BCL6 deletion in CD4 T cells does not affect Th2 effector mediated immunity in the skin

Abstract

Recent studies propose that T follicular helper (Tfh) cells possess a high degree of functional plasticity in addition to their well-defined roles in mediating interleukin-4-dependent switching of germinal center B cells to the production of immunoglobulin (Ig)G1 and IgE antibodies. In particular Tfh cells have been proposed to be an essential stage in Th2 effector cell development that are able to contribute to innate type 2 responses. We used CD4-cre targeted deletion of BCL6 to identify the contribution Tfh cells make to tissue Th2 effector responses in models of atopic skin disease and lung immunity to parasites. Ablation of Tfh cells did not impair the development or recruitment of Th2 effector subsets to the skin and did not alter the transcriptional expression profile or functional activities of the resulting tissue resident Th2 effector cells. However, the accumulation of Th2 effector cells in lung Th2 responses was partially affected by BCL6 deficiency. These data indicate that the development of Th2 effector cells does not require a BCL6 dependent step, implying Tfh and Th2 effector populations follow separate developmental trajectories and Tfh cells do not contribute to type 2 responses in the skin.

Keywords: BCL6; T cell plasticity; Tfh; Th2 differentiation.

Figure 1

BCL6^cKO mice lack Tfh cells, impeding the IgG response. BCL6^fl/fl and BCL6^cKO mice were sensitized with 200 μg HDM or 30 μL PBS i.d. in the ear pinna. (a) Flow cytometry depicting CD44^hi CD4 T cells and Tfh/non‐Tfh cells by CXCR5 and PD‐1 expression day 7 post HDM. (b) Total number of CD4⁺CD44^hi non‐Tfh (CXCR5⁻) cells and Tfh cells (CXCR5^hiPD1⁺) in aLN day 7 post HDM. (c) IgG1 and IgG2a concentration in serum day 14 post HDM sensitization. Black circles represent BCL6^fl/fl, white circles represent BCL6^cKO samples. Data are shown as mean ± s.e.m. for six ears per group from one of five repeats that gave similar results except for ELISA data which shows five mice per group from one of two repeats. *P < 0.05, **P < 0.01, not significant is not shown. P‐values were determined using a Kruskal‐Wallis test applying Dunn's multiple comparison post‐test.

Figure 2

Non‐Tfh cells express reduced levels of IL4‐AC in BCL6^cKO.4C13R mice. BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice were injected in the ear pinna with 200 μg HDM in 30 μL PBS or PBS alone and cells from the draining aLNs were harvested for analysis on day 7. (a) Flow cytometry plots depicting the expression of IL4‐AC and IL13‐DR in non‐Tfh and Tfh cells. (b, c) Frequency and total number of IL4‐AC⁺ non‐Tfh (b) and IL4‐AC⁺ Tfh cells (c). (d) Total number of IL4‐AC⁺ non‐Tfh and Tfh cells. Black circles represent BCL6^fl/fl, white circles represent BCL6^cKO samples. Data are shown as mean ± s.e.m. for six aLN per group from one of five repeats that gave similar results. *P < 0.05, **P < 0.01, ***P < 0.001, not significant is not shown. P‐values were determined using a Kruskal‐Wallis test applying Dunn's multiple comparison post‐test.

Figure 3

Skin Th2eff develop independently of Tfh cells. Mice were sensitized with injected in the ear pinna with 200 μg HDM in 30 μL PBS or PBS alone. Ear skin tissue was collected for analysis on day 7. (a) Frequency and total number of CD4 T cells in ear tissue from BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice. (b) Flow cytometry plots depicting the expression of IL4‐AC and IL13‐DR in CD4 T cells from BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice. (c) Enumerated frequencies and (d) total number of Th2eff cell subsets from BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice. (e) Frequency and total number of CD4 T cells from C57Bl/6J naïve control mice and mice that were injected with 25 μg FTY720 in PBS or PBS alone on days 2 and 4 post HDM sensitization. Black circles represent BCL6^fl/fl, white circles represent BCL6^cKO samples. Data are shown as mean ± s.e.m. for six ears per group from one of five repeats that gave similar results. *P < 0.05, **P < 0.01, ***P < 0.001. P‐values for a and e were determined using a Kruskal‐Wallis test applying Dunn's multiple comparison post‐test. P‐values for c and d were determined using a Mann–Whitney U‐test, independently for each Th2eff subset.

Figure 4

BCL6 deficiency causes minimal transcriptional changes in skin Th2eff cells. Skin Th2eff subsets from BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice were harvested from draining aLN and sorted 7 days after HDM sensitization and ULI‐RNAseq was performed. (a) PCA of skin Th2eff subsets, performed on top 1000 highest variance genes for PC1/PC2 and PC2/PC3 axis. BCL6^fl/fl samples = black; BCL6^cKO samples = white; IL4‐AC SP = square; IL4‐AC.IL13‐DR DP = circle; IL13‐DR SP = triangle. (b) Heatmap showing VSTpk values of canonical Th2 genes in skin Th2eff subsets and naïve LN CD4 T cells. (c) MA plots of skin Th2 subsets comparing BCL6^fl/fl.4C13R and BCL6^cKO.4C13R samples. Black dots represent DEGs with a log₂ FC > 1 or > −1; P‐value < 0.05; and minimum mean read count > 30. Gray dots do not meet differential expression statistical thresholds.

Figure 5

Basophil and eosinophil recruitment to the skin is enhanced in BCL6^cKO.4C13R mice and correlates to reduced frequency of Treg cells. BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice injected in the ear pinna with 200 μg HDM in 30 μL PBS or PBS alone. Ear skin tissue was collected for analysis on day 7 unless indicated. (a) Kinetics of total number of basophils and eosinophils detected in ear tissue. (b) CD11b MFI in basophils and eosinophils. (c) Frequency and total number of FOXP3⁺ Treg cells in ear tissue. (d) GATA3 MFI in FOXP3⁺ Treg cells in ear tissue. Black circles represent BCL6^fl/fl, white circles represent BCL6^cKO samples. Data are shown as mean ± s.e.m. for six ears per group from one of five repeats that gave similar results. *P < 0.05, **P < 0.01. P‐values were determined using a Mann–Whitney U‐test.

Figure 6

BCL6^cKO.4C13R mice show a partial dependence on Tfh cells for Th2eff cell development in the lung. BCL6^fl/fl.4C13R and BCL6^cKO.4C13R mice were infected with 550 live Nb L3 subcutaneously in neck scruff. Lung and draining LN tissues were collected and cells harvested for analysis on day 10. (a) Flow cytometry plots gated on CD4⁺ CD44^hi depicting expression of CXCR5 and PD1 to identify Tfh and non‐Tfh cells. (b) Total number of non‐Tfh and Tfh cells in medLN. (c) Flow cytometry plots depicting the expression of IL4‐AC and IL13‐DR in non‐Tfh and Tfh cells in the medLN. (d, e) Frequency and total number of IL4‐AC⁺ non‐Tfh (d) and IL4‐AC⁺ Tfh cells (e). (f) Frequency of CD4 T cells in the lung. (g) Flow cytometry plots depicting IL4‐AC and IL13‐DR expression in lung CD4 T cells. (h) Frequencies of Th2eff subsets in the lung tissue. Black circles represent BCL6^fl/fl, white circles represent BCL6^cKO samples. Data are shown as mean ± s.e.m. for 2–6 mice per group from one of two repeats that gave similar results. *P < 0.05, **P < 0.01. P‐values were determined for b an d–f using a Kruskal‐Wallis test applying Dunn's multiple comparison post‐test. P‐values for h were determined using a Mann–Whitney U‐test, independently for each Th2eff subset.