Fig 1.
PKC inhibition by AEB071 does not affect the suppressive activity of Treg cells.
(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.
Fig 2.
PKCθ knockdown by siRNA does not affect the suppressive function of iTreg cells.
(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.
Fig 3.
PKCθ is dispensable for iTreg cell differentiation and function.
(A) Naïve CD4+ T cells isolated from PKCθ+/+ and PKCθ-/- mice were differentiated in vitro 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.
Fig 4.
PKCθ deficiency impairs Treg development but does not affect suppressive activity.
(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.