3.7.1 Time-dependent release of cytokines after anti-CD3/CD28 stimulation of CD4+ T cells To determine how human primary CD4+ T cells functionally respond to different anti-CD3/CD28 stimulation conditions, levels of IL-2 (Figure 34A), IFN-γ (Figure 34B), and IL-5 (Figure 34C) were measured in supernatants of stimulated cells at different time points. In untreated cells (time point 0 h), cytokine levels were below the detection limit. After stimulation with different anti-CD3:anti-CD28 ratios, IL-2, IFN-γ, and IL-5 secretion was time-dependently upregulated, however, to a different extent and with different kinetics.
Strong stimulation with 3 µg anti-CD3/well and 3 µg anti-CD28/ml (= 3/3 stimulation) resulted in highest IL-2 levels with maximal concentrations of ~11 ng/ml after 48 h of stimulation (Figure 34A). Treatment with 0.3 µg anti-CD3/well and 3 µg anti-CD28/ml (= 0.3/3 stimulation) led to a faster upregulation but slightly lower peak concentrations of IL-2, when compared to the 3/3 stimulation. Because treatment with 3 µg anti-CD3/well and 0.3 µg anti-CD28/ml (= 3/0.3 stimulation) or with 0.3 µg anti-CD3/well and 0.3 µg anti-CD28/ml (= 0.3/0.3 stimulation) resulted in similar but overall lower IL-2 levels, anti-CD3/CD28 induced IL-2 secretion might be independent of the anti-CD3 strength, but dependent on the costimulatory anti-CD28 stimulus. Using the 3/0.3 stimulation, peak concentrations were reached at 48 h;
using the 0.3/0.3 stimulation at 24 h. Treatment of CD4+ T cells with anti-CD3 alone (= 3/0 stimulation or 0.3/0 stimulation) only weakly induced IL-2 secretion, confirming the anti-CD28 dependency of IL-2 induction. Remarkably, anti-CD3/CD28 induced IL-2 synthesis was transiently induced, because IL-2 levels declined at 72 h and 134 h after stimulation.
Figure 34. Time course of IL-2, IFN-γ, and IL-5 release after treatment of human primary CD4+ T lymphocytes with different anti-CD3/CD28 concentrations. Shown is the time-dependent cytokine secretion of IL-2 (A), IFN-γ (B), and IL-5 (C) after stimulation of CD4+ T cells with different stimulation conditions, as indicated. Supernatants were collected at different time points and cytokine concentrations were determined by enzyme-linked immunosorbent assays. Results are expressed as mean ± SD of 4 donors.
Compared to IL-2 secretion, IFN-γ synthesis was substantially induced at later time points, but was from 24 h onwards steadily upregulated and reached maximal levels at the end of the examined time period (12 - 17 ng/ml at 134 h) (Figure 34B). Because the measured IFN-γ concentrations were similar after treatment with different anti-CD3:anti-CD28 ratios, the upregulation of IFN-γ might be independent of the strength of the anti-CD3 stimulus and independent of the anti-CD28 costimulatory signal.
Similar to IFN-γ secretion, IL-5 release was substantially induced at later time points and was from 24 h onwards steadily upregulated with maximal levels at the end of the examined time period (0.4 - 1.7 ng/ml at 134 h) (Figure 34C). However, IL-5 cytokine levels were dependent on the costimulatory anti-CD28 signal: strong costimulation (3/3 and 0.3/3 stimulation, see above) caused highest IL-5 levels, whereas weaker costimulation (3/0.3 and 0.3/0.3
stimulation) resulted in lower IL-5 levels. IL-5 levels were lowest after stimulation with 3 µg or 0.3 µg anti-CD3/well alone.
3.7.2 Suppression of cytokine release and proliferation by PDE4 and/or PDE3 inhibition To investigate how T cell functions are affected by PDE4 inhibition, CD4+ T cells were treated with the panPDE4 inhibitor RP73401 (piclamilast). Additionally, the PDE3 inhibitor motapizone and both, piclamilast combined with motapizone, were included in the experiments because findings indicate that the effects of PDE4 inhibition can be enforced by simultaneous inhibition of PDE3 (Hatzelmann and Schudt, 2001). In order to compare the effects of PDE inhibitors with the effects of PDE4 subtype-specific siRNAs (see chapter 3.8), transfected cells were used for these experiments, but no siRNA was applied. After a 24 h resting period, transfected cells were stimulated with anti-CD3/CD28. To adjust the control conditions for DMSO concentrations, transfected cells were treated with 0.1% DMSO and the measured cytokine level and proliferation rate of these cells was set to 100%. As in all siRNA validation experiments in CD4+ T cells (see chapter 3.6), the stimulation condition 0.3 µg anti-CD3/well and 0.3 µg anti-CD28/ml was used for all functional studies.
1 µM RP73401 (a maximum concentration that guarantees PDE4 selectivity) significantly inhibited anti-CD3/CD28 induced IL-2 release 24 h and 48 h after stimulation (mean ± SD =
~39 ± 12% and ~65 ± 16% inhibition, respectively), IFN-γ release 48 h and 72 h after stimulation (~45 ± 9% and ~52 ± 11% inhibition, respectively), and IL-5 release 72 h after stimulation (~43 ± 13% inhibition; Figure 35 second bars). Under these conditions, proliferation was also inhibited, but to a lesser extent (~13 ± 4% inhibition 66 h after stimulation). 10 µM motapizone (a maximum concentration that guarantees PDE3 selectivity) significantly inhibited anti-CD3/CD28 induced IL-2 release 24 h and 48 h after stimulation (~30 ± 13% and
~44 ± 11% inhibition, respectively), IFN-γ release 48 h and 72 h after stimulation (~25 ± 11%
and ~31 ± 12% inhibition, respectively), and IL-5 release 72 h after stimulation (~28 ± 14%
inhibition), but hardly affected proliferation (~6 ± 6% inhibition 66 h after stimulation; Figure 35 third bars). The combined application of both 1 µM RP73401 and 10 µM motapizone had drastic effects on anti-CD3/CD28 induced T cell functions and significantly inhibited IL-2 release 24 h and 48 h after stimulation (~85 ± 2% and ~91 ± 9% inhibition, respectively), IFN-γ release 48 h and 72 h after stimulation (~86 ± 7% and ~88 ± 7% inhibition, respectively), IL-5 release 72 h after stimulation (~89 ± 13% inhibition), and proliferation 66 h after stimulation (~50 ± 20% inhibition) to an extent being overall overadditive to the individual inhibition of PDE4 or PDE3 (Figure 35 fourth bars).
Figure 35. Suppression of CD4+ T cell functions by PDE inhibition. Cells treated with the nucleofection procedure alone (NF control cells) were stimulated with 0.3 µg anti-CD3/well in combination with 0.3 µg anti-CD28/ml 24 h after nucleofection and cultured for the indicated periods of time either in the presence of 0.1% DMSO or additionally in the presence of 1 µM RP73401 (= RP, piclamilast), 10 µM motapizone (= Mota), or 1 µM RP73401 combined with 10 µM motapizone (= RP + Mota). Supernatants were collected and cytokine concentrations were determined by enzyme-linked immunosorbent assays. Alternatively, the proliferation rate was measured as detailed in Materials and Methods. Data are shown as mean ± SD (%) of 4 - 6 donors. Significance of differences is indicated: **, p < 0.01; compared to NF control (+ 0.1% DMSO).
3.7.3 Suppression of cytokine release and proliferation by Itk-specific siRNA
To ascertain how siRNAs can affect T cell functions, in addition to the application of PDE4 subtype-specific siRNAs (see chapter 3.8), control experiments were performed with siRNA specific for Itk (IL-2 inducible T cell kinase). Itk is a member of the Tec family kinases and has been shown to be involved in key regulatory signal pathways of T cell activation (Schwartzberg et al., 2005; Berg et al., 2005). Because T cells from mice deficient in Itk have a defective IL-2 secretion and an impaired TCR-mediated proliferation (Liao and Littman, 1995; Liu et al., 1998), siRNA targeting Itk was expected to have also a pronounced functional impact on these functional read-out parameters. The Itk-siRNA (Dharmacon) used in the present study was validated by Dr. O. Steinbach and J. Gilbert at the ALTANA Research Institute (Waltham, USA), who tested the Itk-siRNA for tolerability, efficacy, and off-target effects. The Itk-siRNA was shown to be specific, effective (~60 - 70% knockdown of both mRNA and protein), and well tolerated (personal communication). Thus, in the experiments performed in this study, Itk-siRNA served as positive control. In order to compare the effects of Itk-Itk-siRNA on T cell functions with the effects of the panPDE4 inhibitor RP73401 (see chapter 3.7.2), human primary CD4+ T cells were nucleofected with non-targeting siRNA (NEG_si) or siRNA targeting Itk (Itk_si in Figure 36). After a 24 h resting period, transfected cells were stimulated with anti-CD3/CD28 for the indicated periods of time and IL-2 secretion and the proliferation rate were measured. To adjust for DMSO concentrations, transfected cells were treated with 0.1%
DMSO. The measured cytokine level and proliferation rate of nucleofected control cells (no siRNA applied) was set to 100% (identical with first bar of respective read-out parameters in
Figure 35). Whereas 1.5 µM non-targeting siRNAs did not affect anti-CD3/CD28 induced cytokine release or proliferation, 1.5 µM Itk-siRNA significantly inhibited anti-CD3/CD28 induced IL-2 release 24 h and 48 h after stimulation (mean ± SD = ~74 ± 19% and ~63 ± 17%
inhibition, respectively; Figure 36 fourth bars). Moreover, Itk-siRNA had anti-proliferative effects (~19 ± 13% inhibition 66 h after stimulation). Compared to the suppressive effects of the panPDE4 inhibitor RP73401 (identical with second bar of respective read-out parameters in Figure 35) on IL-2 release and proliferation, siRNA targeting Itk was similar effective (IL-2, 48 h and proliferation, 66 h) or even more effective (IL-2, 24 h) (Figure 36).
These experiments demonstrated that the application of the siRNA technique to human primary CD4+ T cells is a powerful tool to study the functional significance of proteins in T cells.
Figure 36. Suppression of CD4+ T cell IL-2 release and proliferation by RP73401 and Itk-siRNA.
Cells treated with the nucleofection procedure alone (NF control) were stimulated with 0.3 µg anti-CD3/well in combination with 0.3 µg anti-CD28/ml 24 h after nucleofection for the indicated periods of time either in the presence of 0.1% DMSO or additionally in the presence of 1 µM RP73401 (= RP, piclamilast). These data are identical to Figure 35 (first and second bars of functional read-out parameters). Alternatively, cells were nucleofected with 1.5 µM non-targeting siRNA (NEG_si) or with 1.5 µM siRNA targeting Itk (Itk_si) 24 h before stimulation. To adjust for DMSO concentrations, 0.1%
DMSO was added to the siRNA-treated cells. Data are shown as mean ± SD (%) of 4 - 6 donors.
Supernatants were collected and IL-2 concentrations were determined by enzyme-linked immunosorbent assays. The proliferation rate was measured as detailed in Materials and Methods.
Significance of differences is indicated: ns, not significant; **, p < 0.01; ***, p < 0.001; compared to NF control (+ 0.1% DMSO) or Itk treatment compared to RP treatment.