Inhibition of effector function but not T cell activation and increase in FoxP3 expression in T cells differentiated in the presence of PP14

Abstract

Background: T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation.

Methodology/principal findings: Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-γ, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4(+)CD25(high)Foxp3(+) T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway.

Conclusions/significance: These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector functions along with augmenting Treg differentiation.

Figure 1. PP14·Fcγ1-treated cells secrete reduced levels of IFN-γ upon restimulation.

PBMC from healthy donors were stimulated with MBP (20 µg/ml) in the presence or absence of PP14·Fcγ1 (50 µg/ml) for two weeks. After two weeks the cells were split and restimulated with MBP for three days. Conditioned media of responding cells were collected, from one plate, and pooled based on the proliferation measured by H³-thymidine incorporation in the parallel plate. Levels of secreted IFN-γ in conditioned media were measured by ELISA. A, One representative experiment out of 5 is shown. B, The average inhibition of IFN-γ secretion by PP14 in 5 experiments is shown.

Figure 2. PP14·Fcγ1-treated cells proliferate as much as control MBP-stimulated cells upon restimulation.

PBMC from healthy donors were stimulated with MBP (20 µg/ml) in the presence or absence of PP14·Fcγ1 (50 µg/ml) or TGF-β (5 ng/ml) for two weeks. After two weeks the cells were restimulated with MBP for three days and cell proliferation was tested by H³-thymidine incorporation (cpm). One representative experiment out of 5 is shown and each dot represents cell proliferation in a single well of a 96-well plate (p≤0.05).

Figure 3. PP14·Fcγ1 pre-treated cells express the activation marker CD25.

PBMC from healthy donors were stimulated with MBP (20 µg/ml) in the presence or absence of PP14·Fcγ1 (50 µg/ml) or TGF-β (5 ng/ml) for two weeks. After two weeks the cells were restimulated with MBP for three days and then were collected and immunostained for the expression of CD25 on T cells (CD3⁺ cells).

Figure 4. Increased numbers of CD25+FOXP3+ T cells in PP14·Fcγ1 pre-treated cultures.

PBMC from healthy donors were stimulated with MBP (20 µg/ml) in the presence or absence of PP14·Fcγ1 (50 µg/ml) or TGF-β (5 ng/ml) for two weeks. After two weeks the cells were restimulated with MBP for three days and then were collected and immunostained for the expression of FoxP3 by activated CD25⁺ T cells.

Figure 5. PP14·Fcγ1 induces de-novo generation of CD25+FoxP3+ cells.

Naive CD4⁺CD25⁻ T cells were stimulated for one week with beads coated with various anti-CD3 concentrations in combination with soluble anti-CD28 (0.5 µg/ml) in the presence or absence of either PP14·Fcγ1 (50 µg/ml) or TGF-β (5 ng/ml). After one week the cells were collected and expression of CD25 and FoxP3 was analyzed using flow cytometry analysis. Results are presented as fold of increase in the percentage of cells expressing FoxP3 over that induced by TCR triggering alone (dotted line, A). One representative experiment is shown (A) and the average of seven separate experiments is shown in B (p≤0.05).

Figure 6. Retinoic acid enhances TGF-β but not PP14·Fcγ1-induced FoxP3 expression, and abrogates PP14·Fcγ1’s inhibitory activity.

A, Naive CD4⁺CD25⁻ T cells were stimulated with beads coated with various anti-CD3 concentrations and anti-CD28 (0.5 µg/ml) for 1week in the presence or absence of either PP14·Fcγ1 (50 µg/ml) or TGF-β (5 ng/ml), with or without retinoic acid (10 nM). After one week the cells were collected and expression of CD25 and FoxP3 was analyzed using flow cytometry. One representative experiment out of 3 is shown. B, Conditioned media of stimulated cells was collected and analyzed for IFN-γ secretion using ELISA. Data is presented as percentage of inhibition of IFN-γ secretion by PP14. An average of three independent experiments is shown. C, Cells were stimulated with anti-CD3 (0.1 µng/ml) for three days in the presence or absence of g/ml) with or without 10 µnM of All-trans-retinoic acidµ1 (50γPP14•Fc agonist, α(RA). In the indicated wells, RA was replaced by the RAR antagonist GR-110 was added in αAM-580, whereas in other wells the RAR combination with RA. After three days conditioned media were collected and IFN-γ secretion was measured using ELISA. One representative experiment of three independent experiments is shown. The data represent the mean of triplicate samples (p≤.05).

Figure 7. PP14·Fcγ1 inhibits the accumulation of TCR-induced phosphorylated ribosomal protein S6.

Naive CD4⁺CD25⁻ T cells were stimulated with anti-CD3 (5 µg/ml) coated beads and anti-CD28 (0.5 µg/ml) in the presence or absence of PP14·Fcγ1. Cells were collected at the indicated time points after stimulation, lyzed and phosphorylated S6 was analyzed using Western blot analysis. Immunoblotting of actin reveals relative amounts of protein in each lane (lower panels).