Figure 1.
Outline of strategy for performing in vitro suppression assays to measure Treg suppression of Teff.
Treg and Teff subsets were isolated from mouse spleens and evaluated by flow cytometry prior to co-plating. The exact number of CD4+FOXP3- cells co-purifying with Tregs (usually 10–15%; see arrow) was taken into consideration for plating so that the final CD4+FOXP3+ Treg:CD4+FOXP3- Teff ratio was exactly 1∶1 (or 2∶1 or 1∶2 as indicated). The low and consistent number of contaminating CD4- cells in the isolates was not considered in the ratio and are not shown in the dot plots. Prior to co-plating, the Teff subset was labeled with CFSE. The Treg:Teff mixtures were then added together to round bottom 96 well plates and stimulated with anti-CD3/CD28 coated beads at a bead:cell ratio of 1∶1, in triplicate, for three days. Percent suppression was calculated by comparing the proliferation of Teffs co-plated with Tregs to Teffs plated in the same conditions without Tregs.
Figure 2.
The antioxidant n-acetylcysteine (NAC) blocks direct Treg mediated suppression of Teffs in a dose dependent fashion.
Panel (A) shows the proliferation of CD3/CD28 stimulated Teffs (CFSE labeled) plated alone (white bars) or Teff plated at a 1∶1 ratio with Tregs (patterned bars) for 3 days, with and without 1 mM NAC. Data showing flow cytometric evaluation of CFSE staining with the calculated suppression (%) is shown at right. This data is representative of that seen in more than 3 experiments. Panel (B) shows Treg-mediated suppression of Teff under conditions of CD3/CD28 stimulation for three days in increasing concentrations of NAC (0.01 mM, 0.1 mM and 1 mM) with increasing Treg (labeled Tr in figure) to Teff ratios of 1∶2, 1∶1 and 2∶1. The data represents a compilation of data from three identical experiments each performed in triplicate and each yielding similar findings.
Figure 3.
2-mercaptoethanol and inhibitors of NADPH oxidase (VAS-2870 and DPI) block or reduce direct Treg-mediated suppression of Teffs.
Suppression assays were performed as outlined in the presence of 2-mercaptoethanol (2-ME) or the inhibitors of NADPH oxidase, VAS-2870 or diphenyleneiodonium (DPI) at the indicated concentrations, or DMSO alone as a solvent only control, or without any added agents as a positive suppression control. Panel (A) shows representative CFSE data from a single experimental trial. Panel (B) shows a compilation of all data from three separate experiments, each performed in triplicate, and each generating similar results (NS – not significant, ** p≤0.01).
Figure 4.
Suppression by Tregs is partially dependent on TGFβand the suppressive effects of exogenous TGFβ on Teff proliferation can be overcome with NAC.
(A) Suppression assays were performed as outlined with a 1∶1 ratio of Tregs:Teff with and without TGFβ neutralizing antibodies at the indicated concentration, or nonspecific isotype control antibodies (IgG1), and anti-CD3/CD28 beads for 3 days. Similar results were obtained in at least three identical experiments, each performed in triplicate. The data shown represents a compilation of all of these experiments. (B) Purified, CFSE-labeled Teffs, without Tregs, were stimulated in the indicated conditions for 4 days with and without the addition of TGFβ (10 ng/mL) and NAC (1 mM). Proliferation was measured by flow cytometric evaluation of CFSE staining. The data shown is representative of three identical experiments (NS – not significant, ** p≤0.01).
Figure 5.
Treatment of Teffs with TGFβ results in a dose dependent elevation of intracellular oxidants.
(A) Isolated, unlabeled Teffs, without Tregs, were cultured in the indicated conditions with exogenous TGFβ (2 and 10 ng/mL) or with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO; 1 mM) to deplete intracellular glutathione, and anti-CD3/CD28 beads for 24 hours. Subsequently, samples were labeled with the fluorescent, oxidant sensitive cell permeable indicator dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (DCFDA), each for exactly 15 minutes then immediately subjected to flow cytometric evaluation of DCFDA fluorescence. The data shown represents mean fluorescence intensity of DCFDA and is representative of three identical experiments, each performed in triplicate and each yielding similar results. (B) A similar experiment was performed using Teffs isolated from Ncf1-deficient mice. (C) CFSE labeled WT or Ncf1-deficient Teffs were stimulated as indicated for four days with anti-CD3/CD28 beads with and without exogenous TGFβ (2 and 10 ng/mL) then evaluated for proliferation by flow cytometric quantification of CFSE staining. The data shown represents the % reduction in proliferation of TGFβ treated Teffs vs untreated Teff samples and is representative of three identical experiments (NS – not significant, * p<0.05, **p≤0.01).
Figure 6.
Expression of Ncf1, Ncf2 and gp91phox in CD3/CD28 stimulated Teffs with and without TGFβ.
Teffs were purified from WT C57BL/6 mouse spleen suspensions by flow cytometric sorting (>98% pure CD4+CD25- cells after sorting) then subjected to stimulation with CD3/CD28 beads with and without exogenous TGFβ (10 ng/mL) for 1 and 2 days. Total RNA was isolated from freshly sorted but unstimulated cells (Day 0) and from stimulated cells at day 1 and day 2. mRNA expression of three components of the NADPH oxidase complex, Ncf1, Ncf2 and gp91phox, was measured by RT-PCR and normalized to expression of the housekeeping gene, β-actin. The data is graphed as fold-change in expression relative to Day 0 (arbitrarily defined as a relative expression of 1). The figure represents a compilation of data from three separate experiments.
Figure 7.
Suppression of Ncf1−/− Teffs by Ncf1−/− Tregs is markedly decreased.
(A) Tregs were quantified in total spleen cell suspensions from WT C57BL/6 mice (TrWT) and from Ncf1-deficient mice (TrNcf1−/−) by flow cytometric evaluation of CD4 and FOXP3 expression. CD4+FOXP3+ Tregs are shown gated with the percentage of Tregs in total spleen cells and the percentage of Tregs among only CD4+ T cells in parentheses. This data is representative of that observed from three WT and three Ncf1−/− mice. (B) Suppression assays were performed using Ncf1−/− Tregs and Ncf1−/− Teffs co-cultured at the indicated ratios (1∶1 and 2∶1) along with Ncf1−/− Teffs alone as controls. TGFβ neutralizing antibodies (30 µg/mL) were added to one set of samples as indicated. Samples were stimulated for 3 days with CD3/CD28 beads. The data shows proliferation of Ncf1−/− Teffs cultured alone and Ncf1−/− Teffs co-cultured with Ncf1−/− Tregs in suppression assays. CFSE staining data is shown at right along with the calculated suppression for this representative result (%). The data shown is representative of at least 3 identical experiments.
Figure 8.
There is significant but incomplete reconstitution of suppression of Ncf1−/− Teffs by wild type Tregs.
Suppression assays were performed with mixtures (all at a 1∶1 ratio) of wild type Tregs and Teffs (labeled TrWT and Teff WT respectively) and Ncf1−/− Tregs and Teffs (labeled TrNcf1- and TeffNcf1- respectively) as indicated in the figure. As above, samples were stimulated for three days with anti-CD3/CD28 beads and suppression was calculated by measuring Teff proliferation when plated alone and when plated with Tregs. The data shown is a compilation of at least three identical experiments, each performed in triplicate and each yielding similar results (NS – not significant, **p≤0.01).