Figure 1.
Kinetics of spleen-derived Tregs.
The Treg population is increased after experimental myocardial infarction. (A) FACS assay of Tregs after myocardial infarction shows the numbers of Tregs, expressed as a percentage of the total number of CD4 cells, 1 day, 5 days, 14 days and 30 days after myocardial infarction. Treg cell measured by CD4+CD25+FOXP3 (B) Representative FACS results showing kinetics of Tregs 1 day and 30 days after experimental myocardial infarction. p based on Two-way ANOVA; * p<0.05 based on Mann-Whitney test. n (MI) = 18; n(sham) = 20
Figure 2.
Tregs migrate to the injured heart.
(A) FACS analysis records the numbers of Tregs (CD4+CD25hi cells) expressed as a percentage of the total number of CD45 cells after myocardial infarction. (B–C) Representative FACS recordings of the kinetics of CD45 presence in mice with experimentally induced myocardial infarction compared with control mice. (D–E) Representative FACS pictures of CD25+FOXP3 out of CD4 in infarct heart or control heart. *p<0.03 based on Mann-Whitney test, n = 4;
Figure 3.
Suppressive properties of Tregs.
Assay of functional suppression assay (A–B) shows a trend towards decline in the suppressive properties of Tregs, with significant effects at two time points after experimental induction of myocardial infarction at all ratios. p based on Two-way ANOVA; * p<0.05 based on Mann-Whitney test, n = 6–10;
Figure 4.
Homing of Tregs to ischemic hearts after their intravenous injection.
(A–C) Frozen heart sections 1 day after intravenous (i.v.) injection of DiI-labeled Tregs. (D–F) Frozen heart sections 4 days after i.v. injection of DiI-labeled Tregs. (G–I) Frozen heart sections 7 days after i.v. injection of DiI-labeled Tregs. (J–L) Frozen heart sections 4 days after i.v. injection of DiI-labeled no-Tregs. (M–O) Frozen heart sections 4 days after i.v. injection of DiI-labeled Tregs sham mice. (Q) Treg numbers are significantly increased by day 4 post i.v. injection. p based on Kruskal-Wallis; **p<0.001, ***p<0.0001 based on Dunn's multiple comparison test. n (day 1) = 8; n (day 4) = 10; n (day 7) = 8, MI no-Treg cells (n = 8), Sham (n = 8).
Figure 5.
Adoptive transfer of Tregs improves cardiac function.
(A) Treg-treated mice maintain fractional shortening, whereas control (PBS-treated) mice exhibit deterioration in this parameter. (B) Treg-injected mice exhibit no change in LVESA, whereas after 4 weeks the PBS-treated mice show a significant increase in LVESA compared to baseline. p based on Two-way repeated measures ANOVA; **p<0.007 and ***p<0.0005 based on 2-tail paired test.
Table 1.
Comparison of left ventricular remodeling and function in Treg or PBS injected mice by 2-dimensional echocardiography 1 day after MI (baseline) and 4 weeks after first echo.
Table 2.
Percentage of change2 from 1 day after MI (baseline) obtained by echocardiography 4 weeks after first echocardiography.
Figure 6.
Adoptive transfer of regulatory T cells reduces infarct size.
(A) Morphometry shows that treatment with Tregs reduces the proportion of fibrosis. (B) Representative sections of mouse heart treated with Tregs or PBS and labeled by Masson Trichrome staining at x20 magnification compared to control heat. (C) The number of capillaries was 2-fold greater in the Treg-treated group. (D) The number of vessels>10 µm was similar between the groups. (E–F) Serial sections were immunolabeled with antibodies against CD31 at x400 magnification. *p<0.02, **p<0.002 based on Mann-Whitney test.
Figure 7.
Anti-CD25 mAb (PC61) depletes CD4+CD25+FOXP3 cells.
(A) The antibody dosage at three different ratios. Representative FACS picture shows the levels of CD25hiFOXP3 out of CD4+: (B), (D), (F) PBS injection group (C) ACD25 injection group: 800 µg (E) 500 µg and (G) 200 µg. p based on Two-way ANOVA; ***p<0.0001 based on Bonferoni's multiple comparison test.
Figure 8.
No effect on cardiac function after Tregs depletion.
(A) ACD25 mAb or control (IgG isotype control) treated mice exhibit deterioration in FS parameter. (B) ACD25 mAb or control (IgG isotype control) treated mice exhibit significant increase in LVESA after 4 weeks compared to baseline. p based on Two-way repeated measures ANOVA; *p<0.02 and **p<0.002 based on 2-tail paired test.
Table 3.
Comparison of left ventricular remodeling and function in ACD25 or IgG injected mice by 2-dimensional echocardiography 1 day after MI (baseline) and 4 weeks after first echo.
Table 4.
Percentage of change2 from 1 day after MI (baseline) obtained by echocardiography 4 weeks after first echocardiography.
Figure 9.
No effect at infarct size after Tregs depletion.
Representative sections of mouse heart treated with (A) IgG isotype control or PC61 mAb and labeled by Masson Trichrome staining at x20 magnification. (B) The number of capillaries was 2-fold lower in the Treg ablation group. (C) The number of vessels>10 µm was similar between the groups (D–E) Serial sections were immunolabeled with antibodies against CD31 at x400 magnification. **p<0.001 based on Mann-Whitney test; n(ACD25) = 4; n(IgG) = 6.