Fig 1.
Spontaneous germinal center responses in BXD2 mice.
(A) Auto-reactive autoantibody levels against double-strand DNA and histone in the sera of WT and BXD2 mice at the age of 6 months or older were measured by ELISA. (B) Immunofluorescence imaging of PNA+ (red) germinal center area of the spleen from WT control or BXD2 mice (×4 magnification). (C) The number of GC per spleen section in the WT and BXD2 mice was enumerated by fluorescence microscopy. (D) Flow cytometry analysis of the percentage and number of GL7+Fas+ germinal center B cells in the WT and BXD2 mice at the age of 6 months or older. Cells were gated on B220+ B cells. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
Th17 cells are not a major subset of T helper cells in BXD2 mice.
(A) Flow cytometry analysis of the percentage and number of IFN-γ or IL-17A positive CD4+ T cells in the spleen from WT and BXD2 mice at the age of 6 months or older. (B) IL-17A levels in the sera of WT and BXD2 mice at the age of 6 months or older were measured by ELISA. (C) Quantitative RT-PCR analysis of Th1 or Th17 cell related genes from WT and BXD2 mice splenocytes at the age of 6 months or older. Data are representative of the analysis (A~C) indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with BXD2-WT mice.
Fig 3.
Tfh cell responses were increased in the BXD2 mice.
(A) Flow cytometry analysis of the percentage and number of PD-1+CXCR5+ CD4+ T cells in the spleens of WT and BXD2 mice at the age of 6 months or older. (B) Quantitative RT-PCR analysis of Tfh cell related genes from WT and BXD2 mouse splenocytes at the age of 6 months or older. (C) Flow cytometry analysis of the percentage and number of CD4+Foxp3+PD-1+CXCR5+ Tfr cells in the spleens of WT and BXD2 mice at the age of 6 months or older. (D) Ratio of Foxp3- Tfh cells to FoxP3+ Tfr cells in the mouse spleens and germinal center B cells to FoxP3+ Tfr cells in the mouse spleen. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4.
Positive correlation of Tfh cells, not Th17 cells with outputs of GC responses in BXD2 mice.
Linear regression analysis of the frequency of Th17 cells (A) or Tfh cells (D) with that of germinal center B cells, and Th17 cells (B) or Tfh cells (E) with dsDNA specific autoantibody levels. Linear regression analysis of the number of Th17 cells (C) or Tfh cells (F) with that of germinal center B cells. Pearson correlation coefficients (r2) between the percent of T helper cell subset and of germinal center B cells or those of T helper cell subset and dsDNA specific autoantibodies levels are indicated in each graph.
Fig 5.
IL-21-producing CXCR5+CD4+ T cells of BXD2 mice, not IL-17-producing CCR6+CD4+ T cells, provide B cell help for IgG production.
(A) CXCR5+ CD4+ T cells and CCR6+ CD4+ T cells were sorted and subjected to intracellular cytokine staining. (B) Quantitative RT-PCR analysis of Tfh or Th17 cell related gene expression in the CXCR5+ and CCR6+ CD4+ T cells from BXD2 mice (C) Naïve B cells (B220+IgD+GL7-) from BXD2 mice were co-cultured with CXCR5+ or CCR6+ CD4+ T cells from BXD2 mice for 7days. The total IgG levels were measured by ELISA (D) Different cytokine blocking reagents, isotype control antibody (Iso Hu-Fc), Rat anti mouse IL-17A antibody (α-IL-17A), or recombinant mouse IL-21 receptor Fc chimera (IL-21R-Fc) were added (10 μg/ml, every other day) into the cell culture described in (C) and the levels of total IgG were measured by ELISA. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ###p < 0.001 comparing 10:10–10:5 ratio of B:T co-culture condition.