TCR signaling intensity controls CD8+ T cell responsiveness to TGF-β

Abstract

DGK-ζ is a negative regulator of TCR signaling that causes degradation of the second messenger DAG, terminating DAG-mediated activation of Ras and PKCθ. Cytotoxic T cells deficient in DGK-ζ demonstrate enhanced effector functions in vitro and antitumor activity in vivo, perhaps because of insensitivity to inhibitory cytokines. We sought to determine whether the enhanced responsiveness of DGK-ζ-deficient T cells renders them insensitive to the inhibitory cytokine TGF-β and to determine how the loss of DGK-ζ facilitates this insensitivity. We identified decreased transcriptional and functional responses to TGF-β in CD8(+) DGK-ζ(-/-) T cells but preserved TGF-β-mediated conversion of naïve DGK-ζ(-/-) CD4(+) T cells to a regulatory T cell phenotype. Decreased CD8(+) T cell responsiveness to TGF-β did not result from impaired canonical TGF-β signal transduction, because similar levels of TGF-β-R and intracellular Smad components were identified in WT and DGK-ζ(-/-) CD8(+) T cells, and TGF-β-mediated activation of Smad2 was unchanged. Instead, an enhanced TCR signal strength was responsible for TGF-β insensitivity, because (i) loss of DGK-ζ conferred resistance to TGF-β-mediated inhibition of Erk phosphorylation, (ii) TGF-β insensitivity could be recapitulated by exogenous addition of the DAG analog PMA, and (iii) TGF-β sensitivity could be observed in DGK-ζ-deficient T cells at limiting dilutions of TCR stimulation. These data indicate that enhanced TCR signal transduction in the absence of DGK-ζ makes T cells relatively insensitive to TGF-β, in a manner independent of Smads, a finding with practical implications in the development of immunotherapies that target TGF-β.

Keywords: Diacylglycerol; Smad2; diacylglycerol kinase ζ.

Figure 1.. DGK-ζ-deficient CD8⁺ T cells demonstrate decreased sensitivity to TGF-β.

Splenic CD8⁺ T cells from WT or DGK-ζ^−/− mice were labeled with CFSE and incubated in 96-well plates (10⁵ cells/well) precoated with α-CD3 (2C11; 1 μg/ml) for 72 h in the presence or absence of TGF-β. (A) Cells were assessed for proliferation as determined by dilution of CFSE by flow cytometry from representative flow cytometry plots (A, top, without TGF-β [unshaded] or with TGF-β [shaded]) and quantified in the number of cell divisions from replicate wells (A, bottom). Data from one of three representative experiments are shown. (B) Cell culture supernatant from (A) was assayed for the presence of IFN-γ by ELISA, and the amount of IFN-γ (left) or fold-change resulting from the addition of TGF-β was quantified. The mean and standard error are depicted from data pooled from the average values determined in six independent experiments.

Figure 2.. DGK-ζ-deficient CD4⁺ T cells are not impaired in Treg conversion.

Naïve (CD45RB^hi, EGFP-negative) CD4⁺ T cells were isolated from the spleens of WT or DGK-ζ^−/− FoxP3-EGFP mice by flow cytometry, and the cells were incubated with soluble α-CD28 (1 μg/ml) and 2.5 μg plate-bound α-CD3 with or without TGF-β (5 ng/ml) and IL-2 (1000 U/ml). At 72 h, expression of regulatory T cell markers (CD25 and EGFP) was assessed by flow cytometry. (A) Naïve T cells of each genotype were cultured independently. (B) Cells from each genotype were cultured together. Each line indicates a single experiment comparing WT (white circles) to DGK-ζ-deficient (black circles) T cells.

Figure 3.. Loss of DGK-ζ attenuates a subset of TGF-β-mediated transcriptional changes in cytotoxic T cells.

WT or DGK-ζ-deficient CD8⁺ T cells were stimulated for 72 h with plate-bound α-CD3 (1 μg/ml) in the presence or absence of TGF-β (5 ng/ml), and changes in the mRNA levels of selected genes resulting from the presence of TGF-β was determined by RT-PCR. Data shown as the mean and standard error bars generated from data pooled from four independent experiments. The selected genes included TGF-β-RI and -RII, Smad7, INF activated gene 203 (IFI203), integrin-α_e (ITGAE), IFN-γ, and granzyme B (GZMB). *P = 0.035, **P = 0.001, ***P < 0.001.

Figure 4.. Smad signal transduction downstream of TGF-β is unaffected by the loss of DGK-ζ in T cells.

CD8⁺ T cells were isolated from spleens of WT or DGK-ζ-deficient mice and assayed either for TGF-β-RI or TGF-β-RII mRNA levels by RT-PCR (A) or cell surface expression levels of TGF-β-RII by flow cytometry (B). (C) CD8⁺ T cells from the mice of each genotype were stimulated for 1 h with or without TGF-β (10 ng/ml) and harvested (without TCR) or stimulated for an additional 30 min (with TCR) with 2.5 g/ml soluble α-CD3 (TCR). Fractionated lysates were assessed for nuclear levels of phosphorylated Smad2, total Smad2, or DNA polymerase (DNA pol) Representative data (top) and densitometry pooled data from four experiments (bottom) is shown. (D) mRNA levels of Smad7 were determined in WT or DGK-ζ-deficient CD8⁺ T cells was determined by RT-PCR averaged from three experiments. (E) Protein levels of Smad7 from cytosol or nucleus were determined in WT or DGK-ζ-deficient CD8⁺ T cells untreated or treated with 10 ng/ml TGF-β for 1 h. Representative data (left) and data pooled from six experiments (right) are depicted.

Figure 5.. TCR activation of DGK-ζ-deficient CD8⁺ T cells results in enhanced Erk and S6 phosphorylation in the presence or absence of TGF-β.

(A) WT or DGK-ζ-deficient CD8⁺ T cells were stimulated with soluble α-CD3 (2.5 μg/ml) with or without TGF-β (10 ng/ml) for 15 min, and lysates were probed for levels of phosphorylated or total Erk by immunoblotting (above dotted line). Because of artifactual photobleaching, total Erk appears diminished in blots previously probed for phosphorylated Erk. Subsequent immunoblotting of samples for total Erk without previous blotting for phosphorylated Erk demonstrated similar levels of total Erk proteins (below dotted line). Densitometry of phosphorylated Erk normalized to actin was calculated over six experiments (graph). (B) Splenic CD8⁺ T cells were isolated from WT and DGK-ζ-deficient mice, labeled with CFSE, and incubated with plate-bound α-CD3 (2.5 μg/ml) for 72 h in the presence of indicated concentrations of DAG analog (PMA). Proliferation, as assessed by dilution of CFSE, was determined by flow cytometry after gating on live CD8⁺ T cells. The data from one of three representative experiments are shown.

Figure 6.. DGK-ζ-deficient CD8⁺ T cells are sensitive to TGF-β in the presence of low levels of TCR stimulation.

Splenic CD8⁺ T cells from WT and DGK-ζ-deficient mice were labeled with CFSE and stimulated with the indicated concentration of plate-bound α-CD3 for 72 h in the presence or absence of 5 ng/ml TGF-β. (A) T cell proliferation of live CD8⁺ T cells was determined with dilution of CFSE by flow cytometry for cells left untreated (unshaded) or treated (shaded) with TGF-β. (B) Intracellular protein levels of granzyme B was assessed in live CD8⁺ T cells after fixation and permeabilization. Data from one of three representative experiments are shown. Induction of granzyme B was not observed in unstimulated cells of either genotype (data not shown; n = 2).