Reciprocal regulation of T follicular helper cells and dendritic cells drives colitis development

Published scRNA-seq data (GSE125527) from the samples of intestinal biopsies on UC patients were re-analyzed. a, Annotated UMAP plot showing scRNA-seq analysis of CD4⁺ T cells from local lesions in healthy and inflamed sample, colored by clusters (left top) and sample origin (left bottom). Percentages of cluster 4 cells in each sample group were shown (Right). b, Gene expression of classic Tfh cell signatures (CXCR5, PDCD1, BCL6, TOX, TOX2, and SH2D1A) and other subsets markers (FOXP3 and TBX21) on CD4⁺ T cells from combined samples. c, Representative micrographs of colon samples from inflamed sites of patients with UC (left) and healthy control (middle). Sections were stained with DAPI (blue), anti-CD4 (red), anti-CXCR5 (green), anti-PD-1 (yellow), and anti-CD20 (brown). Scale bars, 200 μm. The statistical analysis of lymphoid follicles positive rate showed a significant increase in UC patients (n=19) compared to healthy controls (n=10) (right), as determined by Fisher’s exact test.

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Naïve Bcl6^wt and Bcl6^−/− CD4⁺ T cells were transferred into TCRbd^−/− mice. a, The body weight was monitored weekly, and was calculated relative to original weight (n =4), setting as 100%. b-e, Mice were sacrificed at 4 weeks for analysis. b, Statistics of colon length between two groups. c, Representative images of H&E-stained colon sections and statistics of colitis histological scores. Scale bars, 100 μm. Statistics of total CD4⁺ T cells (d) and IFN-γ-producing T cells (e) in colon LP. Each point represents an individual mouse. Statistical significances were determined by two tailed Mann–Whitney U-test. Data are shown as mean ± SEM and representative of two independent experiments. *P = 0.0286.

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Representative micrographs of IF staining on CPs, ILFs and colon LP from TCRbd^−/− (a) and Rag1^−/− (b) mice receiving Bcl6^RFP naïve CD4⁺ T cells after 4 weeks of transfer. Sections were stained with BCL6-RFP (Yellow), anti-CD3 (red) and anti-B220 (Green) for a; BCL6-RFP (Yellow), anti-CD3 (red) and anti-MHCII (blue) for b. Scale bars, 100 μm. Representative micrographs of IF staining on CPs (c) and ILFs (d) from TCRbd^−/− mice which received Bcl6^RFP or Bcl6^−/− T cells (more than 5 mice for each group). Dashed lines indicate the area of DC-T clusters represented by CD11c⁺CD86⁺ mature DCs and surrounding CD3⁺ T cells in T cell zones. Scale bars, 100 μm. Images were showed as CD3 (red), B220 (blue), CD11c (sky blue) and CD86 (green). e, Images displayed BCL6-RFP (yellow) and CD3 (grey) double-positive Tfh cells. Scale bars, 100 μm. The numbers of CD11c⁺CD86⁺ mature DCs and CD3⁺ T cells in DC-T clusters of T cell zones were calculated from CPs (f) and ILFs (g). Each point represents an individual image. Statistic significances were determined by two tailed t test. The data were representative of two independent experiments and presented as means ± SEM, ****P < 0.0001.

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a-f, Cd4^cre+/Notch1Notch2^(fl/fl) and Cd4^cre-/Notch1Notch2^(fl/fl) naïve CD4⁺ T cells were transferred into Rag1^−/− recipients, respectively. Mice were sacrificed at 4 weeks post T cell transfer for analysis. a, Body weights were calculated relative to original weights (n=4). b, Colon lengths were measured. c, Representative images of H&E-stained colon sections and statistics of colitis histological scores. Scale bars, 200 μm. d, Representative flow-cytometry plots of CXCR5⁺PD-1⁺ Tfh cells (Gating on live CD3⁺CD4⁺ cells) in CPs from wild-type and Notch1^−/−Notch2^−/− CD4⁺ T cells-transferred mice (left). Statistic of percentage of CXCR5⁺PD-1⁺ Tfh cells in total CD4⁺ T cells (middle) and calculation of Tfh cells in CPs (right). Statistic of total CD4⁺ T cells (e) and IFN-γ-producing T cells (f) in colon LP between groups. g-l, Rag1^−/− recipients received the transfer of Il21^−/− or wild-type naïve CD4⁺ T cells, with mice being sacrificed for analysis at 4 weeks post T cell transfer. g, Body weight changes in 4 weeks were shown (n=8). h, Measurement of colon lengths. i, Representative images from H&E-stained colon sections and corresponding statistical data for colitis histological scores. Scale bars, 200 μm. j, Representative flow-cytometry plots illustrating CXCR5⁺PD-1⁺ Tfh cells (Gating on live CD3⁺CD4⁺ cells) in CPs from wild-type and Il21^−/− CD4⁺ T cells-transferred mice (left). Statistical analysis of the percentage of CXCR5⁺PD-1⁺ Tfh cells in total CD4⁺ T cells (middle) and calculation of Tfh cells in CPs (right). Statistical analysis of total CD4⁺ T cells (k) and IFN-γ-producing T cells (l) in colon LP between groups. Each point represents an individual mouse. The data were representative of two independent experiments and presented as means ± SEM. Two tailed Mann–Whitney U-test were used for (a-f) *P = 0.0286, two tailed t test for (g-l).

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(a-c) ScRNA-seq analysis on the T cells from CPs of colitis mice at day 12 post T cell transfer were conducted. a, Heatmap showing differences in apoptosis pathway activities scored by GSVA per cell between Bcl6^wt and Bcl6^−/− CD4⁺ T cells. Violin plots of anti-apoptotic (b) and pro-apoptotic (c) related gene levels. d, Bcl6^wt and Bcl6^−/− naive CD4⁺ T cells were transferred to Rag1^−/− mice, respectively. The total live CD4⁺ T cells in CPs were analyzed at 4 weeks after transfer (n=4). e,f, Bcl6^wt CD45.1/2 and Bcl6^−/− CD45.2 naive CD4⁺ T were co-transferred at a ratio of 1:1 to Rag1^−/− mice. e Representative flow-cytometry plots and statistical analysis of relative WT versus Bcl6^−/− T cells in CPs at Day 14 after transfer (n=3). f, Flow cytometry analysis of Annexin V⁺PI⁺ cells representing apoptotic T cells in colon at day 14 after transfer (n=3). The data were representative of two independent experiments and presented as means ± SEM in (d-f). P values were determined using two-tailed t test for (b) and (c), two-tailed Mann–Whitney U-test for (d) and two-tailed paired t test for (e) and (f).

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Two CD11c expressing clusters of cells were extracted. a, Feature plots showing expression of Cd86, Cd80, Cd40, Cd83, H2-Aa, H2-Ab1, Tnf, and Ccr7 on these two clusters. b, Feature plots showing expression of Icos and Il6 on the CD11c expressing clusters.

Flow cytometry analysis of cDC2s characterized by CD11c⁺MHCII⁺CD172a⁺CD24⁻ cells and cDC1s identified as CD11c⁺MHCII⁺CD172a^-CD24⁺ cells from colon LP of naïve Irf4^+/+Rag1^−/− mice and Irf4^−/−Rag1^−/− mice (a,b). a, Representative of flow-cytometry plot of CD172a⁺CD24⁻ cDC2s and CD172a⁻CD24⁺ cDC1s (gating on CD11c⁺MHCII⁺ cells) in colon LP from indicated group of mice. b, Calculation of ratio of cDC2s/cDC1s (left) and numbers of cDC2s (right) in colon LP from indicated group of mice. c, Wild-type naïve CD4⁺ T cells were transferred to Irf4^+/+Rag1^−/− mice and Irf4^−/−Rag1^−/− mice separately. After 4 weeks, mice were sacrificed for analysis. Calculation of CD11c⁺MHCII^hiCD172a⁺CD11b⁺ mature DCs in CPs in indicated group of mice (left). Calculation of CD4⁺CXCR5⁺PD-1⁺ Tfh cells in indicated group of mice (right). Each point represents one individual mouse. P values were determined using two-tailed Mann–Whitney U-test. The data were representative of two independent experiments and presented as means ± SEM, *P = 0.0286.

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a, Ltbr^−/− or wild-type cDC2s, derived from CD45.1 BMDCs were transferred into Irf4^−/−Rag1^−/− mice at a dose of 0.6 * 10⁶ cells, one day before the administration of CD4⁺ T cells. Mice were euthanized at day 8 post T cell transfer for analysis. b, Calculations of CD45.1⁺CD11c⁺CD11b⁺CD172a⁺MHCII^hi mature DCs and CD3⁺CD4⁺CXCR5⁺PD-1⁺ Tfh cells in CPs by flow-cytometry. c-f, Irf4^−/−Rag1^−/− mice were administered Ltbr^−/− BMDCs-derived CD11b⁺CD24⁻ DCs, wild-type BMDCs-derived CD11b⁺CD24⁻ DCs, or vehicle one day prior to and 14 days after the receipt of 1.5 * 10⁶ wild-type T cells. Each mouse received a dosage of 0.6 * 10⁶ DCs for each administration. c, Colon lengths were measured. d, Body weight changes over a 4-week period were recorded (n=4) and statistical analysis was conducted at 4 weeks. e, Representative images of H&E-stained colon sections and statistics of colitis histological scores. Scale bars, 200 μm. f, Calculations of total CD4⁺ T cells and IFN-γ-producing CD4⁺ T cells in colon LP. Each point represents one individual mouse. Data are means ± SEM and representative of two independent experiments. P values were determined using two-tailed Mann–Whitney U-test for (b) and ANOVA test for (c-f).

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a, The recipient of Rag1^−/− mice underwent surgical excision of spleens and MLNs when receiving T cells, then were subjected to two groups, with one group receiving daily FTY720 at 1 mg/kg via gavage, and the other receiving distilled water. b, Body weight changes in 4 weeks were shown (n=4). c, Images of colons at day 28 after transfer of T cells. Colon lengths were measured. d, Representative images of H&E-stained sections and statistics of colitis histological scores at day 28. Scale bars, 100 μm. e, Total live cells (left) and CD3⁺CD4⁺ cells (right) in colon LP were calculated between FTY720 treatment and the control group (n=4). Representative micrographs of IF staining of CD3 (yellow) and CD11c (blue) on sections of CPs (f) and ILFs (g) together with colon tissues nearby, from water-treated (upper) or FTY720-treated (lower) mice at 28 days after transfer. Scale, 200 μm. h, TCRbd^−/− mice were inoculated with Bcl6^cre-ert2/Rosa-tdTomato naïve CD4⁺ T cells and received tamoxifen injection before being analyzed at specified time points. i, Longitudinal analysis of ratios of tdTomato⁺ (BCL6-fated) CD4⁺ T cells in four weeks (n=3). j, Flow-cytometry analysis of annexin-V⁺ on indicated T cells at day 14 (n=3). k, Representative flow-cytometry plots and statistical analysis of percentages of IFN-γ⁺CD4⁺ T cells from indicated subsets in the total Th1 cells at day 28 (n=3). Each point represents an individual mouse. Data are shown as mean ± SEM and representative of two independent experiments. P values were determined using two-tailed Mann–Whitney U-test for (b), (c), (d), and (e) *P = 0.0286. ANOVA test was used for (i), and two-tailed paired t test for (j) and (k).

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a, Representative images of Multiplex immunohistochemistry staining on lymphoid follicles from patients with UC. Sections were stained with DAPI (blue), anti-CD20 (brown), anti-CD11c (sky blue), anti-CD172a (green), anti-CD4 (red), anti-CXCR5 (purple), and anti-PD-1 (yellow). Scale bars, 100 μm. b, Published scRNA-seq data (GSE125527) from the samples of intestinal biopsies on UC patients were re-analyzed. Trajectory analyses were performed on CD4⁺ T cells. Results were depicted in the UMAP plot, revealing RNA velocity results with directional arrows predicting pseudotime.