Giada Amodio

HLA-G expressing DC-10 and CD4+ T cells accumulate in human decidua during pregnancy

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 tolerogenic DC-10: perspectives for clinical applications

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.

Dendritic cells a double-edge sword in autoimmune responses.

Dendritic cells (DC) are antigen-presenting cells that play a pivotal role in regulating innate and adaptive immune responses. In autoimmunity, DC act as a double-edged sword since on one hand they initiate adaptive self-reactive responses and on the other they play a pivotal role in promoting and maintaining tolerance. Thus, DC are the most important cells in either triggering self-specific responses or in negatively regulating auto-reactive responses. The latter function is mediated by DC in the steady-state or specialized subsets of DC, named tolerogenic DC. Clinical and experimental evidence indicate that prolonged presentation of self-antigens by DC is crucial for the development of destructive autoimmune diseases, and defects in tolerogenic DC functions contribute to eradication of self-tolerance. In recent years, DC have emerged as therapeutic targets for limiting their immunogenicity against self-antigens, while tolerogenic DC have been conceived as therapeutic tools to restore tolerance. The purpose of this review is to give a general overview of the current knowledge on the pathogenic role of DC in patients affected by autoimmune diseases. In addition, the protective role of tolerogenic DC will be addressed. The currently applied strategies to block immune activation or to exploit the tolerogenic potential of DC will be discussed.

Hla-g expression levels influence the tolerogenic activity of human DC-10

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule with known immune-modulatory functions. Our group identified a subset of human dendritic cells, named DC-10, that induce adaptive interleukin-10-producing T regulatory type 1 (Tr1) cells via the interleukin-10-dependent HLA-G/ILT4 pathway. In this study we aimed at defining the role of HLA-G in DC-10-mediated Tr1 cell differentiation. We analyzed phenotype, functions, and genetic variations in the 3′ untranslated region of the HLA-G locus of in vitro-differentiated DC-10 from 67 healthy donors. We showed that HLA-G expression on DC-10 is donor-dependent. Functional studies demonstrated that DC-10, independently of HLA-G expression, secrete interleukin-10 and negligible levels of interleukin-12. Interestingly, DC-10 with high HLA-G promote allo-specific anergic T cells that contain a significantly higher frequency of Tr1 cells, defined as interleukin-10-producing (P=0.0121) or CD49b+LAG-3+ (P=0.0031) T cells, compared to DC-10 with low HLA-G. We found that the HLA-G expression on DC-10 is genetically imprinted, being associated with specific variations in the 3′ untranslated region of the gene, and it may be finely tuned by microRNA-mediated post-transcriptional regulation. These data highlight the important role of HLA-G in boosting DC-10 tolerogenic activity and confirm that interleukin-10 production by DC-10 is necessary but not sufficient to promote Tr1 cells at high frequency. These new insights into the role of HLA-G in DC-10-mediated induction of Tr1 cells provide additional information for clinical use in Tr1- or DC-10-based cell therapy approaches.

HLA-G orchestrates the early interaction of human trophoblasts with the maternal niche

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.

HLA-G expression on blasts and tolerogenic cells in patients affected by acute myeloid leukemia.

Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4+ T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+ T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+ T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the hosts immune system. Further studies on larger populations are required to verify our findings.

DHRS9 Is a Stable Marker of Human Regulatory Macrophages

Background The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs. Methods The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry. Results DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14+ monocytes and was further enhanced by IFN-&ggr; treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. Conclusion DHRS9 is a specific and stable marker of human Mregs.

Association of genetic variants in the 3′UTR of HLA-G with Recurrent Pregnancy Loss

Human Leukocyte Antigen (HLA)-G is involved in reprogramming immune responses at fetal-maternal interface during pregnancy. We evaluated the genetic diversity of the 3′ Un-Translated Region (UTR) of HLA-G, previously associated with HLA-G mRNA post-transcriptional regulation, in women with unexplained Recurrent Pregnancy Loss (RPL), with 2 pregnancy losses (RPL-2, n = 28), or 3 or more pregnancy losses (RPL-3, n = 24), and in 30 women with a history of successful pregnancy. Results showed in RPL-2, but not in RPL-3, women compared to controls: i) higher frequency of the 14 bp Ins allele, in single and in double copy; ii) significantly lower frequency of DelG/X genotype, iii) reduced frequency of the UTR-2, and UTR-3 haplotypes; iv) higher frequencies of the UTR-5, UTR-7, and UTR-8 haplotypes. This pilot study supports the relevance of performing 3′UTR HLA-G genetic screening, not limited to a specific polymorphism, but considering the extended haplotypes, as a possible predictor of pregnancy outcome.

Distinctive Immunological Functions of HLA-G

HLA-G is a non-classical HLA class Ib molecule belonging to the Major Histocompatibility Complex (MHC) located on the short arm of chromosome 6. The tissue-restricted distribution of HLA-G, the low polymorphism in the coding region, the fact that HLA-G primary transcript is alternatively spliced in seven isoforms, and the inhibitory action on immune cells, constitute four hallmarks of HLA-G, which distinguish it from other HLA class I molecules (Carosella et al., 1999). In healthy conditions, a basal level of HLA-G gene transcription is observed in most cells and tissues. However, translation into HLA-G protein is restricted to trophoblasts at the fetal-maternal interface (Carosella et al., 2003), and in adults, to thymic epitelial, cornea, mesenchymal stem cells (MSCs), nail matrix, pancreatic- cells, erythroid and endothelial precursors. HLA-G can be also neo-expressed in pathological conditions including malignant transformation, viral infections, inflammatory and autoimmune diseases, and allogeneic transplantation (Carosella, 2011).

Dendritic Cell Immune Therapy to Break or Induce Tolerance

Dendritic cells (DCs) represent the key regulator of the immune system. The particular milieu in which DCs encounter the antigen together with their intrinsic properties defines their ability to promote an active or a tolerogenic immune response. The development of protocols to differentiate in vitro immunogenic or tolerogenic DCs opened the possibility to employ them as cell therapy in a list of immune-mediated diseases. While a number of clinical trials with immunogenic DCs have proven the safety and the efficacy of the therapy, only few studies have been performed with tolerogenic DCs. In this review, we will discuss major obstacles encountered, and strategies applied, to improve the efficacy of DC-based immunotherapies including lentiviral vector-based approaches.