Downregulation of chemokine receptor 9 facilitates CD4+CD8αα+ intraepithelial lymphocyte development

Abstract

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4⁺ T cells can develop into CD4⁺CD8αα⁺ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4⁺CD8αα⁺ IELs, but CCR9 deficiency results in CD4⁺CD8αα⁺ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4⁺CD8αα⁺ IELs in Ccr9^-/- mice. CD4⁺ T cells isolated from the epithelium of Ccr9^-/- mice also display increased expression of Cbfβ2, and the genomic occupancy modification of Cbfβ2 expression reveals its important function in CD4⁺CD8αα⁺ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4⁺CD8αα⁺ IEL differentiation.

Fig. 1. CCR9 expression is lower in CD4⁺CD8αα⁺ IELs than in other IEL populations.

a Histograms show the CCR9 level among TCRβ⁺CD4⁺CD8α⁺CD8β⁻ Foxp3⁻ (CD4⁺CD8αα⁺; red line), TCRβ⁺CD4⁺CD8α⁻CD8β⁻Foxp3⁻ (CD4⁺CD8αα⁻; green line), and TCRβ⁺CD4⁺CD8α⁻CD8β⁻ Foxp3⁺ (Treg cells; blue line) of IELs and LPLs in the jejunum and ileum. Fluorescence minus one (FMO) control is shown as black line. b Graphs show the mean fluorescence intensity (MFI) of CCR9 among CD4⁺CD8αα⁺, CD4⁺CD8αα⁻, Treg cells, and FMO control of IELs and LPLs in the jejunum and ileum (n = 4 C57BL/6J mice for IEL analysis, n = 5 C57BL/6J mice for LPL analysis, 10 weeks old). c Left; pseudocolor plot shows three populations (ThPOK^lowRunx3^hi, ThPOK^hiRunx3^hi, and ThPOK^hiRunx3^low) among CD4⁺ SI IELs according to the expression of ThPOK and Runx3. Right; representative histogram shows the expression of CCR9 among ThPOK^lowRunx3^hi (red line), ThPOK^hi Runx3^hi (green line), and ThPOK^hiRunx3^low (blue line) CD4⁺ SI IELs. FMO control is shown as black line. d Graph shows the MFI of CCR9 among ThPOK^lowRunx3^hi, ThPOK^hiRunx3^hi, ThPOK^hiRunx3^low and FMO control of CD4⁺ SI IELs (n = 4 Thpok^GFP:Runx3^tdTomato reporter mice, 10 weeks old). e Graphs show relative expression of Zbtb7b, Runx3, Tbx21, and Ccr9 in ThPOK^lowRunx3^hi, ThPOK^hiRunx3^hi, and ThPOK^hiRunx3^low populations among CD4⁺ SI IELs sorted from Thpok^GFP:Runx3^tdTomato reporter mice. Quantitative real-time PCR experiments were performed in duplicate in each sample, and each dots represented as average of duplicate (n = 3). Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparisons post hoc test was applied. Source data are provided as a Source data file.

Fig. 2. CD4⁺CD8αα⁺ IELs are accumulated in Ccr9^−/− mice, but surface CCR9 is dispensable for the induction of CD4⁺CD8αα⁺ IELs.

a Surface CD8α and intracellular Foxp3 expression by TCRβ⁺CD4⁺CD8β^– cells in SI IELs of Ccr9^+/+ and Ccr9^−/− mice (Young; analyzed at 7 weeks old, Adult; analyzed at 10 weeks old). b Graphs show the frequency of CD8α⁺, CD8α⁻ or Foxp3⁺ population among TCRβ⁺CD4⁺CD8β^– cells in SI IELs of Ccr9^+/+ and Ccr9^−/− mice. Data of young and adult mice were shown. Data are presented as mean ± SD. c Graphs show the total cell number of CD8α⁺, CD8α⁻ or Foxp3⁺ population among TCRβ⁺CD4⁺CD8β^– cells in SI IELs of Ccr9^+/+ and Ccr9^−/− mice. Data of young and adult mice were shown. Data are presented as mean ± SD (b, c: n = 8 mice for young mice group, n = 9 mice for adult mice group). d Scheme of in vitro experiment design. Naive CD4⁺ T cells obtained from Ccr9^+/+ and Ccr9^−/− mice were cultured with anti-CD3/CD28, transforming growth factor-β (TGF-β), retinoic acid (RA), IFN-γ, and 2,3,7,8-Tetrachlododibenzodioxin (TCDD). Surface CD8α and intracellular Foxp3 expression were analyzed after culture. e Surface CD8α and intracellular Foxp3 expression among TCRβ⁺CD4⁺CD8β^– cultured cells of Ccr9^+/+ and Ccr9^−/− mice. f Graph shows the frequency of CD8α⁺ population among TCRβ⁺CD4⁺CD8β^– cultured cells of Ccr9^+/+ and Ccr9^−/− mice. Data are presented as mean ± SEM (d–f: three independent experiments were performed in triplicate. Each dot represents the mean of the triplicate). g Schema of experiment design. Cells were obtained from bone marrow of Cd45.1⁺Ccr9^+/+ mice and Cd45.2⁺ Ccr9^−/− mice and mixed 1:1 ratio. Mixed bone marrow cells were transferred to the lethally irradiated (11 Gy) C57BL/6J host mice and mice were analyzed 4 weeks after transfer (n = 6 mice). h Surface CD45.1 and CD45.2 expression among TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs. Graph shows the percentage of CD45.1⁺ and CD45.2⁺ cells in TCRβ⁺CD4⁺CD8β⁻ SI IELs. Data are presented as mean ± SD. i Surface staining of CD8α and intracellular Foxp3 among CD45.1⁺TCRβ⁺CD4⁺CD8β⁻ or CD45.2⁺TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs. j Graphs show the percentage of CD8α and intracellular Foxp3 among CD45.1⁺TCRβ⁺CD4⁺CD8β⁻ or CD45.2⁺TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs. Data are presented as mean ± SD. k Schema of experiment design. TCRβ⁺CD4⁺CD8β⁻ cells obtained from spleen of Cd45.1⁺Ccr9^+/+ mice and Cd45.2⁺ Ccr9^−/− and mixed 1:1 ratio. Mixed cells were transferred to Rag2^−/− mice and mice were analyzed 6 weeks after transfer (n = 4 mice). l Surface staining of CD8α among CD45.1⁺TCRβ⁺CD4⁺CD8β⁻ or CD45.2⁺TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs. m Graph shows the percentage of CD8α among CD45.1⁺TCRβ⁺CD4⁺CD8β⁻ or CD45.2⁺TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs. Data are presented as mean ± SD. n Surface CD8α and intracellular Foxp3 expression by TCRβ⁺CD4⁺CD8β^– cells in SI IELs of Rosa26^lsl-Ccr9-GFP and CD4^Ccr9Tg mice. o Graphs show the frequency of CD8α⁺ or Foxp3⁺ population among TCRβ⁺CD4⁺CD8β⁻ cells in SI IELs of Rosa26^lsl-Ccr9-GFP and CD4^Ccr9Tg mice (n = 4 mice for Rosa26^lsl-Ccr9-GFP group, n = 6 mice for CD4^Ccr9Tg group, 10 weeks old). Data expressed as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons post hoc test (b, c) or the two-sided Student’s t test (f, h, j, m, o) was applied. Source data are provided as a Source data file.

Fig. 3. CD4⁺ T cells in the intraepithelial compartment are differentially distributed between Ccr9^+/+ and Ccr9^−/− mice.

Droplet-based single-cell RNA sequencing (scRNA-seq) was performed using the Chromium 10X platform. CD4⁺ T cells from the spleen, mesenteric lymph nodes (MLNs), LPLs and IELs were sorted from Ccr9^+/+ and Ccr9^−/− mice for scRNA-seq. CD4⁺ T cells from spleen and MLNs were collected from one Ccr9^+/+ and Ccr9^−/− mice, and LPLs and IELs were collected from three Ccr9^+/+ and three Ccr9^−/− mice. Sorted IELs and LPLs were pooled in a 2:1 ratio, and cells of each tissue were pooled in a 1:1 ratio between Ccr9^+/+ and Ccr9^−/− mice. a Cells were positioned by gene expression similarities, and 17 clusters were identified based on their top differentially expressed genes (DEGs), as visualized by uniform manifold approximation and projection (UMAP). b UMAP representation of all sequenced cells, color-coded by cluster and separated by tissue of origin. c Heatmap of the top DEGs in each cluster. d UMAP representation of all sequenced cells and separated by tissue of origin. Cell distribution in each tissue from Ccr9^+/+ mice (middle), Ccr9^−/− mice (bottom), and both (top) is shown. e Bar graph shows the proportion of cells in each cluster originating from the spleen, MLN, LPLs, and IELs of Ccr9^+/+ and Ccr9^−/− mice. f Expression of Cd8a, Runx3, Tbx21 and Gzma in sequenced cells are shown via UMAP. Source data are provided as a Source data file.

Fig. 4. CD4⁺CD8αα⁺ IEL-related genes are abundant in Ccr9^−/− mice.

a Pseudotime analysis of sequenced IELs originating from Ccr9^+/+ mice (left) and Ccr9^−/− mice (right), color-coded by pseudotime gradient. Numbers on the UMAP figure indicate the cluster numbers. b Violin plots show expression of Runx3, Tbx21, and Cbfb among IELs from clusters 0, 4, and 7 (CD4⁺CD8αα⁺ T cells); clusters 1, 2 and 3 (CD4⁺CD8αα⁻ T cells); and clusters 10, 13 and 14 (CD4⁺CD8αα^int T cells). c Violin plots show the expression of Runx3, Tbx21, and Cbfb among IELs from clusters 0, 4, and 7 (CD4⁺CD8αα⁺ T cells); clusters 1, 2 and 3 (CD4⁺CD8αα⁻ T cells); and clusters 10, 13, and 14 (CD4⁺CD8αα^+int T cells) from Ccr9^+/+ and Ccr9^−/− mice (a–c; n = 3 biologically independent IEL samples for each strain). One-way ANOVA with Tukey’s multiple comparisons post-hoc test (b) or the two-sided Student’s t test (c) was applied. Source data are provided as a Source data file.

Fig. 5. Cbfb2 expression is upregulated in CD4⁺CD8αα⁺ IELs of Ccr9^−/− mice.

a Graphs show the relative expression of Runx3, Tbx21, Cbfb1, and Cbfb2 in splenic naive CD4⁺ T cells and in CD4⁺CD8αα⁻ and CD4⁺CD8αα⁺ IELs from Ccr9^+/+ and Ccr9^−/− mice. Quantitative real-time PCR experiments were performed in duplicate in each sample, and each dots represented as average of duplicate (n = 6). b Graphs show the relative expression of Cbfb1 and Cbfb2 in ThPOK^hiRunx3^low, Runx3^hiThPOK^hi, and ThPOK^lowRunx3^hi populations among CD4⁺ SI IELs sorted from Thpok^GFP:Runx3^tdTomato reporter mice. Quantitative real-time PCR experiments were performed in duplicate in each sample, and each dots represented as average of duplicate (n = 3). c Left: Structure of a mouse Cbfb locus that produces two splice variants by distinct splice donor signals within exon 5. cDNA encoding tdTomato or Venus was targeted into exon 6 in the Cbfb locus to monitor Cbfb1 and Cbfb2 splicing by expression of tdTomato and Venus fluorescent protein, respectively. Right: histograms show Cbfb2-Venus and Cbfb1-tdTomato expression in CD4⁺CD8αα⁻ IELs (dotted line) and CD4⁺CD8αα⁺ IELs (red line) from Cbfb2^Venus and Cbfb1^tdTomato reporter alleles, as well as CD4⁺CD8αα⁺ IELs (black line) from wild-type control mice. Data are expressed as mean ± SD. The two-sided Student’s t test (a) or one-way ANOVA with Tukey’s multiple comparisons post hoc test (b) was applied. Source data are provided as a Source data file.

Fig. 6. Cbfβ2 expression regulates the induction of CD4⁺CD8αα⁺ IELs.

a Surface CD8α and intracellular Foxp3 expression of TCRβ⁺CD4⁺CD8β^– SI IELs from Cbfb^+/+ and Cbfb^2m/2m mice. b Graphs show the frequency of CD8α⁺ or Foxp3⁺ populations among TCRβ⁺CD4⁺CD8β^– cells in SI IELs. (n = 7 mice for Cbfb^+/+ group, n = 5 mice for Cbfb^2m/2m group, 10 weeks old). Data are expressed as mean ± SEM. c Surface CD8α and intracellular Foxp3 expression by TCRβ⁺CD4⁺CD8β^– cells in SI IELs of Rosa26^{lsl-Cbfb2-GFP} and CD4^Cbfb2Tg mice analyzed at 7 and 10 weeks of age. d Graphs show the frequency of CD8α⁺ or Foxp3⁺ populations among TCRβ⁺CD4⁺CD8β^– SI IELs of Rosa26^{lsl-Cbfb2-GFP} and CD4^Cbfb2Tg mice (7W; n = 3 mice for each group, 10W; n = 5 mice for Rosa26^{lsl-Cbfb2-GFP} group, n = 4 mice for CD4^Cbfb2Tg group). Data are expressed as mean ± SD. The two-sided Student’s t test (b, d) was applied. Source data are provided as a Source data file.

Fig. 7. Downregulation of Ccr9 in CD4⁺ T cells preferentially develops CD4⁺CD8αα⁺ IELs in a Cbfb-dependent manner.

a Schema of the experimental design. Naive CD4⁺ T cells obtained from Ccr9^+/+ and Ccr9^−/− mice were nucleofected with siRNA targeting negative control, Ccr9, Runx3, Cbfb, and Tbx21 (scramble, siCcr9, siRunx3, siCbfb, and siTbet, respectively). Nucleofected cells were cultured with anti-CD3/CD28, transforming growth factor-β (TGF-β), retinoic acid (RA), 2,3,7,8-tetrachlododibenzodioxin (TCDD), and IFN-ɤ. Surface CD8α and intracellular Foxp3 expression were analyzed 3 days after culture. b, c Graphs show the frequency of CD8α⁺ cells among TCRβ⁺CD4⁺CD8β^– nucleofected naive CD4⁺ T cells obtained from Ccr9^+/+ and Ccr9^−/− mice cultured with anti-CD3/CD28, TGF-β, RA, TCDD, and IFN-γ. Three independent experiments were performed in triplicate. Each dot (n = 3) represents the mean of the triplicate. Data are expressed as mean ± SD. One-way ANOVA with Tukey’s multiple comparisons post hoc test was applied. Source data are provided as a Source data file.