FIG 4.

Increased T_REG/T_H cell ratios protect against AngII-triggered cardiorenal fibrosis. (A) Example of purified iT_REG cells used in these experiments. CD4⁺ cells obtained from homogenized spleens and separated by flow cytometry (two left panels) were cultured with antibodies to CD3, CD28, interleukin 2, and transforming growth factor β₁. After 4 days, CD25 expression was checked by flow cytometry (third panel from left) and injected into mice. In some cases, aliquots of cells were checked for Foxp3 expression (right panel). (B) Ratios of the indicated cell populations in kidneys from animals injected with 2 × 10⁵ Ly5.1⁺ T_REG cells and infused 24 h later with AngII for the indicated periods. (C) Mean numbers of the indicated cell populations in kidneys from mice used in the experiment for which results are shown in panel B (n = 4). (D to F) Cardiorenal fibrosis levels (D and E) and AUC for mean arterial pressure (F) in WT mice maintained under the indicated conditions for 14 days. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) from the control or between the indicated experimental groups (n = 4). (G and H) Representative example (G) and quantification (H) of neutrophils in kidneys from WT mice at the end of the experiments described in the legend to panel B. Asterisks indicate significant differences (**, P ≤ 0.01) from the control or between the indicated experimental groups (n = 4). (I) Quantification of kidney-resident neutrophils in WT mice treated as indicated. *, P ≤ 0.05 (n = 4). (J) Percentages of iT_REG cells generated from splenic WT and Vav1^−/− CD4⁺ CD25⁻ T cells in cell culture (n = 4). (K) Extent of kidney fibrosis in AngII-infused Foxn1^nu mice injected with the indicated iT_REG cells. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01) from the control or between the indicated experimental groups (n = 4).