Tg4 IL-10^−/− mice exhibit spontaneous EAE but no deficiency in Foxp3⁺ Treg cell numbers or phenotype.

<p>A. Tg4 IL-10^−/− mice, untreated, n = 7, or receiving 1.5×10⁶ Tg4 IL-10^+/+ splenic Treg cells i.p. at age 4–5 weeks, n = 9, were monitored daily for the spontaneous development of CNS autoimmune disease. Logrank test, P = 0.25 B. Representative haematoxylin/eosin stained sections of spinal cord (left and middle) and brain (right) from Tg4 IL-10^−/− with established CNS autoimmune disease (n = 6). Arrows indicate lymphocytic infiltrate. Magnifications: left; ×200, middle; ×100, right; ×25. C. Inflammation and demyelination scores of spinal cord and brain sections of Tg4 IL-10^−/− with established disease. Horizontal lines indicate means. n = 6 each. D-J. Tg4 IL-10^+/+ and Tg4 IL-10^−/− mice approximately 5 weeks of age were examined for a number of characteristics; D. Frequency of Foxp3⁺ cells among CD4⁺CD8⁻ (CD4SP) thymocytes or CD4⁺ T cells in the spleen and mesenteric lymph nodes (MLN). Thymus; Tg4 IL-10^+/+ n = 16, Tg4 IL-10^−/− n = 13. Spleen and MLN; Tg4 IL-10^+/+ n = 16, Tg4 IL-10^−/− n = 14. E. Total number of splenocytes. F. Number of CD4⁺Foxp3⁺ cells in the spleen. G. Percentage CD62L expression on splenic CD4⁺ Foxp3⁻ cells. H. Percentage Neuropilin-1 (NRP-1), GITR and CD62L expression on splenic CD4⁺Foxp3⁺ cells. I. Percentage NRP-1, GITR and CD62L expression on CD4⁺Foxp3⁺ cells in the MLN. J. Cumulative frequency of CD4 and CD8 single positive (SP), double positive (DP) or double negative (DN) thymocytes. E–J. Tg4 IL-10^+/+ n = 12, Tg4 IL-10^−/− n = 10. D–I. statistical analysis by unpaired, 2-tailed, t-test. Panels E-I; no significant differences.</p

iTreg cell function is mediated by IL-10 and CTLA-4.

<p>A. CPD-ef450-labeled naive CD4⁺ Tg4 CD45.1⁺ T cells cultured in vitro for 4 days with or without Tg4 CD45.2⁺ iTreg cells and stimulated with 1 µg/ml MBP Ac1–9 and irradiated APCs. Representative overlays gated on CD45.1⁺ cells (left) and proliferation index as means of triplicates + SEM (right). Representative of 3 or more similar experiments. *** P<0.001, one-way ANOVA with Dunnett's post-test. B. Tg4 Rag^−/− mice received 5×10⁶ iTreg cells in PBS or PBS only, i.p., 3 days prior to EAE induction. Data in each line were combined from 2 or more individual experiments and are shown as mean ± SEM. Distribution plot shows disease burden for each individual in each group * P<0.05, Kruskal Wallis with Dunn's post-test comparing all pairs of columns. No statistically significant differences exist between other column pairs. C. Tg4 CD45.1⁺ mice received 5×10⁶ Tg4 CD45.2⁺ iTreg cells, i.p., 72 hrs prior to priming with MBP Ac1–9 in CFA. On day 7 post-prime, spleens, inguinal lymph nodes (ILN) and cervical lymph nodes (CLN) were removed and isolated cells analyzed for Foxp3 (left) and Ki67 (right) expression by FACS. Accumulated results from 4 Tg4 IL-10^+/+ and 3 Tg4 IL-10^−/− mice, gated on CD45.2⁺ cells. ** P<0.01, *** P<0.001, Tukey's multiple comparison test.</p

CTLA-4 but not IL-10 deficiency impairs iTreg development.

<p>A. Foxp3 expression after 3-day (left) or 7-day (right) culture of naive CD4⁺ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of 100 U/ml rhIL-2 and 10 ng/ml rhTGF-β₁. n = 6–14 individual experiments per condition. *** P<0.001, Tukey's multiple comparison test. n.s.  = not significant. Day 3 and day 7 data are not from the same experiments. B. Median fluorescence of Foxp3 staining on CD4⁺Foxp3⁺ T cells after 7-day culture as in A. Matching symbols in either column in each plot correspond to paired samples. n = 6–9. No significant differences; paired, 2-tailed t-test. C + D. Mean expression (C) and representative cytometry plots (D) of transcription factors and surface markers on CD4⁺Foxp3⁺ T cells after 7-day culture as in A. n = 3–12 individual experiments per condition. C * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Dunnett's post-test.</p

Antigen-specific iTreg cells ameliorate EAE progression.

<p>A–C. Mice received 5×10⁶ iTreg cells in PBS or PBS only i.p. 2–3 days prior to EAE induction with MBP Ac1–9 in CFA. A. Tg4 Rag^+/+ recipients. N = 6 per group, combined from 2 individual experiments. ** P = 0.0022, Mann Whitney test. B. Tg4 Rag^−/− recipients. n = 7 per group, combined from 3 individual experiments. *** P = 0.0006, Mann Whitney test. C. Tg4 Rag^−/− recipients. PBS control and B10.PL iTreg n = 5, Tg4 iTreg n = 4, combined from 2 individual experiments. * P< 0.05, Kruskal Wallis with Dunn's post-test. A-C EAE plots depict mean ± SEM. Horizontal lines in disease burden scatter plots indicate mean. D. Tg4 CD45.1 naive CD4⁺ T cells, labeled with CFSE, were cultured alone or co-cultured at a 1 to 1 ratio with B10.PL or Tg4 (both CD45.2⁺) iTreg cells for 4 days and stimulated with 1 µg/ml MBP Ac1–9. CFSE dilution was measured by FACS (gated on CD45.1⁺ cells) and proliferation indexes calculated using FlowJo software. Bar graph shows mean ± SEM. One experiment of 3 similar experiments in triplicate is shown. ** P<0.01, *** P<0.001, Tukey's multiple comparison test.</p

Tg4 FoxP3^gfp iTreg cells are suppressive but unstable in vitro.

<p>A. Representative histograms of cell proliferation dye dilution in labeled CD45.1⁺ naive CD4⁺ T cells cultured for 3 days with/without Tg4 FoxP3^wt or Tg4 FoxP3^gfp iTreg cells at a 1∶1 or 2∶1 ratio and stimulated with 10 µg/ml MBP Ac1-9. One representative of three identical experiments in triplicate. B. Proliferation index of naive CD4⁺ cells after 72 h co-culture with/without iTreg cells. Means of 3 identical experiments carried out in triplicate. * p < 0.05, ** p < 0.001, Tukey's multiple comparison test. C. Frequency of FoxP3 expression and proliferation of CD45.2⁺ iTreg cells after 3-day co-culture with naive CD45.1⁺ CD4⁺ T cells. One experiment representative of three carried out in triplicate. Gated on live CD45.2⁺ CD4⁺ T cells. D. Purity of FoxP3⁺ CD4⁺CD25⁺ Tg4 FoxP3^wt/gfp iTreg cells and frequency of GFP expression therein, prior to in vitro co-culture with naive T cells. E. Proliferation and FoxP3 retention of CD45.2⁺ Tg4 FoxP3^wt/gfp iTreg cells as well as frequency of GFP expression among CD45.2⁺ FoxP3⁺ cells after 3-day co-culture with naive CD45.1⁺ CD4⁺ T cells. F. CD25 expression and cell proliferation dye dilution in naive CD45.1⁺ CD4⁺ T cells after 72-h in vitro co-culture with CD45.2⁺ Tg4 FoxP3^wt/wt or Tg4 FoxP3^wt/gfp iTreg cells at different ratios. Proliferation dye dilution displayed as dot plot versus CD25 expression, or off-set histogram overlays. D-F. One experiment representative of 3 identical experiments.</p

Young Tg4 FoxP3^gfpmice have unaltered levels of FoxP3 expression.

<p>A. FoxP3 expression by CD4⁺CD8⁻ thymocytes or CD4⁺ cells from the spleen and mesenteric lymph nodes of male Tg4 FoxP3^wt and Tg4 FoxP3^gfp mice aged 5–7 weeks, directly ex vivo. Horizontal bar indicates mean. * p = 0.0329, ** p = 0.0084, n.s. = not significant. 2-tailed, unpaired student's t test, n = 7 each. B. Total number of splenocytes, CD4⁺ T cells and FoxP3⁺CD4⁺ Treg cells in the spleen of male Tg4 mice aged 5–7 weeks. Data displayed as mean, error bar indicates SEM. n = 3 each, * p = 0.0108, n.s. = not significant. 2-tailed, unpaired student's t test. C. Mean fluorescence intensity of FoxP3 staining in Treg cells in the thymus, spleen and mLN of 5–7 week old male Tg4 mice. Data displayed as mean, error bar indicates SEM. n = 3 each, no statistical difference, unpaired, 2-tailed student's t test. D. Frequency of FoxP3 expression on CD4⁺CD8⁻ thymocytes or CD4⁺ cells from the spleen or mLN of Tg4 FoxP3^wt/wt, Tg4 FoxP3^wt/gfp or Tg4 FoxP3^gfp/gfp females aged 5–8 weeks. FoxP3^wt/wt n = 8, FoxP3^wt/gfp n = 12, FoxP3^gfp/gfp n = 4. No statistically significant differences, Tukey's multiple comparison test.</p

Tg4 iTreg cells are unstable during homeostatic expansion in vivo.

<p>A. Representative histogram overlay of cell proliferation dye dilution of labeled male Tg4 CD45.1⁺ CD4⁺ Tconv cells (left) and proliferation index (mean±SEM), 5 days after co-transfer with/without CD45.2⁺ Tg4 FoxP3^wt or Tg4 FoxP3^gfp iTreg cells into lymphopenic H2URagKO mice. One representative of three identical experiments. B. Representative histogram overlay of cell proliferation dye dilution in labeled female Tg4 CD45.1⁺ CD4⁺ Tconv cells (left) and proliferation index (mean±SEM), 5 days after co-transfer with/without CD45.2⁺ Tg4 FoxP3^wt/gfp iTreg cells into lymphopenic H2URagKO mice. One representative of three identical experiments. C. Purity of FoxP3⁺ CD4⁺CD25⁺ Tg4 FoxP3^wt/gfp iTreg cells and frequency of GFP expression therein, prior to co-transfer with naive T cells. D. Proliferation and FoxP3 retention of CD45.2⁺ Tg4 FoxP3^wt/gfp iTreg cells (left) as well as frequency of GFP expression among CD45.2⁺ FoxP3⁺ cells (right), 5 days after co-transfer with naive CD45.1⁺ CD4⁺ Tconv cells. C and D; One experiment representative of 3 identical experiments.</p

Tg4 FoxP3gfp mice are not more susceptible to induced disease.

<p>A. Mean EAE disease scores (± SEM) in male and female Tg4 FoxP3 mice, after induction of disease with MBP Ac1-9 in CFA. Male Tg4 FoxP3^wt n = 7, male Tg4 FoxP3^gfp n = 10, female Tg4 FoxP3^wt/wt n = 6, female Tg4 FoxP3^wt/gfp n = 7, female Tg4 FoxP3^gfp/gfp n = 4. B. Mean percentage of initial body weight (on day -1)±SEM, after disease induction. C. disease incidence, mean day of onset (± SD), mean maximum disease grade (± SD) and mortality during the first 21 days following disease induction.</p

Impaired FoxP3 induction in naive Tg4 FoxP3^gfp CD4⁺ T cells.

<p>A. In vitro FoxP3 induction in male, naive CD4⁺CD62L⁺ T cells by stimulation with peptide and antigen-presenting cells or plate-bound anti-CD3 and anti-CD28 antibody in the presence of IL-2 and TGF-β1 for 7 days. n = 6 each for peptide stimulation, n = 8 each for antibody stimulation. B. Mean fluorescence intensity of anti-FoxP3 antibody staining in FoxP3⁺ iTreg cells. Data represented as mean + SEM. n = 6 each for peptide stimulation, n = 8 each for antibody stimulation. C. In vitro FoxP3 induction in female Tg4 FoxP3^wt/wt and Tg4 FoxP3^wt/gfp naive CD4⁺ T cells. Peptide stimulation; FoxP3^wt/wt n = 5, FoxP3^wt/gfp n = 8. Antibody stimulation; FoxP3^wt/wt n = 10, FoxP3^wt/gfp n = 9. D. Frequency of GFP expression in FoxP3⁺CD4⁺ iTreg cells generated from Tg4 FoxP3^wt/gfp naive T cells using peptide or plate-bound antibody. Peptide stimulation; n = 8, antibody stimulation; n = 10. E. Correlation plot of the frequency of GFP expression in antibody-induced FoxP3⁺ iTreg cells and thymic FoxP3⁺ nTreg cells from the same female Tg4 FoxP3^wt/gfp mice. n = 11. F. Proliferation of CD4⁺ T cells during antibody-mediated iTreg cell generation. Naive female CD4⁺CD62L⁺ Tg4 FoxP3^wt/gfp cells were labeled with 5 µM CPD-eFluor450 prior to 7-day culture with IL-2 and TGF-β₁. Plots gated on FoxP3⁺ Treg cells. Shown are proliferation of GFP^neg and GFP^pos Treg cells from mice with a high (top row) or low (bottom row) frequency of GFP expression. Histogram overlays show proliferation of GFP^neg (black, open line) and GFP^pos (grey, filled line) Treg cells. 2 out of 4 identical experiments (each in triplicate) shown. G. Frequency of Helios and PD-1 expression in GFP^pos and GFP^neg FoxP3⁺CD4⁺ iTreg cells generated from splenic, naive CD4⁺ T cells of Tg4 FoxP3^wt/gfp mice. n = 8. A, B and C; 2-tailed, unpaired student's t test. D. 2-tailed, paired t test on actual measurements versus predicted value of 50%. F and G; 2-tailed, paired student's t test.</p

GFP^pos Treg cells display a potentially more suppressive phenotype in Tg4 FoxP3^wt/gfp females.

<p>A. Representative dot plot (splenocytes, left) and distribution graph (right) showing the frequency of GFP expression in CD4⁺FoxP3⁺ cells from the thymus, spleen and mLN of Tg4 FoxP3^wt/gfp females aged 5–8 weeks. Horizontal dotted line represents the predicted frequency. P values show significance of actual vs predicted value. 2-tailed, paired student's t test. n = 10 each. B-H. Representative, smoothed, cytometry plots and distribution graphs showing the frequency of marker expression on GFP^pos and GFP^neg splenic CD4⁺FoxP3⁺ Treg cells in Tg4 FoxP3^wt/gfp females aged 5–10 weeks. P values represent significance determined by 2-tailed, unpaired student's t test. n.s. = not significant. B. Helios, n = 10. C. GITR, n = 10. D. CD103, n = 10. E. PD-1, n = 10. F. CD25, n = 6. G. CD62L, n = 4. H. Ki67, n = 4.</p