Gal1 is expressed and secreted by dNKs but not by pNKs. (A) Western blot of fresh CD56^dim pNK, CD56^bright pNK, and dNK lysates developed with anti-gal1 mAb (Lower) and anti-β-actin mAb (Upper). Gal1 was visualized as a 14-kDa band. Five dNK and and 5 pNK cell samples from different donors were analyzed. Similar results were obtained with decidual macrophages (data not shown). (B) Concentration of gal1 in media conditioned by CD56⁺CD16⁻CD3⁻ dNKs and CD56⁺CD3⁻ pNKs for 60 h (filled bars) and 90 h (open bars). Incubations were done in the presence of 12 ng/mL IL-15. The average of 3 independent experiments is shown. (C) Crossreactivity of the antibody used in B with recombinant human gal1 (diamonds), gal3 (squares), and gal9 (triangles) in ELISA assays. (D) Immunoprecipitation of gal1 from media conditioned by dNKs (CM), control media not exposed to dNKs (M), recombinant human gal1 (gal1), and recombinant human gal3 (gal3) with the same antibody as used in B (anti-gal1) or with an isotype control (Ig). Immunoprecipitates were resolved by Western blotting with anti-gal1 (Left) or anti-gal3 (Right) mAbs. The additional bands are derived from immunoglobulins and protein G. One experiment of 3 is shown.

Medium conditioned by dNKs and containing gal1 has apoptotic activity on T cells and MOLT4 cells. (A) Annexin V staining of MOLT4 cells treated (open histogram) with gal1 (Left) or medium conditioned by dNKs (Right) in the presence (dotted line histogram) or absence (solid line histogram) of 50 mM lactose. Shaded histograms correspond to cells incubated with control medium. Incubations (30 min at 37°C) were done in the presence of 0.5 mM DTT to prevent gal1 oxidative inactivation. (B) Blocking of apoptosis induction of MOLT4 cells (Left) and peripheral T cell blasts (Right) by dNK conditioned medium with anti-gal1 polyclonal antibodies. Control medium (shaded histogram), dNK conditioned medium (solid line histogram), conditioned medium plus nonimmune rabbit serum (dotted line histogram), and conditioned medium plus anti-gal1 rabbit serum (dashed line histogram) are shown.

Differential gal1 binding and glycophenotype of decidual and peripheral blood CD3⁺ T cells. (A) Biosynthetic pathways of tetraantenary N-glycans and core 2 O-glycans indicating the structures recognized by lectins SNA, PNA, MALII, and gal1. Crossed structures are absent from dTs and pTs based on data shown in C. The potential gal1-binding sites in N-glycans (italicized) are masked by α-2,6-sialylation and by the failure to form α-2,3-sialylated N-acetyllactosamine. Core 2 O-glycans are recognized by gal1 on dT cells (in bold). (B) Binding of biotinylated gal1 to dTs (Right) and pTs from pregnant (Center) and nonpregnant (Left) female donors in the presence (dashed line histograms) or absence (solid line histograms) of 50 mM lactose. (C) Binding of biotinylated SNA, MALII, and PNA or anti-core 2 O-glycosylated CD43 mAb 1D4 to dTs (Upper) and pTs (Lower). In B and C, histograms correspond to fresh lymphocyte preparations gated on CD3⁺ cells. Bound biotinylated lectins were detected by staining with fluorescently labeled streptavidin. Gray shaded histograms correspond to CD3⁺ gated cell preparations incubated only with fluorescently labeled streptavidin. Statistics comparing the staining with biotinylated gal1 (D) and biotinylated lectins SNA, PNA, and MALII (E) in dTs and pTs as described in B and C are shown. Asterisks indicate statistical significance in t test comparisons involving the groups denoted by the overlying lines (*, P ≤ 0.05). Results correspond to 8 decidual and 4 peripheral blood samples. Error bars represent standard error. (F) Expression of C2GnT by dTs and pTs evaluated by RT-PCR.

Freshly isolated decidual lymphocytes contain a population of Annexin V-positive T cells. (A) Annexin V and anti-CD56 (NK cells; Left) or anti-CD3 (T cells; Center) staining of freshly isolated decidual lymphocytes, and peripheral blood lymphocytes subjected to an isolation procedure similar to the one used to obtain decidual lymphocytes (Right). Note the presence of CD56⁻ and CD3⁺ dT lymphocytes positive for Annexin V staining. (B and C) BrdU staining for TUNEL analysis (B) and PI staining for hypodiploid DNA content analysis (C) of FACS-sorted CD3⁺ T cells (Left) and FACS-sorted T cells isolated as CD14⁻, CD56⁻ and CD4⁺/CD8⁺ cells (Center) obtained from total decidual lymphocyte (dt; Lower) or from peripheral lymphocyte preparations (pt; Upper). Lower Right panels display results for total decidual lymphocytes. Numbers indicate the percentage of cells with DNA fragmentation in B and of subdiploid cells in C. Panels correspond to independent dT cell preparations. Peripheral T cells shown were obtained from a single preparation. (A′, B′, and C′) Statistics comparing the abundance of Annexin V⁺ (A′), TUNEL⁺ (B′), and hypodiploid cells (C′) in CD3⁺ or CD4⁺/8⁺ T cells preparations from decidual and peripheral blood lymphocytes analyzed as described in A, B, and C. Asterisks indicate statistical significance in t test comparisons involving the groups denoted by the overlying lines (**, P ≤ 0.01; *, P ≤ 0.05). Numbers in parentheses indicate the number of samples analyzed. Error bars represent standard deviation.

Infiltrating T cells form periglandular apoptotic foci in the human decidua. CD3 (A and B), CD56 (B′′), and gal1 (B′′′) immunostaining and TUNEL staining (A′ and B′) of 4-μm serial histological sections (A with A′; and B with B′, B′′, and B′′′) of first-trimester human decidua of 6 weeks' gestational age. Images are representative of 2 samples from 2 different donors. EG, endometrial gland; D, decidua.