Kidney transplant recipients treated with belatacept exhibit increased naïve and transitional B cells.

Phase III clinical studies have shown that kidney transplant (KT) recipients treated with the costimulation blocker belatacept exhibited a better renal allograft function and lower donor-specific anti-HLA immunization when compared to recipients treated with calcineurin inhibitors (CNI). We analyzed B cell phenotype in KT recipients treated with belatacept and stable renal function (N = 13). Results were compared to those observed in stable patients treated with CNI (N = 12), or with chronic antibody-mediated rejection (N = 5). Both transcriptional profile and phenotypic characterization of peripheral B cells were performed by real-time polymerase chain reaction and flow cytometry, respectively. In belatacept group, the frequency and absolute number of transitional B cells as defined by both phenotypes: CD19(+) CD24(hi) CD38(hi) and CD19(+) IgD(hi) CD38(hi) CD27(-) , as well as naïve B cells were significantly higher compared with CNI group. B cell activating factor (BAFF) and BAFF receptor mRNA levels were significantly lower in belatacept group than in CNI group. These results show for the first time that belatacept influences B cell compartment by favoring the occurrence of transitional B cells with potential regulatory properties, as described in operational tolerant patients. This role may explain the lower alloimmunization rate observed in belatacept-treated patients.

Recent advances on the non-classical major histocompatibility complex class I HLA-G molecule.

The human leukocyte antigen (HLA)-G non-classical major histocompatibility complex (MHC) class I molecule was originally described in first-trimester trophoblasts at the fetal-maternal interface in 1990. Eight years later, the First International Conference on this molecule was inaugurated by Prof Jean Dausset, recipient of the Nobel Prize in Medicine. The Fifth International Conference on HLA-G, held in Paris on July 2009, began with a tribute to Prof Jean Dausset who left us recently. This conference was co-chaired by Dr Edgardo D. Carosella and Prof Hans Grosse-Wilde, included 57 oral presentations and was attended by approximately 140 delegates from 16 countries. We summarize here the major advances on the HLA-G molecule that were reported, including findings on its biological activity and characterization of new mechanisms of action, notably through mesenchymal stem cells and regulatory cells, and the previously unexplored role of HLA-G on immune cells such as gammadelta T-cells and B lymphocytes. Furthermore, the role of HLA-G during pregnancy was revisited and its impact in pathologies such as cancer, autoimmune disorders and transplantation was further extended.

HLA-G turns off erythropoietin receptor signaling through JAK2 and JAK2 V617F dephosphorylation: clinical relevance in polycythemia vera.

HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.

Research on HLA-G: an update.

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule from the major histocompatibility complex, which was initially shown to confer protection to the fetus from her mother's immune system. The Third International Conference on HLA-G, held in 2003, showed that beyond its role in fetal-maternal tolerance, HLA-G exerts tolerogenic functions involved in transplant acceptance as well as in tumoral and viral immune escape. The Fourth International Conference, which took place in Paris on July 2006, counted 72 oral presentations and about 200 attendees from 25 countries. The reports presented brought new insight into HLA-G research, and we summarize here the major advances on the HLA-G biology that were reported. Abstracts for all presentations can be found in volume 68 issue number 4 of Tissue Antigens.

Biology and functions of human leukocyte antigen-G in health and sickness.

In 1998, the first International Conference on human leukocyte antigen-G (HLA-G) was held in Paris. At that time, HLA-G was still a new HLA class I molecule, few aspects of its immunological functions were known, and its expression by tumors was just being described. In 1998, tools to properly study HLA-G were lacking, especially monoclonal antibodies, and three conclusions were drawn after the congress: (i) animal models were needed, (ii) the biology of HLA-G isoforms had to be confirmed, and (iii) HLA-G expression by tumors required clarification. Five years later, these three issues have been addressed. HLA-G is now gaining pace and is investigated for its immuno-inhibitory functions in the context of multiple pathologies. Eighty five oral presentations were given this year for more than 200 investigators working on HLA-G by speakers from over 20 countries. The success of the 3rd International Conference on HLA-G reflects the interest and tremendous work of the many research teams which, over the years, contributed to the publication of more than 500 peer-review articles. We summarize the key points that were presented and discussed during this meeting.

HLA-G2, -G3, and -G4 isoforms expressed as nonmature cell surface glycoproteins inhibit NK and antigen-specific CTL cytolysis.

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.

HLA-G1 co-expression boosts the HLA class I-mediated NK lysis inhibition.

It is now acknowledged that the pattern of HLA-G expression is not restricted to extravillous cytotrophoblast cells, as several studies described HLA-G in HLA class I+ cells, such as thymic epithelial cells, cytokine-activated monocytes and some tumors. In these situations, HLA-G may provide an additional inhibitory signal to escape from NK cell-mediated cytotoxicity. Accordingly, the aim of this study was to define the behavior of HLA-G once it is co-expressed into an HLA-A, -B, -C and -E+ cell line. For this purpose, HLA-G1 cDNA was transfected into an HLA class I+ melanoma cell line which was used as a target towards freshly isolated peripheral blood NK cells. Cytotoxic experiments using either anti-HLA-G1 or anti-HLA-G1 inhibitory receptor mAb show that HLA-G1 boosts the HLA class I-mediated inhibition of polyclonal NK cells through interaction with ILT-2, which appears as the major HLA-G1 inhibitory receptor involved. Nevertheless, HLA-G1 is also able to inhibit the cytolytic activity of an ILT-2- NK clone which otherwise expresses another HLA-G1 inhibitory receptor belonging to the KIR103 gene family. In order to more precisely define the relative role exerted by HLA-G1 versus -E on polyclonal NK cells, antibody-blocking assays were carried out using either anti-HLA class I or anti-CD94/NKG2A. Results demonstrate that in the absence of HLA-G1, the naturally expressed HLA class I-mediated NK inhibition is predominantly exerted by HLA-E through binding with CD94/NKG2A. In contrast, once HLA-G1 is expressed, it becomes the major NK inhibitory ligand.

[Fetal cells in the maternal blood and prenatal diagnosis]. / Cellules foetales dans le sang maternel et diagnostic anténatal.

Fetal loss after amniocentesis or chorionic villus sampling is a limit to prenatal diagnosis practice and to its generalization. The existence of fetal cells in the blood of pregnant women is now well established. Recognizing these cells with specific antibodies and isolating them with fluorescent or magnetic systems are the subject of numerous studies. However, to date, neither the sensitivity nor the specificity of these methods are sufficient to allow a non invasive prenatal diagnosis.

HLA-G truncated isoforms can substitute for HLA-G1 in fetal survival.

Pregnancy is considered as an immunologic paradox because the fetus can be viewed as a semiallograft by the mother's immune system. Among the different factors implicated in the maternal-fetal tolerance, a central role has been attributed to HLA-G. The primary HLA-G mRNA is alternatively spliced, encoding four membrane-bound isoforms (HLA-G1, -G2, -G3, and -G4), and three soluble forms (HLA-G5, -G6, and -G7). Whereas HLA-G1 is expressed on trophoblast cells, HLA-G2, -G3, and -G4 isoforms have been only identified as transcripts in trophoblast and term placentas. In this work, we first showed that these HLA-G transcripts are translated into proteins in first trimester cytotrophoblast cells. Then, using a target cell line transfected with HLA-G genomic DNA, we analyzed the functional implication of HLA-G isoforms expression on NK function. Our results show that not only HLA-G1, but also the other HLA-G truncated isoforms, can inhibit NK cytolysis and therefore contribute to immune privilege for the fetus.

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

Role of HLA-G versus HLA-E on NK function: HLA-G is able to inhibit NK cytolysis by itself.

Recent studies have shown that endogenous HLA-E molecules are stabilized on the cell surface upon the expression of HLA-G which contains within its leader sequence, a nonapeptide capable of binding with the HLA-E/beta2m complex. Since HLA-E was found to be the major ligand for the CD94/NKG2A inhibitory receptor, we determined the role of HLA-G versus HLA-E on NK lysis inhibition. We showed that K562 cells transfected with HLA-G1 cDNA are protected from NK lysis by direct interaction between HLA-G1 and killing inhibitory receptor(s). This NK lysis inhibition is not dependent on HLA-E expression, since no HLA-E protein was detected on K562 cells; HLA-G1 is therefore able to inhibit NK lysis by itself.

HLA-G inhibits the allogeneic proliferative response.

HLA-G is a non-classical MHC class I molecule expressed at the feto/maternal interface where it plays a role in materno-fetal tolerance by inhibiting NK cells. Expression of killing inhibitory receptors capable of interacting with HLA-G on T lymphocytes led us to hypothesize that HLA-G molecules could also modulate T cell responses, analyzed here in the context of the allogeneic proliferative response. Using LCL-HLA-G transfectants as stimulators of T cells present among peripheral mononuclear cells and K562-HLA-G1 transfectants as inhibitors in a classical mixed lymphocyte reaction, we showed that HLA-G is able to inhibit T cell allo-proliferation. These findings provide new insight into the role of HLA-G in preventing allograft rejection.

Molecular mechanisms controlling constitutive and IFN-gamma-inducible HLA-G expression in various cell types.

HLA-G molecule is thought to play a major role in down-regulating the maternal immune response by inhibiting NK and T cell cytolytic activities. We examined the molecular regulatory mechanisms that may control the restricted expression pattern of the HLA-G gene. We first analyzed protein interactions between nuclear extracts from the HLA-G-positive JEG-3 choriocarcinoma and the HLA-G-negative NK-like YT2C2 cell lines to a 244 bp regulatory element located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. This allowed characterization of cell-specific DNA-protein interactions that could account for differential cell-specific expression of the HLA-G gene. In particular two DNA-protein complexes were exclusively observed in YT2C2, suggesting that this HLA-G regulatory element is a target for putative cell-specific repressor factors. We further mapped nuclear factor binding sites to a 70 bp fragment in the upstream region of the regulatory element. We then investigated the effect of IFN-gamma on HLA-G gene expression. HLA-G cell surface expression was enhanced by IFN-gamma treatment in JEG-3 and U937 cell lines and peripheral blood monocytes while no effect was observed in tera-2 teratocarcinoma cell line. HLA-G transcriptional activity was increased only in JEG-3 and U937 cell lines. Activity of the 1.4-kb HLA-G promoter region was unchanged after IFN-gamma treatment in JEG-3 and Tera-2. These results suggest that both post-transcriptional and transcriptional mechanisms implicating IFN-responsive regulatory sequences outside the 1.4 kb-region are involved in IFN-gamma gene activation of the HLA-G gene.

HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes.

In the present study, we demonstrate that the non-classical MHC class I molecule HLA-G impairs specific cytolytic T cell functions in addition to its well-established inhibition of NK lysis. The antigen-specific cytotoxic T lymphocyte (CTL) response analyzed was mediated by CD8(+) T cells specific for the influenza virus matrix epitope, M58-66, presented by HLA-A2. The transfection of HLA-G1 cDNA in target cells carrying the M58-66 epitope reduced their lysis by these virus-specific CTL. This HLA-G-mediated inhibition of antigen-specific CTL lysis was (i) peptide dose dependent, (ii) reversed by blocking HLA-G with a specific mAb and (iii) still observed despite the blockade of HLA-E/CD94/NKG2A interaction. By inhibiting both CTL and NK functions, HLA-G appears to have an extensive role in immune tolerance.

IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes.

HLA-G plays an essential role in feto-maternal tolerance by inhibiting lysis by maternal NK cells. The factors that allow tissue-specific activation of HLA-G gene expression in trophoblasts remain to be characterized. We investigated the potential effect of IL-10, a cytokine which is secreted in placenta, on HLA-G gene transcription in trophoblasts. Using Northern blot, RNase protection assay and RT-PCR analysis, we demonstrated that IL-10 enhances steady-state levels of HLA-G transcription in cultured trophoblast cells. We further tested the effect of IL-10 on HLA-G gene transcription and protein expression in peripheral blood monocytes, showing that IL-10 can up-regulate HLA-G cell surface expression in this cell type. This effect of IL-10 is selective, since classical MHC class I products and MHC class II are down-regulated in monocytes following IL-10 treatment. Induction of HLA-G expression by IL-10 on monocytes may thus play a role in down-regulation of the immune response. We propose that IL-10 secretion by trophoblasts during pregnancy may also influence the HLA class I expression pattern at the feto-maternal barrier, thus protecting the fetus from rejection. This should be taken into consideration in the design of treatment for pathologies of pregnancy.

The immunotolerance role of HLA-G.

HLA-G is a non-classical MHC class I molecule involved in immune tolerance. We present our results concerning the effects of HLA-G on the cellular immune response, where it impairs both NK and T cell functions. We describe the NK inhibitory properties of HLA-G ex vivo, demonstrating its role in materno-fetal tolerance, which is supported by our in vitro studies using membrane-bound HLA-G1- and HLA-G2-transfected cells and a full-length soluble HLA-G molecule. We also report how HLA-G interacts at the T cell level, here exemplified by its inhibitory effect on both T cell allogeneic proliferative and Ag-specific CTL responses.

Delineation of human cord blood hematopoietic progenitor cell subsets with a unique monoclonal antibody AY19.

AY19, a unique mAb was used to better characterize the umbilical cord blood hematopoietic progenitor subpopulations. This mAb identifies an 85 kDa cell surface glycoprotein. In this present study we showed that AY19 mAb is reactive with 50-60% of the CD34+ cord blood cells. Extensive phenotypical studies revealed that AY19 mAb defines a novel CD34+ subset different from the ones defined by anti-CD90, anti-CD38, anti-CD33, anti-CD71, anti-CD19, anti-CD7 or anti-HLA-DR mAbs. We show that AY19 mAb reacts with both primitive and committed progenitors including myeloid and lymphoid progenitors. In addition, sorted CD34+high/AY19+ cells contain an increased number of CFU-GM and a decreased number of BFU-E compared with sorted CD34+high/AY19- cells. We show that AY19 mAb exhibits agonistic properties by inducing a significant increase in the size of CFU-GM colonies when added to serum-free liquid cultures of hematopoietic progenitors. This suggests that AY19 mAb identifies a cell surface receptor which may be involved in the regulation of hematopoietic cell proliferation.