Reduced stability of Foxp3 in <i>TRAF6</i>-deficient Treg cells

<p>(A) Foxp3 expression in freshly isolated CD4⁺ T cells from the spleen, mesenteric lymph node (LN) and thymus of the indicated mice. Foxp3 levels were measured using YFP-fluorescence. Data are representative of five independent experiments with similar results. The numbers of YFP⁺ Tregs in each organ are shown in right graphs (n=5). (B) Expression of KLRG1 was measured in CD4⁺Foxp3(YFP)⁺ gated cells. (C) <i>In </i><i>vitro</i> suppression assay. FACS purified 5×10⁴ CD4⁺CD25^-CD62L⁺CD44^- naïve T cells from WT mice were stimulated for 72 hours with 10⁵ T-cell depleted spleen cells and anti-CD3 (2C11) (1µg/ml), in the absence or presence of CD3⁺CD4⁺Foxp3(YFP) + Treg cells from WT (diamond) or cKO (square) mice as indicated concentrations. Cells were pulsed with 0.5 µCi of ³H-thymidine for the final 16 hours before being harvested. Error bars indicate SD; n=3. (D) <i>In </i><i>vivo</i> suppression assay. 3 x 10⁵ naïve T cells from the LN of CD45.1⁺ WT mice together with or without 1.5 x 10⁵ Tregs (CD4⁺YFP ⁺ RFP⁺) from the LN of CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+ (WT-Tregs) or CD45.2 Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl (cKO-Tregs) were transferred into Rag2^-/- mice. Body weight changes were measured daily. Data are representative from four independent experiments. *<i>p</i><0.05 **<i>p</i><0.01; Representative histology of the colon is shown in right panels. Magnification x200 (E) Flow cytometric analysis of Foxp3-YFP and CD45.1 expression on CD3⁺CD4⁺ T cells from the LN in Rag2^-/- mice. Data are representative from four independent experiments. Quantification of the data of the fraction of YFP ⁺ CD45.1^- cells (remaining Foxp3 ⁺ Tregs) is shown in the right panel. **<i>p</i>< 0.01.</p

Activation of T cells in Treg-specific TRAF6-cKO mice.

<p>(A) Flow cytometry for activated/memory phenotypes of freshly isolated CD4⁺ T cells from thymus, spleen and mesenteric lymph nodes (MLNs) in the indicated mice at 22 weeks of age. Representative data from three independent mice are shown. (B) Cytokine production from splenic T cells. Freshly isolated splencytes (1x10⁶) from WT and cKO mice were stimulated with anti-CD3 antibody for 2 days. Cytokine levels in the culture supernatant was measured using ELISA (n=3).</p

Reduced expression of Foxp3 in TRAF6-deficeit Tregs.

<p>(A) Foxp3 protein and YFP expression in Treg cells among gated CD4⁺T cells from the spleen, LN and thymus of female Foxp3 ^YFP-Cre/+ TRAF6^f/f mice and Foxp3 ^YFP-Cre/+ TRAF6^+/+ littermates. Numbers adjacent to the outlined areas indicate percent Foxp3 ⁺ YFP^- cells (top left) or Foxp3+YFP+ cells (top right). These results are representative of three similar experiments. (B) Loss of Foxp3-positivity in lymphopenic conditions. 2 x 10⁵ CD4⁺Foxp3(YFP ⁺ RFP⁺) cells from WT (Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+) or cKO (Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl) mice were transferred Tregs into Rag2^-/- mice. Four weeks after transfer, Foxp3-positivity in CD4⁺RFP⁺ cells was measured. Raito of YFP-/YFP+ is shown in the right panel (N=3). *<i>p</i><0.05.</p

Fate mapping study of <i>TRAF6</i>-deficeint Tregs.

<p>(A) FACS profiles of CD4+ T cells in the spleen of WT (Foxp3 ^YFP-CreROSA26^RFP TRAF6^+/+) or cKO (Foxp3 ^YFP-CreROSA26^RFP TRAF6^fl/fl) mice. A representative profile of WT and cKO mice (16-20 weeks old) with or without severe inflammation. (B) RFP ⁺ YFP^-/RFP ⁺ YFP⁺ ratio in the spleen of WT and cKO mice at 12 to 22 weeks of age (n=5). (C) mRNA levels in each CD4⁺T cell fraction. After sorting using FACS with YFP and RFP, mRNA was isolated from each fraction, and the levels of indicated genes were measured using quantitative real time RT-PCR. The mean ± SD of three independent experiments are shown. All data were analyzed using a Student’s t test. *<i>p</i><0.05, **<i>p</i><0.01vs. WT.</p

CXCR5-deficient B cells fail to access inflamed B cell follicles.

<p>CD21cre-RFP chimeras were untreated (left panel) or injected (right panel) with an emulsion of CFA/PBS in the ears. Three weeks later, recipients were injected with a cohort of CMFDA-labeled Wt-deficient B cells (green) and Celltrace violet–labeled CXCR5-deficient B cells (violet). One day later, ear draining LNs were sectioned, stained for B220 (blue) expression, and imaged by confocal microscopy. RFP⁺ cells appear in red. Data are representative of two different experiments (three mice per experiment).</p

Characterization of <i>Foxp3</i>^Cre-YFP-<i>TRAF6</i>^f/f (cKO) mice.

<p>(A) Representative appearance of a 22-week-old cKO mouse. (B) Incidence of dermatitis in cKO mice (<i>n</i> = 20). (C) Representative macroscopic observations of the LN from WT mice and age (about 20 weeks old)-matched TRAF6 cKO mice. (D) Representative histopathologies of the skin (H&E staining) and joint (Safranin-O staining) lesion of representative cKO mice. (E) Serum titers of immunoglobulin subclasses and anti-dsDNA antibodies from WT and cKO mice measured by ELISA. Each symbol indicates an individual host mouse (^*<i>p</i> < 0.05). (n=7) (F) Spontaneous splenic germinal center formation in cKO mice. Sections of spleen were stained with anti-IgE and anti-CD3 antibodies. Representative data form three independent mice are shown.</p

Induction of CXCL13 secretion in CD21⁻ RFP⁺ stromal cells upon B cell follicle enlargement.

<p>CD21cre-RFP chimeras were untreated (A) or injected (B) with an emulsion of CFA/PBS in the ears. Three weeks later, ear draining LNs were sectioned, stained for collagen IV (green), IgD (white), and CXCL13 expression (blue), and imaged by confocal microscopy. RFP⁺ cells appear in red. Inserts display high magnifications of the boundaries between T and B cell areas (dashed line). In (B), the arrow indicates the area of the enlarged B cell follicle covered by the collagen IV network (conduit system). Single <i>z</i> slices show intracellular staining of CXCL13. (C) CXCL13 mRNA levels in CD21⁻ CD45⁻ RFP⁺ cells purified from resting and CFA-inflamed LNs relative to FRCs. Data are representative of three different experiments (two mice (A and B) and five mice (C) per experiment).</p

Phenotype of CD21⁻ RFP⁺ stromal cells.

<p>(A and E) Confocal images of a LN section from a CD21cre-RFP chimera stained for the indicated markers. RFP⁺ cells appear in red. The dashed line delineates B cell follicle boundary. (B, C, D) CD21⁻ RFP⁻ gp38⁺ CD31⁻ CD45⁻ FRCs and CD21⁻ RFP⁺ gp38⁺ CD31⁻ CD45⁻ cells were sorted by flow cytometry and their transcriptomic profiles were analyzed by microarrays (B and C) or RT-PCR (D). (B) Scatter plot of global comparison of gene expression between CD21⁻ RFP⁺ cells and FRCs. Each gene in the microarray is represented by a dot with coordinates consisting of average gene expression computed from three independent CD21⁻ RFP⁺ samples (<i>y</i>-axis) and from the three matched FRC samples (<i>x</i>-axis). Genes with Log₂ average expression level at least 1.5-fold higher in CD21⁻ RFP⁺ cells are shown in red, while genes with Log₂ average expression level at least 1.5-fold higher in FRCs are shown in blue. These genes are separated from the other genes by colored lines representing these fold change cutoffs. (C) Heat map for the 50 genes with the most significantly higher expression in CD21⁻ RFP⁺ cells. A subset of the genes shown in red in panel B was further selected based on a <i>p</i> value<0.05 for differential expression between CD21⁻ RFP⁺ cells and FRCs, as computed by Limma. Genes (rows) and samples (columns) were clustered by complete linkage hierarchical clustering, using Euclidean distance measure. For each gene, expression levels close to the mean value across all six samples are shown in black, high expression levels in red, and low expression levels in green. (D) Relative expression of <i>Cxcl13</i>, <i>Ccl21</i>, <i>Ccl19</i>, <i>Tgfb1</i>, and <i>Adrb2</i> mRNA levels quantified by RT-PCR. Data are representative of three different experiments (4–5 mice per sample). (E) Confocal images of a LN B cell follicle and its adjacent T cell area from a CD21cre-RFP chimera stained for CXCL13 (green), CD21/35 (blue), and B220 (white). RFP⁺ cells appear in red. The dashed line represents the delineation of the B220 staining. Data are representative of two different experiments (two mice per experiment).</p

CD21⁻ RFP⁺ stromal cells are surrounded by inflamed B cell follicles.

<p>CD21cre-RFP chimeras were untreated (A, upper panel) or injected (A, lower panel and B) with an emulsion of CFA/PBS in the ears. (A) Three weeks later, ear draining LNs were sectioned, stained for collagen IV (green), IgD (white), and FDC-M2 expression (blue) and imaged by confocal microscopy. RFP⁺ cells appear in red. (B) Confocal pictures of an inflamed B cell follicle stained for collagen IV (white), B220 (blue), and CD21/35 (green) expression. Arrows highlight the region of the inflamed B cell follicle enriched in collagen IV. Insert displays high magnification of the boundary between T and B cell areas (dashed line). Data are representative of three different experiments (two mice per experiment).</p

Visualization of a new subset of LN stromal cells.

<p>Confocal images of LN sections from a CD21cre-RFP chimera stained for (A) FDC-M2 (green), CD3 (light blue), and B220 (dark blue) and (B) CD21/35 (green), Collagen IV (white), and B220 (blue). RFP⁺ cells appear in red. In (B), the star (*) signals the position of the collagen-poor central region of the follicle populated by the CD21⁺ RFP⁺ FDC network; arrows indicate the extensions of the conduit network within the follicle and arrowheads point to CD21⁻ RFP⁺ cells attached to these conduits. The dashed line represents the delineation of the B220 staining. No RFP signal was detected in CD21cre⁻RFP⁺ chimeric mice, ruling out a leaky expression of the reporter. Data are representative of three different experiments (two mice per experiment).</p