Immunology and Inflammation

Contrasting roles for IKK regulated inflammatory signalling pathways for development and maintenance of type 1 and adaptive γδ T cells

Farjana Islam
Cayman Williams
Thea Hogan
Louise Webb author has email address
Ines Boal-Carvalho author has email address
Benedict Seddon author has email address

Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, London, United Kingdom

https://doi.org/10.7554/eLife.108940.1

Open access
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Figures and data

Development of type 1 and persistence of adaptive and type 17 γδ T cells depends on IKK expression.
Thymi, lymph nodes and spleen from IKKΔT^CD2 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. (B) Scatter plots are of total cell numbers of the indicated subset from the thymus of IKKΔT^CD2 mice or Cre-ve littermates. (C) Representative flow plots illustrate the 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 from IKKΔT^CD2 mice or Cre-ve littermates. 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.

CASPASE8 ablation does not rescue peripheral γδ T cell compartment of IKKΔT^CD2 mice.
Thymi, lymph nodes and spleen from Casp8.IKKΔT^CD2 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.IKKΔT^CD2 mice or Cre–ve littermates. (B) Representative flow plots from lymph nodes of the indicated mouse strains (rows), with the indicated electronic gates (columns). Scatter plots are of total cell numbers recovered from both spleen and lymph nodes of the indicated subset. Data are pooled from multiple batches of mice analysed. Horizontal lines indicate mean.** p<0.01, **** p<0.0001, Mann Whitney test.

Kinase dead RIPK1 mediates partial rescue of peripheral γδ T cell compartments of IKKΔT^CD2 mice.
Lymph nodes and spleen from IKKΔT^CD2 (n=6) IKKΔT^CD2RIPK1^D138N mice (n=4) and Cre –ve littermates (n=6 and 4 respectively) were enumerated and analysed by flow. (A) Representative flow plots are of lymph node 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 IKKΔT^CD2 strain expressing either WT or kinase dead (KD) RIPK1^D138N. Data are pooled from two independent experiments. Horizontal lines indicate mean.* p < 0.05, ** p<0.01, Mann Whitney test.

CASPASE8 expression is critical for long-term survival of peripheral γδ T cells.
Thymi, lymph nodes and spleen from Casp8ΔT^CD2 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 Casp8ΔT^CD2 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 both spleen and lymph nodes combined, of the indicated subsets. Data are pooled from five independent experiments. Horizontal lines indicate mean. n.s - not significant, **** p<0.0001, Mann Whitney test.

Kinase dead RIPK1 fully restores the peripheral γδ T cell compartments of Casp8ΔT^CD2 mice.
Lymph nodes and spleen from Casp8ΔT^CD2 RIPK1^D138N mice (n=16) and Cre –ve RIPK1^D138N littermates (n=8) were enumerated and analysed by flow. (A) Representative flow plots are of lymph nodes from Casp8ΔT^CD2 RIPK1^D138N mice and Cre –ve RIPK1^D138N 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 Casp8ΔT^CD2 RIPK1^D138N mice and Cre –ve RIPK1^D138N littermates (red symbols) and cell numbers from Casp8ΔT^CD2 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 hours 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 the pooled from 4 independent experiments (C). n.s - not significant, Mann Whitney test.

Alternative NF-κB signalling is redundant for generation and maintenance of γδ T cell compartments.
Thymi, lymph nodes and spleen from IKK1ΔT^CD2 mice (n=5) and Cre –ve littermates (n=6) were enumerated and analysed by flow. (A) Representative flow plots are of thymocytes from IKK1ΔT^CD2 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.

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 RelaΔT^CD2 (n=10), Nfkb1^−/− (n=10), RelaΔT^CD2 Nfkb1^–/– mice (n=5), Rela.RelΔT^CD2 (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 of the indicated subset from mice with different REL subunit deletions. 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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