Synthesis of β₂‐microglobulin‐free, disulphide‐linked HLA‐G5 homodimers in human placental villous cytotrophoblast cells

Abstract

Summary 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 β2‐microglobulin (β2m) light‐chain proteins. Although comparatively low levels of β2m messenger RNA (mRNA) were identified by real‐time reverse transcription–polymerase chain reaction, immunoprecipitation studies failed to detect β2m protein even when specific mRNA was doubled by transduction of a lentivirus‐β2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell β2m mRNA and differentiation into syncytium did not promote β2m synthesis. The failure of vCTB cells to exhibit β2m in vitro was paralleled by a lack of detectable β2m in vCTB cells in vivo. Lack of the β2m protein could be the result of low levels of β2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that β2m‐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 β2m‐free homodimeric form of HLA‐G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal–fetal interface.

HLA-G homodimer-induced cytokine secretion through HLA-G receptors on human decidual macrophages and natural killer cells

“…Notably, none of the available studies of sHLA-G has reported what fraction of the recombinant sHLA-G produced in bacteria or of the naturally occurring sHLA-G reportedly found in fetal placenta and maternal serum is monomer or dimer. Previously full-length recombinant HLA-G (G1) produced in bacteria has been shown to form the dimer (13) as has sHLA-G (G5) reported to be produced by villous trophoblast (41).…”

Embryonic Trophoblasts Induce Decidual Regulatory T Cell Differentiation and Maternal–Fetal Tolerance through Thymic Stromal Lymphopoietin Instructing Dendritic Cells

“…Both isoforms are present at the placental interface on trophoblasts except for HLA-G7, and the HLA-G1 and HLA-G5 are the most frequently observed. HLA-G was described as a key physiological mediator of maternal-fetal tolerance (38)(39)(40)(41). HLA-G inhibits multiple immune cell functions, including cytolytic functions of uterine and peripheral blood NK cells, Ag-specific cytolytic functions of cytotoxic T lymphocytes, allo-responsiveness of CD4 influence induction of DC tolerance (44).…”

HLA-G5 Induces IL-4 Secretion Critical for Successful Pregnancy through Differential Expression of ILT2 Receptor on Decidual CD4+ T Cells and Macrophages

“…HLA-G5 is able to bind b 2 -microglobulin and form dimers (16). Soluble HLA-G5, designated in this study as HLA-G5, has been reported by several groups to be produced by extravillous and villous trophoblast (15,(17)(18)(19)(20). One major HLA-G receptor described to date is ILT2, also designed leukocyte Ig-like receptor (LILR)B1 or CD85j.…”