Involvement of circulating soluble HLA-G after liver transplantation in the low immunogenicity of hepatic allograft.

Graft rejection is a critical risk in solid-organ transplantation. To decrease such risk, an understanding of the factors involved in low immunogenicity of liver allografts could potentially make it possible to transfer this tolerogenic property to other transplanted organs. HLA-G, a natural physiological molecule belonging to the Human Leukocyte Antigen class (HLA) Ib family that induces tolerance, is associated with fewer rejections in solid-organ transplantation. In contrast to HLA-G, HLA antigen incompatibilities between donor and recipient can lead to rejection, except in liver transplantation. We compared HLA-G plasma levels and the presence of anti-HLA antibodies before and after LT to understand the low immunogenicity of the liver. We conducted a large prospective study that included 118 patients on HLA-G plasma levels during a 12-month follow-up and compared them to the status of anti-HLA antibodies. HLA-G plasma levels were evaluated by ELISA at seven defined pre- and post-LT time points. HLA-G plasma levels were stable over time pre-LT and were not associated with patient characteristics. The level increased until the third month post-LT, before decreasing to a level comparable to that of the pre-LT period at one year of follow-up. Such evolution was independent of biological markers and immunosuppressive treatment, except with glucocorticoids. An HLA-G plasma level ≤ 50 ng/ml on day 8 after LT was significantly associated with a higher rejection risk. We also observed a higher percentage of rejection in the presence of donor specific anti-HLA antibodies (DSA) and an association between the increase in HLA-G plasma levels at three months and the absence of DSA. The low immunogenicity of liver allografts could be related to early elevated levels of HLA-G, which lead, in turn, to a decrease in anti-HLA antibodies, opening potential new therapeutic strategies using synthetic HLA-G proteins.

HLA-G Is Widely Expressed by Mast Cells in Regions of Organ Fibrosis in the Liver, Lung and Kidney.

We previously demonstrated that mast cells expressing HLA-G are associated with regions of hepatitis C virus-induced liver fibrosis. Here, we aimed to determine whether HLA-G expression in mast cells is specific to viral etiology, the liver, or to the general process of fibrosis. We enumerated HLA-G+ cells and mast cells by the immunohistochemistry of (i) liver blocks from 41 cases of alcoholic cirrhosis, (ii) 10 of idiopathic pulmonary fibrosis (IPF), and (iii) 10 of renal fibrosis. The nature of the HLA-G+ cells was specified by multiplex immunofluorescence using software. More than half of all HLA-G+ cells were mast cells in fibrotic areas of alcoholic cirrhosis and IPF. In the kidneys, subjected to fibrosis, the HLA-G+ cells were indeed mast cells but could not be counted. Moreover, in certain cases of the liver and lung, we observed a number of cellular nodes, which were secondary or tertiary follicles, in which HLA-G was highly expressed by B lymphocytes. In conclusion, HLA-G+ mast cells could be observed in the fibrotic regions of all organs studied. Previous studies suggest a protective role for HLA-G+ mast cells against inflammation and fibrosis. The observed follicles with B lymphocytes that express HLA-G may also reinforce their antifibrotic role.

Circulating levels of CXCL11 and CXCL12 are biomarkers of cirrhosis in patients with chronic hepatitis C infection.

BACKGROUND & AIMS: The chemokines CXCL10 (interferon ϒ-inducible protein 10 [IP-10]), CXCL11 (Human interferon inducible T cell alpha chemokine [I-TAC]), and CXCL12 (stromal cell derived factor 1 [SDF-1]) contribute to cell recruitment, migration, activation, and homing in liver diseases and their serum levels have been shown to be associated with the degree of liver inflammation or fibrosis in various etiologies. However, the data may be contradictory or insufficient, particularly for CXCL12, in the field of chronic HCV infection. Here, we aimed to provide evidence for these chemokines as biomarkers for chronic HCV infection. METHODS: We analyzed the serum concentration of the three chemokines in healthy donors (n = 39) and patients (n = 87) with chronic HCV infection. Chemokine serum levels were compared to the stage of liver inflammation and fibrosis obtained from liver biopsies. RESULTS: Serum CXCL10 and CXCL11 levels were higher at advanced stages of liver inflammation than at earlier stages, but the results were only of medium significance. Both serum CXCL11 and CXCL12 levels were significantly higher in cirrhotic patients than those with low or medium stages of fibrosis. The AUROCs were 0.8167 and 0.8574, respectively, for the diagnosis of cirrhotic patients. CONCLUSION: These data provide evidence for the value of CXCL10, CXCL11, and CXCL12 as biomarkers of liver inflammation and fibrosis during chronic HCV infection. Serum CXCL10 and CXCL11 levels were associated with liver inflammation, but the level of significance was insufficient. However, serum CXCL11 and CXCL12 levels were elevated in cirrhotic patients, showing equivalent diagnostic accuracy as the existing established single serum fibrosis markers or algorithms.

The anti-fibrotic role of mast cells in the liver is mediated by HLA-G and interaction with hepatic stellate cells.

BACKGROUND & AIMS: We have reported a significant association between HLA-G expression or the number of hepatic mast cells and liver fibrosis. Here, we investigated the role of HLA-G and mast cells in liver fibrosis, focusing, in particular, on interactions between human mast and stellate cells. METHODS: Human mast cells (HMC cell line, CD34-derived mast cells, or tissue-derived mast cells) were co-cultured with purified human hepatic stellate cells (HSCs), and collagen I production by HSCs was evaluated. Mast cells and HSCs were characterized by immunocytochemistry. Various conditions were tested: different times in direct or indirect contact, presence or absence of cytokines, addition or not of HLA-G, and presence or absence of specific protease inhibitors. RESULTS: The reciprocal interaction between HSCs and mast cells led to the attraction of mast cells to HSCs in vivo and in vitro, and to a significant decrease in collagen production, at all times of co-culture, following the direct or indirect contact of mast cells with HSCs alone or in the presence of TGF-ß, IFN-α or IL-10. We identified the diffusible factors involved in collagen I degradation as mast cell proteases. Moreover, HLA-G expression increased during the co-culture of HSCs and mast cells, with HLA-G acting on both mast cells and HSCs, to enhance collagen I degradation. CONCLUSIONS: Mast cells play a beneficial, anti-fibrotic role in liver fibrosis, via the HLA-G-mediated decrease of collagen I. These findings are consistent with high levels of cross-communication between mast cells and hepatic stellate cells and the role of HLA-G.

Serum CXCL10, CXCL11, CXCL12, and CXCL14 chemokine patterns in patients with acute liver injury.

BACKGROUND & AIMS: The chemokines CXCL10 (interferon ϒ-inducible protein 10 [IP-10]), CXCL11 (Human interferon inducible T cell alpha chemokine [I-TAC]), CXCL12 (stromal cell derived factor 1 [SDF-1]), and CXCL14 (breast and kidney-expressed chemokine [BRAK]) are involved in cell recruitment, migration, activation, and homing in liver diseases and have been shown to be upregulated during acute liver injury in animal models. However, their expression in patients with acute liver injury is unknown. Here, we aimed to provide evidence of the presence of circulating CXCL10, CXCL11, CXCL12, and CXCL14 during human acute liver injury to propose new inflammation biomarkers for acute liver injury. METHODS: We analyzed the serum concentration of the studied chemokines in healthy donors (n = 36) and patients (n = 163) with acute liver injuries of various etiologies. RESULTS: Serum CXCL10, CXCL11 and CXCL12 levels were elevated in all the studied groups except biliary diseases for CXCL11. CXCL14 was associated with only acute viral infection and vascular etiologies. The strongest correlation was found between the IFN-inducible studied chemokines (CXCL10 and CXCL11) in all patients and more specifically in the acute viral infection group. CONCLUSION: These data provide evidence for the presence of circulating CXCL10, CXCL11, CXCL12, and CXCL14 during acute liver injury and are consistent with data obtained in animal models. CXCL10, CXCL11 and CXCL12 were the most highly represented and CXCL14 the least represented chemokines. Differential expression patterns were obtained depending on acute liver injury etiology, suggesting the potential use of these chemokines as acute liver injury biomarkers.

Biology of the immunomodulatory molecule HLA-G in human liver diseases.

The non-classical human leukocyte antigen-G (HLA-G), plays an important role in inducing tolerance, through its immunosuppressive effects on all types of immune cells. Immune tolerance is a key issue in the liver, both in liver homeostasis and in the response to liver injury or cancer. It would therefore appear likely that HLA-G plays an important role in liver diseases. Indeed, this molecule was recently shown to be produced by mast cells in the livers of patients infected with hepatitis C virus (HCV). Furthermore, the number of HLA-G-positive mast cells was significantly associated with fibrosis progression. The generation of immune tolerance is a role common to both HLA-G, as a molecule, and the liver, as an organ. This review provides a summary of the evidence implicating HLA-G in liver diseases. In the normal liver, HLA-G transcripts can be detected, but there is no HLA-G protein. However, HLA-G protein is detectable in the liver tissues and/or plasma of patients suffering from hepatocellular carcinoma, hepatitis B or C, or visceral leishmaniasis and in liver transplant recipients. The cells responsible for producing HLA-G differ between diseases. HLA-G expression is probably induced by microenvironmental factors, such as cytokines. The expression of HLA-G receptors, such as ILT2, ILT4, and KIRD2L4, on liver cells has yet to be investigated, but these receptors have been detected on all types of immune cells, and such cells are present in liver. The tolerogenic properties of HLA-G explain its deleterious effects in cancers and its beneficial effects in transplantation. Given the key role of HLA-G in immune tolerance, new therapeutic agents targeting HLA-G could be tested for the treatment of these diseases in the future.

Immunomodulatory properties of HLA-G in infectious diseases.

HLA-G is a nonclassical major histocompatibility complex molecule first described at the maternal-fetal interface, on extravillous cytotrophoblasts. Its expression is restricted to some tissues in normal conditions but increases strongly in pathological conditions. The expression of this molecule has been studied in detail in cancers and is now also beginning to be described in infectious diseases. The relevance of studies on HLA-G expression lies in the well known inhibitory effect of this molecule on all cell types involved in innate and adaptive immunity, favoring escape from immune control. In this review, we summarize the features of HLA-G expression by type of infections (i.e, bacterial, viral, or parasitic) detailing the state of knowledge for each pathogenic agent. The polymorphism, the interference of viral proteins with HLA-G intracellular trafficking, and various cytokines have been described to modulate HLA-G expression during infections. We also discuss the cellular source of HLA-G, according to the type of infection and the potential role of HLA-G. New therapeutic approaches based on synthetic HLA-G-derived proteins or antibodies are emerging in mouse models of cancer or transplantation, and these new therapeutic tools may eventually prove useful for the treatment of infectious diseases.

Expression of HLA-G by mast cells is associated with hepatitis C virus-induced liver fibrosis.

BACKGROUND & AIMS: Infection with hepatitis C virus is a worldwide health problem. An inadequate Th2 cytokine response promotes the fibrosis-cirrhosis fate. Immune-modulating molecules favoring a Th2 profile, such as HLA-G molecules of the HLA class Ib family, may play a role in chronic hepatitis. HLA-G contributes to the escape of tumors, and their involvement in viral infections has been increasingly described. The aim of this work was to study the expression of HLA-G in the liver, its cellular source and its regulation in cases of chronic C hepatitis. METHODS: HLA-G cells in blocks of liver derived from patients infected with HCV were labeled by immunohistochemistry and enumerated. Double immunofluorescence allowed the identification of the cellular source. HLA-G secretion by a human mast cell line was quantified by ELISA after various stimulations. After treatment with IFN-α, real-time PCR was performed to determine the kinetics of cytokine expression profiles, followed by heat map clustering analysis. RESULTS: The number of HLA-G+ cells was significantly associated with the area of fibrosis. For the first time, we identify the HLA-G+ cells as being mast cells. HLA-G secretion was significantly induced in human mast cells stimulated by IL-10 or interferons of class I. The transcriptome of the secretome of this cell line stimulated by IFN-α revealed that (i) the HLA-G gene is upregulated late, and that (ii) T lymphocytes and NK cells are recruited. CONCLUSIONS: These findings suggest an autocrine loop in the genesis of HCV liver fibrosis, based on mast cells expressing HLA-G.

Indoleamine 2,3-dioxygenase activity as a potential biomarker of immune suppression during visceral leishmaniasis.

Leishmania parasites induce an immunomodulation by subverting the host immune response towards a CD4(+) Th2 lymphocytic cell response that favors parasite persistence. Here, we report that after successful treatment of visceral leishmaniasis due to Leishmania infantum, an immune reconstitution syndrome revealing hip septic arthritis was associated with a switch from Th2 towards a Th1 cytokine profile, and a decrease in the level of immunomodulating factors, such as soluble HLA-G and indoleamine 2,3-dioxygenase (IDO) activity. We then measured IDO activity in a cohort of 39 patients and uninfected control subjects. Results showed significantly enhanced IDO activity in patients with visceral Leishmania infection, compared with uninfected control subjects (P < 0.001), but also compared with treated patients (P < 0.05). A decrease in IDO activity could constitute a relevant biomarker for the restoration of the immune response during visceral leishmaniasis.

High level of soluble HLA-G in amniotic fluid is correlated with congenital transmission of Toxoplasma gondii.

The expression of human leukocyte antigen (HLA)-G on cytotrophoblast cells contributes to maternal-fetal tolerance. Soluble forms of HLA-G (sHLA-G) can be detected in amniotic fluid (AF) and a decrease of sHLA-G is known to be correlated to fetal loss. In this work we investigated the role of sHLA-G in the transplacental passage of the protozoan parasite Toxoplasma gondii, responsible for congenital toxoplasmosis in about 30% of fetuses when primary infection (PI) occurs during pregnancy. We determined the sHLA-G concentration in 61 AF from women with PI and 24 controls. Our results showed higher sHLA-G levels in AF from PI than in controls (p<0.001). Moreover sHLA-G level from congenitally infected fetuses (n=12) was higher than in fetus in whom congenital infection was ruled out (n=49, p<0.05). These data suggest that sHLA-G could participate in immunomodulation necessary to avoid fetal loss due to Toxoplasma infection, but that over-expression could favor congenital transmission.

Biology of HLA-G in cancer: a candidate molecule for therapeutic intervention?

Although the expression of the non-classical HLA class I molecule HLA-G was first reported to be restricted to the fetal-maternal interface on the extravillous cytotrophoblasts, the distribution of HLA-G in normal tissues appears broader than originally described. HLA-G expression was found in embryonic tissues, in adult immune privileged organs, and in cells of the hematopoietic lineage. More interestingly, under pathophysiological conditions HLA-G antigens may be expressed on various types of malignant cells suggesting that HLA-G antigen expression is one strategy used by tumor cells to escape immune surveillance. In this article, we will focus on HLA-G expression in cancers of distinct histology and its association with the clinical course of diseases, on the underlying molecular mechanisms of impaired HLA-G expression, on the immune tolerant function of HLA-G in tumors, and on the use of membrane-bound and soluble HLA-G as a diagnostic or prognostic biomarker to identify tumors and to monitor disease stage, as well as on the use of HLA-G as a novel therapeutic target in cancer.

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia.

OBJECTIVES: Waldenstrom Macroglobulinemia (WM) is a B-cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta-2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. METHODS: We assessed the serum levels of the total soluble HLA-I molecules and the HLA-Gs molecules in 105 patients with IgM-related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. RESULTS: We found higher levels of HLA-Is in WM, compared to IgM MGUS and healthy donors. HLA-Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA-Is at the cut-off of 1.8 microg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA-Is level was strongly associated with high serum beta2M level >3 mg/L [OR = 2, (CI 95% 1.1-5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5-4.1), P = 0.06] and haemoglobin < or =11.5 g/dL [OR = 3.3, (CI 95% 1.2-9.7); P = 0.03]. High levels of serum HLA-Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM-MGUS status. CONCLUSION: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells.

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.

Expression of functional soluble human leucocyte antigen-G molecules in lymphoproliferative disorders.

Membrane-bound and soluble human leucocyte antigen-G (sHLA-G) molecules display immunotolerant properties favouring tumour cell escape from immune surveillance. sHLA-G molecules have been detected in several tumour pathologies; this study aimed to evaluate sHLA-G expression in lymphoproliferative disorders. sHLA-G plasma level was significantly increased in 110 of 178 newly diagnosed lymphoid proliferations cases i.e. 59% of chronic lymphocytic leukaemia, 65% of B non-Hodgkin lymphomas (NHL) and 58% of T-NHL. To assess the mechanisms involved in this secretion, the differential effect of cytokines was tested in in vitro cultures of NHL cells. A significant induction of sHLA-G level was shown in T-NHL in contrast with B-NHL and normal equivalent cells, after cytokine stimulation with (i) interferongamma (IFNgamma), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor, (ii) IL-10 and (iii) transforming growth factor beta. An impact of microenvironment on sHLA-G expression was found in B-NHL as shown by the in vitro effect of addition of normal monocytes. Finally, a functional effect of sHLA-G molecules purified from pathologic plasma was demonstrated by their strong capacity to inhibit T-cell proliferation at concentrations currently observed during these disorders. These results suggest that functional sHLA-G molecules are upregulated in lymphoproliferative disorders which can support their potential immunomodulatory role during this pathology.

Spontaneous megakaryocytic colony formation does not discriminate between essential thrombocythemia and polycythemia vera.

Laboratory detection of spontaneous growth of colony-forming unit-megacaryocytes (CFU-MK), allowing us to distinguish essential thrombocythemia (ET) from reactive thrombocytosis, is therefore useful for the diagnostic of this myeloproliferative disorder. Whether CFU-MK assays allow us to discriminate at least partly between ET and other myeloproliferative disorders such as polycythemia vera (PV) remains, however, to be established. To gain insights about this point, we have performed CFU-MK cultures from bone marrow cells of patients diagnosed with ET (n = 42) or PV (n = 50) using a standardized collagen-based serum-free method. Spontaneous growth of CFU-MK was similarly detected in both 40/42 patients with ET and 47/50 patients with PV. These data suggest clearly that the CFU-MK assay is useful to detect not only ET, but also PV, but fails to discriminate, even partly, between these two myeloproliferative disorders.

Soluble HLA-G molecules increase during acute leukemia, especially in subtypes affecting monocytic and lymphoid lineages.

Human leukocyte antigen G (HLA-G) molecules exhibit immunomodulatory properties corresponding to nonclassic class I genes of the major histocompatibility complex. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membrane-bound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-gamma in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.

Total soluble HLA class I and soluble HLA-G in multiple myeloma and monoclonal gammopathy of undetermined significance.

Serum beta2-microglobulin, the light chain of the HLA class I molecular complex, remains one of the best survival prognostic factors in multiple myeloma, but other HLA class I molecules might be of interest in monoclonal gammopathies. In this study, we evaluate total soluble HLA class I (HLA-Is) and soluble HLA-G (HLA-Gs) in 103 patients with newly diagnosed multiple myeloma, 30 patients with monoclonal gammopathy of undetermined significance (MGUS), and 30 healthy subjects, studying their prognostic value in multiple myeloma. In multiple myeloma patients, HLA-Is and HLA-Gs median values were 0.8 microg/mL and 28 ng/mL, respectively. Median HLA-Is concentration was higher in stage II and III multiple myeloma patients than in stage I multiple myeloma, MGUS, and control patients. Median HLA-Gs was significantly lower in healthy controls than in MGUS and multiple myeloma patients. A high level of HLA-Is (> or =2.1 microg/mL) was predictive of short survival (P = 0.017). For each given level of beta2-microglobulin, the relative risk of death was higher for patients with HLA-Is > or = 2.1 microg/mL than in patients with a lower level (P = 0.047). HLA-Gs, a marker of monoclonal gammopathy, was of no prognostic value, but the addition of HLA-Is to beta2-microglobulin produced an efficient prognostic score (P < 0.0001). HLA-Is is a new marker of multiple myeloma tumor load and provides additional survival prognostic information to beta2-microglobulin.

MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells.

Dendritic cells (DC) play a central role in the immune response, linking innate and adaptative responses to pathogens. Myeloid DC (MDC) produce interleukin-12 in response to bacterial stimuli, whereas plasmacytoid DC (PDC) produce high levels of type I interferon upon viral infection. Human leukocyte antigen (HLA)-DR engagement has been shown to induce apoptosis in various antigen-presenting cells (APC). We now report the consequences of HLA-DR molecule engagement in human PDC, which had thus far not been studied as a result of the difficulty in isolating such cells. HLA-DR engagement on PDC, obtained using a two-step, immunomagnetic separation, led to recruitment of HLA-DR molecules at the site of engagement in mature but not immature PDC. In contrast, relocalization of protein kinase C (PKC) isoenzymes, indicating PKC activation, was observed at the site of HLA-DR engagement and was accompanied by relocalization of a lipid raft marker, the ganglioside M1 staining, in immature and mature PDC. Similar to MDC, HLA-DR-mediated apoptosis was regulated throughout PDC maturation. Freshly isolated PDC were resistant, whereas CD40 ligand-matured PDC were sensitive to HLA-DR-mediated apoptosis. Neither caspase activation nor PKC activation was required for HLA-DR-mediated apoptosis. However, the intrinsic pathway of apoptosis was implicated as mature PDC underwent mitochondrial depolarization in response to HLA-DR engagement. These data provide further arguments for considering HLA-DR-mediated apoptosis as a conserved mechanism of regulating survival of diverse APC and support the ongoing development of humanized ligands for HLA class II molecules as therapeutic tools for use in lymphoproliferative disease.

Loss of heterozygosity, a frequent but a non-exclusive mechanism responsible for HLA dysregulation in non-Hodgkin's lymphomas.

The frequent alteration of human leucocyte antigen (HLA) class I molecule expression observed in non-Hodgkin's lymphomas (NHL), similarly to solid tumours, has been reported to favour tumoral escape from the immune system. In order to identify the underlying mechanisms, we analysed 15 HLA defective NHL including partial (n = 10) and total class I (n = 5) loss, as well as HLA class II defects (n = 5). The HLA defect concerned HLA-A and -B antigens in 14 of 15 cases. In the cases with partial defect, the use of specific allelic monoclonal antibodies detected a defect of both alleles of A or B loci in six of seven tested cases. Allelic reverse transcription polymerase chain reaction (RT-PCR) demonstrated defects in six of nine cases, including four alterations of both A and B mRNA alleles. Real-time quantitative RT-PCR (RQ-PCR) did not detect the HLA-DR transcript in the two negative HLA-DR lymphomas, contrasting with the presence of CMH II transactivator (CIITA) transcript. Loss of heterozygosity (LOH) was detected in nine of 14 cases through variable pattern of nine microsatellites markers of the HLA locus. Taken together, these findings demonstrate the complexity and the variability of the mechanisms underlying HLA protein deficiencies with a high frequency of LOH. The diversity of these mechanisms indicates the importance of positive selection of HLA altered clones in the development of these NHL cases.

Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules.

Human leukocyte antigen (HLA-G), a class Ib major histocompatibility complex molecule, is potentially relevant in the immune response through its various immune cell functions. Its expression noticed in some malignancies has also been shown on macrophages and dendritic cells (DC) in tumoral and inflammatory diseases. As DC constitute a key component in the immune response, this work aimed at assessing the expression of HLA-G at transcriptional and proteic levels during differentiation and maturation of the different DC subsets. We show that HLA-G transcription was induced during CD34+-derived DC differentiation and is associated with a cell-surface expression in half of cases and with a substantial secretion of soluble HLA-G in all cases. Results were very similar for monocyte-derived DC, but there was still a weak HLA-G cell-surface expression and a lower level of secretion. On the contrary, HLA-G transcription was weak in plasmacytoid DC without any HLA-G cell-surface expression and with a basal level of secretion. The mechanisms involved in HLA-G expression appear transcriptional and post-transcriptional. However, the amount of HLA-G transcripts and the expression of the protein are not related. HLA-G expression or secretion by DC may have negative consequences on the function of effective immune cells and also on DC themselves via the interaction with inhibitory receptors expressed by these cells. The capacity of DC to express or secrete HLA-G should be studied in the context of cellular therapy using DC in addition to its suppressive action in immune response.