IL‐7‐dependent compositional changes within the γδ T cell pool in lymph nodes during ageing lead to an unbalanced anti‐tumour response

Abstract How the age‐associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the γδ T‐cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell‐intrinsic effects on function and global gene expression of γδ T cells, and γδTCR diversity remains stable. However, ageing alters TCRδ chain usage and clonal structure of γδ T‐cell subsets. Importantly, IL‐17‐producing γδ17 T cells dominate the γδ T‐cell pool of aged mice—mainly due to the selective expansion of Vγ6+ γδ17 T cells and augmented γδ17 polarisation of Vγ4+ T cells. Expansion of the γδ17 T‐cell compartment is mediated by increased IL‐7 expression in the T‐cell zone of old mice. In a Lewis lung cancer model, pro‐tumourigenic Vγ6+ γδ17 T cells are exclusively activated in the tumour‐draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in γδ T‐cell pool in the pLN lead to an unbalanced γδ T‐cell response in the tumour that is associated with accelerated tumour growth.

A The expression of CD45RB and CD44 segregates mature (CD24 neg ) cd T cells in LNs of young (3 months, n = 17), mid-age (12 months, n = 4) and old (> 21 months, n = 16) mice into cd17-committed (CD45RB neg CD44 + ), cd1-committed (CD45RB + CD44 + ), cd1-intermediate (CD45RB + CD44 neg ) and progenitor (CD45RB neg CD44 neg ) cd T-cell subsets. B Percentage of cd17 T cells, as characterised by the lack of CD27 expression, from total cd T cells in old and young pLNs. Results shown are from three independent experiments with seven young and seven old mice. C CD44 and CD62L expression profile of cd17-committed (CD45RB neg CD44 hi ), cd1-committed (CD45RB + CD44 + ), cd1-intermediate (CD45RB + CD44 neg ) and progenitor (CD45RB neg CD44 neg ) cd T cells from pLNs of young mice. D Representative FACS plots and analysis of the memory/activation status of cd T cells in the pLNs of young and old mice. Results shown are collected from three independent experiments using seven young and seven old mice. E Effect of obesity on cd17 bias in the pLNs of old mice. Aged mice with obesity were visually identified across three ageing cohorts in the same animal facility. The proportion of cd17 T cells in the pLN cd T-cell pool of normal and obese old mice was compared. Results shown are collected from 11 independent experiments with 21 old mice (13 normal and eight obese). F-H Cytokine production by total CD3 + T cells (F), cd T cells (G) and CD44 hi memory CD4 + T cells ( Mice were treated with 4 mg/kg IgG2b or anti-IL7 antibody by i.p. injection followed by administration of EdU by i.p. injection and supply in drinking water (as shown in Fig 5I). The level of proliferation was assayed by the level of EdU incorporation over a period of 3 days.

A
The proliferation of cd1 and cd17 T cells within each cd T-cell subsets under treatment with control isotype IgG2b or with anti-IL-7 neutralising antibody. B, C The proliferation of bulk CD4 + T cells and CD44 hi memory CD4 + T cells (B), as well as bulk CD8 + T cells and CD44 hi memory CD8 + T cells (C), under treatment with control isotype IgG2b or with anti-IL-7 neutralising antibody.
Data information: Results shown are collected from two independent experiments with 14 young mice (seven each for control and experimental groups). Statistical significances for changes in expression levels were assessed by two-way ANOVA (A) or Mann-Whitney test (B and C). Error bars represent SD. *P < 0.05; ****P < 0.0001. Young mice were injected every other day by i.p. with FTY720 at a dose of 1 mg/kg or with vehicle control containing 2.5% ethanol and 2% b-cyclodextrin from day À5 to day 13. 3 × 10 6 3LL-A9 cells were given to control and FTY720-treated mice by subcutaneous injection on day 0. Tumours were harvested at day 14 or 15 for FACS analysis.
A, B Number of CD4 + (A) and CD8 + (B) T cells in the tumour of control and FTY720-treated mice. C cd T-cell lineages observed in the tumour of control and FTY720-treated mice. D Composition of cd T cells subsets in the tumour of control and FTY720-treated mice.
Data information: Results shown are obtained from two independent experiments with 11 control and 10 FTY720-treated mice. Statistical significances for the difference in cell densities and cell proportions were assessed by Mann-Whitney test (A and B) or two-way ANOVA (C and D). Error bars represent SD. *P < 0.05; ***P < 0.001; ****P < 0.0001. 3LL-A9 Lewis lung carcinoma cells were injected subcutaneously into young and old mice, and tumours were analysed 14 days after injection.
A Percentage of cd T cells in total tumour-infiltrating CD3 + T lymphocytes. B Density of cd T cells in the tumours of young and of mice. C cd1 and cd17 lineage commitment of tumour-infiltrating Vc1 + , Vc2/3/7, Vc4 + and Vc6 + T cells. D Activation of cd1 and cd17 T-cell subsets in the tumour as determined by their PD-1 and Tim-3 expression profile. Representative FACS plots (left) show the analysis with concatenated FACS data acquired for each individual young mouse. Results shown (right) are obtained from two independent experiments with eight young and five old mice. Cell populations with a total cell number < 10 were excluded from the analysis.   A cd1 and cd17 lineage commitment of cd T cells in the tumour-draining LN of young and old mice. B Proportion of cd T-cell subsets in the tumour-draining LN of young and old mice. C cd1 and cd17 lineage commitment of each cd T-cell subset in the tumour-draining LN was characterised by CD44 and CD45RB expression. D Activation and exhaustion status of each cd T-cell subset in the tumour-draining LN was characterised by PD-1 and Tim-3 expression.
Data information: FACS files acquired for each individual mouse were concatenated for the analysis, and the results are shown as representative dot plots. Results are obtained from two independent experiments with eight young and five old mice. Cell populations with the total cell number < 10 were excluded from analyses. Statistical significances for differences were assessed by two-way ANOVA (A and B). Error bars represent SD. **P < 0.01; ***P < 0.001; ****P < 0.0001.