The altered PD-1/PD-L1 pathway delivers the 'one-two punch' effects to promote the Treg/Th17 imbalance in pre-eclampsia

Abstract

The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical for normal pregnancy by promoting regulatory T (Treg) cell development and inhibiting the Th17 response. However, the relationship between the PD-1/PD-L1 pathway and the Treg/Th17 imbalance in pre-eclampsia (PE) is an enigma. In this study, decreased PD-1 and PD-L1 expression and a Treg/Th17 imbalance were observed at the maternal-fetal interface in PE. The regulatory effects of the PD-1/PD-L1 pathway on the Treg and Th17 cell quantities were determined in vitro by targeting T-cell proliferation, differentiation and transdifferentiation. First, decreased PD-1 expression might contribute to a higher Th17 cell frequency by promoting proliferation in PE. Second, the percentages of Treg but not Th17 cells differentiated from peripheral naive CD4⁺ T cells were increased by PD-L1 Fc administration. This effect was accompanied by decreased PI3K/AKT/m-TOR and increased PTEN mRNA expression and was completely reversed by PD-1 blockade. Finally, the percentage of IL-17-producing Treg cells increased and was positively associated with the Th17 cell frequency in PE. Increased RORγt and IL-17 but not Foxp3 and IL-10 mRNA expression by Treg cells was observed with PD-1 blockade. Similar findings occurred when Treg cells were exposed to IL-6/IL-23/IL-1β and were reversed by PD-L1 Fc. Taken together, our findings indicate that the PD-1/PD-L1 pathway contributes to the Treg/Th17 imbalance via 'one-two punch' approaches: (i) promoting Th17 cell proliferation, (ii) inhibiting Treg cell differentiation and (iii) enhancing Treg cell plasticity into Th17 cells in PE. The therapeutic value of PD-L1 Fc for PE treatment will be explored in the future.

Keywords: Th17 cell; Treg cell; differentiation; proliferation; transdifferentiation.

Figure 1

PD-1, PD-L1, Foxp3 and RORγt expression in placentas from women with NP and women with PE. The placentas were collected from women with NP (n=16) and women with PE (n=10). (a) Structural changes in the placentas were observed by H&E staining (× 200). (b) PD-1 and PD-L1, Foxp3 and RORγt expression in placentas was analyzed by immunohistochemistry (× 200). (c) The PD-1 (32 kDa), PD-L1 (47 kDa), Foxp3 (48 kDa) and RORγt (54 kDa) protein levels in the placenta were analyzed by western blotting. (d) The PD-1, PD-L1, Foxp3 and RORγt mRNA levels in the placenta were analyzed by qRT-PCR. The data are presented as the mean±s.e.m. *P<005, **P<001 versus NP. NP, normal pregnancy; PE, pre-eclampsia.

Figure 2

PD-1 and Ki67 expression in peripheral Treg and Th17 cells and their potential associations. Peripheral blood mononuclear cells were obtained from women with NP (n=15) and women with PE (n=11), and PD-1 and Ki67 expression in Treg and Th17 cells was detected by flow cytometry. (a) The percentages of Treg (CD4⁺CD25^highCD127^low/−) cells, (b) the percentages of Th17 (CD4⁺IL-17A⁺) cells and (c) the Treg/Th17 cell ratios were analyzed. (d) Dot plot of PD-1 and Ki67 detected on Treg and Th17 cells. (e and f) The percentages of PD-1⁺Ki67⁻, PD-1⁺Ki67⁺ and PD-1⁻Ki67⁺ Treg cell subsets. (g and h) The percentages of PD-1⁺Ki67⁻, PD-1⁺Ki67⁺ and PD-1⁻Ki67⁺ Th17 cell subsets. (i) The percentages of PD-1⁺ Treg and PD-1⁺Ki67⁻ Treg cells between NP and PE. (j) The percentages of PD-1⁺ Th17 and PD-1⁺Ki67⁻ Th17 cells between NP and PE. (k and l) Correlation analysis between the percentages of Treg and PD-1⁺ Treg cells. (o and p) Correlation analysis between the percentages of PD-1⁺ Treg cells and PD-1⁺Ki67⁻ Treg cells. (s and t) Correlation analysis between the percentages of Treg and PD-1⁻Ki67⁺Treg cells. (m and n) Correlation analysis between the percentages of Th17 cells and PD-1⁺ Th17 cells. (q and r) Correlation analysis between the percentages of PD-1⁺ Th17 and PD-1⁺Ki67⁻ Th17 cells. (u and v) Correlation analysis between the percentages of Th17 and PD-1⁻Ki67⁺Th17 cells. The data are presented as the mean±s.e.m. * versus NP, # versus PD-1⁺Ki67⁺ subset, $ versus PD-1⁺Ki67⁻ subset, *^/# P<005, **^/##/$$ P<001, ***^/### P<0001 versus NP. NP, normal pregnancy; PE, pre-eclampsia.

Figure 3

The percentages of Treg and Th17 cells differentiated from naive CD4⁺ T cells under different conditions with or without PD-L1 Fc or the anti-PD-L1 mAb. Peripheral naive CD4⁺ T cells freshly isolated from women with NP (n=15) and women with PE (n=15) were seeded at a density of 1 × 10⁶ cells/ml per well in 24-well plates. After 3 days, the cells were washed twice with PBS. (a) Gating strategy for determination of CD4⁺CD25^highCD127^low/− Treg and CD4⁺IL-17A⁺ Th17 cells by flow cytometry. (b) Flow cytometry was used to determine the percentages of Treg and Th17 cells differentiated from naive CD4⁺ T cells under Th0-, Th17- and Treg-prone conditions with or without PD-L1 Fc or an anti-PD-L1 mAb. (c and f) The percentages of Treg cells. (d and g) The percentages of Th17 cells. (e and h) The Treg/Th17 cell ratios. The data are presented as the median (range). * versus Th0-prone condition, $ versus Treg-prone condition, @ versus Th17-prone condition, # versus Th0-prone+anti-PD-L1 mAb condition, & versus Th17-prone+anti-PD-L1 mAb condition, % versus Treg-prone+anti-PD-L1 mAb condition. *^/$/@/&/% P<0.05, **^/$$/## P<0.01. NP, normal pregnancy; PE, pre-eclampsia.

Figure 4

Expression of nuclear transcriptional factors (Foxp3 and RORγt) and potential signaling molecules involved in Treg and Th17 cell differentiation. Naïve CD4⁺ T cells from women with NP (n=15) and women with PE (n=15) were isolated and cultured under Th0-, Treg- and Th17-prone conditions with or without PD-L1 Fc fusion protein or an anti-PD-L1 mAb for 3 days. The Foxp3, RORγt, PI3K/AKT/m-TOR and PTEN mRNA expression levels were determined by quantitative RT-PCR. (a) Molecular mechanism of the PD-1/PD-L1 pathway on T-cell differentiation. The PD-1 pathway inhibits downstream signaling of the PI3K/AKT signaling in naive CD4⁺ T cells, resulting in functional inactivation of naive CD4⁺T cells and inhibition of effector T-cell differentiation and function. In the presence of TGF-β, the PD-1 pathway attenuates the AKT-m-TOR signaling, preferentially biasing naive T-cell programming toward the development of Treg cells and suppressive capacity. (b and c) The mRNA expression of Foxp3. (d and e) The mRNA expression of RORγt. (f–k) The mRNA expression levels of PI3K/AKT/m-TOR. (l and m) The mRNA expression of PTEN. The data are presented as the mean±s.e.m. * versus Th0-prone condition, # versus Th0-prone+PD-L1 Fc condition, & versus Th17-prone condition, % versus Th17-prone+ PD-L1 Fc condition, @ versus Treg-prone condition, $ versus Treg-prone+PD-L1 Fc condition. *^/#/&/%/@/$ P<0.05, **^{/##/&&/%%/@@/$$} P<0.01, ***^/### P<0.001. NP, normal pregnancy; PE, pre-eclampsia.

Figure 5

PD-L1 Fc promoted Treg cell stability by preventing Treg cell transdifferentiation into Th17 cells. PBMCs were isolated from women with NP (n=40) and women with PE (n=23) and stained with antibodies against CD4, IL-17A and Foxp3 to assess IL-17 expression on CD4⁺Foxp3⁺ Treg cells by flow cytometry. (a) The percentage of IL-17-producing Treg cells was determined by flow cytometry. (b) The percentage of IL-17⁺ Treg, IL17⁺ Treg/Treg cell ratios and IL-17⁺ Treg/Th17 cell ratios. (c) Correlation analysis between the percentage of Treg cells and the IL-17-producing Treg cell frequency, between the percentage of Th17 cells and the IL-17⁺ Treg cell frequency, and between the percentage of Th17 cells and the IL-17⁺ Treg/Treg cell ratio. Further investigations were performed to explore the relationship between the PD-1 pathway and Treg cell stability in PE. The regulatory effect of the PD-1/PD-L1 pathway on Treg cell plasticity was studied in vitro. Peripheral CD4⁺CD25⁺ Treg cells freshly isolated from women with NP (n=20) were seeded at a density of 1 × 10⁵ cells/well in 96-well plates in the presence or absence of IL-6/IL-23/IL-1β with or without PD-L1 Fc or an anti-PD-L1 mAb. After 5 days, the cells were collected, and the Foxp3, RORγt, IL-10 and IL-17 mRNA levels were determined by RT-PCR. (d) The Foxp3, RORγt, IL-10 and IL-17 mRNA levels were determined. The data are presented as the mean±s.e.m. * versus anti-CD3 mAb/anti-CD28 mAb/IL-2 condition, # versus IL-6/IL-23/IL-1β condition. *^/# P<0.05, **^/## P<0.01, ***^/### P<0.001. NP, normal pregnancy; PE, pre-eclampsia.

Figure 6

The PD-1/PD-L1 pathway regulated the dynamics of Treg and Th17 cells in PE. This figure illustrates the consequences of these interactions in the peripheral blood or lymphoid organs and the maternal-fetal interface in women with PE. (a) Decreased PD-1 expression in Th17 cells enhanced Ki67 expression, resulting in a higher proliferation capacity, whereas the opposite findings were observed in Treg cells. (b) With PD-1/PD-L1 pathway dysfunction, more Th17 cells were induced from naïve CD4⁺T cells, with increased PI3K/AKT/m-TOR expression. Correspondingly, Treg cells derived from naive CD4⁺T cells were decreased, with lower PTEN expression. (c) In the presence of IL-6/IL-23/IL-1β, Treg cells were more prone to transdifferentiation into IL-17-producing Treg cells and eventually transformed into Th17 cells. Furthermore, PD-1/PD-L1 crosstalk blockade promoted the conversion of Treg cells into Th17 cells. PD-L1 Fc administration reversed this conversion. (d) Thus, more peripheral Th17 than Treg cells migrated to the maternal-fetal interface. The conversion of Treg into Th17 cells might also occur locally. These changes might contribute to a local and systemic Treg/Th17 imbalance in PE and promote the appearance of clinical symptoms such as hypertension, proteinuria, edema and intrauterine growth restriction.