Ncf1 (p47phox) is essential for direct regulatory T cell mediated suppression of CD4+ effector T cells.

BACKGROUND: Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs. METHODOLOGY/PRINCIPAL FINDINGS: Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff. CONCLUSIONS/SIGNIFICANCE: We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function

Treatment of Teffs with TGFβ results in a dose dependent elevation of intracellular oxidants.

<p>(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).</p

Suppression of <i>Ncf1^−/−</i> Teffs by <i>Ncf1^−/−</i> Tregs is markedly decreased.

<p>(A) Tregs were quantified in total spleen cell suspensions from WT C57BL/6 mice (Tr^WT) and from Ncf1-deficient mice (Tr^Ncf1−/−) 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 <i>Ncf1^−/−</i> mice. (B) Suppression assays were performed using <i>Ncf1^−/−</i> Tregs and <i>Ncf1^−/−</i> Teffs co-cultured at the indicated ratios (1∶1 and 2∶1) along with <i>Ncf1^−/−</i> 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 <i>Ncf1^−/−</i> Teffs cultured alone and <i>Ncf1^−/−</i> Teffs co-cultured with <i>Ncf1^−/−</i> 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.</p

2-mercaptoethanol and inhibitors of NADPH oxidase (VAS-2870 and DPI) block or reduce direct Treg-mediated suppression of Teffs.

<p>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).</p

Outline of strategy for performing <i>in vitro</i> suppression assays to measure Treg suppression of Teff.

<p>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.</p

Suppression by Tregs is partially dependent on TGFβand the suppressive effects of exogenous TGFβ on Teff proliferation can be overcome with NAC.

<p>(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).</p

There is significant but incomplete reconstitution of suppression of <i>Ncf1^−/−</i> Teffs by wild type Tregs.

<p>Suppression assays were performed with mixtures (all at a 1∶1 ratio) of wild type Tregs and Teffs (labeled Tr^WT and Teff ^WT respectively) and <i>Ncf1^−/−</i> Tregs and Teffs (labeled Tr^Ncf1- and Teff^Ncf1- 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).</p

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.Fil: Gomez, Eliana B.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Santori, Maria I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Laría, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Engel, Juan C.. University of California; Estados UnidosFil: Swindle, John. Seattle Biomedical Research Institute; Estados UnidosFil: Eisen, Harvey. Fred Hutchinson Cancer Research Institute; Estados UnidosFil: Szankasi, Philippe. Fred Hutchinson Cancer Research Institute; Estados UnidosFil: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin