Multiple mechanisms underlie the surprising willingness of mothers to tolerate the semi-allogeneic fetal tissues during pregnancy. Chief among these is the expression of the HLA-G molecules that has been largely demonstrated to be responsible for reprogramming the local maternal immune response towards tolerance. We recently identified a subset of tolerogenic dendritic cells, DC-10 that secrete high amounts of IL-10 and express high levels of HLA-G and its ligand ILT4. DC-10 are present in the peripheral blood and are essential in inducing adaptive regulatory T cells. We investigated the presence of DC-10 and HLA-G-expressing CD4+ T cells in human decidua in the first trimester of pregnancy. Results showed that these cells are highly represented in human decidua as compared to the peripheral blood. This is the first report describing decidual DC-10 and CD4+HLA-G+ T cells, strongly suggesting that they may accumulate or be induced at the fetal maternal interface to promote tolerance.

Human Leukocyte Antigen G (HLA-G) is a nonclassical HLA class I molecule with well-characterized immunomodulatory activities. HLA-G was first described as a regulatory molecule that allows the fetus to elude the maternal immune response. In the last decade it has become evident that HLA-G is involved in modulating both innate and adaptive immune responses, in maintaining tolerance in autoimmune and inflammatory diseases and after transplantation, and in promoting immune escape in cancer and infectious diseases. HLA-G exerts its modulatory/regulatory functions directly by interacting with specific inhibitory receptors. The expression of HLA-G is finely tuned by genetic variations in the noncoding region of the locus. The recent discovery of dendritic cells-10 (DC-10) as naturally occurring HLA-G-expressing dendritic cells opens new perspectives in the identification of the molecular and cellular mechanisms underlying HLA-G-mediated tolerance. An overview on the HLA-G-mediated inhibition of innate and adaptive immune cells, on the genetic influence on HLA-G expression, and on HLA-G-expressing DC-10 is presented. Moreover, we discuss the central and critical role of DC-10 in the HLA-G-mediated tolerance.

ABSTRACTand CD8 low suppressor T cells, 25 interleukin-10 (IL-10)-producing T regulatory type 1 (Tr1) cells, DC-10 are a subset of human tolerogenic DC that are present in vivo [27][28][29] and can be differentiated in vitro by culturing monocytes in the presence of IL-10. DC-10 secrete IL-10, are CD11c + , express CD14, CD16, HLA-G and ILT4 and, although not activated, display a mature phenotype, being CD86+ and HLA-DR + . The secretion of IL-10 and the expression of membrane-bound HLA-G and ILT4 are critical factors involved in DC-10-mediated induction of Tr1 cells. 27 In the present study, we investigated the role of HLA-G in DC-10-mediated Tr1 cell induction and whether polymorphisms present at the 3'UTR of the gene influence the expression of membrane-bound HLA-G on DC-10. MethodsThe methods are described in full in the Online Supplementary Appendix. Peripheral blood was obtained after informed consent in accordance with the Declaration of Helsinki under protocols approved by the ethical committee of the San Raffaele Telethon Institute for Gene Therapy. Dendritic cell differentiation CD14+ monocytes were isolated from peripheral blood mononuclear cells by positive selection using CD14 MicroBeads (Miltenyi Biotech, Germany) according to the manufacturer's instructions. Cells were cultured in RPMI 1640 (Lonza, Italy) supplemented with 10% fetal bovine serum (FBS) (Lonza, Italy) or with 5% human serum (HS) (EuroClone, Italy), 100 U/mL penicillin/streptomycin (Lonza, Italy), 2 mM L-glutamine (Lonza, Italy), (DC medium) at 37°C in the presence of 10 ng/mL recombinant human (rh)IL-4 (R&D Systems, Minneapolis MN, USA) and 100 ng/mL rhGM-CSF (Genzyme, Seattle, WA, USA) with 10 ng/mL of rhIL-10 (BD, Bioscience, CA, USA) for 7 days to differentiate DC-10. Cells cultured with rhIL-4 and rhGM-CSF on day 5 were matured with 1 mg/mL of lipopolysaccharide (Sigma, CA, USA) for 2 more days to generate mature dendritic cells (mDC). At day 7, DC were collected, phenotypically analyzed, and used to stimulate T cells. Statistical analysisSample mean results were compared using the non-parametric Mann-Whitney U test for continuous variables. HLA-G 3'UTR allele and genotype frequencies were obtained by direct counting. Allele and genotype frequencies between populations were compared using the χ 2 test. The correlation between membranebound HLA-G and ILT4 expression was determined using the Spearman correlation test. FBS and DC-10 HS were compared using a paired t-test. All results are presented as mean values ± standard error of mean (SEM). Differences were regarded as statistically significant at *P<0.05, **P<0.01, and ***P<0.001. The results were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA). Results In vitro differentiated DC-10 express variable levels of membrane-bound HLA-GIndependently of the donor, DC-10 differentiated in vitro as described in the Methods section were Figure 1A,B). High variability in the expression of membrane-bound HLA-G (ranging from 3.5% to 97.7%) and of ILT4 (rangin...

The prominent role of tolerogenic dendritic cells (tolDCs) in promoting immune tolerance and the development of efficient methods to generate clinical grade products allow the application of tolDCs as cell-based approach to dampen antigen (Ag)-specific T cell responses in autoimmunity and transplantation. Interleukin (IL)-10 potently modulates the differentiation and functions of myeloid cells. Our group contributed to the identification of IL-10 as key factor in inducing a subset of human tolDCs, named dendritic cell (DC)-10, endowed with the ability to spontaneously release IL-10 and induce Ag-specific T regulatory type 1 (Tr1) cells. We will provide an overview on the role of IL-10 in modulating myeloid cells and in promoting DC-10. Moreover, we will discuss the clinical application of DC-10 as inducers of Ag-specific Tr1 cells for tailoring Tr1-based cell therapy, and as cell product for promoting and restoring tolerance in T-cell-mediated diseases.

Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.

Extravillous trophoblasts (EVTs) play a central role in educating maternal leukocytes, endometrial stromal and endothelial cells to generate a receptive decidual microenvironment tailored to accept the semi-allogeneic fetus. HLA-G, a non-classical HLA class I molecule endowed with immune-regulatory functions, is primarily expressed on EVTs lining the placenta and on the naturally occurring tolerogenic dendritic cells, named DC-10, which are enriched in the human first trimester decidua. Decidual DC-10 are involved in HLA-G-mediated tolerance at the maternal–fetal interface. EVTs not only establish a tolerogenic microenvironment through the interaction with maternal innate and adaptive cells but also orchestrate placenta vascular and tissue remodeling, leading to a successful pregnancy. Here, we discuss the potential implications of the HLA-G-mediated cross-talk among the cells present at the maternal–fetal interface, and its role in maintaining a positive relationship between the mother and the fetus.

Dendritic cells (DCs) are critically involved in inducing either immunity or tolerance. During the last decades efforts have been devoted to the development of ad hoc methods to manipulate DCs in vitro to enhance or stabilize their tolerogenic properties. Addition of IL-10 during monocyte-derived DC differentiation allows the induction of DC-10, a subset of human tolerogenic DCs characterized by high IL-10/IL-12 ratio and co-expression of high levels of the tolerogenic molecules HLA-G and immunoglobulin-like transcript 4. DC-10 are potent inducers of adaptive type 1 regulatory T cells, well known to promote and maintain peripheral tolerance. In this review we provide an in-depth comparison of the phenotype and mechanisms of suppression mediated by DC-10 and other known regulatory antigen-presenting cells currently under clinical development. We discuss the clinical therapeutic application of DC-10 as inducers of type 1 regulatory T cells for tailoring regulatory T-cell-based cell therapy, and the use of DC-10 as adoptive cell therapy for promoting and restoring tolerance in T-cell-mediated diseases.

The non-classical HLA-G is a well-known immune-modulatory molecule. In physiological condition, HLA-G surface expression is restricted to the maternal-fetal interface and to immune-privileged adult tissues, whereas soluble forms of HLA-G are detectable in various body fluids. HLA-G can be de novo expressed in pathological conditions including tumors, chronic infections, or after allogeneic transplantation. HLA-G exerts positive effects modulating innate and adaptive immune responses and promoting tolerance, or detrimental effects inducing immune escape mechanisms. HLA-G locus, in contrast to classical HLA class I gene, is highly polymorphic in the non-coding 3 ′ untranslated region (UTR) and in the 5 ′ upstream regulatory region (5 ′ URR). Variability in these regions influences HLA-G expression by modifying mRNA stability or allowing posttranscriptional regulation in the case of 3 ′ UTR or by sensing the microenvironment and responding to specific stimuli in the case of HLA-G promoter regions (5 ′ URR). The influence of genetic variations on the expression of HLA-G makes it an attractive biomarker to monitor disease predisposition and progression, or response to therapy. Here, we summarize the current knowledge, efforts, and obstacles to generate a general consensus on the correlation between HLA-G genetic variability, protein expression, and disease predisposition. Moreover, we discuss perspectives for future investigation on HLA-G genotype/expression in association with disease predisposition and progression.