Pedro J. Morales

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

HLA-G in reproduction: studies on the maternal–fetal interface

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.

Recombinant HLA‐G5 and ‐G6 drive U937 myelomonocytic cell production of TGF‐β1

Throughout human pregnancy, activated maternal macrophages producing anti‐inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta‐derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)‐G. Here, we investigated the possibility that two soluble isoforms of HLA‐G, HLA‐G5 and ‐G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12‐myristate 13‐acetate (PMA) and interferon‐γ (IFN‐γ), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin‐like transcript (ILT) receptors for HLA‐G, ILT2 and ILT4. Exposure of the PMA/IFN‐γ‐treated U937 cells to increasing concentrations of recombinant HLA‐G5 or ‐G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)‐10 and dramatically increased transforming growth factor‐β1. Differential effectiveness between the isoforms was demonstrated in dose‐response studies, as was differential binding to ILT2 and ILT4 in receptor‐blocking studies. No effects on production of IL‐4, IL‐1 receptor antagonist, IL‐15, tumor necrosis factor α, IL‐1β, or IL‐6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA‐G‐producing fetal cytotrophoblast cells.

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

BackgroundThe molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.MethodsPC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.ResultsKU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.ConclusionsOverall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

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

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.

Absence of HLA-G Expression in Macrophages of Human Decidua

PROBLEM: Macrophages – together with natural killer (NK) cells – constitute the majority of bone marrow derived infiltrating cells in the decidua. As interferon‐γ (IFN‐γ), a cytokine produced by NK cells, has been reported to induce expression of human leukocyte antigen (HLA‐G) in monocytic cells, suggesting expression of HLA‐G on decidua macrophages potentially stimulated by IFN‐γ, the question arises whether decidua macrophages in normal pregnancy express HLA‐G.

Analysis of the Soluble Isoforms of HLA-G

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, Paan-AG, more closely resemble those in the human MHC class Ia genes than those in the class Ib gene, HLA-G.

The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5′ and 3′ untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon-γ stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon-γ was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3′ untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.

Polymorphisms in the Paan-AG promoter influence NF-κB binding and transcriptional activity

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal–fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-κB (NF-κB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two κB sites. In the current study, binding and activity of the two κB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The κB1 and κB2 elements in AG1 bound NF-κB with similar affinity. In contrast, the κB1 element of AG2 bound NF-κB with a much higher affinity than AG-1 κB1 (a 30-fold increase), whereas κB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of κB1. Similarly, failure of AG2 κB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3′ end of AG1 κB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.

Immunoregulatory properties of decidua macrophages

About 15–20% of bone marrow derived cells in the endometrium and the decidua are CD14‐positive macrophages coexpressing the scavenger receptor CD163. The costimulatory molecule CD86 (B7‐2) but not CD80 (B7‐1) is found expressed on first trimester and term decidua macrophages (D‐Mph). The number of D‐Mph increases at the implantation site in the basal decidua, where they are in contact with the HLA‐G‐positive invading extravillous cytotrophoblast. As D‐Mph express the inhibitory receptors CD85d (ILT4, LIR‐2) and CD85e (ILT2, LIR‐1), they are potentially susceptible to inhibition by class Ia and class Ib molecules. D‐Mph may have specific functions relating to successful pregnancy, and phenotypic and functional properties may differ between early and term pregnancy. A number of data make their molecular repertoire comparable to those of alternatively activated macrophages which are immunosuppressive. For instance, first trimester decidua macrophages express IL‐10 and the MS‐1 high molecular weight antigen on the cell surface fibronectin and F XIIIa are detectable. Functional data are required to complement these studies.