mice (+) or Cre −ve littermates (–). Horizontal bars are mean. Fractional representation of different γδ subsets in different organs is shown in Figure 1—figure supplement 2A. Data are pooled from multiple batches of mice analysed. Horizontal lines indicate mean. *p < 0.05, **p < 0.01, ****p < 0.0001, Mann–Whitney test.
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Figure 1—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
huCD2iCre targets Cre activity to γδ T cells in the thymus.
Thymi and lymph nodes (LNs) from Cre –ve mice were analysed by flow. The density plot number represents the percentage of cells in each gate. (A) Representative flow plots are of thymocyte profiles: Histograms display RFP expression by ɣδ progenitor, effector ɣδ and type 17 ɣδ T cells. (B) Representative flow plots display LNs profiles: Histograms display RFP expression by effector ɣδ and type 17 ɣδ T cells.
Figure 1—figure supplement 2
Representation of different γδ T cell sub-populations in different mouse strains.
Bar charts show the representation of progenitor, adaptive, and type 1 γδ T cell subsets in the thymus, and adaptive, type 1, and type 17 subsets in lymph nodes and spleen. Charts are either of percentage representation amongst total lymphocytes of the specified organ (% total lymphocytes), or the relative representation of the indicated subset amongst total gd T cells in the given organ (% of total TCRδ+ CD3ε+). Data are shown for IKK∆TCD2 mice described in Figure 1, Casp8.IKK∆TCD2 mice described in Figure 2, Casp8∆TCD2 mice described in Figure 4, and Rela∆TCD2, Nfkb1−/−, Rela∆TCD2 Nfkb1–/– mice, Rela.Rel∆TCD2 mice described in Figure 7.
Figure 2
CASPASE8 ablation does not rescue peripheral γδ T cell compartment of IKK∆TCD2 mice.
Thymi, lymph nodes and spleen from Casp8.IKK∆TCD2 mice (n = 13) and Cre –ve littermates (n = 7) were enumerated and analysed by flow. (A) Scatter plots are of total cell numbers of the indicated subset recovered from thymi of Casp8.IKK∆TCD2 mice or Cre –ve littermates. (B) Representative flow plots from lymph nodes of the indicated mouse strains (rows), with the indicated electronic gates (columns). (C) Scatter plots are of total cell numbers recovered from lymph nodes (L), spleen (S), and total combined spleen and lymph nodes (T) of the indicated subset from Cre +ve (+) and Cre –ve (–) littermates. Horizontal bars are mean. Fractional representation of different γδ subsets in different organs is shown in Figure 1—figure supplement 2B. Data are pooled from multiple batches of mice analysed. Horizontal lines indicate mean. **p < 0.01, ****p < 0.0001, Mann–Whitney test.
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Figure 2—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig2-data1-v1.xlsx
Figure 3
Kinase-dead RIPK1 mediates partial rescue of peripheral γδ T cell compartments of IKK∆TCD2 mice.
Lymph nodes and spleen from IKK∆TCD2 (n = 6) IKK∆TCD2RIPK1D138N mice (n = 4) and Cre –ve littermates (n = 6 and 4, respectively) were enumerated and analysed by flow. (A) Representative flow plots are of splenic cells from different strains (rows) with the indicated gates (columns). (B) Scatter plots are of total cell numbers of the indicated subset recovered from lymph nodes and spleen combined of the indicated IKK∆TCD2 strain expressing either WT or kinase-dead (KD) RIPK1D138N. Data are pooled from two independent experiments. Horizontal lines indicate mean.*p < 0.05, **p < 0.01, Mann–Whitney test.
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Figure 3—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig3-data1-v1.xlsx
Figure 4
CASPASE8 expression is critical for long-term survival of peripheral γδ T cells.
Thymi, lymph nodes, and spleen from Casp8∆TCD2 mice (n = 13) and Cre –ve littermates (n = 10) were enumerated and analysed by flow. (A) Representative flow plots are of thymocytes with the indicated gates, showing gates used to identify progenitor, adaptive, and type 1 subsets. (B) Scatter plots are of total cell numbers of the indicated subset from thymus of Casp8∆TCD2 mice (+) and Cre –ve littermates (–). (C) Representative flow plots illustrate gating strategy to identify adaptive, type 1, and type 17 subsets in lymph nodes (shown) and spleen from the indicated strains. (D) Scatter plots are of total cell numbers recovered from lymph nodes (L), spleen (S), and total combined spleen and lymph nodes (T), of the indicated subsets from Cre +ve (+) and Cre –ve (–) littermates. Horizontal bars are means. Fractional representation of different γδ subsets in different organs is shown in Figure 1—figure supplement 2C. Data are pooled from five independent experiments. Horizontal lines indicate mean. n.s. – not significant, ****p < 0.0001, Mann–Whitney test.
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Figure 4—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig4-data1-v1.xlsx
Figure 5
Kinase-dead RIPK1 fully restores the peripheral γδ T cell compartments of Casp8∆TCD2 mice.
Lymph nodes and spleen from Casp8∆TCD2 RIPK1D138N mice (n = 16) and Cre –ve RIPK1D138N littermates (n = 8) were enumerated and analysed by flow. (A) Representative flow plots are of lymph nodes from Casp8∆TCD2 RIPK1D138N mice and Cre –ve RIPK1D138N littermates with the indicated gates applied (columns). (B) Scatter plots are of total cell numbers of the indicated subsets from both lymph nodes and spleen combined from Casp8∆TCD2 RIPK1D138N mice and Cre –ve RIPK1D138N littermates (red symbols) and cell numbers from Casp8∆TCD2 RIPK1WT strains described in Figure 4, for direct comparison. (C) Lymph node cells were stimulated in vitro with calcium ionophore and phorbyl esters for 4 hr with brefeldin A, and then analysed for the indicated intracellular cytokines. Representative flow plots illustrate gating strategy to identify adaptive, type 1, and type 17 subsets on the basis of CD44 and CD122 expression. Quad gates for cytokine detection were set against negative controls of matched unstimulated cells. Bar charts are of total % of cells stained for IFN-gamma or IL-17A. Data are pooled from multiple batches of mice analysed (A, B) or are pooled from four independent experiments (C). n.s. – not significant, Mann–Whitney test.
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Figure 5—source data 1
Cell enumerations of different gamma-delta subsets and representation of cytokine producing cells.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig5-data1-v1.xlsx
Figure 6
Alternative NF-κB signalling is redundant for generation and maintenance of γδ T cell compartments.
Thymi, lymph nodes, and spleen from IKK1∆TCD2 mice (n = 5) and Cre –ve littermates (n = 6) were enumerated and analysed by flow. (A) Representative flow plots are of thymocytes from IKK1∆TCD2 mice and Cre –ve littermate, showing gates used to identify progenitor, adaptive, and type 1 subsets intrathymically. (B) Scatter plots are of total cell numbers of the indicated subset from thymus. (C) Representative flow plots illustrate gating strategy to identify adaptive, type 1, and type 17 subsets in lymph nodes (shown) and spleen. (D) Scatter plots are of total cell numbers recovered from both spleen and lymph nodes of the indicated subset. Data are pooled from two independent experiments.
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Figure 6—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig6-data1-v1.xlsx
Figure 7
Canonical NF-κB signalling is essential for development of type 1 and maintenance of adaptive γδ T cell compartments.
Thymi, lymph nodes, and spleen from Rela∆TCD2 (n = 10), Nfkb1−/− (n = 10), Rela∆TCD2 Nfkb1–/– mice (n = 5), Rela.Rel∆TCD2 (n = 12), and Cre –ve littermates (n = 14) were enumerated and analysed by flow. (A) Scatter plots are of total cell numbers of the indicated subset from thymus, while bar charts show the ratio of progenitor:DN3 subsets for the indicated strains.+ indicates WT allele, – indicates gene deletion for the different REL subunits. (B) Scatter plots are of total cell numbers recovered from both spleen and lymph nodes combined (top row), lymph nodes (middle row), or spleen (bottom row) of the indicated subset from mice with different REL subunit deletions (+ indicates WT, – indicates deletion). Fractional representation of different γδ subsets in different organs is shown in Figure 1—figure supplement 2D. Data are pooled from multiple batches of mice analysed. n.s. – not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 by Mann–Whitney test.
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Figure 7—source data 1
Cell enumerations of different gamma-delta subsets in different lymphoid organs.
- https://cdn.elifesciences.org/articles/108940/elife-108940-fig7-data1-v1.xlsx
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Contrasting roles for IKK-regulated inflammatory signalling pathways for development and maintenance of type 1 and adaptive γδ T cells
eLife 14:RP108940.
https://doi.org/10.7554/eLife.108940.3
