CD4⁺ T cells from [4Y] treated Tg4^KO mice do not suppress the proliferation of naïve Tg4^WT T cell <i>in vitro</i>.

<p>(<b>A</b>) Experimental design. Splenocytes from Tg4^WT and Tg4^KO mice, treated with [4Y] or PBS, were expanded <i>in vitro</i> with MBPAc1-9[4K] and rhIL-2 for seven days before co-culture with CellTrace-labeled CD4⁺ T cells isolated from naïve Tg4^WT mice. (<b>B</b>) Examples of the secretion of IL-10 and IFNγ by expanded CD4⁺ cells from the indicated mice, following restimulation with PMA and ionomycin. (<b>C, D</b>) Representative flow cytometry data and the computed Division Indices of naïve Tg4^WT cells when stimulated with 0.1, 1 or 10μg/ml [4K] and co-cultured with CD4⁺ splenocytes from Tg4^WT and Tg4^KO mice which had been treated with [4Y] or PBS. Gated on live, CD4⁺, CellTrace⁺ cells. All plots show the mean value +/- SEM. Each point represents data from one [4Y] or PBS-treated mouse assayed individually <i>in vitro</i> and is representative of two experiments. *p<0.05, **p<0.01, ns p>0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons.</p

Tg4^KO mice express lower levels of tolerance-associated surface markers following [4Y] treatment.

<p>(<b>A</b>) Representative flow cytometry plots and plots of combined data (<b>B-E</b>) showing production of selected cell surface markers by splenic CD4⁺ T cells from Tg4^WT and Tg4^KO mice treated with [4Y] or PBS. All flow cytometry data is gated on live (viability dye^-) CD4⁺ cells. Shown is the data from two experiments combined, with a total of 6–8 mice per group. All plots show the mean +/- SEM with each point representing data from one animal. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns p>0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons.</p

PKCθ is required for induction of a suppressive environment <i>in vivo</i>.

<p>(<b>A</b>) Experimental design. Cell Proliferation Dye-labeled CD4⁺ T cells from naïve Tg4WT mice were adoptively transferred to Tg4^WT and Tg4^KO mice, pretreated with [4Y] or PBS. After 48 hours, mice were challenged with 80μg of [4Y] and the division of the transferred Tg4^WT cells was measured by flow cytometry after a further 48 hours. (<b>B</b>) Example flow cytometry data and (<b>C</b>) plotted data from all mice showing the proportion of transferred Tg4^WT cells which remained undivided following [4Y] challenge under each pre-treatment condition. Shown is the mean +/- SEM. Each data point represents one [4Y] or PBS-treated recipient mouse which were assayed in a single experiment. *p<0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons.</p

Serum cytokine concentrations in Tg4^WT and Tg4^KO mice over the course of [4Y] treatment.

<p><b>(A)</b> Experimental design. Escalating doses of MBPAc1-9[4Y] peptide were administered subcutaneously to mice every 3–4 days. (<b>B-F</b>) Concentrations of IL-10, IL-2, IFNγ, TNFα and IL-17A in serum from peripheral blood taken two hours after each [4Y] treatment in Tg4^WT (open circles) and Tg4^KO (closed circles) mice. Plots show the average of four animals +/- standard error of the mean (SEM) representing one experiment of three performed. *p<0.01 **p<0.001 by Student’s two-tailed T test.</p

Antibodies used in this study.

<p>Antibodies used in this study.</p

Tg4^KO mice show reduced induction of IL-10⁺ T cells and tolerance-associated transcription factors following [4Y] treatment.

<p>(<b>A</b>) Representative flow cytometry plots and plots of all data (<b>B-G</b>) showing production of selected cytokines by splenic CD4⁺ T cells from Tg4^WT and Tg4^KO mice treated with [4Y] or PBS following <i>ex vivo</i> restimulation with PMA and ionomycin. (<b>H</b>) Representative flow cytometry plots and plots of combined data (<b>I-J</b>) of FoxP3 and cMaf expression in splenic CD4⁺ T cells from Tg4^WT and Tg4^KO mice treated with [4Y] or PBS. All flow cytometry data is gated on live (viability dye^-) CD4⁺ cells. (K-M) Expression of <i>Il10</i>, <i>cmaf</i> and <i>Nfil3</i> in magnetically-isolated CD4⁺ splenocytes from Tg4^WT and Tg4^KO treated with [4Y], restimulated for 18 hours <i>ex vivo</i> with anti-CD3ε and CD28, as measured by RT-PCR. Plots F, G, K-M show data from one experiment with 3–4 mice per group. All other plots show the data from two experiments combined, with a total of 6–8 mice per group. All plots show the mean +/- SEM with each point representing data from one animal. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns p>0.05 assessed by ANOVA with Tukey’s correction for multiple comparisons (plots B-G and I-J). p values shown in K-M were calculated by two-tailed Student’s T 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

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 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

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