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Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.

No abstract

Background/Aims: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and –C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells. Methods: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. Results: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling. Conclusion: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders.