TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells

Abstract

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and cultured in vitro, were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition, Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.

Fig. 1.

Culture of peripheral NK cells with CM from decidual stromal cells causes accumulation of cells showing some characteristics of dNK cells. NK cells purified from peripheral blood (and containing both CD16⁺ and CD16⁻ NK cells) were cultured in normal medium or CM harvested from decidual stromal cells. Both conditioned and normal media were supplemented with IL-15 and SCF. After 2 weeks, expression of CD56, CD16, CD9, and KIR were analyzed by flow cytometry. KIR were detected with pooled HP3-E4, CH-L, and DX9 mAb (recognizing KIR 2DL1, 2DS1, 2DL2, 2DS2, 2DL3, and 3DL1). Staining for these markers at the start of the experiments is shown in the first column. For comparison, a similar staining of cells isolated from decidua is shown in the fourth column. Plots in C were gated at the time of analysis to display CD56⁺ NK cells that were either CD16⁻ or CD16⁺. Plots in B show all purified peripheral NK cells or gated CD56⁺CD16⁻ dNK cells. The fine dotted line in B represents staining with an isotype control mAb. (A, B, and C represent similar but distinct experiments.)

Fig. 2.

Decidual stromal cells, when isolated and cultured in vitro, produce TGFβ1. (A) RNA isolated from in vitro cultured decidual stromal cells was analyzed by RT-PCR. (B) TGFβ1 protein levels were examined by ELISA of CM from decidual stromal cells cultured in the absence or presence of 6α-methyl-17α-hydroxyprogesterone acetate (MPA) and estradiol. Shown are the mean TGFβ1 level and SD (n = 7 and 8), with signals from normal medium subtracted, of samples collected during weeks 3–4 after cell seeding (≈5–6 weeks after tissue isolation).

Fig. 3.

Exogenously added TGFβ1 also causes peripheral NK cells to acquire certain similarities to dNK cells. NK cells isolated from peripheral blood were cultured in normal medium, medium supplemented with 2 ng/ml TGFβ1, or CM harvested from decidual stromal cells. All cultures were additionally supplemented with IL-15 and SCF. After 2–3 weeks, expression of CD56 and CD16 (A), CD9 (B), KIR (C), and CD103 (D) were analyzed by flow cytometry. KIR were detected with pooled HP3-E4, CH-L, and DX9 mAb. Plots in B and C were gated at the conclusion of the experiment to display CD56⁺ NK cells that were either CD16⁺ or CD16⁻. The fine dotted lines in B and D represent staining with an isotype control mAb. At the start of these experiments, enriched NK cells were 3% CD16⁻ (A), completely CD9-negative (B), and completely CD103-negative (D). Initially, KIR were expressed by 5% and 54% of CD16⁻ and CD16⁺ cells, respectively (C). Staining of dNK cells is included for comparison in D only. (A, B, C, and D represent similar but distinct experiments.)

Fig. 4.

TGFβ is responsible for certain effects of decidual stromal CM on peripheral NK cells. NK cells isolated from peripheral blood were cultured in normal medium, decidual stromal cell CM, or decidual stromal CM pretreated with 10 μg/ml anti-TGFβ neutralizing antibody. All cultures were additionally supplemented with IL-15 and SCF. After 2 weeks, expression of CD56 and CD16 (A) and CD9 (B) were analyzed by flow cytometry.

Fig. 5.

TGFβ1 causes conversion of CD16⁺CD9⁻ peripheral NK cells into CD16⁻CD9⁺ NK cells. (A and B) CD56⁺ NK cells from peripheral blood were sorted by flow cytometry into CD16⁺ (A) and CD16⁻ (B) populations, which were subsequently cultured in normal medium, medium supplemented with 2 or 10 ng/ml TGFβ1, or CM from decidual stromal cells. All cultures were additionally supplemented with IL-15 and SCF. After 2 weeks, expression of CD56, CD16, and CD9 were analyzed by flow cytometry. At the start of this experiment, both CD16⁺ and CD16⁻ NK cells were CD9-negative. (C) A similar experiment is shown, showing the purity of sorted CD16⁺ and CD16⁻ NK cells immediately after sorting, and the subsequent change of phenotype upon culture in the presence of 2 ng/ml TGFβ1.

Fig. 6.

Decidual tissue also contains Lin⁻CD34⁺CD45⁺ progenitors, which can differentiate into NK cells. Leukocyte preparations from decidual tissue were stained with anti-CD34, CD45, and Lin (CD3, CD14, CD16, CD19, CD20, and CD56) mAb. Gated Lin⁻ cells are shown in A. Percentages show boxed cells as a proportion of all gated live cells. Decidual Lin⁻CD34⁺CD45⁺ cells were isolated by flow cytometry sorting and cultured in decidual stromal CM supplemented with IL-15 and SCF with or without Flt-3 ligand. After ≈30 days, expression of CD56, CD16, CD9, and CD94 on the resulting cells was examined by flow cytometry (B and C). Plots in C are gated on resulting CD56⁺CD16⁻ cells that resemble dNK cells. Similar cells were observed in three of four experiments.