Detection of HLA-G5 secreting cells

Soluble human leukocyte antigen-G molecules (HLA-G5) can be found in the peripheral blood of healthy females and males, and in other body fluids. To identify cells secreting HLA-G5 we generated a rabbit antiserum against a peptide motif encoded by intron-4 of HLA-G (RaHLA-G/I-4). Utilizing this antiserum as capture and antihuman beta 2-microglobulin as detection reagent, an enzyme-linked immunospot assay specific for HLA-G5 was developed. The results of this enzyme-linked immunospot assay format were proven by the HLA-G1 and soluble HLA-G5 (sHLA-G5) specific mAb MEM/G9 used in parallel as capture reagent. For the choriocarcinoma cell line JEG3 the number of HLA-G5 specific spots was found to be increased after stimulation with IFN-alpha, IFN-beta, and IFN-gamma at various concentrations. In contrast to this, no substantial variation of HLA-G5 specific spots was observed after incubation with lymphokines such as leukemia inhibitory factor, interleukin-10 (IL-10), IL-2, IL-4, and granulocyte-colony-stimulating factor. To clarify the cellular source of secreted HLA-G5 molecules, peripheral blood monocytes, CD4 and CD8 positive T and B cells from healthy donors (n = 14) were tested at a fixed cell number (5000/well) in the absence and presence of IFN-gamma (500 U/ml, 24 hours). In all experiments the number of HLA-G5 specific spots was significantly (p < 0.001) increased primarily in monocytes compared with T and B cells, which suggests that peripheral blood monocytes are the predominant cells secreting HLA-G5.