PKCθ knockdown by siRNA does not affect the suppressive function of iTreg cells.

<p>(A) Experimental setup: siRNA transfection of iTreg cells. (B) On day 3 of differentiation culture, iTreg cells were transfected with PKCθ or scrambled siRNA as a control (co) using the Amaxa transfection system. The silencing efficiency was analyzed 2 days after transfection by intracellular PKCθ staining and flow cytometry in addition by SDS Page and immunoblotting. The percentage of PKCθ-positive cells as well as the mean fluorescent intensity (MFI) of PKCθ staining is depicted in each histogram. Fyn was used as a loading control for the normalization and quantification of PKCθ protein expression (shown below the bands). (C) The suppressive capacity of iTreg cells transfected either with control or PKCθ siRNA was analyzed in co-cultures with CFSE-labeled CD25^-CD4⁺ T cells (Tresp) stimulated with APCs and anti-CD3 antibodies. Histograms of CFSE and CD25 staining (gated on CFSE⁺ CD4⁺ 7AAD^- cells), including values of percentage divided and mean fluorescence intensity (MFI), and bar graphs summarizing results of 4 independent experiments are shown. Proliferation is depicted relative to stimulated Teff in absence of Tregs, whose mean CFSE fluorescence intensity was set to 100 (dotted line). No statistical significant difference (ns) was observed between control and PKCθ-siRNA transfected Tregs with the Friedman test together with Dunn´s multiple comparison test. (D) Knockdown of PKCθ in CD4⁺ Tconv cells (transfected on day3 of culture) was analyzed by SDS Page and immunoblotting 2 days after transfection. The silencing efficiency is depicted below the bands and in the summarizing scatter dot plot. To determine the IL-2 expression transfected CD4⁺ T cells were re-stimulated 2 days after transfection for 4 hours with anti-CD3 antibodies and IL-2 mRNA was determined by quantitative RT-PCR. Expression was normalized to the house keeping gene GAPDH and represented as fold of control siRNA. Dotted line: expression of control siRNA treated cells set to 1. Each symbol represents results obtained with cells isolated from individual mice. Statistical comparison to the control siRNA group was performed using one sample t test. ns = not significant.</p

PKCθ is dispensable for iTreg cell differentiation and function.

<p>(A) Naïve CD4⁺ T cells isolated from PKCθ^+/+ and PKCθ^-/- mice were differentiated <i>in vitro</i> under iTreg-inducing conditions (IL-2/TGF-β) and analyzed for Foxp3 and CD25 expression by flow cytometry on day 5 of culture. Representative FACS dot plots of Foxp3 and CD25 staining together with summarizing bar graphs are shown (n = 8). Statistical significance was determined using the Mann-Whitney U test. (B) The suppressive capacity of PKCθ^+/+ and PKCθ^-/- iTreg cells was analyzed in co-cultures with CFSE-labeled CD25^-CD4⁺ T cells (Tresp) stimulated with APCs and anti-CD3 antibodies. Representative histograms of CFSE staining (gated on CFSE⁺ CD4⁺ 7AAD^- cells) are depicted together with summarizing bar graphs of 4 independent experiments. Statistical analysis was determined using Friedman test together with Dunn´s multiple comparison test. ns = not significant.</p

PKC inhibition by AEB071 does not affect the suppressive activity of Treg cells.

<p>(A) Wild-type CD25^- and CD25⁺CD4⁺ T cells were (pre-)treated for 30 min with 1 μM AEB071 or DMSO and added to the suppression assay after extensive washing. Representative histograms of CFSE staining (gated on CFSE⁺ CD4⁺ 7AAD^- cells) are depicted together with summarizing bar graphs of 3 independent experiments. No statistical significant difference (ns) was observed between DMSO- and AEB-treated wild-type Tregs when analyzed with the Friedman test together with Dunn´s multiple comparison test. (B, C) Wild-type CD4⁺ T cells were either (pre-)treated with DMSO or 1 μM AEB071 before the start of the culture or 1 μM AEB071 was added to the wells (incl.: AEB071 included during the whole period of culture). Activation was assessed by IL-2 expression (determined by Luminex technology and quantitative RT-PCR) as well as by CD25 and CD69 expression (analyzed by flow cytometry) on day 1. Representative FACS histograms of CD69 and CD25 (including the mean fluorescence intensity) are shown together with summarizing scatter dot blots. The data are presented relative to the DMSO control, which was set to 100 (dotted line). Each symbol represents results obtained with individual mice analyzed in at least 4 independent experiments. Statistical comparison to the DMSO control group was performed using one sample t test.</p

PKCθ deficiency impairs Treg development but does not affect suppressive activity.

<p>(A) The frequency and cell counts of Treg cells in the spleen/lymph node (LN) and thymus of PKCθ^-/- and PKCθ^+/+ mice was analyzed by flow cytometry. Representative FACS dot plots of Foxp3 and CD25 staining (spleen/LN cells, gated on CD3⁺CD4⁺) together with summarizing bar graphs are shown (n ≥ 11 for spleen/LN, n ≥ 6 for thymus). Statistical significance was determined using the Mann-Whitney U test. (B) The suppressive capacity of PKCθ^+/+ and PKCθ^-/- CD25⁺CD4⁺ T cells (Treg) was analyzed in co-cultures with CFSE-labeled CD25^-CD4⁺ T cells (Tresp) stimulated with APCs and anti-CD3 antibodies. Representative flow cytometric dot blots of CFSE and CD25 staining (gated on CFSE⁺ CD4⁺ 7AAD^- cells) are depicted together with summarizing bar graphs of 3 independent experiments (duplicates per experiment). Wild-type CD25^-CD4⁺ T cells (non-Treg) were included as controls. Statistical analysis was determined using Friedman test together with Dunn´s multiple comparison test. ns = not significant.</p

Protein Kinase C θ Regulates the Phenotype of Murine CD4⁺ Th17 Cells

<div><p>Protein kinase C θ (PKCθ) is involved in signaling downstream of the T cell antigen receptor (TCR) and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing <i>in vitro</i> (polarizing T cell cultures) and <i>in vivo</i> (experimental autoimmune encephalomyelitis model, EAE) techniques, we demonstrated that PKCθ-deficient CD4⁺ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt), accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ) and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated <i>Stat4</i> mRNA levels in PKCθ^−/− CD4⁺ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the <i>Stat4</i> gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected <i>in vivo</i> by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.</p></div

PKCθ-deficiency does not alter expression of IFN-γ and T-bet under neutral or Th1-polarizing conditions.

<p>Naïve CD4⁺ T cells were cultured under neutral (Th0) and Th1-promoting conditions for 4 days. The expression of IFN-γ (A, flow cytometric staining, Bioplex) and T-bet (B, flow cytometric staining, qRT-PCR) was analyzed. Graphs show combined data from two independent experiments, each with n = 3 per genotype; mean values with error bars indicating +/− SEM are presented. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.</p

Unaltered responses to IL-23 and TGF-β in PKCθ^−/− CD4⁺ T cells.

<p>A) Naïve CD4⁺ T cells were cultured under Th17-promoting conditions with or without IL-23 addition for 4 days. Quantification of flow cytometric analysis of IL-17A, IFN-γ and T-bet expression is shown. Graphs present combined data from two independent experiments, each with n = 3 per genotype. B) Naïve CD4⁺ T cells were cultured under Th1-promoting conditions with and without the addition of TGF-β for 2 days. Quantification of flow cytometric analysis of IFN-γ and T-bet expression is shown. One of two independent experiments with n = 3 per genotype is presented. All graphs represent mean values and error bars indicate +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.</p

PKCθ^−/− CD4⁺ T cells are more potent IFN-γ producers in an autoimmune disease mouse model.

<p>A) Experimental autoimmune encephalomyelitis (EAE) disease progression in wild-type and PKCθ^−/− mice. The graph summarizes data from three independent experiments with total n>20 for each genotype. B) and C) Infiltrating mononuclear cells (MNC) from brain and spinal cord (SC) and lymph node (LN) cells were isolated at onset of the disease (day 11 after EAE-inducing immunisation). The cells were activated with phorbol 12,13-dibutyrate (PDBu)/ionomycin for 4 h and stained intracellulary for cytokine (IFN-γ, IL-17A) and T-bet expression. B) Representative flow cytometric plots of brain-infiltrating CD4⁺ cells. C) Quantification of cytokine-producing CD4⁺ T cell. Graphs depict results from two independent experiments with n = 3 per genotype for brain samples and from one experiment with n = 3 per genotype for SC and LNs. D) Cells from the lymph nodes and spleens were isolated at onset of the disease (day 11 after EAE-inducing immunisation). Cells were cultured <i>in vitro</i> in the presence of MOG_35–55 peptide for 3 days, re-stimulated with PDBu/ionomycin and analyzed by intracellular flow cytometric staining for IFN-γ, IL-17A and T-bet expression. Data from one experiment with n = 12 per genotype are shown. All graphs present mean values with error bars indicating +/− SEM. Statistical significance was assessed by a two-way repeated measures ANOVA (disease progression), a two-way ANOVA with Bonferroni post hoc test (cytokines and T-bet expression in C) or by a two tailed unpaired Student's t-test (gene expression in D).</p

Activation of IFN-γ/STAT1/T-bet axis is enhanced in the PKCθ^−/− CD4⁺ T cells under Th17-polarizing conditions.

<p>Naïve CD4⁺ T cells were cultured under Th17-promoting conditions and analyzed. A) IL-17A, IFN-γ and T-bet expression was measured by intracellular flow cytometry staining on three subsequent days of Th17 differentiation. Graphs show combined data from two independent experiments, each with n = 3 per genotype. B) Early induction of IFN-γ and T-bet mRNA expression in Th17 differentiation cultures was analyzed by qRT-PCR (relative expression normalized to TBP as a reference gene). All graphs summarize data from three independent experiments, each with n = 3 per genotype. C) A representative western blot analysis of T-bet expression and total and phosphorylated STAT1 in Th17-differentiated CD4⁺ T cells (day 1 and 4). One out of three independent experiments, with two biological replicates per genotype, is shown. D) Naïve CD4⁺ T cells were cultured under Th17-promoting conditions in the presence (Th17 sd – Th17 standard conditions) or absence of anti-IFN-γ neutralizing antibodies for 4 days. IFN-γ and T-bet expression was analyzed by intracellular flow cytometric staining. Graphs show combined data from two independent experiments, each with n = 3 per genotype. All the graphs represent mean values with error bars indicating +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.</p

Regulation of STAT4 differs between wild-type and PKCθ^−/− CD4⁺ T cells during the Th17-priming.

<p>A) Western blot analysis of phosphorylated (pSTAT4) and total STAT4 protein. Naïve CD4⁺ T cell were stimulated with indicated cytokines for 45 min (upper panel) or differentiated for 20 h in the presence of anti-CD3/anti-CD28 antibodies into Th17 (IL-6/TGF-β) or Th1 (IL-12) effector cells. One representative experiment is shown. B) Western blot analysis of pSTAT4 and STAT4 in naïve CD4⁺ T cells stimulated for 20 h under Th17-promoting conditions with and without IL-23. One representative experiment, with two biological replicates of each genotype, is shown. C) <i>Stat4</i> mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP as a reference gene) during the Th17 priming of naïve CD4⁺ T cells. D) PKCθ mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP) during the Th17 priming of naïve CD4⁺ T cells. Graphs show representative data of one of three independent experiments, each with n = 3 per genotype. All graphs represent mean values and error bars indicate +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test.</p