Reduced effectiveness of CD4+Foxp3+ regulatory T cells in CD28-deficient NOD.H-2h4 mice leads to increased severity of spontaneous autoimmune thyroiditis

Abstract

NOD.H-2h4 mice given NaI in their drinking water develop iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) with chronic inflammation of the thyroid by T and B cells and production of anti-mouse thyroglobulin (MTg) autoantibody. CD28(-/-) NOD.H-2h4 mice, which have reduced numbers of CD4(+)Foxp3(+) regulatory T cells (Tregs), were developed to examine the role of Tregs in ISAT development. CD28(-/-) NOD.H2-h4 mice develop more severe ISAT than do wild-type (WT) mice, with collagen deposition (fibrosis) and low serum T4. CD28(-/-) mice have increased expression of proinflammatory cytokines IFN-γ and IL-6, consistent with increased mononuclear cell infiltration and tissue destruction in thyroids. Importantly, transferring purified CD4(+)Foxp3(+) Tregs from WT mice reduces ISAT severity in CD28(-/-) mice without increasing the total number of Tregs, suggesting that endogenous Tregs in CD28(-/-) mice are functionally ineffective. Endogenous CD28(-/-) Tregs have reduced surface expression of CD27, TNFR2 p75, and glucocorticoid-induced TNFR-related protein compared with transferred CD28(+/+) Tregs. Although anti-MTg autoantibody levels generally correlate with ISAT severity scores in WT mice, CD28(-/-) mice have lower anti-MTg autoantibody responses than do WT mice. The percentages of follicular B cells are decreased and those of marginal zone B cells are increased in spleens of CD28(-/-) mice, and they have fewer thyroid-infiltrating B cells than do WT mice. This suggests that CD28 deficiency has direct and indirect effects on the B cell compartment. B cell-deficient (B(-/-)) NOD.H-2h4 mice are resistant to ISAT, but CD28(-/-)B(-/-) mice develop ISAT comparable to WT mice and have reduced numbers of Tregs compared with WT B(-/-) mice.

Figure 1

CD28−/− NOD.H-2h4 mice have fewer Treg than WT NOD.H-2h4 mice. CD4+FoxP3+ cells in spleens and lymph nodes of WT and CD28−/− NOD.H-2h4 mice were determined by flow cytometry. Dot plots of representative mice from each group indicating the percentages of splenic CD4+ cells that express FoxP3 are shown (top). Data are shown as mean ± SEM of N = 6(Spleen B−/− and CD28−/−B−/−) and 3(LN B−/− and CD28−/−) per group and are pooled from 2 experiments. ** p<0.01 compared with respective control CD28⁺ group, Student’s t-test.

Figure 2

Comparison of ISAT severity, serum T4, and histology of thyroids from WT and CD28−/−NOD.H-2h4 mice. WT and CD28−/− NOD.H-2h4 were given NaI in their drinking water at 8 wk of age. After 8 weeks, thyroids were removed, fixed, sectioned, and either stained with hematoxylin and eosin or for collagen by Trichrome staining. (A) ISAT severity scores 8 wks after NaI water. p < 0.001 N= 35(WT) and 21(CD28−/−). Results are pooled from 4 experiments and are representative of multiple experiments involving >100 mice. (B) Serum T4 levels from individual mice. p = 0.0005 N= 11(WT) and 21(CD28−/−) from 7 independent experiments. (C) H&E stained thyroid sections demonstrating increased infiltration and follicle destruction in CD28−/− compared with WT mice. Note empty follicles (arrows) in thyroids of CD28−/− mice compared to colloid filled follicles in WT thyroids. ISAT scores (WT) – 2, 3, 2; (CD28−/−) – 4, 4, 4. Trichrome staining shows collagen deposition (blue) in thyroids of CD28−/− mice with severe (4+) ISAT. Fibrosis is absent in thyroids of WT mice with 3+ ISAT. Results are representative of at least eight thyroids per group from five experiments. 100× (bar = 0.01mm); 400× (bar = 0.005mm). (D) SAT severity scores of 4–8 month old WT and CD28−/− mice not given NaI water. N = 13(WT 4–5 mo) and 13(WT 6–8 mo) from 4 experiments, 15(CD28−/− 4–5 mo) from 3 experiments, and 21(CD28−/− 6–8 mo) from 3 experiments.

Figure 3

Differential expression of proinflammatory cytokines in thyroids of CD28−/− and WT NOD.H-2h4 mice. (A) WT and CD28−/− NOD.H-2h4 mice were given NaI in their drinking water at 8 weeks of age. Thyroids were removed 8 wk later, snap frozen and RNA was isolated from individual thyroid lobes. Expression of IFNγ, IL-6, IL-17 and B7-1 were determined by semiquantitative RT PCR as described in Methods. Results represent the ratio of particular cytokine:β-actin densitometric units ± SEM of individual thyroids from 5 mice per group, and are representative of two separate experiments. (B) Thyroid infiltrating cells were stained for the presence of intracellular IFNγ as described in Methods. Dot plots show expression of CD3 and IFNγ by CD45+ thyroid infiltrating cells. Dot plots are representative of the data in the bar graph. The bar graph represents mean ± SEM of CD45+CD3+IFNγ+ cells per thyroid from 4(WT) and 8(CD28−/−) mice per group pooled from two experiments. * p<0.05, Student’s t-test.

Figure 4

CD28+/+ Treg suppress ISAT development in CD28−/− mice. (A) CD28−/− mice were given three i.v. injections of 10⁶ sorted CD4+FoxP3+ Treg or CD4+FoxP3− T cells from CD28+/+ FoxP3−GFP NOD.H-2h4 mice as described in Methods. Mice were given NaI in their drinking water at the time of the first injection of Treg, and ISAT severity was determined 7 wks later. N = 19(Control), 9(+ CD4+FoxP3−), and 20 (+ CD4+FoxP3+) mice per group pooled from 3 experiments. *** p < 0.001, Mann-Whitney nonparametric test. (B, C) Splenocytes from CD4+FoxP3+ Treg, CD4+FoxP3− T cell recipients and control mice were stained for expression of CD4, CD28, and FoxP3. Plots represent the total number of CD4+FoxP3+ Treg (B) or CD4+FoxP3+CD28+ Treg (C) per spleen. Bar graph represents total of CD28−CD4+FoxP3+ and CD28+CD4+FoxP3+ Treg per recipient. N = 12(Control), 9(+ CD4+FoxP3−) and 13(+ CD4+FoxP3+) mice per group pooled from 2 independent experiments. (D) Thyroids from the Treg groups in A–C were stained for expression of CD45, CD4, FoxP3, and CD28 and analyzed by flow cytometry. Plots represent the percentage of CD45+CD4+FoxP3+ cells that express CD28. N = 5(Control), 4(+ CD4+FoxP3−) and 6(+ CD4+FoxP3+) mice per group and are representative of 3 independent experiments) (E) Splenocytes from CD4+FoxP3+ Treg recipients were stained for the presence of CD4, CD28, FoxP3 and CD27, GARP, GITR, or TNFR2 p75. Plots represent the percentage of CD28+CD4+FoxP3+CD28+ or CD28−CD4+FoxP3+ cells positive for the indicated marker. (N=9 mice per group; representative of 3 independent experiments.) (F) Splenocytes from WT or CD28−/− mice given NaI water for 8 wks were stained for the presence of CD4, CD28, FoxP3 and CD27, GARP, GITR, or TNFR2 p75. Plots represent the percentage of CD4+FoxP3+ cells positive for the indicated marker. (N=4 mice per group and are representative of 2 independent experiments) n.s. = not significant, ** p < 0.01; *** p < 0.001, Student’s t-test.

Figure 5

Differences in serum autoantibody levels and changes in the B cell compartment in CD28−/− mice. WT and CD28−/− NOD.H-2h4 mice were given NaI in their drinking water beginning at 8 weeks of age. After 8 weeks, serum anti-MTg autoantibodies were determined (A) and splenocytes were stained for CD19, B220, CD21, and CD23 (B–E) and analyzed by flow cytometry. Thyroids were stained for B220, CD45, and CD138. (F, G) (A) Serum anti-MTg autoantibody production from was determined using 1/50 and 1/100 dilution of serum from individual mice and is expressed as mean OD₄₁₀ ± SEM. * p < 0.05, *** < 0.001, Student’s t-test N= 20(WT), 18(CD28−/−) from 3 experiments. (B) CD19+ cells as a percentage of live gated cells. (C) Dot plots show CD21 and CD23 expression on B220+ cells. Representative dot plots from mice in D and E (D) Follicular B cells as a percentage of B220+ cells. (E) Marginal zone B cells as a percentage of B220+ cells. N = 12 (WT) and 17(CD28−/−) mice pooled from 6 experiments. (F) B220+ cells as a percentage of CD45+ cells. (G) CD138+ cells as a percentage of CD45+B220+ cells. Results are pooled from two independent experiments with N = 5(WT) and 4(CD28−/−) mice per group. Each symbol represents a single mouse and bars represent the mean of each group. * p < 0.05, Student’s t-test.

Figure 6

CD28 deficient B−/− mice have reduced Treg numbers and develop ISAT similar to WT mice. (A) Splenocytes from WT B−/− and CD28−/−B−/− mice were analyzed by flow cytometry for the presence of CD4+FoxP3+ Treg. Dot plots of representative mice from each group indicating the percentages of splenic CD4+ cells that express FoxP3 are shown (top). Data are shown as mean ± SEM of N = 6(Spleen B−/− and CD28−/−B−/−) and 3(LN B−/− and CD28−/−) per group and are pooled from 2 experiments. * p<0.05, ** p<0.01 compared with respective control CD28+ group, Student’s t-test. (B) WT, CD28+/+B−/−, or CD28−/−B−/− mice were given NaI in their drinking water for 8 weeks, and ISAT severity was determined. (N= 19(WT), 13(WT B−/−), and 16(CD28−/−B−/−) mice per group) * p < 0.05, ** p < 0.01, Mann-Whitney nonparametric test. (C) Splenocytes from WT B−/− or CD28−/−B−/− mice given NaI water for 8 wks were stained for the presence of CD4, CD28, FoxP3 and CD27, GARP, GITR, or TNFR2 p75. Plots represent the percentage of CD4+FoxP3+ cells positive for the indicated marker. (N=7 mice per group combined from two experiments) ** p < 0.01; * p < 0.05, Student’s t-test.