Synthesis of beta(2)-microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells

Abstract

Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin-like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA-G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA-G5 heavy (H)-chain proteins are disulphide-bonded homodimers unassociated with beta(2)-microglobulin (beta2m) light-chain proteins. Although comparatively low levels of beta2m messenger RNA (mRNA) were identified by real-time reverse transcription-polymerase chain reaction, immunoprecipitation studies failed to detect beta2m protein even when specific mRNA was doubled by transduction of a lentivirus-beta2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell beta2m mRNA and differentiation into syncytium did not promote beta2m synthesis. The failure of vCTB cells to exhibit beta2m in vitro was paralleled by a lack of detectable beta2m in vCTB cells in vivo. Lack of the beta2m protein could be the result of low levels of beta2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that beta2m-free HLA-G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural beta2m-free homodimeric form of HLA-G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.

Figure 1

Villous CTB cell HLA-G5 consists of disulphide-bonded H-chains. Villous CTB cells were cultured for 6 days to promote synthesis of HLA-G5. (a) Non-reducing conditions. HLA-G5 in vCTB cell lysates consists of ∼74 000 MW dimers. (b) Reducing conditions. vCTB cell HLA-G5 consists of ∼37 000 MW dimers. Fifty micrograms of cell lysate protein was loaded into each lane. Signals were detected by immunoblotting with the mAb to HLA-G intron 4 sequences present only in HLA-G5 and HLA-G6, 16G1, as described in the Materials and methods.

Figure 2

In vitro cultured vCTB cells transcribe the β2m gene but do not demonstrate detectable β2m protein. (a) RT-PCR demonstrates β2m mRNA in lysates of vCTB cells. Semi-quantitative RT-PCR was used to identify β2m mRNA in a lysate of term placental vCTB cells. β2m mRNA was identified based on the generation of a PCR product of expected size (431 bp, arrow). (b) Levels of β2m mRNA in MC and vCTB cells were compared against a standard using real-time PCR. Levels in MC were ∼13-fold greater than levels in matching harvests of term placental vCTB cells. (c) Immunoblotting detected β2m in HLA class I positive placental villous MC cell lysates (lanes 1–3), but failed to detect β2m in three matching lysates of vCTB cells (lanes 4–6). Commercially available purified β2m yielded a positive signal migrating to the same position (∼12 000) on the gel as β2m in placental MC (lane 7). Fifty micrograms of cell lysate protein was loaded into each lane and detected with anti-β2m (3H36) as described in the Materials and methods.

Figure 3

Immunoprecipitation experiments failed to identify β2m in association with HLA class I antigens in vCTB cells. Lysates of PBMC and term placental vCTB cells were immunoprecipitated with W6/32 and 16G1 or control mAb, IgG1 and IgG2a, then subjected to SDS–PAGE and immunoblotted with anti-β2m (2M2). β2m protein was only identified in PBMC.

Figure 4

Transduction of Lenti6-β2m into vCTB cells does not induce detectable β2m. (a) Successful transduction into both placental W6/32 + MC and primary vCTB cells is shown by the presence of β-gal-V5 in immunoblots using anti-V5. The positive control consisted of a lysate of HEK293-FT cells transduced with lentivirus β-gal-V5. (b) RT-PCR identified both transduced Lenti6-β2m (upper panel, signals detectable through 1 : 100 000 dilution of virus) and endogenous β2m (lower panel, signals detected in all samples) mRNAs in vCTB cells. In the absence of reverse transcriptase (no RT), no signals were detected. (c) Immunoblots conducted on vCTB cell lysates using anti-β2m (3H36) failed to reveal β2m at any concentration of Lenti6-β2m virus. Human β2m yielded a positive signal at 2 ng. Fifty micrograms of cell lysate protein was loaded into each lane and signals were detected as described in the Materials and methods.

Figure 5

Culture of vCTB cells in medium containing EGF does not increase β2m mRNA or induce β2m protein. (a) Semi-quantitative RT-PCR shows that β2m, HLA-G5 and HLA-G6 as well as control β-actin mRNAs were readily detected in both vCTB cells cultured in medium alone (No EGF) and in vCTB cells cultured in medium containing EGF (+ EGF). (b) As assessed by scanning densitometry with the data expressed as a ratio against β-actin (which was slightly increased in EGF), all of the messages declined, with HLA-G6 demonstrating a ∼60% decrease. (c) Immunoblots using 3H36 failed to detect any β2m signal in medium-cultured or EGF-cultured cells although following stripping and re-probing, β-actin protein was detected in both. Fifty micrograms of cell lysate protein was loaded into each lane and signals were detected as described in the Materials and methods.

Figure 6

Trophoblast cells in placental villi lack detectable β2m. Immunohistochemistry using 3H36 demonstrates that sTB (large arrows) and underlying vCTB cells (small arrows) do not contain detectable β2m but villous MC (arrowheads) do exhibit the protein. (a) Gestation week 9, first trimester; (b) gestation week 13, second trimester; (c) term placenta, third trimester; (d) isotype-specific control, third trimester. Original magnification, × 400.