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, 198 (7), 2578-2588

Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus

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Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus

Jin-Young Choi et al. J Immunol.

Abstract

Systemic lupus erythematosus (lupus) is characterized by autoantibody-mediated organ injury. Follicular Th (Tfh) cells orchestrate physiological germinal center (GC) B cell responses, whereas in lupus they promote aberrant GC responses with autoreactive memory B cell development and plasma cell-derived autoantibody production. IL-21, a Tfh cell-derived cytokine, provides instructional cues for GC B cell maturation, with disruption of IL-21 signaling representing a potential therapeutic strategy for autoantibody-driven diseases such as systemic lupus erythematosus. We used blockade of IL-21 to dissect the mechanisms by which this cytokine promotes autoimmunity in murine lupus. Treatment of lupus-prone B6.Sle1.Yaa mice with an anti-IL-21 blocking Ab reduced titers of autoantibodies, delayed progression of glomerulonephritis and diminished renal-infiltrating Tfh and Th1 cells, and improved overall survival. Therapy inhibited excessive accumulation of Tfh cells coexpressing IL-21 and IFN-γ, and suppressed their production of the latter cytokine, albeit while not affecting their frequency. Anti-IL-21 treatment also led to a reduction in GC B cells, CD138hi plasmablasts, IFN-γ-dependent IgG2c production, and autoantibodies, indicating that Tfh cell-derived IL-21 is critical for pathological B cell cues in lupus. Normalization of GC responses was, in part, caused by uncoupling of Tfh-B cell interactions, as evidenced by reduced expression of CD40L on Tfh cells and reduced B cell proliferation in treated mice. Our work provides mechanistic insight into the contribution of IL-21 to the pathogenesis of murine lupus, while revealing the importance of T-B cellular cross-talk in mediating autoimmunity, demonstrating that its interruption impacts both cell types leading to disease amelioration.

Figures

Figure 1
Figure 1
Treatment of lupus-prone B6.Sle1.Yaa mice with anti-IL-21 inhibits disease progression and promotes survival. (A, B) Spleen weight (A) and total number of splenocytes (B) from control (closed circles) or anti-IL-21 treated (open circles) B6.Sle1.Yaa mice at indicated ages. (C, D) Scores of glomerulonephritis on a scale of 0 to 6 (C) and representative images of H & E stained kidney sections from 4-month old control (D, left) and anti-IL-21-treated animals (D, right). (E) Correlation between glomerulonephritis and spleen weights from all animals sacrificed at ages 4 and 6 months. Each circle represents an individual animal; controls in closed, and anti-IL-21-treated animals in open, circles. (F) Measurement of creatinine using QuantiChrom Creatinine Assay Kit (BioAssay Systems) in sera from all animals at indicated age (months). (G) Correlation between glomerulonephritis and serum creatinine from all animals sacrificed at ages 4 months. (H) Survival of B6.Sle1.Yaa mice treated with anti-IL-21 (n = 12), compared to PBS-treated controls (n = 9), using Mantel-Cox test. Correlations were determined using Pearson's correlation analyses. (I) Correlation between the amount of anti-IL-21 in sera and spleen weights at 6 months of age.
Figure 2
Figure 2
Anti-IL-21 treatment of B6.Sle1.Yaa mice inhibits GC B cell maturation, plasmablast differentiation, and autoantibody production. (A-C) Representative flow cytometry plots of control (left, closed circles) and anti-IL-21-treated (middle, open circles) animals and scatter plot (right) of the percentage of Ki-67+ among IgDloB220hi activated B cells in mice at 6 months (A) and GL7hiCD95hi GC B cells among B220+ B cells at 4 months (B), and the percentage of TCRβB220loCD138hi plasmablasts among total splenocytes at 4 months (C). Histogram in gray in (A) is IgDloB220hi naive B cells. (D) Representative ELISPOT analysis (left) and number of splenocytes secreting anti-chromatin antibodies in 4-month old animals (right). (E) IgG anti-dsDNA IgG autoantibodies from 4 month old control and anti-IL-21 treated B6.Sle1.Yaa mice. Each dot indicates an individual animal. Each scatter plot is a compilation of 2-3 independent experiments. (F) Immunofluorescent staining for IgG2c and C3 deposits in the glomeruli of control and anti-IL-21-treated mice at 4 months of age. The significance of the difference between two groups was evaluated by two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 3
Figure 3
Altered phenotype of splenic Tfh cells in B6.Sle1.Yaa mice is restored by anti-IL-21 treatment. (A) Quantification of percentage (top) and total numbers (bottom) of CD44loPSGL-1hi naive and CD44hi activated CD4+ T cell subsets from 4 month- and 6 month-old B6.Sle1.Yaa mice. (B, C) Strategy for identification of Tfh cells from control (top, closed circles) or anti-IL-21-treated (bottom, open circles) animals by CD44hiPSGL-1lo gating of CD4+ T cells (left) followed by that of CXCR5hiPD-1hi cells (right), with quantification of percentage (left) and total number (right) of Tfh cells from 4 month- and 6 month-old B6.Sle1.Yaa mice. (D, E) Representative flow cytometry plot from control (top) and anti-IL-21-treated (bottom) animals showing intracellular IL-21 and IFN-γ staining in Tfh cells (D) or Th1 cells (E) in black dot plots overlaid on naive CD4+ T cells staining in gray contour plot. The percentages of total IL-21+ cells (red box) or IL-21+ IFN-γ+ double positive cells (blue box) from control (closed circles) vs. anti-IL-21-treated (open circles) B6.Sle1.Yaa mice are shown at indicated ages. (F) The percentages of CD40Lhi Tfh cells from control compared to anti-IL-21-treated animals at 6 months of age. The gate was set based on CD40L expression on naive CD4+ cells. Each dot indicates an individual animal. Scatter plots are compilations of 2-3 independent experiments. The significance of the difference between two groups was evaluated by a two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4
Figure 4
Altered phenotype of splenic Tfh cells in B6.Sle1.Yaa mice is correlated with disease progression. (A) Representative flow cytometry plots from control (top, closed circles) and anti-IL-21-treated (bottom, open circles) animals showing Bcl6 expressions by Tfh cells (black line) overlaid on naive cells (filled gray) and scatter plots of geometric mean fluorescence intensity (gMFI) of Bcl6 in Tfh cells (middle) and normalized Bcl6 gMFI in Tfh cells (right). (B) Correlation between the ratio of Bcl6 gMFI and spleen weights (left), and the ratio of Bcl6 gMFI and kidney disease scores (right). Correlations were determined using Pearson's correlation analyses. (C, D) Representative flow cytometry plots from control (top) and anti-IL-21-treated (bottom) animals showing T-bet expression by Tfh cells (C) and Th1 cells (D) in black overlaid on naive cells (filled gray) and scatter plots of normalized T-bet gMFI in Tfh cells (C) and Th1 cells (D). Each dot indicates an individual animal. Scatter plots are compilations of 2-3 independent experiments. The significance of the difference between two groups was evaluated by two-tailed Student's t-test. n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5
Figure 5
IL-21 blockade prevents abnormal expansion of leukocytes in B6.Sle1.Yaa mice. (A-C) Quantification of the percentage of TCRβB220 cells (A) from control (closed circles) or anti-IL-21 treated (open circles) mice, total numbers of monocytes (B), and granulocytes (C) among total splenocytes from control (closed circles) or anti-IL-21 treated (open circles) B6.Sle1.Yaa mice at indicated ages. (D, E) The percentages of CD44hi cells among splenic CD8+ T cells (D) and representative flow cytometry plots from control (top) and anti-IL-21-treated (bottom) animals showing PD-1 expression by CD44hiCD8+ cells in black overlaid on naive CD44loCD8+cells (filled gray) and scatter plots of PD-1 gMFI in CD44hiCD8+ cells at age of 6 months (E). (F) Representative flow cytometry plots showing intracellular staining for IFN-γ and granzyme B among splenic CD8+ T cells from 6 months old B6.Sle1.Yaa animals treated with PBS (top) or anti-IL-21 (bottom) and the fraction of the total CD8+ T cells expressing IFN-γ, granzyme B, or both (right). Each dot indicates an individual animal. The significance of the difference between two groups was evaluated by two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 6
Figure 6
Infiltration of inflammatory CD4+ T cells into the kidney is abrogated by treatment with anti-IL-21 in B6.Sle1.Yaa animals. (A, B) Quantification of total number of leukocytes (A), from control (closed circles) or anti-IL-21 treated (open circles), and CD4+ T cell subsets (B) isolated from kidneys from 4 months old B6.Sle1.Yaa mice treated with PBS (closed circle) or anti-IL-21 (open circle). (C, D) Scatter plot of the percentages of IL-21+ IFN-γ+ cells among Th1 cells (C) and PSGL-1lo cells (D) isolated from kidney from control or anti-IL-21 treated B6.Sle1.Yaa mice at 4 months of age. (E) Representative flow cytometry plots from control (top) and anti-IL-21-treated (bottom) animals showing Ki-67 expression in Th1 cells (black line) overlaid on naive cells (filled gray) and the percentage of Ki67+ cells in Th1 cells (right) at 6 months of age. Each dot indicates an individual mouse. Each scatter plot is compiled from 2 independent experiments. The significance of the difference between two groups was evaluated by two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 7
Figure 7
Reduced autoantibody response by a brief treatment with IL-21 blockade in B6.Sle1.Yaa mice with advanced disease. (A, B) Spleen weight (A) and total number of splenocytes (B) from control (closed circles) or anti-IL-21 treated (open circles) B6.Sle1.Yaa mice at age of 4 months. (C) Scatter plots of the percentage of GL7hiCD95hi GC B cells among B220hiIgDlo B cells. (D) Scatter plot of the percentage of TCRβB220loCD138hi plasmablasts among total splenocytes. (E, F) Representative flow cytometry plots from control (top) and anti-IL-21-treated (bottom) animals showing intracellular IgG1 and IgG2a staining among TCRβB220loCD138hi plasmablasts (E) and scatter plots of the percentage of intracellular IgG1+ (left) or IgG2a+ (right) cells among TCRβB220loCD138hi plasmablasts in control vs. anti-IL-21-treated B6.Sle1.Yaa mice at age of 4 month (F). (G) Number of splenocytes secreting anti-dsDNA antibodies in 4-month old animals. (H) Measurement of creatinine in sera from control (closed circles) or anti-IL-21 treated (open circles) B6.Sle1.Yaa mice at indicated ages (months). (I) Scatter plots of the percentage of CD38hiGL7lo memory B cells among B220hiIgDlo B cells. Each dot indicates an individual mouse. The significance of the difference between two groups was evaluated by two-tailed Student's t-test. n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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