Crosstalk between decidual NK and CD14+ myelomonocytic cells results in induction of Tregs and immunosuppression

Abstract

Regulatory T cells (Tregs) are thought to play a major role in pregnancy by inhibiting the maternal immune system and preventing fetal rejection. In decidual tissues, NK cells (dNK) reside in close contact with particular myelomonocytic CD14(+) (dCD14(+)) cells. Here we show that the interaction between dNK and dCD14(+) cells results in induction of Tregs. The interaction is mediated by soluble factors as shown by transwell experiments, and the prominent role of IFN-gamma is revealed by the effect of a neutralizing monoclonal antibody. Following interaction with dNK cells, dCD14(+) cells express indoleamine 2,3-dioxygenase (IDO), which, in turn, induces Tregs. Notably, unlike peripheral blood NK (pNK) cells, dNK cells are resistant to inhibition by the IDO metabolite L-kynurenine. "Conditioned" dCD14(+) cells also may induce Tregs through transforming growth factor-beta (TGF-beta) production or CTLA-4-mediated interactions, as indicated by the effect of specific neutralizing Abs. Remarkably, only the interaction between dNK and dCD14(+) cells results in Treg induction, whereas other coculture combinations involving either NK or CD14(+) cells isolated from peripheral blood are ineffective. Our study provides interesting clues to understanding how the crosstalk between decidual NK and CD14(+) cells may initiate a process that leads to Treg induction and immunosuppression. Along this line, it is conceivable that an impaired function of these cells may result in pregnancy failure.

Fig. 1.

Effect of dCD14⁺ cells cocultured with dNK or pNK cells on T cell proliferation. (A) CD4 and CD8β surface expression in CFSE-labeled T cells cocultured (for 7 d) with dCD14⁺, dCD14⁺/pNK, or dCD14⁺/dNK cells. Analysis was performed by gating on the lymphocyte fraction according to the FSC and SSC parameters. CFSE-negative lymphocytes (i.e., NK cells) were excluded from the analysis. Percentages of proliferating cells are indicated. A shows a representative experiment. (B) Similar results were obtained by analyzing dCD14⁺ and dNK cells derived from 15 different donors: a statistical analysis of the CD4⁺/CD8⁺ proliferating T-cell ratio under different coculture conditions is shown.

Fig. 2.

dCD14⁺/dNK cell interaction induces Tregs. (A) T cells cocultured with the indicated cell combinations were analyzed (at day 7) for the surface expression of CD25, CD127, and CD49d and for the intracellular expression of FOXP3. Two- or three-color immunofluorescence analyses were performed to evaluate cell proliferation and expression of the indicated markers. A shows a representative experiment of six performed. (B) The expression of FOXP3 on proliferating T cells was evaluated in 10 different experiments (i.e., by using pNK, dNK, and dCD14⁺ cells derived from 10 different donors). Bars indicate the percentage mean (±SEM) of proliferating FOXP3⁺ T cells assessed in the indicated coculture conditions (n.s., not significant). (C) T cells cocultured with dCD14⁺/dNK cells were analyzed (at day 7) for the expression of CD39, GITR, and CTLA-4. Two- or three-color immunofluorescence analyses were performed to evaluate the expression of the indicated markers. C shows a representative experiment of three performed. (D) T cells were cocultured as indicated for 35 d and analyzed for the intracellular expression of FOXP3. D shows a representative experiment of three performed.

Fig. 3.

CD4⁺ T cells isolated from dCD14⁺/dNK cell cocultures exhibit suppressive capacity on T cell proliferation. CD4⁺ T cells isolated from the indicated coculture combinations were added to anti-CD3 mAb-stimulated allogeneic CFSE-labeled T cells (1:2 inhibitor:responder ratio) to assess their ability to inhibit T-cell proliferation. The percentages of proliferating cells are indicated. Data are representative of three independent experiments. To exclude dead cells, the experiments were performed in the presence of 7AAD.

Fig. 4.

IFN-γ and IDO production during dNK/dCD14⁺ cell interaction and involvement in Treg induction. The role of factors produced during dCD14⁺/dNK crosstalk in the induction of Tregs was analyzed in the transwell culture system depicted in A. (B) T cells, isolated from cultures in which dCD14⁺_TW cells were conditioned by dNK + dCD14⁺ or by pNK + dCD14⁺ cells, were added to anti-CD3 mAb–stimulated allogeneic CFSE-labeled T cells. Their inhibitory capability was analyzed in a 5-d culture assay. T cells were added at a 1:2 (inhibitor:responder) ratio. The percentage of proliferating CFSE-labeled T cells is indicated. Data are representative of three independent experiments. (C) T cells were cocultured for 7 d with dCD14⁺ and dNK cells in the absence or in the presence of 1MT or anti-IFN-γ mAb. T cells were analyzed for surface expression of CD4. Percentages of proliferating cells are indicated. (D) Cells cultured as in C were analyzed by two- or three-color immunofluorescence by gating on CD4⁺ cells for the expression of CD25 and FOXP3. Similar results were obtained in five different experiments. To exclude dead cells, the experiments were performed in the presence of 7AAD.

Fig. 5.

IDO mRNA induction in dCD14⁺ cells following treatment with IFN-γ or transwell coculture with dCD14⁺/dNK cells. (A) IDO mRNA expression was analyzed in freshly isolated dCD14⁺ cells or in dCD14⁺ cells incubated for 14 h with the indicated IFN-γ concentrations. (B) Freshly isolated dCD14⁺ cells that had been conditioned in transwell cocultures with dCD14⁺/dNK cells (Fig. 4A) or that were further cultured alone for 24 h were analyzed for IDO mRNA expression. RT–PCR was performed with primers specific for IDO and for β-actin as positive control. PCR products were run on a 0.8% agarose gel and visualized by ethidium bromide staining. Similar results were obtained in five independent experiments.

Fig. 6.

Effect of L-kynurenine on dNK and pNK cell function and phenotype and involvement of TGF-β in the induction of Tregs. (A) Freshly isolated NK cells were cultured for 2 d in the presence of IL-2 either alone or in combination with L-kynurenine and then stimulated with anti-NKp46 mAb (or with anti-CD56 mAb as negative control). Supernatants were assessed for their IFN-γ content by ELISA. (B) Treated (black line) or untreated (shaded profile) dNK and pNK cell subsets (CD56^brightCD16⁻ and CD56^dimCD16⁺) were analyzed for the surface expression of NKp46, NKp30, and NKG2D receptors.

Fig. 7.

Involvement of TGF-β in the induction of Tregs. (A) T cells that had been cocultured with conditioned dCD14⁺ _{TW(dCD14/dNK)} in the absence or in the presence of anti-TGF-β mAb were analyzed at day 7 for the expression of CD25 and FOXP3. A shows a representative experiment of three experiments performed. (B) Statistical analysis of three different experiments. Bars indicate the percentage mean (±SEM) of proliferating FOXP3⁺ T cells assessed in the indicated coculture conditions.