Figure 1.
Genetic map of the c1 congenic mouse strains studied.
Thick and thin lines denote NZB and B6 regions, respectively. Dashed lines indicate regions of undefined origin. Polymorphic microsatellite markers and single nucleotide polymorphism (SNP) markers were used to discriminate between NZB and B6 DNA at the termini of the regions according to the NCBI 2007 (m37 release) mouse genome assembly (www.ensembl.org). Potential candidate genes within the interval are indicated above the chromosomal map. Phenotypic features of NZB c1 congenic mouse strains are shown to the right of the c1 congenic mice genetic map [9].
Figure 2.
Expansion of Tfh, Th17 and Th1 cell subsets in c1 congenic mice.
(A) Splenocytes from 4-mo-old mice were stained to assess the proportion of Tfh (CD4+CD44hiCD62LloCXCR5 hiPD1hi) cells. Representative contour plots from B6 and c1(70-100) mice. Thick boxes denote the regions that were used to identify Tfh cells. Cells shown in the right panels were gated on the regions shown in the left panels. (B) Scatter plots showing the proportion of Tfh cells within the CD4+ T cell subset and absolute number of splenic Tfh cells. (C) Representative contour plots and histograms from flow cytometry analysis of IL-17-, IFN-γ-, and IL-4-expressing CD4+ T cells in B6 and c1(70-100) mice. Splenocytes were stimulated with PMA and ionomycin in the presence of GolgiStop for 4 h, and then fixed, stained with anti-CD3 and -CD4, permeabilized, and stained with anti-cytokine Ab. Thick lines outline the regions used to gate CD4+CD3+ T cells. For histograms, the percentage of cells staining positively for each cytokine is indicated. (D) Scatterplots showing the percentages of cytokine-producing cells as a proportion of the CD4+ T cell population. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with **p<0.01, ***p<0.001. Bars with p values above denote significant differences between congenic strains.
Figure 3.
Enhanced differentiation of pro-inflammatory T cell subsets in c1 congenic mice following OVA immunization.
8-wk-old mice were injected i.p. with OVA or PBS in CFA. The proportions of splenic Tfh cells (CD4+CD44hiCD62LloCXCR5 hiPD1hi) and splenic GC B cells (B220 + Fas + PNAhi) were determined by flow cytometry 2 wks later. (A) Scatterplots showing the proportion of Tfh and GC B cells as a proportion of the CD4+ T cell and B220+ B cell populations, respectively. (B) Scatterplots showing the amount of cytokine produced by OVA-primed splenocytes re-stimulated in-vitro with OVA for 72h. Assays were performed in triplicate and the levels of secreted cytokines measured by ELISA or cytokine bead array (see Methods). Each data point represents the mean of the triplicate with background cytokine production in the absence of antigen subtracted. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
Increased differentiation of naïve CD4+ T cells from c1 congenic mice to Th17 and Th1 cells in-vitro.
Naïve T cells from 8-wk-old mice were stimulated under Th0, Th1, Th2, Th17, and IL-21-producing polarizing conditions and cytokine production quantified 5 days later by flow cytometry (see Methods). (A) Representative contour plots gated on CD3+CD4+ T cells from B6 and c1(70-100) mice. For each polarizing condition, plots for relevant cytokine production under Th0 conditions (-) and polarizing conditions (+) are shown. The quadrants used to define positively and negatively staining cells are indicated. (B) Scatterplots showing the percentage of T cells that are IL-21-producing (Tfh), Th17, Th1 and Th2 cells, under relevant polarizing conditions. Horizontal lines indicate the mean for each population examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001. Bars with p values above denote significant differences between congenic strains.
Figure 5.
Intrinsic T cell functional defects together with altered environmental cues promote the enhanced differentiation of OVA-specific T cell subsets in congenic mice.
Naïve T cells from OT-II TCR Tg mice were transferred into pre-autoimmune B6.Thy1.1 or c1(70-100). Thy1.1 mice, that were subsequently immunized with OVA in CFA. Mice were sacrificed 2 wks later and the proportion of transferred T cells differentiating to various T cell subsets was examined by flow cytometry. (A) Representative contour plots following transfer of B6 or c1(70-100) OT-II cells into c1(70-100). Thy1.1 mice. Transferred cells were identified by staining the splenocytes from recipient mice with anti-Thy1.2 mAb. Tfh cells were identified by gating on the CD4+CD44hiPD1 hiCXCR5hi cells (indicated by boxed regions) within this subset. Cytokine-producing cells were identified as outlined in Figures 1 and 2, and the Methods. Scatter plots of the proportion of (B) Tfh and (C) cytokine-producing cells within the transferred T cell population. The open and closed symbols represent cells transferred into B6 or c1(70-100) recipient mice. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05.
Figure 6.
Myeloid DC from c1(88-100) and c1(70-100) mice demonstrate altered function and an enhanced ability to induce differentiation of Th1 cells.
BMDC from 8–12 wk-old mice were expanded with FLT3L for 7 days and then co-cultured with OVA peptide and purified naïve CD4+ T cells from OT-II TCR Tg mice. On day 4, the cells were re-stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop or GolgiPlug, and analyzed by flow cytometry for cell surface DC (CD11c, CD11b, B220, MHC-II, B7.2) or T cell (CD3, CD4) markers and intracellular cytokine levels. (A) Scatterplots showing the percentage of IL-21-, IL-17- and IFN-γ-producing T cells. Results are clustered in groups based on the strain of T cells (top of the figure) with the DC strain shown at the bottom of the figure. Scatterplots showing the percentage of CD11c+CD11b+B220− mDC (B) and CD11c+CD11b-B220+ pDC (C) expressing elevated levels of MHCII and B7.2, or IL-6 and IL-12. Results with the different strains of T cells have been pooled as no differences were noted between strains. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001. Bars with p values above denote significant differences between congenic strains.
Figure 7.
Altered production of IL-6 and/or IL-12 by myeloid DC from c1(88-100) and c1(70-100) mice following stimulation with TLR ligands.
BMDC from 8-12 week-old mice were expanded with FLT3L and then cultured in the presence or absence of Imiquimod R837, Poly(I:C), CpG 2216, or LPS for 18h with GolgiStop (for IL-12) or GolgiPlug (for IL-6) being added for the last 6 h. The cells were then stained as outlined in Figure 5 and the Methods. (A) Left panel shows representative dot plots indicating the regions used to gate B220 + CD11b- pDC (top left box) and B220-CD11b+ mDC (bottom right box) within the CD11c+ population. Shown to the right are representative histogram plots of IL-6 and IL-12 for B6 (solid grey) and c1(70-100) mice (black line) in unstimulated (Media) and stimulated (Poly(I:C) for IL-6 or CpG 2216 for IL-12) conditions. (B) Scatterplots showing the MFI for IL-6 and IL-12 expression on mDC. (C) IFN-α and IL-23 levels in the culture supernatants of BMDC as measured by ELISA. (D) MFI for TNF-α expression in mDC. Horizontal lines indicate the mean. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01. Bars with p values above denote significant differences between congenic strains.